Speciation of Campylobacter coli, C. jejuni, C. helveticus, C. lari, C. sputorum, and C. upsaliensis by matrix-assisted laser desorption ionization-time of flight mass spectrometry

Multiple strains of Campylobacter coli, C. jejuni, C. helveticus, C. lari, C. sputorum, and C. upsaliensis isolated from animal, clinical, or food samples have been analyzed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Whole bacterial cells were harvested from colonies or confluent growth on agar and transferred directly into solvent and then to a spot of dried 3-methoxy-4-hydroxycinnamic acid (matrix). Multiple ions in the 5,000- to 15,000-Da mass range were evident in spectra for each strain; one or two ions in the 9,500- to 11,000-Da range were consistently high intensity. "Species-identifying" biomarker ions (SIBIs) were evident from analyses of multiple reference strains for each of the six species, including the genome strains C. jejuni NCTC 11168 and C. jejuni RM1221. Strains grown on nine different combinations of media and atmospheres yielded SIBI masses within +/-5 Da with external instrument calibration. The highest-intensity C. jejuni SIBIs were cytosolic proteins, including GroES, HU/HCj, and RplL. Multiple intraspecies SIBIs, corresponding probably to nonsynonymous nucleotide polymorphisms, also provided some intraspecies strain differentiation. MALDI-TOF MS analysis of 75 additional Campylobacter strains isolated from humans, poultry, swine, dogs, and cats revealed (i) associations of SIBI type with source, (ii) strains previously speciated incorrectly, and (iii) "strains" composed of more than one species. MALDI-TOF MS provides an accurate, sensitive, and rapid method for identification of multiple Campylobacter species relevant to public health and food safety.     

Species identification of <Emphasis Type="Italic">Campylobacter jejuni</Emphasis> ssp. <Emphasis Type="Italic">jejuni</Emphasis> and <Emphasis Type="Italic">C. coli</Emphasis> by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and PCR

The Campylobacter species strains (n = 42; isolated from clinical samples and deposited in Czech National Collection of Type Cultures, Prague) originally phenotypically (and biochemically) identified as Campylobacter jejuni were re-classified using molecular biological and mass spectrometric methods. Whole-cell MALDI-TOF MS (matrix-assisted laser desorption/ionization time-of-flight mass spectrometry) separated the isolates into two genetically related strains — C. jejuni (n = 26) and C. coli (n = 16) and, moreover, distinguished the intimate details in the group of tested strains. It also made it possible to create the MALDI-TOF MS dendrogram; similarly, the spectral characteristics were used for the 3D cluster analysis. Polymerase chain reaction (PCR) confirmed the results obtained by mass spectrometry. Both methods (PCR and MALDI-TOF MS) gave the same results which supports their suitability in the rapid and accurate Campylobacter-species determination. Part of this work was presented at the 24th Congress of Czechoslovak Society for Microbiology, Liberec (Czech Republic) 2007.     

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).     

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.     

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.     

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.     

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.     

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.     

Survival of Campylobacter jejuni under Conditions of Atmospheric Oxygen Tension with the Support of Pseudomonas spp.

Campylobacter jejuni is a major food-borne pathogen. Despite causing enteritis in humans, it is a well-adapted intestinal microorganism in animals, hardly ever generating disease symptoms. Nevertheless, as a true microaerophilic microorganism it is still puzzling how Campylobacter cells can survive on chicken meat, the main source of human infection. In this study, we demonstrate that C. jejuni is able to withstand conditions of atmospheric oxygen tension when cocultured with Pseudomonas species, major food-spoiling bacteria that are frequently found on chicken meat in rather high numbers. Using an in vitro survival assay, interactions of 145 C. jejuni wild-type strains and field isolates from chicken meat, broiler feces, and human clinical samples with type strains and food isolates of Pseudomonas spp., Proteus mirabilis, Citrobacter freundii, Micrococcus luteus, and Enterococcus faecalis were studied. When inoculated alone or in coculture with Proteus mirabilis, Citrobacter freundii, Micrococcus luteus, or Enterococcus faecalis type strains, Campylobacter cells were able to survive ambient oxygen levels for no more than 18 h. In contrast, Campylobacter bacteria inoculated with type strains or wild-type isolates of Pseudomonas showed a prolonged aerobic survival of up to >48 h. This microbial commensalism was diverse in C. jejuni isolates from different sources; isolates from chicken meat and humans in coculture with Pseudomonas putida were able to use this survival support better than fecal isolates from broilers. Scanning electron microscopy revealed the development of fiberlike structures braiding P. putida and C. jejuni cells. Hence, it seems that microaerophilic C. jejuni is able to survive ambient atmospheric oxygen tension by metabolic commensalism with Pseudomonas spp. This bacterium-bacterium interaction might set the basis for survival of C. jejuni on chicken meat and thus be the prerequisite step in the pathway toward human infection.Campylobacter food-borne infections are the most prevalent bacterial enteric infections in humans in industrialized and developing countries (1). It has been shown that most human infections are related to poultry meat and food produced from cattle or sheep (34, 41). Campylobacter jejuni, the species most frequently causing human disease, can be isolated from the animal intestinal tract at levels of up to 10⁹ CFU per gram of feces and can thus be called a well-adapted intestinal microorganism (30, 37). Nevertheless, because it causes human disease as a food-borne pathogen, it has to survive outside the gut. By cross-contamination at the level of the abattoir, Campylobacter bacteria hit the meat surface and have to adapt to different environmental challenges. C. jejuni is a true microaerophilic bacterium; thus, on the one hand it requires oxygen, but on the other hand it cannot grow under normal atmospheric oxygen tension conditions (15). Despite its sensitivity to high oxygen tension in vitro, viable and culturable Campylobacter bacteria can be isolated from nonskinned chicken meat at frequencies of 10⁴ CFU/g (9, 19). Assumptions on the mechanisms by which Campylobacter cells survive on meat surfaces are diverse, for example, by growing in biofilms, entering a “viable but nonculturable state,” or interacting with other microorganisms.For instance, C. jejuni is able to resist protozoa digestion and can parasitize inside protozoa, e.g., Tetrahymena pyriformis (35). This mechanism provides survival in harsh environments and resistance to antimicrobial substances and thus enhances the potential for transmission. But bacterium-bacterium interaction has also been demonstrated to be of a high level of importance for intestinal survival and uptake (20). Accordingly, members of Campylobacter have been identified to initiate cellular uptake of commensal bacteria into enterocytes (14). However, a bacterial community can also mean competition, e.g., bacteriocin production by Lactobacillus salivarius that is effective against Campylobacter colonization (36).Meat surfaces harbor numerous bacterial species (24). Some of these bacteria have adapted to this specific environmental niche and are well-known spoilage bacteria. Most relevant species belong to the family Pseudomonadaceae. But also different members of the Enterobacteriaceae can be found on meat. To date, information regarding the interaction between spoilage bacteria and pathogens is of increasing importance for public health safety measures.Hence, experimental data on the survival of C. jejuni isolates in the presence of selected meat-spoiling bacteria were analyzed and clearly demonstrated a specific interaction with type strains and isolates of Pseudomonas putida, Pseudomonas fragi, and Pseudomonas fluorescens from chicken meat surfaces.     

Differentiation of Campylobacter Populations as Demonstrated by Flagellin Short Variable Region Sequences

The DNA sequence of the flaA short variable region (SVR) was used to analyze a random population of Campylobacter isolates to investigate the weakly clonal population structure of members of the genus. The SVR sequence from 197 strains of C. jejuni and C. coli isolated from humans, bovine, swine, and chickens identified a group of 43 strains containing disparate short variable region sequences compared to the rest of the population. This group contains both C. jejuni and C. coli strains but disproportionately consisted of bovine isolates. Relative synonymous codon usage analysis of the sequences identified two groups: one group typified C. jejuni, and the second group was characteristic for C. coli and the disparate alleles were not clustered. The data show that there is significant differentiation of Campylobacter populations according to the source of the isolate even without considering the disparate isolates. Even though there is significant differentiation of chicken and bovine isolates, the bovine isolates did not show any difference in ability to colonize chickens. It is possible that disparate sequences were obtained through the lateral transfer of DNA from Campylobacter species other than C. jejuni and C. coli. It is evident that recombination within the flaA SVR occurs rapidly. However, the rate of migration between populations appears to limit the distribution of sequences and results in a weakly clonal population structure.     

High-Resolution Genotyping of Campylobacter Strains Isolated from Poultry and Humans with Amplified Fragment Length Polymorphism Fingerprinting

For epidemiological studies of Campylobacter infections, molecular typing methods that can differentiate campylobacters at the strain level are needed. In this study we used a recently developed genotyping method, amplified fragment length polymorphism (AFLP), which is based on selective amplification of restriction fragments of chromosomal DNA, for genetic typing of Campylobacter jejuni and Campylobacter coli strains derived from humans and poultry. We developed an automated AFLP fingerprinting method in which restriction endonucleases HindIII and HhaI were used in combination with one set of selective PCR primers. This method resulted in evenly distributed band patterns for amplified fragments ranging from 50 to 500 bp long. The discriminatory power of AFLP was assessed with a C. jejuni strain, an isogenic flagellin mutant, and distinct C. jejuni strains having known pulsed-field gel electrophoresis and fla PCR-restriction fragment length polymorphism genotypes. Unrelated C. jejuni strains produced heterogeneous patterns, whereas genetically related strains produced similar AFLP patterns. Twenty-five Campylobacter strains obtained from poultry farms in The Netherlands grouped in three C. jejuni clusters that were separate from a C. coli cluster. The band patterns of 10 C. jejuni strains isolated from humans were heterogeneous, and most of these strains grouped with poultry strains. Our results show that AFLP analysis can distinguish genetically unrelated strains from genetically related strains of Campylobacter species. However, desirable genetically related strains can be differentiated by using other genotyping methods. We concluded that automated AFLP analysis is an attractive tool which can be used as a primary method for subtyping large numbers of Campylobacter strains and is extremely useful for epidemiological investigations.     

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.     

Identification and Characterization of a New Ferric Enterobactin Receptor,CfrB, in Campylobacter

The ferric enterobactin (FeEnt) receptor CfrA is present in the majority of Campylobacter jejuni isolates and is responsible for high-affinity iron acquisition. Our recent work and that of others strongly suggested the existence of another FeEnt uptake system in Campylobacter. Here we have identified and characterized a new FeEnt receptor (designated CfrB) using both in vitro and in vivo systems. CfrB, a homolog of C. jejuni NCTC 11168 Cj0444, shares approximately 34% of amino acid identity with CfrA. Alignment of complete CfrB sequences showed that the CfrB is highly conserved in Campylobacter. Immunoblotting analysis using CfrB-specific antiserum demonstrated that CfrB was dramatically induced under iron-restricted conditions and was produced in the majority of Campylobacter coli (41 out of 45) and in some C. jejuni (8 out of 32) primary strains from various sources and from geographically diverse areas. All of the CfrB-producing C. coli strains also produced CfrA, which was rarely observed in the tested C. jejuni strains. Isogenic cfrB, cfrA, and cfrA cfrB double mutants were constructed in 43 diverse Campylobacter strains. Growth promotion assays using these mutants demonstrated that CfrB has a major role in FeEnt iron acquisition in C. coli. Chicken colonization experiments indicated that inactivation of the cfrB gene alone greatly reduced and even abolished Campylobacter colonization of the intestines. A growth assay using CfrB-specific antiserum strongly suggested that specific CfrB antibodies could block the function of CfrB and diminish FeEnt-mediated growth promotion under iron-restricted conditions. Together, this work reveals the complexity of FeEnt systems in the two closely related Campylobacter species and demonstrates the important role of the new FeEnt receptor CfrB in Campylobacter iron acquisition and in vivo colonization.Campylobacter species have emerged as the leading bacterial cause of food-borne human diseases in many industrialized countries since the late 1970s (25). Two major Campylobacter species, Campylobacter jejuni and Campylobacter coli, cause watery diarrhea and/or hemorrhagic colitis in humans and are also associated with Guillain-Barré syndrome, an acute flaccid paralysis that may compromise respiratory muscle function, resulting in death (24). In parallel to their increased prevalence, members of Campylobacter have become increasingly resistant to antibiotics, including fluoroquinolones and macrolides, the major drugs of choice for treating human campylobacteriosis (10). Therefore, development of new strategies to prevent and control Campylobacter infections in humans and animal reservoirs is urgently needed, which greatly relies on the better understanding of Campylobacter pathogenesis.Despite recent advances in understanding of the pathobiology of C. jejuni (9, 39), the virulence mechanisms of Campylobacter remain poorly understood. Iron is the most abundant transition metal in living organisms, with critical roles in many diverse biological systems (2); thus, iron acquisition is essential for survival and virulence of pathogenic bacteria in the host (5, 31). Examination of iron uptake in Campylobacter began in the 1980s (12), but iron uptake systems, and the associated regulatory systems, in Campylobacter species are now just beginning to be elucidated (reviewed by Miller et al. [22], Stintzi et al. [34], and Wooldridge and van Vliet [37]). Genomic data have shown a large number of genes implicated in iron scavenging, metabolism, storage, and regulation in C. jejuni (22, 34, 37). Several iron uptake systems have been identified and characterized (22, 34); among these, the ferric enterobactin (FeEnt) iron acquisition system is of particular interest because enterobactin (Ent) has the highest affinity for ferric iron of any natural siderophore compound tested (35). Furthermore, Ent is produced by a wide variety of commensal bacteria in the intestines, and this compound is likely to be produced in significant amounts by the resident microflora in the gut (37). Thus, FeEnt may be a significant source of iron for Campylobacter species during intestinal colonization even though Campylobacter species do not appear have the capacity to synthesize Ent (34).A FeEnt acquisition system in C. jejuni was identified which comprises an outer membrane receptor, CfrA, and cognate components, including a TonB-ExbB-ExbD protein complex and an ABC transporter system CeuBCDE (22, 34). The FeEnt receptor CfrA is induced under iron-restricted conditions and plays a critical role in iron acquisition and in vivo colonization by C. jejuni (27). A recent report (40) provides further molecular, antigenic, and functional evidence suggesting that CfrA is a promising subunit vaccine for preventing and controlling C. jejuni infection in humans and animal reservoirs. Interestingly, in this study one C. jejuni strain (JL11), which does not have a gene highly homologous to cfrA, could efficiently utilize FeEnt as a sole iron source for growth (40). An early study also showed that an isogenic cfrA mutant of a human C. coli strain was still fully capable of utilizing FeEnt as a sole iron source for growth (15). These studies strongly suggest that Campylobacter species possess an additional system for FeEnt-mediated iron acquisition.In this study, we demonstrate that a homolog of the C. jejuni NCTC 11168 protein Cj0444 (28) is a FeEnt receptor, designated CfrB, in Campylobacter. CfrB is highly conserved among members of Campylobacter and plays an important role in the colonization of the intestine by both C. jejuni and C. coli.     

The Contribution of ArsB to Arsenic Resistance in Campylobacter jejuni

Arsenic, a toxic metalloid, exists in the natural environment and its organic form is approved for use as a feed additive for animal production. As a major foodborne pathogen of animal origin, Campylobacter is exposed to arsenic selection pressure in the food animal production environments. Previous studies showed that Campylobacter isolates from poultry were highly resistant to arsenic compounds and a 4-gene operon (containing arsP, arsR, arsC, and acr3) was associated with arsenic resistance in Campylobacter. However, this 4-gene operon is only present in some Campylobacter isolates and other arsenic resistance mechanisms in C. jejuni have not been characterized. In this study, we determined the role of several putative arsenic resistance genes including arsB, arsC2, and arsR3 in arsenic resistance in C. jejuni and found that arsB, but not the other two genes, contributes to the resistance to arsenite and arsenate. Inactivation of arsB in C. jejuni resulted in 8- and 4-fold reduction in the MICs of arsenite and arsenate, respectively, and complementation of the arsB mutant restored the MIC of arsenite. Additionally, overexpression of arsB in C. jejuni 11168 resulted in a 16-fold increase in the MIC of arsenite. PCR analysis of C. jejuni isolates from different animals hosts indicated that arsB and acr3 (the 4-gene operon) are widely distributed in various C. jejuni strains, suggesting that Campylobacter requires at least one of the two genes for adaptation to arsenic-containing environments. These results identify ArsB as an alternative mechanism for arsenic resistance in C. jejuni and provide new insights into the adaptive mechanisms of Campylobacter in animal food production environments.     

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.     

Molecular Detection of Campylobacter spp. and Fecal Indicator Bacteria during the Northern Migration of Sandhill Cranes (Grus canadensis) at the Central Platte River

The risk to human health of the annual sandhill crane (Grus canadensis) migration through Nebraska, which is thought to be a major source of fecal pollution of the central Platte River, is unknown. To better understand potential risks, the presence of Campylobacter species and three fecal indicator bacterial groups (Enterococcus spp., Escherichia coli, and Bacteroidetes) was assayed by PCR from crane excreta and water samples collected during their stopover at the Platte River, Nebraska, in 2010. Genus-specific PCR assays and sequence analyses identified Campylobacter jejuni as the predominant Campylobacter species in sandhill crane excreta. Campylobacter spp. were detected in 48% of crane excreta, 24% of water samples, and 11% of sediment samples. The estimated densities of Enterococcus spp. were highest in excreta samples (mean, 4.6 × 10⁸ cell equivalents [CE]/g), while water samples contained higher levels of Bacteroidetes (mean, 5.1 × 10⁵ CE/100 ml). Enterococcus spp., E. coli, and Campylobacter spp. were significantly increased in river water and sediments during the crane migration period, with Enterococcus sp. densities (∼3.3 × 10⁵ CE/g) 2 to 4 orders of magnitude higher than those of Bacteroidetes (4.9 × 10³ CE/g), E. coli (2.2 × 10³ CE/g), and Campylobacter spp. (37 CE/g). Sequencing data for the 16S rRNA gene and Campylobacter species-specific PCR assays indicated that C. jejuni was the major Campylobacter species present in water, sediments, and crane excreta. Overall, migration appeared to result in a significant, but temporary, change in water quality in spring, when there may be a C. jejuni health hazard associated with water and crops visited by the migrating birds.     

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°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.     

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.     

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.     

Prevalence, Antigenic Specificity, and Bactericidal Activity of Poultry Anti-Campylobacter Maternal Antibodies

Poultry are considered the major reservoir for Campylobacter jejuni, a leading bacterial cause of human food-borne diarrhea. To understand the ecology of C. jejuni and develop strategies to control C. jejuni infection in the animal reservoir, we initiated studies to examine the potential role of anti-Campylobacter maternal antibodies in protecting young broiler chickens from infection by C. jejuni. Using an enzyme-linked immunosorbent assay (ELISA), the prevalence of anti-C. jejuni antibodies in breeder chickens, egg yolks, and broilers from multiple flocks of different farms were examined. High levels of antibodies to the organism were detected in serum samples of breeder chickens and in egg yolk contents. To determine the dynamics of anti-Campylobacter maternal antibody transferred from yolks to hatchlings, serum samples collected from five broiler flocks at weekly intervals from 1 to 28 or 42 days of age were also examined by ELISA. Sera from the 1-day and 7-day-old chicks showed high titers of antibodies to C. jejuni. Thereafter, antibody titers decreased substantially and were not detected during the third and fourth weeks of age. The disappearance of anti-Campylobacter maternal antibodies during 3 to 4 weeks of age coincides with the appearance of C. jejuni infections observed in many broiler chicken flocks. As shown by immunoblotting, the maternally derived antibodies recognized multiple membrane proteins of C. jejuni ranging from 19 to 107 kDa. Moreover, in vitro serum bactericidal assays showed that anti-Campylobacter maternal antibodies were active in antibody-dependent complement-mediated killing of C. jejuni. Together, these results highlight the widespread presence of functional anti-Campylobacter antibodies in the poultry production system and provide a strong rationale for further investigation of the potential role of anti-C. jejuni maternal antibodies in protecting young chickens from infection by C. jejuni.