Effects of DNA extraction methods on the real time PCR quantification of Campylobacter jejuni, Campylobacter coli, and Campylobacter lari in chicken feces and ceca contents.

Polymerase chain reaction (PCR) method was coupled with a DNA extraction to enumerate Campylobacter spp. from poultry gastrointestinal tract samples. Three experiments were conducted that included: 1) Development of a DNA standard curve related to bacterial DNA primers; 2) Design of a cell/genomic DNA extraction protocol to isolate Campylobacter spp. DNA from complex samples such as poultry feces; and 3) Comparison of PCR quantification to standard plate count methodology. The standard curve using primers for Campylobacter spp. was created for DNA extracted from environmental isolates with a linear range (R2 > 0.95) and with a high specificity for C. coli and C. jejuni recovered from poultry, swine and laboratory isolates. A 2-step extraction process of bacterial DNA from poultry feces was developed in which the cells were first concentrated using a gradient-centrifugation step followed by comparison of 4 DNA extraction methods. Two commercial DNA extraction methods (Zymo Research Quick DNA, and Invitrogen magnetic separation), a traditional phenol-chloroform DNA extraction method using proteinase K to inactivate DNAses, and an in-house isolation method for DNA extraction based on chaotropic salts were used. The middle gradient layer recovered 89% to 98% of the bacteria cells from the sample, with recovery dependent upon the Campylobacter genus. The 4 DNA extractions methods recovered 112 to 302 ug/nL of DNA. Finally, the qPCR and standard plate methods were highly correlated for enumerating Campylobacter spp. in the 2.0 to 8.0-log CFU range. Analyses of the results from this study demonstrate that the combination of the standard curve for Campylobacter spp. DNA primers, the gradient cell concentration method and DNA extraction techniques with qPCR can be used to enumerate Campylobacter spp. from poultry samples with findings similar those of traditional plate count methodology.

Genomic Diversity of Campylobacter lari Group Isolates from Europe and Australia in a One Health Context.

Members of the Campylobacter lari group are causative agents of human gastroenteritis and are frequently found in shellfish, marine waters, shorebirds, and marine mammals. Within a One Health context, we used comparative genomics to characterize isolates from a diverse range of sources and geographical locations within Europe and Australia and assess possible transmission of food, animal, and environmental isolates to the human host. A total of 158 C. lari isolates from Australia, Denmark, France, and Germany, which included 82 isolates from human stool and blood, 12 from food, 14 from domestic animal, 19 from waterbirds, and 31 from the environment were analyzed. Genome-wide analysis of the genetic diversity, virulence, and antimicrobial resistance (AMR) traits was carried-out. Most of the isolates belonged to C. lari subsp. lari (Cll; 98, 62.0%), while C. lari subsp. concheus and C. lari urease-positive thermotolerant Campylobacter (UPTC) were represented by 12 (7.6%) and 15 (9.5%) isolates, respectively. Furthermore, 33 (20.9%) isolates were not assigned a subspecies and were thus attributed to distant Campylobacter spp. clades. Whole-genome sequence-derived multilocus sequence typing (MLST) and core-genome MLST (cgMLST) analyses revealed a high genetic diversity with 97 sequence types (STs), including 60 novel STs and 14 cgMLST clusters (≤10 allele differences), respectively. The most prevalent STs were ST-21, ST-70, ST-24, and ST-58 (accounting for 13.3%, 4.4%, 3.8%, and 3.2% of isolates, respectively). A high prevalence of the 125 examined virulence-related loci (from 76.8 to 98.4% per isolate) was observed, especially in Cll isolates, suggesting a probable human pathogenicity of these strains. IMPORTANCE Currently, relatedness between bacterial isolates impacting human health is easily monitored by molecular typing methods. These approaches rely on discrete loci or whole-genome sequence (WGS) analyses. Campylobacter lari is an emergent human pathogen isolated from diverse ecological niches, including fecal material from humans and animals, aquatic environments, and seafood. The presence of C. lari in such diverse sources underlines the importance of adopting an integrated One Health approach in studying C. lari population structure for conducting epidemiological risk assessment. This retrospective study presents a comparative genomics analysis of C. lari isolates retrieved from two different continents (Europe and Australia) and from different sources (human, domestic animals, waterbirds, food, and environment). It was designed to improve knowledge regarding C. lari ecology and pathogenicity, important for developing effective surveillance and disease prevention strategies.

Validation of RapidChek®Campylobacter Test System for the Detection of C. jejuni, C. coli, and C. lari in Poultry Samples: AOAC Performance Tested MethodSM 052201.

BACKGROUND: Campylobacter is one of the leading causes of human bacterial gastroenteritis worldwide. Campylobacter infections are most often associated with the consumption of raw milk, undercooked poultry, and contaminated water. OBJECTIVE: The RapidChek®Campylobacter test system (PTM number 052201) was validated for the detection of Campylobacter jejuni, C. coli, and C. lari in raw ground chicken, chicken carcass rinse, and turkey carcass sponges. METHODS: The method uses a proprietary enrichment medium. Following aerobic enrichment, an immunochromatographic test strip is inserted into the tube containing the enrichment, developed for 20 min, and interpreted. Campylobacter-inoculated food samples were tested by the method, as well as the USDA/FSIS cultural reference method; Isolation and Identification of Campylobacter jejuni/coli/lari from Poultry Rinse, Sponge and Raw Product Samples MLG 41.04. The candidate method was also confirmed by an alternative cultural method. The RapidChek method was tested with 50 Campylobacter strains comprised of C. jejuni, C. coli, and C. lari, and 30 non-target strains. RESULTS: A total of 80 low-level spiked samples were tested by both methods in the study. The candidate method yielded 49 presumptive positives: all presumptive results were confirmed culturally. The reference method produced a total of 41 confirmed positive results. No difference between the alternate confirmation method and reference confirmation method was observed. Probability of detection analysis demonstrated no significant differences in the number of positive samples detected by the candidate method and cultural reference method. The RapidChek method detected all 50 Campylobacter strains and none of the 30 non-target strains, including Campylobacter spp. other than C. jejuni, C. coli, and C. lari. CONCLUSION: The candidate method performed as well as the reference method in the detection of C. jejuni, C. coli, and C. lari in raw ground chicken, chicken carcass rinse, and turkey carcass sponges. HIGHLIGHTS: Aerobic enrichment of selected matrixes for 48 h yielded reliable presumptive results for Campylobacter.

Campylobacter lari Vertebral Osteomyelitis.

We report a case of Campylobacter lari vertebral osteomyelitis with iliopsoas abscess. This is the first case report of vertebral osteomyelitis due to C. lari, which was identified from a vertebral biopsy sample collected using CT-guided percutaneous needle biopsy in a patient without obvious episodes of immunodeficiency. Cultureing using the HK semisolid medium aided in pathogen ideutification. It is important to make every possible effort to identify the causative pathogen in vertebral osteomyelitis.

A Multiplex Quantitative Polymerase Chain Reaction Using Applied Biosystems 7500 Fast System for Simultaneous Identification of Three Campylobacter Species with Potential Applications to Food Analysis.

Consumption of Campylobacter-contaminated food is one of the most common causes of bacterial diarrhea. A previously developed quantitative polymerase chain reaction (qPCR) utilizing the SmartCycler instrument platform for identification of Campylobacter jejuni, Campylobacter coli, and Campylobacter lari had to be modified to address the recent discontinuation of the SmartCycler system. In this study, a multiplex qPCR assay was optimized on the Applied Biosystems 7500 Fast (AB7500F) platform to continue using qPCR for the identification of three target Campylobacter spp. AB7500F qPCR efficiencies obtained by testing reference genomic DNA (gDNA) were 90.9%, 86.4%, and 94.6% for C. jejuni, C. coli, and C. lari, respectively, with all correlation coefficient values >0.99. The qPCR results exhibited 100% specificity by testing gDNA samples from 37 non-target reference strains and 86 target strains (50 C. jejuni, 27 C. coli, and 9 C. lari strains) in this study. The lowest detection level using gDNA was 4, 7, and 2 genome copies per reaction for C. jejuni, C. coli, and C. lari, respectively. With a 2-day enrichment procedure, the qPCR method correctly detected target species in a spiked food matrix (frog leg, an aquaculture product). The sensitivity in 25 g food matrix was 4 colony-forming units (CFUs) for C. jejuni, 3 CFUs for C. coli, and 2 CFUs for C. lari. The results suggest that this AB7500F-based qPCR has potential applications for the identification of C. jejuni, C. coli, and C. lari in contaminated food.

Identification and Antimicrobial Susceptibility Testing of Campylobacter Using a Microfluidic Lab-on-a-Chip Device.

Campylobacter spp. have been recognized as major foodborne pathogens worldwide. An increasing frequency of antibiotic-resistant pathogens, including Campylobacter spp., have been identified to transmit from food products to humans and cause severe threats to public health. To better mitigate the antibiotic resistance crisis, rapid detection methods are required to provide timely antimicrobial resistance surveillance data for agri-food systems. Herein, we developed a polymer-based microfluidic device for the identification and antimicrobial susceptibility testing (AST) of Campylobacter spp. An array of bacterial incubation chambers were created in the microfluidic device, where chromogenic medium and antibiotics were loaded. The growth of Campylobacter spp. was visualized by color change due to chromogenic reactions. This platform achieved 100% specificity for Campylobacter identification. Sensitive detection of multiple Campylobacter species (C. jejuni, C. coli, and C. lari) was obtained in artificially contaminated milk and poultry meat, with detection limits down to 1 × 102 CFU/ml and 1 × 104 CFU/25 g, respectively. On-chip AST determined Campylobacter antibiotic susceptibilities by the lowest concentration of antibiotics that can inhibit bacterial growth (i.e., no color change observed). High coincidences (91% to 100%) of on-chip AST and the conventional agar dilution method were achieved against several clinically important antibiotics. For a presumptive colony, on-chip identification and AST were completed in parallel within 24 h, whereas standard methods, including biochemical assays and traditional culture-based AST, take several days for multiple sequential steps. In conclusion, this lab-on-a-chip device can achieve rapid and reliable detection of antibiotic-resistant Campylobacter spp.IMPORTANCE Increasing concerns of antibiotic-resistant Campylobacter spp. with regard to public health emphasize the importance of efficient and fast detection. This study described the timely identification and antimicrobial susceptibility testing of Campylobacter spp. by using a microfluidic device. Our developed method not only reduced the total analysis time, but it also simplified food sample preparation and chip operation for end users. Due to the miniaturized size of the lab-on-a-chip platform, the detection was achieved by using up to 1,000 times less of the reagents than with standard reference methods, making it a competitive approach for rapid screening and surveillance study in food industries. In addition, multiple clinically important Campylobacter species (C. jejuni, C. coli, and C. lari) could be tested by our device. This device has potential for wide application in food safety management and clinical diagnostics, especially in resource-limited regions.

Structure of bacterial oligosaccharyltransferase PglB bound to a reactive LLO and an inhibitory peptide.

Oligosaccharyltransferase (OST) is a key enzyme of the N-glycosylation pathway, where it catalyzes the transfer of a glycan from a lipid-linked oligosaccharide (LLO) to an acceptor asparagine within the conserved sequon N-X-T/S. A previous structure of a ternary complex of bacterial single subunit OST, PglB, bound to a non-hydrolyzable LLO analog and a wild type acceptor peptide showed how both substrates bind and how an external loop (EL5) of the enzyme provided specific substrate-binding contacts. However, there was a relatively large separation of the substrates at the active site. Here we present the X-ray structure of PglB bound to a reactive LLO analog and an inhibitory peptide, revealing previously unobserved interactions in the active site. We found that the atoms forming the N-glycosidic bond (C-1 of the GlcNAc moiety of LLO and the -NH2 group of the peptide) are closer than in the previous structure, suggesting that we have captured a conformation closer to the transition state of the reaction. We find that the distance between the divalent metal ion and the glycosidic oxygen of LLO is now 4 Å, suggesting that the metal stabilizes the leaving group of the nucleophilic substitution reaction. Further, the carboxylate group of a conserved aspartate of PglB mediates an interaction network between the reducing-end sugar of the LLO, the asparagine side chain of the acceptor peptide, and a bound divalent metal ion. The interactions identified in this novel state are likely to be relevant in the catalytic mechanisms of all OSTs.

Molecular basis of lipid-linked oligosaccharide recognition and processing by bacterial oligosaccharyltransferase.

Oligosaccharyltransferase (OST) is a membrane-integral enzyme that catalyzes the transfer of glycans from lipid-linked oligosaccharides (LLOs) onto asparagine side chains, the first step in protein N-glycosylation. Here, we report the X-ray structure of a single-subunit OST, PglB from Campylobacter lari, trapped in an intermediate state bound to an acceptor peptide and a synthetic LLO analog. The structure reveals the role of the external loop EL5, present in all OST enzymes, in substrate recognition. Whereas the N-terminal half of EL5 binds LLO, the C-terminal half interacts with the acceptor peptide. The glycan moiety of LLO must thread under EL5 to access the active site. Reducing EL5 mobility decreases the catalytic rate of OST when full-size heptasaccharide LLO is provided, but not for a monosaccharide-containing LLO analog. Our results define the chemistry of a ternary complex state, assign functional roles to conserved OST motifs, and provide opportunities for glycoengineering by rational design of PglB.

Simultaneous Identification of Campylobacter jejuni, Campylobacter coli, and Campylobacter lari with SmartCycler-Based Multiplex Quantitative Polymerase Chain Reaction.

BACKGROUND: Consumption of Campylobacter contaminated food or water is a leading cause of human acute gastroenteritis. Campylobacter jejuni, Campylobacter coli, and Campylobacter lari account for over 95% of total Campylobacter infections. A multiplex quantitative polymerase chain reaction (qPCR) for simultaneous identification of C. jejuni, C. coli, and C. lari was developed for use with the SmartCycler II system. MATERIALS AND METHODS: We evaluated and combined previously described primers and probes for Campylobacter detection, designed a new internal amplification control, and optimized the multiplex qPCR for the detection of C. jejuni, C. coli, and C. lari. RESULTS: This method was 100% specific when tested against a panel of 32 target Campylobacter strains and 31 non-Campylobacter reference strains. Furthermore, there was no cross-reactivity with seven strains from four nontarget Campylobacter species. The amplification efficiency of each target in this multiplex qPCR was over 90%, and each coefficient of linearity was greater than 0.99. With artificially mixed genomic DNA, this method detected as few as two, three, and two genome copies of C. jejuni, C. coli, and C. lari, respectively. This method was also able to detect these three Campylobacter species in artificially contaminated milk with a sensitivity of five spiked cells of each target per reaction. CONCLUSION: The three Campylobacter targets were simultaneously identified using artificially mixed genomic DNA and spiked raw milk. This SmartCycler-based multiplex qPCR is a rapid, specific, and sensitive method to identify C. jejuni, C. coli, and C. lari.

A Cytolethal Distending Toxin Gene-Based Multiplex PCR Assay for Campylobacter jejuni, C. fetus, C. coli, C. upsaliensis, C. hyointestinalis, and C. lari.

In this study, we devised a multiplex PCR assay based on the gene of cytolethal distending toxin (cdt) B subunit to simultaneously detect and discriminate Campylobacter jejuni, C. fetus, C. coli, C. upsaliensis, C. hyointestinalis, and C. lari. Species-specific PCR products were successfully obtained from all 38 C. jejuni, 12 C. fetus, 39 C. coli, 22 C. upsaliensis, 24 C. hyointestinalis, and 7 C. lari strains tested. On the other hand, no specific PCR products were obtained from other campylobacters and bacterial species tested (41 strains in total). The proposed multiplex PCR assay is a valuable tool for detection and descrimination of 6 major Campylobacter species, that are associated with gastrointestinal diseases in humans.

Transformation and characterization of an arsenic gene operon from urease-positive thermophilic Campylobacter (UPTC) in Escherichia coli.

An arsenate susceptibility test was performed with transformed and cultured Escherichia coli DH5α cells, which carried recombinant DNA of full-length arsenic (ars) operon, namely a putative membrane permease, ArsP; a transcriptional repressor, ArsR; an arsenate reductase, ArsC; and an arsenical-resistance membrane transporter, Acr3, from the Japanese urease-positive thermophilic Campylobacter lari (UPTC) CF89-12. The E. coli DH5α transformant showed reduced susceptibility to arsenate (~1536 µg/mL), compared to the control. Thus, these ars four-genes from the UPTC CF89-12 strain cells could confer a reduced susceptibility to arsenate in the transformed and E. coli DH5α cells. E. coli transformants with truncated ars operons, acr3 (acr3) and arsC-acr3 (∆arsC-acr3), of the ars operon, showed an MIC value of 384 µg/mL (~384 µg/mL), similar to the E. coli cells which carried the pGEM-T vector (control). Reverse transcription PCR confirmed in vivo transcription of recombinant full-length ars operon and deletion variants (∆acr3 and ∆arsC-acr3) in the transformed E. coli cells.

Rapid Detection of Campylobacter jejuni, Campylobacter coli, and Campylobacter lari in Fresh Chicken Meat and By-Products in Bangkok, Thailand, Using Modified Multiplex PCR.

A multiplex PCR assay for simultaneous detection and differentiation of Campylobacter jejuni, Campylobacter coli, and Campylobacter lari was developed and validated to assess the occurrence of these bacteria in fresh chicken meat and by-products in Bangkok, Thailand, by using a new combination of four previously published PCR primers for C. jejuni, C. coli, C. lari, and a universal 16S rDNA gene as an internal control. The specificity was determined by using 13 strains of other bacteria. With pure culture DNA, the detection limit was 0.017 ng/PCR for C. jejuni and C. coli and was 0.016 ng/PCR for C. lari. It can detect 10 CFU of C. jejuni, C. coli, and C. lari in 2 g of chicken meat within a 16-h enrichment time. Our multiplex PCR assay was applied for identification of Campylobacter spp. in 122 supermarket samples and 108 fresh market samples. Of the 230 samples evaluated by multiplex PCR, 54.0, 3.3, and 10.7% of supermarket samples were positive for C. jejuni, C. coli, and mixed C. jejuni and C. coli, respectively, and 56.5 and 33.3% of fresh market samples were positive for C. jejuni and mixed C. jejuni and C. coli, respectively. No sample was positive for C. lari. Fresh market samples had significantly higher C. jejuni and C. coli contamination than those from supermarkets (relative risk: 1.3; P = 0.0001). Compared with the culture method (a gold standard), the sensitivity, specificity, positive predictive value, negative predictive value, and diagnostic accuracy of multiplex PCR were 97.7, 86.8, 96.1, 92.0, and 95.2%, respectively. No significant difference was observed between results from two methods (P = 0.55). Therefore, the established multiplex PCR was not only rapid and easy to perform but had a high sensitivity and specificity to distinguish between C. jejuni, C. coli, and C. lari, even in samples containing mixed contamination. Our study indicated that fresh chicken meat and by-products from fresh markets were significantly less hygienic than those from supermarkets.

Molecular analysis of the tlyA gene in Campylobacter lari.

Full-length tlyA gene and its adjacent genetic loci from the urease-positive thermophilic Campylobacter (UPTC) CF89-12 [approximately 15,000 base pairs (bp) in length], as well as a reference strain Campylobacter lari RM2100 (approximately 9,000 bp), were analyzed. The possible open-reading frame of tlyA from UPTC CF89-12 was shown to have 720 bp with a calculated molecular mass of approximately 26.7 kDa. Using a primer pair designed in silico, a total of approximately 1.1 kbp consisting of putative promoter region, structural gene for tlyA, and its adjacent genetic loci were identified in all 17 C. lari isolates [n = 13 for UPTC; n = 4 for urease-negative (UN) C. lari]. Although sequence differences were demonstrated at approximately 20 loci within the 90 bp non-coding (NC) region, including the putative promoter structure candidates immediately upstream of the tlyA gene among the 18 isolates including C. lari RM2100, no sequence differences were identified within the NC region among the five UN C. lari isolates examined. A start codon ATG and a probable ribosome-binding site, AGGC(T)GG(A), for the tlyA gene were identified in all 18 isolates, including C. lari RM2100. The putative intrinsic ρ-independent transcriptional terminator structure candidate was also identified for the tlyA gene in both UPTC CF89-12 and C. lari RM2100. Additionally, the hemolysis assay was performed with some of the C. lari isolates. The tlyA gene nucleotide sequence data may possibly be useful for discrimination between UN C. lari and UPTC organisms, as well as for the differentiation among the four thermophilic Campylobacter species.

Evaluation of antimicrobial resistance of Campylobacter spp. isolated from broiler carcasses.

1. The aim of the present study was to evaluate the antimicrobial resistance of Campylobacter strains (C. jejuni, C. coli and C. lari) isolated from broiler carcasses processed in the State of Paraná, Brazil. 2. Rates of microbial resistance and susceptibility were assessed by both Disk Diffusion (DD) and Etest (Minimum Inhibitory Concentration) techniques. Antibiotics were tested using DD (12 antibiotics) and/or MIC (7 antibiotics) methods. 3. A total of 95.8% of the strains were resistant to at least two agents. In terms of multidrug resistance, 75% of strains were resistant to three or more groups of antibiotics. The highest rates of resistance were detected for cefalotin, ciprofloxacin, tetracycline and nalidixic acid. A high rate of susceptibility of the strains to erythromycin (95.8%) was found confirming that this is considered the agent of choice for treating campylobacteriosis. Comparison of the microbial resistance and susceptibility, as determined simultaneously by the two methods, found the techniques to be statistically equivalent for 5 out of the 6 antibiotics tested. 4. The results of this study suggest the need for adopting measures to control the use of antibiotics in broiler production to prevent multidrug resistance of Campylobacter strains and reduce the risk of serious human diseases caused by the consumption of contaminated chicken meat.

Comparative genomics of the Campylobacter lari group.

The Campylobacter lari group is a phylogenetic clade within the epsilon subdivision of the Proteobacteria and is part of the thermotolerant Campylobacter spp., a division within the genus that includes the human pathogen Campylobacter jejuni. The C. lari group is currently composed of five species (C. lari, Campylobacter insulaenigrae, Campylobacter volucris, Campylobacter subantarcticus, and Campylobacter peloridis), as well as a group of strains termed the urease-positive thermophilic Campylobacter (UPTC) and other C. lari-like strains. Here we present the complete genome sequences of 11 C. lari group strains, including the five C. lari group species, four UPTC strains, and a lari-like strain isolated in this study. The genome of C. lari subsp. lari strain RM2100 was described previously. Analysis of the C. lari group genomes indicates that this group is highly related at the genome level. Furthermore, these genomes are strongly syntenic with minor rearrangements occurring only in 4 of the 12 genomes studied. The C. lari group can be bifurcated, based on the flagella and flagellar modification genes. Genomic analysis of the UPTC strains indicated that these organisms are variable but highly similar, closely related to but distinct from C. lari. Additionally, the C. lari group contains multiple genes encoding hemagglutination domain proteins, which are either contingency genes or linked to conserved contingency genes. Many of the features identified in strain RM2100, such as major deficiencies in amino acid biosynthesis and energy metabolism, are conserved across all 12 genomes, suggesting that these common features may play a role in the association of the C. lari group with coastal environments and watersheds.

A national investigation of the prevalence and diversity of thermophilic Campylobacter species in agricultural watersheds in Canada.

The occurrence and diversity of thermophilic Campylobacter species (C. jejuni, coli, and lari) were studied in water samples from four river basins located across Canada. These basins located in Quebec (Bras d'Henri), Alberta (Oldman), Ontario (South Nation), and British Columbia (Sumas) represented some of the most intensive farming areas in Canada for hog, beef cattle, dairy cattle, and poultry, respectively. This study analyzed 769 water samples collected from 23 monitoring sites with agricultural influence, and four reference sites with limited or no agricultural influence. Water samples were collected bi-weekly over two years and analyzed for Campylobacter using a semi-quantitative minimum probable number (MPN) enrichment protocol. Putative isolates were confirmed by genus- and species-specific multiplex polymerase chain reaction (PCR) assays. A total of 377 (49%) water samples were positive for campylobacters with 355 samples having a cell density ranging from 4 to 4000 MPN L(-1). Campylobacters were more common at agricultural than reference sites in each river basin, although this difference was not significant in the Oldman and South Nation (p > 0.05). Campylobacter was significantly more common in the Bras d'Henri and Sumas (63%) compared to the South Nation (45%) and Oldman (33%) River basins (p < 0.05). C. jejuni, C. coli and C. lari were detected in each river basin, and these species occurred in 45% (n = 168), 34% (n = 128) and 19% (n = 73), of all Campylobacter positive samples, respectively. The remaining Campylobacter positive water samples without these three species (n = 67; 18%) were identified as other Campylobacter species. C. jejuni was the predominant species occurring in the Sumas, Oldman and South Nation River basins. However, in the Bras d'Henri River basin with intensive hog production, C. coli was the predominant species. This study found campylobacters to be common in some agricultural systems with intensive livestock farming activities, and different river basins could have strikingly different profiles of either C. jejuni or C. coli as the predominant waterborne thermophilic Campylobacter species.

Molecular structural analysis of major outer membrane protein (MOMP) gene clusters in Campylobacter lari.

Southern hybridisation shows that urease-negative (UN) Campylobacter lari JCM2530(T) carries two putative major outer membrane protein (MOMP) genes. Sequences of approximately 2.1 kbp, encoding non-coding (NC) regions, with possible open reading frames (ORFs) for MOMP (porA1 or porA2) of approximately 1.2 kbp, NC regions and partial and putative Cla_0435 or Cla_1109 ORFs were identified in all five UN C. lari isolates examined, following polymerase chain reaction (PCR) cloning and sequencing. Each putative MOMP structural gene carried start and stop codons and ribosome binding sites of 1236-1278 bp in length. The putative sigma70 transcriptional promoter and the hypothetical rho-independent transcription terminator structures were also seen. Using Northern hybridisation, there was in vivo monocistronic MOMP gene transcription. In addition, in a Japanese urease-positive thermophilic Campylobacter (UPTC) CF89-12 strain, the porA1 gene locus, including an extra gene (approximately 2000 bp in length) was identified. The extra gene may occur within the porA1 gene locus in the eight UPTC isolates of the 23 C. lari isolates examined. Thus, a genetic heterogeneity occurred within the porA1 gene locus from some of the C. lari organisms including the UPTC CF89-12.

Validation according to ISO 16140:2003 of a commercial real-time PCR-based method for detecting Campylobacter jejuni, C. coli, and C. lari in foods.

Campylobacteriosis was the most frequently reported zoonosis in the European Union (EU) in 2010, with Campylobacter jejuni, Campylobacter coli, and Campylobacter lari as the most frequently reported species in foodborne outbreaks (FBOs). Relatively sensitive to environmental factors, these species may be present in low numbers. In line with EU policy for food control and FBO detection and in view of the need to reduce response time, we validated an alternative molecular method according to ISO 16140:2003 which establishes the general principle and technical protocol for the validation of alternative methods in the microbiological analysis of food. We used a qualitative real-time PCR commercial kit for the detection of C. jejuni, C. coli, and C. lari in two food categories "fruit and vegetable-based products" and "dairy products". The validation protocol comprises two phases: the first is a method comparison study of the alternative method against the reference method, and the second is an interlaboratory study of each of the two methods. In the first step, ISO 16140:2003 validation examines the following parameters: limit of detection (LOD); relative accuracy, relative specificity and sensitivity; relative detection level (RDL); and inclusivity and exclusivity. Except for LOD, inclusivity and exclusivity, the other steps were performed against the reference method (ISO 10272:2006). The LOD of the real-time PCR method was set at 4CFU/25g or mL for both food categories. Relative accuracy (98.33%), specificity (96.77%), and sensitivity (100%) were recorded for the food category "fruit and vegetable-based products" and 93.3%, 88.24%, 100%, respectively, for "dairy products". The RDL according to Fisher's exact test was p=1 for both food categories, for each level, and each food/strain combination. The interlaboratory study results showed correct identification of all 24 blind samples with both methods by all the participating laboratories. The results show that this commercial kit is suitable for the rapid detection of C. jejuni, C. coli, and C. lari on fruit, vegetables and dairy products and may aid in official controls. In conclusion, the use of alternative methods is recommended for the rapid identification of positive samples and the identification of the possible bacterial source in a FBO within 48 h.