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The challenge of enumerating Listeria monocytogenes in food
Accepted Manuscript The challenge of enumerating Listeria monocytogenes in food Anaïs Auvolat, Nathalie Gnanou Besse PII:
S0740-0020(15)00172-0
DOI:
10.1016/j.fm.2015.09.003
Reference:
YFMIC 2447
To appear in:
Food Microbiology
Received Date: 19 March 2015 Revised Date:
28 August 2015
Accepted Date: 3 September 2015
Please cite this article as: Auvolat, A., Besse, N.G., The challenge of enumerating Listeria monocytogenes in food, Food Microbiology (2015), doi: 10.1016/j.fm.2015.09.003. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT 1
The challenge of enumerating Listeria monocytogenes in food
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Anaïs Auvolat and Nathalie Gnanou Besse*.
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French Agency for Food, Environmental and Occupational Health & Safety (ANSES),
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Laboratory for Food Safety.
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(ANSES, Laboratoire de Sécurité des Aliments, 14 Rue Pierre et Marie Curie, 94701 Maisons
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Alfort, France)
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ABSTRACT
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Listeria monocytogenes is recognised as a serious foodborne pathogen in humans.
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However, food products are usually contaminated at low levels (i.e. <100 CFU/g) and there is
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still no adequate enumeration method for testing food. Much research has been carried out to
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improve Listeria enumeration methods, leading to several proposed alternative methods such
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as the most probable number technique, molecular-based methods and bacterial cell
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concentration techniques. Here, we catalogue the current knowledge concerning L.
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monocytogenes enumeration, with a particular focus on the problem of enumerating low
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level contamination.
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Keywords: Listeria monocytogenes, microbiological methods, enumeration, MPN,
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quantitative PCR, filtration, digital PCR.
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Running title: Current research on Listeria enumeration.
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* Corresponding author: Tel. +33 1 49 77 28 32; fax: +33 1 49 77 46 66; E-mail address:
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[email protected] (N. Gnanou Besse).
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INTRODUCTION
Despite its low incidence, foodborne listeriosis is characterised by life-threatening
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symptoms and a high mortality rate of up to 30% (Anonymous, 2000). In addition, the the
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presence of Listeria monocytogenes in food has important economic consequences, such as
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the withdrawal of products from the consumer marketplace and a decrease in sales for the
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incriminated products. Since 2000, an increase in listeriosis cases has been observed in
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several European countries, but reasons for this increase remain unclear. According to the
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European Union Summary Reports, there was a significant increase in the notification rate of
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listeriosis cases in humans between 2002 and 2006 (Anonymous, 2007). This notification rate
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remained at the same level in 2007, with 1558 such cases recorded in 26 Member States
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(Anonymous, 2009a). During the 2008-2012 period, the number of reported confirmed cases
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of human listeriosis in the European Union (EU) increased substantially (Anonymous, 2014).
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In 2012, there were 1642 confirmed human cases, which was a 10.5% increase compared
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with 2011 (1486). Moreover, 198 deaths were reported (case fatality rate of 17.8%)
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(Anonymous, 2014).
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Improvement of L. monocytogenes enumeration techniques is thus a subject of major
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concern in the field of food hygiene. Adequate enumeration methods are essential to provide
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reliable data for research studies in predictive microbiology, epidemiology, quantitative risk
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assessment, and for routine analysis or monitoring programmes in food processing plants.
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However, despite this need, there has been no adequate, sensitive enumeration method
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identified to date. Food products are usually contaminated at very low levels, i.e. at less than
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100 colony forming units (CFU) per gram of food product (Anonymous, 2012). Accordingly,
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the International Commission on Microbiological Specifications for Foods (1994) set the
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critical food-safety threshold at 100 CFU/g, for non “at-risk” populations. The recent
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ACCEPTED MANUSCRIPT European Community (EC) Regulation 2073/2005 (Anonymous, 2005) on microbiological
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criteria for foodstuffs established a quantitative limit for L. monocytogenes of 100 CFU/g,
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which is applicable to certain categories of ready-to-eat food products within their quality-
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based shelf-life. The manufacturer must be able to demonstrate that its product will not
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exceed this limit during storage, based on various types of data and studies, such as challenge
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tests, durability studies and predictive microbiology. Improving L. monocytogenes
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enumeration has inspired extensive research, leading to the proposal of several methods
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alternative to the standard reference method, most of them being described in the review of
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Gnanou Besse and Colin (2004). In most cases, L. monocytogenes is enumerated using the
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most probable number (MPN) technique, although there have been attempts to enumerate L.
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monocytogenes with molecular biology-based methods. Another approach to improve the
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sensitivity of the enumeration method has been to use a concentration technique, such as
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membrane filtration, which is already widely used for the microbiological analysis of water
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and other liquids.
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More recently, there have been changes and improvements in the above-cited
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methods: MPN methods have been combined with polymerase chain reaction (PCR)
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techniques to avoid numerous and fastidious confirmation steps, quantitative PCR methods
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have been improved by the inclusion of pre-amplification steps that purify and concentrate
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DNA better, and eliminate non-viable cells. More recently, digital PCR combining the
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principles of the MPN and molecular based methods allow accurate quantification of target
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cells, but its application in microbiology is still very limited.
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Comprehensive reviews of rapid methods and automation in microbiology for L.
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monocytogenes include advances in detection, typing, total viable cell count methodologies,
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instrumental analysis, PCR, biosensors, etc. (Churchill et al., 2006; Jasson et al., 2010;
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Rossmanith and Wagner 2010; Jadhav et al., 2012; Postollec et al., 2011. The objective of
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this paper is to review the most recent knowledge regarding L. monocytogenes enumeration in
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food, focusing in particular on the problem of low level enumeration.
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1. CONVENTIONAL METHODS: CULTURE-BASED TECHNIQUES
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1.1.
REFERENCE STANDARD METHOD:
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Direct plate counts are quite simple and fast, but are characterised by poor
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performance in terms of sensitivity, reproducibility, recovery of stressed cells, and sometimes
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selectivity (Jasson et al., 2010). This relatively high variability is a common feature to all
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conventional enumeration methods used in food microbiology. This variability needs to be
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taken into account, and should be considered when establishing tolerance limits and sampling
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plans for food control. Automation, such as the spiral plate method, can be an improvement
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on direct plate counts and can significantly reduce required manpower and material costs
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(Jasson et al., 2010).
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The European and International Standard method for enumeration of L.
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monocytogenes EN ISO 11290-2 (Anonymous, 1998, 2004) is cited as the reference standard
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method in the quantitative criteria of EC Regulation no. 2073/2005 for L. monocytogenes.
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This method consists of spreading decimally diluted samples and further decimal dilutions on
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selective agar plates. PALCAM agar, long prescribed by the reference method, cannot
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distinguish L. monocytogenes from other Listeria spp. colonies. Therefore, five typical
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colonies must be confirmed per plate and counts are based on the ratio of colonies confirmed
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as L. monocytogenes. This dramatically increased method variability. The specificity has
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however improved since 2004 with the introduction of a more specific agar, agar Listeria
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according to Ottaviani and Agosti (LOA agar), which distinguishes L. monocytogenes from
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ACCEPTED MANUSCRIPT other species of Listeria and thus better identifies the L. monocytogenes colonies for
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confirmation. LOA agar was adopted by the International Organization for Standardization
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(ISO) as the standard medium for L. monocytogenes detection and enumeration methods. This
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medium performs well, as demonstrated in terms of productivity ratio (at least 0.95),
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selectivity and detection ratio (Vlaemynck et al., 2000).
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Meanwhile, it has been shown that the precision of this standard method is relatively
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poor, especially at low counts. The method still lacks sufficient sensitivity to reliably quantify
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L. monocytogenes at 100 CFU/g and does not seem to be optimal for the examination of food
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products that are usually contaminated at low levels. In fact, plate counts are characterised by
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an enumeration limit of 100 CFU/g of original non-blended sample when spreading a total of
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1 mL of the blended (1/10 diluted) sample onto three 90 mm plates (or one 140 mm plate).
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Liquid products do not need any additional suspension thus eliminating the initial dilution
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that results from making a homogeneous suspension of the food sample and the theoretical
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limit of enumeration becomes 10 CFU/g. Furthermore, the variability of the method at this
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level is particularly high. According to the general EN ISO 7218 standard “Microbiology of
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food and animal feeding stuffs – General requirements and guidance for microbiological
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examinations”, (Anonymous, 2013):
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- the limit of detection, based on the formation of a single CFU, of the method, for a non-
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liquid sample, is 10 CFU/g when plating 1 mL of the blended sample.
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- the theoretical limit of quantitation is four times the limit of detection or 40 CFU/g when
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plating a total volume of 1 mL of the blended sample. Below this limit, the microorganism is
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considered to be present, but its concentration cannot be reliably quantified.
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- when fewer than 10 CFU combined total are present on the plates, the result must be
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expressed as an estimated result or with its measurement uncertainty.
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In addition, the EN ISO 7218 standard specifies that two successive dilutions must be
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analysed (1 plate per dilution), and when only one dilution can be analysed, then two plates
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per dilution should be used. Consequently, when enumerating a contamination level of 100 CFU/g (e.g.
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approximately 20 CFU in total when spreading in duplicate 1 mL of the decimally diluted
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sample on three plates), the result is associated with a 95 % confidence interval of 60 to 150
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CFU/g (EN ISO 7218 standard, 1996, Annex A: Table for confidence intervals for low
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number estimation).
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validation studies:
A validation study of EN ISO 11290-2 in its initial version (without the current
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amendment which replaced PALCAM agar with LOA agar) implementing an inter-laboratory
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trial was funded by the European Commission (Standards, Measurement and Testing Fourth
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Framework Programme Project SMT4-CT96-2098). The objective was to determine the
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precision of the methods in terms of repeatability (r) and reproducibility (R) using different
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sample types (meat, cheese, dried egg powder and reference material) inoculated at different
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levels. r is the difference, expressed as a ratio, between two results obtained on the same
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sample and in exactly the same conditions, in the same laboratory, whereas R is the
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difference, expressed as a ratio, between two results obtained on the same sample in two
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different laboratories. The overall repeatability was r = log10 0.58, and the overall
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reproducibility was R = log10 0.81 (Scotter et al., 2001). This means that for a sample having
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a true contamination of 100 CFU/g, a laboratory may find a result as low as 15 CFU/g, or as
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high as 646 CFU/g.
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The objective of the recent validation study of the revised Nordic Committee on Food
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Analysis (NMKL) method no. 136 “Listeria monocytogenes. Detection and enumeration in
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standard, was to determine the precision of the standard method in terms of repeatability and
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reproducibility using different food sample types. For a contamination level close to 100
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CFU/g (2.2 log CFU/g), the overall repeatability (r) of the method was 0.44, 0.91, and 0.66
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and the overall reproducibility (R) was 0.48, 1.08, and 0.54 for Brie cheese made from
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pasteurized milk, hot-smoked salmon, and cooked vacuum-packed ham, respectively
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(Loncarevic et al., 2008). In the presence of L. innocua the corresponding values reached
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r=0.76, 0.52, and 0.70 and R=0.87, 0.68,and 0.87 for the same three products, respectively.
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This means that for a sample having a true contamination level of 100 CFU/g, if R = 0.87 a
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laboratory may find a result as low as 13 CFU /g, or as high as 741 CFU/g.
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More recently, a new validation study of EN ISO 11290-2 (in its new version, with
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LOA agar) through inter-laboratory trial has been fully funded by the European Commission
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(CEN Mandate M381). The European Committee for Standardization (CEN)/Technical
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Committee 275/Working Group 6, in charge of standardisation in food microbiology methods
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at the European level, received a mandate from the European Commission to standardise
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and/or validate using inter-laboratory studies (ILS), a set of reference methods in food
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microbiology, including the reference methods for the detection and enumeration of L.
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monocytogenes in food. The ILS were conducted in 2013, and standards will be published by
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the end of 2016. Since Standards EN ISO 11290-1&2, under revision are applicable to all
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food, feed and food processing environments, the ILS were conducted on five matrices (cold-
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smoked salmon, milk powder, vegetables, environmental samples and cheese), representative
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of categories cited in EC Regulation no. 2073/2005 on microbiological criteria for foodstuffs.
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For quantitative studies, and for each matrix, four levels of contamination including a blank
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(0, 100-150, 1000-1500, 10 000-15 000 CFU/g), and two blind replicates per level were used.
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In total, eight samples per matrix were sent to each laboratory. These contamination levels are
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different countries participated in each trial. Results of the ILS were generally satisfactory,
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comparable, if not better in comparison with former validation studies. The revised version of
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EN ISO 11290-1&2, will include these results, and the estimation of the performances of the
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method, in terms of the standard deviations of repeatability and reproducibility, sensitivity,
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specificity and limit of detection.
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1.2.
CULTURING METHODS THAT INCLUDE A RESUSCITATION STEP:
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When enumerating low numbers of L. monocytogenes, the recovery of injured bacteria
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cells is an issue of great importance. In food, L. monocytogenes is often affected by one or
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several sources of stress caused by a variety of processing treatments including heating,
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freezing, drying, exposure to acids, exposure to disinfectants, and exposure to high osmotic
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pressure. Kang and Fung (1999) developed a solid media resuscitation method (thin agar
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layer method, TAL) to recover heat-injured L. monocytogenes. They overlaid 5 mL of non-
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selective tryptic soy agar (TSA) medium onto pre-poured and solidified modified Oxford
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medium (MOX). The MOX selective agents diffuse slowly to the top layer, allowing the cells
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on the TSA to undergo repair during the first hours of incubation. When enumerating heat-
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injured L. monocytogenes from milk, significantly higher counts were obtained with this
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protocol than with direct plating on MOX agar. Wu and Fung (2002, 2003, 2004) improved
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the TAL method by the simultaneous recovery and detection of four pathogens (including L.
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monocytogenes) from foods, the use of the ISOGRID to enumerate very low pathogen levels
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on TAL, and the use of Oxyrase® in the TAL system to stimulate recovery of pathogens in
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foods. Miller et al. (2010) evaluated the TAL procedure for the recovery of injured L. innocua
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cells exposed to thermal treatments. The TAL procedure (PALCAM agar with TSA+Yeast
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Extract agar overlay) recovered more bacteria (>1 log10) than the PALCAM direct count
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when used on parsley or meat products.
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1.3.
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CULTURING METHODS THAT INCLUDE A CELL CONCENTRATION STEP
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According to NF EN ISO 7218 “Microbiology of food and animal feeding stuffs –
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General requirements and guidance for microbiological examinations”, section 9.2.2: for the
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enumeration of low numbers of microorganisms, it is possible to concentrate the bacteria
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present by centrifugation or membrane filtration to improve enumeration. However, it is
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recommended to check the performance of the complete method in terms of sensitivity,
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selectivity, linearity and repeatability.
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Membrane filtration has been applied to the detection and enumeration of L.
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monocytogenes, with the detection of target strains by culturing the membrane on selective
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agar (Entis and Lerner, 2000; Gnanou Besse and Lafarge, 2001; Jehanno et al., 1999) or
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through various rapid techniques such as, for example, the direct epifluorescence filter
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technique (DEFT, Tortorello et al., 1997), electronic microscopy (Hale et al., 1990), or
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immunoassays (Carrol et al., 2000). The necessity of processing the food in a filterable form
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limits the applicability of membrane filtration because volumes that can be filtered depend on
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the sample type. The above-cited filtration protocols are generally quite labour-intensive, and,
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in most cases, they have been developed and evaluated for specific types of products.
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The hydrophobic grid membrane filter (HGMF) technique, including a filtration step,
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has been applied to L. monocytogenes enumeration (Entis and Lerner, 2000). This technique
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has been widely tested and has already been validated by AOAC International for several
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ACCEPTED MANUSCRIPT bacterial species in food (Entis, 1986, 1989, 1990, 2000). The HGMF is a membrane upon
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which is applied a hydrophobic grid to divide it into a large number of individual growth
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compartments (generally about 1600). The HGMF count is determined by MPN calculation.
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Thus, the counting range of the HGMF is expanded well beyond the actual number of
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observed growth units (effective MPN counting range of about three logarithmic cycles), and
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consequently covers a much larger range than is obtained with direct plate counts or
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membrane filtration, and with greater precision. HGMF consequently minimises both labour
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(needed for diluting) and the probability of losing data by misjudging platable dilution ranges.
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For the enumeration of L. monocytogenes in cold-smoked salmon, a sensitive
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enumeration method based on membrane filtration, followed by transfer of the filter to a
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selective medium, has been recently developed and standardised in France (Anonymous,
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2009b; Gnanou Besse et al., 2004). Briefly, the operating protocol consists of three filtration
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steps conducted in parallel, using respectively 5, 15 and 30 mL of a 1:10 dilution of the
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salmon suspension (previously treated with an enzyme surfactant) through 0.45 µm pore-size
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cellulose ester membranes. The filters are then transferred to LOA agar for cultivation.
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Colonies are counted on filters containing less than 100 colonies. Typical L. monocytogenes
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colonies need to be reisolated and confirmed according to the EN ISO 11290-2 method.
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When contamination levels are unknown, the standard ISO 11290-2 method should also be
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conducted to be able to enumerate samples contaminated at levels higher than 200 CFU/g.
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The results obtained with this newly developed method have been compared with those
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obtained with the reference EN ISO 11290-2 method. The membrane filtration method
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provided more precise results for the enumeration of L. monocytogenes from both artificially
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and naturally contaminated cold-smoked salmon (Gnanou Besse et al., 2004). It improved the
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enumeration sensitivity to 0.2 CFU/g, instead of 10 to 100 CFU/g with the reference EN ISO
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11290-2 method. The method has been validated through an ILPT (Gnanou Besse et al.,
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0.6 and 1.6 log10 CFU/g) was used as matrix. Unlike most validation studies, very low
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contamination levels were chosen to be more representative of actual foodborne
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contamination levels. Reproducibility standard deviations were 0.23 log10 CFU/g and 0.15
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log10 CFU/g at the lower level and the higher level, respectively. Under certain conditions, the
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measurement uncertainty can be derived from the method’s reproducibility standard
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deviation, calculated to be 0.46 log10 CFU/g for the lower contamination level and 0.30 log10
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CFU/g for the higher contamination level. These values can be considered as satisfactory for
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such low contamination levels. This sensitive enumeration method is quite simple to use, is
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based on classical microbiology and relies on the use of the same selective agar as the
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reference EN ISO 11290-2 method. This method can be useful for precise estimation at
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densities of around 100 CFU/g or to conduct challenge tests, durability studies and
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quantitative risk assessment studies under more realistic conditions, at low contamination
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levels. The method has already been successfully used to monitor the growth of L.
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monocytogenes in cold-smoked salmon at various initial inoculum densities (Gnanou Besse et
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al., 2006), and to estimate contamination levels and growth of L. monocytogenes in naturally
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contaminated cold-smoked salmon (Beaufort et al., 2007). Nevertheless, the filtration method
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requires the use of a specific apparatus and is more labour-intensive than the reference
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method. The use of a very detailed protocol and a training period is necessary before the
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method can be performed satisfactorily and to avoid technical difficulties such as filtering
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problems (Gnanou Besse et al., 2008). Moreover, this method cannot be implemented on
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other food products without additional validation studies.
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The applicability of the membrane filtration method to other categories of matrices in
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comparison with the EN ISO 11290-2 modified reference method was evaluated mainly with
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artificially contaminated foods (Baudouin et al. 2010). In that study, all foods could be
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meat and seafood products being the most easily filtered). However, even for those products
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that were difficult to filter, approximately 5 mL could be concentrated on a filter, enabling
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analysis of a greater quantity of food. However, the proposed method seems to be difficult to
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apply to some food products, due to background microflora interfering with colony reading.
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This was the case for several cheese and delicatessen matrices. Finally, an evaluation of this
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method was performed with several categories of foods naturally contaminated with L.
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monocytogenes (Barre et al., 2015). The results obtained with this technique were compared
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with those obtained from the modified reference EN ISO 11290-2 method for the
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enumeration of L. monocytogenes in food, and provided more precise results. In most cases,
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the filtration method makes it possible to examine a greater quantity of food, thus greatly
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improving the sensitivity of the enumeration. In particular, the method provides good results
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for seafood products, vegetables, and some meat products (minced or unprocessed meat,
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some sausages, etc.). However, it was unsuitable for some food categories (cheese, pastries,
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delicatessen) due to filtration problems and background microflora interference.
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In conclusion, the membrane filtration method is an improvement over the reference
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standard method for enumerating low levels of L. monocytogenes in certain food matrices,
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with satisfactory sensitivity. The sensitivity of the method is critical when enforcing the
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European regulatory contamination limit of 100 CFU/g as well as conducting shelf-life
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studies under realistic conditions of low level contamination.
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1.4.
THE MOST PROBABLE NUMBER TECHNIQUE
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The MPN technique relies on probability statistics; results are directly related to the
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frequency of occurrence in a dilution series of a sample, which is used to calculate the
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calculated from the number of positive tubes out of a total number of tubes tested, for a series
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of consecutive dilutions. This technique has two main assumptions. First, it is assumed that
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organisms are distributed randomly throughout the sample, and that cell clumping does not
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occur . This is an important parameter to take into account, considering the analytical test
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portion is likely to be much smaller than the food sample and one wants to apply the results to
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the entire food sample and not just to the portion that was tested. Second, it is assumed that
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each dilution, when properly incubated, will exhibit growth when one or more organisms are
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present (Oblinger and Koburger, 1975). In most cases, L. monocytogenes MPN procedures
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rely on conventional detection methods for media, incubation times and isolation protocols,
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such as those published by, for example, the US Food and Drug Administration (FDA), the
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US Department of Agriculture’s Food Safety and Inspection Service (USDA-FSIS), the
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International Standard Organization (ISO) or the Netherlands Government Food Inspection
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Service (NGFIS) (see Anonymous, 1992; Aymerich et al., 2004; Blysick-McKennal and
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Schaffner, 1994; Capita and Alonso-Calleja, 2003; Gombas et al., 2003; Swaminathan et al.,
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1994; Tortorello et al., 1997; Tran and Hitchins, 1996).. This method of estimating bacterial
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densities is still widely used for L. monocytogenes, both for research studies and routine
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analysis. Some of these MPN methods have already been reviewed in Gnanou Besse and
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Colin (2004): Blysick-McKennal and Schaffner, 1994; Capita and Alonso-Calleja, 2003;
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Cortesi et al., 1997; Dalgaard and Jorgensen, 1998; De Buyser et al., 1990; Farber and Daley,
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1994; Hayes et al., 1992; Inoué et al., 2000; Jemmi and Keusch, 1992; Jorgensen and Huss,
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1998; Tran and Hitchins, 1996; Yu and Fung, 1993; Yu et al., 1995., etc. The MPN technique
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has numerous useful applications. When the contamination level is unknown, it is important
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to use an efficient method for a wide range of bacterial contamination levels. With the MPN
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method, estimates of a population can be made using any number of dilutions, as in the plate
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ACCEPTED MANUSCRIPT count method. Yu and Fung (1993) showed that a five-tube MPN method using Fung-Yu
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tubes was effective in enumerating both low and high levels of L. monocytogenes (from 10 to
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106 CFU/mL) in cooked chopped hams, with a correlation coefficient of 0.99 when compared
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with direct plate counts. However, Carroll et al. (2000) showed that, in spiked meat products,
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when the inoculum level was high, the MPN procedure tends to underestimate the number of
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L. monocytogenes, whereas, when the inoculum is low, MPN tends to overestimate the
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number of L. monocytogenes. Hildebrandt and Schott (2001) compared the direct plate count
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method with the MPN method for quantification of L. monocytogenes in artificially and
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naturally contaminated beef samples sold at retail. The detection limit of MPN was 66%
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lower than the plate count, and detected more positive samples. Nevertheless, the plate count
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method is preferred for routine use, due to its slightly higher productivity and much lower
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variation in results.
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However, when contamination levels are suspected of being low (<1 CFU/g) then the
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MPN method is the more appropriate choice for enumeration. Large sample size is what
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makes this technique extremely powerful. No other enumeration technique allows
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enumeration of a few cells per 100 g or covers such a large range of samples.
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The fundamental disadvantage of using the MPN method is that L. monocytogenes
343
must be recovered and confirmed from each presumptive positive tube. Other disadvantages
344
often associated with the MPN method include various aspects of the technique, such as time,
345
materials, cost, space, precision and selectivity.
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MPN estimates of bacterial densities generally lack precision. Proper interpretation of
347
the “confidence interval” around a given value, as reported in the tabular MPN data, is
348
fundamental. The tabular MPN value actually represents a range, not an absolute value. MPN
349
estimates are often credited with a precision they do not live up to (Oblinger and Koburger,
15
ACCEPTED MANUSCRIPT 350
1975). However, the measurement uncertainty of MPN methods is often of the same order as
351
that of direct plate count methods. The presence of competitive microflora in the food sample may affect MPN
353
performance (Blysick-McKennal and Schaffner, 1994; Tran and Hitchins, 1996). The
354
problem of competition between bacterial strains must be considered when several Listeria
355
species are present in food samples. From a practical perspective, overgrowth by a non-
356
pathogenic species of Listeria may mask the presence of low numbers of L. monocytogenes in
357
the original food sample, and thus result in false-negative results (Gnanou Besse et al., 2005,
358
2010). An enrichment step may lead either to the non-detection of L. monocytogenes or to an
359
underestimation of L. monocytogenes counts when combined with an MPN procedure.
360
Although conflicting results have been reported, in the case of induced stress, the selective
361
enrichment procedure of most standard methods may also lead to lower recovery rates of
362
injured or stressed bacterial cells, compared to non-selective enrichment procedures, for
363
example in cheeses (Rijpens et al., 2004). Moreover, common food industry sources of stress
364
may have a high impact on single-cell lag time and growth probabilities of L. monocytogenes
365
in half-Fraser broth, meaning that the risk of false-negative samples may be high for very low
366
contamination levels, depending on the type of stress (Dupont et al., 2009).
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All of the enrichment, isolation and identification steps are generally included in the
368
MPN protocol, and are performed and replicated for each dilution. To counteract the above
369
mentioned problems, the MPN technique has recently been combined with PCR and real-time
370
PCR for confirmation of positive results.
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PCR analysis was used by Aymerich et al. (2004) to confirm MPN tubes for
372
enumerating L. monocytogenes in spiked cooked and fermented meat products (about 2 to 3
373
log10 CFU/g). The prfA gene was chosen for amplification, and the method produced final
374
results in two days.
16
ACCEPTED MANUSCRIPT Martin et al. (2004) compared MPN with PCR confirmation (MPN-PCR) with direct
376
plating on PALCAM agar for enumerating L. monocytogenes in fermented sausage (chorizo).
377
The MPN analysis was performed using half-Fraser broth for 40 h at 30 °C. The PCR reaction
378
targeted the prfA gene. The counts determined by MPN-PCR were generally higher than plate
379
counts, which were impossible in some cases, due to very abundant interfering bacteria.
380
However, the technique remained labour-intensive because pre-PCR treatments for
381
confirmation of positive results included centrifugation, and filtration or resin-based
382
techniques (Chelex-100), and visualization of the PCR products required gel electrophoresis.
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In 2010, Jeyaletchumi et al. evaluated a MPN-PCR technique, based on the
384
combination of the FDA-Bacteriological Analytical Manual (BAM) selective enrichment and
385
PCR detection of the listeriolysin O gene, to detect L. monocytogenes in salad vegetables.
386
This method proved to be sensitive, accurate, linear and significantly reduced time and
387
labour: results are obtained within two days, compared with the traditional five days.
388
However, although the correlation between spiking concentration and microbial levels was
389
greater than that of the EN ISO reference method and FDA-BAM method, it was only 67%
390
for the artificially contaminated samples. Moreover, several centrifugation steps were
391
necessary for DNA extraction and purification, and gel electrophoresis was required to
392
visualise the PCR products, making the technique very labour-intensive.
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de Martinis et al. (2007) developed a SYBR green based real-time PCR method,
394
targeting the 16S rRNA gene, to accelerate MPN tube confirmation of L. monocytogenes in
395
several foods (spiked milk, lettuce, smoked salmon, Brie cheese, ice cream, pork pâté, salami,
396
ready-to-eat shrimp, raw ground beef and fresh soft cheese). The use of real-time PCR
397
produced results in two days instead four by reducing the time needed to confirm the presence
398
of L. monocytogenes in the MPN tubes. For contamination levels from 70 to 100 CFU/g,
399
there was good agreement between the conventional and PCR MPN methods. Some foods
17
ACCEPTED MANUSCRIPT required diluted enrichment sample extracts to obtain positive PCR results, comparable with
401
those of the standard MPN culture method. This suggests a concentration-dependent matrix
402
interference in the real-time PCR assay. Moreover, the boiling-water DNA extraction method
403
requires successive centrifugations at 8000 ×g for 10 min, increasing laboratory time for this
404
assay.
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de Oliveira et al. (2010) also evaluated the combination of the MPN enumeration
406
method with real-time PCR confirmation of L. monocytogenes. The products tested included
407
minimally processed leafy vegetables samples. L. monocytogenes was enumerated in six
408
artificially and two naturally contaminated samples using the MPN technique with detection
409
and confirmation by conventional culturing-based methods and with real-time PCR targeting
410
the16S rRNA gene of L. monocytogenes. The MPN assay, with real-time PCR confirmation,
411
detected 1-5 CFU per 50 g with no interference from the background microflora. There was
412
generally good agreement between conventional MPN and the real-time PCR method, which
413
was easy to perform and produced results in 48 h, instead of seven days for the conventional
414
method. However, for each MPN tube, the culture DNA must be extracted (boiling-water
415
method) which included several rounds of centrifugation. Another problem is the interference
416
with inhibitors contained in the product. Since this phenomenon occurred more frequently in
417
leafy vegetables with a stronger pigmentation, the authors suggested that the green pigment
418
(chlorophyll) contained in the leaves could be the inhibitor; thus the largest quantities of
419
sample showed the lowest number of positive reactions. Dilution of the DNA extracts (1:50)
420
was therefore needed before PCR analysis. This pigment interference makes this protocol
421
cumbersome and demonstrates an important limitation of this method.
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Methods for confirming MPN tubes, other than PCR, have also been investigated.
423
Cruz et al. (2012) compared six commercial kits (Clearview™, Reveal®, TECRA®,
424
VIDAS® LDUO, VIP™ and Petrifilm™ kits) for detecting Listeria species in seafood and
18
ACCEPTED MANUSCRIPT environmental samples using the standard FDA BAM enrichment protocol with the MPN
426
technique. The commercial kits gave similar results as the BAM Listeria confirmation
427
protocol for the seafood products, but showed lower sensitivity than the BAM protocol for
428
environmental samples. However, for the environmental surface samples, none of the tested
429
MPN methods gave accurate quantification, and results were largely underestimated. This
430
study illustrates the difficulties in detecting and quantifying Listeria in environmental
431
samples, in which cells are subjected to stressful conditions (dehydration, nutrient limitation,
432
etc.).
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In conclusion, culture-based techniques include very different methods, and are
434
generally characterised by high variability in precision (Table 1). They are relatively simple
435
and rapid to perform, confirm the presence of only viable cells, but are generally time-
436
consuming, and sometimes labour-intensive. They may require individual biochemical
437
confirmation of the species on isolated colonies. In addition, there is no detection of viable,
438
but non-culturable bacteria (VBNC), injured or stressed cells, and overgrowth of other
439
bacteria can alter the results (D’Urso et al., 2009; Jadhav et al., 2012; Jasson et al., 2010).
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2. MOLECULAR BIOLOGY METHODS
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2.1.
FLUORESCENCE IN SITU HYBRIDISATION METHODS
444 445
The main drawback of the conventional plate count method is its long time to results
446
(4-7 days). FISH (fluorescence in situ hybridisation) is a cytogenetic technique used to detect
447
and localise the presence or absence of specific DNA sequences. FISH uses fluorescent
19
ACCEPTED MANUSCRIPT probes that bind to complementary sequences and bound probes are then visualised using
449
fluorescence microscopy. Fuchizawa et al. (2008) designed a FISH technique combined with
450
filter cultivation (FISHFC) to rapidly enumerate viable Listeria spp. The fluorescent labelled
451
probe was designed from the 23S rRNA region of Listeria spp. genome. The FISHFC
452
technique was chosen to limit interference of food particles and to facilitate counts under an
453
epi-fluorescence microscope. Their method used 1 mL of sample homogenate which was
454
filtered through 0.45 µm pore size polypropylene membranes and incubated for 12 h at 37°C,
455
followed by 2 h of FISH treatment. At a level of about 2 log10 CFU/g, there were no
456
significant differences in enumeration between direct plate counts and the FISHFC method in
457
various foods (smoked salmon, cheese, ham). In 2009, Fuchizawa et al. improved the
458
specificity of the FISHFC method to quantify L. monocytogenes in food within 16 h.
459
461
2.2.
PCR METHODS
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In the early 1990s, PCR was only used for identification of pure bacterial cultures or
463
colonies on agar plates. Thereafter, PCR was recognised as one of the most promising rapid
464
microbiological methods for the detection and identification of bacteria in a wide range of
465
foods (Garrido et al., 2013). More recently, PCR techniques have been applied to
466
quantification. Although PCR has been used indirectly for enumeration applications such as
467
MPN-PCR methods (see above), the use of PCR to quantitate directly from samples initially
468
relied on competitive PCR techniques.
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PCR is a technique that amplifies a specific DNA sequence, producing thousands to
470
millions of copies. PCR reactions are characterised by a three-step cycle: denaturation of the
471
double-stranded sample DNA, annealing of primers that target DNA by complementarity, and 20
ACCEPTED MANUSCRIPT finally, synthesis of a new, complementary DNA strand by DNA polymerase (extension).
473
Typically, PCR consists of a series of 20 to 40 cycles. In an efficient cycle, the quantity of
474
DNA depends on the initial quantity of DNA (de Martinis et al., 2007; Jadhav et al., 2012;
475
Ryser and Marth, 2007). Commonly targeted L. monocytogenes genes are the listeriolysin O
476
gene (hlyA), the invasion-associated protein p60 gene (iap), internalin genes (inlA, inlB),
477
aminopeptidase C genes, and 16S rRNA (Churchill et al., 2006).
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After amplification, agarose gel electrophoresis is performed to separate and visualise
479
the PCR products according to their size. Conventional, end-point PCR methods provide only
480
qualitative results, because the amount of PCR product does not reflect the initial amount of
481
DNA. PCR products increase exponentially during the early cycles of the PCR, but level off
482
in the final cycles (Rijpens and Herman, 2002). However, a variety of quantitative PCR
483
techniques based on the indirect MPN-PCR technique (see above), or direct PCR
484
quantification have been recently developed. Using these techniques, semi-quantification of
485
the initial amount of bacteria in a sample is possible. However, the short period for sampling
486
PCR products, the lack of automation and poor gel sensitivity, together with high risk of
487
carry-over contamination are major drawbacks to these techniques. PCR approaches have
488
benefited from the introduction of real-time PCR techniques (discussed below) that detect
489
amplification during the reaction itself, as opposed to end-point detection used in PCR. In
490
quantitative competitive PCR, a known amount of internal competitor (for example, a
491
deletion mutant) is co-amplified with the target sequence in the same reaction. In general,
492
target and competitor have different sizes so as to be easily discriminated by gel
493
electrophoresis. To determine the level of contamination, extracted test DNA is serially
494
diluted and added to a constant amount of competitor. The reaction tube that yields identical
495
amounts of both types of amplicon at the end of the PCR is assumed to contain initially
496
identical amounts of target and competitor. The amount of target can be reliably inferred from
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ACCEPTED MANUSCRIPT 497
this technique provided that the targets and competitor have the same amplification
498
efficiency, a condition rarely observed with amplicons of different size. The advantage of this
499
method compared to other qPCR methods is that no expensive fluorophores or radioactive
500
labels are required (Churchill et al., 2006). Today, use of competitive PCR is very limited. Wang and Hong (1999) developed a competitive PCR protocol combined with an
502
enzyme-linked immunosorbent assay (ELISA) for quantification of L. monocytogenes in milk
503
or fish samples. The target gene was iap, a virulence factor in L. monocytogenes. The
504
detection limits in artificially contaminated milk and catfish fillets were respectively 20
505
CFU/mL and 2 CFU/g. There were slight differences (<0.52 log) between expected and
506
detected numbers of L. monocytogenes. Results were obtained in two days, and there were
507
few cross-reactions with other bacteria. Choi and Hong (2003) developed a 5 h competitive
508
PCR for quantification of L. monocytogenes in milk, using the hlyA gene. The detection limit
509
was 103 CFU per 0.5 mL of inoculated milk and the method appeared to be selective for the
510
tested strains. Quantifications were approximately equal to conventional plate counts.
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In fluorescent resonance energy transfer (FRET)-PCR, the DNA product is analysed
512
directly after PCR by measuring a fluorescence signal (Churchill et al., 2006). Two DNA
513
probes of the gene of interest are used, one with a reporter, fluorescein label, and the other
514
with a quencher label. During the annealing and primer extension steps of PCR, the first
515
probe hybridises to the gene of interest, and is then hydrolysed by the 5’3’ exonuclease
516
activity of the DNA polymerase during amplification, resulting in the release of the
517
fluorophore from the probe. The probe with the quencher label can only anneal to the
518
fluorescein-labelled probe at the end of PCR, when the mixture cools to room temperature.
519
Only unbound fluorescein fluoresces; the fluorescence signal read at the end of the reaction
520
thus indicates the number of cells in the sample (Churchill et al., 2006). Koo and Jaykus
521
(2003) used this method, focusing on the hlyA gene, to detect 103-104 CFU of L.
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ACCEPTED MANUSCRIPT 522
monocytogenes in 25 g of skim milk. In 2008, O’Grady et al. used FRET-PCR combined with
523
an enrichment step to qualitatively detect 1-5 CFU of L. monocytogenes in 25 g of food, as
524
part of a real-time PCR assay.
526 527
2.3.
REAL-TIME QUANTITATIVE PCR (qPCR)
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In qPCR, the PCR products are detected as they accumulate. The amount of generated
529
PCR product is proportional to the increase in a fluorescent signal, which is monitored during
530
the exponential phase. The number of cells can be estimated using standard regression curves
531
of the quantification cycle (Cq) values, previously known as threshold cycle (CT) values (or
532
crossing point, take-off point), based on samples at known concentrations. The absence of any
533
essential post-PCR step simplifies the method, and allows high throughput and automation.
534
Several fluorescence systems based on various dyes can be used for detecting the signal
535
(Rijpens and Herman, 2002). For example, SYBR Green, a fluorescent dye, has an emission
536
spectrum that is 50- to 100-fold brighter when the dye is bound to double-stranded DNA. The
537
increase in fluorescence can be followed as the DNA amplifies. The fluorescent molecule can
538
also be a target specific oligonucleotide probe labelled with a fluorescent dye along with a
539
quencher. The hydrolysis probes (TaqMan probes) contain the reporter and quencher dye on
540
the same oligonucleotide, resulting in quenching. During the annealing and primer extension
541
step of PCR, the probe hybridizes to the gene of interest, and is then hydrolyzed by the
542
5’3’exonuclease activity of the DNA polymerase during amplification. Once released, the
543
reporter dye is no longer quenched, and the increase in fluorescence can be measured (Jadhav
544
et al., 2012). Nowadays, a great variety of fluorescence reporting systems have been
545
developed, and may be classified as non-specific dye binding (e.g. SYBR Green, SYBR gold,
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ACCEPTED MANUSCRIPT and EVA green), non-specific universal labeled primer (i.e. Amplifluor System from
547
Milipore), or specific (hydrolysis probes, dual labelled probes, Molecular Beacon, Scorpion
548
uniprobes or biprobes, Mediator probes system, LNA or PNA probes). Regarding the
549
comparison of dye-binding chemistries versus probe-binding chemistries, many benefits and
550
drawbacks can be listed for each. Non-specific dyes have high signal/low background, and
551
consequently are highly sensitive. They are inexpensive and can be rapidly developed and
552
thus are attractive for initial exploratory stages. However, with this system, specificity
553
depends entirely on the primers, and the melting curves must be analysed in each experiment.
554
In contrast, with specific probes, systems are more specific, avoid primer-dimer problems,
555
and enable multiplex PCR, but are more expensive.
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Sensitivity of qPCR applied to a food matrix is generally quite limited when compared
557
with other enumeration methods. Consequently, in most cases, qPCR is not suitable for the
558
accurate enumeration of low levels of L. monocytogenes in food (Rijpens and Herman, 2002).
559
To provide enumeration results, qPCR must be applied directly to the food sample (i.e.
560
without any enrichment step), which can be a source of technical difficulty. Appropriate
561
sample preparation is needed to eliminate PCR-inhibiting components from the sample, and
562
to concentrate bacteria to minimum PCR detection limits. A very sensitive PCR system is
563
thus required for qPCR (Rijpens and Herman, 2002). The quantitative range of qPCR is
564
generally over 100 copies of DNA. Although multiple measurements can help improve
565
precision of the standard curve, concentrations of DNA from one to several tens of copies are
566
generally considered as (sometimes) detectable, but non-quantifiable. In fact, at low numbers
567
of target, the PCR reaction results follow a Poisson distribution and primer hybridisation is
568
stochastic.
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For quantification purposes, standard calibration curves generally have to be
570
established using an external standard (absolute quantification). In this case, the choice and
24
ACCEPTED MANUSCRIPT quantification of the standard itself are critical. The capacity of the method to determine
572
accurately the concentration of the target bacteria depends on the linearity, efficiency and
573
limit of quantification of the PCR. Linearity is the ability of the method to generate results
574
proportional to the concentration, and is represented by the regression coefficient r2 of the
575
(linear) relationship between the initial number of L. monocytogenes DNA molecules, and the
576
Cq values. Efficiency (E) is the capacity of the PCR to duplicate the amplicon molecules in
577
each cycle, and is calculated from the slope of the linear regression curve. The limit of
578
quantification is established at the lowest sample dilution in which the resulting confidence
579
interval does not overlap with that of the next dilution (Rodríguez-Lázaro et al., 2005, 2010).
580
The MIQE (minimum information for publication of qPCR experiments) guidelines specify
581
the information that must be reported for a qPCR experiment to ensure its relevance,
582
accuracy, correct interpretation, repeatability, and assessment (Bustin et al., 2009). However,
583
the ongoing lack of consensus in the literature may have adverse consequences on the
584
reliability of these methods and researchers’ confidence in them.
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Viability is another variable that must be considered when evaluating bacterial
586
densities. Conventional PCR detects both viable and non-viable cells. The inability to
587
selectively detect only viable bacterial cells has impaired the implementation of this method
588
in routine food microbiology laboratories.
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Underestimation of the contamination load is also possible, because PCR inhibitors
590
(components of food samples, culture media, nucleic acid extraction reagents, etc.) can lead
591
to false-negative results making the use of qPCR difficult for routine food analysis
592
(Rodríguez-Lázaro et al., 2005). To avoid this problem, pre-treatment procedures can be
593
developed, but in all cases, PCR efficiency should be assessed with an internal amplification
594
control (IAC). To date, however, only a few qPCR assays have included IACs, despite the
595
general opinion that it should be mandatory (Rodríguez-Lázaro et al., 2005). IACs are non-
25
ACCEPTED MANUSCRIPT target DNA fragments that are co-amplified simultaneously with a target sequence, ideally
597
with the same primers. The simultaneous use of two different labelled fluorescent probes
598
(including an IAC probe) makes it possible to detect and quantify the target and, at the same
599
time, assess PCR efficiency (Rodríguez-Lázaro et al., 2005). Problems of lysis and extraction
600
efficiency may also occur before PCR. Additionally, though there have been important
601
advances in technologies, heterogeneity in temperatures among PCR thermocyclers has
602
sometimes been reported, leading to variability and underestimation of results due to
603
inadequate temperature incubation zones and large differences according to the materials and
604
apparatuses used (http://www.realtimepcr.dk/). Another problem likely to compromise the
605
applicability of qPCR is the existence of inter-strain variability in the target DNA sequence,
606
leading to weak signals and underestimation of the amount of DNA (Rodríguez-Lázaro et al.,
607
2004b).
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Nevertheless, the major advantage of qPCR methods is their rapidity compared with
609
culture methods. The absence of any essential post-PCR step simplifies the qPCR method,
610
allows high throughput and automation and decreases the risk of cross-contamination in the
611
laboratory. Moreover, PCR can detect the presence of sub-dominant populations, sometimes
612
unable to grow in current culture conditions, in the presence of large background microflora.
614 615
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2.3.1. RECENTLY DEVELOPPED VARIATIONS ON THE qPCR METHOD In 2001, Hein et al. developed a qPCR method for detection and enumeration of L.
616
monocytogenes or L. innocua in artificially contaminated milk using the iap gene (a Listeria
617
species-specific gene) as the target. The method required several rounds of centrifugation,
618
heating and enzyme treatments for DNA isolation before qPCR could be performed. Results
26
ACCEPTED MANUSCRIPT 619
obtained were 1 to 2 log higher than CFU obtained by the plate count method using
620
PALCAM agar. Rodríguez-Lázaro et al. (2004b) evaluated the usefulness of the iap and hly genes as
622
targets for quantification using qPCR. The hly-based assay resulted in specific, sensitive and
623
accurate quantification of L. monocytogenes, whereas the iap-based assay resulted in
624
heterogeneous and unreliable quantification, due to serovar-related target sequence
625
variability.
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Rodríguez-Lázaro et al. (2004a) developed a duplex qPCR assay, for the simultaneous
627
detection and quantification of L. monocytogenes and Listeria spp. using the hly gene, and the
628
23S rDNA gene. Simultaneous quantification was possible over a 5 log range, and the limit of
629
quantification was 30 target molecules per reaction. The hly and 23S rDNA duplex reaction
630
showed the same results as the monoplex reaction, indicating that multiplexing did not
631
negatively influence the real-time PCR assay.
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Rodríguez-Lázaro et al. (2005) developed a novel qPCR for L. monocytogenes that
633
monitors analytical performance via the use of an IAC. In this study, the target sequence was
634
the hly gene, and the IAC was a chimeric DNA sequence flanked by the hly-specific target
635
sequences. A second hydrolysis probe, labelled with a specific fluorochrome detects the IAC
636
sequence. The results show that this assay is as specific, sensitive and quantitative as the
637
monoplex assay, and could be used for routine analysis for the detection and quantification of
638
L. monocytogenes DNA in food products. The quantification limit was 30 genome
639
equivalents per reaction, and the coefficients for PCR linearity and efficiency were high: r2 =
640
0.997 and E = 0.80. At high contamination levels (i.e. from 3×105 to 3×107 CFU/g), the
641
system accurately quantified L. monocytogenes in processed meat samples. However, Fraser
642
and half-Fraser broths and some food (raw pork, raw and cold-smoked salmon) can be
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ACCEPTED MANUSCRIPT 643
strongly PCR-inhibitory, leading to underestimation of contamination loads, due to PCR
644
failure. A hydrolysis-probe PCR-based method for the rapid detection and quantification of L.
646
monocytogenes, developed by Oravcová et al. (2005), targeted a specific sequence of the actA
647
gene encoding for a protein involved in the actin filament assembly. This highly sensitive and
648
specific assay for L. monocytogenes can detect 102 CFU/mL after 45 PCR cycles, and can
649
produce linear calibration curves from 109 to 102 CFU/mL, even in the presence of 106
650
CFU/mL of L. innocua.
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A qPCR method targeting the hlyA gene for quantification of L. monocytogenes in
652
biofilms (Guilbaud et al., 2005) had a reported sensitivity estimated at 6×102 CFU/cm2, but
653
suffered from a lack of precision, considering the dispersal of the points. Detection below a
654
concentration of 104 CFU/cm2 was less regular and more difficult.
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In 2006, Berrada et al. performed a qPCR on salad samples, quantifying DNA in a
656
LightCycler system with the LightCycler L. monocytogenes Detection Kit from Roche. The
657
method was linear in a range of 10 to 105 CFU L. monocytogenes. At 102 CFU/g, accuracy
658
(expressed as % bias) was as high as 26% and precision (expressed as % coefficient of
659
variation) as high as 22%. At 102 CFU/g, intra-day and inter-day variability were 12 and 14%
660
respectively. Based on the precision of this method, 10 CFU/g was the reported limit of
661
detection. The method provided accurate quantification of L. monocytogenes in three
662
naturally contaminated salads (around 1 to 7×102 CFU/g), with results similar to plate counts.
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In 2008, Long et al. developed a novel qPCR for L. monocytogenes that monitors
664
analytical performance via the use of a live bacterium as the IAC. Their purpose was to
665
develop a system able to indicate either inhibitors of the PCR amplification, or poor recovery
666
of microorganisms during the DNA extraction step. The target gene for the detection of L.
667
monocytogenes was the hly gene. The IAC had similar length, flanking regions and G+C
28
ACCEPTED MANUSCRIPT 668
content as the target, as well as a resistance gene to select L. monocytogenes cells with
669
successful chromosomal integration of the IAC. Quantification by this method gave a 5 log
670
linearity range, i.e. up to 105 CFU. Rosmanith et al. (2010) used internal sample process control (ISPC) to investigate
672
cheeses from an outbreak in Austria. The ISPC quantitatively determined the efficiency of the
673
target bacteria during pre-PCR steps. A known number of ISPC cells were added to the
674
sample prior to analysis, and pathogen load was deduced from the efficiency of the whole
675
process. In this case, recovery of the control was 43%, and the corrected contamination values
676
of investigated samples corroborated those of the EN ISO 11290-2 standard method, with no
677
significant differences (Rosmanith and Wagner, 2010).
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Reagent cost is one of the key factors that influence routine adoption of qPCR in
679
diagnostic laboratories. Traunšek et al. (2011) optimised 5 µL qPCR reactions for L.
680
monocytogenes based on the amplification of the hlyA gene and the use of a commercial IAC
681
(via second qPCR). Limits of detection and quantification were as low as 165 genome
682
equivalents.
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685
2.3.2.1.
686
qPCR PRE-ANALYTICAL PROCESSING STEPS
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qPCR METHODS WITH A CELL CONCENTRATION STEP
687
Sample preparation has become an important topic in molecular food pathogen
688
detection, as it is increasingly recognised as the crucial prerequisite for reliable qPCR
689
(Rossmanith and Wagner, 2010). To improve sensitivity, target cells can be concentrated
690
before performing PCR.
29
ACCEPTED MANUSCRIPT 691
In most qPCR studies, a solid food product is generally analyzed after first blending
692
with a buffer (usually in a 1:10 dilution) and taking an aliquot of this mixture (typically 1 mL)
693
that is then centrifuged, washed and re-suspended; a supplementary centrifugation step can be
694
added to the protocol to improve sensitivity. Hough et al. (2002) developed a qPCR method including a centrifugation step for the
696
enumeration of L. monocytogenes in cabbage. Using a region of the hlyA gene, the method
697
provided quantitative results within 8 h for artificially contaminated samples. A linear
698
response over 7 log cycles, from 1.4 ×102 to 1.4×109 CFU of L. monocytogenes per 25 g of
699
cabbage and analysis of the assay showed that log differences could reliably be distinguished.
700
There were no cross-reactions with other microorganisms.
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A filtration-based qPCR method was developed by D’Urso et al. (2009) to quantify
702
viable L. monocytogenes and S. enterica, down to a limit of 10 CFU/10 g of yogurt. In this
703
case, an initial centrifugation step of the diluted food sample before PCR led to higher
704
sensitivity.
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Rodríguez-Lázaro et al. (2010) developed a novel qPCR for L. ivanovii that monitors
706
analytical performance via the use of an IAC. It targeted a specific region of the
707
sphingomyelinase C (smcL) gene, which is located on a L. ivanovi-specific pathogenicity
708
island and encodes a membrane-damaging virulence factor. The method detected as little as
709
one genome equivalent in 45% of the reactions. In raw milk, PCR accuracy was excellent
710
over a 6 log range, with high r2 and E values, and a limit of quantification of approximately
711
530 CFU in 25 ml. Given the centrifugation/concentration step, the method detected as few as
712
50 CFU in 25 mL of raw milk (5 genome equivalents per reaction) with a 66% probability.
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Sample preparation has recently become an important topic in molecular-based
714
foodborne pathogen detection, and new approaches have been described with selective
30
ACCEPTED MANUSCRIPT 715
solubilisation of the food matrix in a lysis buffer, and subsequent centrifugation to harvest
716
bacterial cells. Some of these protocols were reviewed in Rosmanith and Wagner (2010).
717
2.3.2.2.
METHODS WITH IMPROVED PURIFICATION/EXTRACTION STEPS
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Inhibition of PCR and real-time PCR by food components, such as fat, selective
721
enrichment media, or large amounts of non-target DNA, can occur, and pre-amplification
722
steps may be needed to eliminate theses. Many different protocols are used for DNA
723
extraction/purification. Typically, they rely on mechanical disruption or enzymatic digestion
724
of the cell membrane, followed by cell solubilisation, using reagents such as guanidine
725
isothyocianate, and subsequent clean-up steps using organic solvents and alcohol
726
precipitation, suspended silica, affinity or ion exchange purification columns, ultrafiltration,
727
among others.
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Most available PCR methods require selective enrichment steps to overcome the
729
problem of PCR inhibitors, especially at low pathogen concentrations. To avoid enrichment,
730
Rodriguez-Lazaro et al. (2004c) used filtration and Chelex-100-based DNA purification, and
731
qPCR for the quantitative detection of L. monocytogenes in meat products. Chelex-100 is an
732
ion-exchange resin, specifically designed for extraction of PCR-ready template DNA. The
733
removal of PCR inhibitors is accomplished by scavenging contaminant metal ions that
734
catalyze the cleavage of DNA. Pre-PCR filtration treatments were performed on 11 µm or 22-
735
25 µm pore size filters, which should not retain L. monocytogenes cells but remove larger
736
particles. Based on this strategy as few as 103 CFU/g L. monocytogenes were quantified in
737
meat products (ham, sausages, raw pork) with excellent accuracy.
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31
ACCEPTED MANUSCRIPT Similarly, Rodriguez-Lazaro et al. (2005) accurately quantified as few as 103 CFU/g L.
739
monocytogenes in smoked salmon products, with a qPCR method involving pre-PCR
740
filtration through 22-25 µm pore size filters and DNA purification with a commercial kit
741
(Wizard genomic DNA purification kit, Promega). However, results were less conclusive for
742
raw salmon.
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Rodriguez-Lazaro and Hernandez (2006) showed that the Wizard kit (Promega) was
744
more suitable than the Chelex kit (Bio-Rad) for L. monocytogenes DNA extraction from meat
745
samples requiring sensitive and accurate results. Many other extraction/purification kits are
746
now commercially available. Rosmanith et al. (2007) developed a protocol for the
747
concentration of Gram-positive bacteria from food, with the removal of fat, carbohydrates and
748
proteins, compatible with DNA-based quantification methods.
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743
Immunomagnetic separation (IMS) allows the specific capture of L. monocytogenes
750
from food or culture media, and consequently the removal of PCR inhibitors. Large magnetic
751
microbeads (>1 µm in diameter) coated with specific antibodies have been used for
752
separation and concentration of L. monocytogenes in food samples. More recently,
753
nanoparticles coated with pathogen-specific antibodies have been shown to have higher
754
capture efficiency due to a higher surface-to-volume ratio and faster reaction kinetics (Yang
755
et al. (2007).
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749
Probably due to the presence of PCR inhibitors, detection of DNA extracted directly
757
from dairy products for PCR sometimes shows very poor sensitivity. Nogva et al. (2000)
758
developed a qPCR method for the quantitative detection of L. monocytogenes in water, skim
759
milk and unpasteurised whole milk, based on the use of a 113 bp amplicon from the hlyA
760
gene as the target and a hydrolysis probe. The method was highly selective and could be
761
completed within 3 h. It included non-specific binding of bacteria to paramagnetic beads,
762
with subsequent DNA purification using the same beads. In the Nogva et al. study,
32
ACCEPTED MANUSCRIPT 763
quantification in artificially contaminated samples was linear over 7 log units, and the
764
detection limit was approximately 200-2000 CFU/mL. Amagliani et al. (2006) developed a PCR assay for direct detection of L.
766
monocytogenes in milk samples, with a magnetic capture hybridisation procedure for
767
selective DNA purification, using nanoparticles. The sensitivity of the assay was 10 CFU/mL.
768
Yang et al. (2007) developed an assay based on nanoparticle-based IMS in
769
combination with qPCR, using a portion of the hlyA gene as a target, to quantify L.
770
monocytogenes in artificially contaminated milk. Purification of bacterial DNA with IMS
771
detected > 102 CFU per 0.5 mL. In contrast, no detection was possible without IMS even
772
when L. monocytogenes numbers where greater than 107 CFU in 0.5 mL, and with
773
Dynabeads®, detection was possible only for concentrations greater than 105 CFU in 0.5 mL.
774
With Dynabeads®, cell numbers derived from Cq values were lower than those determined
775
from direct plate counts of the beads. In contrast, for nanoparticles, results were 1.5 to 7 times
776
higher than those derived from plate counts. Cell aggregation or lower culturability may be
777
possible explanations for such discrepancies. The method improved the sensitivity of L.
778
monocytogenes detection (226 CFU/0.5 mL) and was rapid and quantitative. However, it is
779
labour-intensive, requiring the synthesis and coating of specific nanoparticles. Moreover,
780
nanoparticles had to be removed by centrifugation after DNA extraction, because they can
781
reduce PCR efficiency in a dose-dependent manner. An excessive quantity of nanoparticles
782
present on cell surfaces, improving cell separation, can also affect DNA extraction efficiency
783
and thus the limit of detection by PCR.
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784
Amagliani et al. (2010) developed a multiplex qPCR assay including an IAC for the
785
simultaneous detection of L. monocytogenes and Salmonella spp. in seafood samples. A
786
magnetic capture hybridisation procedure was performed for selective DNA purification,
787
using nanoparticles. The sensitivity of the assay was 102 -103 CFU/g. One can note that
33
ACCEPTED MANUSCRIPT 788
Amagliani et al. (2006) and (2010) used magnetic particles coupled to oligonucleotids (not
789
antibodies) as a means to purify and concentrate nucleic acids, and not whole bacterial cells,
790
directly from samples before PCR. Walcher et al. (2010) compared direct plate counts and qPCR combined with
792
paramagnetic beads for separation and quantification of L. monocytogenes in raw milk. A
793
high correlation was found between the inocula and counts obtained both by direct plating or
794
qPCR. Recovery rates ranged from 46.6 to 122.8% for plate counts, and from 64.7 to 95.1%
795
for qPCR. The detection limit of the latter method was 102-103 CFU/mL.
SC
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791
Commercially available nanoparticles were coated and used by Yang et al. (2013) to
797
develop a multiplex non-quantitative PCR for simultaneous detection of L. monocytogenes,
798
Escherichia coli O157:H7 and Salmonella typhimurium in food (lettuce, tomato, ground
799
beef), with a sensitivity of about 1000 CFU/g for L. monocytogenes. The overall assay time
800
was 4.5 h. To detect only the viable cells, propidium monoazide was applied to selectively
801
suppress the detection of DNA from dead cells.
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802
2.3.3. qPCR METHODS THAT ASSESS VIABILITY
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803
The determination of bacterial viability is a key issue for the application of food risk
805
management. Reichert-Schwillinsky et al. (2009) showed that under poor growth conditions,
806
as well as during the lag phase, the qPCR data overestimated the actual viable L.
807
monocytogenes counts. This result was attributed to the possible accumulation of dead cells,
808
extracellular genomic DNA or VBNC bacteria. On the other hand, these effects are the result
809
of reduced growth within the culture when the conditions worsen.
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810 811
2.3.3.1.
REVERSE TRANSCRIPTION PCR (RT-PCR)
34
ACCEPTED MANUSCRIPT One alternative is to use an mRNA-based PCR method. This type of method has been
813
successfully applied by Klein and Juneja (1997) for detection of viable L. monocytogenes in
814
food. However, these systems must be used judiciously, because mRNA itself can be stable
815
for a quite long time after cell death (Birch et al., 2001; Rijpens and Herman, 2002). One
816
technique, reverse transcription PCR (RT-PCR) can be used for the detection of mRNA;
817
however this technique has some disadvantages, such as the necessity to eliminate all traces
818
of DNA, limited sensitivity, reproducibility and specificity (d’Urso et al., 2009). Nucleic acid
819
sequence-based amplification (NASBA) is a promising method for the analysis of viable
820
cells, through continuous amplification of RNA at isothermal conditions (41.5°C). At this
821
temperature, DNA remains double-stranded, and does not become a substrate for
822
amplification (Rodríguez-Lázaro et al., 2007).
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In 2008, Rantsiou et al. developed a qPCR protocol to detect, quantify and determine
824
the viability of L. monocytogenes in food samples (meat and milk products, and vegetables).
825
A couple of primers and a hydrolysis probe were designed on the intergenic region spacer
826
between the 16S and 23S rRNA genes. To define the number of viable cells, this sequence
827
was preferred to specific virulence genes, which are controlled by environmental factors
828
(medium components, temperature, etc.). Amplification is performed at the DNA and RNA
829
level using RT-qPCR to quantify total or only viable cells. Standard curves were constructed
830
from artificially contaminated matrices, and the method had a high quantification limit of 103-
831
104 CFU/g or mL, and of 104-105 CFU/g or mL, when considering viability, which makes this
832
assay unsuitable for the enumeration of L. monocytogenes at low contamination levels.
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833
Molinos et al. (2010) developed an RT-qPCR method based on bacterial mRNA to
834
quantify L. monocytogenes in food after bacteriocin treatment. For concentrations greater than
835
102 CFU/g, viable cell count results were better than PALCAM agar counts. For RT-qPCR
836
determination of RNA expression levels — a multi-step procedure involving lysis of bacteria
35
ACCEPTED MANUSCRIPT cells, RNA extraction, DNase treatment, cDNA synthesis, and finally qPCR —, the detection
838
of scarce target RNA from a low number of bacteria remains a challenge. Werbrouck et al.
839
(2007) evaluated and optimised each step of this method for L. monocytogenes. Their results
840
showed that many factors can influence the outcome of the assays, which thus require careful
841
assay design and reaction optimisation to maximise the sensitivity and obtain reliable
842
quantification.
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843
2.3.3.2.
OTHER APPROACHES TO ASSESS VIABILITY
SC
844
Another approach for the detection of only viable cells is the use of ethidium
846
monoazide bromide (EMA) or propidium monoazide (PMA) for staining the cells before
847
DNA extraction and PCR to inhibit DNA amplification of dead cells. These dyes
848
preferentially penetrate cells with compromised membranes. For lack of other viability
849
measurements, membrane integrity is still a well-accepted criterion for distinguishing viable
850
from dead cells (Yang et al., 2013). However, in some cases, dyes can also penetrate viable
851
cell membranes and covalently link DNA, resulting in PCR inhibition and underestimation of
852
counts, mainly when using EMA (Pan and Breidt, 2007; d’Urso et al., 2009). The high
853
discrepancies in the same samples between quantified culturable cells and the much higher
854
level of Listeria population estimated by PCR with a PMA viability protocol (Overney, 2014)
855
also raises the issue of how to interpret the results and what they mean in terms of food
856
safety, cells’ ability to recover, cross-contamination and growth.
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857
A filtration-based qPCR method was developed by D’Urso et al. (2009) to eliminate
858
dead or severely damaged L. monocytogenes and S. enterica from food samples (yogurt).
859
Firstly, after a centrifugation step, samples undergo an initial lysis-filtration pre-treatment,
860
which retains only viable bacteria on the filter in less than 30 min. The discrimination buffer
36
ACCEPTED MANUSCRIPT can completely solubilise protein complexes, eukaryotic cells and other contaminants, letting
862
them pass through the filter used for pre-treatment. Briefly, pre-treatment filtration consisted
863
of addition of discrimination buffer (4 M guanidine thiocyanate, 2 M NaCl, 25 mM Tris-HCl
864
pH 7.0 ) incubated at 60°C for 15 min with shaking after prior enzymatic treatment with 10
865
mg/mL lysozyme for 15 min at 37°C. The suspensions were filtered on filter paper (0.4 µm),
866
and washed with 500 µL of washing buffer (2 M NaCl, 25 mM Tris-HCl pH 7) at 60°C, then
867
with another washing buffer (25 mM Tris-HCl pH 7.0, 0.5% SDS), and with 300 µL of
868
double-distilled water to ensure all dead cells pass through the filter. Secondly, after
869
resuspension of the viable cells from the filter, the bacterial DNA was extracted on silica-
870
based columns, and was finally amplified by qPCR. Quantitative results clearly match those
871
obtained by standard plating, over a wide dynamic range (at least 5 log). Though several
872
labour-intensive steps were necessary, the filtration-based qPCR assay quantitatively detected
873
living forms of L. monocytogenes and S. enterica, at concentrations as low as 10 CFU/10 g of
874
yogurt.
876
2.4.
DIGITAL PCR
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Digital PCR (dPCR) is a new approach that directly quantifies, detects and amplifies a
878
target nucleic acid sequence. It was first developed by Vogelstein and Kinzler (1999) to
879
extend the applications of conventional PCR. dPCR partitions a sample into several hundreds
880
or thousands individual PCRs: some of these reactions receive the target molecule (positive),
881
whilst others do not (negative). The reaction volume of dPCR is several picoliters to
882
nanoliters. The average target nucleic acid concentration is determined by the proportion of
883
negative PCR reactions, based on the Poisson distribution. It is then possible to determine a
884
copy number of the target gene without the need for internal controls or standards for
AC C
877
37
ACCEPTED MANUSCRIPT quantification and assessing reaction efficiency that are required for qPCR (Straub et al.,
886
2013). dPCR has benefited from progress in microfluidic technologies, and is used for
887
clinical PCR applications requiring extreme accuracy of copy number variation. It has been
888
used to detect cancer by quantification of mutant alleles and detection of allelic imbalance
889
(Pohl and Shih, 2004). Although applications for biodetection in environmental samples are
890
starting to appear, to our knowledge, no trials of dPCR have yet been reported for the
891
quantification of L. monocytogenes. Recently, Straub et al. (2013) used dPCR to directly
892
enumerate plasmid and chromosome copies in three strains of Bacillus anthracis. According
893
to their results, dPCR appears to be an attractive, accurate and rapid alternative method that
894
provides for direct quantification of target genes. Recently, the performance of microfluidic
895
digital RT-PCR (RT-dPCR) was compared with RT-qPCR for detecting the main viruses
896
responsible for foodborne outbreaks (human noroviruses and hepatitis A virus) in spiked
897
lettuce and bottled water (Coudray-Meunier et al., 2015). MIQE guidelines are now also
898
available for dPCR (Huggett et al., 2013).
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Today, various commercially available kits (for example BioMark and EP from
900
Fluidigm, QuantStudio 3D Digital PCR Instrument from Life Technologies, Q100 from Bio
901
Rad, RainDrop from RainDance) make it possible to conduct dPCR experiments with high
902
accuracy and low cost per sample. The kits can collect real-time amplification data from each
903
reaction. For Fluidigm, the digital array with an integrated fluidic circuit precisely partitions a
904
sample pre-mixed with PCR reagents into hundreds of individual PCR reactions. However,
905
for food analysis, sample preparation, DNA concentration, extraction and purification may be
906
limiting factors. Although measurement precision has improved, sensitivity is likely to be
907
similar to qPCR. A potential application is the quantification of L. monocytogenes in
908
environmental samples, because these samples require a lower amount of organic matrix.
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909
38
ACCEPTED MANUSCRIPT In conclusion, although the major advantage of PCR-based methods is their rapidity,
911
all share a main drawback: detection limits generally far greater than contamination levels in
912
food. Molecular methods are not simple single-step methods, but complex and sometimes
913
labour-intensive multi-step systems, for which all steps need careful evaluation and
914
optimisation. However, recent advances have significantly improved their performance
915
(Table 2).
RI PT
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916
CONCLUSION
SC
917
Enumeration of L. monocytogenes in food faces many difficulties due to low
919
contamination levels, the presence of numerous background microorganisms, and the great
920
variety of matrices to be considered. None of the enumeration methods mentioned above is
921
ideal for any one purpose, and potential users must choose a technique according to their
922
needs, available products and materials (Table 3). For example, there are several
923
circumstances under which semi-quantitative results can be sufficient.
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918
Culture-based techniques (Table 1) include very different methods, characterised by
925
high variability in precision. They have numerous advantages: they are relatively simple and
926
rapid to perform, they confirm the presence of only viable cells. Nevertheless, they are
927
generally time-consuming and sometimes labour-intensive. Moreover, these methods may
928
require individual biochemical confirmation of the species for some isolated colonies. In
929
addition, there is no detection of VBNC bacteria, injured or stressed cells, and the growth of
930
other bacteria can alter the results (D’Urso et al. 2009, Jadhav et al., 2012; Jasson et al.,
931
2010).
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932
Molecular methods (Table 2), in particular qPCR, with their capacity to measure 933
amounts of nucleic acids in a wide range of samples and sources, with their conceptual and
39
ACCEPTED MANUSCRIPT 934
practical simplicity, and their combined speed, sensitivity and specificity in a homogenous 935
assay, can now be considered as the technology par excellence for diagnostic applications, 936
including microbial quantification (Bustin et al., 2009). However, PCR-based methods have 937
detection limits generally much greater than the contamination levels in food. In fact, for L. 938
RI PT
monocytogenes, qPCR may not be the method of choice for detecting a few bacteria (and 939
consequently low quantities of bacterial DNA or RNA) in a large amount of food matrix, 940
because detection limits are far above contamination levels in naturally contaminated samples 941
SC
(Rossmanith and Wagner, 2010). A number of issues such as methods to concentrate bacteria, 942
assess viability or improve purification or extraction steps have been addressed. Although
M AN U
943
many of these studies do examine the linearity of the assay over several order of magnitude to 944
assess the overall efficiency of the PCR and the limit of detection for the PCR assay, the 945
variability of food matrices often makes it very difficult to use these assays for quantification 946
because each sample must be individually assessed for inhibition and assay efficiency. These 947 948
TE D
methods are currently more likely to be used to reliably detect the presence of microorganisms in the food sample, not enumerate them. Moreover, molecular-based 949 950
EP
methods are not simple single-step methods, but complex and sometimes labour-intensive multi-step procedures. However, recent advances have significantly improved their 951 952
AC C
effectiveness. As underlined by the very high number of published studies involving these techniques (Table 3), they remain very attractive due in particular to their rapidity. Recent 953
development in standardised protocols of diagnostic PCR for the detection of foodborne 954
pathogens may favour efficient transfer of PCR technology from expert research to routine 955
laboratories. The international standard EN ISO 22118 (Anonymous, 2011) regarding the 956
performance characteristics of PCR for the detection and quantification of foodborne 957
pathogens is part of a series of documents on PCR used for the detection of pathogenic 958
microorganisms in food and specifies the minimum requirements for the performance
40
ACCEPTED MANUSCRIPT 959
characteristics for the detection of nucleic acid sequences (DNA or RNA) by molecular 960
methods. However, the lack of validation data may hinder the adoption of molecular methods 961
for routine analysis. It is widely accepted that the use of validated methods is an essential part of any sound
963
laboratory quality assurance program. The use of methods that have documented
964
effectiveness based on protocols issued by the major international and regional
965
standardisation bodies (i.e. ISO and CEN) is essential. Evaluation of enumeration methods
966
should be performed according to the EN ISO 16140 standard (Anonymous, 2003), which
967
defines validation procedures for an alternative method compared to a reference method (case
968
of quantitative analysis methods). The EN ISO 11290-2 standard method for enumeration of
969
L. monocytogenes in food (Anonymous, 1998, 2004) is cited as the reference method in the
970
quantitative criteria stated in EC Regulation 2073/2005 for L. monocytogenes. However, in
971
various countries such as France for example, the competent authority allows the network of
972
accredited laboratories to use alternative methods, even for official controls, if they have been
973
validated according to the EN ISO 16140 standard, and certified by a third party
974
(Anonymous, 2008). For practical reasons, it is very difficult for one laboratory to perform
975
such a validation procedure (in particular, the ILPTs ), and most of the studies presented in
976
this review involve the development of methods that have not yet been validated. However,
977
the extent of validation and verification depends also on the context within which the method
978
is to be used. Several certified commercial enumeration methods are available on AFNOR
979
certification site (www.afnor-validation.org). Interestingly, an international ISO 16140-based
980
validation trial of a non-proprietary qPCR-based methodology for detection of L.
981
monocytogenes in soft cheese has been recently described (Gianfranceschi et al., 2014).
982
Barbaud-Piednoir et al. (2013) proposed a new validation procedure to specifically validate
983
qPCR assays for detection applied to food microbiology according to two guidelines: the ISO
AC C
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41
ACCEPTED MANUSCRIPT 22118 standard and the "Definition of minimum performance requirements for analytical
985
methods of GMO testing". For assessing the performance of enumeration methods, it is
986
always far preferable to use naturally contaminated samples, to take into account potential
987
physical entrapment of naturally occurring pathogens, interaction between the target pathogen
988
and the food matrix and the background microflora, which occur in naturally contaminated
989
food matrices, and various types of stress that may be not properly considered when using
990
artificially contaminated samples. In the case of L. monocytogenes, it is sometimes very
991
difficult to find appropriate naturally contaminated samples, from various origins and food
992
types, because of extremely low and heterogeneous contamination levels. Many studies
993
presented here have been conducted using artificially contaminated samples.
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984
In conclusion, the improvement of L. monocytogenes enumeration method has
995
resulted in a considerable amount of research, leading to several alternative methods. Many
996
show satisfactory performance, and are suitable for their intended purposes. However, these
997
new techniques are not widely used. Their low popularity can be attributed to several reasons.
998
Practicability is important to consider when choosing an analytical method. This includes
999
ease of use (preparation of the sample, automation, adaptation to data processing,
1000
obstruction), speed (delay of obtaining the results and cadences of analyses) and cost.
1001
Manufacturer guarantees include validation by a third party (such as by AFNOR Certification,
1002
MicroVal, NordVal, AOAC), reputation of the materials and reagent supplier, reliability of
1003
the material and the reagents (stability, regularity, availability) and after-sales service, user
1004
training. New methods including various steps and unusual materials and reagents may be
1005
difficult to adapt to any routine analysis laboratory. Research in L monocytogenes
1006
enumeration often leads to in-house development of new methods characterised by
1007
insufficient evaluation and validation. Standard methods, and more generally traditional
1008
microbiological analysis methods, based on the observation of microbial growth in adapted
AC C
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994
42
ACCEPTED MANUSCRIPT 1009
culture media and biochemical identification of the microorganisms, are still standard. One of
1010
the main problems is the interpretation of results of new techniques based on a type of
1011
measurement different from that of the conventional techniques. Finally, the use of a completely verified enumeration method is not itself a guarantee
1013
that analytical data are reliable. In particular, sampling plans and the overall competence of
1014
the laboratory must be reliable.
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ACCEPTED MANUSCRIPT 1016 1017
ACNOWLEDGEMENTS
1018
The authors would like to thank Robert Debuchy, Christophe Soumet, Frédéric Auvray,
1020
Patrick Fach and Bertrand Lombard for helpful advice and information.
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REFERENCES
1023
Amagliani, G., Omiccioli, E., Brandi, G., Bruce, I.J., Magnani, M. (2010). A multiplex
1025
magnetic capture hybridisation and multiplex real-time PCR protocol for pathogen detection
1026
in seafood. Food Microbiology, 27, 580-585.
1027
Amagliani, G., Omiccioli, E., del Campo, A., Bruce, I.J., Brandi, G., Magnani, M. (2006) A
1028
multiplex magnetic capture hybridisation and multiplex real-time PCR protocol for pathogen
1029
detection in seafood. Journal of Applied Microbiology, 100, 375-383.
1030
Anonymous (1992). FDA bacteriological analytical manual, Food and Drug Administration,
1031
7th edition. The Journal of AOAC International. Washington, District of Columbia.
1032
Anonymous (1998). Microbiology of food and animal feeding stuffs - horizontal method for
1033
detection and enumeration of Listeria monocytogenes, Part 2. Enumeration Method.
1034
International Standard ISO 11290-2. International Organisation for Standardisation, Geneva,
1035
Switzerland.
1036
Anonymous (2000). Rapport de la commission d’étude des risques liés à Listeria
1037
monocytogenes, Juillet 2000, AFFSA. Agence Française de Sécurité Sanitaire des Aliments,
1038
Maisons Alfort, France.
1039
Anonymous (2003). Microbiology of food and animal feeding stuffs – Protocol for the
1040
validation of alternative methods. International Standard ISO 16140. International
1041
Organisation for Standardisation, Geneva, Switzerland.
1042
Anonymous (2004). International Standard ISO 11290-2/A1. Microbiology of food and
1043
animal feeding stuffs – Horizontal method for the detection and enumeration of Listeria
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Rossmanith, P., Süß, B., Wagner, M., Hein, I. (2007). Development of matrix lysis for
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concentration of gram positive bacteria from food and blood. Journal of Microbiological
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Roussel, S., Félix, B., Colanéri, C., Vignaud, M.L., Dao, T.T., Marault, M., Brisabois, A.
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Bruckner-Lea, C.J. (2013). Estimated copy number of Bacillus anthracis plasmids pXO1 and
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Wang, C. and Hong, C. (1999). Quantitative PCR for Listeria monocytogenes with
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colorimetric detection. Journal of Food Protection, 62, 35-39.
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Werbrouck, H., Botterldoom, N., Uyttendaele, M., Herman, L., Van Coillie, E. (2007).
1381
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transcription PCR: optimization, evaluation and pitfalls. Journal of Microbiological Methods,
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69, 306-314.
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Wu, V.C.H., Fung, D.Y.C. (2002). Evaluation of the thin agar layer Oxyrase® method for
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recovery of heat-injured foodborne pathogens in liquid medium and food systems. Food
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Wu, V.C.H., Fung, D.Y.C. (2003). Simultaneous recovery of four injured foodborne
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pathogens in the four-compartment thin agar layer plate. Journal of Food Science. 68, 646-
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Wu, V.C.H., Fung, D.Y.C. (2004). An improved method for Iso-Grid Hydrophobic Grid
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Membrane Filter (HGMF) system to detect heat-injured foodborne pathogens in ground beef.
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Journal of Food Science. 69, 85-89.
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Yang, H., Qu, L., Wimbrow, A.N., Jiang, X., Sun, Y. (2007). Rapid detection of Listeria
1394
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International Journal of Food Microbiology , 118, 132-138.
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Yang, Y., Xu, F., Xu, H., Aguilar, Z.P., Niu, R., Yuan, Y., Sun, J., You, X., Lai, W., Xiong,
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ACCEPTED MANUSCRIPT viable Salmonella Typhimurium, Escherichia coli O157:H7 and Listeria monocytogenes in
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food products. Food Microbiology, 34, 418-424.
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Yu, L.S, Prasai, R.K., Fung, Y.C.,(1995). Most probable numbers of Listeria species in raw
1402
meats detected by motility enrichment. Journal of Food Protection, 58, 943-945.
1403
Yu, L.S., Fung, Y.C. (1993). Five-tube most-probable-number method using the Fung-Yu
1404
tube for enumeration of Listeria monocytogenes in restructured meat products during
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refrigerated storage. International Journal of Food Microbiology, 18, 97-106.
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ACCEPTED MANUSCRIPT List of Tables
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Table 1: Main characteristics of published culture-based methods.
1410
Table 2: Main characteristics of published molecular methods (when not indicated precisely
1411
in the publication, time to result is generally less than 1 day).
1412
Table 3: Compared advantages/disadvantages of the main methods used, based on the
1413
literature review.
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ACCEPTED MANUSCRIPT Table 1. Limit of quantification /Levels tested
Matrix
Culture methods based on colony counts
40 CFU/g
TAL (Palcam overlain with TSAYE)
≥5 days
40 CFU/g
Filtration+ LOA culture
2 to 5 days
0.8 CFU/g
Food, environment of food production
References
Long time to results
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2 to 4 days
Parsley, meat
Long time to results Recovers stressed cells
Miller et al. (2010)
Cold smoked salmon
Long time to results Specific apparatus Training Not applicable to all foods Sensitive
Gnanou Besse et al. (2004, 2008) Barre et al. (2015)
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EN ISO 11290-2
Advantages/ disadvantages
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Time to results
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Method
Culture methods based on MPN
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MPN+ PCR (prfA)
2 days
100-1000/g
Cooked and fermented meat products
Conventional PCR
Aymerich et al. (2004)
MPN+ PCR (prfA)
2 days
>2-3 log10/g
Chorizo
Fastidious preamplification steps; Quantification
Martin et al. (2004)
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ACCEPTED MANUSCRIPT possible with high levels of background flora 2 days
70-100/g
Milk, lettuce, smoked salmon, ice cream, pâté, salami, shrimp, ground beef, soft cheese
Interference with food component varies with dilution
MPN+ qPCR (16S rDNA)
2 days
10-1000/g.
Minimally processed leafy vegetables
Interference with chlorophyll varies with dilution
de Oliveira et al. (2010)
MPN+ PCR (hlyA gene)
2 days
300/g
Salads
Conventional PCR
Jeyaletchumi et al. (2010)
MPN+ 6 rapid kits
1-2 days.
Large underestimation (environment)
Cruz et al. (2012)
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de Martinis et al. (2007)
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MPN+ qPCR (16S rDNA)
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ACCEPTED MANUSCRIPT Table 2. Time to results
Limit of quantificatio n
Matrix
Competitive PCR (hlyA gene)
5h
103 CFU per 0.5 mL (detection limit)
Milk
Competitive PCR (iap gene)
2 days
20 CFU/mL and 2 CFU/g (detection limit)
FISHFC
16 h
200 CFU/g
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Molecular methods based on FISH and conventional PCR
Advantages/ Disadvantages
Matches plate counts Conventional PCR
Choi and Hong (2003)
Milk, catfish fillet
Matches plate counts Conventional PCR
Wang and Hong (1999)
Smoked salmon, mozzarella, cabbage
Rapid epifluorescence microscopy
Fuchizawa et al. (2008, 2009)
Milk
Results vary with plate counts (1 log)
Hein et al. (2001)
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References
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Method
qPCR (iap gene)
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Molecular methods based on qPCR
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Duplex qPCR (hly gene, and 23S rDNA)
30 target molecules per reaction
Simultaneous quantification of L. monocytogenes and Listeria spp.
RodríguezLázaro et al. (2004)
qPCR (actA gene)
>102 CFU/mL
Quantification possible with high levels of L. innocua
Oravcová et al. (2005)
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ACCEPTED MANUSCRIPT qPCR (hly gene)
104 CFU/cm2
Biofilms
Guilbaud et al. (2005)
qPCR
102-7102 CFU/g
Salads
Berrada et al. (2006)
qPCR+IAC
qPCR (hly gene)
105 CFU
Processed meat samples
Cheese
165 genome equivalents
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qPCR (hly gene)
IAC, PCR inhibition (raw pork, raw and smoked salmon)
RodríguezLázaro et al. (2005)
Viable bacterium as IAC
Long et al. (2008)
Internal sample process control
Rosmanith et al. (2010)
Commercial IAC
Traunšek et al. (2011)
Viability
Pan and Breidt (2007)
Viability
Werbrouck et al. (2007)
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30 genome equivalents per reaction
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qPCR (hly gene)
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Molinos et al. (2010)
qPCR+viability assessment
103 CFU/cm2
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qPCR +PMA
Biofilms
RT-qPCR
RT-qPCR (intergenic region spacer between 16S and 23S rDNA)
104-105 CFU/g or mL
Meat and milk products, vegetables
Viability
Rantsiou et al. (2008)
qPCR +lysis-filtration +centrifugation
10 CFU/10 g
Yogurt
Viability Laborious
D’Urso et al. (2009)
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ACCEPTED MANUSCRIPT qPCR+sample concentration
8h
qPCR +lysis-filtration +centrifugation
>102 CFU/25 g
Cabbage
Higher sensitivity
Hough et al. (2002)
10 CFU/10 g
Yogurt
Viability Labourintensive
D’Urso et al. (2009)
qPCR+improved purification/extraction
200-2000 CFU/mL (detection limit )
qPCR +pre-PCR treatments (filtration+resins)
1000 CFU/g
1419 1420
Pre-PCR treatments Poor results with raw salmon
EP
> 102 CFU per 0.5 mL
102-103 CFU/mL (detection limit)
Milk
Raw milk
Nogva et al. (2000)
RodriguezLazaro et al. (2004, 2005) RodriguezLazaro and Hernandez (2006)
Labourintensive
Yang et al. (2007)
Walcher et al. (2010)
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qPCR +IMS
ham, sausages, raw pork, smoked salmon
Meat samples
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qPCR +resins
qPCR (hly gene) +nanoparticlebased IMS
Water, skim milk, unpasteurised whole milk
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3h
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qPCR (hly gene) +IMS
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qPCR (hly gene)+ centrifugation
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ACCEPTED MANUSCRIPT
Table 3. Time to results
Limit of quantification /Levels tested
Measurement uncertainty
Matrix /flora interference
Viability
Plate counts methods
-(2-4 days)
+/(40/g)
(<0.87 log10 CFU/g)
+/-
+
Plate counts + concentration step
-(3-5 days)
+ (0.8/g)
+/(<0.46 log10 CFU/g)
+/-
MPN
-(2-6 days)
++
(similar to plate counts for ~100/g)
+
Molecular techniques (quantitative PCR+FISH)
++ (<1 day)
(100/?1000/g)
Infrastructure /equipment
Validation
+/-
+
+
+
+/-
+/-
+
5
+
+
+/-
-
6
-
+/-
-
- /+
23
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Approximate number of references
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+/-
Labourintensive
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Method
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ACCEPTED MANUSCRIPT Highlights
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None new method is ideal for any purpose, and users must choose them according to their needs
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• •
We summed up the current knowledge concerning L. monocytogenes enumeration Enumeration difficulties are linked to low contamination levels in foods the improvement of L. monocytogenes enumeration gave place to a considerable amount of research projects Both new culture based techniques and molecular methods are not largely used
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S0740-0020(15)00172-0
DOI:
10.1016/j.fm.2015.09.003
Reference:
YFMIC 2447
To appear in:
Food Microbiology
Received Date: 19 March 2015 Revised Date:
28 August 2015
Accepted Date: 3 September 2015
Please cite this article as: Auvolat, A., Besse, N.G., The challenge of enumerating Listeria monocytogenes in food, Food Microbiology (2015), doi: 10.1016/j.fm.2015.09.003. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT 1
The challenge of enumerating Listeria monocytogenes in food
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Anaïs Auvolat and Nathalie Gnanou Besse*.
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French Agency for Food, Environmental and Occupational Health & Safety (ANSES),
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Laboratory for Food Safety.
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(ANSES, Laboratoire de Sécurité des Aliments, 14 Rue Pierre et Marie Curie, 94701 Maisons
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Alfort, France)
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ABSTRACT
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Listeria monocytogenes is recognised as a serious foodborne pathogen in humans.
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However, food products are usually contaminated at low levels (i.e. <100 CFU/g) and there is
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still no adequate enumeration method for testing food. Much research has been carried out to
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improve Listeria enumeration methods, leading to several proposed alternative methods such
17
as the most probable number technique, molecular-based methods and bacterial cell
18
concentration techniques. Here, we catalogue the current knowledge concerning L.
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monocytogenes enumeration, with a particular focus on the problem of enumerating low
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level contamination.
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Keywords: Listeria monocytogenes, microbiological methods, enumeration, MPN,
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quantitative PCR, filtration, digital PCR.
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Running title: Current research on Listeria enumeration.
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* Corresponding author: Tel. +33 1 49 77 28 32; fax: +33 1 49 77 46 66; E-mail address:
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[email protected] (N. Gnanou Besse).
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INTRODUCTION
Despite its low incidence, foodborne listeriosis is characterised by life-threatening
32
symptoms and a high mortality rate of up to 30% (Anonymous, 2000). In addition, the the
33
presence of Listeria monocytogenes in food has important economic consequences, such as
34
the withdrawal of products from the consumer marketplace and a decrease in sales for the
35
incriminated products. Since 2000, an increase in listeriosis cases has been observed in
36
several European countries, but reasons for this increase remain unclear. According to the
37
European Union Summary Reports, there was a significant increase in the notification rate of
38
listeriosis cases in humans between 2002 and 2006 (Anonymous, 2007). This notification rate
39
remained at the same level in 2007, with 1558 such cases recorded in 26 Member States
40
(Anonymous, 2009a). During the 2008-2012 period, the number of reported confirmed cases
41
of human listeriosis in the European Union (EU) increased substantially (Anonymous, 2014).
42
In 2012, there were 1642 confirmed human cases, which was a 10.5% increase compared
43
with 2011 (1486). Moreover, 198 deaths were reported (case fatality rate of 17.8%)
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(Anonymous, 2014).
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Improvement of L. monocytogenes enumeration techniques is thus a subject of major
47
concern in the field of food hygiene. Adequate enumeration methods are essential to provide
48
reliable data for research studies in predictive microbiology, epidemiology, quantitative risk
49
assessment, and for routine analysis or monitoring programmes in food processing plants.
50
However, despite this need, there has been no adequate, sensitive enumeration method
51
identified to date. Food products are usually contaminated at very low levels, i.e. at less than
52
100 colony forming units (CFU) per gram of food product (Anonymous, 2012). Accordingly,
53
the International Commission on Microbiological Specifications for Foods (1994) set the
54
critical food-safety threshold at 100 CFU/g, for non “at-risk” populations. The recent
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ACCEPTED MANUSCRIPT European Community (EC) Regulation 2073/2005 (Anonymous, 2005) on microbiological
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criteria for foodstuffs established a quantitative limit for L. monocytogenes of 100 CFU/g,
57
which is applicable to certain categories of ready-to-eat food products within their quality-
58
based shelf-life. The manufacturer must be able to demonstrate that its product will not
59
exceed this limit during storage, based on various types of data and studies, such as challenge
60
tests, durability studies and predictive microbiology. Improving L. monocytogenes
61
enumeration has inspired extensive research, leading to the proposal of several methods
62
alternative to the standard reference method, most of them being described in the review of
63
Gnanou Besse and Colin (2004). In most cases, L. monocytogenes is enumerated using the
64
most probable number (MPN) technique, although there have been attempts to enumerate L.
65
monocytogenes with molecular biology-based methods. Another approach to improve the
66
sensitivity of the enumeration method has been to use a concentration technique, such as
67
membrane filtration, which is already widely used for the microbiological analysis of water
68
and other liquids.
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More recently, there have been changes and improvements in the above-cited
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methods: MPN methods have been combined with polymerase chain reaction (PCR)
71
techniques to avoid numerous and fastidious confirmation steps, quantitative PCR methods
72
have been improved by the inclusion of pre-amplification steps that purify and concentrate
73
DNA better, and eliminate non-viable cells. More recently, digital PCR combining the
74
principles of the MPN and molecular based methods allow accurate quantification of target
75
cells, but its application in microbiology is still very limited.
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Comprehensive reviews of rapid methods and automation in microbiology for L.
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monocytogenes include advances in detection, typing, total viable cell count methodologies,
78
instrumental analysis, PCR, biosensors, etc. (Churchill et al., 2006; Jasson et al., 2010;
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Rossmanith and Wagner 2010; Jadhav et al., 2012; Postollec et al., 2011. The objective of
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ACCEPTED MANUSCRIPT 80
this paper is to review the most recent knowledge regarding L. monocytogenes enumeration in
81
food, focusing in particular on the problem of low level enumeration.
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1. CONVENTIONAL METHODS: CULTURE-BASED TECHNIQUES
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1.1.
REFERENCE STANDARD METHOD:
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Direct plate counts are quite simple and fast, but are characterised by poor
86
performance in terms of sensitivity, reproducibility, recovery of stressed cells, and sometimes
87
selectivity (Jasson et al., 2010). This relatively high variability is a common feature to all
88
conventional enumeration methods used in food microbiology. This variability needs to be
89
taken into account, and should be considered when establishing tolerance limits and sampling
90
plans for food control. Automation, such as the spiral plate method, can be an improvement
91
on direct plate counts and can significantly reduce required manpower and material costs
92
(Jasson et al., 2010).
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The European and International Standard method for enumeration of L.
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monocytogenes EN ISO 11290-2 (Anonymous, 1998, 2004) is cited as the reference standard
96
method in the quantitative criteria of EC Regulation no. 2073/2005 for L. monocytogenes.
97
This method consists of spreading decimally diluted samples and further decimal dilutions on
98
selective agar plates. PALCAM agar, long prescribed by the reference method, cannot
99
distinguish L. monocytogenes from other Listeria spp. colonies. Therefore, five typical
100
colonies must be confirmed per plate and counts are based on the ratio of colonies confirmed
101
as L. monocytogenes. This dramatically increased method variability. The specificity has
102
however improved since 2004 with the introduction of a more specific agar, agar Listeria
103
according to Ottaviani and Agosti (LOA agar), which distinguishes L. monocytogenes from
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ACCEPTED MANUSCRIPT other species of Listeria and thus better identifies the L. monocytogenes colonies for
105
confirmation. LOA agar was adopted by the International Organization for Standardization
106
(ISO) as the standard medium for L. monocytogenes detection and enumeration methods. This
107
medium performs well, as demonstrated in terms of productivity ratio (at least 0.95),
108
selectivity and detection ratio (Vlaemynck et al., 2000).
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Meanwhile, it has been shown that the precision of this standard method is relatively
110
poor, especially at low counts. The method still lacks sufficient sensitivity to reliably quantify
111
L. monocytogenes at 100 CFU/g and does not seem to be optimal for the examination of food
112
products that are usually contaminated at low levels. In fact, plate counts are characterised by
113
an enumeration limit of 100 CFU/g of original non-blended sample when spreading a total of
114
1 mL of the blended (1/10 diluted) sample onto three 90 mm plates (or one 140 mm plate).
115
Liquid products do not need any additional suspension thus eliminating the initial dilution
116
that results from making a homogeneous suspension of the food sample and the theoretical
117
limit of enumeration becomes 10 CFU/g. Furthermore, the variability of the method at this
118
level is particularly high. According to the general EN ISO 7218 standard “Microbiology of
119
food and animal feeding stuffs – General requirements and guidance for microbiological
120
examinations”, (Anonymous, 2013):
121
- the limit of detection, based on the formation of a single CFU, of the method, for a non-
122
liquid sample, is 10 CFU/g when plating 1 mL of the blended sample.
123
- the theoretical limit of quantitation is four times the limit of detection or 40 CFU/g when
124
plating a total volume of 1 mL of the blended sample. Below this limit, the microorganism is
125
considered to be present, but its concentration cannot be reliably quantified.
126
- when fewer than 10 CFU combined total are present on the plates, the result must be
127
expressed as an estimated result or with its measurement uncertainty.
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In addition, the EN ISO 7218 standard specifies that two successive dilutions must be
129
analysed (1 plate per dilution), and when only one dilution can be analysed, then two plates
130
per dilution should be used. Consequently, when enumerating a contamination level of 100 CFU/g (e.g.
132
approximately 20 CFU in total when spreading in duplicate 1 mL of the decimally diluted
133
sample on three plates), the result is associated with a 95 % confidence interval of 60 to 150
134
CFU/g (EN ISO 7218 standard, 1996, Annex A: Table for confidence intervals for low
135
number estimation).
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Regardless of the contamination level, results have been highly variable in some
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validation studies:
A validation study of EN ISO 11290-2 in its initial version (without the current
139
amendment which replaced PALCAM agar with LOA agar) implementing an inter-laboratory
140
trial was funded by the European Commission (Standards, Measurement and Testing Fourth
141
Framework Programme Project SMT4-CT96-2098). The objective was to determine the
142
precision of the methods in terms of repeatability (r) and reproducibility (R) using different
143
sample types (meat, cheese, dried egg powder and reference material) inoculated at different
144
levels. r is the difference, expressed as a ratio, between two results obtained on the same
145
sample and in exactly the same conditions, in the same laboratory, whereas R is the
146
difference, expressed as a ratio, between two results obtained on the same sample in two
147
different laboratories. The overall repeatability was r = log10 0.58, and the overall
148
reproducibility was R = log10 0.81 (Scotter et al., 2001). This means that for a sample having
149
a true contamination of 100 CFU/g, a laboratory may find a result as low as 15 CFU/g, or as
150
high as 646 CFU/g.
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The objective of the recent validation study of the revised Nordic Committee on Food
152
Analysis (NMKL) method no. 136 “Listeria monocytogenes. Detection and enumeration in
7
ACCEPTED MANUSCRIPT foods” (Loncarevic et al., 2008), which is very similar to the current EN ISO 11290-2
154
standard, was to determine the precision of the standard method in terms of repeatability and
155
reproducibility using different food sample types. For a contamination level close to 100
156
CFU/g (2.2 log CFU/g), the overall repeatability (r) of the method was 0.44, 0.91, and 0.66
157
and the overall reproducibility (R) was 0.48, 1.08, and 0.54 for Brie cheese made from
158
pasteurized milk, hot-smoked salmon, and cooked vacuum-packed ham, respectively
159
(Loncarevic et al., 2008). In the presence of L. innocua the corresponding values reached
160
r=0.76, 0.52, and 0.70 and R=0.87, 0.68,and 0.87 for the same three products, respectively.
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This means that for a sample having a true contamination level of 100 CFU/g, if R = 0.87 a
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laboratory may find a result as low as 13 CFU /g, or as high as 741 CFU/g.
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More recently, a new validation study of EN ISO 11290-2 (in its new version, with
164
LOA agar) through inter-laboratory trial has been fully funded by the European Commission
165
(CEN Mandate M381). The European Committee for Standardization (CEN)/Technical
166
Committee 275/Working Group 6, in charge of standardisation in food microbiology methods
167
at the European level, received a mandate from the European Commission to standardise
168
and/or validate using inter-laboratory studies (ILS), a set of reference methods in food
169
microbiology, including the reference methods for the detection and enumeration of L.
170
monocytogenes in food. The ILS were conducted in 2013, and standards will be published by
171
the end of 2016. Since Standards EN ISO 11290-1&2, under revision are applicable to all
172
food, feed and food processing environments, the ILS were conducted on five matrices (cold-
173
smoked salmon, milk powder, vegetables, environmental samples and cheese), representative
174
of categories cited in EC Regulation no. 2073/2005 on microbiological criteria for foodstuffs.
175
For quantitative studies, and for each matrix, four levels of contamination including a blank
176
(0, 100-150, 1000-1500, 10 000-15 000 CFU/g), and two blind replicates per level were used.
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In total, eight samples per matrix were sent to each laboratory. These contamination levels are
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different countries participated in each trial. Results of the ILS were generally satisfactory,
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comparable, if not better in comparison with former validation studies. The revised version of
181
EN ISO 11290-1&2, will include these results, and the estimation of the performances of the
182
method, in terms of the standard deviations of repeatability and reproducibility, sensitivity,
183
specificity and limit of detection.
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1.2.
CULTURING METHODS THAT INCLUDE A RESUSCITATION STEP:
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When enumerating low numbers of L. monocytogenes, the recovery of injured bacteria
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cells is an issue of great importance. In food, L. monocytogenes is often affected by one or
189
several sources of stress caused by a variety of processing treatments including heating,
190
freezing, drying, exposure to acids, exposure to disinfectants, and exposure to high osmotic
191
pressure. Kang and Fung (1999) developed a solid media resuscitation method (thin agar
192
layer method, TAL) to recover heat-injured L. monocytogenes. They overlaid 5 mL of non-
193
selective tryptic soy agar (TSA) medium onto pre-poured and solidified modified Oxford
194
medium (MOX). The MOX selective agents diffuse slowly to the top layer, allowing the cells
195
on the TSA to undergo repair during the first hours of incubation. When enumerating heat-
196
injured L. monocytogenes from milk, significantly higher counts were obtained with this
197
protocol than with direct plating on MOX agar. Wu and Fung (2002, 2003, 2004) improved
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the TAL method by the simultaneous recovery and detection of four pathogens (including L.
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monocytogenes) from foods, the use of the ISOGRID to enumerate very low pathogen levels
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on TAL, and the use of Oxyrase® in the TAL system to stimulate recovery of pathogens in
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foods. Miller et al. (2010) evaluated the TAL procedure for the recovery of injured L. innocua
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cells exposed to thermal treatments. The TAL procedure (PALCAM agar with TSA+Yeast
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Extract agar overlay) recovered more bacteria (>1 log10) than the PALCAM direct count
204
when used on parsley or meat products.
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1.3.
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CULTURING METHODS THAT INCLUDE A CELL CONCENTRATION STEP
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According to NF EN ISO 7218 “Microbiology of food and animal feeding stuffs –
209
General requirements and guidance for microbiological examinations”, section 9.2.2: for the
210
enumeration of low numbers of microorganisms, it is possible to concentrate the bacteria
211
present by centrifugation or membrane filtration to improve enumeration. However, it is
212
recommended to check the performance of the complete method in terms of sensitivity,
213
selectivity, linearity and repeatability.
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Membrane filtration has been applied to the detection and enumeration of L.
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monocytogenes, with the detection of target strains by culturing the membrane on selective
216
agar (Entis and Lerner, 2000; Gnanou Besse and Lafarge, 2001; Jehanno et al., 1999) or
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through various rapid techniques such as, for example, the direct epifluorescence filter
218
technique (DEFT, Tortorello et al., 1997), electronic microscopy (Hale et al., 1990), or
219
immunoassays (Carrol et al., 2000). The necessity of processing the food in a filterable form
220
limits the applicability of membrane filtration because volumes that can be filtered depend on
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the sample type. The above-cited filtration protocols are generally quite labour-intensive, and,
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in most cases, they have been developed and evaluated for specific types of products.
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The hydrophobic grid membrane filter (HGMF) technique, including a filtration step,
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has been applied to L. monocytogenes enumeration (Entis and Lerner, 2000). This technique
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has been widely tested and has already been validated by AOAC International for several
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ACCEPTED MANUSCRIPT bacterial species in food (Entis, 1986, 1989, 1990, 2000). The HGMF is a membrane upon
227
which is applied a hydrophobic grid to divide it into a large number of individual growth
228
compartments (generally about 1600). The HGMF count is determined by MPN calculation.
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Thus, the counting range of the HGMF is expanded well beyond the actual number of
230
observed growth units (effective MPN counting range of about three logarithmic cycles), and
231
consequently covers a much larger range than is obtained with direct plate counts or
232
membrane filtration, and with greater precision. HGMF consequently minimises both labour
233
(needed for diluting) and the probability of losing data by misjudging platable dilution ranges.
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For the enumeration of L. monocytogenes in cold-smoked salmon, a sensitive
235
enumeration method based on membrane filtration, followed by transfer of the filter to a
236
selective medium, has been recently developed and standardised in France (Anonymous,
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2009b; Gnanou Besse et al., 2004). Briefly, the operating protocol consists of three filtration
238
steps conducted in parallel, using respectively 5, 15 and 30 mL of a 1:10 dilution of the
239
salmon suspension (previously treated with an enzyme surfactant) through 0.45 µm pore-size
240
cellulose ester membranes. The filters are then transferred to LOA agar for cultivation.
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Colonies are counted on filters containing less than 100 colonies. Typical L. monocytogenes
242
colonies need to be reisolated and confirmed according to the EN ISO 11290-2 method.
243
When contamination levels are unknown, the standard ISO 11290-2 method should also be
244
conducted to be able to enumerate samples contaminated at levels higher than 200 CFU/g.
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The results obtained with this newly developed method have been compared with those
246
obtained with the reference EN ISO 11290-2 method. The membrane filtration method
247
provided more precise results for the enumeration of L. monocytogenes from both artificially
248
and naturally contaminated cold-smoked salmon (Gnanou Besse et al., 2004). It improved the
249
enumeration sensitivity to 0.2 CFU/g, instead of 10 to 100 CFU/g with the reference EN ISO
250
11290-2 method. The method has been validated through an ILPT (Gnanou Besse et al.,
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ACCEPTED MANUSCRIPT 2008). Cold-smoked salmon artificially contaminated at two different levels (approximatively
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0.6 and 1.6 log10 CFU/g) was used as matrix. Unlike most validation studies, very low
253
contamination levels were chosen to be more representative of actual foodborne
254
contamination levels. Reproducibility standard deviations were 0.23 log10 CFU/g and 0.15
255
log10 CFU/g at the lower level and the higher level, respectively. Under certain conditions, the
256
measurement uncertainty can be derived from the method’s reproducibility standard
257
deviation, calculated to be 0.46 log10 CFU/g for the lower contamination level and 0.30 log10
258
CFU/g for the higher contamination level. These values can be considered as satisfactory for
259
such low contamination levels. This sensitive enumeration method is quite simple to use, is
260
based on classical microbiology and relies on the use of the same selective agar as the
261
reference EN ISO 11290-2 method. This method can be useful for precise estimation at
262
densities of around 100 CFU/g or to conduct challenge tests, durability studies and
263
quantitative risk assessment studies under more realistic conditions, at low contamination
264
levels. The method has already been successfully used to monitor the growth of L.
265
monocytogenes in cold-smoked salmon at various initial inoculum densities (Gnanou Besse et
266
al., 2006), and to estimate contamination levels and growth of L. monocytogenes in naturally
267
contaminated cold-smoked salmon (Beaufort et al., 2007). Nevertheless, the filtration method
268
requires the use of a specific apparatus and is more labour-intensive than the reference
269
method. The use of a very detailed protocol and a training period is necessary before the
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method can be performed satisfactorily and to avoid technical difficulties such as filtering
271
problems (Gnanou Besse et al., 2008). Moreover, this method cannot be implemented on
272
other food products without additional validation studies.
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The applicability of the membrane filtration method to other categories of matrices in
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comparison with the EN ISO 11290-2 modified reference method was evaluated mainly with
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artificially contaminated foods (Baudouin et al. 2010). In that study, all foods could be
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ACCEPTED MANUSCRIPT filtered, but large differences were observed between food-matrix categories (vegetables,
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meat and seafood products being the most easily filtered). However, even for those products
278
that were difficult to filter, approximately 5 mL could be concentrated on a filter, enabling
279
analysis of a greater quantity of food. However, the proposed method seems to be difficult to
280
apply to some food products, due to background microflora interfering with colony reading.
281
This was the case for several cheese and delicatessen matrices. Finally, an evaluation of this
282
method was performed with several categories of foods naturally contaminated with L.
283
monocytogenes (Barre et al., 2015). The results obtained with this technique were compared
284
with those obtained from the modified reference EN ISO 11290-2 method for the
285
enumeration of L. monocytogenes in food, and provided more precise results. In most cases,
286
the filtration method makes it possible to examine a greater quantity of food, thus greatly
287
improving the sensitivity of the enumeration. In particular, the method provides good results
288
for seafood products, vegetables, and some meat products (minced or unprocessed meat,
289
some sausages, etc.). However, it was unsuitable for some food categories (cheese, pastries,
290
delicatessen) due to filtration problems and background microflora interference.
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In conclusion, the membrane filtration method is an improvement over the reference
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standard method for enumerating low levels of L. monocytogenes in certain food matrices,
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with satisfactory sensitivity. The sensitivity of the method is critical when enforcing the
294
European regulatory contamination limit of 100 CFU/g as well as conducting shelf-life
295
studies under realistic conditions of low level contamination.
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1.4.
THE MOST PROBABLE NUMBER TECHNIQUE
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The MPN technique relies on probability statistics; results are directly related to the
299
frequency of occurrence in a dilution series of a sample, which is used to calculate the
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ACCEPTED MANUSCRIPT likelihood of the presence of a given number of bacteria cells in the sample. The statistics are
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calculated from the number of positive tubes out of a total number of tubes tested, for a series
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of consecutive dilutions. This technique has two main assumptions. First, it is assumed that
303
organisms are distributed randomly throughout the sample, and that cell clumping does not
304
occur . This is an important parameter to take into account, considering the analytical test
305
portion is likely to be much smaller than the food sample and one wants to apply the results to
306
the entire food sample and not just to the portion that was tested. Second, it is assumed that
307
each dilution, when properly incubated, will exhibit growth when one or more organisms are
308
present (Oblinger and Koburger, 1975). In most cases, L. monocytogenes MPN procedures
309
rely on conventional detection methods for media, incubation times and isolation protocols,
310
such as those published by, for example, the US Food and Drug Administration (FDA), the
311
US Department of Agriculture’s Food Safety and Inspection Service (USDA-FSIS), the
312
International Standard Organization (ISO) or the Netherlands Government Food Inspection
313
Service (NGFIS) (see Anonymous, 1992; Aymerich et al., 2004; Blysick-McKennal and
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Schaffner, 1994; Capita and Alonso-Calleja, 2003; Gombas et al., 2003; Swaminathan et al.,
315
1994; Tortorello et al., 1997; Tran and Hitchins, 1996).. This method of estimating bacterial
316
densities is still widely used for L. monocytogenes, both for research studies and routine
317
analysis. Some of these MPN methods have already been reviewed in Gnanou Besse and
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Colin (2004): Blysick-McKennal and Schaffner, 1994; Capita and Alonso-Calleja, 2003;
319
Cortesi et al., 1997; Dalgaard and Jorgensen, 1998; De Buyser et al., 1990; Farber and Daley,
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1994; Hayes et al., 1992; Inoué et al., 2000; Jemmi and Keusch, 1992; Jorgensen and Huss,
321
1998; Tran and Hitchins, 1996; Yu and Fung, 1993; Yu et al., 1995., etc. The MPN technique
322
has numerous useful applications. When the contamination level is unknown, it is important
323
to use an efficient method for a wide range of bacterial contamination levels. With the MPN
324
method, estimates of a population can be made using any number of dilutions, as in the plate
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ACCEPTED MANUSCRIPT count method. Yu and Fung (1993) showed that a five-tube MPN method using Fung-Yu
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tubes was effective in enumerating both low and high levels of L. monocytogenes (from 10 to
327
106 CFU/mL) in cooked chopped hams, with a correlation coefficient of 0.99 when compared
328
with direct plate counts. However, Carroll et al. (2000) showed that, in spiked meat products,
329
when the inoculum level was high, the MPN procedure tends to underestimate the number of
330
L. monocytogenes, whereas, when the inoculum is low, MPN tends to overestimate the
331
number of L. monocytogenes. Hildebrandt and Schott (2001) compared the direct plate count
332
method with the MPN method for quantification of L. monocytogenes in artificially and
333
naturally contaminated beef samples sold at retail. The detection limit of MPN was 66%
334
lower than the plate count, and detected more positive samples. Nevertheless, the plate count
335
method is preferred for routine use, due to its slightly higher productivity and much lower
336
variation in results.
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However, when contamination levels are suspected of being low (<1 CFU/g) then the
338
MPN method is the more appropriate choice for enumeration. Large sample size is what
339
makes this technique extremely powerful. No other enumeration technique allows
340
enumeration of a few cells per 100 g or covers such a large range of samples.
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The fundamental disadvantage of using the MPN method is that L. monocytogenes
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must be recovered and confirmed from each presumptive positive tube. Other disadvantages
344
often associated with the MPN method include various aspects of the technique, such as time,
345
materials, cost, space, precision and selectivity.
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MPN estimates of bacterial densities generally lack precision. Proper interpretation of
347
the “confidence interval” around a given value, as reported in the tabular MPN data, is
348
fundamental. The tabular MPN value actually represents a range, not an absolute value. MPN
349
estimates are often credited with a precision they do not live up to (Oblinger and Koburger,
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ACCEPTED MANUSCRIPT 350
1975). However, the measurement uncertainty of MPN methods is often of the same order as
351
that of direct plate count methods. The presence of competitive microflora in the food sample may affect MPN
353
performance (Blysick-McKennal and Schaffner, 1994; Tran and Hitchins, 1996). The
354
problem of competition between bacterial strains must be considered when several Listeria
355
species are present in food samples. From a practical perspective, overgrowth by a non-
356
pathogenic species of Listeria may mask the presence of low numbers of L. monocytogenes in
357
the original food sample, and thus result in false-negative results (Gnanou Besse et al., 2005,
358
2010). An enrichment step may lead either to the non-detection of L. monocytogenes or to an
359
underestimation of L. monocytogenes counts when combined with an MPN procedure.
360
Although conflicting results have been reported, in the case of induced stress, the selective
361
enrichment procedure of most standard methods may also lead to lower recovery rates of
362
injured or stressed bacterial cells, compared to non-selective enrichment procedures, for
363
example in cheeses (Rijpens et al., 2004). Moreover, common food industry sources of stress
364
may have a high impact on single-cell lag time and growth probabilities of L. monocytogenes
365
in half-Fraser broth, meaning that the risk of false-negative samples may be high for very low
366
contamination levels, depending on the type of stress (Dupont et al., 2009).
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All of the enrichment, isolation and identification steps are generally included in the
368
MPN protocol, and are performed and replicated for each dilution. To counteract the above
369
mentioned problems, the MPN technique has recently been combined with PCR and real-time
370
PCR for confirmation of positive results.
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PCR analysis was used by Aymerich et al. (2004) to confirm MPN tubes for
372
enumerating L. monocytogenes in spiked cooked and fermented meat products (about 2 to 3
373
log10 CFU/g). The prfA gene was chosen for amplification, and the method produced final
374
results in two days.
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ACCEPTED MANUSCRIPT Martin et al. (2004) compared MPN with PCR confirmation (MPN-PCR) with direct
376
plating on PALCAM agar for enumerating L. monocytogenes in fermented sausage (chorizo).
377
The MPN analysis was performed using half-Fraser broth for 40 h at 30 °C. The PCR reaction
378
targeted the prfA gene. The counts determined by MPN-PCR were generally higher than plate
379
counts, which were impossible in some cases, due to very abundant interfering bacteria.
380
However, the technique remained labour-intensive because pre-PCR treatments for
381
confirmation of positive results included centrifugation, and filtration or resin-based
382
techniques (Chelex-100), and visualization of the PCR products required gel electrophoresis.
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In 2010, Jeyaletchumi et al. evaluated a MPN-PCR technique, based on the
384
combination of the FDA-Bacteriological Analytical Manual (BAM) selective enrichment and
385
PCR detection of the listeriolysin O gene, to detect L. monocytogenes in salad vegetables.
386
This method proved to be sensitive, accurate, linear and significantly reduced time and
387
labour: results are obtained within two days, compared with the traditional five days.
388
However, although the correlation between spiking concentration and microbial levels was
389
greater than that of the EN ISO reference method and FDA-BAM method, it was only 67%
390
for the artificially contaminated samples. Moreover, several centrifugation steps were
391
necessary for DNA extraction and purification, and gel electrophoresis was required to
392
visualise the PCR products, making the technique very labour-intensive.
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de Martinis et al. (2007) developed a SYBR green based real-time PCR method,
394
targeting the 16S rRNA gene, to accelerate MPN tube confirmation of L. monocytogenes in
395
several foods (spiked milk, lettuce, smoked salmon, Brie cheese, ice cream, pork pâté, salami,
396
ready-to-eat shrimp, raw ground beef and fresh soft cheese). The use of real-time PCR
397
produced results in two days instead four by reducing the time needed to confirm the presence
398
of L. monocytogenes in the MPN tubes. For contamination levels from 70 to 100 CFU/g,
399
there was good agreement between the conventional and PCR MPN methods. Some foods
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ACCEPTED MANUSCRIPT required diluted enrichment sample extracts to obtain positive PCR results, comparable with
401
those of the standard MPN culture method. This suggests a concentration-dependent matrix
402
interference in the real-time PCR assay. Moreover, the boiling-water DNA extraction method
403
requires successive centrifugations at 8000 ×g for 10 min, increasing laboratory time for this
404
assay.
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de Oliveira et al. (2010) also evaluated the combination of the MPN enumeration
406
method with real-time PCR confirmation of L. monocytogenes. The products tested included
407
minimally processed leafy vegetables samples. L. monocytogenes was enumerated in six
408
artificially and two naturally contaminated samples using the MPN technique with detection
409
and confirmation by conventional culturing-based methods and with real-time PCR targeting
410
the16S rRNA gene of L. monocytogenes. The MPN assay, with real-time PCR confirmation,
411
detected 1-5 CFU per 50 g with no interference from the background microflora. There was
412
generally good agreement between conventional MPN and the real-time PCR method, which
413
was easy to perform and produced results in 48 h, instead of seven days for the conventional
414
method. However, for each MPN tube, the culture DNA must be extracted (boiling-water
415
method) which included several rounds of centrifugation. Another problem is the interference
416
with inhibitors contained in the product. Since this phenomenon occurred more frequently in
417
leafy vegetables with a stronger pigmentation, the authors suggested that the green pigment
418
(chlorophyll) contained in the leaves could be the inhibitor; thus the largest quantities of
419
sample showed the lowest number of positive reactions. Dilution of the DNA extracts (1:50)
420
was therefore needed before PCR analysis. This pigment interference makes this protocol
421
cumbersome and demonstrates an important limitation of this method.
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Methods for confirming MPN tubes, other than PCR, have also been investigated.
423
Cruz et al. (2012) compared six commercial kits (Clearview™, Reveal®, TECRA®,
424
VIDAS® LDUO, VIP™ and Petrifilm™ kits) for detecting Listeria species in seafood and
18
ACCEPTED MANUSCRIPT environmental samples using the standard FDA BAM enrichment protocol with the MPN
426
technique. The commercial kits gave similar results as the BAM Listeria confirmation
427
protocol for the seafood products, but showed lower sensitivity than the BAM protocol for
428
environmental samples. However, for the environmental surface samples, none of the tested
429
MPN methods gave accurate quantification, and results were largely underestimated. This
430
study illustrates the difficulties in detecting and quantifying Listeria in environmental
431
samples, in which cells are subjected to stressful conditions (dehydration, nutrient limitation,
432
etc.).
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In conclusion, culture-based techniques include very different methods, and are
434
generally characterised by high variability in precision (Table 1). They are relatively simple
435
and rapid to perform, confirm the presence of only viable cells, but are generally time-
436
consuming, and sometimes labour-intensive. They may require individual biochemical
437
confirmation of the species on isolated colonies. In addition, there is no detection of viable,
438
but non-culturable bacteria (VBNC), injured or stressed cells, and overgrowth of other
439
bacteria can alter the results (D’Urso et al., 2009; Jadhav et al., 2012; Jasson et al., 2010).
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2. MOLECULAR BIOLOGY METHODS
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2.1.
FLUORESCENCE IN SITU HYBRIDISATION METHODS
444 445
The main drawback of the conventional plate count method is its long time to results
446
(4-7 days). FISH (fluorescence in situ hybridisation) is a cytogenetic technique used to detect
447
and localise the presence or absence of specific DNA sequences. FISH uses fluorescent
19
ACCEPTED MANUSCRIPT probes that bind to complementary sequences and bound probes are then visualised using
449
fluorescence microscopy. Fuchizawa et al. (2008) designed a FISH technique combined with
450
filter cultivation (FISHFC) to rapidly enumerate viable Listeria spp. The fluorescent labelled
451
probe was designed from the 23S rRNA region of Listeria spp. genome. The FISHFC
452
technique was chosen to limit interference of food particles and to facilitate counts under an
453
epi-fluorescence microscope. Their method used 1 mL of sample homogenate which was
454
filtered through 0.45 µm pore size polypropylene membranes and incubated for 12 h at 37°C,
455
followed by 2 h of FISH treatment. At a level of about 2 log10 CFU/g, there were no
456
significant differences in enumeration between direct plate counts and the FISHFC method in
457
various foods (smoked salmon, cheese, ham). In 2009, Fuchizawa et al. improved the
458
specificity of the FISHFC method to quantify L. monocytogenes in food within 16 h.
459
461
2.2.
PCR METHODS
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In the early 1990s, PCR was only used for identification of pure bacterial cultures or
463
colonies on agar plates. Thereafter, PCR was recognised as one of the most promising rapid
464
microbiological methods for the detection and identification of bacteria in a wide range of
465
foods (Garrido et al., 2013). More recently, PCR techniques have been applied to
466
quantification. Although PCR has been used indirectly for enumeration applications such as
467
MPN-PCR methods (see above), the use of PCR to quantitate directly from samples initially
468
relied on competitive PCR techniques.
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PCR is a technique that amplifies a specific DNA sequence, producing thousands to
470
millions of copies. PCR reactions are characterised by a three-step cycle: denaturation of the
471
double-stranded sample DNA, annealing of primers that target DNA by complementarity, and 20
ACCEPTED MANUSCRIPT finally, synthesis of a new, complementary DNA strand by DNA polymerase (extension).
473
Typically, PCR consists of a series of 20 to 40 cycles. In an efficient cycle, the quantity of
474
DNA depends on the initial quantity of DNA (de Martinis et al., 2007; Jadhav et al., 2012;
475
Ryser and Marth, 2007). Commonly targeted L. monocytogenes genes are the listeriolysin O
476
gene (hlyA), the invasion-associated protein p60 gene (iap), internalin genes (inlA, inlB),
477
aminopeptidase C genes, and 16S rRNA (Churchill et al., 2006).
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After amplification, agarose gel electrophoresis is performed to separate and visualise
479
the PCR products according to their size. Conventional, end-point PCR methods provide only
480
qualitative results, because the amount of PCR product does not reflect the initial amount of
481
DNA. PCR products increase exponentially during the early cycles of the PCR, but level off
482
in the final cycles (Rijpens and Herman, 2002). However, a variety of quantitative PCR
483
techniques based on the indirect MPN-PCR technique (see above), or direct PCR
484
quantification have been recently developed. Using these techniques, semi-quantification of
485
the initial amount of bacteria in a sample is possible. However, the short period for sampling
486
PCR products, the lack of automation and poor gel sensitivity, together with high risk of
487
carry-over contamination are major drawbacks to these techniques. PCR approaches have
488
benefited from the introduction of real-time PCR techniques (discussed below) that detect
489
amplification during the reaction itself, as opposed to end-point detection used in PCR. In
490
quantitative competitive PCR, a known amount of internal competitor (for example, a
491
deletion mutant) is co-amplified with the target sequence in the same reaction. In general,
492
target and competitor have different sizes so as to be easily discriminated by gel
493
electrophoresis. To determine the level of contamination, extracted test DNA is serially
494
diluted and added to a constant amount of competitor. The reaction tube that yields identical
495
amounts of both types of amplicon at the end of the PCR is assumed to contain initially
496
identical amounts of target and competitor. The amount of target can be reliably inferred from
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this technique provided that the targets and competitor have the same amplification
498
efficiency, a condition rarely observed with amplicons of different size. The advantage of this
499
method compared to other qPCR methods is that no expensive fluorophores or radioactive
500
labels are required (Churchill et al., 2006). Today, use of competitive PCR is very limited. Wang and Hong (1999) developed a competitive PCR protocol combined with an
502
enzyme-linked immunosorbent assay (ELISA) for quantification of L. monocytogenes in milk
503
or fish samples. The target gene was iap, a virulence factor in L. monocytogenes. The
504
detection limits in artificially contaminated milk and catfish fillets were respectively 20
505
CFU/mL and 2 CFU/g. There were slight differences (<0.52 log) between expected and
506
detected numbers of L. monocytogenes. Results were obtained in two days, and there were
507
few cross-reactions with other bacteria. Choi and Hong (2003) developed a 5 h competitive
508
PCR for quantification of L. monocytogenes in milk, using the hlyA gene. The detection limit
509
was 103 CFU per 0.5 mL of inoculated milk and the method appeared to be selective for the
510
tested strains. Quantifications were approximately equal to conventional plate counts.
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In fluorescent resonance energy transfer (FRET)-PCR, the DNA product is analysed
512
directly after PCR by measuring a fluorescence signal (Churchill et al., 2006). Two DNA
513
probes of the gene of interest are used, one with a reporter, fluorescein label, and the other
514
with a quencher label. During the annealing and primer extension steps of PCR, the first
515
probe hybridises to the gene of interest, and is then hydrolysed by the 5’3’ exonuclease
516
activity of the DNA polymerase during amplification, resulting in the release of the
517
fluorophore from the probe. The probe with the quencher label can only anneal to the
518
fluorescein-labelled probe at the end of PCR, when the mixture cools to room temperature.
519
Only unbound fluorescein fluoresces; the fluorescence signal read at the end of the reaction
520
thus indicates the number of cells in the sample (Churchill et al., 2006). Koo and Jaykus
521
(2003) used this method, focusing on the hlyA gene, to detect 103-104 CFU of L.
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monocytogenes in 25 g of skim milk. In 2008, O’Grady et al. used FRET-PCR combined with
523
an enrichment step to qualitatively detect 1-5 CFU of L. monocytogenes in 25 g of food, as
524
part of a real-time PCR assay.
526 527
2.3.
REAL-TIME QUANTITATIVE PCR (qPCR)
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In qPCR, the PCR products are detected as they accumulate. The amount of generated
529
PCR product is proportional to the increase in a fluorescent signal, which is monitored during
530
the exponential phase. The number of cells can be estimated using standard regression curves
531
of the quantification cycle (Cq) values, previously known as threshold cycle (CT) values (or
532
crossing point, take-off point), based on samples at known concentrations. The absence of any
533
essential post-PCR step simplifies the method, and allows high throughput and automation.
534
Several fluorescence systems based on various dyes can be used for detecting the signal
535
(Rijpens and Herman, 2002). For example, SYBR Green, a fluorescent dye, has an emission
536
spectrum that is 50- to 100-fold brighter when the dye is bound to double-stranded DNA. The
537
increase in fluorescence can be followed as the DNA amplifies. The fluorescent molecule can
538
also be a target specific oligonucleotide probe labelled with a fluorescent dye along with a
539
quencher. The hydrolysis probes (TaqMan probes) contain the reporter and quencher dye on
540
the same oligonucleotide, resulting in quenching. During the annealing and primer extension
541
step of PCR, the probe hybridizes to the gene of interest, and is then hydrolyzed by the
542
5’3’exonuclease activity of the DNA polymerase during amplification. Once released, the
543
reporter dye is no longer quenched, and the increase in fluorescence can be measured (Jadhav
544
et al., 2012). Nowadays, a great variety of fluorescence reporting systems have been
545
developed, and may be classified as non-specific dye binding (e.g. SYBR Green, SYBR gold,
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ACCEPTED MANUSCRIPT and EVA green), non-specific universal labeled primer (i.e. Amplifluor System from
547
Milipore), or specific (hydrolysis probes, dual labelled probes, Molecular Beacon, Scorpion
548
uniprobes or biprobes, Mediator probes system, LNA or PNA probes). Regarding the
549
comparison of dye-binding chemistries versus probe-binding chemistries, many benefits and
550
drawbacks can be listed for each. Non-specific dyes have high signal/low background, and
551
consequently are highly sensitive. They are inexpensive and can be rapidly developed and
552
thus are attractive for initial exploratory stages. However, with this system, specificity
553
depends entirely on the primers, and the melting curves must be analysed in each experiment.
554
In contrast, with specific probes, systems are more specific, avoid primer-dimer problems,
555
and enable multiplex PCR, but are more expensive.
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Sensitivity of qPCR applied to a food matrix is generally quite limited when compared
557
with other enumeration methods. Consequently, in most cases, qPCR is not suitable for the
558
accurate enumeration of low levels of L. monocytogenes in food (Rijpens and Herman, 2002).
559
To provide enumeration results, qPCR must be applied directly to the food sample (i.e.
560
without any enrichment step), which can be a source of technical difficulty. Appropriate
561
sample preparation is needed to eliminate PCR-inhibiting components from the sample, and
562
to concentrate bacteria to minimum PCR detection limits. A very sensitive PCR system is
563
thus required for qPCR (Rijpens and Herman, 2002). The quantitative range of qPCR is
564
generally over 100 copies of DNA. Although multiple measurements can help improve
565
precision of the standard curve, concentrations of DNA from one to several tens of copies are
566
generally considered as (sometimes) detectable, but non-quantifiable. In fact, at low numbers
567
of target, the PCR reaction results follow a Poisson distribution and primer hybridisation is
568
stochastic.
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For quantification purposes, standard calibration curves generally have to be
570
established using an external standard (absolute quantification). In this case, the choice and
24
ACCEPTED MANUSCRIPT quantification of the standard itself are critical. The capacity of the method to determine
572
accurately the concentration of the target bacteria depends on the linearity, efficiency and
573
limit of quantification of the PCR. Linearity is the ability of the method to generate results
574
proportional to the concentration, and is represented by the regression coefficient r2 of the
575
(linear) relationship between the initial number of L. monocytogenes DNA molecules, and the
576
Cq values. Efficiency (E) is the capacity of the PCR to duplicate the amplicon molecules in
577
each cycle, and is calculated from the slope of the linear regression curve. The limit of
578
quantification is established at the lowest sample dilution in which the resulting confidence
579
interval does not overlap with that of the next dilution (Rodríguez-Lázaro et al., 2005, 2010).
580
The MIQE (minimum information for publication of qPCR experiments) guidelines specify
581
the information that must be reported for a qPCR experiment to ensure its relevance,
582
accuracy, correct interpretation, repeatability, and assessment (Bustin et al., 2009). However,
583
the ongoing lack of consensus in the literature may have adverse consequences on the
584
reliability of these methods and researchers’ confidence in them.
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Viability is another variable that must be considered when evaluating bacterial
586
densities. Conventional PCR detects both viable and non-viable cells. The inability to
587
selectively detect only viable bacterial cells has impaired the implementation of this method
588
in routine food microbiology laboratories.
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Underestimation of the contamination load is also possible, because PCR inhibitors
590
(components of food samples, culture media, nucleic acid extraction reagents, etc.) can lead
591
to false-negative results making the use of qPCR difficult for routine food analysis
592
(Rodríguez-Lázaro et al., 2005). To avoid this problem, pre-treatment procedures can be
593
developed, but in all cases, PCR efficiency should be assessed with an internal amplification
594
control (IAC). To date, however, only a few qPCR assays have included IACs, despite the
595
general opinion that it should be mandatory (Rodríguez-Lázaro et al., 2005). IACs are non-
25
ACCEPTED MANUSCRIPT target DNA fragments that are co-amplified simultaneously with a target sequence, ideally
597
with the same primers. The simultaneous use of two different labelled fluorescent probes
598
(including an IAC probe) makes it possible to detect and quantify the target and, at the same
599
time, assess PCR efficiency (Rodríguez-Lázaro et al., 2005). Problems of lysis and extraction
600
efficiency may also occur before PCR. Additionally, though there have been important
601
advances in technologies, heterogeneity in temperatures among PCR thermocyclers has
602
sometimes been reported, leading to variability and underestimation of results due to
603
inadequate temperature incubation zones and large differences according to the materials and
604
apparatuses used (http://www.realtimepcr.dk/). Another problem likely to compromise the
605
applicability of qPCR is the existence of inter-strain variability in the target DNA sequence,
606
leading to weak signals and underestimation of the amount of DNA (Rodríguez-Lázaro et al.,
607
2004b).
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Nevertheless, the major advantage of qPCR methods is their rapidity compared with
609
culture methods. The absence of any essential post-PCR step simplifies the qPCR method,
610
allows high throughput and automation and decreases the risk of cross-contamination in the
611
laboratory. Moreover, PCR can detect the presence of sub-dominant populations, sometimes
612
unable to grow in current culture conditions, in the presence of large background microflora.
614 615
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2.3.1. RECENTLY DEVELOPPED VARIATIONS ON THE qPCR METHOD In 2001, Hein et al. developed a qPCR method for detection and enumeration of L.
616
monocytogenes or L. innocua in artificially contaminated milk using the iap gene (a Listeria
617
species-specific gene) as the target. The method required several rounds of centrifugation,
618
heating and enzyme treatments for DNA isolation before qPCR could be performed. Results
26
ACCEPTED MANUSCRIPT 619
obtained were 1 to 2 log higher than CFU obtained by the plate count method using
620
PALCAM agar. Rodríguez-Lázaro et al. (2004b) evaluated the usefulness of the iap and hly genes as
622
targets for quantification using qPCR. The hly-based assay resulted in specific, sensitive and
623
accurate quantification of L. monocytogenes, whereas the iap-based assay resulted in
624
heterogeneous and unreliable quantification, due to serovar-related target sequence
625
variability.
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Rodríguez-Lázaro et al. (2004a) developed a duplex qPCR assay, for the simultaneous
627
detection and quantification of L. monocytogenes and Listeria spp. using the hly gene, and the
628
23S rDNA gene. Simultaneous quantification was possible over a 5 log range, and the limit of
629
quantification was 30 target molecules per reaction. The hly and 23S rDNA duplex reaction
630
showed the same results as the monoplex reaction, indicating that multiplexing did not
631
negatively influence the real-time PCR assay.
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Rodríguez-Lázaro et al. (2005) developed a novel qPCR for L. monocytogenes that
633
monitors analytical performance via the use of an IAC. In this study, the target sequence was
634
the hly gene, and the IAC was a chimeric DNA sequence flanked by the hly-specific target
635
sequences. A second hydrolysis probe, labelled with a specific fluorochrome detects the IAC
636
sequence. The results show that this assay is as specific, sensitive and quantitative as the
637
monoplex assay, and could be used for routine analysis for the detection and quantification of
638
L. monocytogenes DNA in food products. The quantification limit was 30 genome
639
equivalents per reaction, and the coefficients for PCR linearity and efficiency were high: r2 =
640
0.997 and E = 0.80. At high contamination levels (i.e. from 3×105 to 3×107 CFU/g), the
641
system accurately quantified L. monocytogenes in processed meat samples. However, Fraser
642
and half-Fraser broths and some food (raw pork, raw and cold-smoked salmon) can be
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ACCEPTED MANUSCRIPT 643
strongly PCR-inhibitory, leading to underestimation of contamination loads, due to PCR
644
failure. A hydrolysis-probe PCR-based method for the rapid detection and quantification of L.
646
monocytogenes, developed by Oravcová et al. (2005), targeted a specific sequence of the actA
647
gene encoding for a protein involved in the actin filament assembly. This highly sensitive and
648
specific assay for L. monocytogenes can detect 102 CFU/mL after 45 PCR cycles, and can
649
produce linear calibration curves from 109 to 102 CFU/mL, even in the presence of 106
650
CFU/mL of L. innocua.
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A qPCR method targeting the hlyA gene for quantification of L. monocytogenes in
652
biofilms (Guilbaud et al., 2005) had a reported sensitivity estimated at 6×102 CFU/cm2, but
653
suffered from a lack of precision, considering the dispersal of the points. Detection below a
654
concentration of 104 CFU/cm2 was less regular and more difficult.
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In 2006, Berrada et al. performed a qPCR on salad samples, quantifying DNA in a
656
LightCycler system with the LightCycler L. monocytogenes Detection Kit from Roche. The
657
method was linear in a range of 10 to 105 CFU L. monocytogenes. At 102 CFU/g, accuracy
658
(expressed as % bias) was as high as 26% and precision (expressed as % coefficient of
659
variation) as high as 22%. At 102 CFU/g, intra-day and inter-day variability were 12 and 14%
660
respectively. Based on the precision of this method, 10 CFU/g was the reported limit of
661
detection. The method provided accurate quantification of L. monocytogenes in three
662
naturally contaminated salads (around 1 to 7×102 CFU/g), with results similar to plate counts.
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In 2008, Long et al. developed a novel qPCR for L. monocytogenes that monitors
664
analytical performance via the use of a live bacterium as the IAC. Their purpose was to
665
develop a system able to indicate either inhibitors of the PCR amplification, or poor recovery
666
of microorganisms during the DNA extraction step. The target gene for the detection of L.
667
monocytogenes was the hly gene. The IAC had similar length, flanking regions and G+C
28
ACCEPTED MANUSCRIPT 668
content as the target, as well as a resistance gene to select L. monocytogenes cells with
669
successful chromosomal integration of the IAC. Quantification by this method gave a 5 log
670
linearity range, i.e. up to 105 CFU. Rosmanith et al. (2010) used internal sample process control (ISPC) to investigate
672
cheeses from an outbreak in Austria. The ISPC quantitatively determined the efficiency of the
673
target bacteria during pre-PCR steps. A known number of ISPC cells were added to the
674
sample prior to analysis, and pathogen load was deduced from the efficiency of the whole
675
process. In this case, recovery of the control was 43%, and the corrected contamination values
676
of investigated samples corroborated those of the EN ISO 11290-2 standard method, with no
677
significant differences (Rosmanith and Wagner, 2010).
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Reagent cost is one of the key factors that influence routine adoption of qPCR in
679
diagnostic laboratories. Traunšek et al. (2011) optimised 5 µL qPCR reactions for L.
680
monocytogenes based on the amplification of the hlyA gene and the use of a commercial IAC
681
(via second qPCR). Limits of detection and quantification were as low as 165 genome
682
equivalents.
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2.3.2.
685
2.3.2.1.
686
qPCR PRE-ANALYTICAL PROCESSING STEPS
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qPCR METHODS WITH A CELL CONCENTRATION STEP
687
Sample preparation has become an important topic in molecular food pathogen
688
detection, as it is increasingly recognised as the crucial prerequisite for reliable qPCR
689
(Rossmanith and Wagner, 2010). To improve sensitivity, target cells can be concentrated
690
before performing PCR.
29
ACCEPTED MANUSCRIPT 691
In most qPCR studies, a solid food product is generally analyzed after first blending
692
with a buffer (usually in a 1:10 dilution) and taking an aliquot of this mixture (typically 1 mL)
693
that is then centrifuged, washed and re-suspended; a supplementary centrifugation step can be
694
added to the protocol to improve sensitivity. Hough et al. (2002) developed a qPCR method including a centrifugation step for the
696
enumeration of L. monocytogenes in cabbage. Using a region of the hlyA gene, the method
697
provided quantitative results within 8 h for artificially contaminated samples. A linear
698
response over 7 log cycles, from 1.4 ×102 to 1.4×109 CFU of L. monocytogenes per 25 g of
699
cabbage and analysis of the assay showed that log differences could reliably be distinguished.
700
There were no cross-reactions with other microorganisms.
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A filtration-based qPCR method was developed by D’Urso et al. (2009) to quantify
702
viable L. monocytogenes and S. enterica, down to a limit of 10 CFU/10 g of yogurt. In this
703
case, an initial centrifugation step of the diluted food sample before PCR led to higher
704
sensitivity.
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Rodríguez-Lázaro et al. (2010) developed a novel qPCR for L. ivanovii that monitors
706
analytical performance via the use of an IAC. It targeted a specific region of the
707
sphingomyelinase C (smcL) gene, which is located on a L. ivanovi-specific pathogenicity
708
island and encodes a membrane-damaging virulence factor. The method detected as little as
709
one genome equivalent in 45% of the reactions. In raw milk, PCR accuracy was excellent
710
over a 6 log range, with high r2 and E values, and a limit of quantification of approximately
711
530 CFU in 25 ml. Given the centrifugation/concentration step, the method detected as few as
712
50 CFU in 25 mL of raw milk (5 genome equivalents per reaction) with a 66% probability.
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713
Sample preparation has recently become an important topic in molecular-based
714
foodborne pathogen detection, and new approaches have been described with selective
30
ACCEPTED MANUSCRIPT 715
solubilisation of the food matrix in a lysis buffer, and subsequent centrifugation to harvest
716
bacterial cells. Some of these protocols were reviewed in Rosmanith and Wagner (2010).
717
2.3.2.2.
METHODS WITH IMPROVED PURIFICATION/EXTRACTION STEPS
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Inhibition of PCR and real-time PCR by food components, such as fat, selective
721
enrichment media, or large amounts of non-target DNA, can occur, and pre-amplification
722
steps may be needed to eliminate theses. Many different protocols are used for DNA
723
extraction/purification. Typically, they rely on mechanical disruption or enzymatic digestion
724
of the cell membrane, followed by cell solubilisation, using reagents such as guanidine
725
isothyocianate, and subsequent clean-up steps using organic solvents and alcohol
726
precipitation, suspended silica, affinity or ion exchange purification columns, ultrafiltration,
727
among others.
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Most available PCR methods require selective enrichment steps to overcome the
729
problem of PCR inhibitors, especially at low pathogen concentrations. To avoid enrichment,
730
Rodriguez-Lazaro et al. (2004c) used filtration and Chelex-100-based DNA purification, and
731
qPCR for the quantitative detection of L. monocytogenes in meat products. Chelex-100 is an
732
ion-exchange resin, specifically designed for extraction of PCR-ready template DNA. The
733
removal of PCR inhibitors is accomplished by scavenging contaminant metal ions that
734
catalyze the cleavage of DNA. Pre-PCR filtration treatments were performed on 11 µm or 22-
735
25 µm pore size filters, which should not retain L. monocytogenes cells but remove larger
736
particles. Based on this strategy as few as 103 CFU/g L. monocytogenes were quantified in
737
meat products (ham, sausages, raw pork) with excellent accuracy.
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ACCEPTED MANUSCRIPT Similarly, Rodriguez-Lazaro et al. (2005) accurately quantified as few as 103 CFU/g L.
739
monocytogenes in smoked salmon products, with a qPCR method involving pre-PCR
740
filtration through 22-25 µm pore size filters and DNA purification with a commercial kit
741
(Wizard genomic DNA purification kit, Promega). However, results were less conclusive for
742
raw salmon.
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Rodriguez-Lazaro and Hernandez (2006) showed that the Wizard kit (Promega) was
744
more suitable than the Chelex kit (Bio-Rad) for L. monocytogenes DNA extraction from meat
745
samples requiring sensitive and accurate results. Many other extraction/purification kits are
746
now commercially available. Rosmanith et al. (2007) developed a protocol for the
747
concentration of Gram-positive bacteria from food, with the removal of fat, carbohydrates and
748
proteins, compatible with DNA-based quantification methods.
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Immunomagnetic separation (IMS) allows the specific capture of L. monocytogenes
750
from food or culture media, and consequently the removal of PCR inhibitors. Large magnetic
751
microbeads (>1 µm in diameter) coated with specific antibodies have been used for
752
separation and concentration of L. monocytogenes in food samples. More recently,
753
nanoparticles coated with pathogen-specific antibodies have been shown to have higher
754
capture efficiency due to a higher surface-to-volume ratio and faster reaction kinetics (Yang
755
et al. (2007).
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Probably due to the presence of PCR inhibitors, detection of DNA extracted directly
757
from dairy products for PCR sometimes shows very poor sensitivity. Nogva et al. (2000)
758
developed a qPCR method for the quantitative detection of L. monocytogenes in water, skim
759
milk and unpasteurised whole milk, based on the use of a 113 bp amplicon from the hlyA
760
gene as the target and a hydrolysis probe. The method was highly selective and could be
761
completed within 3 h. It included non-specific binding of bacteria to paramagnetic beads,
762
with subsequent DNA purification using the same beads. In the Nogva et al. study,
32
ACCEPTED MANUSCRIPT 763
quantification in artificially contaminated samples was linear over 7 log units, and the
764
detection limit was approximately 200-2000 CFU/mL. Amagliani et al. (2006) developed a PCR assay for direct detection of L.
766
monocytogenes in milk samples, with a magnetic capture hybridisation procedure for
767
selective DNA purification, using nanoparticles. The sensitivity of the assay was 10 CFU/mL.
768
Yang et al. (2007) developed an assay based on nanoparticle-based IMS in
769
combination with qPCR, using a portion of the hlyA gene as a target, to quantify L.
770
monocytogenes in artificially contaminated milk. Purification of bacterial DNA with IMS
771
detected > 102 CFU per 0.5 mL. In contrast, no detection was possible without IMS even
772
when L. monocytogenes numbers where greater than 107 CFU in 0.5 mL, and with
773
Dynabeads®, detection was possible only for concentrations greater than 105 CFU in 0.5 mL.
774
With Dynabeads®, cell numbers derived from Cq values were lower than those determined
775
from direct plate counts of the beads. In contrast, for nanoparticles, results were 1.5 to 7 times
776
higher than those derived from plate counts. Cell aggregation or lower culturability may be
777
possible explanations for such discrepancies. The method improved the sensitivity of L.
778
monocytogenes detection (226 CFU/0.5 mL) and was rapid and quantitative. However, it is
779
labour-intensive, requiring the synthesis and coating of specific nanoparticles. Moreover,
780
nanoparticles had to be removed by centrifugation after DNA extraction, because they can
781
reduce PCR efficiency in a dose-dependent manner. An excessive quantity of nanoparticles
782
present on cell surfaces, improving cell separation, can also affect DNA extraction efficiency
783
and thus the limit of detection by PCR.
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784
Amagliani et al. (2010) developed a multiplex qPCR assay including an IAC for the
785
simultaneous detection of L. monocytogenes and Salmonella spp. in seafood samples. A
786
magnetic capture hybridisation procedure was performed for selective DNA purification,
787
using nanoparticles. The sensitivity of the assay was 102 -103 CFU/g. One can note that
33
ACCEPTED MANUSCRIPT 788
Amagliani et al. (2006) and (2010) used magnetic particles coupled to oligonucleotids (not
789
antibodies) as a means to purify and concentrate nucleic acids, and not whole bacterial cells,
790
directly from samples before PCR. Walcher et al. (2010) compared direct plate counts and qPCR combined with
792
paramagnetic beads for separation and quantification of L. monocytogenes in raw milk. A
793
high correlation was found between the inocula and counts obtained both by direct plating or
794
qPCR. Recovery rates ranged from 46.6 to 122.8% for plate counts, and from 64.7 to 95.1%
795
for qPCR. The detection limit of the latter method was 102-103 CFU/mL.
SC
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Commercially available nanoparticles were coated and used by Yang et al. (2013) to
797
develop a multiplex non-quantitative PCR for simultaneous detection of L. monocytogenes,
798
Escherichia coli O157:H7 and Salmonella typhimurium in food (lettuce, tomato, ground
799
beef), with a sensitivity of about 1000 CFU/g for L. monocytogenes. The overall assay time
800
was 4.5 h. To detect only the viable cells, propidium monoazide was applied to selectively
801
suppress the detection of DNA from dead cells.
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802
2.3.3. qPCR METHODS THAT ASSESS VIABILITY
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803
The determination of bacterial viability is a key issue for the application of food risk
805
management. Reichert-Schwillinsky et al. (2009) showed that under poor growth conditions,
806
as well as during the lag phase, the qPCR data overestimated the actual viable L.
807
monocytogenes counts. This result was attributed to the possible accumulation of dead cells,
808
extracellular genomic DNA or VBNC bacteria. On the other hand, these effects are the result
809
of reduced growth within the culture when the conditions worsen.
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2.3.3.1.
REVERSE TRANSCRIPTION PCR (RT-PCR)
34
ACCEPTED MANUSCRIPT One alternative is to use an mRNA-based PCR method. This type of method has been
813
successfully applied by Klein and Juneja (1997) for detection of viable L. monocytogenes in
814
food. However, these systems must be used judiciously, because mRNA itself can be stable
815
for a quite long time after cell death (Birch et al., 2001; Rijpens and Herman, 2002). One
816
technique, reverse transcription PCR (RT-PCR) can be used for the detection of mRNA;
817
however this technique has some disadvantages, such as the necessity to eliminate all traces
818
of DNA, limited sensitivity, reproducibility and specificity (d’Urso et al., 2009). Nucleic acid
819
sequence-based amplification (NASBA) is a promising method for the analysis of viable
820
cells, through continuous amplification of RNA at isothermal conditions (41.5°C). At this
821
temperature, DNA remains double-stranded, and does not become a substrate for
822
amplification (Rodríguez-Lázaro et al., 2007).
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In 2008, Rantsiou et al. developed a qPCR protocol to detect, quantify and determine
824
the viability of L. monocytogenes in food samples (meat and milk products, and vegetables).
825
A couple of primers and a hydrolysis probe were designed on the intergenic region spacer
826
between the 16S and 23S rRNA genes. To define the number of viable cells, this sequence
827
was preferred to specific virulence genes, which are controlled by environmental factors
828
(medium components, temperature, etc.). Amplification is performed at the DNA and RNA
829
level using RT-qPCR to quantify total or only viable cells. Standard curves were constructed
830
from artificially contaminated matrices, and the method had a high quantification limit of 103-
831
104 CFU/g or mL, and of 104-105 CFU/g or mL, when considering viability, which makes this
832
assay unsuitable for the enumeration of L. monocytogenes at low contamination levels.
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833
Molinos et al. (2010) developed an RT-qPCR method based on bacterial mRNA to
834
quantify L. monocytogenes in food after bacteriocin treatment. For concentrations greater than
835
102 CFU/g, viable cell count results were better than PALCAM agar counts. For RT-qPCR
836
determination of RNA expression levels — a multi-step procedure involving lysis of bacteria
35
ACCEPTED MANUSCRIPT cells, RNA extraction, DNase treatment, cDNA synthesis, and finally qPCR —, the detection
838
of scarce target RNA from a low number of bacteria remains a challenge. Werbrouck et al.
839
(2007) evaluated and optimised each step of this method for L. monocytogenes. Their results
840
showed that many factors can influence the outcome of the assays, which thus require careful
841
assay design and reaction optimisation to maximise the sensitivity and obtain reliable
842
quantification.
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843
2.3.3.2.
OTHER APPROACHES TO ASSESS VIABILITY
SC
844
Another approach for the detection of only viable cells is the use of ethidium
846
monoazide bromide (EMA) or propidium monoazide (PMA) for staining the cells before
847
DNA extraction and PCR to inhibit DNA amplification of dead cells. These dyes
848
preferentially penetrate cells with compromised membranes. For lack of other viability
849
measurements, membrane integrity is still a well-accepted criterion for distinguishing viable
850
from dead cells (Yang et al., 2013). However, in some cases, dyes can also penetrate viable
851
cell membranes and covalently link DNA, resulting in PCR inhibition and underestimation of
852
counts, mainly when using EMA (Pan and Breidt, 2007; d’Urso et al., 2009). The high
853
discrepancies in the same samples between quantified culturable cells and the much higher
854
level of Listeria population estimated by PCR with a PMA viability protocol (Overney, 2014)
855
also raises the issue of how to interpret the results and what they mean in terms of food
856
safety, cells’ ability to recover, cross-contamination and growth.
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857
A filtration-based qPCR method was developed by D’Urso et al. (2009) to eliminate
858
dead or severely damaged L. monocytogenes and S. enterica from food samples (yogurt).
859
Firstly, after a centrifugation step, samples undergo an initial lysis-filtration pre-treatment,
860
which retains only viable bacteria on the filter in less than 30 min. The discrimination buffer
36
ACCEPTED MANUSCRIPT can completely solubilise protein complexes, eukaryotic cells and other contaminants, letting
862
them pass through the filter used for pre-treatment. Briefly, pre-treatment filtration consisted
863
of addition of discrimination buffer (4 M guanidine thiocyanate, 2 M NaCl, 25 mM Tris-HCl
864
pH 7.0 ) incubated at 60°C for 15 min with shaking after prior enzymatic treatment with 10
865
mg/mL lysozyme for 15 min at 37°C. The suspensions were filtered on filter paper (0.4 µm),
866
and washed with 500 µL of washing buffer (2 M NaCl, 25 mM Tris-HCl pH 7) at 60°C, then
867
with another washing buffer (25 mM Tris-HCl pH 7.0, 0.5% SDS), and with 300 µL of
868
double-distilled water to ensure all dead cells pass through the filter. Secondly, after
869
resuspension of the viable cells from the filter, the bacterial DNA was extracted on silica-
870
based columns, and was finally amplified by qPCR. Quantitative results clearly match those
871
obtained by standard plating, over a wide dynamic range (at least 5 log). Though several
872
labour-intensive steps were necessary, the filtration-based qPCR assay quantitatively detected
873
living forms of L. monocytogenes and S. enterica, at concentrations as low as 10 CFU/10 g of
874
yogurt.
876
2.4.
DIGITAL PCR
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Digital PCR (dPCR) is a new approach that directly quantifies, detects and amplifies a
878
target nucleic acid sequence. It was first developed by Vogelstein and Kinzler (1999) to
879
extend the applications of conventional PCR. dPCR partitions a sample into several hundreds
880
or thousands individual PCRs: some of these reactions receive the target molecule (positive),
881
whilst others do not (negative). The reaction volume of dPCR is several picoliters to
882
nanoliters. The average target nucleic acid concentration is determined by the proportion of
883
negative PCR reactions, based on the Poisson distribution. It is then possible to determine a
884
copy number of the target gene without the need for internal controls or standards for
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37
ACCEPTED MANUSCRIPT quantification and assessing reaction efficiency that are required for qPCR (Straub et al.,
886
2013). dPCR has benefited from progress in microfluidic technologies, and is used for
887
clinical PCR applications requiring extreme accuracy of copy number variation. It has been
888
used to detect cancer by quantification of mutant alleles and detection of allelic imbalance
889
(Pohl and Shih, 2004). Although applications for biodetection in environmental samples are
890
starting to appear, to our knowledge, no trials of dPCR have yet been reported for the
891
quantification of L. monocytogenes. Recently, Straub et al. (2013) used dPCR to directly
892
enumerate plasmid and chromosome copies in three strains of Bacillus anthracis. According
893
to their results, dPCR appears to be an attractive, accurate and rapid alternative method that
894
provides for direct quantification of target genes. Recently, the performance of microfluidic
895
digital RT-PCR (RT-dPCR) was compared with RT-qPCR for detecting the main viruses
896
responsible for foodborne outbreaks (human noroviruses and hepatitis A virus) in spiked
897
lettuce and bottled water (Coudray-Meunier et al., 2015). MIQE guidelines are now also
898
available for dPCR (Huggett et al., 2013).
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Today, various commercially available kits (for example BioMark and EP from
900
Fluidigm, QuantStudio 3D Digital PCR Instrument from Life Technologies, Q100 from Bio
901
Rad, RainDrop from RainDance) make it possible to conduct dPCR experiments with high
902
accuracy and low cost per sample. The kits can collect real-time amplification data from each
903
reaction. For Fluidigm, the digital array with an integrated fluidic circuit precisely partitions a
904
sample pre-mixed with PCR reagents into hundreds of individual PCR reactions. However,
905
for food analysis, sample preparation, DNA concentration, extraction and purification may be
906
limiting factors. Although measurement precision has improved, sensitivity is likely to be
907
similar to qPCR. A potential application is the quantification of L. monocytogenes in
908
environmental samples, because these samples require a lower amount of organic matrix.
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38
ACCEPTED MANUSCRIPT In conclusion, although the major advantage of PCR-based methods is their rapidity,
911
all share a main drawback: detection limits generally far greater than contamination levels in
912
food. Molecular methods are not simple single-step methods, but complex and sometimes
913
labour-intensive multi-step systems, for which all steps need careful evaluation and
914
optimisation. However, recent advances have significantly improved their performance
915
(Table 2).
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916
CONCLUSION
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917
Enumeration of L. monocytogenes in food faces many difficulties due to low
919
contamination levels, the presence of numerous background microorganisms, and the great
920
variety of matrices to be considered. None of the enumeration methods mentioned above is
921
ideal for any one purpose, and potential users must choose a technique according to their
922
needs, available products and materials (Table 3). For example, there are several
923
circumstances under which semi-quantitative results can be sufficient.
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918
Culture-based techniques (Table 1) include very different methods, characterised by
925
high variability in precision. They have numerous advantages: they are relatively simple and
926
rapid to perform, they confirm the presence of only viable cells. Nevertheless, they are
927
generally time-consuming and sometimes labour-intensive. Moreover, these methods may
928
require individual biochemical confirmation of the species for some isolated colonies. In
929
addition, there is no detection of VBNC bacteria, injured or stressed cells, and the growth of
930
other bacteria can alter the results (D’Urso et al. 2009, Jadhav et al., 2012; Jasson et al.,
931
2010).
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932
Molecular methods (Table 2), in particular qPCR, with their capacity to measure 933
amounts of nucleic acids in a wide range of samples and sources, with their conceptual and
39
ACCEPTED MANUSCRIPT 934
practical simplicity, and their combined speed, sensitivity and specificity in a homogenous 935
assay, can now be considered as the technology par excellence for diagnostic applications, 936
including microbial quantification (Bustin et al., 2009). However, PCR-based methods have 937
detection limits generally much greater than the contamination levels in food. In fact, for L. 938
RI PT
monocytogenes, qPCR may not be the method of choice for detecting a few bacteria (and 939
consequently low quantities of bacterial DNA or RNA) in a large amount of food matrix, 940
because detection limits are far above contamination levels in naturally contaminated samples 941
SC
(Rossmanith and Wagner, 2010). A number of issues such as methods to concentrate bacteria, 942
assess viability or improve purification or extraction steps have been addressed. Although
M AN U
943
many of these studies do examine the linearity of the assay over several order of magnitude to 944
assess the overall efficiency of the PCR and the limit of detection for the PCR assay, the 945
variability of food matrices often makes it very difficult to use these assays for quantification 946
because each sample must be individually assessed for inhibition and assay efficiency. These 947 948
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methods are currently more likely to be used to reliably detect the presence of microorganisms in the food sample, not enumerate them. Moreover, molecular-based 949 950
EP
methods are not simple single-step methods, but complex and sometimes labour-intensive multi-step procedures. However, recent advances have significantly improved their 951 952
AC C
effectiveness. As underlined by the very high number of published studies involving these techniques (Table 3), they remain very attractive due in particular to their rapidity. Recent 953
development in standardised protocols of diagnostic PCR for the detection of foodborne 954
pathogens may favour efficient transfer of PCR technology from expert research to routine 955
laboratories. The international standard EN ISO 22118 (Anonymous, 2011) regarding the 956
performance characteristics of PCR for the detection and quantification of foodborne 957
pathogens is part of a series of documents on PCR used for the detection of pathogenic 958
microorganisms in food and specifies the minimum requirements for the performance
40
ACCEPTED MANUSCRIPT 959
characteristics for the detection of nucleic acid sequences (DNA or RNA) by molecular 960
methods. However, the lack of validation data may hinder the adoption of molecular methods 961
for routine analysis. It is widely accepted that the use of validated methods is an essential part of any sound
963
laboratory quality assurance program. The use of methods that have documented
964
effectiveness based on protocols issued by the major international and regional
965
standardisation bodies (i.e. ISO and CEN) is essential. Evaluation of enumeration methods
966
should be performed according to the EN ISO 16140 standard (Anonymous, 2003), which
967
defines validation procedures for an alternative method compared to a reference method (case
968
of quantitative analysis methods). The EN ISO 11290-2 standard method for enumeration of
969
L. monocytogenes in food (Anonymous, 1998, 2004) is cited as the reference method in the
970
quantitative criteria stated in EC Regulation 2073/2005 for L. monocytogenes. However, in
971
various countries such as France for example, the competent authority allows the network of
972
accredited laboratories to use alternative methods, even for official controls, if they have been
973
validated according to the EN ISO 16140 standard, and certified by a third party
974
(Anonymous, 2008). For practical reasons, it is very difficult for one laboratory to perform
975
such a validation procedure (in particular, the ILPTs ), and most of the studies presented in
976
this review involve the development of methods that have not yet been validated. However,
977
the extent of validation and verification depends also on the context within which the method
978
is to be used. Several certified commercial enumeration methods are available on AFNOR
979
certification site (www.afnor-validation.org). Interestingly, an international ISO 16140-based
980
validation trial of a non-proprietary qPCR-based methodology for detection of L.
981
monocytogenes in soft cheese has been recently described (Gianfranceschi et al., 2014).
982
Barbaud-Piednoir et al. (2013) proposed a new validation procedure to specifically validate
983
qPCR assays for detection applied to food microbiology according to two guidelines: the ISO
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41
ACCEPTED MANUSCRIPT 22118 standard and the "Definition of minimum performance requirements for analytical
985
methods of GMO testing". For assessing the performance of enumeration methods, it is
986
always far preferable to use naturally contaminated samples, to take into account potential
987
physical entrapment of naturally occurring pathogens, interaction between the target pathogen
988
and the food matrix and the background microflora, which occur in naturally contaminated
989
food matrices, and various types of stress that may be not properly considered when using
990
artificially contaminated samples. In the case of L. monocytogenes, it is sometimes very
991
difficult to find appropriate naturally contaminated samples, from various origins and food
992
types, because of extremely low and heterogeneous contamination levels. Many studies
993
presented here have been conducted using artificially contaminated samples.
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984
In conclusion, the improvement of L. monocytogenes enumeration method has
995
resulted in a considerable amount of research, leading to several alternative methods. Many
996
show satisfactory performance, and are suitable for their intended purposes. However, these
997
new techniques are not widely used. Their low popularity can be attributed to several reasons.
998
Practicability is important to consider when choosing an analytical method. This includes
999
ease of use (preparation of the sample, automation, adaptation to data processing,
1000
obstruction), speed (delay of obtaining the results and cadences of analyses) and cost.
1001
Manufacturer guarantees include validation by a third party (such as by AFNOR Certification,
1002
MicroVal, NordVal, AOAC), reputation of the materials and reagent supplier, reliability of
1003
the material and the reagents (stability, regularity, availability) and after-sales service, user
1004
training. New methods including various steps and unusual materials and reagents may be
1005
difficult to adapt to any routine analysis laboratory. Research in L monocytogenes
1006
enumeration often leads to in-house development of new methods characterised by
1007
insufficient evaluation and validation. Standard methods, and more generally traditional
1008
microbiological analysis methods, based on the observation of microbial growth in adapted
AC C
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994
42
ACCEPTED MANUSCRIPT 1009
culture media and biochemical identification of the microorganisms, are still standard. One of
1010
the main problems is the interpretation of results of new techniques based on a type of
1011
measurement different from that of the conventional techniques. Finally, the use of a completely verified enumeration method is not itself a guarantee
1013
that analytical data are reliable. In particular, sampling plans and the overall competence of
1014
the laboratory must be reliable.
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ACCEPTED MANUSCRIPT 1016 1017
ACNOWLEDGEMENTS
1018
The authors would like to thank Robert Debuchy, Christophe Soumet, Frédéric Auvray,
1020
Patrick Fach and Bertrand Lombard for helpful advice and information.
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ACCEPTED MANUSCRIPT 1022
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1141
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1142
de Oliveira, M.A., Ribeiro, E.G.A.,
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Quantification of Listeria monocytogenes in minimally processed leafy vegetables using a
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Listeria
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Bergamini, A.M.M., De Martinis, E.C.P. (2010).
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1155
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Chemists, 73, 734-742.
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1158
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137 agar. Journal of Food Protection, 63, 354-363.
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Farber, J.M., Daley, E. (1994). Presence and growth of Listeria monocytogenes in naturally-
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1163
of viable Listeria monocytogenes using fluorescence in situ hybridization in combination with
1164
filter cultivation. Journal of Applied Microbiology, 105, 502-509.
1165
Fuchizawa, I., Shimizu, S., Ootsubo, M., Kawai, Y., Yamazaki, K. (2009). Specific and rapid
1166
quantification of viable Listeria monocytogenes using fluorescence in situ hybridization in
1167
combination with filter cultivation. Microbes and Environments, 24, 273-275.
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Garrido, A., Chapela, M.J., Román, B., Fajardo, P., Lago, J., Vieites, J.M., Gabado, A.G.
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ACCEPTED MANUSCRIPT List of Tables
1409
Table 1: Main characteristics of published culture-based methods.
1410
Table 2: Main characteristics of published molecular methods (when not indicated precisely
1411
in the publication, time to result is generally less than 1 day).
1412
Table 3: Compared advantages/disadvantages of the main methods used, based on the
1413
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ACCEPTED MANUSCRIPT Table 1. Limit of quantification /Levels tested
Matrix
Culture methods based on colony counts
40 CFU/g
TAL (Palcam overlain with TSAYE)
≥5 days
40 CFU/g
Filtration+ LOA culture
2 to 5 days
0.8 CFU/g
Food, environment of food production
References
Long time to results
M AN U
2 to 4 days
Parsley, meat
Long time to results Recovers stressed cells
Miller et al. (2010)
Cold smoked salmon
Long time to results Specific apparatus Training Not applicable to all foods Sensitive
Gnanou Besse et al. (2004, 2008) Barre et al. (2015)
EP
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EN ISO 11290-2
Advantages/ disadvantages
RI PT
Time to results
SC
Method
Culture methods based on MPN
AC C
1415
MPN+ PCR (prfA)
2 days
100-1000/g
Cooked and fermented meat products
Conventional PCR
Aymerich et al. (2004)
MPN+ PCR (prfA)
2 days
>2-3 log10/g
Chorizo
Fastidious preamplification steps; Quantification
Martin et al. (2004)
65
ACCEPTED MANUSCRIPT possible with high levels of background flora 2 days
70-100/g
Milk, lettuce, smoked salmon, ice cream, pâté, salami, shrimp, ground beef, soft cheese
Interference with food component varies with dilution
MPN+ qPCR (16S rDNA)
2 days
10-1000/g.
Minimally processed leafy vegetables
Interference with chlorophyll varies with dilution
de Oliveira et al. (2010)
MPN+ PCR (hlyA gene)
2 days
300/g
Salads
Conventional PCR
Jeyaletchumi et al. (2010)
MPN+ 6 rapid kits
1-2 days.
Large underestimation (environment)
Cruz et al. (2012)
AC C
EP
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1416 1417
SC
M AN U Seafood products and environment
de Martinis et al. (2007)
RI PT
MPN+ qPCR (16S rDNA)
66
ACCEPTED MANUSCRIPT Table 2. Time to results
Limit of quantificatio n
Matrix
Competitive PCR (hlyA gene)
5h
103 CFU per 0.5 mL (detection limit)
Milk
Competitive PCR (iap gene)
2 days
20 CFU/mL and 2 CFU/g (detection limit)
FISHFC
16 h
200 CFU/g
SC
Molecular methods based on FISH and conventional PCR
Advantages/ Disadvantages
Matches plate counts Conventional PCR
Choi and Hong (2003)
Milk, catfish fillet
Matches plate counts Conventional PCR
Wang and Hong (1999)
Smoked salmon, mozzarella, cabbage
Rapid epifluorescence microscopy
Fuchizawa et al. (2008, 2009)
Milk
Results vary with plate counts (1 log)
Hein et al. (2001)
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References
RI PT
Method
qPCR (iap gene)
EP
Molecular methods based on qPCR
AC C
1418
Duplex qPCR (hly gene, and 23S rDNA)
30 target molecules per reaction
Simultaneous quantification of L. monocytogenes and Listeria spp.
RodríguezLázaro et al. (2004)
qPCR (actA gene)
>102 CFU/mL
Quantification possible with high levels of L. innocua
Oravcová et al. (2005)
67
ACCEPTED MANUSCRIPT qPCR (hly gene)
104 CFU/cm2
Biofilms
Guilbaud et al. (2005)
qPCR
102-7102 CFU/g
Salads
Berrada et al. (2006)
qPCR+IAC
qPCR (hly gene)
105 CFU
Processed meat samples
Cheese
165 genome equivalents
TE D
qPCR (hly gene)
IAC, PCR inhibition (raw pork, raw and smoked salmon)
RodríguezLázaro et al. (2005)
Viable bacterium as IAC
Long et al. (2008)
Internal sample process control
Rosmanith et al. (2010)
Commercial IAC
Traunšek et al. (2011)
Viability
Pan and Breidt (2007)
Viability
Werbrouck et al. (2007)
SC
30 genome equivalents per reaction
M AN U
qPCR (hly gene)
RI PT
Molinos et al. (2010)
qPCR+viability assessment
103 CFU/cm2
EP
AC C
qPCR +PMA
Biofilms
RT-qPCR
RT-qPCR (intergenic region spacer between 16S and 23S rDNA)
104-105 CFU/g or mL
Meat and milk products, vegetables
Viability
Rantsiou et al. (2008)
qPCR +lysis-filtration +centrifugation
10 CFU/10 g
Yogurt
Viability Laborious
D’Urso et al. (2009)
68
ACCEPTED MANUSCRIPT qPCR+sample concentration
8h
qPCR +lysis-filtration +centrifugation
>102 CFU/25 g
Cabbage
Higher sensitivity
Hough et al. (2002)
10 CFU/10 g
Yogurt
Viability Labourintensive
D’Urso et al. (2009)
qPCR+improved purification/extraction
200-2000 CFU/mL (detection limit )
qPCR +pre-PCR treatments (filtration+resins)
1000 CFU/g
1419 1420
Pre-PCR treatments Poor results with raw salmon
EP
> 102 CFU per 0.5 mL
102-103 CFU/mL (detection limit)
Milk
Raw milk
Nogva et al. (2000)
RodriguezLazaro et al. (2004, 2005) RodriguezLazaro and Hernandez (2006)
Labourintensive
Yang et al. (2007)
Walcher et al. (2010)
AC C
qPCR +IMS
ham, sausages, raw pork, smoked salmon
Meat samples
TE D
qPCR +resins
qPCR (hly gene) +nanoparticlebased IMS
Water, skim milk, unpasteurised whole milk
SC
3h
M AN U
qPCR (hly gene) +IMS
RI PT
qPCR (hly gene)+ centrifugation
69
ACCEPTED MANUSCRIPT
Table 3. Time to results
Limit of quantification /Levels tested
Measurement uncertainty
Matrix /flora interference
Viability
Plate counts methods
-(2-4 days)
+/(40/g)
(<0.87 log10 CFU/g)
+/-
+
Plate counts + concentration step
-(3-5 days)
+ (0.8/g)
+/(<0.46 log10 CFU/g)
+/-
MPN
-(2-6 days)
++
(similar to plate counts for ~100/g)
+
Molecular techniques (quantitative PCR+FISH)
++ (<1 day)
(100/?1000/g)
Infrastructure /equipment
Validation
+/-
+
+
+
+/-
+/-
+
5
+
+
+/-
-
6
-
+/-
-
- /+
23
SC
M AN U
TE D
Approximate number of references
AC C
EP
+/-
Labourintensive
RI PT
Method
71
ACCEPTED MANUSCRIPT Highlights
TE D
M AN U
SC
RI PT
None new method is ideal for any purpose, and users must choose them according to their needs
EP
• •
We summed up the current knowledge concerning L. monocytogenes enumeration Enumeration difficulties are linked to low contamination levels in foods the improvement of L. monocytogenes enumeration gave place to a considerable amount of research projects Both new culture based techniques and molecular methods are not largely used
AC C
• • •