Alexandre Thibodeau

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.

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.

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.

Survival in water of Campylobacter jejuni strains isolated from the slaughterhouse

Campylobacter jejuni cause gastroenteritis in humans. The main transmission vector is the consumption or handling of contaminated chicken meat, since chicken can be colonized asymptomatically by C. jejuni. However, water has been implicated as the transmission vector in a few outbreaks. One possibility is the contamination of water effluent by C. jejuni originating from chicken farm. The ability of C. jejuni to be transmitted by water would be closely associated to its ability to survive in water. Therefore, in this study, we have evaluated the ability of reference strains and chicken-isolated strains to survive in water. Defined water media were used, since the composition of tap water is variable. We showed that some isolates survive better than others in defined freshwater (Fraquil) and that the survival was affected by temperature and the concentration of NaCl. By comparing the ability of C. jejuni to survive in water with other phenotypic properties previously tested, we showed that the ability to survive in water was negatively correlated with autoagglutination. Our data showed that not all chicken isolates have the same ability to survive in water, which is probably due to difference in genetic content.

Broiler chicken carcasses and their associated abattoirs as a source of enterotoxigenic Clostridium perfringens : Prevalence and critical steps for contamination

Clostridium perfringens ranks among the three most frequent bacterial pathogens causing human foodborne diseases in Canada, and poultry meat products are identified as a source of infection for humans. The objective of the current study was to estimate the proportion of broiler chicken flocks, carcasses and various environmental samples from critical locations of the slaughter plant positive for the presence of C. perfringens enterotoxin encoding gene (cpe). From the 16 visits conducted, 25% of the 79 flocks sampled, 10% of the 379 carcasses sampled and 5% of the 217 environmental samples collected were found positive for cpe. The proportion of cpe-positive carcasses was statistically different between surveyed plants, with 17.0% for one abattoir and 2.2% for the other. For the most contaminated plant, cpe-positive carcasses were identified at each step of the processing line, with prevalence varying between 10.0% and 25.0%, whereas this prevalence varied between 0% and 25.0% for the environmental surfaces sampled. Based on the results obtained, enterotoxigenic C. perfringens strains could potentially represent a risk for the consumer.

Salmonella shedding status of the sow affects the microbiota of their piglets at weaning

To observe the transfer of the digestive microbiota from sow to piglet, describe the impact of the sows Salmonella shedding on this transfer and identify transferred populations that could be associated with the future Salmonella status of the piglets.

Salmonella spp. and Campylobacter spp. in poultry feces and carcasses in Ouagadougou, Burkina Faso

Abstract The importance of Salmonella and Campylobacter as foodborne pathogens is well recognized worldwide. Poultry and poultry products are commonly considered as the major vehicles of Salmonella and Campylobacter infection in humans. The aim of this study was to investigate the hygienic status of poultry facilities and determine the prevalence of Salmonella and Campylobacter in slaughtered poultry feces and carcasses in four different markets in Ouagadougou, capital city of Burkina Faso. A total of 103 poultry feces and 20 carcasses were analyzed using microbiological standard methods. Among the 103 fecal samples, 70 were positive for Campylobacter ssp (67.96%) and 54 for Salmonella ssp (52.42%). The hippurate hydrolysis test revealed that among the 70 Campylobacter strains isolated from feces, 49 were C. jejuni (70%) and 21 were C. coli (30%). From the 20 carcasses analyzed, 18 were contaminated with Salmonella (90%) and 10 with Campylobacter ssp (50%). Among the 10 Campylobacter ssp samples isolated from poultry carcasses, all were identified as C. jejuni using the API CAMPY system and the hippurate hydrolysis test. The assessment of markets hygienic practices for production, transportation, display, and vending of meat revealed unhygienic conditions. To complete the observation of unhygienic practices, we have sampled chicken‐washing solution from the study sites and microbiological analysis of these samples revealed the presence of Salmonella spp in 100% of the samples. This study highlighted that poultry products on sale in Ouagadougou are highly contaminated with Salmonella and Campylobacter. To the best of our knowledge, this is the first report describing Campylobacter presence in the poultry industry of Burkina Faso. Our findings might help to better understand the epidemiology of Salmonella and Campylobacter.

Reduction of Salmonella Shedding by Sows during Gestation in Relation to Its Fecal Microbiome

Pork meat is estimated to be responsible for 10–20% of human salmonellosis cases in Europe. Control strategies at the farm could reduce contamination at the slaughterhouse. One of the targeted sectors of production is maternity, where sows could be Salmonella reservoirs. The aim of this study was to assess the dynamics of shedding of Salmonella in terms of variation in both shedding prevalence and strains excreted during gestation in Quebec’s maternity sector. The evolution of the fecal microbiota of these sows during gestation was also assessed to detect bacterial populations associated with these variations. A total of 73 sows both at the beginning and the end of the gestation were randomly selected and their fecal matter was analyzed. Salmonella detection was conducted using a method that includes two selective enrichment media (MSRV and TBG). Nine isolates per positive samples were collected. Among the 73 sows tested, 27 were shedding Salmonella. Sows in the first third of their gestation shed Salmonella significantly more frequently (21/27) than those in the last third (6/46) (χ2 P < 0.05). The shedding status of 19 of the sows that were previously sampled in the first third of their gestation was followed, this time in the last third of their gestation, which confirmed reduction of shedding. Using 16S rRNA gene sequencing and qPCR, significant differences between the fecal flora of sows at the beginning and the end of the gestation, shedding Salmonella or not and with different parity number were detected. Using MaAsLin, multiple OTUs were found to be associated with the time of gestation, the status of Salmonella excretion and parity number. Some of the identified taxa could be linked to the reduction of the shedding of Salmonella at the end of gestation. In this study, we showed that the level of Salmonella shedding was variable during gestation with significantly higher shedding at the beginning rather than at the end of gestation. We also observed for the first time a significant change in the microbiota during sow gestation and identified interesting taxa which could be linked to a reduced Salmonella shedding.

Phenotypic and Transcriptomic Responses of Campylobacter jejuni Suspended in an Artificial Freshwater Medium

Campylobacter jejuni is the leading cause of campylobacteriosis in the developed world. Although most cases are caused by consumption of contaminated meat, a significant proportion is linked to ingestion of contaminated water. The differences between C. jejuni strains originating from food products and those isolated from water are poorly understood. Working under the hypothesis that water-borne C. jejuni strains are better equipped at surviving the nutrient-poor aquatic environment than food-borne strains, the present study aims to characterize these differences using outbreak strains 81116 and 81-176. Strain 81116 caused a campylobacteriosis outbreak linked to consumption of water, while strain 81-176 was linked to consumption of raw milk. CFU counts and viability assays showed that 81116 survives better than 81-176 at 4°C in a defined freshwater medium (Fraquil). Moreover, 81116 was significantly more resistant to oxidative stress and bile salt than strain 81-176 in Fraquil. To better understand the genetic response of 81116 to water, a transcriptomic profiling study was undertaken using microarrays. Compared to rich broth, strain 81116 represses genes involved in amino acid uptake and metabolism, as well as genes involved in costly biosynthetic processes such as replication, translation, flagellum synthesis and virulence in response to Fraquil. In accordance with the observed increase in stress resistance in Fraquil, 81116 induces genes involved in resistance to oxidative stress and bile salt. Interestingly, genes responsible for cell wall synthesis were also induced upon Fraquil exposure. Finally, twelve unique genes were expressed in Fraquil; however, analysis of their distribution in animal and water isolates showed that they are not uniquely and ubiquitously present in water isolates, and thus, unlikely to play a major role in adaptation to water. Our results show that some C. jejuni strains are more resilient than others, thereby challenging current water management practices. The response of 81116 to Fraquil serves as a starting point to understand the adaptation of C. jejuni to water and its subsequent transmission.