THE PRESENCE OF LISTERIA SPP . AND LISTERIA MONOCYTOGENES IN A CHOSEN FOOD PROCESSING ESTABLISHMENT IN SERBIA

The aim of the study was to establish a protocol to evaluate the presence of Listeria spp. in food processing environments. The presence of Listeria spp. was evaluated in a selected restaurant in Serbia on three occasions. Samples were collected from 47 sampling spots in the commercial kitchen equipment and environment. The presence of Listeria spp. and Listeria monocytogenes were detected by conventional culture methods and by the PCR method. The obtained results showed that 23 swab samples were positive for Listeria spp. Interestingly, the swabs from the bread-cutting board and meat defrosting sink were positive for L. monocytogenes.


INTRODUCTION
Listeria spp.are ubiquitous in nature and can be isolated from soil, vegetables and natural waters as well as from healthy animals and man (Roberts and Wiedmann, 2003).Listeria monocytogenes is the most pathogenic species of this genus, although infections mainly occur in neonates, pregnant women, the elderly and immunocompromised individuals (McLauchlin, 1997).The primary mode of transmission of L. monocytogenes to humans is the consumption of contaminated, minimally processed food (Schlech, 2000;Kathariou, 2002;Shen et al., 2006).Accessibility to the public and relatively limited control interventions at food service establishments and the lack of a specific regulatory framework increase the likelihood of introduction of this pathogen into some foods in these establishments.
Various studies have indicated that certain strains of L. monocytogenes survive well within the food-processing environment (Kathariou, 2002;Tompkin, 2002;Pan et al., 2006) and the persistence of such strains is of concern as they have the potential to act as a continual source of contamination (Pan et al., 2006).Moreover, the detection of nonpathogenic Listeria spp.can be considered as a useful indicator of a deterioration in hygiene or process conditions during food production (PHLS, 2000), leading to an increased risk of contamination with pathogenic Listeria spp.Therefore, the detection of all Listeria spp. is necessary when testing food and environmental samples.
Advances in molecular technologies, particularly the PCR methodology, have allowed reliable microbial identification and surveillance.PCR techniques are sensitive, highly specific and allow rapid processing times, and they also enhance the likelihood of detecting Listeria spp.without the need for isolating pure cultures (Aznar & Alarcón, 2003;Cocolin et al., 2002).Moreover, PCR methods have been applied successfully to the detection and identification of pathogenic organisms in clinical and environmental samples (Simon, 1999;Soumet et al., 1994;Kaur et al., 2007).
Since studies on the prevalence of Listeria spp. in food processing establishments in Serbia are lacking, the aim of the present study was to establish a molecular protocol to evaluate the presence of Listeria spp.The samples were analyzed by either culture method or by PCR.

Bacterial strains and growth conditions
The standard strains of Listeria monocytogenes 4b ATCC 19115 and Listeria innocua ATCC 33090 were obtained from the American Type Culture Collection (ATCC; Manassas, Va., USA).Standard strains were grown on brain-heart agar (BHA) (Merck, GmbH Darmstadt, Germany) and buffered peptone water (Merck) at 37°C.

Samples
For the analysis, 141 environmental samples from 47 sampling areas in a restaurant in Belgrade, Serbia, were selected.The samples were taken from different locations (goods reception area, raw material storage, food processing area) from drains, floors and food contact surfaces, including the equipment used to transport (carts), store (display case, coolers, freezers), or prepare food (slicers, knives, choppers, cutting boards, strainers).Samples (10 x 10 cm) were collected by using sterile commercial cotton swabs, on three independent visits.Whenever possible, swabs were collected from the same sites on each visit.All samples were kept refrigerated and analyzed within 2 h.

Enrichment and isolation of Listeria spp. from environmental swabs
The conventional culture method was carried out by two-step enrichment (Anonymous, 1996).For primary enrichment, commercial cotton swabs were homogenized in 20 ml of half-concentrated Fraser broth and incubated at 30°C for 24 h, followed by a secondary enrichment.After that the primary enrichment broth (100 μl) was transferred into 10 ml of Fraser broth and incubated for a further 48 h at 37°C.After 48 h, a loopful of the enrichment broth culture was plated onto polymyxin-acriflavine-lithium chlorideceftazidime-aesculin-mannitol (PALCAM) (Merck) and OXFORD (Oxoid, UK) agar plates, and incubated for 48 h at 37°C.For each sample Listeria presumptive colonies were tested for catalase and oxidase reactions, Gram staining and motility.

Biochemical identification
Hemolysis was assessed by streaking isolated colonies on 5% sheep blood agar (Base: meat peptone 15 g l -1 , liver digest 2.5 g l -1 , yeast extract 5 g l -1 , sodium chloride 5 g l -1 , agar 9 g l -1 .To 100 ml base, 5 ml of defibrinated sheep blood was added).After 24 h of incubation at 37°C the plates were examined for the presence of a zone of hemolysis.Isolates that were small, Gram-positive rods, catalase positive, oxidase negative, with motility, were further identified by using the API-Listeria identification system (BioMerieux, France), and confirmed by PCR analysis.

DNA extraction
DNA was extracted by using the DNeasy Tissue Kit (Qiagen GmbH, Germany) according to the manufacturer's protocol for Gram-positive bacteria.

Sequencing of 16S rDNA amplicons
The 16S rDNA amplicons were purified by QIAquick PCR Purification KIT/250 (QIAGEN GmbH, Hilden, Germany), and sequenced by CRIBI-BMR Genomics Service sequencing of DNA (University of Padua, Italy).The BLAST algorithm was used to determine the close-matching sequence relatives in the NCBI nucleotide sequence database (http://www.ncbi.nlm.nih.gov/BLAST).

RESULTS
In this study, 141 environmental samples were taken from 47 sampling spots in a restaurant in Belgrade on three independent visits.The samples were analyzed by either culture method or by PCR.Results obtained by culture method showed that during the first visit 4 out of 47 swabs were positive for the presence of Listeria spp.(Table 1).
The results showed that the same sample S2-08-1/II was positive for the presence of Listeria spp. in direct identification from the swab samples, isolated during the second visit (Table 1).According to the length of the PCR products obtained with List-univ.1/List-univ.2 primers, it could be concluded that the sample contained two different Listeria spp., identified as L. innocua and L. welshimeri (Fig. 4).The results of the PCR analyses by using LM1/LM2 primers confirmed that there were no L. monocyto-genes on the swab samples taken during the second visit (Table 1).
Results obtained by culture method showed that during the third visit 5 out of 47 swabs were positive for the presence of Listeria spp.(Table 1).L. welshimeri was detected in three samples isolated from the floor (S2-05-1/III) and drain (S2-08-1/III and S3-05-2/III), and L. innocua was identified in the sample isolated from the corner of the wall (S2-06-2/III).The results of the PCR analyses showed the presence of L. innocua in all samples (Table 1).Moreover, L. monocytogenes was identified in the sample isolated from the meat defrosting sink (S3-01-2/III), by either culture method or PCR analyses (Fig. 1).
The results of the direct PCR detection by using List-univ.1/ List-univ.2 primers from the swab samples taken during the third visit showed the presence of Listeria spp. in 11 samples.Moreover, the results of the PCR analyses by using LM1/LM2 primers showed the presence of L. monocytogenes in samples isolated from the drain (S2-08-1/III), meat defrosting sink (S3-01-2/III) and floor drain (S3-05-2/III) (Table 1).

DISCUSSION
The detection and identification of Listeria spp.have attracted the attention of many authors.For the purposes of public health protection, it is important to recover nonpathogenic Listeria spp., as these act as markers for the likelihood of the presence of L. monocytogenes and allow preventive action to be taken.
In total, 23 samples were identified as positive for the presence of Listeria spp.(Table 1).Out of them, 17 samples were found to be positive for the presence of Listeria spp. in direct identification from the swab samples.Among them, Listeria spp. was most commonly isolated from the drains and floors, as was shown previously.The results of Gudmundsdóttir et al. (2005) suggested that the cleaning procedures were not sufficient to eliminate cross-contamination from drains and floors and that the in-house flora represents a potential health risk.Several other studies have concluded that the plant equipment and the processing environment (in-house flora) rather than the raw material is the source of L. monocytogenes (Rørvik et al., 1995;Autio et al., 1999;Giovannacci et al., 1999;Miettinen and Wirtanen, 2006).However, this does not exclude the possibility that the raw material may be an important, initial source for contaminating the processing equipment and environment (Vogel et al., 2001).This is in concordance with our findings that the swabs from the bread-cutting board and meat defrosting sink were positive for the presence of L. monocytogenes.Furthermore, definitive identification, revealed by both culture and PCR methods, was obtained for 10 samples, and all but two samples were correctly identified.One discordant sample (S3-05-2/I) was misidentified as L. monocytogenes by PCR assay by using primers complementary to the iap gene (Fig. 2).On the other hand, the results of the culture method showed that it could be L. innocua, thus underscoring the biases of traditional methods.The results obtained by PCR using List-univ.1/List-univ.2 primers showed that the other discordant sample (S2-02-2/I), misidentified by culture method as L. welshimeri, belonged to L. innocua (Fig. 2).These results underscore the reliability and accuracy of the PCR method.Finally, ambiguous identification was resolved by direct sequencing of the 16S rRNA and nucleotide sequence analysis revealed homology with L. innocua (92%).The obtained results suggest the need to use combined methods in order to obtain accurate identification of Listeria spp.

Table 1 .
Natural swabs, positive for the presence of Listeria spp., collected during three visits