Gilles Salvat

Listeria monocytogenes in pork slaughtering and cutting plants: use of RAPD, PFGE and PCR–REA for tracing and molecular epidemiology

In order to determine the origin of pork cuts contamination by Listeria monocytogenes, 287 isolates, collected from five French pork slaughtering and cutting plants, from live pigs to pork cuts, were characterised using three molecular typing methods: random amplification of polymorphic DNA (RAPD) carried out with five different primers, genomic macrorestriction using ApaI with pulsed-field gel electrophoresis (PFGE) and a PCR-restriction enzyme analysis (PCR-REA) based on the polymorphism existing within the inlA and inlB genes. Results obtained from RAPD and PFGE were closely related and distinguished respectively 17 RAPD types (r1-r17) and 17 PFGE types (a1-a17) among the 287 isolates, whereas the PCR-REA analysis only yielded two profiles (p1 and p2). Considering the combined results obtained with the three molecular typing methods, 19 Listeria monocytogenes genotypes (1-19) were distinguished. Serotyping led at least four serotypes being distinguished: 1/2a, 3a, 1/2c and 3c. The application of genotyping identified the predominance of a Listeria monocytogenes strain of type (1) and other very closely related ones (5, 9, 10, 12, 13, 14, 16 and 19) which were present on pork as well as in the environment within the five investigated plants. This study also pointed out the presence of these closely related Listeria monocytogenes strains over a 1-year period in the environments of two plants, even after cleaning and disinfection procedures. This highlights the possibility for some Listeria monocytogenes strains to persist in pork processing environments and raises the problem of the efficiency of cleaning and disinfection procedures used in pork slaughterhouses, chilling and cutting rooms.

Molecular epidemiology of Listeria monocytogenes isolates collected from the environment, raw meat and raw products in two poultry‐ and pork‐processing plants

Aims: In order to study the transmission of Listeria monocytogenes in a poultry and a pork meat plant, we analysed the contamination by this pathogen over several months.

Campylobacter contamination in French chicken production from farm to consumers. Use of a PCR assay for detection and identification of Campylobacter jejuni and Camp. coli.

Aims: Campylobacter contamination in French chicken production from the farm to the consumer was determined using a PCR assay for bacteria detection and identification.

Epidemiological analysis of Salmonella enterica ssp. enterica serovars Hadar, Brancaster and Enteritidis from humans and broiler chickens in Senegal using pulsed‐field gel electrophoresis and antibiotic susceptibility

Aims:  Salmonella Hadar, Salmonella Brancaster and Salmonella Enteritidis are the main Salmonella enterica ssp. enterica serovars isolated from poultry in Senegal. Our objective was to analyse the pulsed‐field gel electrophoresis (PFGE) and antibioresistance patterns of strains belonging to these serovars and to assess the significance of broiler‐chicken meat as a source of human infection.

Tracing of Salmonella spp. in two pork slaughter and cutting plants using serotyping and macrorestriction genotyping.

I. GIOVANNACCI, S. QUEGUINER, C. RAGIMBEAU, G. SALVAT, J. L. VENDEUVRE, V. CARLIER AND G. ERMEL. 2001.

Campylobacter contamination of broiler caeca and carcasses at the slaughterhouse and correlation with Salmonella contamination

In order to estimate the prevalence of Campylobacter spp. and Salmonella spp. on broiler chicken carcasses and the prevalence of Campylobacter spp. in caeca, 58 French slaughterhouses were investigated in 2008. Enumeration of Campylobacter spp. was also performed in order to study the relation between caeca and carcass contamination. A pool of 10 caeca and one carcass were collected from 425 different batches over a 12-month period in 2008. Salmonella was isolated on 32 carcasses leading to a prevalence of 7.5% ([5.0-10.0](95%CI)). The prevalence of Campylobacter was 77.2% ([73.2-81.2](95%CI)) in caeca and 87.5% ([84.4-90.7](95%CI)) on carcasses. No significant correlation was found between Campylobacter and Salmonella. Positive values of Campylobacter were normally distributed and the average level was 8.05 log(10) cfu/g ([7.94-8.16](95%CI)) in caeca and 2.39 cfu/g ([2.30-2.48](95%CI)) on carcasses. A positive correlation (r = 0.59) was found between the mean of Campylobacter in caeca and on carcasses (p < 0.001). Thus, carcasses from batches with Campylobacter-positive caeca had significantly (p < 0.001) higher numbers of Campylobacter per gram than batches with negative caeca. These results show that Campylobacter can be present in both matrices and reduction in caeca could be a possible way to reduce the amount of bacteria on carcasses. Of the 2504 identifications performed, 3 species of Campylobacter (Campylobacter jejuni, Campylobacter coli and Campylobacter lari) were identified. The main species recovered were C. jejuni and C. coli, which were isolated in 55.3% and 44.5% of positive samples, respectively. These two species were equally represented in caeca but C. jejuni was the most frequently isolated on carcasses with 57.1% and 42.5% of positive carcasses for C. jejuni and C. coli, respectively. This study underlines that target a reduction of Campylobacter on final products requires a decrease of contamination in caeca.

Campylobacter transfer from naturally contaminated chicken thighs to cutting boards is inversely related to initial load.

Foods prepared in the kitchen can become cross-contaminated with Campylobacter by contacting raw products, particularly skinned poultry. We measured the percent transfer rate from naturally contaminated poultry legs purchased in supermarkets. Transfer of Campylobacter from skin (n = 43) and from meat (n = 12) to high-density polyethylene cutting board surfaces was quantitatively assessed after contact times of 1 and 10 min. The percent transfer rate was defined as the ratio between the number of Campylobacter cells counted on the cutting board surface and the initial numbers of Campylobacter naturally present on the skin (i.e., the sum of Campylobacter cells on the skin and board). Qualitative transfer occurred in 60.5% (95% confidence interval, 45.5 to 75.4) of the naturally contaminated legs studied and reached 80.6% (95% confidence interval, 63.0 to 98.2) in the subpopulation of legs that were in contact with the surface for 10 min. The percent transfer rate varied from 5 x 10(-2)% to 35.7% and was observed as being significantly different (Kruskall-Wallis test, P < 0.025) and inversely related to the initial counts on poultry skin. This study provides quantitative data describing the evolution of the proportion of Campylobacter organisms transferred from naturally contaminated poultry under kitchen conditions. We emphasize the linear relationship between the initial load of Campylobacter on the skin and the value of the percent transfer rate. This work confirms the need for modeling transfer as a function of initial load of Campylobacter on leg skin, the weight of poultry pieces, and the duration of contact between the skin and surface.

Physicochemical surface properties of five Listeria monocytogenes strains from a pork-processing environment in relation to serotypes, genotypes and growth temperature

Physicochemical surface properties, related to electrostatic, van der Waals and Lewis acid–base interactions, of five Listeria monocytogenes strains isolated from pork‐processing environments were determined after two subcultures at 37 °C and a final culture at three temperatures: 37, 10 and 4 °C. Three strains (Lm1, Lm114 and Lm191) were genetically related while two were unrelated (Lm25 and Lm74) according to ApaI‐macrorestriction and pulsed‐field gel electrophoresis (PFGE) typing.

Colonization and organ invasion in chicks experimentally infected with Dermanyssus gallinae contaminated by Salmonella Enteritidis

The poultry red mite (Dermanyssus gallinae) is the most important and common ectoparasite of laying hens in Europe. This haematophagous mite has been experimentally demonstrated to be a vector of Salmonella Enteritidis by acquiring bacteria through the blood meal or cuticular contact. We have evaluated another route of infection by orally inoculating chicks with mites previously infected by S. Enteritidis. Two methods of infecting the mites were tested: mites contaminated by cuticular contact or during the blood meal. After the washing of mites with paraformaldehyde, groups of 10 Salmonella-contaminated mites were inoculated individually into 1-day-old chicks. The titre of the inoculum suspension was evaluated by crushing mites and followed by bacteriological counting. It was 3×104 colony-forming units/chick and 2.7×106 colony-forming units/chick, respectively, for cuticular contact and orally mediated contamination of mites. Each bird was found to be positive 12 days post-inoculation. Salmonella colonized the intestinal tracts and invaded the livers and spleens. The caecal content concentration reached a mean level of S. Enteritidis of 8.5×104 most probable number (MPN) Salmonella/g. This experiment demonstrated the ability of mites to orally infect 1-day-old chicks with subsequent colonization and multiplication of Salmonella. Consequently, mites infected by S. Enteritidis constitute potential reservoir hosts of this bacterium, allowing it to persist in the poultry house as a source of infection for newly introduced animals. If contaminated mites are found in poultry facilities, effective red mite control should be performed before new batches are introduced into the facility.

Use of pulsed-field gel electrophoresis to characterize the heterogeneity and clonality of Salmonella serotype Enteritidis, Typhimurium and Infantis isolates obtained from whole liquid eggs

Salmonella is a well-documented pathogen known to occur in a wide range of foods, especially poultry products. The most frequently reported food-sources of human infection are eggs and egg products. In this study, in order to describe Salmonella contamination of egg products, 144 liquid egg samples were collected from 3 different egg-breaking plants during the 3 sampling periods. Salmonella detection was performed on raw samples stored at 2 degrees C for 2 days (D+2) and on pasteurised samples stored at 2 degrees C at D+2 and at shelf-life date. Salmonella was detected in 130 of the 144 raw egg samples collected and in 11 of the 288 pasteurised egg samples analysed. 740 Salmonella isolates were collected and serotyped: 14 serovars were demonstrated. A great diversity, particularly during summer, was noted. The dominant serovars were S. Enteritidis, S. Typhimurium and S. Infantis, mainly found in whole raw egg products. Typing of 325 isolates of S. Enteritidis, 54 isolates of S. Typhimurium and 58 isolates of S. Infantis was carried out by macrorestriction of the genomic DNA with XbaI and SpeI enzymes followed by pulsed field gel electrophoresis (PFGE). The Salmonella Enteritidis isolates could be grouped into 3 clusters. Cluster 1 was predominant at all 3 egg-breaking companies during the different sampling periods. This cluster seemed to be adapted to the egg-breaking plants. Cluster 2 was linked to plant 1 and cluster 3 to plant 3. Two main clusters of Salmonella Typhimurium were demonstrated. Cluster A was mainly found at plant 2 during autumn. Plant 3 was contaminated by all the Salmonella Typhimurium genotypes but in a more sporadic manner during the three seasons studied. Plant 1 seemed to be less contaminated by Salmonella Typhimurium than the others. Three clusters and 2 genotypes of Salmonella Infantis were shown. The main cluster, cluster alpha, consisted of 75% of the S. Infantis isolates and was mainly found during summer at plants 1 and 3. Plant 2 seemed to be less contaminated by S. Infantis. In this study, molecular typing demonstrated that, although certain clusters were common to all three companies, specific clusters, notably of S. Enteritidis were present at each plant.