Simultaneous detection of food safety hazards (Salmonella enterica and Listeria monocytogenes) in table eggs using multiplex PCR

aResearch Scholar, Department of Livestock Products and Technology, Veterinary College, Bengaluru-560024, Karnataka, INDIA bProfessor and Head, Department of Livestock Products and Technology, Veterinary College, Bengaluru-560024, Karnataka, INDIA cAssistant Professor, Department of Veterinary Public Health & Epidemiology, Veterinary College, Shivamogga, 577203, Karnataka, INDIA. dProfessor, Department of Veterinary Public Health & Epidemiology, Veterinary College, Gokula Campus, Vidyanagar, Hassan – 573 201, Karnataka, INDIA. Corresponding author: C. O. Vinayananda | email: vinvet22@gmail.com Co-authors: MNF: prof.nadeem@gmail.com ; CBM: cbmadhava@rediffmail.com ; NSK: pub.nag@gmail.com Received: 19-10-2020, Accepted: 29-11-2020, Published online: 21-12-2020


Introduction
Salmonella enterica and Listeria monocytogenes are the major food safety hazards of public health concern often implicated in foodborne disease outbreaks (Singh et al., 2012). Salmonellosis, caused by Salmonella enterica, a disease of economic significance having implications on the food industry and it is primarily associated with poultry and poultry products. Genus Salmonella comprises of over 2,600 serovars and it is the second largest cause of food poisoning in the world (Gal-Mor et al., 2014); Salmonellae can also be transmitted from animals to humans through food; occasionally, person to person transmission also occurs through the faeco-oral route in addition to prevalent sporadic cases and it also cause severe extra-intestinal infections such as bacteremia and meningitis (Borges et al., 2018). Salmonellae gets transmitted vertically among poultry, has longer persistence in the environment and poultry products are often contaminated with Salmonellae (Afshari et al., 2018). Likewise, L. monocytogenes is a ubiquitous gram-positive bacterium widespread in the environment such as soil, water, animal gut, etc.
L. monocytogenes grows over wide pH (4.39 to 9.40) range, refrigeration temperatures and is associated with listeriosis in human and animals (Singh et al., 2012). Among 17 Listeria species, L. monocytogenes has been identified as the most significant member owing to its pathogenic nature to humans and animals responsible for food-borne infections, meningitis, encephalitis and febrile gastroenteritis (Barbuddhe et al., 2002).
A rapid and sensitive method for detection of these pathogens will be absolutely useful for taking decision at field level about acceptance or the rejection of table egg lots. In the last decade, molecular techniques have appeared as most promising alternatives than conventional cultural methods in food microbiology. Conventional culture methods require series of procedure which consumes more time and are laborious. In such cases, use of molecular-based techniques thwart the delay and permits to detect the pathogens with greater sensitivity and reliability than conventional culture methods (Germini et al., 2009;Rattanachaikunsopon and Phumkhachorn, 2012). A rapid and sensitive assay like polymerase chain reaction (PCR) based method for identification of the food borne pathogens is proven and even several pathogens can be detected in a single reaction by multiplex PCR or mPCR (Soumet et al., 1999). Therefore, present study was designed with the objectives of simultaneous detection of S. enterica and L. monocytogenes using mPCR assay targeting invA and prfA genes respectively and comparison of mPCR with that of conventional method.

Materials and Methods
Sample collection: Table egg (360) were collected from different markets comprising of commercial layer farms (240) and backyard rearing (120) systems. Egg samples were collected from retail markets into sterile polybags and transported to the laboratory immediately. Samples were collected on four occasions at an interval of 15 days and analyzed for S. enterica and L. monocytogenes using the multiplex PCR.
Sample preparation: Pool of ten eggs selected from each batch were surface sterilized with 70% ethanol, broken under aseptic conditions and whole egg contents were mixed in bag for 3 minutes using stomacher (Bagmixer ® , Interscience). Ten milliliters of resultant egg homogenates were then transferred into two separate sterile polybags containing 90 milliliters of buffered peptone water (BPW) or half Fraser Broth (hFB) for the primary enrichment (1:9) of S. enterica or L. monocytogenes, respectively. Bags containing BPW or hFB were mixed well in stomacher for 4 minutes and incubated at 37 ºC and 30 ºC for 24 hours for Salmonella and Listeria, respectively. DNA isolation from primary enrichment: DNA was extracted from pre-enriched samples using snap chill method (Manoj et al., 2014). Briefly, 2 mL preenriched broth (BPW or hFB) were centrifuged at 6,000 rpm for 10 minutes; resultant pellet containing bacterial cells was washed with sterile phosphate buffered saline and re-suspended in 50 µL nuclease free water. After boiling for 100 °C for 10 minutes, putative suspensions were subjected for one freezethaw cycle (-20 °C for 15 min.). Thawed suspension was centrifuged at 6,000 rpm for 10 minutes and supernatant collected into a fresh tube was used as template DNA for PCR. invA (invasion 'A' gene) of S. enterica (Rahn et al., 1992) and prfA (transcriptional activator of virulence factor) of L. monocytogenes (Wernars et al., 1992) as described by Germini et al., (2009) with minor modifications.

Isolation and identification of S. enterica and L. monocytogenes:
Samples showing positive by mPCR amplicons from the primary enrichment were subjected for conventional cultural isolation of S. enterica species and L. monocytogenes as per standard protocols (ISO 6579:2002 andISO 11290:1998). Identification of S. enterica species and L. monocytogenes was carried out using a battery of biochemical tests (Blodgett, 2010).

Results and Discussion
Presence of S. enterica and L. monocytogenes was detected based on the amplification of the 284 and 217 base pairs PCR products in Salmonella and Listeria species, respectively (Figure 1). Among the table egg samples screened using the mPCR, Salmonella and Listeria were detected in 19.4 and 2.7 per cent, respectively. Distribution of these food safety hazards among different categories of table egg samples is given in Table 1. Occurrence of S. enterica was highest in backyard eggs followed commercial table eggs. Primary enrichment samples of commercial eggs were found negative for L. monocytogenes using prfA gene based mPCR. However, one sample of backyard eggs showed amplification for prfA gene.
Use of cultural media and methodology for detection and isolation of pathogens will vary and even sensitivity is also low and difficult to detect non-viable cells or injured cells of pathogens by cultural method, but they may have potential to recover and grow when the food is consumed, therefore molecular assay may be more appropriate. Several simplex or multiplex PCR assays have been developed for the rapid detection of of Salmonella and Listeria species using specific primers (Germini et al., 2009;Manning et al., 2015;Liu et al., 2015;Afshari et al., 2018;Heymans et al., 2018;Dzieciol et al., 2016). Of the several amplification targets used for detection of Salmonella such as agfA, fimA, viaB, fliC-d, virulence-associated plasmids, etc the invA gene has been one of the most widely gene targets for the specific detection of Salmonellae (Phumkhachorn and Rattanachaikunsopon, 2017;Afshari et al., 2018;Heymans et al., 2018;Borges et al., 2018). Likewise, L. monocytogenes could be specifically detected using virulence factors such as actin polymerization protein (actA), phosphatidylinositol phospholipase C (plcA), hemolysin (hlyA) and invasive associated protein (iap) genes that are involved in the pathogenesis (Liu et al., 2015;Dzieciol et al., 2016); of these, prfA has been the accepted target for the specific detection of L.  (Wernars et al., 1992).
The mPCR adopted in the present study was found to detect both pathogens simultaneously and the results of the mPCR were in complete concordance with the conventional cultural methods. However, separate primary enrichment broth for Salmonella and Listeria monocytogens is preferred over universal primary enrichment medium while screening for pathogens in food samples using mPCR, where Salmonella will predominant over the Listeria monocytogens multiplication irrespective of initial counts (Jofre et al., 2005).
Similar studies by other investigators could detect few pathogen (Nagappa et al., 2007;Gole et al., 2013, Vinay et al., 2016Rajashekhara et al., 2017) or have shown discrepancies (Al-Obaidi et al., 2011;Safaei et al., 2011;Paul et al., 2016). The mPCR employed in the present study with minor modification was found reliable for the simultaneously detection of two major food safety hazards in table eggs with an anticipation of food safety compliance. Keeping in view, the limitations of laborious and time consuming conventional cultural methods, results of the present study support to propose application of mPCR is for the rapid and specific detection of S. enterica and Listeria monocytogenes in table eggs. Nevertheless, multi-centric validations involving large samples are warranted for the wide scale acceptance of results.

Conclusions
The applications of PCR have revolutionized the molecular diagnostics including detection of food safety hazards. In the present study, mPCR assay adopted for the rapid and simultaneous detection of S. enterica and L. monocytogenes in table egg contents. The results of the mPCR were in complete concordance with the conventional cultural methods. Therefore, the mPCR assay has been proposed as alternative tool for the rapid and simultaneous detection of S. enterica and L. monocytogenes in table eggs for the purpose of food safety compliance testing of samples.