Campylobacter insulaenigrae isolates from northern elephant seals (Mirounga angustirostris) in California

There are only two reports in the literature demonstrating the presence of Campylobacter spp. in marine mammals. One report describes the isolation of a new species, Campylobacter insulaenigrae sp. nov., from three harbor seals (Phoca vitulina) and a harbor porpoise (Phocoena phocoena) in Scotland, and the other describes the isolation of Campylobacter jejuni, Campylobacter lari, and an unknown Campylobacter species from northern elephant seals (Mirounga angustirostris) in California. In this study, 72 presumptive C. lari and unknown Campylobacter species strains were characterized using standard phenotypic methods, 16S rRNA PCR, and multilocus sequence typing (MLST). Phenotypic characterization of these isolates showed them to be variable in their ability to grow either at 42 degrees C or on agar containing 1% glycine and in their sensitivity to nalidixic acid and cephalothin. Based on both 16S rRNA PCR and MLST, all but 1 of the 72 isolates were C. insulaenigrae, with one isolate being similar to but distinct from both Campylobacter upsaliensis and Campylobacter helveticus. Phylogenetic analysis identified two C. insulaenigrae clades: the primary clade, containing exclusively California strains, and a secondary clade, containing some California strains and all of the original Scottish strains. This study demonstrates the inability of phenotypic characterization to correctly identify all Campylobacter species and emphasizes the importance of molecular characterization via 16S rRNA sequence analysis or MLST for the identification of Campylobacter isolates from marine mammals.     

Absence of intervening sequences and point mutations in the V domain within 23S rRNA in Campylobacter lari isolates

Although the absence of intervening sequences (IVSs) within the 23S rRNA genes in Campylobacter lari isolates has been described, there are apparently no reports regarding correlations between the nucleotide sequences of 23S rRNA genes and erythromycin (Ery) susceptibility in C. lari isolates. Here, we determined the minimum inhibitory concentrations of 35 C. lari isolates [n?=?19 for urease-positive thermophilic Campylobacter (UPTC); n?=?16 urease-negative (UN) C. lari] obtained from Asia, Europe, and North America. We found that the 18 isolates were resistant to the Ery (defined as ≧8 μg/mL), and three isolates, UPTC A1, UPTC 92251, and UPTC 504, showed increased resistance (16 μg/mL). No correlations between the IVSs in the helix 45 region within the 23S rRNA gene sequences and Ery resistance were identified in the C. lari isolates examined. In addition, no point mutations occurred at any expected or putative position within the V domain in the isolates. In conclusion, antibiotic resistance against the macrolide erythromycin is mediated through an alternative pathway to that described above.     

Campylobacter spp. Recovered from the Upper Oconee River Watershed, Georgia in a 4-Year Study

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.     

Effect of Incubation Temperature on the Detection of Thermophilic Campylobacter Species from Freshwater Beaches,Nearby Wastewater Effluents,and Bird Fecal Droppings

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.     

Isolation and Characterization of Campylobacter spp. from Antarctic Fur Seals (Arctocephalus gazella) at Deception Island,Antarctica

The presence of Campylobacter spp. was investigated in 41 Antarctic fur seals (Arctocephalus gazella) and 9 Weddell seals (Leptonychotes weddellii) at Deception Island, Antarctica. Infections were encountered in six Antarctic fur seals. The isolates, the first reported from marine mammals in the Antarctic region, were identified as Campylobacter insulaenigrae and Campylobacter lari.The Antarctic and sub-Antarctic regions are often regarded as pristine landscapes, unaffected by human activity. A limited number of surveys have been carried out to investigate the possible occurrence of zoonotic enteropathogens and if certain bacteria could be used as tools for evaluating biological pollution in this area (4, 11). In the case of Campylobacter species, there have been only three reports in the literature, but in all of them Campylobacter was isolated from marine seabirds but not from marine mammals. Campylobacter jejuni was isolated in Antarctic and sub-Antarctic areas from Macaroni penguins (Eudyptes chrysolophus) (4), and Campylobacter lari was isolated from Brown skuas, South Polar skuas, and Adelie penguins (2, 11).Reports of Campylobacter species isolated from marine mammals are rare. Campylobacter insulaenigrae was isolated from three harbor seals (Phoca vitulina) and a harbor porpoise (Phocoena phocoena) in Scotland (7). The isolation of C. jejuni, C. lari, and an unknown Campylobacter species from juvenile northern elephant seals (Mirounga angustirostris) in California was also reported (22). Finally, 71 isolates of C. insulaenigrae and 1 isolate similar to but distinct from both Campylobacter upsaliensis and Campylobacter helveticus were isolated from northern elephant seals in California (23). In the South Georgia Archipelago, fecal swabs were taken from 206 Antarctic fur seal pups, but no isolates could be obtained (4). In this study, we successfully isolated C. lari from 7.3% of Antarctic fur seals (Arctocephalus gazella) sampled and C. insulaenigrae from a further 7.3%. On the other hand, Campylobacter was not detected in the nine Weddell seals (Leptonychotes weddellii) sampled. To our knowledge, this is the first report on the isolation of C. lari and C. insulaenigrae from marine mammals in the Antarctic region.Fieldwork was conducted at Deception Island (latitude of 62°58′S and longitude of 60°40′W), in the South Shetland Islands. During January to February 2007, Antarctic fur seals (Arctocephalus gazella) and Weddell seals (Leptonychotes weddellii) were captured and fecal samples were collected by insertion of sterile cotton wool swabs into the rectum of the marine mammals. A total of 41 Antarctic fur seals and 9 Weddell seals were sampled. The distribution by ages was of 7 adults (over 4 years of age with breeding activity), 19 subadults (2 to 4 years of age), and 15 juvenile Antarctic fur seals (less than 2 years of age), and 8 adult Weddell seals and 1 juvenile. All animals presented a good body condition and showed no symptoms at the time of sampling.Three swabs were taken from each animal and were placed in FBP medium (8) with 0.5% active charcoal (Sigma Ltd.), Amies transport medium with charcoal, and Cary Blair transport medium, respectively. All samples were kept at +4 to 8°C until culture in the lab. The number of days between sampling and cultivation varied from 96 to 124 days, with a median value of 105 days.Each swab was placed in 10 ml of Campylobacter enrichment broth (Lab M) with 5% laked horse blood and CAT supplement (cefoperazone [8 μg/ml], teicoplanin [4 μg/ml], and amphotericin B [10 μg/ml]) at 37°C. The broth was incubated at 37°C for 48 h and 5 days in 3.5-liter anaerobic containers using CampyGen sachets (Oxoid), before an aliquot of 100 μl was plated onto CAT agar and the plates were incubated at 37°C for 72 h in a microaerobic atmosphere. In addition, a 47-mm-diameter cellulose membrane with 0.60-μm pores was placed on the surface of an anaerobe agar base (Oxoid) with 5% laked horse blood. Eight to 10 drops of enrichment broth (200 μl) were placed onto the surface of the membrane. The membrane was left for 20 to 30 min on the agar surface at room temperature until all of the fluid had passed through (20). The plates were incubated as described above, but for 5 days to isolate the less common, slower growing species.Isolates were examined by dark-field microscopy to determine morphology and motility and tested to determine whether oxidase was produced. For each sample, five isolates from each of the solid media that had typical morphology and motility and for which the oxidase test was positive were frozen at −80°C in FBP medium (8) until they were tested by phenotypic and genotypic methods.Original Campylobacter identification was done by Gram staining, catalase activity, hippurate hydrolysis, ability to hydrolyze indoxyl acetate, urease activity, H₂S production on triple-sugar iron slants, growth at 25°C and 42°C in a microaerophilc environment, growth at 37°C in an aerobic atmosphere, and agglutination with Microscreen latex (Microgen, Camberley, United Kingdom).No differences between the strains were observed in any of the phenotypic tests used. All isolates showed a Gram-negative, slender, curved, seagull wing-like morphology under light microscopy and positive reactions in the catalase test. They were negative for hippurate and indoxyl acetate hydrolysis and urease and did not show H₂S production. In addition, they grew at 42°C but did not grow at 25°C or 37°C in an aerobic atmosphere. Finally, all of them were positive in the agglutination test.Because phenotypic results commonly lead to misidentification of Campylobacter species, it is recommended that a molecular method be included in the identification scheme for Campylobacter (5, 15). Identification of the isolates was performed using 16S rRNA gene PCR and sequence analysis (15, 21). Forward and reverse conserved 16S rRNA eubacterial primers 8F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′) were used to amplify the 16S rRNA according to the protocol described by Jang et al. (9). Forward and reverse sequencing reactions were performed by the Laboratorio Central de Veterinaria''s DNA sequencing facility (LCV Algete, Madrid, Spain). Three strains were identified as C. lari and the other three as C. insulaenigrae based on both forward and reverse sequence analysis.Molecular characterization of strains was carried out using a combination of pulsed-field gel electrophoresis (PFGE) using KpnI enzyme and multilocus sequence typing (MLST). Preparation of intact Campylobacter DNA for PFGE was performed following the Pulsenet protocol (17, 24). PFGE for the restriction enzyme KpnI (Takara, Conda, Spain) was performed following the protocol described by Ribot et al. (17). DNA fragments were resolved on 0.9% Seakem Gold agarose gels (Iberlabo, Spain) with a Bio-Rad CHEF DRIII system (Bio-Rad, Spain) at 14°C and 6 V/cm. Electrophoresis was carried out for 22 h with pulse times ramping from 4 s to 20 s. The fingerprinting experiments were analyzed using the InfoQuest FP software (Bio-Rad, Spain), and the dendrogram was constructed using the unweighted-pair group method using average linkages (UPGMA).MLST of C. lari strains was performed as described by Miller et al. (13). In the case of C. insulaenigrae strains, MLST was performed following the protocol described by Stoddard et al. (23). All amplicons were sequenced by the Sequencing Service of the Instituto de Salud Carlos III (Madrid, Spain). Sequence data were collated, and alleles were assigned using the Campylobacter PubMLST database (http://pubmlst.org/campylobacter/). Novel alleles and sequence types were submitted for allele and sequence type (ST) designations when appropriate.Regarding the age distribution of animals, C. lari was isolated from 1 of 7 adult (14.3%), 1 of 19 subadult (5.3%), and 1 of 15 juvenile (6.6%) Antarctic fur seals. C. insulaenigrae was isolated from 1 of 7 adults (14.3%) and 2 of 19 of subadults (10.5%) but not from juvenile animals (Table ​(Table1).1). All strains were obtained from the swabs kept in FBP transport medium.

TABLE 1.

Source of Campylobacter isolates

Molecular analysis of superoxide dismutase in Campylobacter lari

The superoxide dismutase (SOD) gene clusters, sodB and sodC, and their adjacent genetic loci from a urease-positive thermophilic Campylobacter (UPTC) CF89-12 strain were analyzed molecularly, and compared with those of thermophilic campylobacters. The UPTC CF89-12 strain carried sodB [structural gene 654 base pairs (bp)] and sodC (540 bp) genes, as did the Campylobacter lari RM2100 reference strain. However, the other three thermophilic Campylobacter jejuni, C. coli and C. upsaliensis reference strains carried only a single sodB gene, and no sodC. Although sodB and sodC in the UPTC strain shared relatively high nucleotide sequence similarities (92.9 % and 91.7 %, respectively) with the corresponding genes in the C. lari RM2100 strain, the sodB gene in the UPTC CF89-12 and C. lari RM2100 strains shared relatively low nucleotide sequence similarities with those in C. jejuni NCTC11168 (80.8 % and 81.7 %), C. coli RM2228 (82.0 % and 83.1 %) and C. upsaliensis RM3195 (75.9 % and 77.0 %), respectively. All PCR amplifications of sodB and sodC gene segments with 28 C. lari isolates, including 14 UPTC isolates, gave positive results. C. lari organisms were shown to carry both the sodB and sodC genes with extremely high frequency. More high-SOD activity was seen with the C. lari isolates (n?=?9), including UPTC, than was seen with the other three thermophilic Campylobacter and Helicobacter pylori organisms.     

Molecular Epidemiology and Characterization of Campylobacter spp. Isolated from Wild Bird Populations in Northern England

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).Infection with Campylobacter spp. continues to be the leading cause of human infectious intestinal disease in the United Kingdom and has a significant economic impact (39). Consequently, there is a continuing effort to identify effective control methods. The majority of human infections (∼90%) are caused by Campylobacter jejuni subsp. jejuni (46). Other Campylobacter species, including Campylobacter coli and Campylobacter lari, can also cause enteritis in humans, but their prevalence is lower. Most C. jejuni infections are believed to result from consumption of contaminated food, including poultry meat (27, 40), red meat (52), and milk (13), which is thought to be contaminated primarily by feces. It is well established that most livestock species, including poultry, ruminants, and pigs, carry C. jejuni asymptomatically (27), making control at the farm level difficult. However, the epidemiology of C. jejuni cannot be explained solely by food-borne exposure; C. jejuni has also been isolated from a range of environmental samples, including samples of soil, water, sand, and the feces of a number of wildlife species, including wild birds (1-3). However, the role that non-food-borne exposure plays in the epidemiology of C. jejuni is currently not well defined.High prevalences of Campylobacter species infections have been found in a wide range of wild bird species, although there is great variation between taxa (2, 4, 7, 16, 35, 47, 48). Given their ability to fly long distances and their ubiquity, wild birds have the potential to play an important role in the epidemiology and evolution of Campylobacter spp. However, whether wild birds are a source of infection for humans or domestic livestock or are mainly recipients of domestic animal strains or, indeed, whether separate cycles of infection occur remain unknown. These questions remain unanswered in part because investigations of the epidemiology of Campylobacter spp. have been complicated by their high inter- and intraspecies genetic diversity (6).The methods that have been routinely used to characterize Campylobacter isolates are restricted due to genomic instability in Campylobacter populations (10, 38, 45). Multilocus sequence typing (MLST) is a method that has the advantage of being objective since it is sequence based, which allows comparison of isolates from different laboratories and accurate determination of relationships between isolates from diverse sources (11). MLST studies of C. jejuni in farm animals and the environment, including wildlife, suggest that some strains may be associated with particular host groups (6, 10, 15, 30). However, in the same studies other strains were found to occur in several host species or habitats. Few studies have investigated the molecular epidemiology of Campylobacter infection in wild bird populations using MLST, and because only a relatively small number of isolates from wild birds have been characterized by MLST, conclusions have not been drawn yet about how wild bird isolates fit into the overall phylogenetic scheme or whether wild birds act as reservoirs, amplifiers, or merely indicators of infection of domestic animals with zoonotic genotypes.In the current study a large cross-sectional survey of wild bird populations in northern England was undertaken to investigate the epidemiology of Campylobacter infection. Previous studies that have focused on the epidemiology of Campylobacter spp. solely in wild birds have investigated either a narrow range of taxonomic groups (2, 5, 17, 23, 29, 33, 43, 50) or wild birds from a limited range of habitats (18, 25, 48). Studies that have investigated a broad range of wild bird species have used Campylobacter characterization techniques that do not allow conclusions about possible host associations to be drawn or comparison of the genetic diversity of isolates between studies (21, 25, 34, 47, 53). Therefore, the aims of this study were (i) to determine the host range and prevalence of Campylobacter spp. in a wild bird population and (ii) through molecular characterization of isolates to determine whether wild birds were a likely source of infection in humans or domestic livestock and whether separate cycles of infection with host-adapted strains of Campylobacter spp. were maintained in the wild bird population.     

Acanthamoeba-Campylobacter Coculture as a Novel Method for Enrichment of Campylobacter Species

In this study, we present a novel method to isolate and enrich low concentrations of Campylobacter pathogens. This method, Acanthamoeba-Campylobacter coculture (ACC), is based on the intracellular survival and multiplication of Campylobacter species in the free-living protozoan Acanthamoeba polyphaga. Four of the Campylobacter species relevant to humans and livestock, Campylobacter jejuni, C. coli, C. lari, and C. hyointestinalis, were effectively enriched by the coculture method, with growth rates comparable to those observed in other Campylobacter enrichment media. Studying six strains of C. jejuni isolated from different sources, we found that all of the strains could be enriched from an inoculum of fewer than 10 bacteria. The sensitivity of the ACC method was not negatively affected by the use of Campylobacter-selective antibiotics in the culture medium, but these were effective in suppressing the growth of seven different bacterial species added at a concentration of 10⁴ CFU/ml of each species as deliberate contamination. The ACC method has advantages over other enrichment methods as it is not dependent on a microaerobic milieu and does not require the use of blood or other oxygen-quenching agents. Our study found the ACC method to be a promising tool for the enrichment of Campylobacter species, particularly from water samples with low bacterial concentrations.     

DNA identification and characterization of Campylobacter jejuni and Campylobacter coli isolated from caecal samples of chickens in Grenada

Aims: To speciate Campylobacter strains from the caeca of chickens in Grenada using PCR and to evaluate DNA‐based typing methods for the characterization of these isolates. Methods and Results: Isolates were speciated with two multiplex PCR assays and were typed with flaA‐RFLP, pulsed‐field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Results confirmed that Campylobacter coli strains were more predominant than Campylobacter jejuni strains. From 56 isolates, 18 were misidentified using biochemical tests. PFGE typing gave the highest discriminatory power among the methods used (Simpson’s index of diversity, D = 0·9061). However, the combination of flaA‐RFLP, PFGE and MLST results gave the highest discrimination for subtyping of these isolates (D = 0·9857). A band position tolerance of 4% in Bio Numerics was the most appropriate for the analysis of this database. MLST profiles were generally concordant with PFGE and/or flaA‐RFLP types. Several isolates exhibited new MLST sequence types (STs), and 43 of the 49 Camp. coli strains belonged to the ST‐828 clonal complex. Conclusions: Campylobacter coli was the most prevalent species isolated from broilers and layers in Grenada, and a combination of restriction and sequence methods was most appropriate for the typing of Camp. coli isolates. Campylobacter coli STs clustered with described poultry‐associated Camp. coli STs by phylogenetic analysis. Significance and Impact of the Study: Further studies to understand the predominance of Camp. coli within Campylobacter spp. from chickens in Grenada may help elucidate the epidemiology of these pathogens in chickens.     

Comparison of PCR Binary Typing (P-BIT), a New Approach to Epidemiological Subtyping of Campylobacter jejuni,with Serotyping,Pulsed-Field Gel Electrophoresis,and Multilocus Sequence Typing Methods

To overcome some of the deficiencies with current molecular typing schema for Campylobacter spp., we developed a prototype PCR binary typing (P-BIT) approach. We investigated the distribution of 68 gene targets in 58 Campylobacter jejuni strains, one Campylobacter lari strain, and two Campylobacter coli strains for this purpose. Gene targets were selected on the basis of distribution in multiple genomes or plasmids, and known or putative status as an epidemicity factor. Strains were examined with Penner serotyping, pulsed-field gel electrophoresis (PFGE; using SmaI and KpnI enzymes), and multilocus sequence typing (MLST) approaches for comparison. P-BIT provided 100% typeability for strains and gave a diversity index of 98.5%, compared with 97.0% for SmaI PFGE, 99.4% for KpnI PFGE, 96.1% for MLST, and 92.8% for serotyping. Numerical analysis of the P-BIT data clearly distinguished strains of the three Campylobacter species examined and correlated somewhat with MLST clonal complex assignations and with previous classifications of “high” and “low” risk. We identified 18 gene targets that conferred the same level of discrimination as the 68 initially examined. We conclude that P-BIT is a useful approach for subtyping, offering advantages of speed, cost, and potential for strain risk ranking unavailable from current molecular typing schema for Campylobacter spp.Campylobacter species, particularly C. jejuni subsp. jejuni (hereafter C. jejuni), represent the most commonly reported bacterial cause of gastroenteritis in humans in the developed world (47), with New Zealand having one of the highest rates of infection (55). The sheer scale of infection makes concerted epidemiological studies difficult, as does the extremely wide distribution of the organism, found in all major avian and mammalian food animals, their products, and indeed environments. Moreover, many Campylobacter spp. are susceptible to spontaneous genetic change through a variety of mechanisms that can result in conflicting data for genetic typing methods aiming to establish a molecular epidemiological link between strains (reviewed by On and colleagues [47]).The poor discrimination of phenotypic typing methods led to intense developments in molecular epidemiological tools for more accurate data. Although a wide range of genotypic methods have been described (47), two methods are now more commonly used by laboratories worldwide. The availability of standardized protocols for macrorestriction profiling with pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) have facilitated major contributions to our understanding of the epidemiology of these bacteria. Nonetheless, issues remain, notably relating to the speed, cost, and ease of data analysis from these methods. Furthermore, although MLST has proven useful in evaluating the original host of a given strain, no current methods provide information on the relative risk to human health from individual strains. Various studies, including those identifying stable clones found in humans and various animals as well as strain types only in a particular animal host (5, 13, 38, 48, 61), and whole-genome microarray-based comparisons revealing a correlation between genome content and stress survival (46) indicate that not all strains are of equal risk to humans.In this study, we designed a range of specific PCR assays and investigated the distribution of 68 genes associated with epidemicity factors in C. jejuni, to establish the basis of a novel PCR binary typing (P-BIT) system that is inexpensive, rapid, and highly portable. We compared our data with MLST and PFGE (using restriction enzymes SmaI and KpnI) results for the same isolates of C. jejuni (n = 58), C. coli (n = 2), and C. lari (n = 1).     

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.     

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.     

Source Attribution of Human Campylobacter Isolates by MLST and Fla-Typing and Association of Genotypes with Quinolone Resistance

Campylobacteriosis is the most frequent zoonosis in developed countries and various domestic animals can function as reservoir for the main pathogens Campylobacter jejuni and Campylobacter coli. In the present study we compared population structures of 730 C. jejuni and C. coli from human cases, 610 chicken, 159 dog, 360 pig and 23 cattle isolates collected between 2001 and 2012 in Switzerland. All isolates had been typed with multi locus sequence typing (MLST) and flaB-typing and their genotypic resistance to quinolones was determined. We used complementary approaches by testing for differences between isolates from different hosts with the proportion similarity as well as the fixation index and by attributing the source of the human isolates with Bayesian assignment using the software STRUCTURE. Analyses were done with MLST and flaB data in parallel and both typing methods were tested for associations of genotypes with quinolone resistance. Results obtained with MLST and flaB data corresponded remarkably well, both indicating chickens as the main source for human infection for both Campylobacter species. Based on MLST, 70.9% of the human cases were attributed to chickens, 19.3% to cattle, 8.6% to dogs and 1.2% to pigs. Furthermore we found a host independent association between sequence type (ST) and quinolone resistance. The most notable were ST-45, all isolates of which were susceptible, while for ST-464 all were resistant.     

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.     

Dissemination of Fluoroquinolone-Resistant Campylobacter spp. within an Integrated Commercial Poultry Production System

While characterizing the intestinal bacterial community of broiler chickens, we detected -proteobacterial DNA in the ilea of 3-day-old commercial broiler chicks (J. Lu, U. Idris, B. Harmon, C. Hofacre, J. J. Maurer, and M. D. Lee, Appl. Environ. Microbiol. 69:6816-6824, 2003). The sequences exhibited high levels of similarity to Campylobacter jejuni and Campylobacter coli sequences, suggesting that chickens can carry Campylobacter at a very young age. Campylobacter sp. was detected by PCR in all samples collected from the ilea of chicks that were 3 to 49 days old; however, it was detected only in the cecal contents of chickens that were at least 21 days old. In order to determine whether the presence of Campylobacter DNA in young chicks was due to ingestion of the bacteria in food or water, we obtained commercial broiler hatching eggs, which were incubated in a research facility until the chicks hatched. DNA sequencing of the amplicons resulting from Campylobacter-specific 16S PCR performed with the ileal, cecal, and yolk contents of the day-of-hatching chicks revealed that Campylobacter DNA was present before the chicks consumed food or water. The 16S rRNA sequences exhibited 99% similarity to C. jejuni and C. coli sequences and 95 to 98% similarity to sequences of other thermophilic Campylobacter species, such as C. lari and C. upsaliensis. The presence of C. coli DNA was detected by specific PCR in the samples from chicks obtained from a commercial hatchery; however, no Campylobacter was detected by culturing. In order to determine whether the same strains of bacteria were present in multiple levels of the integrator, we cultured Campylobacter sp. from a flock of broiler breeders and their 6-week-old progeny that resided on a commercial broiler farm. The broiler breeders had been given fluoroquinolone antibiotics, and we sought to determine whether the same fluoroquinolone-resistant strain was present in their progeny. The isolates were typed by pulsed-field gel electrophoresis, which confirmed that the parental and progeny flocks contained the same strain of fluoroquinolone-resistant C. coli. These data indicate that resistant C. coli can be present in multiple levels of an integrated poultry system and demonstrated that molecular techniques or more sensitive culture methods may be necessary to detect early colonization by Campylobacter in broiler chicks.     

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.     

LuxS distribution and AI-2 activity of Campylobacter spp

Aims: This study investigates the distribution of LuxS within Campylobacter (Camp.) species and Autoinducer (AI)‐2 activity of Camp. jejuni NCTC 11168 in food matrices. Methods and Results: LuxS (S‐ribosylhomocysteinase) sequences of different Campylobacter spp. were compared, and AI‐2 activity was measured with an AI‐2 reporter assay. Highest LuxS homologies were shared by Camp. jejuni, Camp. coli and Camp. upsaliensis, and their LuxS sequences had more similarities to the analysed Arcobacter and Vibrio harveyi strains than to all other analysed Campylobacter species. Of 15 analysed species only Camp. lari, Camp. peloridis and Camp. insulaenigrae did not produce AI‐2 molecules. Cultivation of Camp. jejuni NCTC 11168 in chicken juice reduced AI‐2 activity, and this reduction is not because of lower luxS expression or functionality. Conclusion: Not all Campylobacter species encode luxS. Food matrices can reduce AI‐2 activity in a LuxS‐independent manner. Significance and Impact of the Study: Besides, Camp. lari, Camp. peloridis and Camp. insulaenigrae do not show AI‐2 activity. Further investigations should clarify the function of AI‐2 in Campylobacter spp. and how species lacking luxS could overcome this alteration. Furthermore, the impact of food matrices on these functions needs to be determined as we could show that chicken juice reduced AI‐2 activity.     

Typing of Campylobacter jejuni and Campylobacter coli isolated from live broilers and retail broiler meat by flaA-RFLP, MLST, PFGE and REP-PCR

We analyzed 100 Campylobacter spp. isolates (C. jejuni and C. coli) from Grenada, Puerto Rico and Alabama, which were collected from live broilers or retail broiler meat. We analyzed these isolates with four molecular typing methods: restriction fragment length polymorphism of the flaA gene (flaA-RFLP), multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE), and automated repetitive extragenic palindromic polymerase chain reaction (REP-PCR) using the DiversiLab system. All methods performed similarly for the typing of C. jejuni and C. coli. The DNA extraction method appears to influence the results obtained with REP-PCR. This method was better for the typing of C. jejuni than C. coli, however both REP-PCR and flaA-RFLP generated types that were indistinguishable between C. jejuni and C. coli and appeared to be random, without any relationship to species, location, or source of isolates. PFGE and MLST generated typing results that had a better correlation with the geographic location of the isolates and showed higher concordance with the Wallace coefficient. The adjusted Rand coefficient did not show higher concordance among the methods, although the PFGE/MLST combination exhibited the highest concordance. PFGE and MLST revealed a better discriminatory power for C. coli isolates than REP-PCR or flaA-RFLP. The use of readily available online tools to calculate the confidence interval of the Simpson's index of diversity and the adjusted Rand and Wallace coefficients helped estimate the discriminatory power of typing methods. Further studies using different C. jejuni and C. coli strains may expand our understanding of the benefits and limitations of each of these typing methods for epidemiological studies of Campylobacter spp.     

Practicalities of Using Non-Local or Non-Recent Multilocus Sequence Typing Data for Source Attribution in Space and Time of Human Campylobacteriosis

In this study, 1208 Campylobacter jejuni and C. coli isolates from humans and 400 isolates from chicken, collected in two separate periods over 12 years in The Netherlands, were typed using multilocus sequence typing (MLST). Statistical evidence was found for a shift of ST frequencies in human isolates over time. The human MLST data were also compared to published data from other countries to determine geographical variation. Because only MLST typed data from chicken, taken from the same time point and spatial location, were available in addition to the human data, MLST datasets for other Campylobacter reservoirs from selected countries were used. The selection was based on the degree of similarity of the human isolates between countries. The main aim of this study was to better understand the consequences of using non-local or non-recent MLST data for attributing domestically acquired human Campylobacter infections to specific sources of origin when applying the asymmetric island model for source attribution. In addition, a power-analysis was done to find the minimum number of source isolates needed to perform source attribution using an asymmetric island model. This study showed that using source data from other countries can have a significant biasing effect on the attribution results so it is important to carefully select data if the available local data lack in quality and/or quantity. Methods aimed at reducing this bias were proposed.     

Comprehensive Genomic Characterization of Campylobacter Genus Reveals Some Underlying Mechanisms for its Genomic Diversification

Campylobacter species.are phenotypically diverse in many aspects including host habitats and pathogenicities, which demands comprehensive characterization of the entire Campylobacter genus to study their underlying genetic diversification. Up to now, 34 Campylobacter strains have been sequenced and published in public databases, providing good opportunity to systemically analyze their genomic diversities. In this study, we first conducted genomic characterization, which includes genome-wide alignments, pan-genome analysis, and phylogenetic identification, to depict the genetic diversity of Campylobacter genus. Afterward, we improved the tetranucleotide usage pattern-based naïve Bayesian classifier to identify the abnormal composition fragments (ACFs, fragments with significantly different tetranucleotide frequency profiles from its genomic tetranucleotide frequency profiles) including horizontal gene transfers (HGTs) to explore the mechanisms for the genetic diversity of this organism. Finally, we analyzed the HGTs transferred via bacteriophage transductions. To our knowledge, this study is the first to use single nucleotide polymorphism information to construct liable microevolution phylogeny of 21 Campylobacter jejuni strains. Combined with the phylogeny of all the collected Campylobacter species based on genome-wide core gene information, comprehensive phylogenetic inference of all 34 Campylobacter organisms was determined. It was found that C. jejuni harbors a high fraction of ACFs possibly through intraspecies recombination, whereas other Campylobacter members possess numerous ACFs possibly via intragenus recombination. Furthermore, some Campylobacter strains have undergone significant ancient viral integration during their evolution process. The improved method is a powerful tool for bacterial genomic analysis. Moreover, the findings would provide useful information for future research on Campylobacter genus.