Philippe Fravalo

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.

Chicken Caecal Microbiome Modifications Induced by Campylobacter jejuni Colonization and by a Non-Antibiotic Feed Additive

Campylobacter jejuni is an important zoonotic foodborne pathogen causing acute gastroenteritis in humans. Chickens are often colonized at very high numbers by C. jejuni, up to 109 CFU per gram of caecal content, with no detrimental effects on their health. Farm control strategies are being developed to lower the C. jejuni contamination of chicken food products in an effort to reduce human campylobacteriosis incidence. It is believed that intestinal microbiome composition may affect gut colonization by such undesirable bacteria but, although the chicken microbiome is being increasingly characterized, information is lacking on the factors affecting its modulation, especially by foodborne pathogens. This study monitored the effects of C. jejuni chicken caecal colonization on the chicken microbiome in healthy chickens. It also evaluated the capacity of a feed additive to affect caecal bacterial populations and to lower C. jejuni colonization. From day-0, chickens received or not a microencapsulated feed additive and were inoculated or not with C. jejuni at 14 days of age. Fresh caecal content was harvested at 35 days of age. The caecal microbiome was characterized by real time quantitative PCR and Ion Torrent sequencing. We observed that the feed additive lowered C. jejuni caecal count by 0.7 log (p<0.05). Alpha-diversity of the caecal microbiome was not affected by C. jejuni colonization or by the feed additive. C. jejuni colonization modified the caecal beta-diversity while the feed additive did not. We observed that C. jejuni colonization was associated with an increase of Bifidobacterium and affected Clostridia and Mollicutes relative abundances. The feed additive was associated with a lower Streptococcus relative abundance. The caecal microbiome remained relatively unchanged despite high C. jejuni colonization. The feed additive was efficient in lowering C. jejuni colonization while not disturbing the caecal microbiome.

Prevalence of Listeria monocytogenes in Poultry Production in France

This study aimed to update and create a data set from laying hens and broilers regarding contamination by Listeria monocytogenes. Two hundred laying-hen flocks were sampled, with 88 flocks reared in cages and 112 reared on the floor. One hundred forty-five broiler flocks were sampled, with 85 conventional and 60 free-range flocks. A total of 774 and 725 samples were analyzed from laying hens and broilers, respectively. L. monocytogenes was detected in 31 of 200 flocks, yielding an estimated prevalence of 15.5% in laying-hen flocks. Among positive flocks, there appeared a significant (P = 0.004) difference between caged and floor-reared hens, with a higher detection in dust samples from floor-reared hens. In positive caged hen flocks, significant (P = 0.028) differences between dust and fecal samples appeared, with a higher detection in feces than in dust samples. In broiler flocks, L. monocytogenes was isolated in 46 of 145 flocks, yielding an estimated prevalence of 32% (28% in conventional flocks versus 37% in the free-range flocks). L. monocytogenes was isolated in samples taken from conventional flocks with a lower frequency than in free-range flocks (13 versus 18%, respectively). The serotyping of L. monocytogenes strains showed that the majority belonged to type 1/2a in laying-hen flocks (74.3%) and in broiler flocks (40.5%). A significant difference (P = 0.007) between laying hens and broilers was shown for serogroup 4 and for serovar 1/2b (P = 0.007); these serogroups were more prevalent in broilers (40%) than in laying hens (5.7%).

Isolation of Salmonella enterica in Laying-Hen Flocks and Assessment of Eggshell Contamination in France

The present investigation was conducted in conjunction with the European Union baseline study for the estimation of Salmonella prevalence in laying-hen flocks. It aimed at evaluating eggshell contamination in farms positive for Salmonella, characterizing the genetic patterns of Salmonella strains and identifying the factors associated with Salmonella contamination of eggshells. For this purpose, a total of 4,200 eggs were collected from 28 positive flocks and analyzed according to draft Annex D of International Organization for Standardization Method 6579. Molecular characterization of the Salmonella strains was obtained by the pulsed-field gel electrophoresis method with two restriction enzymes, XbaI and BlnI. The relationship between the presence of Salmonella on eggshells and rearing practices was studied by using multiple correspondence analysis. Results showed that 39.3% of the positive flocks had at least one positive eggshell, with a total of 1.05% of eggshells testing positive for Salmonella. We detected the same serovars on samples taken from the farm and from eggshells within a given flock, with isolates sharing the same genetic pattern in 7 of 11 flocks. Eggshells tested positive for Salmonella in flocks (i) located where delivery trucks pass near air entrances of the poultry house, (ii) with high holding capacity (>30,000 laying hens), and (iii) with more than five positive samples coming from the farm environment, as well as in cases of flocks with a maximum egg-laying rate of >96% and in cases where farmers worked in other animal production. This study provided valuable information that could be used for risk management and risk assessment studies.

Genetic diversity of group A rotavirus in swine in Canada

Abstract Group A rotaviruses (RVA) in pigs have been poorly investigated in Canada. In a continued effort to fill this gap, ten finisher swine farms in Quebec, Canada, were sampled over a nine-month period. The presence of RVA was detected in healthy pigs on all farms investigated during the entire sampling period. The genotypes detected included G2, G5, G9 and G11; P[6], P[7], P[13], P[27] and P[34]; and I5 and I14. The predominant types were G2, P[13] and I5, which is different from previous global reports. Various fomites were consistently contaminated by RVA, suggesting that a resident viral flora remains in the farm environment and may play a role in the infection of incoming pigs. The results also suggest temporal or geographical specificities regarding strain distribution on pig farms.

Analysis of Listeria monocytogenes Strain Distribution in a Pork Slaughter and Cutting Plant in the Province of Quebec

Following the 2008 Canadian listeriosis outbreak associated with ready-to-eat (RTE) meat products, regulations on the presence of Listeria monocytogenes in RTE food production facilities were modified by Health Canada, confirming the need to control this pathogen, not only in the final product but also in the plant environment. Information on the occurrence of this microorganism during the early steps of production, such as the slaughtering process and in the cutting area, is scarce in Canada. In this study, we sampled different production steps in a slaughtering and cutting plant in the province of Quebec over a 2-year period. The lairage pens, representative areas of the slaughter line, and cutting zones were targeted after their respective cleaning procedures. A total of 874 samples were analyzed for the presence of L. monocytogenes. Characterization was done by first genoserogrouping the isolates using multiplex PCR and then using a pulsed-field gel electrophoresis approach. L. monocytogenes was detected throughout all production stages. The 108 positive samples found were analyzed further, and we established that there were 4 different serogroups, with serogroup IIb being the most prevalent. The results of pulsed-field gel electrophoresis analysis showed a significant decrease in the diversity of strains from the first areas of the plant to the cutting room (10 pulsotypes in 13 positive samples in lairage and 9 in 86 positive samples in cutting) and also showed the overrepresentation of a single predominant strain in the cutting room environment (type 1, representing 96.1% of the isolates). Biofilm formation analysis of the strains cannot exclusively explain the transitions we observed. A strong genotypic similarity between strains isolated in the early production areas and some strains in the cutting room was shown. These results support the need for better surveillance of L. monocytogenes prior to RTE food production in order to design control strategies that are better adapted from a public health perspective.

Extensive characterization of Campylobacter jejuni chicken isolates to uncover genes involved in the ability to compete for gut colonization

BackgroundCampylobacter jejuni is responsible for human foodborne enteritis. This bacterium is a remarkable colonizer of the chicken gut, with some strains outcompeting others for colonization. To better understand this phenomenon, the objective of this study was to extensively characterize the phenotypic performance of C. jejuni chicken strains and associate their gut colonizing ability with specific genes.ResultsC. jejuni isolates (n = 45) previously analyzed for the presence of chicken colonization associated genes were further characterized for phenotypic properties influencing colonization: autoagglutination and chemotaxis as well as adhesion to and invasion of primary chicken caecal cells. This allowed strains to be ranked according to their in vitro performance. After their in vitro capacity to outcompete was demonstrated in vivo, strains were then typed by comparative genomic fingerprinting (CGF). In vitro phenotypical properties displayed a linear variability among the tested strains. Strains possessing higher scores for phenotypical properties were able to outcompete others during chicken colonization trials. When the gene content of strains was compared, some were associated with different phenotypical scores and thus with different outcompeting capacities. Use of CGF profiles showed an extensive genetic variability among the studied strains and suggested that the outcompeting capacity is not predictable by CGF profile.ConclusionThis study revealed a wide array of phenotypes present in C. jejuni strains, even though they were all recovered from chicken caecum. Each strain was classified according to its in vitro competitive potential and its capacity to compete for chicken gut colonization was associated with specific genes. This study also exposed the disparity existing between genetic typing and phenotypical behavior of C. jejuni strains.

Impact of Nutrient Restriction on the Structure of Listeria monocytogenes Biofilm Grown in a Microfluidic System

Biofilm formation by the pathogen Listeria monocytogenes is a major concern in food industries. The aim of this work was to elucidate the effect of nutrient limitation on both biofilm architecture and on the viability of the bacteria in microfluidic growth conditions. Biofilm formation by two L. monocytogenes strains was performed in a rich medium (BHI) and in a 10-fold diluted BHI (BHI/10) at 30°C for 24 h by using both static conditions and the microfluidic system Bioflux. In dynamic conditions, biofilms grown in rich and poor medium showed significant differences as well in structure and in the resulting biovolume. In BHI/10, biofilm was organized in a knitted network where cells formed long chains, whereas in the rich medium, the observed structure was homogeneous cellular multilayers. Biofilm biovolume production in BHI/10 was significantly higher than in BHI in these dynamic conditions. Interestingly, biovolume of dead cells in biofilms formed under limited nutrient conditions (BHI/10) was significantly higher than in biofilms formed in the BHI medium. In the other hand, in static conditions, biofilm is organized in a multilayer cells and dispersed cells in a rich medium BHI and poor medium BHI/10 respectively. There was significantly more biomass in the rich medium compared to BHI/10 but no difference was noted in the dead/damaged subpopulation showing how L. monocytogenes biofilm could be affected by the growth conditions. This work demonstrated that nutrient concentration affects biofilm structure and the proportion of dead cells in biofilms under microfluidic condition. Our study also showed that limited nutrients play an important role in the structural stability of L. monocytogenes biofilm by enhancing cell death and liberating extracellular DNA.

Distribution of Colonization and Antimicrobial Resistance Genes in Campylobacter jejuni Isolated from Chicken

Campylobacter jejuni is an important worldwide foodborne pathogen commonly found as a commensal organism in poultry that can reach high numbers within the gut after colonization. Although information regarding some genes involved in colonization is available, little is known about their distribution in strains isolated specifically from chickens and whether there is a linkage between antimicrobial resistance (AMR) and colonization genes. To assess the distribution and relevance of genes associated with chicken colonization and AMR, a C. jejuni microarray was created to detect 254 genes of interest in colonization and AMR including variants. DNA derived from chicken-specific Campylobacter isolates collected in 2003 (n=29) and 2008 (n=28) was hybridized to the microarray and compared. Hybridization results showed variable colonization-associated gene presence. Acquired AMR genes were low in prevalence whereas chemotaxis receptors, arsenic resistance genes, as well as genes from the cell envelope and flagella functional groups were highly variable in their presence. Strains clustered into two groups, each linked to different control strains, 81116 and NCTC11168. Clustering was found to be independent of collection time. We also show that AMR weakly associated with the CJ0628 and arsR genes. Although other studies have implicated numerous genes associated with C. jejuni chicken colonization, our data on chicken-specific isolates suggest the opposite. The enormous variability in presumed colonization gene prevalence in our chicken isolates suggests that many are of lesser importance than previously thought. Alternatively, this also suggests that combinations of genes may be required for natural colonization of chicken intestines.

Lack of Evidence That Selenium-Yeast Improves Chicken Health and Modulates the Caecal Microbiota in the Context of Colonization by Campylobacter jejuni

Faced with ever-increasing demand, the industrial production of food animals is under pressure to increase its production. In order to keep productivity, quality, and safety standards up while reducing the use of antibiotics, farmers are seeking new feed additives. In chicken production, one of these additives is selenium. This element is expected to confer some advantages in terms of animal health and productivity, but its impact on chicken intestinal microbiota as well as on the carriage of foodborne pathogens is unknown. In this study, chickens raised in a level 2 animal facility were fed or not 0.3 ppm of in-feed selenium-yeast until 35 days of age and were inoculated or not with the foodborne pathogen Campylobacter jejuni at the age of 14 days. At the end of the study, body weight, seric IgY, intestinal IgA, seric gluthatione peroxydase activity, the caecal microbiota (analyzed by MiSeq 16S rRNA gene sequencing), and C. jejuni caecal levels were analyzed. The experiment was completely replicated twice, with two independent batches of chickens. This study revealed that, for healthy chickens raised in very good hygienic conditions, selenium-yeast does not influence the bird’s body weight and lowers their seric gluthatione peroxidase activity as well as their intestinal IgA concentrations. Furthermore, selenium-yeast did not modify the caecal microbiota or the colonization of C. jejuni. The results also showed that C. jejuni colonization does not impact any of the measured chicken health parameters and only slightly impacts the caecal microbiota. This study also clearly illustrated the need for true biological replication (independent animal trials) when assessing the microbiota shifts associated with treatments as the chickens microbiotas clearly clustered according to study replicate.