Valérie Rose

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.

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.

Genetic Diversity and Antimicrobial Resistance Profiles of Salmonella enterica Serotype Derby Isolated from Pigs, Pork, and Humans in France

In France, Salmonella enterica serotypes Typhimurium and Derby are the most often isolated serotypes in pigs. Moreover, serotype Derby usually ranks between third and fourth in prevalence among human isolates in France. The aim of this study was to evaluate the genetic relationships between human and pig Salmonella Derby isolates based on their pulsed-field gel electrophoresis (PFGE) patterns after XbaI, BlnI, and SpeI restriction and on their antimicrobial resistance profiles. The 196 studied isolates were isolated in 2006 and 2007: 73 from fattening pigs, 27 from pork, and 96 from humans. Forty-four PFGE XbaI patterns were identified. A major pattern (SDX01) was identified for 96 isolates (49%). This pattern was common to pig, pork, and human isolates. Among the 146 isolates tested for their antimicrobial resistance, 84.2% (n=123) showed resistance to at least one antibiotic and 69.2% (n=101) were simultaneously resistant to at least streptomycin, sulfonamides, and tetracycline. Most of the isolates that are resistant to these three antibiotics also displayed the major SDX01 pattern. The use of two other restriction enzymes on a part of the panel (155 isolates) brought a significant increase in the discriminatory index, in particular for SDX01 strains. As Salmonella Derby is essentially isolated from pigs, and major resistance and PFGE patterns of isolates from pigs and pork were very similar to human isolates, human salmonellosis due to Salmonella Derby may be related to pigs.

Genome-wide identification of host-segregating epidemiological markers for source attribution in Campylobacter jejuni

ABSTRACT Campylobacter is among the most common worldwide causes of bacterial gastroenteritis. This organism is part of the commensal microbiota of numerous host species, including livestock, and these animals constitute potential sources of human infection. Molecular typing approaches, especially multilocus sequence typing (MLST), have been used to attribute the source of human campylobacteriosis by quantifying the relative abundance of alleles at seven MLST loci among isolates from animal reservoirs and human infection, implicating chicken as a major infection source. The increasing availability of bacterial genomes provides data on allelic variation at loci across the genome, providing the potential to improve the discriminatory power of data for source attribution. Here we present a source attribution approach based on the identification of novel epidemiological markers among a reference pan-genome list of 1,810 genes identified by gene-by-gene comparison of 884 genomes of Campylobacter jejuni isolates from animal reservoirs, the environment, and clinical cases. Fifteen loci involved in metabolic activities, protein modification, signal transduction, and stress response or coding for hypothetical proteins were selected as host-segregating markers and used to attribute the source of 42 French and 281 United Kingdom clinical C. jejuni isolates. Consistent with previous studies of British campylobacteriosis, analyses performed using STRUCTURE software attributed 56.8% of British clinical cases to chicken, emphasizing the importance of this host reservoir as an infection source in the United Kingdom. However, among French clinical isolates, approximately equal proportions of isolates were attributed to chicken and ruminant reservoirs, suggesting possible differences in the relative importance of animal host reservoirs and indicating a benefit for further national-scale attribution modeling to account for differences in production, behavior, and food consumption. IMPORTANCE Accurately quantifying the relative contribution of different host reservoirs to human Campylobacter infection is an ongoing challenge. This study, based on the development of a novel source attribution approach, provides the first results of source attribution in Campylobacter jejuni in France. A systematic analysis using gene-by-gene comparison of 884 genomes of C. jejuni isolates, with a pan-genome list of genes, identified 15 novel epidemiological markers for source attribution. The different proportions of French and United Kingdom clinical isolates attributed to each host reservoir illustrate a potential role for local/national variations in C. jejuni transmission dynamics.

Campylobacter coli in Organic and Conventional Pig Production in France and Sweden: Prevalence and Antimicrobial Resistance.

The purpose of the study was to evaluate and compare the prevalence and antimicrobial resistance of Campylobacter coli in conventional and organic pigs from France and Sweden. Fecal or colon samples were collected at farms or at slaughterhouses and cultured for Campylobacter. The minimum inhibitory concentrations of ciprofloxacin, nalidixic acid, streptomycin, tetracycline, erythromycin, and gentamicin were determined by microdilution for a total of 263 French strains from 114 pigs from 50 different farms and 82 Swedish strains from 144 pigs from 54 different farms. Erythromycin resistant isolates were examined for presence of the emerging rRNA methylase erm(B) gene. The study showed that within the colon samples obtained in each country there was no significant difference in prevalence of Campylobacter between pigs in organic and conventional productions [France: conventional: 43/58 (74%); organic: 43/56 (77%) and Sweden: conventional: 24/36 (67%); organic: 20/36 (56%)]. In France, but not in Sweden, significant differences of percentages of resistant isolates were associated with production type (tetracycline, erythromycin) and the number of resistances was significantly higher for isolates from conventional pigs. In Sweden, the number of resistances of fecal isolates was significantly higher compared to colon isolates. The erm(B) gene was not detected in the 87 erythromycin resistant strains tested.

First Complete Genome Sequence of a Salmonella enterica subsp. enterica Serovar Derby Strain Associated with Pork in France

ABSTRACT In France, Salmonella enterica subsp. enterica serovar Derby is one of the most often isolated serovars in pigs. Here, we describe the draft genome sequence of a strain isolated from a pig. This strain had the most frequent pulsed-field gel electrophoresis (PFGE) and antimicrobial patterns (S, SSU, T) usually observed in pig production in France. Those patterns have been also highlighted in human isolates.

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.

Prevalence of Thermophilic Campylobacter in Cattle Production at Slaughterhouse Level in France and Link Between C. jejuni Bovine Strains and Campylobacteriosis

Campylobacter is the leading cause of bacterial gastroenteritis in industrialized countries, with poultry reservoir as the main source of infection. Nevertheless, a recent study on source attribution showed that cattle could be a source of human contamination in France (Thépault et al., 2017). However, few data are available on thermophilic Campylobacter epidemiology in cattle in France. The aim of this study is to collect new data of thermophilic Campylobacter prevalence in these animals and to subtype C. jejuni isolates to assess the potential implication of cattle in campylobacteriosis. A 6-month survey was carried out in one of the largest European slaughterhouse of cattle. Based on a statistical representative sampling plan, 959 intestinal content samples (483 adult cattle and 476 calves) were collected. An adapted version of the ISO 10272 standard and Maldi-Tof were used for detection and speciation of thermophilic Campylobacter isolates. Within more than 2000 thermophilic Campylobacter isolates collected, a selection of 649 C. jejuni isolates was typed with Comparative Genomic Fingerprinting (CGF40) and a subset of 77 isolates was typed using Multilocus Sequence Typing (MLST). Simultaneously, clinical isolates occurred in France were genotyped. Prevalence of thermophilic Campylobacter in the global cattle population was 69.1% (CI95% = 66.1, 72.1) at slaughterhouse level. In adult cattle, the prevalence was 39.3%, while 99.4% of calves were contaminated, and C. jejuni was the most prevalent species with prevalence of 37.3 and 98.5%, respectively and a higher genetic diversity in adult cattle. The prevalence of C. coli was lower with 3% in adult cattle and 12.5% in calves. MLST and CGF40 genotyping did not showed a high number of clusters within cattle isolates but the predominance of few clusters accounted for a large part of the population (CC-21, CC-61, CC-48, and CC-257). By comparison with clinical genotypes, genetic diversity was significantly lower in cattle. Moreover, significant overlap was observed between genotypes from both origins, with 3 of the 4 main cattle clusters present in human isolates. This study provides new insights on the epidemiology of thermophilic Campylobacter and C. jejuni in cattle production in France and their potential implication in human infection.

Ruminant and chicken: important sources of campylobacteriosis in France despite a variation of source attribution in 2009 and 2015

Pathogen source attribution studies are a useful tool for identifying reservoirs of human infection. Based on Multilocus Sequence Typing (MLST) data, such studies have identified chicken as a major source of C. jejuni human infection. The use of whole genome sequence-based typing methods offers potential to improve the precision of attribution beyond that which is possible from 7 MLST loci. Using published data and 156 novel C. jejuni genomes sequenced in this study, we performed probabilistic host source attribution of clinical C. jejuni isolates from France using three types of genotype data: comparative genomic fingerprints; MLST genes; 15 host segregating genes previously identified by whole genome sequencing. Consistent with previous studies, chicken was an important source of campylobacteriosis in France (31–63% of clinical isolates assigned). There was also evidence that ruminants are a source (22–55% of clinical isolates assigned), suggesting that further investigation of potential transmission routes from ruminants to human would be useful. Additionally, we found evidence of environmental and pet sources. However, the relative importance as sources varied according to the year of isolation and the genotyping technique used. Annual variations in attribution emphasize the dynamic nature of zoonotic transmission and the need to perform source attribution regularly.

No Clear Differences between Organic or Conventional Pig Farms in the Genetic Diversity or Virulence of Campylobacter coli Isolates

To evaluate the impact of pig farm management on the genetic diversity and on the virulence of Campylobacter coli, we characterized isolates from 19 organic pig farms (62 isolates) and from 24 conventional pig farms (58 isolates). The 120 C. coli isolates were typed using pulsed field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) and the presence of nine virulence genes was screened using real-time PCR. The capacity of adhesion and invasion of 61 isolates (32 from organic and 29 from conventional farms) were then tested on human intestinal Caco-2 cells. A total of 59 PFGE types and of 50 sequence types (STs) were identified. Twelve PFGE types and nine STs, accounting for 34 and 41.6% of the isolates, respectively, were common between the two production systems with ST854 dominating (18.3% of the isolates). Twenty-nine PFGE types and 25 STs were only found in isolates from organic farms, and 18 PFGE types and 16 STs from conventional farms. No significant differences were found in diversity despite the differences in rearing systems, except at the locus level for the glnA, gltA, and uncA genes. All isolates, regardless of their origin, carried the ceuE, iam, ciaB, and flaA genes and more than 95% of the isolates carried the cadF and cdtABC genes. No significant differences were found in pathogenicity between the two farming systems. The pathogenicity of the C. coli isolates was low compared to C. jejuni control strains tested. The plasmid gene virb11 was detected in only 13 isolates from organic farms; these isolates showed greater invasion capacity than those without this gene. Our study indicates that pig farm management does not significantly affect the diversity and the virulence of Campylobacter coli isolated from pigs. The common genotypes between conventional and organic farms may indicate that some genotypes are adapted to pigs.