Campylobacter coli of porcine origin exhibits an open pan-genome within a single clonal complex: insights from comparative genomic analysis.

Introduction: Although Campylobacter spp., including Campylobacter coli, have emerged as important zoonotic foodborne pathogens globally, the understanding of the genomic epidemiology of C. coli of porcine origin is limited. Methods: As pigs are an important reservoir of C. coli, we analyzed C. coli genomes that were isolated (n = 3) from pigs and sequenced (this study) them along with all other C. coli genomes for which pig intestines, pig feces, and pigs were mentioned as sources in the NCBI database up to January 6, 2023. In this paper, we report the pan-genomic features, the multi-locus sequence types, the resistome, virulome, and mobilome, and the phylogenomic analysis of these organisms that were obtained from pigs. Results and discussion: Our analysis revealed that, in addition to having an open pan-genome, majority (63%) of the typeable isolates of C. coli of pig origin belonged to a single clonal complex, ST-828. The resistome of these C. coli isolates was predominated by the genes tetO (53%), blaOXA-193 (49%), and APH (3')-IIIa (21%); however, the virulome analysis revealed a core set of 37 virulence genes. Analysis of the mobile genetic elements in the genomes revealed wide diversity of the plasmids and bacteriophages, while 30 transposons were common to all genomes of C. coli of porcine origin. Phylogenomic analysis showed two discernible clusters comprising isolates originating from Japan and another set of isolates comprising mostly copies of a type strain stored in three different culture collections.

Age-related presence and genetic diversity of Campylobacter spp. in young and adult yellow-legged gulls (Larus michahellis) in Croatia.

The epidemiology of Campylobacter species in wild birds is still poorly understood. This study describes the occurrence and genetic diversity of Campylobacter in adult and nestlings of yellow-legged gulls, highlighting differences between breeding locations. The gulls were captured in Croatia between 2021 and 2023. A cloacal swab was taken from each individual and tested for the presence of Campylobacter. Isolated Campylobacter species were genotyped using the multilocus sequence typing (MLST) method. A total of 1071 gulls were captured and sampled, of which 152 samples were identified as Campylobacter species, with Campylobacter jejuni (9.90%) being the most frequently isolated bacterium, followed by Campylobacter lari (3.36%) and Campylobacter coli (0.93%). Complete sequence type (ST) profiles were generated for 141 isolates: 100 C. jejuni, 33 C. lari, and 8 C. coli. A significant difference in the occurrence of positive Campylobacter species was found depending on the sampling sites, while both sampling site and age were significant for the occurrence of C. jejuni. Adults and nestlings showed high genetic diversity for C. jejuni and C. lari, and there were no significant differences between strains isolated from adults and nestlings or between sites, suggesting a high genotype flow in the studied gull population.

Quadruplex qPCR for detection and discrimination of C. Coli,C. fetus, and C. Jejuni from other Campylobacter species in chicken and sheep meat.

Campylobacter is gram-negative bacteria considered the predominant genera isolated from poultry samples and associated with gastroenteritis. Due to the problems in conventional cultural methods of time-consuming and technically demanding requirements, a rapid and feasible method for their identification and discrimination of the closely related spp. Including Campylobacter coli, Campylobacter fetus, and Campylobacter jejuni is needed. This study analyzes the chicken and sheep meats samples (n = 125) using culture and pre-enrichment-based Quadraplex real-time PCR by targeting OrfA, CstA, HipO, and 16 S rRNA genes of C. coli, C. fetus, C. jejuni and Campylobacter spp. Respectively. The analysis of 125 chicken and sheep meat samples by culture and real-time PCR showed high concordance between the results of the two methods. The present study show high prevalence of Campylobacter species (35% and 32% from chicken and meat respectively) of which C. jejuni were the most abundant. Reaction efficiencies were between 90 and 110%, and detect as low as 8.9 fg in C. jejuni. The need for quick detection and discrimination methods in sheep and chicken meat can be met using the described Quadraplex real-time PCR methodology.

Molecular and in silico typing of the lipooligosaccharide biosynthesis gene cluster in Campylobacter jejuni and Campylobacter coli.

The extensive genetic variation in the lipooligosaccharide (LOS) core biosynthesis gene cluster has led to the development of a classification system; with 8 classes (I-VIII) for Campylobacter coli (C. coli) LOS region and with 23 classes (A-W) or four groups (1-4) for Campylobacter jejuni (C. jejuni) LOS region. PCR based LOS locus type identification for C. jejuni clinical isolates from a UK hospital as well as in silico LOS locus analysis for C. jejuni and C. coli genome sequences from GenBank was carried out to determine the frequencies of various LOS genotypes in C. jejuni and C. coli. Analysis of LOS gene content in 60 clinical C. jejuni isolates and 703 C. jejuni genome sequences revealed that class B (Group 1) was the most abundant LOS class in C. jejuni. The hierarchy of C. jejuni LOS group prevalence (group 1 > group 2 > group 3 > group 4) as well as the hierarchy of the frequency of C. jejuni LOS classes present within the group 1 (B > C > A > R > M > V), group 2 (H/P > O > E > W), group 3 (F > K > S) and group 4 (G > L) was identified. In silico analysis of LOS gene content in 564 C. coli genome sequences showed class III as the most abundant LOS locus type in C. coli. In silico analysis of LOS gene content also identified three novel LOS types of C. jejuni and previously unknown LOS biosynthesis genes in C. coli LOS locus types I, II, III, V and VIII. This study provides C. jejuni and C. coli LOS loci class frequencies in a smaller collection of C. jejuni clinical isolates as well as within the larger, worldwide database of C. jejuni and C. coli.

Genomic analysis of the diversity, antimicrobial resistance and virulence potential of clinical Campylobacter jejuni and Campylobacter coli strains from Chile.

Campylobacter jejuni and Campylobacter coli are the leading cause of human gastroenteritis in the industrialized world and an emerging threat in developing countries. The incidence of campylobacteriosis in South America is greatly underestimated, mostly due to the lack of adequate diagnostic methods. Accordingly, there is limited genomic and epidemiological data from this region. In the present study, we performed a genome-wide analysis of the genetic diversity, virulence, and antimicrobial resistance of the largest collection of clinical C. jejuni and C. coli strains from Chile available to date (n = 81), collected in 2017-2019 in Santiago, Chile. This culture collection accounts for more than one third of the available genome sequences from South American clinical strains. cgMLST analysis identified high genetic diversity as well as 13 novel STs and alleles in both C. jejuni and C. coli. Pangenome and virulome analyses showed a differential distribution of virulence factors, including both plasmid and chromosomally encoded T6SSs and T4SSs. Resistome analysis predicted widespread resistance to fluoroquinolones, but low rates of erythromycin resistance. This study provides valuable genomic and epidemiological data and highlights the need for further genomic epidemiology studies in Chile and other South American countries to better understand molecular epidemiology and antimicrobial resistance of this emerging intestinal pathogen.

Prevalence, molecular typing and antimicrobial susceptibility of Campylobacter spp. isolates in northern Spain.

AIM: To analyse the prevalence, genetic diversity and antimicrobial susceptibility of Campylobacter spp. in northern Spain. METHODS AND RESULTS: Campylobacter was isolated from 139 samples of broiler meat and faecal dropping of broiler and swine with a prevalence of 35·4, 62 and 42·8%, respectively. Campylobacter jejuni (n = 55) and Campylobacter coli (n = 31) were identified by multiplex-PCR in meat, faeces and human clinical samples while Campylobacter fetus (n = 3) was exclusively detected in the latter. Fingerprinting by flaA-RFLP and PFGE revealed 68 different genotypes from the 89 isolates with a Biodiversity Simpson's index of 0·98. The 86·5% of the isolates were resistant to ciprofloxacin, 85·4% to tetracycline and 49·4% to erythromycin; only three genotypes were susceptible to the three antimicrobial drugs. Multidrug resistance was detected in the 40·7% of the isolates. CONCLUSIONS: Campylobacter remains prevalent in northern Spain with a high biodiversity degree. About 93·3% of the isolates were resistant to one or more drugs. SIGNIFICANCE AND IMPACT OF THE STUDY: Although different measures are taken to control Campylobacter, the detection of isolates resistant to the drugs used in the treatment of campylobacteriosis is still high, including different species and genotypes. This evidences the need of additional strategies against this pathogen.

Genetic environments and related transposable elements of novel cfr(C) variants in Campylobacter coli isolates of swine origin.

The cfr(C) is a cfr-like gene that confers cross-resistance to antibiotics targeting the 23S rRNA through methylation of nucleotide A2503. Here, we identified 7 C. coli isolates containing 4 novel cfr(C) variants from swine farm and slaughterhouses samples. Of the 7 cfr(C)-carrying isolates, one had a frame-shift mutation, while the other 6 had intact genes. However, one of the 6 intact genes did not show a PhLOPSA phenotype in the original isolate, but was fully functional when cloned into C. jejuni NCTC 11168. Cloning of cfr(C) variants into C. jejuni NCTC 11168 and conjugative transfer of the two cfr(C)-containing plasmids further confirmed their role in conferring resistance to PhLOPSA antimicrobials, and resulted in an 8-128-fold increase in their MICs. In all cfr(C)-carrying isolates, cfr(C) genes were located in the downstream of the kanamycin resistant gene aphA3. IS607* and IS1595-like were located immediately upstream of aphA3 gene and seemed to play a role in its recombination. A novel transposable element named ISCco7, which located immediately downstream of cfr(C) in two isolates, was probably associated with the integration of cfr(C). However, neither insertion sequence nor other transposable elements were identified near cfr(C) in the remaining five cfr(C)-positive isolates, indicating the mechanism underlying the integration of cfr(C) into plasmids or chromosomal DNA requires further investigation. These results reveal novel cfr(C) variants and their associated genetic environments in C. coli isolates and indicate the flexibility of C. coli in acquiring new antibiotic resistance genes.

Laboratory Study on the Gastroenteritis Outbreak Caused by a Multidrug-Resistant Campylobacter coli in China.

Three severe acute gastroenteritis patients were identified within a 5-h period in a sentinel hospital enrolled in the foodborne pathogen surveillance project in Beijing. All patients had high fever (over 38.5°C), diarrhea, abdominal pain, vomiting, and headache. Ten grams of fresh patient stool sample and 25 g of six suspected foods were collected for real-time PCR screening for 10 major pathogens. Bacterial isolation was performed. Pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and antibiotic susceptibility tests were conducted for all the isolates. Whole-genome sequences of the three Campylobacter coli isolates were compared using whole-genome MLST. All stool samples were positive for C. coli, as revealed by PCR. Eleven of the C. coli isolates had the same PFGE and ST type. All isolates were resistant to nalidixic acid, ciprofloxacin, streptomycin, and tetracycline, consistent with the findings of the in silico antibiotic resistance gene profiling. Most coding sequences (99%, 1736/1739) were identical among the three sequenced isolates, except for three frameshift-mutated genes caused by the simple sequence repeats (poly-Gs). This was likely a single-source outbreak caused by a group of highly clonal C. coli. This was the first outbreak of severe gastroenteritis caused by C. coli in China.

Pulsed-field gel electrophoresis fingerprinting of Campylobacter jejuni and Campylobacter coli strains isolated from clinical specimens, Iran.

The aim of this study was to determine the clonal correlation of Campylobacter strains isolated from diarrheal children under 5 years of age in Iran using the PFGE method and to determine the antimicrobial susceptibility and virulence gene content of strains. Of 750 patients with bacterial diarrhea, 33 (4%) Campylobacter spp., including 31 C. jejuni (94%) and 2 C. coli (6%), were isolated during 18-month period in Tehran, Iran. Except for one strain, remaining Campylobacter strains were positive for the flaA gene. A complete set of cytolethal distending toxin (CDT) encoding genes (cdtABC) were detected in 52% of the C. jejuni strains, while the 2 C. coli isolates under study only harbored cdtA and cdtB of the CDT cluster. All strains were resistant to at least three antibiotic classes. Resistance to ampicillin among C. coli and C. jejuni strains was 100% and 84%, respectively, and 80% of all strains were susceptible to gentamicin. PFGE genotyping generated 19 pulsotypes with two major clusters, displaying the maximum and minimum similarity of 100% and 26%, respectively. The C. coli strains showed clearly distinct pulsotypes and each fell within separate clusters. A very homogeneous Campylobacter population was detected among Iranian patients with 33 % of strains showing identical banding patterns and no clear correlation was observed between antibiotic resistance profiles and PFGE patterns of the isolates.

Proteotyping as alternate typing method to differentiate Campylobacter coli clades.

Besides Campylobacter jejuni, Campylobacter coli is the most common bacterial cause of gastroenteritis worldwide. C. coli is subdivided into three clades, which are associated with sample source. Clade 1 isolates are associated with acute diarrhea in humans whereas clade 2 and 3 isolates are more commonly obtained from environmental waters. The phylogenetic classification of an isolate is commonly done using laborious multilocus sequence typing (MLST). The aim of this study was to establish a proteotyping scheme using MALDI-TOF MS to offer an alternative to sequence-based methods. A total of 97 clade-representative C. coli isolates were analyzed by MALDI-TOF-based intact cell mass spectrometry (ICMS) and evaluated to establish a C. coli proteotyping scheme. MLST was used as reference method. Different isoforms of the detectable biomarkers, resulting in biomarker mass shifts, were associated with their amino acid sequences and included into the C. coli proteotyping scheme. In total, we identified 16 biomarkers to differentiate C. coli into the three clades and three additional sub-clades of clade 1. In this study, proteotyping has been successfully adapted to C. coli. The established C. coli clades and sub-clades can be discriminated using this method. Especially the clinically relevant clade 1 isolates can be differentiated clearly.

Phylogenetic Diversity and Antimicrobial Resistance of Campylobacter coli from Humans and Animals in Japan.

The phylogenetic diversity and antimicrobial resistance (AMR) of Campylobacter coli from humans and animals in Japan between 2008 and 2014 were investigated. A total of 338 foodborne campylobacterioses were reported in Osaka, and C. coli was isolated from 38 cases (11.2%). In the present study, 119 C. coli strains (42 from humans, 25 each from poultry, cattle, and swine, and 2 from wild mallard) were examined by multilocus sequence typing (MLST). MLST assigned 36 sequence types (STs), including 14 novel STs; all human strains and 91% of animal strains (70/77) were assigned to the ST-828 clonal complex. The predominant human ST was ST-860 (18/42, 43%), followed by ST-1068 (8/42, 19%); these STs were also predominant in poultry (ST-860, 9/25, 36%) and cattle (ST-1068, 18/25, 72%). ST-1562 was only predominant in swine (11/25, 44.0%). Swine strains showed the greatest resistance to erythromycin (EM; 92.0%), while EM resistance was only found in 2 out of the 42 human strains examined (4.8%). All EM-resistant swine strains (n=15) exhibited a common point mutation in the 23S rRNA sequence (A2085G), and the tetO gene was detected in 22 out of the 23 TET-resistant swine strains. A whole genome sequencing analysis of four representative swine ST-1562 strains revealed abundant AMR-associated gene clusters in their genomes, suggesting horizontal gene transfer events during host adaptation. This is the first study to demonstrate the phylogenetic diversity and AMR profiles of C. coli in Japan. The present results suggest that poultry and cattle are major reservoirs, improving our knowledge on the epidemiological and ecological traits of this pathogen.

Whole-Genome Sequence Analysis of Multidrug-Resistant Campylobacter Isolates: a Focus on Aminoglycoside Resistance Determinants.

A whole-genome sequencing (WGS) approach was conducted in order to identify the molecular determinants associated with antimicrobial resistance in 12 multidrug-resistant Campylobacter jejuni and Campylobacter coli isolates, with a focus on aminoglycoside resistance determinants. Two variants of a new aminoglycoside phosphotransferase gene [aph(2″)-Ii1 and aph(2″)-Ii2 ] putatively associated with gentamicin resistance were found. In addition, the following new genes were identified for the first time in Campylobacter: a lincosamide nucleotidyltransferase gene [lnu(G)], likely associated with lincomycin resistance, and two resistance enzyme genes (spw and apmA) similar to those found in Staphylococcus aureus, which may confer spectinomycin and gentamicin resistance, respectively. A C1192T mutation of the 16S rRNA gene that may be involved in spectinomycin resistance was also found in a C. coli isolate. Genes identified in the present study were located either on the bacterial chromosome or on plasmids that could be transferred naturally. Their role in aminoglycoside resistance remains to be supported by genetic studies. Regarding the other antimicrobial agents studied, i.e., ampicillin, ciprofloxacin, erythromycin, and tetracycline, a perfect correlation between antimicrobial phenotypes and genotypes was found. Overall, our data suggest that WGS analysis is a powerful tool for identifying resistance determinants in Campylobacter and can disclose the full genetic elements associated with resistance, including antimicrobial compounds not tested routinely in antimicrobial susceptibility testing.

Multilocus sequence typing of Campylobacter jejuni and Campylobacter coli to identify potential sources of colonization in commercial turkey farms.

Poultry are the main reservoir for thermophilic Campylobacter spp., which is the most common causative agent of human bacterial gastroenteritis. The epidemiology of Campylobacter in poultry, particularly in turkeys, is not completely understood. This study aimed at identifying potential sources and transmission routes of thermophilic Campylobacter spp. in commercial turkey farms. C. jejuni and C. coli isolates from breeders (n = 29, 20 C. jejuni and 9 C. coli) and their progeny (n = 51, 18 C. jejuni and 33 C. coli) reared in two different farms for three sequential production cycles were analysed by multilocus sequence typing (MLST). Strains (n = 88, 42 C. jejuni and 46 C. coli) isolated from environmental (i.e. anteroom and in-house overshoes), water (i.e. drinkers and water line), and pest (i.e. flies, Alphitobius diaperinus, and mice) sources were also examined. MLST of C. jejuni and C. coli isolates resulted in 13 and 12 different sequence types (STs) belonging to six and one previously-described clonal complexes (CCs), respectively. Three novel STs were identified. Genetic similarities were detected between isolates from fattening turkeys and the considered environmental, water, and pest sources, and with the breeders to a lesser extent. Source attribution analysis estimated that environmental and water sources accounted for most (∼75%) of fattening turkey isolates and were therefore identified as the most likely sources of flock colonization, followed by pests (∼20%) and breeders (∼5%). These sources may thus be targeted by control measures to mitigate the risk of Campylobacter colonization in commercial turkeys. RESEARCH HIGHLIGHTS High occurrence of C. jejuni and C. coli in commercial turkey flocks. High genetic diversity of C. jejuni and C. coli in commercial turkey flocks. Horizontal transmission responsible for Campylobacter colonization of commercial turkey flocks. Environmental and water sources involved in Campylobacter colonization of commercial turkey flocks. Strategies for prevention and control of Campylobacter colonization of commercial turkey flocks are needed.

High survival rates of Campylobacter coli under different stress conditions suggest that more rigorous food control measures might be needed in Brazil.

Campylobacter spp. have been the most commonly reported gastrointestinal bacterial pathogen in many countries. Consumption of improperly prepared poultry meat has been the main transmission route of Campylobacter spp. Although Brazil is the largest exporter of poultry meat in the world, campylobacteriosis has been a neglected disease in the country. The aim of this study was to characterize 50 Campylobacter coli strains isolated from different sources in Brazil regarding the frequency of 16 virulence genes and their survival capability under five different stress conditions. All strains studied presented the cadF, flaA, and sodB genes that are considered essential for colonization. All strains grew at 4 °C and 37 °C after 24 h. High survival rates were observed when the strains were incubated in BHI with 7.5% NaCl and exposed to acid and oxidative stress. In conclusion, the pathogenic potential of the strains studied was reinforced by the presence of several important virulence genes and by the high growth and survival rates of the majority of those strains under different stress conditions. The results enabled a better understanding of strains circulating in Brazil and suggest that more rigorous control measures may be needed, given the importance of contaminated food as vehicles for Campylobacter coli.

Characterization of Swedish Campylobacter coli clade 2 and clade 3 water isolates.

Campylobacter jejuni and Campylobacter coli are important bacterial enteropathogens. Poultry is the best-known reservoir for Campylobacter infection but natural bodies of water have also been shown to be important pathways for transmission. Campylobacter can survive in cold water but most of the studies have focused on C. jejuni only. In this paper, we take a closer look at the biology and water survival strategies of C. coli. Eight C. coli isolates cultivated from raw (incoming) surface water at water plants in Sweden were characterized using whole-genome sequencing and phenotypical assays. Phylogenetic analysis assigned the Swedish water isolates to clades 2 and 3, known to include C. coli of environmental origin. In addition, 53 earlier published sequences of C. coli clade 2 and 3 from environmental waters were included for in silico analyses. Generally, clade 2 isolates had larger genomes, which included a functional tricarballylate utilization locus, while clade 3 isolates contained different genes involved in oxidative stress as well as putative virulence factors. The Swedish water isolates of clade 2 formed large, blurry bacterial colonies on agar, whereas clade 3 colonies were smaller. All Swedish isolates were motile, but clade 3 isolates formed larger motility zones on soft agar, and none of these isolates produced biofilm. Although water survival varied between the analyzed isolates, there were hardly any clade-specific significant differences. Our results highlight the diversity of C. coli in general, and show differences in metabolic capabilities and ways to handle oxidative stress between clade 2 and 3 water isolates.

High Prevalence of Resistance to Fluoroquinolones and Tetracycline Campylobacter Spp. Isolated from Poultry in Poland.

Campylobacter spp. is a major cause of foodborne diseases in humans, particularly when transmitted by the handling or consumption of undercooked poultry meat. Most Campylobacter infections are self-limiting, but antimicrobial treatment (e.g., fluoroquinolones and macrolides) is necessary in severe or prolonged cases. The indiscriminate use of these drugs, both in clinical medicine and animal production, has a major impact on public health. The aim of the present study was to identify Campylobacter strains, isolated from turkey and broilers, using both PCR and the matrix-assisted laser desorption-ionization time-of-flight (MALDI-TOF) methods to reveal the accuracy of identification, as well to evaluate the antimicrobial and genetic resistance of the investigated strains. MALDI-TOF and PCR methods were used to show differences, if any, in the specificity of that test. In this study, MALDI-TOF mass spectrometry gave the same results as multiplex PCR, in all cases. The highest rate of resistance (i.e., 100% of turkey and broiler strains) was detected against ciprofloxacin, whereas 58.1% of turkey and 78.6% of broiler strains were resistant to tetracycline. Multidrug-resistant isolates were not found in the study. All ciprofloxacin-resistant strains had a mutation in the gyrA gene, at the Thr-86 position. The presence of the tetO gene was found in 71% of turkey and in 100% of broiler strains. All resistant to tetracycline strains included tetO gene. Additionally, in five turkey and three broiler strains, susceptible to tetracycline, tetO gene was present. These results indicate the high prevalence of Campylobacter strains, which are phenotypically and genetically resistant to fluoroquinolones and tetracycline.

Core Genome Multilocus Sequence Typing Scheme for Stable, Comparative Analyses of Campylobacter jejuni and C. coli Human Disease Isolates.

Human campylobacteriosis, caused by Campylobacter jejuni and C. coli, remains a leading cause of bacterial gastroenteritis in many countries, but the epidemiology of campylobacteriosis outbreaks remains poorly defined, largely due to limitations in the resolution and comparability of isolate characterization methods. Whole-genome sequencing (WGS) data enable the improvement of sequence-based typing approaches, such as multilocus sequence typing (MLST), by substantially increasing the number of loci examined. A core genome MLST (cgMLST) scheme defines a comprehensive set of those loci present in most members of a bacterial group, balancing very high resolution with comparability across the diversity of the group. Here we propose a set of 1,343 loci as a human campylobacteriosis cgMLST scheme (v1.0), the allelic profiles of which can be assigned to core genome sequence types. The 1,343 loci chosen were a subset of the 1,643 loci identified in the reannotation of the genome sequence of C. jejuni isolate NCTC 11168, chosen as being present in >95% of draft genomes of 2,472 representative United Kingdom campylobacteriosis isolates, comprising 2,207 (89.3%) C. jejuni isolates and 265 (10.7%) C. coli isolates. Validation of the cgMLST scheme was undertaken with 1,478 further high-quality draft genomes, containing 150 or fewer contiguous sequences, from disease isolate collections: 99.5% of these isolates contained ≥95% of the 1,343 cgMLST loci. In addition to the rapid and effective high-resolution analysis of large numbers of diverse isolates, the cgMLST scheme enabled the efficient identification of very closely related isolates from a well-defined single-source campylobacteriosis outbreak.

Genetic diversity and antimicrobial resistance profiles of Campylobacter coli and Campylobacter jejuni isolated from broiler chicken in farms and at time of slaughter in central Italy.

AIMS: Genetic diversity and antimicrobial resistance of Campylobacter coli and Campylobacter jejuni were investigated along the broiler chicken production chain in central Italy. METHODS AND RESULTS: Campylobacter sp. isolated from cloacal swabs in farms (n = 116) and from the neck skin of chilled and eviscerated carcasses at slaughter (n = 24) were identified as C. coli (n = 99) and C. jejuni (n = 41) by multiplex PCR. Characterization by single amplified fragment length polymorphism (s-AFLP) revealed a specific genotype of Campylobacter for each farm. Minimal inhibitory concentration showed high prevalence of fluoroquinolones (70%), tetracycline (70%) and erythromycin (30%) resistance among C. coli isolates. Campylobacter jejuni isolates showed lower prevalence of fluoroquinolone (39%) and tetracycline (10%) resistance, and all isolates were susceptible to erythromycin. The S-AFLP types of the C. coli and C. jejuni isolates were associated with their antimicrobial resistance profiles (P < 0·001). CONCLUSIONS: The genetic diversity detected in Campylobacter isolates suggested that a specific genotype was harboured in each farm. A considerable number of C. coli isolates were resistant to erythromycin. SIGNIFICANCE AND IMPACT OF THE STUDY: Campylobacter coli was detected more frequently than C. jejuni in contrast to common findings for poultry. The high prevalence of 30% resistance to erythromycin in C. coli strains isolated from poultry is worrisome, as this is the first antibiotic of choice to treat human campylobacteriosis.

Exploiting Bacterial Whole-Genome Sequencing Data for Evaluation of Diagnostic Assays: Campylobacter Species Identification as a Case Study.

The application of whole-genome sequencing (WGS) to problems in clinical microbiology has had a major impact on the field. Clinical laboratories are now using WGS for pathogen identification, antimicrobial susceptibility testing, and epidemiological typing. WGS data also represent a valuable resource for the development and evaluation of molecular diagnostic assays, which continue to play an important role in clinical microbiology. To demonstrate this application of WGS, this study used publicly available genomic data to evaluate a duplex real-time PCR (RT-PCR) assay that targets mapA and ceuE for the detection of Campylobacter jejuni and Campylobacter coli, leading global causes of bacterial gastroenteritis. In silico analyses of mapA and ceuE primer and probe sequences from 1,713 genetically diverse C. jejuni and C. coli genomes, supported by RT-PCR testing, indicated that the assay was robust, with 1,707 (99.7%) isolates correctly identified. The high specificity of the mapA-ceuE assay was the result of interspecies diversity and intraspecies conservation of the target genes in C. jejuni and C. coli Rare instances of a lack of specificity among C. coli isolates were due to introgression in mapA or sequence diversity in ceuE The results of this study illustrate how WGS can be exploited to evaluate molecular diagnostic assays by using publicly available data, online databases, and open-source software.