Enrichment cultivation in detection of food-borne Salmonella

Food Control 26 (2012) 369e377

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Food Control journal homepage: www.elsevier.com/locate/foodcont

Review

Enrichment cultivation in detection of food-borne Salmonella Sanna Taskila*, Mika Tuomola, Heikki Ojamo Bioprocess Engineering Laboratory, Department of Process and Environmental Engineering, University of Oulu, P.O. Box 4300, Linnanmaa, FI-90014 Oulu, Finland

a r t i c l e i n f o

a b s t r a c t

Article history: Received 10 February 2011 Received in revised form 16 January 2012 Accepted 21 January 2012

Food-borne Salmonellosis is a significant risk to public health. Despite wide variety of rapid molecular methods, the detection of Salmonella still requires at least one enrichment cultivation step. Altough the adjustment in the enrichment cultivation may significantly improve the sensitivity and specificity of Salmonella diagnostics, the majority of current research focuses on the development of novel detection methods adapting existing cultivation principles without consideration. However, the understanding of enrichment cultivation is essential for the success in detection. This review aims to summarize the main factors that affect the enrichment cultivation of food-borne Salmonella, in order to provide tools for further improvement of both enrichment and detection methods. Ó 2012 Elsevier Ltd. All rights reserved.

Keywords: Enrichment Cultivation Detection Food Salmonella

Contents 1. 2. 3.

4.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 369 Enrichment media for Salmonella . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .370 Selection of the enrichment procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .371 3.1. Regulations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 371 3.2. Sample matrix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 371 3.3. Background microbiota . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 372 3.4. Concentration and condition of target cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 373 3.5. Environmental stress factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 373 3.6. Detection method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 374 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .374 Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 374 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 374

1. Introduction Food-borne salmonellosis is an important public health problem worldwide, the annual cost of salmonellosis in the USA being over $2600 million (Economic Research Service, 2010). The major sources of salmonellosis outbreaks include eggs, meats and dairy products (EFSA, 2008). Although novel rapid detection methods and more effective sample processing techniques are frequently

* Corresponding author. þ3588 553 2579. E-mail address: sanna.taskila@oulu.fi (S. Taskila). 0956-7135/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodcont.2012.01.043

reported, the need for enrichment cultivation still exists. This is due to possibly low concentration and sub-lethal injuries of target bacteria, and the high amount of other bacteria and inhibitory food components in samples. The duration of the cultivation step mostly depends on the length of the bacterial lag-time, which can extend the detection procedure up to one week. Therefore, the word rapid usually refers only to the molecular detection method, and in order to speed up the total analytical procedure enhanced enrichment cultivation is needed. A large number of rapid detection methods have been reported for Salmonella, and many of them are commercially available. Salmonella screening kits range from complex procedures including

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sophisticated techniques, such as thin agar underlay method or immunomagnetic separation, to simple lateral flow assays incorporating immunochromatographic technology. To date, over thirty alternative Salmonella detection methods have been validated, the majority of them being based on either immunoassays or PCR (Anonymous, 2010a, 2010b, 2010c). High-throughput immunoassays for Salmonella are mainly based on the heterogeneous noncompetitive ELISA format where the target cells are bound between antibodies directed against somatic and flagellar antigens (Cudjoe, Hagtvedt, & Dainty, 1995; Magliulo et al., 2007; ValdiviesoGarcia, Riche, Abubakar, Waddell, & Brooks, 2001). Enriched samples can be analysed by immunoassays directly or after a short boiling step, and methods can be automated, which improves testing efficiency and reduces possibilities for human error. Several nucleic acid hybridization methods have been developed for detecting Salmonella specific rRNA, because the target molecules are abundant in growing bacterial cells and their use ensures that only viable cells are detected (Oerther, Pernthaler, Schramm, Amann, & Raskin, 2000). These methods include e.g., fluorescence in situ hybridization (FISH) (Almeida, Azevedo, Fernandes, Keevil, & Vieira, 2010; Fang, Brockmann, Botzenhart, & Wiedenmann, 2003), DNA-hybridization (Ahn & Walt, 2005; Giannino et al., 2009), and sandwich hybridization (Taskila, Osmekhina, Tuomola, Ruuska, & Neubauer, 2011). Nucleic acid in vitro amplification techniques, such as the predominant DNA polymerase chain reaction (PCR) technology (Saiki et al., 1985), have improved the analytical detection limits further. Tens of Salmonella specific genes have been utilized for different PCR applications, including targets such as rRNA genes, genes coding for toxins and enzymes, and repetitive elements (Levin, 2009). Contamination problems associated with older PCR methods requiring post-amplification manipulations, such as gel electrophoresis or heterogeneous hybridization, have been solved by changing into closed assays formats, which eliminate carryover risks. Technical complexity has been reduced and robustness increased by the introduction of real-time PCR, where amplification is monitored at every reaction cycle by using fluorescent reporter system. Two reporter systems, the DNA binding dye technology (intercalating SYBRÒ Green assay) and the 50 nuclease assay (TaqManÒ chemistry), have been particularly popular in the realtime PCR analysis of Salmonella (Levin, 2009; Postollec, Falentin, Pavan, Combrisson, & Sohier, 2011), but other detection systems such as Scorpion probes (Reynisson, Josefsen, Krause, & Hoorfar, 2006) and Molecular beacons (Chen, Martinez, & Mulchandani, 2000) have also been used for the purpose. Real-time PCR together with enrichment cultivation has been applied for the detection of Salmonella in various food matrices, such as eggs (Chen

et al., 2010; Malorny, Bunge, & Helmuth, 2007), tomatoes (Warren, Yuk, & Schneider, 2007), and different meats (McGuinness et al., 2009; O’Regan et al., 2008). Alternative amplification-based nucleic acid detection methods have also been developed for food-borne Salmonella, including isothermal nucleic acid sequencebased amplification (NASBA) (D’Souza & Jaykus, 2003), and loopmediated isothermal amplification (LAMP) (Ye et al., 2011). In addition to the presented assays based on immunochemistry or nucleic acid detection, other novel approaches have also been used which allow sensitive and specific detection of Salmonella. These include bacteriophage assays that are based either on invasion of phage to the host cells (Chen & Griffiths, 1996; Mosier-Boss et al., 2003), direct and specific lysis of target bacteria by the phages (Favrin, Jassim, & Griffiths, 2001), or using phages in biosensors as recognition elements (Olsen et al., 2006).

2. Enrichment media for Salmonella The enrichment media for food-borne Salmonella can be divided into non-selective and selective media. Since the molecular detection is usually done after cultivation in broth media, the solid media are not described here. The most frequently used nonselective enrichment broth is buffered peptone water (BPW). A universal pre-enrichment broth (UPB) and nutrient broth (NB) are also widely used, although they are not common in the validated alternative methods. The use of the listed media is based on the maximal recovery of small amounts of sub-lethally injured cells (de Boer, 1998). The drawback of the non-selective broths is that they also support the growth of various other microbes. The nonselective broths typically contain only peptones as sources of nitrogen, carbon, vitamin, and minerals. BPW and UPB also contain sodium chloride for maintaining osmotic balance and phosphates for ensuring buffering capacity. In addition to those, UPB contains salts as ion sources and glucose as an energy source. The pH of the non-selective media is initially adjusted near to neutral. A variety of enrichment media have been developed and evaluated for the selective isolation of Salmonella. The most often reported include the RappaporteVassiliadis soy broth (RVS) (Rappaport, Konforti, & Navon, 1956; van Schothorst & Renaud, 1983; van Schothorst & Renaud, 1985), selenite cystine broth (SC) (Leifson, 1939; Silva et al., 2010), tetrathionate broth (TT)(Müller, 1923; Schrank et al., 2001), tetrathionate brilliant green broth (TGB), and the MüllereKauffmann tetrathionate novobiocin broth (MKTTn)(Kauffmann, 1930, 1935; Müller, 1923). Summary of selectivity agents used for the cultivation of Salmonella is presented in Table 1.

Table 1 Summary of the selectivity agents used in the enrichment cultivation of food-borne Salmonella and media in which they are used. Factor

Media

Acriflavine Bile salts Brilliant green

SEL MKTTn, RVS, TGB, TT MKTTn, RVS, TGB

Target microbes

Cycloheximide Fosfomycin Lithium chloride Malachite green Nalidixic acid

SEL SEL SSL RVS

Novobiocin

MKTTn

Gram-positive bacteria Gram-positive bacteria, Gram-negative bacilli Eukaryotic cells Several bacteria Several bacteria Several bacteria Other Enterobacteria, some Gram-negative bacteria Proteus

Potassium tellurite Sodium selenite Tetrathionate

SSL SC MKTTn, TGB, TT

Coliforms and intestine bacteria Gram-positive bacteria, coliforms Coliforms and intestine bacteria

References (Kim & Bhunia, 2008) (Kauffmann, 1930, Kauffmann, 1935; Müller, 1923) (Kauffmann, 1930, Kauffmann, 1935) (Kim & Bhunia, 2008) (Kim & Bhunia, 2008) (Yu et al., 2010) (Rappaport et al., 1956) (Kim & Bhunia, 2008; Yu et al., 2010) (Kauffmann, 1930, Kauffmann, 1935; Restaino, Grauman, Mccall, & Hill, 1977) (Yu et al., 2010) (Leifson, 1939) (Müller, 1923)

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RVS has been shown to be superior in the selective enrichment cultivation of Salmonella in several studies (June, Sherrod, Hammack, Amaguana, & Andrews, 1995; Maijala, Johansson, & Hirn, 1992; Rhodes, Quesnel, & Collard, 1985; Schönenbrucher, Mallinson, & Bülte, 2008; van Schothorst & Renaud, 1983) and it is included in the ISO standard method (Anonymous, 2002a). The selectivity of RVS is based on high concentration of magnesium chloride (decreases water activity, acts as inhibitory compound for other bacteria), low pH and high concentration of malachite green (also inhibits other bacteria). Despite these selectivity agents, some other enteric bacteria are still able to grow in RVS (Krascsenicsova, Kaclikova, & Kuchta, 2006). The enrichment media used in some examples of alternative methods are summarized in Table 2. The reported performances of Salmonella-selective media are controversial. However, following conclusions are obtained in the majority of studies: i) none of the developed media is 100% selective for Salmonella; ii) the selectivity of medium depends on the characteristics and concentration of background microbes (Beckers, vd Heide, Fenigsen-Narucka, & Peters, 1987; Chen, Fraser, & Yamazaki, 1994); iii) a certain minimum concentration of cells is required for Salmonella to survive in the selective conditions (Chen, Fraser, & Yamazaki, 1993); and iv) the performance of selective media may vary depending on the target strain (Besse et al., 2010; Huang, Garcia, Brooks, Nielsen, & Ng, 1999). Currently, a major trend is to develop enrichment media for the simultaneous isolation of several pathogenic bacteria; for example selective enrichment broths SEL and SSL may be used for detection of both Salmonella and Listeria (Kim & Bhunia, 2008; Yu et al., 2010). However, simultaneous detection of pathogens may constitute a risk of overgrowth of certain bacteria (Besse et al., 2010) which can lead to false negative results.

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tightly regulated, it has been reported that the use of case-specific enrichment media could be beneficial (Besse et al., 2010). The factors that should be taken into consideration in selection of the media include the regulations laid by the authorities, the sample matrix, the concentration of the background microbiota in the sample, the concentration and the physiological condition of the target bacteria, and the applied detection method. These are discussed in the following chapters. Additionally, the applicability and the cost-efficiency of the selected enrichment procedure should be at an acceptable level. It is notable that the suitable media may also depend on the target Salmonella strain. 3.1. Regulations The detection of food-borne Salmonella is essential for the safety of the consumers. Therefore, the use and validation of the monitoring procedures are regulated by the authorities. In the EU, the regulations are given by the European Commission (Anonymous, 2005). Due to a low prevalence of Salmonella in Finland and Sweden, each batch of meat, minced meat and eggs imported to Finland and Sweden must be tested for Salmonella using either the ISO reference method (Anonymous, 2002a) or an alternative method that has been compared against the ISO method, validated according to the ISO 16140 method and certified by a third party (Anonymous, 2002b). 3.2. Sample matrix The composition of the enrichment broth and the cultivation conditions should be selected based on the sample matrix. Food components can hinder or slow down the nutrient transfer in the enrichment culture, and thereby affect the growth of bacteria. Food particles may also challenge the pre-processing of the samples, or inhibit the growth of target bacteria. Additionally, the enrichment cultivation may be needed for the removal or dilution of food components that might hamper molecular detection.

3. Selection of the enrichment procedure Although the standard media for the enrichment of foodcontaminating bacteria are widely accepted and in some cases also

Table 2 Summary of the enrichment cultivation broths included in examples of validated alternative methods for the detection of food-borne Salmonella (Anonymous, 2010a, b, c). In case that the enrichment procedure depends on the food type, the procedures for meat/meat products are presented. BPW e Buffered peptone water, RVS e Rappaport Vassiliadis soy broth, BHI e Brain heart infusion broth, TGB e Tetrathionate brilliant green broth. Method (manufacturer)

Immunoassays Oxoid Salmonella Rapid Test (Oxoid Thermo Fisher Scientific) RapidChekÔ SELECTÔ (Strategic Diganostics Inc.) RayAl Salmonella assays (RayAl) RIDASCREENÒ Salmonella (R-Biopharm AG) TAG 24 Salmonella (BioControl Systems) 3MÔ TecraÔ Unique Salmonella (3M Company) TRANSIAÒ Plate Salmonella Gold (BioControl Systems) VIDAS Easy Salmonella (Biomerieux) PCR-based methods ADIAFOOD Salmonella (AES Chemunex) Assurance GDS Salmonella (BioControl Systems) BAX Salmonella (DuPont Qualicon) GeneDisc Salmonella spp. (Pall GeneSystems) iQ-CheckÔ Salmonella II (BIO-RAD) TaqMan Salmonella (Life Technologies Corporation) Hybridization-based methods GeneQuence Salmonella (Neogen Corporation) Lumiprobe 24 Salmonella (EUROPROBE SA) a b c d

Enrichment cultivation procedure broth time [h] temperature [ C] Non-selective

Selective

BPW 18 35-38 16-22 40.5e42.5b BPW 16-20 36-38 BPW 16-20 37 BPWc,d 16-20 36-38 BPW 16-20 36-38 BPW 16-20 36-38 BPW 16-22 36-38

SRTEMa 24 40.5e41.5 6-8 40.5e42.5b RVS 18-24 40.5e42.5 e BHIc 4-5 40.5e42.5 RVS 18-24 40.5e42.5 RVS 18-24 40.5e42.5 SX2 22-26 40.5e42.5

BPW BPW BPW BPW BPW BPW

e BHI 2-4 35-38 e e

16-20 18-24 16-20 16-20 20-22 16-20

36-38 36-38 36-38 36-38 36-38 36-38

NB or BPW 18-24 35 RM 6-8 36-38

Salmonella Rapid Test Elective Medium, included in the test kit. Primary and secsondary enrichment broths utilizing Salmonella specific bacteriophages are included in the test kit. Specific TAG supplement included in the test kit is added. Parallel selective enrichment cultivations in RVS and TBG.

e RVS þ TBGd 22-26 42.5 RVS 17-19 40.5e42.5

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Pre-processing of food samples usually aims to increase the concentration of target bacteria prior to enrichment cultivation (pre-concentration) and change the structure of the sample (homogenization). The increase in the concentration of target cells may shorten the lag-time of the cells in the enrichment culture and hasten the detection, while the removal of food particles allows decreasing the cultivation volume. Pre-processing may also remove inhibitory components that could hamper the detection. Immunomagnetic separation (IMS) can be used directly with samples or after short enrichment cultivations step, depending on the sample matrix and background microbiota. A major advantage of IMS is the possibility for combination of selective separation of Salmonella and subsequent detection assay, which may both increase the sensitivity and specificity, and decrease the duration of molecular assays (Cudjoe et al., 1995; Hagren et al., 2008; Moreira et al., 2008). The interference of magnetic particles with sample components may hamper the use of IMS. Differential centrifugation is suitable for a large variety of samples (McClelland & Pinder, 1994). The use of pre-concentration methods is sometimes hampered due to sample components. Examples include clogging of membranes, co-flocculation of bacteria with sample components in differential centrifugation, or non-specific binding of immunomagnetic beads with other than target proteins (Payne & Kroll, 1991; Stevens & Jaykus, 2004a). When the use of pre-concentration methods is not possible, the concentration of bacteria in the sample remains low and the enrichment is more challenging. In such cases the structure of the sample material may be changed by homogenization. The attachment to the food particles has been shown to improve the survival of Salmonella under stressful conditions (Kinsella, Rowe, Blair, McDowell, & Sheridan, 2007), and homogenization may be needed for the separation of attached cells from the food particles. However, it may also cause excessive elution of PCR inhibiting food components (Kanki et al., 2009). Certain antibacterial components in foods can cause the induction of the so-called viable but not-culturable status of the target bacteria and thereby lead to failure in the enrichment cultivation. For example, the growth of Salmonella has been shown to be inhibited by certain bacteriocins of lactic acid bacteria (Alakomi et al., 2007; Rubin, Nerad, & Vaughan, 1982; Tatsadjieu, Njintang, Sonfack, Daoudou, & Mbofung, 2009). Muniesa, Blanch, Lucena, and Jofre (2005) have shown that the enrichment cultivation of target bacteria can also fail due to bacteriophages in food samples. The inhibitory effects of food components may vary in the sample and in the enrichment broth (McCann et al., 2005), which can lead into false negative results in the detection. The effect of inhibitory compounds can be reduced by sufficient dilution of samples for the enrichment cultivation (Ramnani, Jarvis, & Mackey, 2010). Molecular detection methods may also be inhibited by certain food components. Especially biosensor-based detection tools have been reported to have a low tolerance for interference by contaminating food particles (Gomez et al., 2001; Radke & Alocilja, 2005; Terry, White, & Tigwell, 2005). Several sources report the inhibition of PCR detection by food components (Chen et al., 2010; Rossen, Norskov, Holmstrom, & Rasmussen, 1992; Rådström, Löfström, Lövenklev, Knutsson, & Wolffs, 2003). The common solution is to use internal amplification controls in PCR reactions, which enables the recognition of potentially false negative results due to the presence of inhibitory compounds (Chen et al., 2010). 3.3. Background microbiota Other microbes present in foods can overgrow the target bacteria or inhibit their growth (Thomas & Wimpenny, 1996). For

example, meat products carry lactic acid bacteria that have been shown to inhibit the growth of enteric bacteria (Vold, Holck, Wasteson, & Nissen, 2000). The presence of Gram-negative background microflora can lead to a so-called Jameson effect, in which the growth of minor population is suppressed by other populations. The effect is related to RpoS induction (quorum sensing) and it increases when the redox potential in the culture decreases (Komitopoulou, Bainton, & Adams, 2004). In order to suppress the growth of background microbiota, the selectivity of the medium can be increased by the addition of antibiotics or selective growth factors (Ha, Nisbet, Corrier, Deloach, & Ricke, 1995), or by adjusting the pH and temperature. The use of elevated temperature is a simple way for increasing the selectivity of cultivation (Krascsenicsova et al., 2006; Rhodes et al., 1985; Taskila et al., 2011), but it may also slow down the recovery of injured Salmonella cells. The enrichment cultivation of Salmonella is usually carried out in two-steps, of which the latter takes place in selective media. The reports on the necessity for selective cultivation are controversial. Some detection methods allow the detection of Salmonella directly after cultivation in non-selective media (Chen et al., 2010; Gibbs, Patterson, & Early, 1979; Löfström, Krause, Josefsen, Hansen, & Hoorfar, 2009; Matias et al., 2010; Tatavarthy et al., 2009). However, several case studies have shown that selective enrichment cultivation enhances the accuracy of molecular detection (Myint, Johnson, Tablante, & Heckert, 2006; Upadhyay et al., 2010). In rRNA targeting methods the timing of sampling is crucial due to large variation of the concentration of target molecules between growth phases; the use of over-night culture that has reached the stationary phase is not feasible for the detection (Hsu, Shih, & Zee, 1994). The use of selective media is not advisable if the concentration of the cells is low (Chen et al., 1993). Therefore, a non-selective enrichment cultivation period is usually preferred at the beginning of analysis. On the other hand, the introduction of some selectivity in the non-selective enrichment cultivation has been shown to minimize the overgrowth of the background microbiota. Antibiotics, such as novobiocin or malachite green, can significantly reduce the amount of background bacteria and therefore, benefit the recovery of Salmonella (Jensen, Sørensen, Baggesen, Bødker, & Hoorfar, 2003; van Schothorst & Renaud, 1985). However, the addition of antibiotics at an early stage of enrichment cultivation also causes stress to Salmonella cells, especially in case of sublethally injured cells, and can therefore risk their recovery (Chen et al., 1993). Test kit manufactured by Strategic Diagnostics Inc. (RapidChekÔ SELECTÔ) utilizes selective bacteriophages for the suppression of the background microbiota and thereby improved enrichment cultivation of Salmonella (Stave & Teaney, 2009). Techniques that allow the release of selective supplements in the non-selective culture have been utilized for the cultivation of Salmonella. Sveunm and Harman (1977) used wax-coated gelatin capsules for a gradual release of iodine and selenite into a nonselective broth. So-called solid-repair methods have been used for the two-step recovery of injured bacteria (Kang, 2002). The recovery takes place initially inside or on top of the non-selective agar plate. The selective agents are then diffused to the nonselective agar, changing the cultivation conditions to selective. Disadvantages of solid-repair methods include formation of small colonies that are difficult to pick under the selective medium layer, and the negative effect of the melted selective overlay agar on the injured cells. The direct pour-plating of the sample also incorporates inhibitors present in the food sample into the culture, which may negatively influence the enrichment procedure. Oxoid Ltd. markets a test that incorporates the use of special growth facilitators for an improved recovery of injured cells,

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followed by the timed release of selective agents into the recovery medium. The method has been shown to improve the rate of detection of low numbers of injured salmonellae after 24 h of enrichment cultivation (Baylis, MacPhee, & Betts, 2000a; 2000b).

enrichment cultivation (Hurst, 1977). Tween 80 is a nonionic surfactant and emulsifier, that has been shown to increase the recovery of injured bacteria by repairing damaged cells membranes (Murthy & Gaur, 1987).

3.4. Concentration and condition of target cells

3.5. Environmental stress factors

The concentration and physiological condition of bacterial cells affect their recovery in laboratory media. Harvey and Price (1975) showed that the performance of different medium brands can vary depending on the inoculation concentration of Salmonella. Inoculation concentration also affects the lag-time of Salmonella Typhimurium (Stephens et al., 1997). Food-borne bacteria may be injured due to food processing and handling procedures, such as thermal treatment, refrigeration, freezing, drying, irradiation, from exposure to preservatives, acidity, starvation, and low water activity. The presence of injured cells needs to be considered when enrichment cultivation conditions are selected (Shintani, 2006). Different stress factors can cause large variations in the lag-time between individual cells (Mackey & Derrick, 1984), and when the concentration of cells is low, the effect of stress factors on the bacterial lag-time tends to increase (Shida, Komagata, & Mitsugi, 1975b). It is known, that sublethally injured cells become temporarily susceptible to many selective compounds in the media, and that they do not repair or multiply in the presence of the selective compounds (Busta, 1976). Therefore, the primary recovery of injured cells should take place in non-selective conditions. The enrichment cultivation of Salmonella is usually challenging due to the poor physiological status of the cells. Sub-lethal injury of Salmonella cells has been reported to cause changes in the metabolism leading to for example degradation of ribosomal DNA and RNA (Chambliss, Narang, Juneja, & Harrison, 2006; Gomez, Blais, Herrero, & Sinskey, 1976; Gomez & Sinskey, 1973; Tomlins & Ordal, 1971). This can lead in failure in recovery of the cells, especially on nutrient rich media (Clark & Ordal, 1969; Gomez, Sinskey, Davies, & Labuza, 1973). The tolerance of Salmonella enterica against different stress factors varies between serotypes (Sherry, Patterson, & Madden, 2004). The recovery of injured cells can be improved using repair methods, based on either solid or liquid media. These methods have been recently reviewed in detail (Wu, 2008). A liquid-repair method for Salmonella cultivation reported by Kang and Siragusa (2001) includes a two-fold serial dilution of samples in a 96-well microtiter plate containing BPW, followed by incubation at 37  C for 3 h for resuscitation of sub-lethally injured cells. After that an equal volume of double strength selective broth is added to each well, and the cultures are further incubated for 13 h at 37  C in the dark. Solid-repair methods that are used in the release of selective agents in the non-selective culture (see previous chapters) have been reported to improve the recovery of injured bacteria, including S. typhimurium (Kang & Fung, 2000; Wu & Fung, 2006). The use of growth factors, such as iron supplements in the medium, can improve the recovery of sub-lethally injured cells (Gast & Holt, 1995). Siderophores, high-affinity iron chelating compounds secreted by bacteria, have been shown to improve the recovery of injured bacteria in enrichment cultivation. Ferrioxamines are selective siderophores that have been used for resuscitation of injured Salmonella (Reissbrodt, Heier, Tschape, Kingsley, & Williams, 2000; Reissbrodt, Vielitz, Kormann, Rabsch, & Kuhn, 1996). Some Salmonella cells may even be nonrecoverable without the addition of such sole iron supplement (Thammasuvimol, Seo, Song, Holt, & Brackett, 2006). Since magnesium is necessary for the recovery of injured bacteria, the addition of magnesium sulphate may be beneficial in

The effects of low concentration and poor physiological condition of the target cells on enrichment cultivations are further intensified if the growth conditions in the sample and the media are different (Gomez & Sinskey, 1973; Gomez et al., 1973; Zwietering, Dekoos, Hasenack, deWit, & Vantriet, 1991; Zwietering, deWit, Cuppers, & Vantriet, 1994). The recovery and the lag-time of especially injured bacteria depend largely on the cultivation conditions. Important factors include osmolarity, i.e. the concentration of nutrients and salts (Clark & Ordal, 1969; Shida, Komagata, & Mitsugi, 1975a,b; Chambliss et al., 2006); pH (Chambliss et al., 2006; Shida et al., 1975b); oxygen concentration (Lushchak, 2001); and temperature (Mattick, Jorgensen, Legan, Lappin-Scott, & Humphrey, 2001; Shida et al., 1975b; Zwietering et al., 1994). The stress responses of bacteria can vary between species and even within the species (van de Guchte et al., 2002; Sherry, Patterson, & Madden, 2009). Changes of osmolarity in the medium can cause osmotic stress to bacteria. Osmotic stress can affect both lag-time and growth rate in bacterial cultures (Shida et al., 1975a; Shida et al., 1975b). Viable Salmonella cells tolerate high osmolarity relatively well compared to other Enterobacteriaceae. Therefore, elevated salt concentration may be used to suppress the growth of competing bacteria in enrichment cultivation of food-borne Salmonella (van Schothorst & Renaud, 1983). However, sub-lethal injury of cells reduces the tolerance to high osmolarity (Gomez & Sinskey, 1973), and the use of media with low osmolarity is therefore recommended for the recovery of Salmonella in foods (Clark & Ordal, 1969; Gomez et al., 1973). The addition of nutrients or other growth factors into nonselective medium may have an effect on both recovery and growth of Salmonella (Bailey & Cox, 1992). The selection of suitable initial glucose concentration is somewhat complex since the provided glucose may actually end up accelerating the growth of fast-recovering background microbiota. In our recent study we showed that enzyme controlled release of glucose to BPW medium can facilitate the recovery of heat-injured S. typhimurium in minced meat background (Taskila et al., 2011). The stress caused by a sudden decrease or increase in pH is called acidic or alkaline stress, respectively. Bacteria can tolerate acidic and alkaline stresses by applying various mechanisms (Flahaut, Hartke, Giard, & Auffray, 1997; Humphrey, Richardson, Statton, & Rowbury, 1993), but in the case of enrichment cultivation pH shifts can extend the lag-time of the culture (CheroutreVialette, Lebert, Hebraud, Labadie, & Lebert, 1998), and even cause the death of individual cells. The appropriate pH of the enrichment broth depends therefore on the pH of the sample. Besides during inoculation, pH may also shift during the enrichment cultivation; the metabolites produced by bacteria often decrease the pH in the bacterial cultures. Therefore, the buffering capacity of enrichment medium should be sufficient to slow down the acidification. Oxidative stress is a condition in which the production of reactive oxygen species results in adverse effects on bacteria. Formation of reactive oxygen radicals in the broth media has been shown to affect the recovery of injured bacteria (Mizunoe, Wai, Takade, & Yoshida, 1999). The major enzymes protecting the bacterial cells against reactive oxygen species are superoxide dismutase, catalase, and peroxidases (Morris, 1977). The activity of enzymes involved in degradation of reactive oxygen species in

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bacteria can diminish due to sub-lethal injury, which may explain the toxic effects of relatively low peroxide levels in enrichment media (Andrews & Martin, 1979; Kobayashi et al., 2005). The addition of antioxidants into the enrichment medium has been shown to improve the growth of Salmonella. Antioxidants used in the cultivation of Salmonella include catalase (Rayman, Aris, & Elderea, 1978), sodium puryvate (Rayman et al., 1978), and OxyraseÒ (Niroomand & Fung, 1992). Suitable candidates include at least ascorbic acid (Elli, Zink, Marchesini-Huber, & Renioro, 2002) and thiol compounds (Elli et al., 2002). Thermal stress is caused by a shift in the cultivation temperature. The temperature has been shown to influence both lag-time and growth rate of bacteria (Shida et al., 1975b). The influence may be further increased if the cells are in low concentrations (Robinson et al., 2001; Smelt & Haas, 1978). The effect of the cultivation temperature on the lag-times and growth rates of Salmonella are not expected to largely vary between serotypes (Oscar, 2000), but some difference may occur depending on the sample matrix and enrichment medium (Quintavalla, Larini, Mutti, & Burbuti, 2001). The recovery from thermal stress may relate to other stress responses. According to Kobayashi et al. (2005), the recovery of Salmonella induces the expression of both heat-inducible and oxidative stress related genes. The selection of enrichment medium requires therefore holistic analysis of possible stress factors and their interactions. 3.6. Detection method The performance of enrichment broths is linked to the selected detection method in several ways. The medium components can directly interfere with the detection and therefore the optimal media for the recovery of bacteria may not always be the optimal choice with respect to the subsequent analytical steps. PCR is known to be relatively sensitive to interference, and inhibition by medium components, such as agar or antibiotics, has been reported (Faraq, Gomah, & Balabel, 2010; Gibb & Wong, 1998). Enrichment medium may also affect the detection by decreasing the concentration of marker molecules in the cells. For example, the cultivation of sub-lethally injured Salmonella in unsuitable medium can cause denaturation of certain antigens (Hahm & Bhunia, 2006) and repression of virulence marker genes (Cochrane & O’Connor, 2002). The expression of target genes may directly relate to e.g. osmolarity of media (Pratt, Hsing, Gibson, & Silhavy, 1996). The duration and the number of the enrichment cultivation steps depend on the selectivity and the specificity of the detection method. Due to the need for detecting low levels of food-borne Salmonella and the non-even distribution of the cells in the samples, almost all rapid test protocols include at least one enrichment cultivation step. For example, PCR methods generally require 8e24 h of non-selective enrichment cultivation. Some bacteriophage assays are claimed to allow the detection already after 6 h of cultivation in BPW, which could cause the risk of missing some slowly recovering cells (Beckers et al., 1987; Zhao & Doyle, 2001). Some types of foods may allow the PCR detection directly from the samples (Stevens & Jaykus, 2004b). However, nearly all of the validated alternative methods include enrichment cultivation and take at least 24 h to complete (Anonymous, 2010a; Anonymous, 2010c). In the direct detection without cultivation especially nucleic acid based applications may be prone to produce false results due to partial degradation of ribosomal DNA and RNA in Salmonella cells (Chen & Deutscher, 2010; Deutscher, 2009).

4. Conclusions Enrichment cultivation is a critical step in the detection of foodcontaminating Salmonella. The proper selection of enrichment conditions allows higher specificity and sensitivity of detection, while the use of non-suitable media may lead even to total failure and result in risk to the consumers. Despite controversial reports, the direct detection of Salmonella in food samples is risky, even with the most sensitive methods. The two-step cultivation including recovery in non-selective broth and selective cultivation in more stringent conditions is currently considered as the most feasible procedure for the enrichment of Salmonella. The length of the procedure can be theoretically affected by shortening the enrichment cultivation periods by enabling faster and more efficient recovery, and accelerating the growth of the target cells while suppressing the growth of background microbiota. However, when the workflow in the industrial laboratories is considered, a couple of hours’ suppression is of minor relevance. In the future, the use of automated detection systems that carry out the sample pretreatments during the night could perhaps change this situation. Generally, the over-night cultivation is also practical due to its flexibility; the samples taken at different times during the day are ready for the detection step in the morning all at the same time. The progress in the development of novel Salmonella media has usually focused on the second, selective or indicative enrichment cultivation, while the non-selective enrichment cultivation has been left to be still carried out in BPW. This is probably because the use of commonly accepted enrichment procedure is safe and allows the saving of resources in research and development. The influence of the growth of competing microflora requires further investigation, but further developments in the enrichment cultivation of Salmonella should address in particular the enhanced recovery of injured cells. This could be done for example via screening for components that would inactivate the growth inhibitors present in the cultures. Efforts should be put into development of the total analytical procedure as a whole, instead of just focussing on the features of the chosen detection method. Acknowledgements Prof. Atte von Wright, Prof. Peter Neubauer, Doc. Maria Saarela and Dr. Tadgh O’Sullivan are acknowledged for the useful discussions. The work was financed by EnSTe graduate school and Tauno Tönning foundation. References Ahn, S., & Walt, D. R. (2005). Detection of Salmonella spp. using microsphere-based, fiber-optic DNA microarrays. Analytical Chemistry, 77, 5041e5047. Alakomi, H. L., Puupponen-Pimiä, R., Aura, A. M., Helander, I. M., Nohynek, L., Oksman-Caldentey, K. M., et al. (2007). Weakening of Salmonella with selected microbial metabolites of berry-derived phenolic compounds and organic acids. Journal of Agricultural and Food Chemistry, 55, 3905e3912. Almeida, C., Azevedo, N. F., Fernandes, R. M., Keevil, C. W., & Vieira, M. J. (2010). Fluorescence in situ hybridization method using a peptide nucleic acid probe for identification of Salmonella spp. in a broad spectrum of samples. Applied and Environmental Microbiology, 76, 4476e4485. Andrews, G. P., & Martin, S. E. (1979). Catalase activity during the recovery of heatstressed Staphylococcus aureus Mf-31. Applied and Environmental Microbiology, 38, 390e394. Anonymous. (2002a). ISO 6579:2002 microbiology of food and animal feeding stuffs e Horizontal method for the detection of Salmonella spp. International Organization for Standardization. Anonymous. (2002b). Microbiology of food and animal feeding stuffs. Protocol for the validation of alternative methods (EN ISO 16140). Paris: European Committee for Standardization. Anonymous. (2005). European Commission regulation (EC) No 1688/2005 of 14 October 2005 implementing regulation (EC) No 853/2004 of the European Parliament and of the council as regards special guarantees concerning

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