Search

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.

We investigate the role of black-headed gulls (Chroicocephalus ridibundus), an omnivorous species that is among the most likely wild bird candidates for transmission of zoonotic agents, as a potential reservoir ofCampylobacterspp. Colonies with different anthropogenic pressures were studied to examine differences in exposure to sources ofCampylobacterbetween rural and urban birds. We recordedCampylobacterspp. in 4.87% of adult black-headed gulls and 2.22% of their chicks after analysing 1036 cloacal swabs collected over two breeding seasons in three colonies in northern Poland.Campylobacter jejuniwas found most frequently (85.72%), andCampylobacter lariandCampylobacter coliwere much scarcer. Prevalence ofCampylobacterdid not differ significantly between black-headed gulls breeding in urban (4.27%) and rural (3.80%) habitats. Almost all isolates from chicks and adults were susceptible to azithromycin (97.62%) and erythromycin (95.24%), but fewer to tetracycline (50.00%) and ciprofloxacin (47.62%).Campylobacterprevalence was unrelated to the date of sampling. Our study indicates that black-headed gulls are carriers of resistant to antibioticsCampylobacterand they can contaminate natural waterbodies with their faeces, which poses a threat to human and farm animal health.

The aim of this work was the establishment of a national laboratory sentinel surveillance service for human clinical Campylobacter in Ireland. This included detailed genomic molecular epidemiology of Campylobacter for 2019. For February-December 2019, 24 clinical microbiology laboratories in Ireland submitted all PCR/culture-positive clinical Campylobacter spp. specimens to Public Health Laboratory (PHL) Dublin one week out of every four. Antimicrobial susceptibility testing (AST) according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) criteria was carried out for Campylobacter spp. isolates for ciprofloxacin, tetracycline and erythromycin. Batch whole genome sequencing (WGS) was carried out on cultures and analysis was performed to determine species, genotype, identify antimicrobial resistance (AMR) and virulence determinants and identify clusters. A total of 75 isolates and 366 PCR-positive stools were received, and 277 isolates recovered (55.7% recovery from stools). Of 257 isolates characterized by WGS, 86.4% (n = 222) were Campylobacter jejuni, 11.7% (n = 30) Campylobacter coli and 1.9% (n = 5) Campylobacter lari. There were 20 clonal complexes with ST-21 clonal complex most prevalent at 26.8% (n = 69). 50.5% (n = 140) of isolates were susceptible to all three antimicrobials tested. 39.3% (n = 109) isolates were ciprofloxacin resistant, 26.3% (n = 73) tetracycline resistant and two isolates erythromycin resistant. Congruence between phenotypic and genotypic AST was observed. There was 95.9% and 95.6% sensitivity and specificity for WGS to predict ciprofloxacin sensitivity and 98.6% and 99.5% sensitivity and specificity for WGS to predict tetracycline sensitivity. Virulence factors flaA, racR, ciaB and cdtB were detected in all isolates. WGS identified 31 potential clusters for public health alert. This sentinel surveillance of human campylobacteriosis in Ireland establishes the basis for a national reference service. Linking with other partners in a 'One Health' framework will help us better understand sources of infection to reduce disease burden and the threat of AMR.

Campylobacter is a major agent of gastroenteritis worldwide. The incidence and prevalence of campylobacteriosis have been increasing in both developed and developing countries over the last decade. In this study, 197 strains of successive Campylobacter-like were identified in French laboratories in September 2018. Bacterial isolates from clinical samples were identified with a mass spectrometer (Ultraflex III TOF/TOF and the BIOTYPER database from Bruker Daltonics). Of the 197 isolates tested, 143 were identified as Campylobacter jejuni (72.59%), 28 as Campylobacter coli (14.21%), 2 as Arcobacter butzleri (1.02%), 1 as Campylobacter fetus and 1 as Campylobacter lari with (0.51% each) by the MALDI-TOF mass spectrometry. Isolation rate of Campylobacter was highest in the 0 - 9 age group (22%). The proportion of male and female patients was 59.4% (CI 95% = 52.2-66.3) and 40.6% (CI 95% = 33.7-42.8) respectively. Sixty strains (30.5%) were resistant to tetracycline and 52 (26.4%) resistant to ampicillin. This study showed that the MALDI-TOF mass spectrometry is a rapid and accurate identification method of Campylobater spp in patients treated in private French laboratories. Key words: Campylobacter, identification, MALDI-TOF, patients, France.

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.

Consumption of

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.

Campylobacter spp. are one of the most important food-borne pathogens, which are quite susceptible to environmental or technological stressors compared to other zoonotic bacteria. This might be due to the lack of many stress response mechanisms described in other bacteria. Nevertheless, Campylobacter is able to survive in the environment and food products. Although some aspects of the heat stress response in Campylobacter jejuni are already known, information about the stress response in other Campylobacter species are still scarce. In this study, the stress response of Campylobacter coli and Campylobacter lari to elevated temperatures (46°C) was investigated by survival assays and whole transcriptome analysis. None of the strains survived at 46°C for more than 8 h and approximately 20% of the genes of C. coli RM2228 and C. lari RM2100 were differentially expressed. The transcriptomic profiles showed enhanced gene expression of several chaperones like dnaK, groES, groEL, and clpB in both strains, indicating a general involvement in the heat stress response within the Campylobacter species. However, the pronounced differences in the expression pattern between C. coli and C. lari suggest that stress response mechanisms described for one Campylobacter species might be not necessarily transferable to other Campylobacter species.

SUMMARYThe Foodborne Diseases Active Surveillance Network (FoodNet) conducts population-based surveillance forCampylobacterinfection. For 2010 through 2015, we compared patients withCampylobacter jejuniwith patients with infections caused by otherCampylobacterspecies.Campylobacter colipatients were more often >40 years of age (OR = 1·4), Asian (OR = 2·3), or Black (OR = 1·7), and more likely to live in an urban area (OR = 1·2), report international travel (OR = 1·5), and have infection in autumn or winter (OR = 1·2).Campylobacter upsaliensispatients were more likely female (OR = 1·6), Hispanic (OR = 1·6), have a blood isolate (OR = 2·8), and have an infection in autumn or winter (OR = 1·7).Campylobacter laripatients were more likely to be >40 years of age (OR = 2·9) and have an infection in autumn or winter (OR = 1·7).Campylobacter fetuspatients were more likely male (OR = 3·1), hospitalized (OR = 3·5), and have a blood isolate (OR = 44·1). International travel was associated with antimicrobial-resistantC. jejuni(OR = 12·5) andC. coli(OR = 12) infections. Species-level data are useful in understanding epidemiology, sources, and resistance of infections.

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.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.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.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.

Poultry meat and products are major transmission routes of human campylobacteriosis. The aim of this study was to determine the numbers and antibiogram profile of Campylobacter isolates from slaughtered broiler and layer birds. One hundred and sixty caecal and one hundred and thirty two carcasses were randomly sampled at the Kejetia poultry slaughter, isolated on charcoal-cefoperazone-deoxycholate agar (CCDA) and confirmed by API CAMPY and their resistance profiles assessed by Kirby-Bauer disk diffusion. Prevalence was 22.5 and 21.9% in the faecal and carcasses, respectively with no significant differences. Species identified among faecal isolates were Campylobacter jejuni (42%), Campylobacter coli (28%), Campylobacter lari (22%) and Campylobacter hyo-intestinalis (8%) while 79% C. jejuni, 14% C. coli, 4% C. jejuni sub sp. doylei, and 3% C. lari were obtained from the carcasses. Resistance to the β-lactams ranged from 75 to 100%, 41 to 86% to the quinolones, 14 to 36% to the aminoglycosides, 100% to erythromycin, 97 to 100% to tetracycline, 72 to 83% to chloramphenicol and 90 to 94% to trimethoprim sulfamethoxazole. All species were sensitive to imipenem, but 100% of isolates were multidrug resistant. Contamination of carcasses with multidrug resistant strains of Campylobacter is a threat to handlers and consumers and of major public health issue. Key words: Multidrug resistance, faeces, carcass, poultry, Kumasi, Ghana.

The aim of this study was to identify and characterize thermophilic Campylobacter species in faecal samples from goats in Khartoum State, Sudan, by application of multiplex polymerase chain reaction. Campylobacteriosis is a zoonotic disease of global concern, and the organisms can be transmitted to human via food, water and through contact with farm animals and pets. There are five clinically related Campylobacter species: Campylobacter jejuni (C. jejuni). Campylobacter coli, Campylobacter lari, Campylobacter upsaliensis and Campylobacter fetus. Conventional cultural methods to diagnose campylobacteriosis are tedious and time consuming. Wide ranges of genes have been reported to be used for PCR-based identification of Campylobacter spp. We used a multiplex PCR assay to simultaneously detect genes from the major five clinically significant Campylobacter spp. The genes selected were hipO (hippuricase) and 23S rRNA from glyA (serine hydroxymethyl transferase) from each of C. jejuni. C. coli, C. lari, and C. upsaliensis; and sapB2 (surface layer protein) from C. fetus subsp. fetus. The assay was used to identify Campylobacter isolates recovered from 336 cultured faecal samples from goats in three localities in Khartoum State. C. coli was the most predominant isolate (234; 69.6%), followed by C. jejuni (19; 5.7%), C. upsaliensis (13; 3.9%), C. fetus subsp. fetus (7; 2.1%) and C. lari (6; 1.8%). Twenty-nine goats showed mixed infection with Campylobacter spp., 21 of which harbored two Campylobacter spp., while eight animals were infected with three species. Ten out of twelve goats that displayed diarrhea harbored C. coli only. C. coli, C. jejuni and C. upsaliensis showed significant variation with localities. The prevalence of C. coli was significantly higher (87; 25.9%) in goats from Omdurman, whereas C. jejuni and C. upsaliensis were significantly higher (11; 3.3%, 9; 2.7%) in goats from Khartoum. The multiplex PCR assay was found to be rapid and easy to perform and had a high sensitivity and specificity for characterizing the isolates, even in mixed cultures. The study demonstrated the significance of goats as reservoirs in the dissemination of Campylobacter spp. which could be considered as potential agent of caprine enteritis and abortion as well as contamination of the wider environment posing serious public health concern in Khartoum State.

To better understand public health implications of waterfowl as reservoirs for zoonotic sources of Campylobacter in recreational waters, we developed a Gallus gallus (chick) model of infection to assess the pathogenicity of environmental isolates of Campylobacter. This method involved exposure of 1-day-old chicks through ingestion of water, the natural route of infection. Viable Campylobacter from laboratory-infected animals were monitored by using a modified non-invasive sampling of fresh chick excreta followed by a passive polycarbonate-filter migration culture assay. The method was used to evaluate the infectivities of three laboratory strains of Campylobacter spp. (Campylobacter coli, Campylobacter jejuni, and Campylobacter lari), three clinical isolates of C. jejuni, and four environmental Campylobacter spp. isolated from California gulls (Larus californicus). The results revealed that chicks were successfully infected with all laboratory and clinical isolates of Campylobacter spp. through ingestion of Campylobacter-spiked water, with infection rates ranging from 90% in a dose-dependent manner. More importantly, exposure of chicks with Campylobacter spp. isolated from Gallus gallus excreta also resulted in successful establishment of infection (≤90%). Each monitored Campylobacter spp. contained ≥7.5 × 104 CFU⋅g–1 of feces 7 days post-exposure. These results suggest that a G. gallus model can be used to assess infectivity of Campylobacter isolates, including gull and human clinical isolates. Use of an avian animal model can be applied to assess the importance of birds, such as the G. gallus, as potential contributors of waterborne-associated outbreaks of campylobacteriosis.

A DNA hybridization test was investigated for application to the detection of Campylobacter spp. in poultry samples. The test chemistry involves solution phase hybridization and detection by means of an enzymatically generated colorimetric endpoint. DNA probes used in the test system are targeted to unique sequences of ribosomal RNA and are specific for Campylobacter jejuni , Campylobacter coli , Campylobacter lari , and Campylobacter fetus subsp. fetus . Initial experiments with pure cultures of C. jejuni established the sensitivity limit of the DNA hybridization assay at approximately 106-7 CFU per sample. Experiments were designed to define optimal conditions for recovery and selective enrichment of Campylobacter spp. from chicken carcasses for use in conjunction with the DNA hybridization assay. Following overnight enrichment, cultures were swabbed onto Campy-Cefex agar plates and allowed to incubate for 24 h. This overnight growth was then suspended and assayed with the DNA probe. The remainder of the overnight enrichment was centrifuged and the resulting pellet was analyzed. Thirty-eight chicken carcasses were assayed for Campylobacter spp. by DNA probe and culture methodology employing culture enrichment and selective plating. Culture procedures isolated Campylobacter spp. from 23 carcasses, while the DNA probe assay detected the organism from 21 carcasses. The DNA probe registered five "false" positives and seven "false" negatives relative to the cultural bacteriologic approach.

Aim : The study was conducted to determine the prevalence and viability of Campylobacter species isolates from different species of poultry and humans in Sokoto State, Nigeria. Materials and Methods : A cross-sectional study was performed in the live birds markets, humans on admission and at outpatient clinics in the randomly selected hospitals in Sokoto State. Isolation and characterization of Campylobacter species were performed using standard culture isolation techniques and biochemical characterization. A total of 798 (506 cloacal and 292 fecal) swabs from poultry and humans, respectively, were collected and analyzed. The viability of 307 isolates stored in 15% glycerol and 85% tryptone broth at −20°C was determined after 7-13 months. Results : A total of 312 (39%) were positive for Campylobacter species which comprises 119 (30%), 20 (30%), 3 (14%), 9 (56%), 1 (50%), and 160 (55%) in chicken, guinea fowls, pigeons, ducks, turkey, and humans, respectively. The total of 38 (24%), 63 (39%), and 59 (37%) humans and 29 (19%), 79 (52%), and 44 (29%) poultry isolates were positive for Campylobacter jejuni , Campylobacter Coli , and Campylobacter Lari, respectively. A total of 261 (85%) of the stored isolates were still viable on re-isolation with the viability rates of 41 (95%), 67 (85%), and 17 (59%) at 7, 9, and 13 months of storage, respectively. There was a negative correlation between months of storage and viability rates. However, there was no significant statistical association (p>0.05) between prevalence rate and species of poultry. Conclusion : Campylobacter species have been detected with varying degree of prevalence in both poultry and humans and their ability to survive freezing at −20°C (95%) for up to 7 months has been revealed in the study. This is not only a concern to food and livestock industries but also a concern to the public health at large, especially, in view of the study area being considered one of the largest livestock producers in Nigeria. Campylobacteriosis is known to be associated with the cost of gastroenteritis management, antimicrobial resistance, food contamination, and complications such as a paralytic condition called Guillain-Barre syndrome.

Background: The objective of this study was to determine the prevalence of thermophilic Campylobacter spp. in asymptomatic school-going children and establish the antibiotic resistance patterns of the isolates towards the drugs used to treat campylobacteriosis, including macrolides, quinolones and tetracycline. Campylobacter spp. are a leading cause of enteric illness and have only recently shown resistance to antibiotics. Methods: This study isolated Campylobacter spp., including Campylobacter coli, Campylobacter jejuni and Campylobacter lari, in stool samples from asymptomatic school-going children in one of the biggest urban slums in Kenya. The disc diffusion method using EUCAST breakpoints was used to identify antibiotic-resistant isolates, which were further tested for genes encoding for tetracycline resistance using primer-specific polymerase chain reaction. Results: In total, 580 stool samples were collected from 11 primary schools considering both gender and age. Subjecting 294 biochemically characterized Campylobacter spp. isolates to genus-specific PCR, 106 (18.27% of stool samples) isolates were confirmed Campylobacter spp. Out of the 106 isolates, 28 (4.83%) were Campylobacter coli, 44 (7.58%) were Campylobacter jejuni while 11 (1.89%) were Campylobacter lari. Campylobacter jejuni had the highest number of isolates that were multi-drug resistant, with 26 out of the 28 tested isolates being resistant to ciprofloxacin (5 mg), nalidixic acid (30 mg), tetracycline (30 mg) and erythromycin (15 mg). Conclusions: In conclusion, asymptomatic school going children in the study area were found to be carriers of multidrug resistant Campylobacter coli, Campylobacter jejuni and Campylobacter lari at 84%. A one-health approach, which considers overlaps in environment, animals and human ecosystems, is recommended in addressing multidrug resistane in Campylobacter, since animals are the main reservoirs and environmental contamination is evident.

The objective of this study was to examine the efficacy of three antibiotic mixtures for use as supplements in a selective medium that could be used to isolate Campylobacter from mixed bacterial cultures during aerobic incubation. A non-selective, basal broth medium was prepared and supplemented with Bolton, Cefex, or Skirrow antibiotic mixtures. The ability of pure cultures of Campylobacter coli, Campylobacter fetus, Campylobacter jejuni, and Campylobacter lari to grow in basal broth and basal broth supplemented with each one of the antibiotic mixtures after aerobic incubation for 24 and 48 h at 37°C was determined. Also, the ability of pure cultures of Escherichia coli, Enterococcus faecalis, Listeria monocytogenes, Pseudomonas aeruginosa, Salmonella Kentucky, and Staphylococcus aureus to grow in basal and supplemented broth medium was determined. Additionally, the recovery of bacteria from mixed cultures containing one Campylobacter isolate with the other 6 non-Campylobacter isolates after aerobic incubation for 48 h at 37°C in basal broth and broth supplemented with the Bolton antibiotic mixture was examined. Results indicated that there was significant (p ≤ 0.05) growth of most Campylobacter isolates in basal broth and in broth supplemented with Bolton, Cefex, or Skirrow antibiotic mixtures after 24 or 48 h of aerobic incubation. Although, there was significant growth non-Campylobacter isolates in basal broth, growth of most these bacteria was inhibited in media supplemented with antibiotic mixtures. Also, Campylobacter growth was generally significantly less than other bacteria in mixed bacterial cultures grown in basal broth for 48 h at 37°C, but significantly more of most Campylobacter isolates were recovered from mixed bacterial cultures grown in media supplemented with the Bolton antibiotic mixture. Findings indicate that basal medium containing this supplement should be studied as a selective medium that may be utilized with aerobic incubation to isolate Campylobacter from environmental samples containing other bacteria.

Campylobacter spp. is one of the main cause of bacterial gastroenteritis in the world. The increase of antibiotic resistance in this species is a threat to public health. A Campylobacter spp. surveillance study was performed in Italy in the 2013–2016 period by the Enter-Net Italia network. The most prevalent Campylobacter species identified causing gastroenteritis was Campylobacter jejuni (73.4%) and 45% of all the annual cases of campylobacteriosis were reported in the summer period. High rates of ciprofloxacin and tetracycline resistance in Campylobacter spp. have been observed. An increasing percentage of Campylobacter coli strains simultaneously resistant to ciprofloxacin, tetracycline and erythromycin has been found. Molecular mechanisms of resistance have been investigated and the role of efflux pumps evaluated. Antibiotic resistance in Campylobacter spp. is an increasing serious threat that requires coordinated action to minimize the emergence and spread of antimicrobial resistant strains from animals to humans throughout the food chain.

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.

Aim: The study was carried out to survey and determine the prevalence of Campylobacter species among pigs and humans within the pig rearing areas of Zuru Kebbi State, Nigeria. Materials and Methods: A cross-sectional study was conducted among household pigs and humans between September, 2013 and February, 2014. Isolation and characterization of Campylobacter species were performed using standard culture isolation techniques and biochemical characterization. A total of 450 fecal samples comprised of 300 from pigs and 150 from humans was collected and analyzed. Results: Prevalence of Campylobacter were 278 (92.67%) and 94 (62.67%) for pigs and humans respectively. The most encountered Campylobacter species in both cases was Campylobacter coli (276 [74.19]) followed by Campylobacter jejuni (62 [16.66]). The least isolated species in pigs was Campylobacter hyointestinalis 5 (1.8%) while Campylobacter lari 2 (2.13%) was least isolated in humans. C. lari was not found in pigs. No significant association (p>0.05) existed between Campylobacter isolates and the age and sex of both pigs and humans in this study. Conclusion: Both pigs and humans within the pig rearing areas of Zuru have been shown to harbor Campylobacter species and this might be due to extensive system of pig farming with indiscriminate defecation by pigs coupled with unhygienic disposal of human wastes in the environment and poor personal hygiene.

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.

Campylobacter is one of the most common bacterial enteropathogens of food borne origin in industrialized countries with C. jejuni being the most common species followed by C. coli. The prevalence of Campylobacters in and around Chandigarh, India was studied by phenotypic and genotypic methods. Fecal samples from 1145 diarrheal patients and 102 healthy subjects from hospital and community were cultured on Campylobacter media and identified by Gram stain, biochemical investigations and serotyping. Molecular identification of Campylobacter isolates was done using specific primers to unique regions of 16S rRNA, Campylobacter jejuni (hipO), Campylobacter coli (aspK), Campylobacter lari (glyA) and Campylobacter upsaliensis (lpxA) genes. Identification of specific genes to look for resistance to nalidixic acid, ciprofloxacin, tetracyclin and streptomycin was also done. Campylobacters were isolated from 2.6% of patients with diarrhea. Campylobacteriosis was more prevalent in children ≤5 years old and during summer season. The most frequent serotypes were S:27, B:2, Z5:52 and V:32. All the Campylobacters isolated by culture were confirmed genotypically by identification of 16S rRNA, hipO and aspK genes. Of the 30 isolates, 27 were C. jejuni and 3 were C. coli. No C. lari or C. upsaliensis were detected. Antibiotic resistance was 40% for nalidixic acid, 23.3% for ciprofloxacin, 50% for tetracyclin and 20% for streptomycin. Campylobacter prevalence is low in the region with C. jejuni being the most common species. A high degree of resistance was found for nalidixic acid and tetracyclin but moderate for ciprofloxacin and streptomycin.

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.

The thermotolerant species Campylobacter jejuni, Campylobacter coli, Campylobacter lari and Campylobacter upsaliensis are the causative agents of the human illness called campylobacteriosis. This infection represents a threat for the health of consumers in Europe. It is well known that poultry meat is an important food vehicle of Campylobacter infection. As emerged from the reported scientific literature published between 2006 and 2016, poultry meat sold at retail level in Europe represents an important source of the pathogen. The contamination level of poultry meat sold at retail can vary depending on pre- and post-harvest factors. Among the pre-harvest measures, strict biosecurity practices must be guaranteed; moreover, among post-harvest control measures scalding, chilling and removal of faecal residues can reduce the contamination level of Campylobacter. An additional issue is represented by increasing proportion of Campylobacter isolates resistant to tetracyclines, ciprofloxacin, and nalidixic acid, thus feeding a serious concern on the effectiveness of antibiotic treatment for human campylobacteriosis in a near future.

Summary Campylobacter causes acute gastroenteritis in people worldwide and is frequently isolated from food, animals and the environment. The disease is predominately food-borne but many routes of transmission and sources of infection have been described, including contact with pets. The prevalence of Campylobacter spp. in dogs and cats varies widely, and data on New Zealand pets are limited. This study aimed to investigate the prevalence of Campylobacter spp. in dogs, cats and retail raw meat pet food products in New Zealand and to characterize Campylobacter jejuni isolates using multilocus sequence typing (MLST). Ninety dogs and 110 cats examined at the Massey University Veterinary Teaching Hospital for elective procedures, and fifty locally purchased retail raw meat pet diets were sampled. Two culture protocols combining Bolton broth enrichment and mCCDA and CAT agars in a microaerobic atmosphere at 42°C and 37°C with species identification using PCR were performed. The prevalence of Campylobacter spp., C. jejuni, Campylobacter upsaliensis and Campylobacter helveticus was 36%, 13%, 23% and 1% in dogs and 16%, 5%, 5% and 7% in cats, respectively. One dog had Campylobacter lari confirmed, and three dogs and one cat had multiple Campylobacter spp. detected. Significantly more animals tested positive using CAT than mCCDA agar (P

This large-scale study compared incubation temperatures (37°C versus 42°C) to study the detection of thermophilic Campylobacter species, including Campylobacter jejuni , C. coli , and C. lari , in various surface water samples and bird fecal droppings around Hamilton Harbor, Lake Ontario. The putative culture isolates obtained from incubation temperatures of 37 and 42°C were confirmed by Campylobacter genus- and species-specific triplex PCR assays targeting the 16S rRNA gene and the 16S-23S rRNA gene internal transcribed spacer (ITS) region. A total of 759 water, wastewater, and bird fecal dropping samples were tested. Positive amplification reactions for the genus Campylobacter were found for 454 (60%) samples incubated at 37°C, compared to 258 (34%) samples incubated at 42°C. C. jejuni (16%) and C. lari (12%) were detected significantly more frequently at the 42°C incubation temperature than at 37°C (8% and 5%, respectively). In contrast, significantly higher rates of C. coli (14%) and other Campylobacter spp. (36%) were detected at the 37°C incubation temperature than at 42°C (8% and 7%, respectively). These results were consistent across surface water, wastewater, and bird fecal dropping samples. At times, Campylobacter spp. were recovered and detected at 37°C (3% for C. jejuni , 10% for C. coli , and 3% for C. lari ) when the same samples incubated at 42°C were negative. A significantly higher rate of other Campylobacter spp. was detected only at 37°C (32%) than only at 42°C (3%). These results indicate that incubation temperature can significantly influence the culturability and detection of thermophilic and other fastidious Campylobacter spp. and that a comprehensive characterization of the Campylobacter spp. in surface water, wastewaters, or bird fecal droppings will require incubation at both 37 and 42°C.

A real time quantitative PCR combined with propidium monoazide (PMA) treatment of samples was implemented to quantify live C. jejuni, C. coli and C. lari on broiler chicken carcasses at selected processing steps in the slaughterhouse. The samples were enumerated by culture for comparison. The Campylobacter counts determined with the PMA-qPCR and the culture method were not concordant. We conclude that the qPCR combined with PMA treatment of the samples did not fully reduce the signal from dead cells.

The objectives of this study were to determine the prevalence of thermophilic Campylobacter spp. in turkey meat samples by conventional cultivation method, to confirm the Campylobacter jejuni, Campylobacter coli, and Campylobacter lari isolates by the detection of specific genes with polymerase chain reaction (PCR), and to find out the antimicrobial susceptibility of the C. jejuni isolates using disc diffusion method. In the present study, thermophilic Campylobacter spp. were in 123 of 270 (45.6%) turkey meat samples. Among the isolates, 109 (40.4%) were C. jejuni and 11 (4.1%) were C. coli as identified by conventional cultivation technique and verified by PCR assay. Prevalence of thermophilic Campylobacter spp. was higher in warm months than cold months. In the study, 104 (95.4%) of the C. jejuni isolates were found resistant to azithromycin, while 103 (94.5%) to erythromycin, 40 (36.7%) to tetracycline, 19 (17.4%) to ciprofloxacin and 10 (9.2%) to nalidixic acid. Practical Applications Thermophilic Campylobacter is one of the major causes of human gastroenteritis worldwide and the present study emphasizes the importance of turkey meat as an important thermophilic Campylobacter spp. source from a public health point of view. Although antimicrobial therapy is suggested for human campylobacteriosis, an increase in antibiotic resistance of Campylobacter jejuni, isolates from human cases and animals were reported. In the present study, prevalence of thermophilic Campylobacter spp. in turkey meat and the antibiotic resistance profiles of the C. jejuni isolates were reported.

Waterways should be considered in the migration routes of Campylobacter, and the genus has been isolated from several water sources. Inferences on migration routes can be made from tracking genetic types in populations found in specific habitats and testing how they are linked to other types. Water samples were taken over a 4-year period from waterways in the Upper Oconee River Watershed, Georgia, to recover isolates of thermophilic Campylobacter. The isolates were typed by multilocus sequence typing (MLST) and analyzed to determine the overall diversity of Campylobacter in that environment. Forty-seven independent isolates were recovered from 560 samples (8.4 %). Two (~4 %) isolates were Campylobacter coli, three (~6 %) isolates were putatively identified as Campylobacter lari, and the remaining 42 (~90 %) were Campylobacter jejuni. The C. jejuni and C. coli isolates were typed by the Oxford MLST scheme. Thirty sequence types (STs) were identified including 13 STs that were not found before in the MLST database, including 24 novel alleles. Of the 17 previously described STs, 10 have been isolated from humans, 6 from environmental water, and 6 from wild birds (five types from multiple sources). Seven sites had multiple positive samples, and on two occasions, the same ST was isolated at the same site. The most common type was STST61 with four isolates, and the most common clonal complex was CC179 with nine isolates. CC179 has been commonly associated with environmental water. Although some Campylobacter STs that were found in the Oconee River engage in widespread migration, most are tightly associated with or unique to environmental water sources.

The objectives of this study were to determine the prevalence of antimicrobial resistance (AMR) in faecal Campylobacter spp. from lambs and adult sheep and associations between antimicrobial use (AMU) and AMR. A total of 275 faecal samples collected during initial and final visits from 51 sheep flocks, including one feedlot, across southern Ontario were tested for the presence of Campylobacter spp. Campylobacter jejuni was detected in 52% (143/275) of the faecal samples, Campylobacter coli in 7% (19/275), Campylobacter lari in 1% (2/275) and 2% (4/275) were non-speciated Campylobacter. Broth microdilution was used to test antimicrobial susceptibility of 162 isolates to nine antimicrobials. Campylobacter jejuni isolates (n = 142) were resistant to tetracycline (39%), ciprofloxacin (4%), nalidixic acid (4%) and telithromycin (1%). C. coli isolates (n = 19) were resistant to tetracycline (74%), and azithromycin, clindamycin, erythromycin, and telithromycin (5%). The C. lari isolate displayed resistance to nalidixic acid. No statistically significant associations were found between AMU and AMR during multivariate modelling in this study.

Presence or absence of Campylobacter spp. in water of five rivers upstream from an intake point for drinking water production was investigated, and isolates genetically compared with human, pig and poultry isolates in order to determine their source. River water and drinking water obtained from these rivers were sampled one time per month, over a period of one year, and tested for Campylobacter. Isolates were typed by PFGE. Campylobacter was not detected in treated drinking water, but 50% of the river samples were contaminated. Contamination was observed on the four seasons. In total, 297 Campylobacter isolates were collected and generated 46 PFGE profiles. Campylobacter jejuni was the most frequently detected species in samples (74.1% of the isolates), followed by Campylobacter coli (17.8%) and Campylobacter lari (8.1%). Forty-two of the 46 PFGE profiles were unique. Only one genotype was detected three times in a river during the year and four genotypes in two different rivers. When compared to animal and human Campylobacter PFGE profiles, 14, 11 and one Campylobacter genotypes from water were genetically closed to human, poultry, and pig Campylobacter genotypes, respectively. The Campylobacter population displayed a high level of genetic diversity, suggesting that contamination originated from various origins. Human, poultry and pig were sources of contamination of the river by Campylobacter. Finally, no Campylobacter were detected in drinking water, indicating that the risk of outbreaks due to consumption of drinking water is low.

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).

The antibacterial activity of ethanolic extracts of selected Thai medicinal plants (Rhodomyrtus tomentosa (Aiton) Hassk., Quercus infectoria G. Olivier, and Eleutherine americana Merr.) against Campylobacter spp. was investigated. Sixty-five Campylobacter, including 39 isolates from humans and 26 isolates from chicken samples, were tested. Reference Campylobacter spp. that are commonly encountered in gastroenteritis were included. The ethanolic extract of E. americana demonstrated good antibacterial activity against all the tested isolates. Inhibition zones ranged from 10 to 37 mm. Minimum inhibitory concentration (MIC) of the extract against Campylobacter isolates from humans and chicken samples ranged from 31.25 to 500 μg/mL and 62.50 to 1,000 μg/mL, respectively. The minimum bactericidal concentration ranged from 31.25 to 1,000 μg/mL for isolates from humans and 125 to 1,000 μg/mL from chicken isolates. The bactericidal activity of the ethanolic extracts of E. americana against important Campylobacter spp., including Campylobacter coli MUMT 18630, Campylobacter fetus ATCC 27374, Campylobacter jejuni ATCC 81176, Campylobacter lari ATCC 43675, and Campylobacter upsaliensis DMST 19055, were assessed at MIC, 2 MIC, and 4 MIC by counting viable cells after various time intervals. At 4 MIC, the level of the tested isolates decreased by 2 to 5 log-fold within 8 h. The ethanolic extract of E. americana demonstrated antibacterial activity against all Campylobacter spp. from both human and chicken isolates. Further investigation of this plant species may provide an alternative medicine for Campylobacter infection and an effective food additive to prevent the infection.

Infections caused by thermotolerant Campylobacter spp. are among the most frequent bacterial diseases from food in Europe. An important source of infection is poultry. The aim was to perform the first nationwide study on the prevalence of Campylobacter spp. at retail in the Czech Republic. Samples of chilled and frozen broiler chickens were obtained from supermarkets localized in the eight largest cities in the Czech Republic. From February to November 2009, a total of 120 samples of chilled and 120 samples of frozen poultry were collected and assessed. Qualitative and quantitative analyses were performed according to the ISO 10272-1:2006 and ISO 10272-2:2006 methods. The microdilution method was used to test the resistance of Campylobacter spp. isolates to selected antibiotics. High prevalence of Campylobacter spp. was detected in chilled poultry (75%). The prevalence was significantly lower (37%) in frozen poultry. The most prevalent species was Campylobacter jejuni detected in 70% of positive findings, followed by Campylobacter coli (18%). In 12% of cases, the samples were contaminated by both C. jejuni and C. coli. The species Campylobacter lari and Campylobacter upsaliensis were not detected. The highest resistance rates were to quinolone antibiotics and ampicillin.

Salmonella and Campylobacter are common bacterial pathogens associated with human gastro-enteritis; and raw poultry is considered to be an important source of these bacteria. To evaluate whether the Salmonella serovars and Campylobacter spp. bacteria could be monitored for the purpose of microbial presence, enumeration and antimicrobial resistance in raw poultry, 152 poultry carcasses were randomly selected from 10 markets in retail outlets of Phnom Penh during March 2006 to February 2007. The majority of poultry samples was contaminated by Salmonella serovars (88.2%) and Campylobacter spp. (80.9%). A very high contamination of Salmonella was found at 3-4 log₁₀ CFU/g for 22.4% of samples and of Campylobacter at 7-8 log₁₀ CFU/g for 1.3% of samples. Fifty nine different Salmonella serovars contaminated 134 poultry carcasses; five most prevalent serovars covered 29.1% of serovars isolates (Anatum, Typhimurium, Corvallis, Stanley and Enteritidis). Three Campylobacter species contaminating 123 raw poultry were Campylobacter jejuni (50.0%), Campylobacter coli (29.0%) and Campylobacter lari (21.0%). High antibiotic resistance percentages were found among Salmonella serovars and Campylobacter spp. isolates. This study revealed that raw poultry at the retail outlets in Phnom Penh markets are contaminated with high prevalences of food-borne pathogens, and communicating the importance of minimizing this risk in reducing human infections.

Several species of free-living amoebae can cause disease in humans. However, in addition to the direct pathogenicity of e.g. Acanthamoebae and Naegleria species, they are recognized as environmental hosts, indirectly involved in the epidemiology of many pathogenic bacteria. Although several studies have demonstrated intracellular survival of many different bacteria in these species, the extent of such interactions as well as the implications for the epidemiology of the bacterial species involved, are largely unknown and probably underestimated. In this study, we evaluated eight different unicellular eukaryotic organisms, for their potential to serve as environmental hosts for Campylobacter species. These organisms include four amoebozoas (Acanthamoeba polyphaga, Acanthamoeba castellanii, Acanthamoeba rhysodes and Hartmanella vermiformis), one alveolate (Tetrahymena pyriformis), one stramenopile (Dinobryon sertularia), one eugoenozoa (Euglena gracilis) and one heterolobosea (Naegleria americana). Campylobacter spp. including Campylobacter jejuni, Campylobacter coli and Campylobacter lari are the most common cause of gastroenteritis in the western world. Survival and replication of these three species as well as Campylobacter hyointestinalis were assessed in co-cultures with the eukaryotic organisms. Campylobacter spp. generally survived longer in co-cultures, compared to when incubated in the corresponding growth media. The eukaryotic species that best promoted bacterial survival was the golden algae D. sertularia. Three species of amoebozoas, of the genus Acanthamoeba promoted both prolonged survival and replication of Campylobacter spp. The high abundance in lakes, ponds and water distribution networks of these organisms indicate that they might have a role in the epidemiology of campylobacteriosis, possibly contributing to survival and dissemination of these intestinal pathogens to humans and other animals. The results suggest that not only C. jejuni, but a variety of Campylobacter spp. can interact with different eukaryotic unicellular organisms.

A multiplex real-time PCR (qPCR) assay was developed for simultaneous detection and differentiation of the three most important Campylobacter species in chickens. Three novel sets of PCR primers and TaqMan probes were designed to amplify the unique DNA sequences within the hipO, cdtA, and pepT genes which are specific to Campylobacter jejuni, Campylobacter coli, and Campylobacter lari, respectively. To avoid competition in the multiple target amplifications, the concentrations of primers and probes were optimized. By using the optimized qPCR conditions together with a minor-groove binding probe of pepT, amplification efficiency greater than 92% and detection sensitivity of 38 genome copies/reaction have been achieved for all three targets. The assay was highly specific for C. jejuni, C. coli, and C. lari with testing of 33 Campylobacter strains and 20 non-Campylobacter strains. In chicken samples spiked with known quantities of Campylobacter cells, the assay was able to detect 1 CFU/g after a 24-h enrichment. Application of the assay in food was further evaluated using 21 fresh chicken samples obtained from local supermarkets. The results revealed that, after a 24-h or 48-h enrichment, 14 samples (66.7%) were positive for C. jejuni, five samples (23.8%) were positive for C. coli, and none of the samples was contaminated by C. lari. Taken together, the multiplex qPCR assay combined with an enrichment step is a sensitive, species-specific, and non-labor-intensive method suitable for rapid detection of C. jejuni, C. coli, and C. lari in chicken samples.

The aim of the present study was to monitor the occurrence of Campylobacter spp. in broiler chicken production in Estonia from 2002 to 2007. Campylobacter spp. was isolated in 163 (12.3%) of 1320 broiler chicken meat samples form 2002 to 2007 and in 115 (6.3%) of 1819 cecal samples in 2005–2007. Campylobacter jejuni was the most commonly isolated species (98.2%), followed by Campylobacter coli (1.4%) and Campylobacter lari (0.4%). The seasonal peak of Campylobacter contamination was from July to September. Our findings showed that Campylobacter contamination at all levels of broiler chicken production in Estonia was low.

A multiplex real-time PCR assay based on four differently labeled TaqMan probes for detection and differentiation of the thermophilic Campylobacter species C. jejuni, C. coli, and C. lari was established and validated in food products. This assay combines two previously published PCR assays for C. jejuni and C. coli with a newly developed detection assay for C. lari and an internal amplification control system. The selectivity of the method was determined by analyzing 70 Campylobacter strains and 43 strains of other bacteria. The sensitivity was 50 fg of C. jejuni and C. lari DNA and 500 fg of C. coli DNA per PCR. It was possible to detect 1 to 10 CFU/25 g of food before preenrichment of all three species. More than 400 samples of various foods (poultry, seafood, and meat) were analyzed after 48 h of preenrichment parallel to the conventional diagnostic method of culture and biochemical identification. Using the established real-time PCR assay, 55.4% of the samples were recognized as positive for thermophilic Campylobacter species, whereas with the conventional method only 40.3% of the samples were positive. The real-time PCR assay also detected contaminations with two different Campylobacter species in 32.6% of the analyzed poultry samples, a finding of epidemiological interest. Compared with the original PCR method, which was established for the differentiation of bacterial isolates of C. jejuni and C. coli, this new method also detects and distinguishes C. lari, was validated as an analytical tool for food analysis, and provides reliable and extensive results within 2 days.

A new rapid method based on real-time PCR was developed to detect four thermophilic Campylobacter species (Campylobacter jejuni, Campylobacter coli, Campylobacter lari, and Campylobacter upsaliensis) in food samples. The assay targeted the bipA gene for C. upsaliensis and C. lari, whereas the gene encoding the ATP-binding protein CJE0832 was used to detect C. coli and C. jejuni. These genes were chosen for this assay due to their low variability and mutation rate at a species level. The multiplex PCR showed 100% inclusivity for all 25 thermophilic Campylobacter strains tested and 100% exclusivity for 38 non-targeted strains belonging to closely related species. The newly developed real-time PCR could detect down to 102 genomes/reaction and displayed efficiency above 97% for all species except for C. upsaliensis (90.1%). The method proved to be a reliable tool for food analysis, showing 100% sensitivity, 96% efficiency, and 92.45% specificity when validated against the gold standard method UNE-EN ISO 10272:2006 using 200 diverse food samples (meat, fish, fruits and vegetables, and raw milk). In artificially spiked samples, the detection limit of the method was 10 cfu/g in salad, 5 cfu/g in turkey meat, and 1 cfu/g in the rest of meat samples tested. Consequently, the newly designed molecular tool represents a quick and safe alternative to obtain reliable results concerning the presence/absence of the main thermophilic Campylobacter in any food sample.

Campylobacter infections have been reported at prevalences ranging from 2 to 50% in a range of wild bird species, although there have been few studies that have investigated the molecular epidemiology of Campylobacter spp. Consequently, whether wild birds are a source of infection in humans or domestic livestock or are mainly recipients of domestic animal strains and whether separate cycles of infection occur remain unknown. To address these questions, serial cross-sectional surveys of wild bird populations in northern England were carried out over a 2-year period. Fecal samples were collected from 2,084 wild bird individuals and screened for the presence of Campylobacter spp. A total of 56 isolates were recovered from 29 birds sampled at 15 of 167 diverse locales. Campylobacter jejuni , Campylobacter lari , and Campylobacter coli were detected by PCR, and the prevalences of different Campylobacter spp. in different avian families ranged from 0% to 33%. Characterization of 36 C. jejuni isolates by multilocus sequence typing revealed that wild birds carry both livestock-associated and unique strains of C. jejuni . However, the apparent absence of unique wild bird strains of C. jejuni in livestock suggests that the direction of infection is predominantly from livestock to wild birds. C. lari was detected mainly in wild birds sampled in an estuarine or coastal habitat. Fifteen C. lari isolates were analyzed by macrorestriction pulsed-field gel electrophoresis, which revealed genetically diverse populations of C. lari in Eurasian oystercatchers ( Haematopus ostralegus ) and clonal populations in magpies ( Pica pica ).

A polyphasic study was undertaken to clarify the taxonomic position of Campylobacter lari-like strains isolated from shellfish and humans. The diversity within the strain collection was initially screened by means of fluorescent amplified fragment length polymorphism analysis and whole-cell protein electrophoresis, revealing the existence of two clusters distinct from C. lari and other Campylobacter species. The divergence of these clusters was confirmed by phenotypic analysis and by 16S rRNA and hsp60 gene sequence analysis. Phylogenetic analysis identified C. lari, Campylobacter jejuni, Campylobacter coli and Campylobacter insulaenigrae as the closest phylogenetic neighbours of both taxa. DNA-DNA hybridizations revealed that one cluster, comprising 10 strains, represented a novel Campylobacter species, for which the name Campylobacter peloridis sp. nov. is proposed, with 2314BVA(T) (=LMG 23910(T) =CCUG 55787(T)) as the type strain. The second cluster, comprising six strains, represents a novel subspecies within the species C. lari, for which the name Campylobacter lari subsp. concheus subsp. nov. is proposed, with 2897R(T) (=LMG 21009(T) =CCUG 55786(T)) as the type strain. The description of C. lari subsp. concheus has the effect of automatically creating the subspecies Campylobacter lari subsp. lari subsp. nov. (type strain LMG 8846(T)=NCTC 11352(T)).

Conventional procedures for isolation of thermophilic Campylobacter spp. from chicken are complex, labor intensive, and time-consuming. The objective of this study was to create a novel Campylobacter culturing apparatus. A main concept of the device was based on the ability of Campylobacter to pass through a 0.45 microm pore size filter in viscous media. Preliminary study demonstrated that only viable Campylobacter moved through the membrane filter and could multiply in the enrichment culture. C. jejuni, C. coli, C. lari, and C. upsaliensis in the chicken samples were detected at cell concentrations as low as 10 cfu/g, after 24 h incubation at 42 degrees C. In total, 84 retail chicken samples were comparatively studied using both conventional method and apparatus. Sixteen samples (19.05%) were positive by the apparatus method; 14 (16.66%) of these positive samples contained C. coli and 2 (2.38%) contained C. jejuni. With the conventional method, 7 (8.33%) samples were positive 7 (8.33%) with C. coli. In conclusion, the apparatus detected more positive samples than did the conventional culture method.

Background/Aim. One of the most important bacterial zoonosis is campylobacteriosis. Human disease is mostly caused by thermophilic Campylobacter spp: Campylobacter jejuni (C. jejuni), Campylobacter coli (C. coli), Campylobacter lari (C. lari) and Campylobacter upsaliensis (C. upsaliensis). Campylobacteriosis is a mild and self-healing disorder. In patients with more severe and prolonged forms, an antibiotic treatment is recommended. Recommended drugs are erythromycin, ciprofloxacin, tetracyclin, chloramphenicol and ampicillin. Lately, an increase of Campylobacter genus resistance to antibiotics mostly used in therapy is an annoying evidence. The rise coincided with the beginning of antibiotic use, especially quinolones, in veterinary medicine. The aim of the study was to isolate and identify thermophilic Campylobacter spp. from human feces and to determine their sensitivity to antibiotics and hemotherapeutics mostly used in campylobacteriosis treatment. Methods. Sensitivity to erythromycin, tetracyclin, ampicillin, chloramphenicol and ciprofloxacin of 24 strains of Campylobacter spp. isolated from humans was investigated by E-test. Results. Seventeen C. jejuni and seven C. coli strains were investigated. Six (25%) out of 24 C. jejuni and C. coli investigated strains were resistant to ampicillin and seven (29.2%) were resistant to tetracycline. Twelve (50%) C. jejuni and C. coli investigated strains were resistant to ciprofloxacin. C. jejuni was more resistant (52.9%). Four (23.5%) C. jejuni strains were resistant to erythromycin and 11.7% to choramphenicol. None of C. coli strains were resistant to both chloramphenicol and erytromycin. Conclusons. Testing sensitivity to erythromycin, tetracycline, ampicillin, chloramphenicol and ciprofloxacin demonstrated a rather high resistance frequency of C. jejuni and C. coli strains isolated from humans. C. jejuni strains were more resistant than those of C. coli.

The thermophilic Campylobacters are enteropathogenic for humans. We recently showed that Omp50 is a Campylobacter species-specific porin produced in Campylobacter jejuni and Campylobacter lari but not in Campylobacter coli. In the present study, we investigated regulation of the omp50 gene and found that its expression in C. jejuni was temperature-dependent, but independent of growth phase or medium viscosity. The use of RT-PCR and omp50::lacZ fusions showed that growth temperature control occurred at the transcriptional level. The promoter and the coding sequence were cloned in an Escherichia coli-Campylobacter shuttle plasmid and transferred to E. coli and to a C. jejuni Omp50-deficient strain. Regulation of omp50 gene expression by growth temperature was observed in the recombinant C. jejuni strain, but not in E. coli. The same regulation was also observed in wild-type C. lari strains and in a C. coli strain supplemented by the plasmid, suggesting that omp50 expression is controlled by a mechanism conserved among Campylobacter species.