Molecular Epidemiology of Campylobacter Isolates from Poultry Production Units in Southern Ireland

This study aimed to identify the sources and routes of transmission of Campylobacter in intensively reared poultry farms in the Republic of Ireland. Breeder flocks and their corresponding broilers housed in three growing facilities were screened for the presence of Campylobacter species from November 2006 through September 2007. All breeder flocks tested positive for Campylobacter species (with C. jejuni and C. coli being identified). Similarly, all broiler flocks also tested positive for Campylobacter by the end of the rearing period. Faecal and environmental samples were analyzed at regular intervals throughout the rearing period of each broiler flock. Campylobacter was not detected in the disinfected house, or in one-day old broiler chicks. Campylobacter jejuni was isolated from environmental samples including air, water puddles, adjacent broiler flocks and soil. A representative subset of isolates from each farm was selected for further characterization using flaA-SVR sub-typing and multi-locus sequence typing (MLST) to determine if same-species isolates from different sources were indistinguishable or not. Results obtained suggest that no evidence of vertical transmission existed and that adequate cleaning/disinfection of broiler houses contributed to the prevention of carryover and cross-contamination. Nonetheless, the environment appears to be a potential source of Campylobacter. The population structure of Campylobacter isolates from broiler farms in Southern Ireland was diverse and weakly clonal

Functional Characterization of a Lipoprotein-Encoding Operon in Campylobacter jejuni

Background: Bacterial lipoproteins have important functions in bacterial pathogenesis and physiology. In Campylobacter jejuni, a major foodborne pathogen causing gastroenteritis in humans, the majority of lipoproteins have not been functionally characterized. Previously, we showed by DNA microarray that CmeR, a transcriptional regulator repressing the expression of the multidrug efflux pump CmeABC, modulates the expression of a three-gene operon (cj0089, cj0090, and cj0091) encoding a cluster of lipoproteins in C. jejuni. Methodology/Principal Findings: In this work, we characterized the function and regulation of the cj0089-cj0090-cj0091 operon. In contrast to the repression of cmeABC, CmeR activates the expression of the lipoprotein genes and the regulation is confirmed by immunoblotting using anti-Cj0089 and anti-Cj0091 antibodies. Gel mobility shift assay showed that CmeR directly binds to the promoter of the lipoprotein operon, but the binding is much weaker compared with the promoter of cmeABC. Analysis of different cellular fractions indicated that Cj0089 was associated with the inner membrane, while Cj0091 was located on the outer membrane. Inactivation of cj0091, but not cj0089, significantly reduced the adherence of C. jejuni to INT 407 cells in vitro, indicating that Cj0091 has a function in adherence. When inoculated into chickens, the Cj0091 mutant also showed a defect in early colonization of the intestinal tract, suggesting that Cj0091 contributes to Campylobacter colonization in vivo. It was also shown that Cj0091 was produced and immunogenic in chickens that wer

Distribution and Genetic Profiles of Campylobacter in Commercial Broiler Production from Breeder to Slaughter in Thailand

Poultry and poultry products are commonly considered as the major vehicle of Campylobacter infection in humans worldwide. To reduce the number of human cases, the epidemiology of Campylobacter in poultry must be better understood. Therefore, the objective of the present study was to determine the distribution and genetic relatedness of Campylobacter in the Thai chicken production industry. During June to October 2012, entire broiler production processes (i.e., breeder flock, hatchery, broiler farm and slaughterhouse) of five broiler production chains were investigated chronologically. Representative isolates of C. jejuni from each production stage were characterized by flaA SVR sequencing and multilocus sequence typing (MLST). Amongst 311 selected isolates, 29 flaA SVR alleles and 17 sequence types (STs) were identified. The common clonal complexes (CCs) found in this study were CC-45, CC-353, CC-354 and CC-574. C. jejuni isolated from breeders were distantly related to those isolated from broilers and chicken carcasses, while C. jejuni isolates from the slaughterhouse environment and meat products were similar to those isolated from broiler flocks. Genotypic identification of C. jejuni in slaughterhouses indicated that broilers were the main source of Campylobacter contamination of chicken meat during processing. To effectively reduce Campylobacter in poultry meat products, control and prevention strategies should be aimed at both farm and slaughterhouse levels

Evaluation of flaA short variable region sequencing, multilocus sequence typing and Fourier transform infrared spectroscopy for discrimination between Campylobacter jejuni strains

Discriminatory and robust typing methods are needed to improve the understanding of the dynamics of food-borne Campylobacter infections and epidemiology in primary animal production. To evaluate the strain discriminatory potential of typing methods, flaA short variable region (SVR) sequencing and Fourier transform infrared (FTIR) spectroscopy were applied on a collection of 102 epidemiologically related and unrelated Campylobacter jejuni strains. Previous application of FTIR spectroscopy for subtyping of Campylobacter has been limited. A subset of isolates, initially discriminated by flaA SVR sequencing, were further subjected to multilocus sequence typing (MLST). It was found that flaA SVR sequencing had a slightly higher discriminatory power than FTIR spectroscopy, based on the Simpson diversity index. The clustering of strains indicated that FTIR spectroscopy is indeed a suitable method for discrimination of Campylobacter. The isolates were assigned to six clusters based on flaA SVR sequences and nine clusters based on the FTIR spectroscopy profiles. Furthermore, the cluster analysis of flaA SVR sequences, MLST, and FTIR spectroscopy profiles showed a high degree of congruence, assigning the isolates to similar cluster structures. In conclusion, FTIR spectroscopy can be applied for subtyping of Campylobacter, and the high discriminatory potential of both flaA SVR sequencing and FTIR spectroscopy render them suitable screening methods for large numbers of strains

Differentiation of Campylobacter jejuni and Campylobacter coli Using Multiplex-PCR and High Resolution Melt Curve Analysis

Campylobacter spp. are important causes of bacterial gastroenteritis in humans in developed countries. Among Campylobacter spp. Campylobacter jejuni (C. jejuni) and C. coli are the most common causes of human infection. In this study, a multiplex PCR (mPCR) and high resolution melt (HRM) curve analysis were optimized for simultaneous detection and differentiation of C. jejuni and C. coli isolates. A segment of the hippuricase gene (hipO) of C. jejuni and putative aspartokinase (asp) gene of C. coli were amplified from 26 Campylobacter isolates and amplicons were subjected to HRM curve analysis. The mPCR-HRM was able to differentiate between C. jejuni and C. coli species. All DNA amplicons generated by mPCR were sequenced. Analysis of the nucleotide sequences from each isolate revealed that the HRM curves were correlated with the nucleotide sequences of the amplicons. Minor variation in melting point temperatures of C. coli or C. jejuni isolates was also observed and enabled some intraspecies differentiation between C. coli and/or C. jejuni isolates. The potential of PCR-HRM curve analysis for the detection and speciation of Campylobacter in additional human clinical specimens and chicken swab samples was also confirmed. The sensitivity and specificity of the test were found to be 100% and 92%, respectively. The results indicated that mPCR followed by HRM curve analysis provides a rapid (8 hours) technique for differentiation between C. jejuni and C. coli isolates