Occurrence and diversity of Listeria spp. in seafood processing plant environments

Accepted Manuscript Occurrence and diversity of Listeria spp. in seafood processing plant environments Kitiya Vongkamjan, Janejira Fuangpaiboon, Sirasa Jirachotrapee, Matthew P. Turner PII:

S0956-7135(14)00499-X

DOI:

10.1016/j.foodcont.2014.09.001

Reference:

JFCO 4050

To appear in:

Food Control

Received Date: 24 May 2014 Revised Date:

25 August 2014

Accepted Date: 2 September 2014

Please cite this article as: Vongkamjan K., Fuangpaiboon J., Jirachotrapee S. & Turner M.P., Occurrence and diversity of Listeria spp. in seafood processing plant environments, Food Control (2014), doi: 10.1016/j.foodcont.2014.09.001. 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

Occurrence and diversity of Listeria spp. in seafood processing plant environments

2 Kitiya Vongkamjan1*, Janejira Fuangpaiboon2, Sirasa Jirachotrapee3, and Matthew P. Turner4

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Department of Food Technology, Prince of Songkla University, Hat Yai, Thailand 90112

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3M Food Safety, 3M Thailand Ltd., Asoke-Montri Rd., Wattana, Bangkok, Thailand 10110

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Health Care, DKSH Thailand Ltd., Sukhumvit Rd., Bangjak, Prakanong, Bangkok, Thailand 10260

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3M Asia Pacific Pte Ltd., 1 Yishun Ave. 7, Singapore 768923

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*Corresponding author:

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Department of Food Technology

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Faculty of Agro-Industry

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Prince of Songkla University

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Hat Yai, Thailand 90112

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Phone: +66 74-286-337

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Fax: +6674-228-866

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Email: [email protected]

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Running title: Listeria spp. in seafood processing plant environments

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To be submitted to Food Control

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Abstract Listeria contamination in processing plant environments is a major issue for the seafood industry worldwide; faster and more reliable results are therefore desired for early detection and

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monitoring of environmental Listeria spp. This study aimed to gain a better understanding of the

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prevalence and diversity of Listeria spp., and to evaluate a rapid detection method, the 3M

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Molecular Detection Assay (MDA) Listeria, for its ability to detect Listeria spp. in environmental

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samples from seafood processing plants. Duplicate environmental sponge samples (n = 444) were

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collected from 152 different sites within three seafood processing plants, and analyzed for Listeria

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spp. by the MDA method (after 26 and 48 h of enrichment) and the U.S. Food and Drug

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Administration Bacteriological Analytical Manual method. Overall, detection of Listeria spp. by the

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two methods did not differ significantly (p>0.05); 11 (4.9%) and 13 (5.9%) samples were positive for

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Listeria spp. by the MDA and FDA-BAM method, respectively. The sensitivity of the MDS was 87.0%

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(95% CI: 77.4–96.6%), specificity was 97.6% (95% CI: 95.5–99.7%), accuracy was 95.3%, and the

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positive predictive value was 89.4% (95% CI: 80.5–98.2%). Classification of 19 Listeria isolates by

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partial SigB sequencing analysis identified three allelic types. Twelve of these isolates were ATs 58

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and 60 which were classified as L. monocytogenes lineage I and serotypes 1/2b, 3b, 4b, 4d, 4e, by

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multiplex-PCR serotyping. Six Listeria isolates were classified as L. innocua (AT31). Our data show

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that the 3M Molecular Detection Assay Listeria provides rapid and reliable results for detection and

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monitoring of Listeria spp., which are important for seafood processing plants. Effective Listeria

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monitoring programs will allow for improved development of Listeria control measures in order to

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minimize cross-contamination in finished products.

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1. Introduction Listeria spp. is ubiquitous in nature and commonly found in diverse sources, e.g., soil,

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manure, silage, raw agricultural commodities (Fenlon, 1999; Wesley, 1999; Vongkamjan et al., 2012;

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den Bakker et al., 2014). Among these diverse species of Listeria, Listeria monocytogenes is a serious

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pathogen that can cause a severe human disease, listeriosis. Although this disease is rare, 20 to 30%

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of listeriosis cases are fatal, and the majority of those who have an invasive L. monocytogenes

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infection typically require hospitalization (CDC, 2013). Consumption of food contaminated with this

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pathogen has been reported as the main route of transmission to humans (Farber and Peterkin,

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1991). Listeria spp., including L. monocytogenes have also been isolated from the environment of

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various types of food processing facilities, e.g., meat, poultry, dairy, and seafood processing facilities

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(Ojeniyi et al., 1996; Lappi et al., 2004; Eifert et al., 2005; Williams et al., 2011; Vongkamjan et al.,

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2013) as well as retail establishments (Sauders et al., 2004). Control of L. monocytogenes is a

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continuous challenge for the food industry [reviewed in (Ferreira et al., 2014)], including the seafood

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industry. Processing facilities manufacturing seafood products are common in Thailand as seafood is

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a major food category for international markets. Listeria contamination of final products in seafood

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processing facilities may occur from various sources, for example, contamination of raw materials

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(Norton et al., 2001) or contamination of products during processing from the process environments

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(Rørvik et al., 1995; Dauphin et al., 2001; Fonnesbech Vogel et al., 2001; Hoffman et al., 2003).

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Several previous studies have also found that the processing environment was the major

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contamination source for final seafood products, including Ready-To-Eat (RTE) foods (Lappi et al.,

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2004; Rørvik et al., 1995; Autio et al., 1999; Thimothe et al., 2004; Kornacki and Gurtler, 2007).

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Prevalence of Listeria can fluctuate over time in a given plant, dependent upon the

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manufacturing loads and disinfection schedules between the shifts (Fonnesbech Vogel et al., 2001;

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Hu et al., 2006). Early detection and monitoring of environmental Listeria spp. are thus crucial for

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the seafood industry to prevent foodborne outbreaks and to comply with regulatory requirements.

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The US FDA has recommended that each processing facility establish and implement a written plan

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for collection of environmental samples from high risk surfaces and areas, and for the testing of

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those samples for the presence of Listeria spp. or L. monocytogenes (FDA, 2008). Although testing

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ACCEPTED MANUSCRIPT for Listeria spp. as an indicator for L. monocytogenes contamination is recommended and routinely

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practiced, the relationship between the L. monocytogenes and Listeria spp. has not been well

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elucidated. A previous study has reported that prevalence of Listeria spp., on food and nonfood

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contact surfaces, was not shown to be a good indicator for L. monocytogenes prevalence in seafood

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processing plants by analysis of nine sets of peer-reviewed published data (Alali and Schaffner,

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2013). Data from periodic sampling and testing can be used as a reference for determining routes of

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Listeria contamination to the plants that may lead to post-processing contamination in finished

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products. Data are also valuable for developing and evaluating control measures of Listeria in a given

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processing plant over time.

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The 3M Molecular Detection System (MDS) is an advanced rapid method for the detection of

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Listeria spp. The MDS utilizes an innovative technique called loop-mediated isothermal amplification

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(LAMP) that has been shown to be a simple method to perform, which yields high specificity,

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sensitivity, and rapid results (Mori and Notomi, 2009). LAMP has been previously deployed for the

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detection of pathogens in food as well as a diagnostic method of infectious diseases (Mori and

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Notomi, 2009; Parida et al., 2008). Faster and more reliable results are typically desired for early

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detection and monitoring of environmental Listeria spp. in order to validate effective control

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measures and prevent further post-processing contamination in food products. The current study

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was conducted to (i) gain a better understanding of the prevalence and diversity of Listeria spp., and

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(ii) evaluate the 3M Molecular Detection Assay (MDA) Listeria for its ability to detect Listeria spp. in

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environmental samples from seafood processing plants in Thailand.

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2. Materials and methods

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2.1 Collection of environmental samples

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Duplicate environmental sponges were collected from 152 different sites within three

seafood processing plants in Southern Thailand. For each pair of samples collected from a given site,

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one was used for enrichment with the 3M Modified Listeria Recovery Broth (mLRB) and one for

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enrichment with the standard U.S. Food and Drug Administration Bacteriological Analytical Manual

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(FDA BAM) procedures, as detailed below. Sterile pre-wetted 3M Sponge-Sticks in Letheen Buffer

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were used to swab areas of approximately 10 x 10 cm2 five times vertically and five times

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horizontally. Sponges were then placed aseptically in their original sterile bags. Sticks were

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detached from the sponges before sealing the bags. Samples were kept on ice during transport to

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the laboratory, and analysis of samples was performed within 24 h of sample collection. To increase the likelihood of finding Listeria in environmental samples, the busy operation

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schedules of the processing plants were chosen, i.e., the last two weeks of December to the first

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week of January (production for Christmas and New Year’s sell cycles). Overall, 444 sponge samples

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were collected from three plants between November 2012 and January 2013. Two sampling visits

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were conducted for each plant. For plant A, duplicate sponge samples were collected from the same

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40 sites at each of the two visits, which were 10 days apart for a total of 160 sponges collected from

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this plant. For plant B, 120 sponges were collected from the same 30 sites during two visits which

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were two days apart. Eight sponges were also collected from four additional sites of plant B during

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visit 1. For plant C, 48 sponges were collected from 24 sites during visit 1, and 60 samples from 30

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sites during visit 2. See Table 1 on details of the sites in each seafood processing plant that were

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included for environmental sample collection. Of 152 sites within three seafood processing plants,

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64 sites were food contact surfaces and 88 sites were non-food contact surfaces (see Table2).

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2.2 Preparation of artificially contaminated sponges

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Cultures of three Listeria spp. [L. monocytogenes, FSL J1-208 (Food Safety Laboratory,

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Cornell University, Ithaca, NY); L. innocua (DMST 9011), and L. ivanovii (DMST 9012; obtained from

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the Culture Collection of the Department of Medical Science Thailand)] were used separately to

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prepare artificially contaminated samples. An isolated colony of a given species was used to prepare

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an overnight in 5 ml of BHI (Brain Heat Infusion) broth followed by incubation at 37°C for 18–20 h.

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Overnight culture of each species were diluted in PBS (Phosphate Buffered Saline, pH 7.4) to achieve

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approximately 5 CFU/ml in a final volume of 10 ml. For each study, two artificial contamination and

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sampling procedures were performed in duplicate: (i) direct addition onto sponges and (ii) swabbing

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the artificially inoculated surfaces (polystyrene and stainless steel plates). The prepared inoculum of

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a given species (1 ml, representing 5 CFU) was directly added into a bag containing pre-wetted 3M

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Sponge-Sticks in Letheen Buffer. Surfaces of sterile lids of the 96-well plates (polystyrene material)

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of each given species. The prepared inoculum (1 ml) was spotted, by dropping 10 drops of

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approximately 100 µl, onto the surfaces of polystyrene lids and stainless steel plates, covering the

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areas of approximately 10 x 10 cm2. Applied inoculum on the surfaces was allowed to air-dry for 3

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hours in the laminar flow, followed by swabbing using the pre-wetted 3M Sponge-Sticks in Letheen

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Buffer as the procedures described above. Overall, the artificial contamination and sampling

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procedures with three species of Listeria were performed on 36 samples in two independent studies.

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Samples were tested by both methods as described below.

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2.3 Detection of Listeria spp. using the 3M Molecular Detection Assay (MDA) Listeria

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Environmental sponges were processed for enrichment; 90 ml of the 3M Modified Listeria

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Recovery Broth (mLRB) was added directly to each collected sample. Samples were homogenized

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using a stomacher (220 rpm) for 60 s (Stomacher 400, Seward Medical, UK), and incubated at 37°C

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for 26 h and 48 h. For each set of samples tested, positive and negative process control samples

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were prepared. For a positive control, 1 ml of the diluted overnight culture of L. monocytogenes FSL

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J1-208 was added into a sample bag with sterile enrichment media to achieve the level of 105

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CFU/ml. The overnight cultures of L. monocytogenes were prepared and diluted following the

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procedures described above. Uninoculated enrichment media was used as a negative control. The

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same enrichments were used after 26 h and 48 h of incubation to prepare lysate to be tested with

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the 3M MDA, following the manufacturer’s protocol. Sample lysate (20 µl) was transferred to a

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reagent tube and its corresponding 3M Molecular Detection Matrix Control tube, and mixed by

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pipetting. Reagent and Matrix Control tubes were loaded into the 3M Molecular Detection Speed

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Loader Tray, and analysis was performed using the instrument and software (3M MDS). For each test

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run, one positive process control, one negative process control, one reagent control (provided with

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the kit), one negative assay kit control (provided with the kit) were also included. Presumptive

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positive results were reported in real-time, while negative results were displayed at the end of the

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75-min run.

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To confirm the results obtained by the 3M MDS, the FDA BAM plating procedures were followed. An aliquot (50 µl) of the same enrichments used to prepare the lysate mentioned earlier 6

ACCEPTED MANUSCRIPT was streaked onto Chromogenic Listeria agar (OCLA; Oxoid, Basingstoke, Hampshire, UK). OCLA

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plates were incubated at 37°C for 48 h. Typical colonies on OCLA of Listeria spp. (bluish-green,

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without halo zone) and L. monocytogenes or L. ivanovii (bluish-green, with halo zone) were

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substreaked onto brain heart infusion agar plates (BHI; Oxoid). BHI agar plates were incubated for

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37°C for 24 h. Presumptive Listeria colonies were confirmed by PCR and partial SigB gene sequencing

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analysis as detailed below (section 2.5). All Listeria isolates were kept at -80°C in BHI with 15%

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glycerol.

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2.4 Detection of Listeria spp. using the FDA Bacteriological Analytical Manual (BAM) method

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Duplicate environmental samples collected from the 152 sampling sites (Table 2) were processed for Listeria enrichment and testing according to the FDA-BAM standard method (Hitchins

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and Jinneman, 2011) with minor modifications. Briefly, 90 ml of Buffered Listeria Enrichment Broth

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Base (BLEBB; Oxoid) was added to each bag containing a sponge. Samples were homogenized using a

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stomacher (220 rpm) for 60 s, followed by incubation at 30°C for 4 h. After 4 h of incubation, Listeria

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Selective Enrichment Supplement (SR 141E; Oxoid) was added into each sample according to the

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manufacturer’s instruction, followed by continued incubation at 30°C. At 24 h and 48 h of

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incubation, 50 µl of enrichment was streaked onto each Modified Listeria Selective Oxford Agar

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(MOX; Oxoid) and OCLA plates. MOX plates were incubated at 30°C for 48 h, and OCLA plates were

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incubated at 37°C for 48 h.

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2.5 Confirmation of Listeria spp. and serotyping by multiplex PCR

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For each sample that tested positive for Listeria, one putative colony was selected from BHI

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plate obtained as described in section 2.3. To confirm Listeria spp., PCR amplification of a 370-bp

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region in the prs gene (Doumith et al., 2004) was performed, using the primer pairs: 5’GCT GAA GAG

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ATT GCG AAA GAA G3’ and 5’CAA AGA AAC CTT GGA TTT GCG G3’. Additional primer pairs were used

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to amplify lmo0737 (691 bp), lmo1118 (906 bp), ORF2819 (471 bp), and ORF2110 (597 bp) for

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classification into groups of serotypes, as reported by Doumith et al. (2004). Oligonucleotide primers

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used in this study were synthesized by Macrogen, Korea. Each bacterial lysate used for the PCR

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reaction was prepared following the boiling method [modified from Kim et al. (2007)]. Briefly, an 7

ACCEPTED MANUSCRIPT aliquot (1 ml) of the overnight culture was subjected to centrifugation at 10,000 X g for 10 min to

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pellet the cells, followed by resuspension in 500 µl of sterile distilled water and boiling at 100 °C for

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10 min. Samples were cooled immediately at -20°C for 5 min, and centrifuged at 10,000 X g for 10

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min. Lysates were kept at -20°C, followed by thawing and centrifugation at 4,000 X g to pellet the

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debris before using as template. Two microlitters of the supernatant was used as the template DNA

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for a 25-µl PCR reaction mixture containing regents of the same concentrations and the same PCR

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cycling conditions as described in Doumith et al. (2004). PCR products were resolved on

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electrophoresis on 1.0 % agarose gel. Agarose gels were run at 100 V for 30 min, and visualized

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under UV light. A DNA-molecular ladder (100-bp ladder; Vivantis Technologies) was included in each

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gel.

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2.6 Identification of Listeria species by SigB sequencing

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Partial sequences of the SigB gene were used to classify isolates into Listeria spp. based on allelic type (AT) classification as in previous studies (den Bakker et al., 2010; Milillo et al., 2012). PCR

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amplification of a 780-bp region of SigB gene was performed following the protocols described in

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(Nightingale et al., 2005). PCR products were submitted to Macrogen, Korea for purification and

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sequencing. Sequences were assembled and edited with DNA Baser Sequence Assembler v3.x

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(2012), Heracle BioSoft SRL Romania (http://www.DnaBaser.com), followed by AT assignment. ATs

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were assigned by comparison of SigB sequences to an existing reference database (provided by H.

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den Bakker, Cornell University, Ithaca, NY) comprising of 153 previously identified unique SigB allelic

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types from L. monocytogenes, L. marthii, L. innocua, L. welshimeri, L. seeligeri, and L. ivanovii.

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2.7 Statistical analysis

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To determine the differences between the proportions of Listeria-positive samples obtained

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from the 3M MDA Listeria, and the reference BMA-FDA method, a Chi-square test was performed.

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The 95% confidence intervals for the performance of the 3M MDA Listeria as compared to the

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reference BAM-FDA method for the detection of Listeria spp. were calculated. JMP software version

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11 (SAS Institute Inc., SAS Campus Drive, Cary, North Carolina) was used for statistical analysis in this

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study.

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3. Results

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3.1 Detection of Listeria spp. from environmental and artificially contaminated samples Two detection methods, the standard FDA BAM and 3M MDA Listeria, were performed on

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each set of 222 environmental samples (n = 444). Of 222 environmental samples tested following

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the FDA BAM method, 13 samples (5.9%), all from plant A, were positive for Listeria spp. The other

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222 samples (collected in parallel with those 222 samples) duplicate tested following the 3M MDA

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Listeria, 11 samples (4.9%) were positive for Listeria spp. All positive samples tested by this method

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were also collected from plant A. Overall, detection of Listeria spp. by the two methods did not differ

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significantly (p > 0.05).

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Of the 13 samples from plant A that tested positive for Listeria spp. by the FDA BAM method, nine positive samples were found among samples collected during sampling visit 1, while

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four other positive samples were from sampling visit 2 (Table 2). Among the nine positive samples

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recovered from visit 1, eight represented food contact surface (FCS) samples and only one was non-

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food contact surface (NFCS) sample (Table 3). Samples from FCS were collected from three

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processing departments of plant A (i.e., Gyoza processing, shrimp tempura processing, and squid

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sushi processing). The only one NFCS sample that tested positive was swabbed from the drain of the

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preparation room of the squid sushi processing department. Among the four positive samples

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recovered from visit 2, three were FCS samples from two processing departments (i.e., Gyoza

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processing and shrimp tempura processing), and one was swabbed from the drain of the preparation

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room of the squid sushi processing department. Among the 222 environmental samples tested by

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the 3M MDA method, seven and four positive samples represented visit 1 and 2, respectively, to

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plant A. For visit 1, six positive samples were collected from FCS of three processing departments,

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and one positive sample was swabbed from the drain of the tempura frying room (NFCS). For visit 2,

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one positive sample was swabbed from the bucket for soaking shrimps (FCS), and three positive

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samples were sampled from the cleaning sponge and the drains of the tempura frying room, as well

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as the drain of the Gyoza steaming room.

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3.2 Recovery of diverse Listeria spp. in seafood processing plant environments Listeria isolates recovered from each positive sample were further identified to the species

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level by SigB sequencing analysis. These isolates included (i) 12 isolates recovered from 12 positive

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samples that were detected by a single method (five isolates by the MDA only and seven isolates by

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the FDA BAM only); (ii) six isolates from samples positive by both methods (12 samples were positive

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by both methods and these isolates were selected from six positive samples by the FDA BAM

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method for further analysis). Overall, 18 Listeria isolates included in the analysis generated three

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allelic types (Table 3). Ten Listeria isolates were classified as AT 60 and two other Listeria isolates

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were classified as AT 58. These two allelic types could classify these 12 isolates as L. monocytogenes

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lineage I. Six Listeria isolates were identified as AT 31 and classified as L. innocua. Three Listeria

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isolates, recovered from FCS of the Gyoza processing department during visit 1 and 2, were

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identified as L. monocytogenes lineage I (AT 60), while one isolate recovered from the drain of the

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Gyoza steaming room was identified as L. innocua (AT 31). L. monocytogenes isolates from FCS of the

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tempura processing department were classified as L. monocytogenes lineage I (AT 58 and AT 60),

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while two isolates from the drain of the tempura frying room were classified as L. monocytogenes

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lineage I (AT 60). Isolates from sushi department were identified as L. monocytogenes lineage I (AT

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60) and L. innocua (AT 31). PCR amplification of a 370-bp region in the prs gene was positive for 18

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Listeria isolates. Multiplex PCR serotyping showed that 12 L. monocytogenes could be classified into

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the same serogroup (1/2b, 3b, 4b, 4d, 4e), based on the classification scheme reported by Doumith

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et al. (2004).

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3.3 Correlation of the 3M MDA Listeria and the standard FDA BAM method for the detection of

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Listeria spp. on environmental samples

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Of 222 environmental samples (collected in duplicate), six samples showed concordant

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results, while 12 samples showed discrepant results (Table 4). Five samples tested positive by the

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3M MDA only; three samples showed positive after 26–48 h of enrichment, and two samples

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showed positive after 48 h of enrichment. Seven samples tested positive by the FDA BAM only (after

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26–48 h of enrichment). All 36 samples that were artificially contaminated (spiked directly into

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ACCEPTED MANUSCRIPT sponges or spiked onto surfaces of stainless steel and plastic, followed by surface swabbing) showed

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concordant results by the two detection methods. The 3M MDA Listeria was able to detect as low as

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5 CFU of each of the three species of Listeria (L. monocytogenes, L. innocua, and L. ivanovii) in

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environmental sponge samples collected from stainless steel and plastic surfaces. The overall

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comparison, including both artificially contaminated and environmental samples, between the two

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methods revealed that the sensitivity of the 3M MDA was 87.0% (95% CI: 77.4–96.6%), specificity

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was 97.6% (95% CI: 95.5–99.7%), accuracy was 95.3% (95% CI: 92.8–97.9%), and the positive

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predictive value was 89.4% (95% CI: 80.5–98.2%; Table 5). A matrix control of the 3M MDA was used

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with the lysate of a given sample. None of the matrix controls were inhibited by sample lysates,

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suggesting that the performance of the reagents of the 3M MDA was not affected by components

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present in these environmental samples.

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4. Discussion

To understand the prevalence of Listeria spp. in food processing plant environments, we

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collected samples from 152 location sites of the seafood processing plants, including food contact

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surfaces and non-food contact surfaces. Culture-based method (FDA-BAM) and a molecular-based

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rapid detection method for the detection of Listeria spp. in environmental samples were compared.

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Overall, we found that (i) Listeria spp. were recovered on both food contact surfaces and non-food

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contact surfaces in one of the three seafood processing plants tested, with all isolates representing

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L. monocytogenes and L. innocua; and (ii) the 3M Molecular Detection Assay Listeria yields results

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that were comparable to those obtained by the FDA BAM method on environmental samples.

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4.1 Listeria spp. were recovered on both food contact surfaces and non-food contact surfaces in one

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of the three seafood processing plants tested, with all isolates representing L. monocytogenes or L.

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innocua in the seafood processing plant environments

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Occurrence of Listeria spp., especially L. monocytogenes in food processing plant environments has been a major concern for the food industry. Several studies have shown that food

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ACCEPTED MANUSCRIPT processing environment is a key source of Listeria contamination in finished products, typically at

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the post-processing stage, or packaging of foods (Thimothe et al., 2004; Kornacki and Gurtler, 2007).

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Listeria spp., including L. monocytogenes have often been found in food processing plants

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manufacturing different food products such as smoked fish (Hoffman et al., 2003; Vongkamjan et al.,

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2013), poultry (Kim et al., 2008), meat (Ferreira et al., 2011), and dairy (Kabuki et al., 2004). Our data

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showed that Listeria spp., including L. monocytogenes were common in different environments of

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the seafood processing plant. Although Listeria spp., including L. monocytogenes are ubiquitous in

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nature with each type of food processing facility may encounter differences in their prevalence. For

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example, one previous study (Kovačević et al., 2012) has tested samples collected from processing

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facilities producing three different types of products, and found that Listeria spp. contamination was

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more common in fish processing environments sampled as compared to dairy and meat facilities

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sampled. A study by Thimothe at el. (2003) reported that Listeria spp. were commonly found not

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only in raw and finished products, but also in environmental samples collected from four different

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smoked fish processing plants.

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contact surfaces and non-food contact surfaces, while the prevalence of Listeria spp. was higher in

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food contact surfaces. These environmental samples were collected from three different processing

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departments, manufacturing Gyoza, shrimp tempura, and squid sushi within a given seafood

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processing plant. A previous study also found that Listeria spp. were most commonly recovered from

312

close-to-food contact and food contact surfaces (e.g., cutting boards and work tables) of the fish

313

processing facilities as compared to meat and dairy facilities (Kovačević et al., 2012). However,

314

several previous studies reported the higher prevalence of Listeria spp. and L. monocytogenes in

315

drains and other non-food contact sites, and Listeria from these locations were found to be

316

associated with contamination of the finished products (Lappi et al., 2004; Thimothe et al., 2004). In

317

addition, a study by Vongkamjan et al. (2013) reported that environmental samples (mainly from

318

drain locations) collected from a smoked fish plant over approximately 1 year yielded higher

319

prevalence of Listeria spp. (30.3%) as compared to that of L. monocytogenes (18.2%).

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Recovery of Listeria spp. and especially the foodborne pathogen L. monocytogenes in the environments of the processing departments manufacturing RTE food (i.e., squid sushi) is of a

322

particular concern. Our findings may indicate a risk of (i) cross-contamination directly from food

323

contact surfaces to finished products, and (ii) cross-contamination from non-food contact surfaces to

324

food contact surfaces with subsequent contamination of finished products. Listeria were specifically

325

detected in environmental swabs from the splitter and on the towel for absorbing squids that were

326

directly contacted onto food products during production, thus increasing the chance of Listeria

327

contamination in finished RTE products. RTE seafood products, especially sushi and sashimi, mainly

328

include raw seafood such as minced or sliced tuna, fish roe, shrimps, and squids. Previous studies

329

have reported high contamination rate of L. monocytogenes in raw RTE seafood products (Handa et

330

al., 2005; Miya et al., 2010). In addition, all L. monocytogenes isolates from our study showed

331

serotypes 1/2b, 3b, 4b, 4d, 4e which included the common serotypes that have been previously

332

isolated from foods and human listeriosis cases (Graves et al., 2007).

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Our study showed that L. monocytogenes and L. innocua were commonly found among environmental samples from one seafood processing plant. Both Listeria spp. and L. monocytogenes

335

could be present in environmental samples from food processing plants in previous studies (Lappi et

336

al., 2004; Kovačević et al., 2012; Fortes et al., 2013). Another study by Vongkamjan et al. (2013)

337

demonstrated that both Listeria spp. and L. monocytogenes could be detected in environmental

338

samples collected from a smoked fish processing plant over approximately 2 years of sampling.

339

Different species of Listeria, especially the three most common species, L. innocua, L. welshimeri,

340

and L. seeligeri have been commonly recovered together with L. monocytogenes in food processing

341

plant environments (Muller et al., 2010; Keeratipibul and Techaruwichit, 2012; Fortes et al., 2013). It

342

is necessary to control both Listeria spp. and L. monocytogenes in food processing plant

343

environments in order to reduce an incidence of finished product contamination. Our study found

344

that both Listeria innocua and L. monocytogenes could be recovered from the same processing

345

department (i.e., shrimp tempura processing), suggesting common favorable conditions that may

346

allow for survival and persistence of other Listeria spp. together with the pathogenic L.

347

monocytogenes in the same given environments. Our study showed that Listeria spp. or L.

348

monocytogenes were mostly recovered from FCS samples, effective monitoring programs for Listeria

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ACCEPTED MANUSCRIPT spp. and L. monocytogenes are thus necessary for seafood processing plants. Effective monitoring

350

will allow for further development of Listeria control programs, including sanitation plans in order to

351

minimize cross-contamination in finished products.

352

4.2 The 3M Molecular Detection Assay Listeria yields results that were comparable to those obtained

353

by the FDA BAM method on environmental samples

354

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Our study showed that the 3M MDA Listeria revealed high sensitivity, specificity, and

accuracy for the detection of Listeria spp. in environmental samples. Overall, the 3M MDA Listeria

356

yielded results that were comparable to those obtained by the standard FDA BAM method. Some

357

samples tested positive by the 3M MDA Listeria only. This was likely due to the presence at low

358

levels of possibly injured and stressed Listeria spp. Enrichment of these injured and stressed cells of

359

Listeria spp. may require up to 48 h or even beyond 2 days to detect positive results by the

360

traditional culture-based method (Warburton et al., 1991; Warburton et al., 1992). Seven samples

361

tested positive by the FDA BAM method only (after 26–48 h of enrichment). However, the 3M mLRB

362

enrichment of these samples were confirmed by culture-based isolation and found negative on this

363

isolation method, suggesting a true negative result obtained by the 3M MDA Listeria. The matrix

364

controls were not inhibited by these sample lysates, suggesting that the performance of the

365

reagents of the 3M MDA Listeria was not affected by components present in these environmental

366

samples. Overall, in this study, the sensitivity of the 3M MDA Listeria was not found to be affected

367

by various components of environmental samples as found in a previous study for LAMP assays

368

(Kaneko et al., 2007).

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The traditional culture-based method requires several procedures for sample analysis and

370

result confirmation. For detection of Listeria spp., the culture-based method requires a plating step

371

onto selective and differential media. The incubation step after plating requires up to 48 h, followed

372

by a confirmation of presumptive colonies or biochemical test (FDA, 2011). While both the standard

373

FDA-BAM and the 3M MDA Listeria methods require enrichment step, the enrichment step for the

374

3M MDA is only 26 h as compared to up to 48 h in selective media for Listeria spp. detection by the

375

standard method. For the 3M MDA Listeria method, enriched samples are used in the lysis steps for

14

ACCEPTED MANUSCRIPT isothermal amplification to be measured by the Molecular Detection System. The Molecular

377

Detection System can yield rapid results (within 75 min) as compared to the traditional culture-

378

based method. The lysis and detection steps can be performed with a large set of environmental

379

samples which is less time-consuming as compared to the traditional method. Overall, high-

380

throughput screening of samples can be achieved by the 3M MDA Listeria and the Molecular

381

Detection System.

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382 5. Conclusions

384

Our study shows that Listeria spp. are widely distributed on both food contact surfaces and non-food

385

contact surfaces, and that L. monocytogenes and L. innocua are common species found in the

386

seafood processing plant environments. The 3M Molecular Detection Assay Listeria is a powerful

387

method for rapid detection and monitoring of Listeria spp. in food processing plant environments.

388

Data suggest that the 3M Molecular Detection Assay Listeria yields comparable detection results to

389

those obtained by the cultural-based method with environmental samples. Detection of Listeria spp.

390

after enrichment using the 3M MDS method is simple to perform and less labor intensive as

391

compared to the routine traditional culture-based method, suggesting an operating alternative for

392

high-throughput screening of Listeria in environmental samples for the food industry. Moreover, the

393

assay is able to detect various species of Listeria, and was not inhibited by various components of

394

environmental samples, with results that could be generated in less than 75 min (after enrichment).

395

Molecular-based detection methods have increasingly gained more interest among the food

396

industries for pathogen detection in foods and environmental samples. However, further studies can

397

be performed to include environmental samples from different processing departments of the

398

seafood facilities as well as other facilities manufacturing different food products. Other Listeria spp.

399

that are likely to be present in food processing plant environments can also be spiked into food

400

samples to investigate the performance of the 3M Molecular Detection Assay Listeria. Overall,

401

monitoring of Listeria spp. in food processing plant environments is considerably important in order

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15

ACCEPTED MANUSCRIPT 402

to obtain valuable data for further development of effective control measures to reduce occurrence

403

of Listeria spp. in foods before distribution to retail establishments.

404

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405 ACKNOWLEDGEMENT

407

This study was supported by a sponsored research agreement between 3M Asia Pacific Pte Ltd. and

408

the department of Food Technology, Prince of Songkla University (with K.V.), and the funding from

409

the Prince of Songkla University.

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411 412

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Graves, L. M., Swaminathan, B., Hunter, S. B., 2007. Subtyping Listeria monocytogenes. In: Ryser, E. T. and Marth, E. H. (Eds.), Listeria, Listeriosis and Food Safety. Marcel Dekker Inc., New York, NY, pp. 283-304.

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Hu, Y., Gall, K., Ho, A., Ivanek, R., Grohn, Y. T., Wiedmann, M., 2006. Daily variability of Listeria contamination patterns in a cold-smoked salmon processing operation. J. Food Prot. 69, 2123-2133.

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Kabuki, D. Y., Kuaye, A. Y., Wiedmann, M., Boor, K. J., 2004. Molecular subtyping and tracking of Listeria monocytogenes in Latin-style fresh-cheese processing plants. J. Dairy Sci. 87, 2803-2812.

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Kim, J. W., Siletzky, R. M., Kathariou, S., 2008. Host ranges of Listeria-specific bacteriophages from the turkey processing plant environment in the United States. Appl. Environ. Microbiol. 74, 66236630.

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Kovačević, J., McIntyre, L. F., Henderson, S. B., Kosatsky, T., 2012. Occurrence and distribution of Listeria species in facilities producing ready-to-eat foods in British Columbia, Canada. J Food Prot 75, 216-224.

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Lappi, V. R., Thimothe, J., Nightingale, K. K., Gall, K., Scott, V. N., Wiedmann, M., 2004. Longitudinal studies on Listeria in smoked fish plants: impact of intervention strategies on contamination patterns. J. Food Prot. 67, 2500-2514.

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Milillo, S., Stout, J., Hanning, I., Clement, A., Fortes, E., den Bakker, H., Wiedmann, M., Ricke, S., 2012. Listeria monocytogenes and hemolytic Listeria innocua in poultry. Poult. Sci. 91, 2158-2163.

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Miya, S., Takahashi, H., Ishikawa, T., Fujii, T., Kimura, B., 2010. Risk of Listeria monocytogenes contamination of raw ready-to-eat seafood products available at retail outlets in Japan. Appl. Environ. Microbiol. 76, 3383-3386.

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525 526

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527 528 529

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530 531 532

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533 534 535

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536 537 538

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539 540

Wesley, I. V. , 1999. Listeriosis in animals. In: Ryser, E. T. and Marth, E. H. (Eds.), Listeria listeriosis and food safety. Marcel Dekker, Inc, New York, NY, pp. 39-73.

541 542 543

Williams, S. K., Roof, S., Boyle, E. A., Burson, D., Thippareddi, H., Geornaras, I., Sofos, J. N., Wiedmann, M., Nightingale, K., 2011. Molecular ecology of Listeria monocytogenes and other Listeria species in small and very small Ready-to-Eat meat processing plants. J. Food Prot. 74, 63-77.

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Table 1. Sites in each seafood processing plant included for environmental sample collection.

(1) Frozen cooked shrimps

• • • • • • • •

Non-food contact surfaces

• Conveyor belt to freezer • Working table, sizing station • Cutting board • Sieve containing shrimps • Stainless steel, peeling station • Working table, sizing station

• Drain, boiling room • Conveyor belt from boiling pot • Cart wheel • Top of glazing machine • Conveyor, boiling section • Conveyor belt, peeling station • Conveyor belt, auto weigh • Side of glazing machine

• Sieve containing shrimps, cooling section • Bucket for weighing shrimps

• Floor, cooling section • Cart wheel, cooling section • Drain, cooling section • Conveyor belt, peeling station • Working table, freezing section • Scale

SC

• Drain, steaming room

Food contact surfaces

Plant C

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• Employee's gloves (production room) • Splitter • Working table • Knife • Stainless steel block containing shrimps • Towel for absorbing squid • Employee's gloves (prep room)

Processing department

Towel spinner Conveyor belt Forceps Bucket for seasoning Cleaning sponge Scale Drain, production room Drain, prep room

(2) Shrimp (cooked) sushi

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(2) Squid sushi

• Bucket containing shrimps • Employee's gloves (prep room) • Gyoza mold • Blender for Gyoza stuffing

Non-food contact surfaces

EP

(1) Gyoza

Food contact surfaces

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Processing department

Plant B

RI PT

Plant A

1

Processing department (1) Frozen cooked shrimps

Food contact surfaces Raw material receiving section: • Bucket containing shrimps • Tray containing shrimps Sizing station: • Tray containing shrimps • Work table

Preparation (splitting dissecting, cleaning): • Bucket containing shrimps • Tray containing washed shrimps • Work table • Knife blade

Non-food contact surfaces Preparation (splitting dissecting, cleaning): • Drain • Cart wheel/handle of the cart • Conveyor belt • Ice scoop • Ice bucket • Back of the freezer • Sink • Bucket for washing shrimps • Spatula Packing: • Drain • Sink • Conveyor belt of a scale

ACCEPTED MANUSCRIPT

• • • •

Drain Scale Conveyor belt 2 Conveyor belt 3

(3) Shrimp (raw) sushi

• Sieve containing shrimps • Cutting board • Stick, chilling section

• Drain, production room • Scale • Cart containing ice • Employee's apron, peeling station • Conveyor belt, peeling station • Conveyor belt • Working table, chilling section • Cart wheel • Conveyor belt, peeling station

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• Employee's gloves (splitting shrimps) • Basket • Bowl containing shrimps • Employee's gloves (packaging) • Bucket containing shrimps • Working table

• Conveyor belt • Drain, frying room • Cleaning sponge

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Batter blender Bread crumbs tray Stainless steel sieve Bucket for soaking shrimps • Stainless steel tray containing shrimps • Employee's hand (dipping batter) • Employee's gloves (prep)

EP

• • • •

AC C

(4) Shrimp tempura

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(3) Shrimp sushi

2

Production (boiling, chilling, freezing, glazing): • Bucket containing shrimps • Weighing basket • Stainless steel block containing shrimps

Packing: • Work table • Funnel • Weighing basket

ACCEPTED MANUSCRIPT Table 2. Recovery of Listeria spp. from environmental samples collected from seafood processing plants.

Visit 1

Plant A Plant B Plant C Total

Visit 2

b

c

No. (%) of samples positive for Listeria spp. detected by:

Total samples (sites)

FDA BAM method

FCS

NFCS

FCS

NFCS

Visit 1

24 10 (1) 5

16 20 (3) 19

24 10 24

16 20 30

80 (40) 64 (34) 78 (78)

9 0 0

4 0 0

40

58

58

66

222 (152)

9

4

a

Visit 2

Total

3M MDA method

Visit 1

Visit 2

RI PT

a

No. of samples collected during each sampling visit:

13 0 0

7 0 0

4 0 0

11 0 0

13

7

4

11

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Samples were collected in duplicate from a given sampling site; numbers shown in this table represent single sponge from a given site. One of each duplicate was tested by the corresponding detection method. b Sampling sites of only plant A and B were kept constant during sampling visits 1 and 2. For visit 1 to plant B, additional 4 sampling sites (indicated in parentheses) were also included. C Listeria spp. includes L. monocytogenes.

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ACCEPTED MANUSCRIPT

Table 3. Distribution of Listeria spp. in environmental locations of plant A.

Allelic Type (classification)

b

Detected by: FDA BAM

d

Serotype

3M MDA

FDA BAM

Food Contact Surfaces N/D

AT60 (LM lineage I)

(G) Blender for Gyoza stuffing

Listeria spp.

N/D

AT60 (LM lineage I)

(G) Gyoza mold

Listeria spp.

Listeria spp.

AT60 (LM lineage I)

(T) Bread crumbs tray

Listeria spp.

Listeria spp.

AT31 (L. innocua)

(T) Stainless steel sieve

Listeria spp.

Listeria spp.

AT31 (L. innocua)

(T) Stainless steel tray containing shrimps

Listeria spp.

Listeria spp.

AT58 (LM lineage I)

(T) Bucket for soaking shrimps

N/D

N/D

(T) Batter blender

Listeria spp.

N/D

(S) Stainless steel block containing squid

Listeria spp.

N/D

d

Serotype

3M MDA

1/2b, 3b, 4b, 4d, 4e

N/D

N/D

—

—

1/2b, 3b, 4b, 4d, 4e

N/D

N/D

—

—

1/2b, 3b, 4b, 4d, 4e

Listeria spp.

N/D

AT60 (LM lineage I)

1/2b, 3b, 4b, 4d, 4e

—

N/D

N/D

—

—

—

N/D

N/D

—

—

1/2b, 3b, 4b, 4d, 4e

Listeria spp.

N/D

AT58 (LM lineage I)

1/2b, 3b, 4b, 4d, 4e

—

—

Listeria spp.

Listeria spp.

AT60 (LM lineage I)

1/2b, 3b, 4b, 4d, 4e

AT31 (L. innocua)

—

N/D

N/D

—

—

AT60 (LM lineage I)

1/2b, 3b, 4b, 4d, 4e

N/D

N/D

—

—

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Listeria spp.

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EP

TE D

(G) Bucket containing shrimps

c

Allelic Type (classification)

b

Detected by:

SC

(Processing department ) Environmental sites

Sampling Visit 2 c

RI PT

Sampling Visit 1 a

1

ACCEPTED MANUSCRIPT

N/D

Listeria spp.

AT60 (LM lineage I)

1/2b, 3b, 4b, 4d, 4e

N/D

N/D

—

—

(S) Towel for absorbing squid

N/D

Listeria spp.

AT60 (LM lineage I)

1/2b, 3b, 4b, 4d, 4e

N/D

N/D

—

—

(S) Drain, prep. room

Listeria spp.

N/D

AT31 (L. innocua)

—

N/D

N/D

—

—

(T) Drain, tempura frying room

N/D

Listeria spp.

AT60 (LM lineage I)

1/2b, 3b, 4b, 4d, 4e

Listeria spp.

Listeria spp.

AT60 (LM lineage I)

1/2b, 3b, 4b, 4d, 4e

(T) Cleaning sponge

N/D

N/D

—

—

N/D

Listeria spp.

AT31 (L. innocua)

—

(G) Drain, steaming room

N/D

N/D

—

—

N/D

Listeria spp.

AT31 (L. innocua)

—

M AN U TE D

a

SC

Non Food Contact Surfaces

RI PT

(S) Splitter

AC C

EP

Processing departments of plant A that showed Listeria-positive results; (G) is Gyoza processing; (T) is tempura-shrimp processing; (S) is sushi-squid processing. b Duplicate samples were detected for Listeria spp. by the FDA BAM and the 3M Molecular Detection Assay Listeria methods. ‘N/D’ indicates not detected. c Allelic type and classification of Listeria species by the analysis of partial sequences of the SigB gene. d Serotypes classified by a multiplex-PCR serotyping based on the classification scheme reported by Doumith et al. (2004).

2

ACCEPTED MANUSCRIPT Table 4. Correlation of the detection results by the 3M Molecular Detection Assay Listeria and the FDA-BAM standard method after enrichment. No. of samples (source)

24–26 h of enrichment 3M MDA

48 h of enrichment

FDA-BAM

3M MDA FDA-BAM

AC C

EP

TE D

M AN U

SC

RI PT

Concordant results N = 204 – – – – N = 6 (Environmental) + + + + N = 36 (Artificially contaminated) + + + + Discrepant results (All environmental) N=3 + – + – N=2 – – + – N=7 – + – + a Number of samples that showed positive (+) or negative (–) results by both the 3M MDA and the standard FDA-BAM methods (concordant results), or (ii) only one of the two methods (discrepant results) after (i) 24–26 h of enrichment and (ii) 48 h of enrichment.

ACCEPTED MANUSCRIPT Table 5. Overall comparison of the 3M Molecular Detection Assay Listeria to the FDA-BAM method. The 3M MDA compared to FDA-BAM method

95% Confidence Interval (CI)

Sensitivity Specificity Accuracy True Positive

87 97.6 95.3

77.4–96.6 95.5–99.7 92.8–97.9

(Positive Predictive Value)

89.4

RI PT

Value (%)

AC C

EP

TE D

M AN U

SC

80.5–98.2

ACCEPTED MANUSCRIPT Highlights We find Listeria spp. from environmental samples from seafood processing plant.

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Listeria spp. can be recovered from both food and non-food contact surfaces.

•

Isolates recovered from a seafood plant are L. monocytogenes or L. innocua.

•

The 3M Molecular Detection Assay Listeria provides rapid detection results.

•

Monitoring of Listeria spp. in food processing environments is important.

AC C

EP

TE D

M AN U

SC

RI PT

•