Diversity Of Campylobacter Jejuni Research Articles

Whole genome-based characterisation of antimicrobial resistance and genetic diversity in Campylobacter jejuni and Campylobacter coli from ruminants

Campylobacter, a leading cause of gastroenteritis in humans, asymptomatically colonises the intestinal tract of a wide range of animals.Although antimicrobial treatment is restricted to severe cases, the increase of antimicrobial resistance (AMR) is a concern. Considering the significant contribution of ruminants as reservoirs of resistant Campylobacter, Illumina whole-genome sequencing was used to characterise the mechanisms of AMR in Campylobacter jejuni and Campylobacter coli recovered from beef cattle, dairy cattle, and sheep in northern Spain. Genome analysis showed extensive genetic diversity that clearly separated both species. Resistance genotypes were identified by screening assembled sequences with BLASTn and ABRicate, and additional sequence alignments were performed to search for frameshift mutations and gene modifications. A high correlation was observed between phenotypic resistance to a given antimicrobial and the presence of the corresponding known resistance genes. Detailed sequence analysis allowed us to detect the recently described mosaic tet(O/M/O) gene in one C. coli, describe possible new alleles of blaOXA-61-like genes, and decipher the genetic context of aminoglycoside resistance genes, as well as the plasmid/chromosomal location of the different AMR genes and their implication for resistance spread. Updated resistance gene databases and detailed analysis of the matched open reading frames are needed to avoid errors when using WGS-based analysis pipelines for AMR detection in the absence of phenotypic data.

Detection of Genetic Diversity in Campylobacter jejuni Isolated from a Commercial Turkey Flock Using flaA Typing, MLST Analysis and Microarray Assay

Campylobacter is genetically highly diverse and undergoes frequent intraspecific recombination. Turkeys have been identified as an important reservoir for Campylobacter jejuni which is of public health significance. The assessment of the genetic diversity among Campylobacter population is critical for our understanding of the epidemiology of this bacterium. The genetic profiles were different according to the molecular typing methods used. The performance of established flaA genotyping, multilocus sequencing typing (MLST) and DNA microarray assay based on the ArrayTube™ technology was evaluated using 14 Campylobacter jejuni isolated from a commercial turkey flock. The flaA typing was performed using PCR-RFLP with restriction enzymes Sau3AI, AluI, a ‘composite’ flaA analysis of AluI and Sau3AI and DdeI. The 14 isolates were differentiated into 3, 5, 7 and 9 genotypes, respectively. Entire flaA gene and short variable region (SVR) sequences were analysed. Sequencing of the entire flaA provided 11 different genotypes. flaA-SVR sequence analysis detected 8 flaA alleles and 4 flaA peptides. One new flaA allele type (528) was identified. MLST analysis represented 10 different sequence types (STs) and 5 clonal complexes (CCs). The microarray assay recognised 14 different genotypes. The discriminatory indices were 0.560, 0.802, 0.857, and 0.912 for flaA-RFLP depending on the used enzymes, 0.890 for flaA-SVR, 0.967 for entire flaA sequencing, 0.945 for MLST and 1.00 for the DNA microarray assay. The flaA gene was genetically stable over 20 passages on blood agar. In conclusion, the different typing tools demonstrated a high level of genetic heterogeneity of Campylobacter jejuni in a turkey flock, indicating that a single flock can be infected by multiple genotypes within one rearing cycle. DNA microarray-based assays had the highest discriminatory power when compared with other genotyping tools.

Genetic diversity in Campylobacter jejuni is associated with differential colonization of broiler chickens and C57BL/6J IL10-deficient mice

Previous studies have demonstrated that Campylobacter jejuni, the leading causative agent of bacterial food-borne disease in the USA, exhibits high-frequency genetic variation that is associated with changes in cell-surface antigens and ability to colonize chickens. To expand our understanding of the role of genetic diversity in the disease process, we analysed the ability of three C. jejuni human disease isolates (strains 11168, 33292 and 81-176) and genetically marked derivatives to colonize Ross 308 broilers and C57BL/6J IL10-deficient mice. C. jejuni colonized broilers at much higher efficiency (all three strains, 23 of 24 broilers) than mice (11168 only, 8 of 24 mice). C. jejuni 11168 genetically marked strains colonized mice at very low efficiency (2 of 42 mice); however, C. jejuni reisolated from mice colonized both mice and broilers at high efficiency, suggesting that this pathogen can adapt genetically in the mouse. We compared the genome composition in the three wild-type C. jejuni strains and derivatives by microarray DNA/DNA hybridization analysis; the data demonstrated a high degree of genetic diversity in three gene clusters associated with synthesis and modification of the cell-surface structures capsule, flagella and lipo-oligosaccharide. Finally, we analysed the frequency of mutation in homopolymeric tracts associated with the contingency genes wlaN (GC tract) and flgR (AT tracts) in culture and after passage through broilers and mice. C. jejuni adapted genetically in culture at high frequency and the degree of genetic diversity was increased by passage through broilers but was nearly eliminated in the gastrointestinal tract of mice. The data suggest that the broiler gastrointestinal tract provides an environment which promotes outgrowth and genetic variation in C. jejuni; the enhancement of genetic diversity at this location may contribute to its importance as a human disease reservoir.

Diversity of Campylobacter jejuni and Campylobacter coli Genotypes from Human and Animal Sources from Rio de Janeiro, Brazil

To compare the genotypes of Campylobacter jejuni and Campylobacter coli isolates of human and animal origin collected in Rio de Janeiro City, 30 C. jejuni and 35 C. coli isolates from animal sources ( n = 45) and human patients with gastroenteritis ( n = 20) were genotyped by PCR-based techniques, namely random amplified polymorphic DNA (RAPD-PCR) and enterobacterial repetitive intergenic consensus sequence (ERIC-PCR). RAPD-PCR identified 50 types and ERIC-PCR identified 22 genotypes, among the 65 Campylobacter isolates. Both PCR methods discriminated the C. jejuni and C. coli groups of isolates. Combining the results of both methods, no single genotype was shared between isolates from human and animal sources. Two groups of two C. coli isolates each with identical genotypes were found among poultry and pig isolates. A high level of genetic diversity observed among the Campylobacter isolates suggests lack of overlap between isolates from different sources.

Genotyping of Campylobacter jejuni from Broiler Carcasses and Slaughterhouse Environment by Amplified Fragment Length Polymorphism

We examined the occurrence and diversity of Campylobacter jejuni on broiler carcasses during slaughter of an infected flock and in the slaughterhouse environment during slaughter and postdisinfection before a new production run. During the slaughter of a known C. jejuni infected broiler flock, samples were taken from broiler carcasses at 7 different stages during the process. Thirty-seven sites in the slaughterhouse environment were sampled both during process and postdisinfection. The samples were analyzed for C. jejuni, and genetic fingerprinting was performed using amplified fragment length polymorphism. All carcass samples were positive. Of the environmental samples collected during slaughter, 89% were positive; 100% of those from the arrival, stunning, scalding, defeathering, and evisceration facilities and 67% of those from the cooling and sorting facilities. Postdisinfection, 41% of the samples were positive; 71% of those from the arrival and stunning area, 60% of those from the scalding and defeathering area, and 20% of those from the evisceration, cooling, and sorting area. The C. jejuni isolates (n = 60) recovered were grouped into 4 different amplified fragment length polymorphism clones with a similarity index of 95% or greater. All isolates obtained from the flock and 94% of the isolates obtained from the environment during slaughtering belonged to clone A, whereas 1 environmental isolate belonged to each of the clones B and C. Isolates from clones A, B, and D were present postdisinfection. Only clone B was detected on flocks slaughtered during the previous week. The high level and continuous presence of Campylobacter in the environment constitutes a risk for transmission to negative carcasses. In Norway, where above 96% of the broiler flocks are Campylobacter-negative, this aspect is of special importance. The ability of Campylobacter to remain in the slaughterhouse environment through washing and disinfection is associated with constructional conditions of equipment and buildings, complicating cleaning and providing sufficient moisture. To reduce the probability of the workers acquiring campylobacteriosis, precautions should be taken when slaughtering Campylobacter-positive flocks.

Prevalence and Diversity of Campylobacter jejuni in Pig Herds on Farms with and without Cattle or Poultry

The prevalence and diversity of Campylobacter jejuni was investigated in pig herds on farms with and without cattle or poultry production. A bacteriological screening of pig cecal samples from 247 finisher herds was carried out at the slaughterhouse. Subsequently, a follow-up study was conducted in 24 herds (either with or without prior C. jejuni isolation from pigs) in which fecal samples were collected from pigs and, if present, cattle and poultry. Samples were analyzed for presence of Campylobacter, and subsequent analysis included species identification, serotyping, and, for selected strains, pulsed-field gel electrophoresis typing. In the slaughterhouse screening, C. jejuni was isolated from pigs in 21 (8.5%) herds, but no significant difference in prevalence was found between herd types (pigs, pigs and cattle, pigs and poultry). At the slaughterhouse, C. jejuni and Campylobacter coli prevalence in pigs was 2.3 and 90.1%, respectively. In the follow-up study, herd prevalence of C. jejuni was 8.3%, whereas C. jejuni and C. coli were isolated from 0.8 and 92.0% of pigs, respectively. In mixed production herds, C. jejuni predominated in cattle (42.7%) and poultry (31.6%), whereas C. jejuni was only isolated from 1.3 to 2.5% of pigs in these herds. There were no significant differences in C. jejuni or C. coli prevalence in pigs, cattle, and poultry between herds with and without prior C. jejuni isolation at the slaughterhouse. Pulsed-field gel electrophoresis typing did not yield evidence of C. jejuni transmission between cattle or poultry and pigs in mixed production herds. In contrast, pulsed-field gel electrophoresis analysis showed indistinguishable serotypes of C. coli in pigs and cattle in two herds. Verification of C. jejuni–positive pig samples showed that individual pigs can excrete high levels of C. jejuni and that mixed infection with C. jejuni and C. coli was common in C. jejuni–positive pigs. The results of our study suggest that transmission of C. jejuni between pigs and cattle or poultry in mixed production herds occurs infrequently. Detection of indistinguishable C. coli isolates in two herds, however, might indicate the existence of low-level transmission between pigs and cattle in herds of mixed production.

Prevalence and clonal diversity of Campylobacter jejuni from dairy farms and urban sources

AIM: To investigate the role of free-living animals such as spar- rows, rodents and flies as potential reservoirs of Campylobacter spp on a dairy farm, and to assess the genetic diversity among Campylobacter isolates from the farm and an urban source. METHODS: A total of 290 samples (bovine, passerine and ro- dent faeces, and whole flies) were collected from a large com- mercial dairy farm in the Manawatu district in New Zealand, and from faeces from urban sparrows in a nearby city. Other samples collected from the dairy farm included five from silage, two from aprons worn by workers during milking, two from workers' boots and two from water in troughs in a paddock. Isolates of thermophilic Campylobacter spp were identified mor- phologically and phenotypically and further characterised mo- lecularly using pulsed-field gel electrophoresis (PFGE) and the restriction enzyme SmaI. RESULTS: Campylobacter jejuni was the only Campylobacter species isolated from all samples. The highest prevalence was found in faeces from dairy cows (54%), followed by faeces from sparrows from the urban area (40%) and the farm (38%), and from rodents (11%) and whole flies (9%). Other samples from the farm environment such as silage, trough water, and work- ers' aprons and boots were also positive for C. jejuni. Of the 22 restriction patterns obtained, seven were common to more than one source. CONCLUSIONS: Cattle, sparrows, rodents and flies are po- tential reservoirs of C. jejuni on dairy farms. Identical clones of C. jejuni carried by cattle, sparrows, flies and rodents prob- ably indicate a common source of infection. The high level of asymptomatic carriage of C. jejuni by healthy dairy cows could be sufficient to maintain infections within the dairy farm sur- roundings via environmental contamination.

Structural and antigenic properties of lipopolysaccharides from serotype reference strains of Campylobacter jejuni.

To investigate the molecular basis for heat-stable antigenic diversity in Campylobacter jejuni, lipopolysaccharides (LPSs) from serotype reference strains and serotyped isolates were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis coupled with silver staining and immunoblotting. By silver staining, only low-Mr components, consisting of one major band and as many as three minor bands ranging in Mr from 4,500 to 5,000, were detected. However, by immunoblotting with homologous antisera, 10 of 34 strains were shown to have a series of high-Mr LPS components characteristic of molecules with O side chains of various lengths. Isolates of the same serotype as the reference strain that had high-Mr LPS molecules were also found to have high-Mr LPS and in one case of cross-reacting strains it was found that the cross-reaction was associated with antibodies against high-Mr LPS. The reactions of LPSs with homologous and heterologous antisera indicated that both high- and low-Mr-type LPSs were strain-specific antigens, but in some cases cross-reactions were noted. Evidently, all C. jejuni strains possess low-Mr LPS that is readily detectable by silver staining, but some serotypes also possess high-Mr LPS components that can be visualized by immunoblotting.