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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.

Background The Thermo Scientific SureTect™ Campylobacter jejuni, C. coli, and C. lari PCR Kit is a real-time PCR assay for the detection and differentiation of C. jejuni, C. coli, and C. lari from raw poultry, ready-to-cook poultry products, and environmental samples. Objective The Thermo Scientific SureTect Campylobacter jejuni, C. coli, and C. lari PCR Kit was evaluated for AOAC®Performance Tested MethodsSM certification. Methods Inclusivity/exclusivity, matrix studies, product consistency and stability, and robustness testing were conducted to assess the method’s performance. In the matrix studies, the method was validated against United States and international reference methods for Campylobacter detection. Results There were no statistically significant differences found in the matrix studies between the candidate and reference methods when analyzed by probability of detection. All 52 inclusivity strains and none of the 51 exclusivity strains tested were detected by the assay. Robustness testing demonstrated that the assay gave reliable performance with specific method deviations outside of the recommended parameters, and the real-time stability testing demonstrated that there were no statistically significant differences between kit lots, validating the stated shelf life of the kit. Conclusion The data presented support the product claims that the Thermo Scientific SureTect Campylobacter jejuni, C. coli, and C. lari PCR assay is suitable for the detection and differentiation of C. jejuni, C. coli, and C. lari from raw poultry, ready-to-cook poultry products, and environmental samples. Highlights Presumptive results can be obtained in as little as 23 h. Microaerophilic incubators are not required for enrichment.

Background: The Bruker MALDI Biotyper® method utilizes matrix-assisted laser desorption/ionization time-of-flight MS for the rapid and accurate identification and confirmation of Gram-negative bacteria from select media types. The alternative method was evaluated in a method extension study of AOAC INTERNATIONAL First Action Official MethodSM 2017.09 using nonselective and selective agars to identify Cronobacter spp., Salmonella spp., Campylobacter spp., and select Gram-negative bacteria. Results obtained by the Bruker MALDI Biotyper were compared to the traditional biochemical methods as prescribed in the appropriate reference methods. Methods: Two collaborative studies were organized, one in the United States focusing on Cronobacter spp. and other Gram-negative bacteria and one in Europe focusing on Salmonella spp. and other Gram-negative bacteria. Fourteen collaborators from seven laboratories located within the United States participated in the first collaborative study for Cronobacter spp. Fifteen collaborators from 15 service laboratories located within Europe participated in the second collaborative study for Salmonella spp. For each target organism (either Salmonella spp. or Cronobacter spp.), a total of 24 blind-coded isolates were evaluated. In each set of 24 organisms, there were 16 inclusivity organisms (Cronobacter spp. or Salmonella spp.) and 8 exclusivity organisms (non-Cronobacter spp. and non-Salmonella spp. closely related Gram-negative organisms). For the Campylobacter spp. method extension, 17 collaborators from eight laboratories located within the United States (seven laboratories) and Canada (one laboratory) participated in the collaborative study. A total of 24 blind-coded isolates were evaluated. In each set of 24 organisms, there were 16 inclusivity organisms (Campylobacter spp.) and 8 exclusivity organisms (non-Campylobacter spp. closely related Gram-negative organisms). Results: After testing was completed, the total percentage of correct identifications from each agar type for each strain was determined at a percentage of 100.0% to the genus level for the Cronobacter study and a percentage of 100.0% to the genus level for the Salmonella study. For the Campylobacter method extension, a correct identification and confirmation rate of 100.0% was obtained for the Campylobacter organisms at the species level. For non-Cronobacter, non-Salmonella, and non-Campylobacter organisms, 100.0% were correctly identified. Conclusions: The results indicated that the alternative method produced equivalent results when compared to the confirmatory procedures specified by each reference method. Highlights: The method extension can be modified to include the identification and confirmation of Campylobacter jejuni, Campylobacter coli, and Campylobacter lari.

Infections with Campylobacter spp. pose a significant health burden worldwide. The significance of Campylobacter jejuni/Campylobacter coli infection is well appreciated but the contribution of non-C. jejuni/C. coli spp. to human gastroenteritis is largely unknown. In this study, we employed a two-tiered molecular study on 7194 patient faecal samples received by the Microbiology Department in Cork University Hospital during 2009. The first step, using EntericBio(®) (Serosep), a multiplex PCR system, detected Campylobacter to the genus level. The second step, utilizing Campylobacter species-specific PCR identified to the species level. A total of 340 samples were confirmed as Campylobacter genus positive, 329 of which were identified to species level with 33 samples containing mixed Campylobacter infections. Campylobacter jejuni, present in 72.4% of samples, was the most common species detected, however, 27.4% of patient samples contained non-C. jejuni/C. coli spp.; Campylobacter fetus (2.4%), Campylobacter upsaliensis (1.2%), Campylobacter hyointestinalis (1.5%), Campylobacter lari (0.6%) and an emerging species, Campylobacter ureolyticus (24.4%). We report a prominent seasonal distribution for campylobacteriosis (Spring), with C. ureolyticus (March) preceeding slightly C. jejuni/C. coli (April/May).

Aims: Quantitative PCR and a culture method were used to investigate Campylobacter occurrence over 3 years in a watershed located in southern Ontario, Canada that is used as a source of drinking water. Methods and Results: Direct DNA extraction from river water followed by quantitative PCR analysis detected thermophilic campylobacters at low concentrations (

Aims: To develop a PCR-denaturing gradient gel electrophoresis (PCR-DGGE) method for the detection and identification of Campylobacter, Helicobacter and Arcobacter species (Epsilobacteria) in clinical samples and evaluate its efficacy on saliva samples from humans and domestic pets. Methods and Results: A semi-nested PCR was developed to allow sensitive detection of all Epsilobacteria, with species separation undertaken by DGGE. A database was constructed in BioNumerics using 145 strains covering 51 Campylobacter, Arcobacter and Helicobacter taxa; Nineteen distinct DGGE profile-groups were distinguished. This approach detected Epsilobacteria in all saliva samples collected from humans, cats and dogs, and identified Campylobacter concisus and/or Campylobacter gracilis in the human samples. The pet animal samples were taken from individuals with oral/dental diseases; PCR-DGGE identified up to four different species in each sample. The most common species detected included Wolinella succinogenes, Arcobacter butzleri and two hitherto uncultured campylobacters. The enteropathogen Campylobacter lari was also found. Conclusions: PCR combined with DGGE is a useful tool for direct detection and preliminary identification of Epsilobacteria in the oral cavity of humans and small animals. Significance and Impact of the Study: The PCR-DGGE method should allow determination of the true prevalence and diversity of Epsilobacteria in clinical and other samples. Contact with the oral cavity of domestic pets may represent a route of transmission for epsilobacterial enteric diseases.

Aim: To examine the occurrence, diversity and transmission of Campylobacter in a poultry slaughterhouse. Methods and Results: During a 4-week period, a slaughterhouse was sampled alternately during slaughtering and the following mornings post-disinfection. Samples were taken from poultry at six stages in the slaughter process and from 25 environmental sites. For positive broiler flocks slaughtered on one occasion, 92% and 48% of the environmental sites were positive during slaughter and post-disinfection, respectively. For positive laying hen flocks slaughtered on three occasions, 8–56% and 12–20% of the environmental sites were positive during slaughter and post-disinfection, respectively. Genetic fingerprinting by amplified fragment length polymorphism (AFLP) of the 109 isolates obtained resulted in 28 different AFLP clones. Five AFLP clones were present for more than 1 week. Conclusions: Slaughtering of Campylobacter-positive broilers resulted in extensive contamination of the slaughterhouse, including the air. A high proportion of the laying hen flocks were Campylobacter positive, but these caused less environmental contamination than the broilers. This, together with the freezing of all layer carcasses, results in a lower public health risk from laying hens, when compared with broilers. Significance and Impact of the Study: When slaughtering Campylobacter-positive broilers, the implementation of preventive measures is important to reduce contamination of negative carcasses and to protect the workers against infection.

Consecutive isolates of quinolone-resistant campylobacter isolated over a 5 year period (1990-1995) from the faeces of patients with enteritis in Plymouth, UK, were examined for the epidemiology of mutations in gyrA (n = 127). In addition, clinical isolates and poultry isolates from Germany, The Netherlands and other regions of the UK collected before 1995 were examined for mutations in the quinolone resistance-determining region of gyrA by single- stranded conformational polymorphism analysis and direct sequencing of a 270 bp fragment of PCR-generated DNA. The majority of isolates (173/208) carried a mutation at codon 86 in gyrA resulting in substitution of Ile for Thr; all of these were resistant to ciprofloxacin (MIC ≥ 2 mg/L). One isolate of Campylobacter jejuni had a mutation at Asp-90, and another had a double muta- tion at Thr-86 and Pro-104. Only two resistant isolates showed no mutation in gyrA. A novel gyrA sequence was amplified from two Campylobacter lari and one C. jejuni, which exhibited a valine at codon 86. Only 8/192 isolates had changes in gyrB; all were shown to relate to silent mutations in gyrB and presumably reflect natural polymorphisms in the gene.

Aims: The differences between phenotyping and genotyping (polymerase chain reaction– restriction fragment length polymorphism) of Campylobacter jejuni, Campylobacter coli, Campylobacter lari and Campylobacter upsaliensis were assessed. Methods and Results: A total of 51, 63 and 88 strains from dogs, pigs and humans, respectively, were examined. The strains were first typed by biochemical methods, then by PCR–RFLP using AluI and Tsp509I. None of the strains were typed as Camp. lari by the PCR–RFLP. The biggest differences were found in the identification of Camp. jejuni and Camp. coli. The main discrepancies were caused with the hippurate hydrolysis test and sensitivity to cephalothin and nalidixic acid. Strains which were identified biochemically as Camp. coli and by digestion with AluI as Camp. jejuni (eight strains) were tested for the presence of the hippuricase gene. Conclusion: The PCR typing results showed the presence of the hippuricase gene as unique to Camp. jejuni. Significance and Impact of the Study: A reliable identification of Campylobacter spp. should be supplemented with a molecular method.

Aim: To estimate the prevalence of thermotolerant Campylobacter spp. in commercially reared partridges (Perdix perdix) in southern Italy. Methods and Results: Cloacal swabs of partridges (n = 240), equally distributed between male and female birds, from a game bird farm located in the Southern Italy were examined for the prevalence of thermotolerant Campylobacter spp. The samples were processed in order to detect thermotolerant Campylobacter spp. by culture methods. The positive samples were then confirmed by multiplex polymerase chain reaction. Thermotolerant Campylobacter spp. were isolated from 118 (49·2%) of the 240 cloacal swabs examined. As proved by PCR, 100% of the strains were identified as Campylobacter coli (118/118), and 15 (12·7%) out of the 118 positive samples were also positive for Campylobacter jejuni. In contrast, Campylobacter lari was not identified. Adult partridges showed a significantly higher prevalence (P

A survey of the isolation rate and population size of thermophilic campylobacters in lambs at slaughter was carried out to determine the seasonal variation of thermophilic campylobacters in ovine hosts. Isolation rates determined by enrichment methods were always higher than those using direct plating onto selective agar and showed that Campylobacter could be isolated from 91·7% (n = 360) of samples from the small intestine of the lambs. Enumerations (MPN), done monthly over a 2-year period, averaged 4·00 log 10 (n = 1080, S.D. 0·16) campylobacters g−1 fresh weight (fw) intestinal contents with some samples giving values higher than 7 log MPN gfw−1. These results show that the prevalence of thermophilic campylobacters in sheep intended for slaughter is much higher than previously reported. Statistical analyses showed that there was a significant seasonal periodicity in the Campylobacter populations in the small intestines of lambs at slaughter (P = 0·004) but that there was no statistical relationship with environmental parameters such as minimum and maximum temperature, rainfall or hours of sunshine. In an analysis along the length of the intestinal tract of lambs, campylobacters were isolated from 80% of small intestine and 30% of rumen samples, but not from the true stomach, large intestine or caecal samples. A survey of sheep grazing on salt marsh, fell (upland) and farm pasture showed that the isolation rate of thermophilic campylobacters in fresh faeces was substantially lower (29·3% (n = 420)) than that from the small intestine of lambs at slaughter. No significant difference (>0·05) was found between the isolation rate of campylobacters from faeces sampled in late spring/early summer and autumn, nor between the different types of grazing.

Two strains of urease-positive thermophilic Campylobacter (UPTC), CF89–12 and CF89–14, which were identified as UPTC by biochemical characterization, were found for the first time in river water in the Far East, namely, in Japan. The biochemical characteristics were identical to those of strains described previously by Bolton and colleagues. Furthermore, these two strains were positive for arylsulphatase. Consequently, it was demonstrated that UPTC may possibly be differentiated phenotypically from Campylobacter lari by the arylsulphatase test, as well as urease and nalidixic acid tests. Analysis by pulsed-field gel electrophoresis (PFGE) after digestion with Apa I, Sal I and Sma I, which were found to produce distributions of DNA fragments to be suitable for analysis of the genomic DNA from the thermophilic Campylobacter, respectively, demonstrated that these three restriction enzymes produced distributions of a relatively limited number of genomic DNA fragments and also demonstrated that the PFGE profiles obtained with the three restriction enzymes were indistinguishable between the two strains, respectively. The PFGE analysis and conventional fixed-field agarose gel electrophoresis suggested that the both genomes were approximately 1862 kb in length. Even though the two isolates of UPTC were isolated from water in different rivers in Japan, the results suggested that a single strain. as opposed to two distinct strains, was isolated. PFGE profiles after digestion with Sal I and Sma I, respectively, were also demonstrated to be distinctly different among strains isolated in Japan and previously in Europe. This is the first example of the isolation of UPTC from natural sources in countries other than those in Europe.

From the end of April 1991 until the end of 1991, 2209 isolates of Campylobacter spp. have been collected in Plymouth PHL of which 91 (4.1%) were resistant to ciprofloxacin. None of the 91 patients involved had taken a quinolone, but 30/91 (33%) had travelled abroad (16 to the Iberian peninsula) in the three months preceding isolation of the ciprofloxacin-resistant Campylobacter spp. In the case-control study 12/15 (80%) of the cases had recently consumed poultry as had 20/24 (83%) of controls with enteritis due to ciprofloxacin-susceptible Campylobacter spp. A small study of poultry purchased from the supermarket revealed that only 1/37 campylobacters isolated from 64 UK bred chickens was resistant to ciprofloxacin, whereas 7/26 campylobacters isolated from 50 imported chickens were ciprofloxacin-resistant. Of the 75 clinical isolates of ciprofloxacin-resistant Campylobacter spp. subjected to detailed analysis, 68 were Campylobacter jejuni, six were Campylobacter lari, and one was Campylobacter coli. All isolates from man and poultry were resistant to ciprofloxacin, norfloxacin, sparfloxacin and tosufloxacin, and there was an association between fluoroquinolone-resistance and increased MICs of tetracycline. The range of susceptibility to erythromycin and kanamycin were typical of the species. gyrA from C. jejuni P6 (a case with history of travel to Spain) and C. jejuni P16 (isolate from imported chicken) contained point mutations corresponding to an amino acid substitution of isoleucine for threonine at codon 86. It has been suggested that veterinary use of quinolones, notably enrofloxacin, is providing a selective pressure for emergence of resistance to ciprofloxacin amongst human isolates. Now that enrofloxacin has been licensed for use in broiler flocks in the UK, it will be interesting to monitor the prevalence of resistance of campylobacters to quinolones in UK-produced poultry and in UK-acquired human infection.

A total of 209 well-defined Campylobacter, Helicobacter , and Wolinella strains were tested with a Gen-Probe oligodeoxyribonucleotide probe for Campylobacter jejuni , Campylobacter coli , and Campylobacter lari . Released ribosomal RNA was hybridized with the acridinium-ester-labeled probe. The amount of light proportional to the number of DNA-RNA hybrid molecules was captured by a photo-multiplier and converted to a digital signal. A reading of more than 50,000 relative light units (RLU) was considered positive. After isolates were cultivated, lysis and hybridization procedures were completed in 30 minutes. All 104 strains within the target species were positive, as well as two Campylobacter hyointestinalis strains. The remaining 103 strains, including 15 C hyointestinalis strains, were negative. Our data indicate a sensitivity of 1.0 and specificity of 0.98 (P