Detection of cytolethal distending toxin activity and cdt genes in Campylobacter spp. isolated from chicken carcasses

This study was designed to determine whether isolates from chicken carcasses, the primary source of Campylobacter jejuni and Campylobacter coli in human infections, commonly carry the cdt genes and also whether active cytolethal distending toxin (CDT) is produced by these isolates. Campylobacter spp. were isolated from all 91 fresh chicken carcasses purchased from local supermarkets. Campylobacter spp. were identified on the basis of both biochemical and PCR tests. Of the 105 isolates, 70 (67%) were identified as C. jejuni, and 35 (33%) were identified as C. coli. PCR tests amplified portions of the cdt genes from all 105 isolates. Restriction analysis of PCR products indicated that there appeared to be species-specific differences between the C. jejuni and C. coli cdt genes, but that the restriction patterns of the cdt genes within strains of the same species were almost invariant. Quantitation of active CDT levels produced by the isolates indicated that all C. jejuni strains except four (94%) had mean CDT titers greater than 100. Only one C. jejuni strain appeared to produce no active CDT. C. coli isolates produced little or no toxin. These results confirm the high rate of Campylobacter sp. contamination of fresh chicken carcasses and indicate that cdt genes may be universally present in C. jejuni and C. coli isolates from chicken carcasses.     

Prevalence of Campylobacter spp., Escherichia coli, and Salmonella Serovars in Retail Chicken, Turkey, Pork, and Beef from the Greater Washington, D.C., Area

A total of 825 samples of retail raw meats (chicken, turkey, pork, and beef) were examined for the presence of Escherichia coli and Salmonella serovars, and 719 of these samples were also tested for Campylobacter spp. The samples were randomly obtained from 59 stores of four supermarket chains during 107 sampling visits in the Greater Washington, D.C., area from June 1999 to July 2000. The majority (70.7%) of chicken samples (n = 184) were contaminated with Campylobacter, and a large percentage of the stores visited (91%) had Campylobacter-contaminated chickens. Approximately 14% of the 172 turkey samples yielded Campylobacter, whereas fewer pork (1.7%) and beef (0.5%) samples were positive for this pathogen. A total of 722 Campylobacter isolates were obtained from 159 meat samples; 53.6% of these isolates were Campylobacter jejuni, 41.3% were Campylobacter coli, and 5.1% were other species. Of the 212 chicken samples, 82 (38.7%) yielded E. coli, while 19.0% of the beef samples, 16.3% of the pork samples, and 11.9% of the turkey samples were positive for E. coli. However, only 25 (3.0%) of the retail meat samples tested were positive for Salmonella. Significant differences in the bacterial contamination rates were observed for the four supermarket chains. This study revealed that retail raw meats are often contaminated with food-borne pathogens; however, there are marked differences in the prevalence of such pathogens in different meats. Raw retail meats are potential vehicles for transmitting food-borne diseases, and our findings stress the need for increased implementation of hazard analysis of critical control point (HACCP) and consumer food safety education efforts.     

Prevalence and Pathogenic Potential of Campylobacter Isolates from Free-Living,Human-Commensal American Crows

Recent studies have suggested a potential role for wild birds in zoonotic transmission of Campylobacter jejuni, the leading cause of gastroenteritis in humans worldwide. In this study, we detected Campylobacter spp. in 66.9% (85/127) of free-ranging American crows (Corvus brachyrhyncos) sampled in the Sacramento Valley of California in 2012 and 2013. Biochemical testing and sequence analysis of 16S rRNA revealed that 93% of isolates (n = 70) were C. jejuni, with cytolethal distending toxin (CDT) and flagellin A genes detected by PCR in 20% and 46% of the C. jejuni isolates (n = 59), respectively. The high prevalence of C. jejuni, coupled with the occurrence of known virulence markers CDT and flagellin A, demonstrates that crows shed Campylobacter spp. in their feces that are potentially pathogenic to humans. Crows are abundant in urban, suburban, and agricultural settings, and thus further study to determine their role in zoonotic transmission of Campylobacter will inform public health.     

Prevalence of Campylobacter spp. in Cattle in Finland and Antimicrobial Susceptibilities of Bovine Campylobacter jejuni Strains

The study investigated the prevalence of Campylobacter spp. in Finnish cattle at slaughter and carcass contamination after slaughter. During the period January to December 2003, bovine rectal fecal samples (n = 952) and carcass surface samples (n = 948) from 12 out of 15 Finnish slaughterhouses were examined. In total, campylobacters were detected in 31.1% of fecal samples and in 3.5% of carcass surface samples. Campylobacter jejuni was isolated from 19.5%, Campylobacter coli from 2.2%, and presumptive Campylobacter hyointestinalis from 10.8% of fecal samples. Campylobacters were detected in 4.4% and 37.4% of the fecal samples examined both by direct culture and by enrichment (n = 730), respectively, suggesting a low level of campylobacters in the intestinal content. A slightly increasing trend was observed in the overall prevalence of campylobacters towards the end of summer and autumn. Seventeen different serotypes were detected among the fecal C. jejuni isolates using a set of 25 commercial antisera for serotyping heat-stable antigens (Penner) of C. jejuni by passive hemagglutination. The predominant serotypes, Pen2 and Pen4-complex, were isolated from 52% of the fecal samples. Subtyping by pulsed-field gel electrophoresis (SmaI) yielded 56 and 20 subtypes out of 330 fecal and 70 carcass C. jejuni isolates, respectively. MICs of ampicillin, enrofloxacin, erythromycin, gentamicin, nalidixic acid, and oxytetracycline for 187 C. jejuni isolates were determined using a commercial broth microdilution method. Sixteen (9%) of the isolates were resistant to at least one of the antimicrobials tested. Resistance to nalidixic acid was most commonly detected (6%). No multiresistance was observed.     

Genotyping and PCR detection of potential virulence genes in <Emphasis Type="Italic">Campylobacter jejuni</Emphasis> and <Emphasis Type="Italic">Campylobacter coli</Emphasis> isolates from different sources in Poland

The prevalence of potential virulence markers was determined among the population of Polish Campylobacter jejuni and Campylobacter coli isolates from children, chickens, pigs and dogs. The presence of the flaA, flaB, cdtA, cdtB, cdtC, cdtABC, virB11, and cj0588 genes among 74 C. jejuni and 15 C. coli isolates was detected by PCR. High prevalence of five different putative virulence and toxin genes (flaA, cdtA, cdtB, cdtC, and cj0588) was found among isolates obtained from children, chickens and dogs. The occurrence of these genes among isolates obtained from pigs was significantly different than for strains isolated from other sources. Two methods for genotyping Campylobacter spp. strains were applied — flaA-typing, and ADSRRS-fingerprinting method, which was used for the first time for Campylobacter spp. strains. Similarity of the genetic profiles was demonstrated in strains isolated from chickens and dogs, and in isolates from chickens and children. Strains isolated from pigs, both C. jejuni as well as C. coli, did not group with isolates from other sources.     

Stable Concentrated Emulsions of the 1-Monoglyceride of Capric Acid (Monocaprin) with Microbicidal Activities against the Food-Borne Bacteria Campylobacter jejuni, Salmonella spp., and Escherichia coli

Of 11 fatty acids and monoglycerides tested against Campylobacter jejuni, the 1-monoglyceride of capric acid (monocaprin) was the most active in killing the bacterium. Various monocaprin-in-water emulsions were prepared which were stable after storage at room temperature for many months and which retained their microbicidal activity. A procedure was developed to manufacture up to 500 ml of 200 mM preconcentrated emulsions of monocaprin in tap water. The concentrates were clear and remained stable for at least 12 months. They were active against C. jejuni upon 160- to 200-fold dilution in tap water and caused a >6- to 7-log₁₀ reduction in viable bacterial count in 1 min at room temperature. The addition of 0.8% Tween 40 to the concentrates as an emulsifying agent did not change the microbicidal activity. Emulsions of monocaprin killed a variety of Campylobacter isolates from humans and poultry and also killed strains of Campylobacter coli and Campylobacter lari, indicating a broad anticampylobacter activity. Emulsions of 1.25 mM monocaprin in citrate-lactate buffer at pH 4 to 5 caused a >6- to 7-log₁₀ reduction in viable bacterial counts of Salmonella spp. and Escherichia coli in 10 min. C. jejuni was also more susceptible to monocaprin emulsions at low pH. The addition of 5 and 10 mM monocaprin emulsions to Campylobacter-spiked chicken feed significantly reduced the bacterial contamination. These results are discussed in view of the possible utilization of monocaprin emulsions in controlling the spread of food-borne bacteria from poultry to humans.     

Prevalence and Antimicrobial Resistance of Thermophilic Campylobacter spp. from Cattle Farms in Washington State

The prevalence of thermophilic Campylobacter spp. was investigated in cattle on Washington State farms. A total of 350 thermophilic Campylobacter isolates were isolated from 686 cattle sampled on 15 farms (eight dairies, two calf rearer farms, two feedlots, and three beef cow-calf ranches). Isolate species were identified with a combination of phenotypic tests, hipO colony blot hybridization, and multiplex lpxA PCR. Breakpoint resistance to four antimicrobials (ciprofloxacin, nalidixic acid, erythromycin, and doxycycline) was determined by agar dilution. Campylobacter jejuni was the most frequent species isolated (34.1%), followed by Campylobacter coli (7.7%) and other thermophilic campylobacters (1.5%). The most frequently detected resistance was to doxycycline (42.3% of 350 isolates). Isolates from calf rearer facilities were more frequently doxycycline resistant than isolates from other farm types. C. jejuni was most frequently susceptible to all four of the antimicrobial drugs studied (58.8% of 272 isolates). C. coli isolates were more frequently resistant than C. jejuni, including resistance to quinolone antimicrobials (89.3% of isolates obtained from calves on calf rearer farms) and to erythromycin (72.2% of isolates obtained from feedlot cattle). Multiple drug resistance was more frequent in C. coli (51.5%) than in C. jejuni (5.1%). The results of this study demonstrate that C. jejuni is widely distributed among Washington cattle farms, while C. coli is more narrowly distributed but significantly more resistant.     

Production of a Monoclonal Antibody Specific for the Major Outer Membrane Protein of Campylobacter jejuni and Characterization of the Epitope

Campylobacter species are important enteric pathogens causing disease in humans and animals. There is a lack of a good immunological test that can be used routinely to separate Campylobacter jejuni from other Campylobacter species. We produced monoclonal antibodies (MAbs) directed against the major outer membrane protein (MOMP) of C. jejuni using recombinant MOMP as the antigen. One MAb, designated MAb5C4 and of the immunoglobulin G1 isotype, was found to be potentially specific for C. jejuni. Dot blots demonstrated that MAb5C4 reacted with all 29 isolates of C. jejuni tested but did not react with 2 C. jejuni isolates, 26 other Campylobacter spp. isolates, and 19 non-Campylobacter isolates. Western blotting showed that MAb5C4 bound to a single protein band approximately 43 kDa in size, corresponding to the expected size of C. jejuni MOMP. The detection limit of MAb5C4 in a dot blot assay was determined to be about 5 × 10³ bacteria. The epitope on the MOMP was mapped to a region six amino acids in length with the sequence ²¹⁶GGQFNP²²¹, which is 97% conserved among C. jejuni strains but divergent in other Campylobacter spp.; a GenBank search indicated that 95% of C. jejuni isolates will be able to be detected from non-Campylobacter spp. based on the highly specific and conserved region of the GGQFNP polypeptide. The epitope is predicted to be located in a region that is exposed to the periplasm. MAb5C4 is a potentially specific and sensitive MAb that can be used for the specific detection and identification of C. jejuni.     

Antimicrobial-Resistant Campylobacter Species from Retail Raw Meats

The antimicrobial susceptibilities of 378 Campylobacter isolates were determined. Resistance to tetracycline was the most common (82%), followed by resistance to doxycycline (77%), erythromycin (54%), nalidixic acid (41%), and ciprofloxacin (35%). Campylobacter coli displayed significantly higher rates of resistance to ciprofloxacin and erythromycin than Campylobacter jejuni, and Campylobacter isolates from turkey meat showed a greater resistance than those from chicken meat.     

Evaluation of novel agars for the enumeration of Campylobacter spp. in poultry retail samples

The purpose of this study was to examine the performance of novel agars for the identification and enumeration of Campylobacter species. The analytical sensitivity and specificity of Campylobacter Selective agar (CASA), Brilliance CampyCount agar (BCCA) and CampyFoodIDagar (CFA) for 84 Campylobacter spp. isolates and 50 non-Campylobacter spp. isolates from 37 distinct genera were of 100% sensitivity, with a 98% specificity for BCCA and CFA, and a 100% specificity for CASA. The application of these selective agars for Campylobacter spp. enumeration in comparison to the conventional agars, modified charcoal cefoperazonedeoxycholate agar (mCCDA) and Campy-Cefex (CCA) was examined using Campylobacter jejuni and Campylobacter coli inoculated samples. From C. jejuni inoculated samples, recovery on BCCA was significantly greater than other media (p < 0.05). Recovery on CASA was not significantly different from mCCDA and CCA (p > 0.05). With C. coli inoculated samples, recovery was significantly greater on BCCA and CASA than with other media (p < 0.05). The recovery of both C. jejuni and C. coli from inoculated samples with CFA was significantly less than with other media (P < 0.05). CASA was able to effectively inhibit and differentiate Campylobacter spp. from background microflora while false positive organisms occurred with BCCA and CFA. An examination of 483 randomly selected suspect Campylobacter colonies from naturally contaminated samples demonstrated a colony confirmation rate for CCA, CFA, BCCA, mCCDA, and CASA, of 84%, 87%, 88%, 90%, and 100%, respectively. The media evaluated present an alternative to conventional selective agars for the identification and enumeration of thermotolerant Campylobacter spp. from samples of poultry origin through the farm to fork continuum.     

Genomic Insights into the Convergence and Pathogenicity Factors of Campylobacter jejuni and Campylobacter coli Species

Whether or not bacteria form coherent evolutionary groups via means of genetic exchange and, hence, elicit distinct species boundaries remains an unsettled issue. A recent report implied that not only may the former be true but also, in fact, the clearly distinct Campylobacter jejuni and Campylobacter coli species may be converging as a consequence of increased interspecies gene flow fostered, presumably, by the recent invasion of an overlapping ecological niche (S. K. Sheppard, N. D. McCarthy, D. Falush, and M. C. Maiden, Science 320:237-239, 2008). We have reanalyzed the Campylobacter multilocus sequence typing database used in the previous study and found that the number of interspecies gene transfer events may actually be too infrequent to account, unequivocally, for species convergence. For instance, only 1 to 2% of the 4,507 Campylobacter isolates examined appeared to have imported gene alleles from another Campylobacter species. Furthermore, by analyzing the available Campylobacter genomic sequences, we show that although there seems to be a slightly higher number of exchanged genes between C. jejuni and C. coli relative to other comparable species (∼10% versus 2 to 3% of the total genes in the genome, respectively), the function and spatial distribution in the genome of the exchanged genes are far from random, and hence, inconsistent with the species convergence hypothesis. In fact, the exchanged genes appear to be limited to a few environmentally selected cellular functions. Accordingly, these genes may represent important pathogenic determinants of pathogenic Campylobacter, and convergence of (any) two bacterial species remains to be seen.High-throughput sequencing studies during the last decade have revealed that bacterial genomes are much more diverse and “fluid” than previously anticipated (14, 31). This genomic fluidity is primarily attributable to the great pervasiveness and promiscuity of horizontal gene transfer (HGT) in the bacterial world (5, 17). Nonetheless, evidence of any two distinct bacterial species or lineages merging due to directed (as opposed to promiscuous) interspecies genetic exchange was reported, probably for the first time ever, by the recent study of Sheppard et al. (26). Species convergence, if occurring, has major theoretical implications for the bacterial species concept (reviewed extensively elsewhere [9, 10, 14, 24, 30]) and important practical consequences for accurate identification of bacterial pathogens in the clinical setting.Sheppard and colleagues reported that as many as ∼18.6% of the unique alleles of housekeeping genes found in Campylobacter coli isolates may have been recently imported (through HGT) from a close relative, Campylobacter jejuni (26). The results were based on the analysis of 4,507 Campylobacter spp. isolates, which were genotyped at seven genes (loci), available though the Campylobacter multilocus sequence typing (MLST) database (4). In brief, the 4,507 genotyped isolates contained a total of 2,917 unique sequence types (STs). A unique ST represents the concatenated sequence of the seven genes present in the genome of an isolate and contains a unique sequence (allele) for at least one of the seven genes when compared against any other unique ST in the database (different isolates may be characterized by the same ST). The unique STs were assigned to either C. coli or C. jejuni species by using the program STRUCTURE (6). Neighbor-joining phylogenetic trees of all available unique alleles for each individual gene were subsequently built. Instances where the ST assignment to a species differed from the assignment of an individual gene sequence, which comprised the ST, were attributed to interspecies transfer of the gene, and the number of such instances was reported (26).Here, we have reevaluated the available Campylobacter MLST data set and show that the predominant STs, i.e., the STs characterizing >98% of the isolates, do not contain imported alleles and, hence, do not support the species convergence hypothesis. In agreement with these findings, analyses of the available Campylobacter genomic sequences indicate that the interspecies genetic exchange is limited and heavily biased toward a few genes under positive selection. In fact, housekeeping genes (such as those used in MLST) were found to be exchanged between the two species only in (rare) hitchhiking events associated with the horizontal transfer of adaptive genes. Accordingly, a clear species boundary between the C. jejuni and C. coli species is evident and it is unlikely that this boundary is being eroded.     

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

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

Molecular Epidemiology of Campylobacter jejuni in Broiler Flocks Using Randomly Amplified Polymorphic DNA-PCR and 23S rRNA-PCR and Role of Litter in Its Transmission

Poultry has long been cited as a reservoir for Campylobacter spp., and litter has been implicated as a vehicle in their transmission. Chicks were raised on litter removed from a broiler house positive for Campylobacter jejuni. Litter was removed from the house on days 0, 3, and 9 after birds were removed for slaughter. Chicks were raised on these three litters under controlled conditions in flocks of 25. None of these birds yielded C. jejuni in their cecal droppings through 7 weeks. Two successive flocks from the same Campylobacter-positive broiler house were monitored for Campylobacter colonization. Campylobacter jejuni prevalence rates were determined for each flock. Randomly amplified polymorphic DNA (RAPD)-PCR and 23S rRNA-PCR typing methods were used to group isolates. A high prevalence (60%) of C. jejuni in flock 1 coincided with the presence of an RAPD profile not appearing in flock 2, which had a lower rate of prevalence (28%). A 23S rRNA-PCR typing method was used to determine if strains with different RAPD profiles and different prevalence rates contained different 23S sequences. RAPD profiles detected with higher prevalence rates contained a spacer in the 23S rRNA region 100% of the time, while RAPD profiles found with lower prevalence rates contained an intervening sequence less than 2% of the time. Data suggest varying colonizing potentials of different RAPD profiles and a source other than previously used litter as a means of transmission of C. jejuni. These molecular typing methods demonstrate their usefulness, when used together, in this epidemiologic investigation.     

Prevalence and Genetic Diversity of Campylobacter spp. in Environmental Water Samples from a 100-Square-Kilometer Predominantly Dairy Farming Area

Water samples were taken systematically from a 100-km² area of mainly dairy farmland in northwestern England and examined for Campylobacter spp. Pulsed-field gel electrophoresis-restriction fragment length polymorphism (PFGE-RFLP) and flaA strain typing of Campylobacter jejuni and Campylobacter coli isolates were done. Data on the water source and the adjacent environment were recorded and examined as explanatory variables. Campylobacter spp. were isolated from 40.5% (n = 119) of the water samples tested. C. jejuni was isolated from 14.3%, C. coli was isolated from 18.5%, and Campylobacter lari was isolated from 4.2% of the samples. Campylobacter hyointestinalis was not isolated from any water source. The difference in prevalence between water types (trough, running, and standing) was significant (P = 0.001). C. jejuni was the species most commonly isolated from trough-water and running-water sources, while C. coli was the most frequently isolated from standing water (P < 0.001). No association was found between the presence of Escherichia coli and that of Campylobacter spp. The final multivariable logistic regression model for Campylobacter spp. included the following variables: water source, soil type, aspect, and amount of cattle fecal material in the environment (fecal pat count). Strain typing demonstrated a diverse population of C. jejuni and the presence of a common C. coli flaA type that was widely distributed throughout the area. Most of the isolates within the common flaA type were discriminated by PFGE-RFLP. These findings suggest a possible role for environmental water in the epidemiology of Campylobacter spp. in a farming environment.     

Effect of Preslaughter Events on Prevalence of Campylobacter jejuni and Campylobacter coli in Market-Weight Turkeys

The effects of events which occur prior to slaughter, such as loading, transport, and holding at an abattoir, on the prevalence of Campylobacter species, including Campylobacter jejuni and Campylobacter coli, were examined. Cloacal swabs from market-weight turkeys in each of five flocks were obtained on a farm prior to loading (time 1; 120 swabs per flock) and after transport and holding at the abattoir (time 2; 120 swabs per flock). A statistically significant increase in the overall prevalence of Campylobacter spp. was observed for cloacal swabs obtained from farm 3 following transport (P < 0.01). At time 2, an increase in the prevalence of C. coli was also noted for cloacal swabs from farms 3, 4, and 5 (P < 0.01). Neither the minimum time off of feed nor the distance transported from the farm to the abattoir was correlated with the increase in C. coli prevalence. Similarly, responses to an on-farm management questionnaire failed to detect any factors contributing to the observed changes in Campylobacter sp. prevalence. A SmaI macrorestriction analysis of Campylobacter sp. isolates recovered from flock 5 indicated that C. coli was more diverse than C. jejuni at both time 1 and time 2 (P < 0.01), based on a comparison of the Shannon indices of diversity and evenness.     

Genotype and Antibiotic Resistance Analyses of Campylobacter Isolates from Ceca and Carcasses of Slaughtered Broiler Flocks

To obtain genetic information about Campylobacter jejuni and Campylobacter coli from broilers and carcasses at slaughterhouses, we analyzed and compared 340 isolates that were collected in 2008 from the cecum right after slaughter or from the neck skin after processing. We performed rpoB sequence-based identification, multilocus sequence typing (MLST), and flaB sequence-based typing; we additionally analyzed mutations within the 23S rRNA and gyrA genes that confer resistance to macrolide and quinolone antibiotics, respectively. The rpoB-based identification resulted in a distribution of 72.0% C. jejuni and 28.0% C. coli. The MLST analysis revealed that there were 59 known sequence types (STs) and 6 newly defined STs. Most of the STs were grouped into 4 clonal complexes (CC) that are typical for poultry (CC21, CC45, CC257, and CC828), and these represented 61.8% of all of the investigated isolates. The analysis of 95 isolates from the cecum and from the corresponding carcass neck skin covered 44 different STs, and 54.7% of the pairs had matching genotypes. The data indicate that cross-contamination from various sources during slaughter may occur, although the majority of Campylobacter contamination on carcasses appeared to originate from the slaughtered flock itself. Mutations in the 23S rRNA gene were found in 3.1% of C. coli isolates, although no mutations were found in C. jejuni isolates. Mutations in the gyrA gene were observed in 18.9% of C. jejuni and 26.8% of C. coli isolates, which included two C. coli strains that carried mutations conferring resistance to both classes of antibiotics. A relationship between specific genotypes and antibiotic resistance/susceptibility was observed.Campylobacteriosis is the leading food-borne bacterial gastroenteritis worldwide (12, 15). In Switzerland, the number of registered campylobacteriosis cases has rapidly increased to more than 100 per 100,000 inhabitants in the past few years (14), and this trend has also been observed in the European Union (EU) (12). However, the real number of cases is likely higher, because not all cases are reported due to the self-limiting nature of the disease and its potentially mild symptoms.Campylobacter jejuni and Campylobacter coli are commonly associated with human infection, and they can be detected in up to 85% and 15% of cases, respectively (33). Despite the important role that C. jejuni and C. coli play as zoonotic pathogens worldwide, there is little information regarding the route(s) of transmission (17). Numerous case-control and modeling studies on the infection sources of C. jejuni and C. coli have suggested that handling and consumption of contaminated poultry meat are associated with a risk of human campylobacteriosis (17, 45, 47, 49, 51). Initial meat contamination with C. jejuni or C. coli from the chicken intestine may occur during commonly used automated slaughter processing through several routes, such as the air, water, previously slaughtered flocks, or machinery (19, 36, 37).Precise genotyping and continuous comparison of the strains obtained from, e.g., the production site, flocks, slaughterhouse, retail meat, and infected humans would help to trace the source of infection and might indicate possible intervention strategies for the contaminated site.DNA sequence-based typing methods, such as multilocus sequence typing (MLST), are well suited for this purpose (28), and MLST has become the method of choice for genotyping of Campylobacter (6, 8). Moreover, extension of the classical MLST technique for C. jejuni and C. coli with sequencing of the short variable region (SVR) within the flagellin-encoding gene flaB allows a more precise differentiation among strains that have the same MLST sequence type (ST) (9, 29). An extended MLST work flow was recently developed that reduces the associated time and cost (24). In addition, the new approach allows genetic determination of antibiotic resistance to quinolones and macrolides. Resistance to these antibiotics is a worldwide issue of concern, as an increasing number of Campylobacter isolates are resistant to them. Strikingly, a number of strains are resistant to ciprofloxacin (a quinolone) and, to a lesser extent, erythromycin (a macrolide), which is problematic, because these drugs are typically used to treat campylobacteriosis. Resistance to quinolones is mainly associated with a point mutation in the DNA gyrase gene (gyrA) at position C257T, and a transition in the 23S rRNA gene at position A2075G is commonly responsible for macrolide resistance (1). Simple sequence-based analysis of these common mutational positions can therefore provide information about the antibiotic susceptibility or resistance of a strain. Besides the prudent use of antibiotics, knowledge about the genetic composition of the infectious agent can be helpful to both treat the disease and prevent the spread of resistant strains.In the current study, MLST, flaB typing, and sequence-based determination of quinolone and macrolide resistances were used to investigate the genetic background of C. jejuni and C. coli isolates collected from Swiss broilers in a spatiotemporal study in 2008. We addressed the following three aspects: (i) the diversity of Campylobacter isolates that were recovered from pooled cecum samples and the carcass neck skin, (ii) the possible impact of cross- and self-contamination during slaughter, and (iii) the antibiotic resistance of Campylobacter strains from the broiler flocks and chicken carcasses. All of the data, including the strain information and trace files, were entered into a commercial Web-based Campylobacter MLST database (SmartGene, Zug, Switzerland). This database allows users to retrieve and compare information for any analyzed strain for monitoring purposes (24).     

Rapid host switching in generalist Campylobacter strains erodes the signal for tracing human infections

Campylobacter jejuni and Campylobacter coli are the biggest causes of bacterial gastroenteritis in the developed world, with human infections typically arising from zoonotic transmission associated with infected meat. Because Campylobacter is not thought to survive well outside the gut, host-associated populations are genetically isolated to varying degrees. Therefore, the likely origin of most strains can be determined by host-associated variation in the genome. This is instructive for characterizing the source of human infection. However, some common strains, notably isolates belonging to the ST-21, ST-45 and ST-828 clonal complexes, appear to have broad host ranges, hindering source attribution. Here whole-genome sequencing has the potential to reveal fine-scale genetic structure associated with host specificity. We found that rates of zoonotic transmission among animal host species in these clonal complexes were so high that the signal of host association is all but obliterated, estimating one zoonotic transmission event every 1.6, 1.8 and 12 years in the ST-21, ST-45 and ST828 complexes, respectively. We attributed 89% of clinical cases to a chicken source, 10% to cattle and 1% to pig. Our results reveal that common strains of C. jejuni and C. coli infectious to humans are adapted to a generalist lifestyle, permitting rapid transmission between different hosts. Furthermore, they show that the weak signal of host association within these complexes presents a challenge for pinpointing the source of clinical infections, underlining the view that whole-genome sequencing, powerful though it is, cannot substitute for intensive sampling of suspected transmission reservoirs.     

Antimicrobial Susceptibility Profiles and Molecular Typing of Campylobacter jejuni and Campylobacter coli Isolates from Ducks in South Korea

Campylobacter is a food-borne zoonotic pathogen that causes human gastroenteritis worldwide. Campylobacter bacteria are commensal in the intestines of many food production animals, including ducks and chickens. The objective of the study was to determine the prevalence of Campylobacter species in domestic ducks, and the agar dilution method was used to determine resistance of the isolates to eight antibiotics. In addition, multilocus sequence typing (MLST) was performed to determine the sequence types (STs) of selected Campylobacter isolates. Between May and September 2012, 58 duck farms were analyzed, and 56 (96.6%) were positive for Campylobacter. Among the isolates, 82.1% were Campylobacter jejuni, 16.1% were C. coli, and one was unidentified by PCR. Of the 46 C. jejuni isolates, 87.0%, 10.9%, and 21.7% were resistant to ciprofloxacin, erythromycin, and azithromycin, respectively. Among the C. coli isolates, all 9 strains were resistant to ampicillin, and 77.8% and 33.3% were resistant to ciprofloxacin and azithromycin, respectively. The majority of the Campylobacter isolates were classified as multidrug resistant. Twenty-eight STs were identified, including 20 STs for C. jejuni and 8 STs for C. coli. The most common clonal complexes in C. jejuni were the ST-21 complex and the ST-45 complex, while the ST-828 complex predominated in C. coli. The majority of isolates were of STs noted in ducks and humans from earlier studies, along with seven STs previously associated only with human disease. These STs overlapped between duck and human isolates, indicating that Campylobacter isolates from ducks should be considered potential sources of human infection.     

Spatiotemporal Homogeneity of Campylobacter Subtypes from Cattle and Sheep across Northeastern and Southwestern Scotland

Source attribution using molecular subtypes has implicated cattle and sheep as sources of human Campylobacter infection. Whether the Campylobacter subtypes associated with cattle and sheep vary spatiotemporally remains poorly known, especially at national levels. Here we describe spatiotemporal patterns of prevalence, bacterial enumeration, and subtype composition in Campylobacter isolates from cattle and sheep feces from northeastern (63 farms, 414 samples) and southwestern (71 farms, 449 samples) Scotland during 2005 to 2006. Isolates (201) were categorized as sequence type (ST), as clonal complex (CC), and as Campylobacter jejuni or Campylobacter coli using multilocus sequence typing (MLST). No significant difference in average prevalence (cattle, 22%; sheep, 25%) or average enumeration (cattle, 2.7 × 10⁴ CFU/g; sheep, 2.0 × 10⁵ CFU/g) was found between hosts or regions. The four most common STs (C. jejuni ST-19, ST-42, and ST-61 and C. coli ST-827) occurred in both hosts, whereas STs of the C. coli ST-828 clonal complex were more common in sheep. Neither host yielded evidence for regional differences in ST, CC, or MLST allele composition. Isolates from the two hosts combined, categorized as ST or CC, were more similar within than between farms but showed no further spatiotemporal trends up to 330 km and 50 weeks between farm samples. In contrast, both regions yielded evidence for significant differences in ST, CC, and allele composition between hosts, such that 65% of isolates could be attributed to a known host. These results suggest that cattle and sheep within the spatiotemporal scales analyzed are each capable of contributing homogeneous Campylobacter strains to human infections.Campylobacter species are the largest cause of bacterial intestinal infection in the developed and developing world (3). Almost all reported human Campylobacter infections in the United Kingdom are caused by Campylobacter jejuni, which accounts for approximately 92% of cases, and Campylobacter coli, which accounts for most of the rest (8). Campylobacter species are carried asymptomatically in a wide range of host animals and excreted into the environment in feces (23). Humans can be infected by several routes including consumption of contaminated water (14) or food (23); indeed, case control studies indicate that consumption of poultry meat is a risk factor (11, 12, 28), but other foods including unpasteurized milk (33) and meat from cattle and sheep contaminated at the abattoir might be important (30).Cattle and sheep on farms are major hosts of Campylobacter, with up to 89% of cattle herds (31) and up to 55% of sheep flocks (26) being infected. The prevalence of C. jejuni and C. coli combined, estimated at the level of individual animals from fecal specimens, is 23 to 54% in cattle (22, 25) and up to 20% in sheep (37). Campylobacter enumeration in feces shed from individual animals ranges from <10² to 10⁷ CFU/g in both hosts (31), and the concentration shed varies with time. Meat products of cattle and sheep, by contrast, have generally lower levels of Campylobacter contamination. Prevalence values are 0.5 to 4.9% in surveys of retail beef (11a, 17, 36) and 6.9 to 12.6% in surveys of retail lamb and mutton (17, 35).Clinical Campylobacter strains can be attributed to infection sources in animals by comparing subtype frequencies in clinical cases with those in different candidate sources, including cattle, sheep, pigs, and the physical environment. Campylobacter subtype data sets are most transferable when subtypes are defined as sequence type (ST) using multilocus sequence typing (MLST). Three recent MLST-based studies based in northwestern England (34), mainland Scotland (29), northeastern Scotland (32), and New Zealand (24) have used source attribution models to infer the source of human clinical infection. The results suggest that retail chicken is the source with the highest (55 to 80%) attribution while cattle and sheep combined are ranked second (20 to 40%). These attribution models require further empirical validation but appear to be showing broadly similar results.Attribution of human Campylobacter infections to cattle and sheep raises the question of whether Campylobacter subtypes infecting farm cattle and sheep are generally homogeneous or tend to have spatiotemporal structure. Regarding spatial differences, isolates of C. jejuni from a 100-km² study of farmland area with dairy cattle and sheep in northwestern England displayed increased genetic similarity up to 1 km apart but no further trend over distances of 1 to 14 km apart (7), and isolates from three dairy cattle farms 2 or 5 km apart in the same area demonstrated differences in the frequencies of strains of clonal complexes (CCs) ST-42 and ST-61 (15). Regarding temporal differences, isolates of C. jejuni from five dairy cattle farms in the same area demonstrated differences in the frequency of strains of CC ST-61 between the spring and summer of 2003 (15). Lastly, regarding host-associated strains, STs of CCs ST-21, ST-42, and ST-61 are associated with cattle, and the more limited data for STs from sheep also show the presence of ST-21 and ST-61 (7, 15).The larger-scale spatiotemporal structure of Campylobacter strains from cattle and sheep is poorly known. The main aims of the present study were (i) to characterize C. jejuni and C. coli from cattle and sheep from two distinct geographical Scottish regions in terms of Campylobacter prevalence and enumeration and C. jejuni and C. coli ST composition and (ii) to estimate the extent of host association of C. jejuni and C. coli STs from cattle versus sheep.     

Comparative Analysis and Limitations of Ethidium Monoazide and Propidium Monoazide Treatments for the Differentiation of Viable and Nonviable Campylobacter Cells

The lack of differentiation between viable and nonviable bacterial cells limits the implementation of PCR-based methods for routine diagnostic approaches. Recently, the combination of a quantitative real-time PCR (qPCR) and ethidium monoazide (EMA) or propidium monoazide (PMA) pretreatment has been described to circumvent this disadvantage. In regard to the suitability of this approach for Campylobacter spp., conflicting results have been reported. Thus, we compared the suitabilities of EMA and PMA in various concentrations for a Campylobacter viability qPCR method. The presence of either intercalating dye, EMA or PMA, leads to concentration-dependent shifts toward higher threshold cycle (C_T) values, especially after EMA treatment. However, regression analysis resulted in high correlation coefficient (R²) values of 0.99 (EMA) and 0.98 (PMA) between Campylobacter counts determined by qPCR and culture-based enumeration. EMA (10 μg/ml) and PMA (51.10 μg/ml) removed DNA selectively from nonviable cells in mixed samples at viable/nonviable ratios of up to 1:1,000. The optimized EMA protocol was successfully applied to 16 Campylobacter jejuni and Campylobacter coli field isolates from poultry and indicated the applicability for field isolates as well. EMA-qPCR and culture-based enumeration of Campylobacter spiked chicken leg quarters resulted in comparable bacterial cell counts. The correlation coefficient between the two analytical methods was 0.95. Nevertheless, larger amounts of nonviable cells (>10⁴) resulted in an incomplete qPCR signal reduction, representing a serious methodological limitation, but double staining with EMA considerably improved the signal inhibition. Hence, the proposed Campylobacter viability EMA-qPCR provides a promising rapid method for diagnostic applications, but further research is needed to circumvent the limitation.