Polynucleotide phosphorylase has an impact on cell biology of Campylobacter jejuni

Polynucleotide phosphorylase (PNPase), encoded by the pnp gene, is known to degrade mRNA, mediating post-transcriptional regulation and may affect cellular functions. The role of PNPase is pleiotropic. As orthologs of the two major ribonucleases (RNase E and RNase II) of Escherichia coli are missing in the Campylobacter jejuni genome, in the current study the focus has been on the C. jejuni ortholog of PNPase. The effect of PNPase mutation on C. jejuni phenotypes and proteome was investigated. The inactivation of the pnp gene reduced significantly the ability of C. jejuni to adhere and to invade Ht-29 cells. Moreover, the pnp mutant strain exhibited a decrease in C. jejuni swimming ability and chick colonization. To explain effects of PNPase on C. jejuni 81-176 phenotype, the proteome of the pnp mutant and parental strains were compared. Overall, little variation in protein production was observed. Despite the predicted role of PNPase in mRNA regulation, the pnp mutation did not induce profound proteomic changes suggesting that other ribonucleases in C. jejuni might ensure this biological function in the absence of PNPase. Nevertheless, synthesis of proteins which are involved in virulence (LuxS, PEB3), motility (N-acetylneuraminic acid synthetase), stress-response (KatA, DnaK, Hsp90), and translation system (EF-Tu, EF-G) were modified in the pnp mutant strain suggesting a more specific role of PNPase in C. jejuni. In conclusion, PNPase deficiency induces limited but important consequences on C. jejuni biology that could explain swimming limitation, chick colonization delay, and the decrease of cell adhesion/invasion ability

Genome-wide identification of host-segregating epidemiological markers for source attribution in <i>Campylobacter jejuni</i>

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 multi-locus sequence typing (MLST), have been used to attribute the source of human campylobacteriosis by quantifying the relative abundance of alleles, at 7 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 1810 genes identified through gene-by-gene comparison of 884 genomes of C. 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 UK 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 UK. 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 modelling to account for differences in production, behaviour 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 UK clinical isolates attributed to each host reservoir illustrates a potential role for local/national variations in C. jejuni transmission dynamics.</p

LES CAMPYLOBACTER DANS LA FILIERE AVICOLE. CARACTERISATION GENOMIQUE ET ORIGINE DE LA CONTAMINATION DES ELEVAGES

BREST-BU Droit-Sciences-Sports (290192103) / SudocSudocFranceF

Dogs and Cats: Reservoirs for Highly Diverse Campylobacter jejuni and a Potential Source of Human Exposure

Assessing the carriage of Campylobacter in animal reservoirs is essential to better understand Campylobacter epidemiology. Here, we evaluated the prevalence of thermophilic Campylobacter spp. in dogs and cats, hereafter defined as pets, and characterized Campylobacter jejuni (C. jejuni) isolates to assess their genetic diversity and their potential link with isolates from other animals or human cases. During a 6-month period, 304 feces samples were collected from pets. A significantly higher prevalence of thermophilic Campylobacter spp. was found in dogs compared with cats, as well as in dogs &le; 1-year-old compared with older dogs. C. jejuni was the predominant species found in pets, and its genomic characterization revealed a high genetic diversity. Genotypes comparison with previously characterized isolates revealed a partial overlap between C. jejuni isolates from pets, chicken, cattle, and clinical cases. This overlap suggests the potential role of livestock and humans in pets&rsquo; exposure to Campylobacter, or vice versa. The isolation of pets&rsquo; specific profiles may suggest the existence of other sources of pet contamination or imply that pets may constitute a reservoir for Campylobacter. Because of the proximity between humans and pets, along with their frequent carriage of C. jejuni, human exposure to Campylobacter from pets can be more important than previously thought

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

Detection of Listeria monocytogenes in raw and pasteurized liquid whole eggs and characterization by PFGE.

International audienceListeria monocytogenes has been recognized as a human pathogen for decades and is known to be an important foodborne pathogen. There have been no documented foodborne L. monocytogenes illnesses due to the consumption of eggs or egg products, even though the bacterium has been isolated from faeces, body fluid, and oviducts of asymptomatic laying hens. In order to describe L. monocytogenes contamination of egg products, 144 liquid whole egg samples were collected from 3 different egg-breaking plants during 3 sampling periods. L. monocytogenes detection was performed on raw samples stored at 2 degrees C for two days (D+2) and on pasteurized samples stored at 2 degrees C at D+2 and at shelf-life date (SLD). L. monocytogenes was detected in 25 of the 144 raw egg samples collected, in 4 of the 144 pasteurized egg samples at D+2 and in 2 of the 144 ones analysed at SLD. Contamination of raw egg products appeared to be season dependant and was higher during summer and winter than during autumn. One hundred and ninety-six L. monocytogenes isolates were collected and serotyped; 3 serovars were demonstrated. The dominant serovar was L. monocytogenes 1/2a which was presented by 94.4% of the isolates. Typing of 196 L. monocytogenes isolates was carried out by macrorestriction of the genomic DNA with ApaI and AscI enzymes followed by pulsed field gel electrophoresis (PFGE). A large diversity was observed with 21 genotypes of L. monocytogenes, even for a given manufacturer. Nevertheless, most of the egg product samples were contaminated by one genotype, except for five samples which were contaminated by two or three distinct genotypes. The genotypes seem to be specific to each manufacturer. No cluster of L. monocytogenes was found to recur in the different plants over successive seasons

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

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 multi-locus sequence typing (MLST), have been used to attribute the source of human campylobacteriosis by quantifying the relative abundance of alleles, at 7 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 1810 genes identified through gene-by-gene comparison of 884 genomes of C. 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 UK 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 UK. 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 modelling to account for differences in production, behaviour and food consumption

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.

RA2007ACL09-RIVInternational audienceSalmonella 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

Genome-Wide Identification of Host-Segregating Epidemiological Markers for Source Attribution in Campylobacter jejuni

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 multi-locus sequence typing (MLST), have been used to attribute the source of human campylobacteriosis by quantifying the relative abundance of alleles, at 7 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 1810 genes identified through gene-by-gene comparison of 884 genomes of C. 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 UK 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 UK. 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 modelling to account for differences in production, behaviour 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 UK clinical isolates attributed to each host reservoir illustrates a potential role for local/national variations in C. jejuni transmission dynamics.</p

Inactivation of the LysR regulator Cj1000 of Campylobacter jejuni affects host colonization and respiration.

International audienceTranscriptional regulation mediates adaptation of pathogens to environmental stimuli and is important for host colonization. The Campylobacter jejuni genome sequence reveals a surprisingly small set of regulators, mostly of unknown function, suggesting an intricate regulatory network. Interestingly, C. jejuni lacks the homologues of ubiquitous regulators involved in stress response found in many other Gram-negative bacteria. Nonetheless, cj1000 is predicted to encode the sole LysR-type regulator in the C. jejuni genome, and thus may be involved in major adaptation pathways. A cj1000 mutant strain was constructed and found to be attenuated in its ability to colonize 1-day-old chicks. Complementation of the cj1000 mutation restored the colonization ability to wild-type levels. The mutant strain was also outcompeted in a competitive colonization assay of the piglet intestine. Oxygraphy was carried out for what is believed to be the first time with the Oroboros Oxygraph-2k on C. jejuni and revealed a role for Cj1000 in controlling O2 consumption. Furthermore, microarray analysis of the cj1000 mutant revealed both direct and indirect regulatory targets, including genes involved in energy metabolism and oxidative stress defences. These results highlight the importance of Cj1000 regulation in host colonization and in major physiological pathways