Muriel Guyard-Nicodème

Prevalence and characterization of Campylobacter jejuni from chicken meat sold in French retail outlets.

Campylobacter was detected in 76% of broiler meat products collected in retail outlets during a monitoring plan carried out in France throughout 2009. Campylobacter jejuni was the most prevalent species (64.7% of products being contaminated). The 175 C. jejuni isolates collected were characterized. MLST typing results confirmed substantial genetic diversity as the 175 C. jejuni isolates generated 76 sequence types (STs). The ST-21, ST-45 and ST-464 complexes predominated accounting for 43% of all isolates. A class-specific PCR to screen the sialylated lipooligosaccharide (LOS) locus classes A, B and C showed that 50.3% of the C. jejuni isolates harbored sialylated LOS. The antimicrobial resistance profiles established using a subset of 97 isolates showed that resistance to tetracycline was the most common (53.6%), followed with ciprofloxacin and nalidixic acid (32.9%, and 32.0% respectively). All the tested isolates were susceptible to erythromycin, chloramphenicol and gentamicin. Clear associations were demonstrated between certain clonal complexes and LOS locus classes and between certain clonal complexes and antimicrobial resistance. This work paints a representative picture of C. jejuni isolated from poultry products circulating in France, providing data on STs, LOS locus classes and antibiotic resistance profiles in isolates recovered from products directly available to the consumer.

Control strategies against Campylobacter at the poultry production level: biosecurity measures, feed additives and vaccination.

Campylobacteriosis is the most prevalent bacterial foodborne gastroenteritis affecting humans in the European Union, and ranks second in the United States only behind salmonellosis. In Europe, there are about nine million cases of campylobacteriosis every year, making the disease a major public health issue. Human cases are mainly caused by the zoonotic pathogen Campylobacter jejuni. The main source of contamination is handling or consumption of poultry meat. Poultry constitutes the main reservoir of Campylobacter, substantial quantities of which are found in the intestines following rapid, intense colonization. Reducing Campylobacter levels in the poultry chain would decrease the incidence of human campylobacteriosis. As primary production is a crucial step in Campylobacter poultry contamination, controlling the infection at this level could impact the following links along the food chain (slaughter, retail and consumption). This review describes the control strategies implemented during the past few decades in primary poultry production, including the most recent studies. In fact, the implementation of biosecurity and hygiene measures is described, as well as the immune strategy with passive immunization and vaccination trials and the nutritional strategy with the administration of organic and fatty acids, essential oil and plant‐derived compound, probiotics, bacteriocins and bacteriophages.

Characterization of Campylobacter spp. transferred from naturally contaminated chicken legs to cooked chicken slices via a cutting board

Campylobacter represents the leading cause of gastroenteritis in Europe. Campylobacteriosis is mainly due to C. jejuni and C. coli. Poultry meat is the main source of contamination, and cross-contaminations in the consumers kitchen appear to be the important route for exposure. The aim of this study was to examine the transfer of Campylobacter from naturally contaminated raw poultry products to a cooked chicken product via the cutting board and to determine the characteristics of the involved isolates. This study showed that transfer occurred in nearly 30% of the assays and that both the C. jejuni and C. coli species were able to transfer. Transfer seems to be linked to specific isolates: some were able to transfer during separate trials while others were not. No correlation was found between transfer and adhesion to inert surfaces, but more than 90% of the isolates presented moderate or high adhesion ability. All tested isolates had the ability to adhere and invade Caco-2 cells, but presented high variability between isolates. Our results highlighted the occurrence of Campylobacter cross-contamination via the cutting board in the kitchen. Moreover, they provided new interesting data to be considered in risk assessment studies.

Identification of Novel Vaccine Candidates against Campylobacter through Reverse Vaccinology

Campylobacteriosis is the most prevalent bacterial foodborne gastroenteritis affecting humans in the European Union. Human cases are mainly due to Campylobacter jejuni or Campylobacter coli, and contamination is associated with the handling and/or consumption of poultry meat. In fact, poultry constitutes the bacterias main reservoir. A promising way of decreasing the incidence of campylobacteriosis in humans would be to decrease avian colonization. Poultry vaccination is of potential for this purpose. However, despite many studies, there is currently no vaccine available on the market to reduce the intestinal Campylobacter load in chickens. It is essential to identify and characterize new vaccine antigens. This study applied the reverse vaccinology approach to detect new vaccine candidates. The main criteria used to select immune proteins were localization, antigenicity, and number of B-epitopes. Fourteen proteins were identified as potential vaccine antigens. In vitro and in vivo experiments now need to be performed to validate the immune and protective power of these newly identified antigens.

Recent Advances in Screening of Anti-Campylobacter Activity in Probiotics for Use in Poultry

Campylobacteriosis is the most common cause of bacterial gastroenteritis worldwide. Campylobacter species involved in this infection usually include the thermotolerant species Campylobacter jejuni. The major reservoir for C. jejuni leading to human infections is commercial broiler chickens. Poultry flocks are frequently colonized by C. jejuni without any apparent symptoms. Risk assessment analyses have identified the handling and consumption of poultry meat as one of the most important sources of human campylobacteriosis, so elimination of Campylobacter in the poultry reservoir is a crucial step in the control of this foodborne infection. To date, the use of probiotics has demonstrated promising results to reduce Campylobacter colonization. This review provides recent insights into methods used for probiotic screening to reduce the prevalence and colonization of Campylobacter at the farm level. Different eukaryotic epithelial cell lines are employed to screen probiotics with an anti-Campylobacter activity and yield useful information about the inhibition mechanism involved. These in vitro virulence models involve only human intestinal or cervical cell lines whereas the use of avian cell lines could be a preliminary step to investigate mechanisms of C. jejuni colonization in poultry in the presence of probiotics. In addition, in vivo trials to evaluate the effect of probiotics on Campylobacter colonization are conducted, taking into account the complexity introduced by the host, the feed, and the microbiota. However, the heterogeneity of the protocols used and the short time duration of the experiments lead to results that are difficult to compare and draw conclusions at the slaughter-age of broilers. Nevertheless, the combined approach using complementary in vitro and in vivo tools (cell cultures and animal experiments) leads to a better characterization of probiotic strains and could be employed to assess reduced Campylobacter spp. colonization in chickens if some parameters are optimized.

Use of the potential probiotic strain Lactobacillus salivarius SMXD51 to control Campylobacter jejuni in broilers.

Campylobacteriosis is the most frequently reported zoonotic disease in humans in the EU since 2005. As chicken meat is the main source of contamination, reducing the level of Campylobacter in broiler chicken will lower the risk to consumers. The aim of this project was to evaluate the ability of Lactobacillus salivarius SMXD51 to control Campylobacter jejuni in broilers and to investigate the mechanisms that could be involved. Thirty broilers artificially contaminated with C. jejuni were treated by oral gavage with MRS broth or a bacterial suspension (107CFU) of Lb. salivarius SMXD51 (SMXD51) in MRS broth. At 14 and 35days of age, Campylobacter and Lb. salivarius loads were assessed in cecal contents. The impact of the treatment on the avian gut microbiota at day 35 was also evaluated. At day 14, the comparison between the control and treated groups showed a significant reduction (P<0.05) of 0.82 log. After 35days, a significant reduction (P<0.001) of 2.81 log in Campylobacter loads was observed and 73% of chickens treated with the culture exhibited Campylobacter loads below 7log10CFU/g. Taxonomic analysis revealed that SMXD51 treatment induced significant changes (P<0.05) in a limited number of bacterial genera of the avian gut microbiota and partially limited the impact of Campylobacter on Anaerotruncus sp. decrease and Subdoligranulum sp. increase. Thus, SMXD51 exhibits an anti-Campylobacter activity in vivo and can partially prevent the impact of Campylobacter on the avian gut microbiota.

Comparison of Adhesion, Invasion, Motility, and Toxin Production of Campylobacter Strains and Their Resistant Mutants

The objectives of this study were to compare the in vitro adhesion and invasion of human epithelial cells, motility, and toxin production characteristics of Campylobacter-susceptible strains and their fluoroquinolone- or macrolide-resistant mutants. Susceptible strains and resistant mutants demonstrated similar adhesion capacities to epithelial cells. For Campylobacter coli, fluoroquinolone-resistant mutants with Thr86Ile or Asp90Asn substitutions showed a higher rate of invasion of Caco-2 cells than their isogenic parental strain. Fluoroquinolone resistance did not impact C. coli motility. Mutants harboring Asp90Asn had greater cytotoxic activity than the parental strain. Macrolide resistance had no impact on the studied characteristics of C. coli. For Campylobacter jejuni, fluoroquinolone-resistant mutants had slightly different invasiveness levels and significantly lower motility than the isogenic parental strain. C. jejuni macrolide-resistant mutants with A2074G substitution in the 23S rRNA gene had a higher invasiveness level than its parental strain, but mutants with A2074C in 23S rRNA and G221A in rplD showed reduced motility and similar invasion levels to the susceptible strains. Neither fluoroquinolone nor macrolide resistance appears to affect C. jejuni cytotoxicity. In conclusion, mutations that are frequently encountered in Campylobacter-resistant strains can enhance the invasiveness in Caco-2 cells.

Promising new vaccine candidates against Campylobacter in broilers

Campylobacter is the leading cause of human bacterial gastroenteritis in the European Union. Birds represent the main reservoir of the bacteria, and human campylobacteriosis mainly occurs after consuming and/or handling poultry meat. Reducing avian intestinal Campylobacter loads should impact the incidence of human diseases. At the primary production level, several measures have been identified to reach this goal, including vaccination of poultry. Despite many studies, however, no efficient vaccine is currently available. We have recently identified new vaccine candidates using the reverse vaccinology strategy. This study assessed the in vivo immune and protective potential of six newly-identified vaccine antigens. Among the candidates tested on Ross broiler chickens, four (YP_001000437.1, YP_001000562.1, YP_999817.1, and YP_999838.1) significantly reduced cecal Campylobacter loads by between 2 and 4.2 log10 CFU/g, with the concomitant development of a specific humoral immune response. In a second trial, cecal load reductions results were not statistically confirmed despite the induction of a strong immune response. These vaccine candidates need to be further investigated since they present promising features.

A representative overview of the genetic diversity and lipooligosaccharide sialylation in Campylobacter jejuni along the broiler production chain in France and its comparison with human isolates

Campylobacter jejuni is the most common cause of bacterial gastroenteritis worldwide and is associated with post-infectious neuropathies. Moreover, the chicken reservoir is described as the main source of human infection and C. jejuni sialylated lipooligosaccharides seem to play an important role in the pathogenesis of neuropathies. In this study, MultiLocus Sequence Typing (MLST) and Comparative Genomic Fingerprinting using 40 assay genes (CGF40) were used to describe C. jejuni populations within clinical isolates and a representative collection of isolates from French poultry production. In addition, the sialylation of C. jejuni LOS was assessed. Here, we report high levels of genetic diversity among both chicken and human disease C. jejuni populations. The predominance of the ST-21, ST-45, and ST-464 complexes in chicken isolates and of the ST-21, ST-206, and ST-48 complexes in the clinical isolates was observed as were correlations between some MLST and CGF40 genotypes. Furthermore, some C. jejuni genotypes were frequently isolated among clinical cases as well as all along the broiler production chain, suggesting a potentially high implication of chicken in human campylobacteriosis in France. Finally, the LOS classes A, B and C were predominant within clinical C. jejuni isolates supporting the hypothesis of a benefit in infectivity for C. jejuni isolates showing sialylated LOS.

Effect of Feed Additives on Productivity and Campylobacter spp. Loads in Broilers Reared under Free Range Conditions

The poultry reservoir, especially broiler meat, is generally recognized as one of the most-important sources for human Campylobacteriosis. The measures to control Campylobacter targeted essentially the primary production level. The aim of this work was to evaluate the effectiveness of different treatments against natural Campylobacter colonization in a French experimental farm of free-range broilers during the whole rearing period. Five commercial products and a combination of two of them were tested and all the products were added to feed or to water at the dose recommended by the suppliers. Campylobacter loads in caeca and on carcasses of broilers at the slaughter were determined by culture methods. Natural contamination of the flock occurred at the end of the indoor rearing period between day 35 and day 42. At day 42, the multispecies probiotic added to the feed reduced the contamination of 0.55 log10 CFU/g (p = 0.02) but was not significant (p > 0.05) at the end of rearing at day 78. However, another treatment, a combination of a cation exchange clay-based product in feed and an organic acid mixture (formic acid, sodium formate, lactic acid, propionic acid) in water, led to a slight but significant reduction of 0.82 ± 0.25 log10 CFU/g (p = 0.02) compared to the control group at day 78. Testing this combination in field conditions in several flocks is needed to determine if it is biologically relevant and if it could be a valuable measure to reduce Campylobacter in broiler flocks.