Campylobacter, Third Edition

This chapter presents an overview of the biological diversity of Campylobacter species and Arcobacter species and also addresses the taxonomic information that has become available through whole-genome sequence analysis. Classical biochemical tests routinely used for the identification of clinical bacteria often yielded negative or variable results within Campylobacter species. This poor biochemical reactivity and lack of clear-cut differential characters led to the wide application of vernacular names for many groups of Campylobacter-like organisms (CLOs). Some of the CLO groups were later classified as novel species, but several were identified as biochemical variants of well-known species. The first isolated Campylobacter was almost certainly Campylobacter fetus. In 1914, a researcher observed a vibrio, later classified as Vibrio sputorum, in sputum of a patient with bronchitis. Similar bacteria isolated from the bovine vagina and semen were classified as V. bubulus. During a study of the bacterial flora of the cloacae of whooping cranes, 10 atypical Campylobacter isolates were recovered on two separate occasions and were classified as C. canadensis. Strain NP4, isolated from groundwater with high arsenic concentrations, is classified as a Sulfurospirillum species on the basis of 16S rRNA gene sequence analysis, and it differs from the other Sulfurospirillum isolates in its carbon source and electron acceptor usage profiles. Representative strains of Bacteroides ureolyticus species were included in a polyphasic taxonomic study to elucidate their taxonomic status. Strains have been isolated from superficial ulcers and soft tissue infections, urethritis, vaginosis, and periodontal disease.

This chapter explores the contribution that population studies have made to one's understanding of the biology of the Campylobacter, and the authors argue that such studies have a central role to play in understanding the epidemiology and pathogenesis of this important group of gram-negative bacteria. Campylobacter jejuni and Campylobacter coli cause the majority of human cases of Campylobacter-associated gastroenteritis; these two organisms are associated with approximately 90 and 10% of cases, respectively. This chapter discusses variation within the genus Campylobacter. Although human infection is one of the most important practical applications of studies of Campylobacter populations, in terms of Campylobacter population dynamics and evolution, infection is probably irrelevant. Understanding the population biology of Campylobacter is, however, crucial in understanding the transmission to humans and developing means for its control. It is instructive to reflect that the first study of C. jejuni and C. coli population structure by multilocus enzyme electrophoresis provided many insights that have proved to be correct and that have been extended and deepened by multilocus sequence typing (MLST) studies. Ongoing nucleotide sequence-based studies involving large numbers of isolates and improved genealogical analysis tools provide the highly attractive prospect that well within the next 10 years, the population biology of these organisms, at least insofar as it relates to human infection, will be effectively resolved.

This chapter reviews the current knowledge on some of the metabolic aspects of Campylobacter jejuni physiology, with emphasis on those features of carbon, nitrogen, and electron flow that are likely to be of importance in understanding growth in the environment and in vivo. It focuses on catabolic pathways, i.e., those involved in the breakdown of extracellular solutes, yielding energy and key intracellular intermediates that are the building blocks for new cell growth. It describes the transport systems that relate to the major metabolic pathways in C. jejuni for which some functional data are available. C4-dicarboxylate transport (malate, succinate, fumarate, and possibly also aspartate) seems to be particularly important in C. jejuni. These substrates can feed directly into the citric acid cycle (CAC), malate and succinate can act as direct electron donors for aerobic respiration, and fumarate is an alternative electron acceptor under oxygen-limiting conditions. The chapter also focuses on central carbon metabolism in C. jejuni, and amino acid catabolism and nitrogen assimilation. A variety of primary dehydrogenases can be identified that feed electrons to a menaquinone pool. C. jejuni contains a proton-translocating cytochrome bc 1 complex feeding electrons to a periplasmic c-type cytochrome (probably Cj1153 in NCTC 11168) and then to a high-affinity cb-type oxidase, which allows efficient energy conservation when oxygen is used as electron acceptor. One conclusion that can be drawn is that many campylobacters show an unexpected metabolic versatility, which is particularly reflected in the complexity of the electron transport chains in C. jejuni.

The use of comparative genomics combined with robust methods for data analysis will continue and will form the basis for the development of rational intervention strategies to reduce Campylobacter jejuni in the food chain. This chapter reviews the salient comparative features of the four fully sequenced genomes and reveals highlights from selected whole-genome microarray studies. The publication of the first C. jejuni genome paved the way for comparative genomics of this species. The relative genome diversity of bacterial species varies from clonal (genetically uniform) to genetically highly variable. The majority of genes on CJIE3 are of unknown function, although 23% share homology with Helicobacter hepaticus ATCC 51449 genomic island (HHGI1). Recent comparative phylogenomics studies have been undertaken on increasingly large collections of strains from defined origins. The chapter discusses case studies of C. jejuni genomic comparisons by microarray. More recently, the authors studied over 230 C. jejuni strains from diverse origin and have further demonstrated the split of strains into two clades. Approximately half of the human isolates from this study are not associated with the livestock clade. DNA microarrays represent a powerful enabling technology for the whole-scale comparison of bacterial genomes. This, coupled with new methods to model DNA microarray data, is facilitating the development of robust comparative phylogenomics analyses. The next challenge for microbiologists in this postgenomic era is to correlate C. jejuni genome to phenome. This will provide a clear and more comprehensive understanding of the biology of C. jejuni.

The best characterized of the campylobacters is Campylobacter jejuni subsp. jejuni, a common commensal of warm-blooded animals, especially birds. The availability of eight new nonjejuni Campylobacter (NJC) genomes permits genomic comparisons between the NJC group and between the NJC genomes and the C. jejuni subsp. jejuni genomes. This chapter compares and contrasts the genomes of Campylobacter species other than C. jejuni subsp. jejuni. The primary phenotypic marker associated with Campylobacter plasmids is antibiotic resistance. Genes encoding resistance to tetracycline, kanamycin, and chloramphenicol have been identified on Campylobacter plasmids. Campylobacter plasmids fall into two basic groups: cryptic plasmids and megaplasmids. Homopolymeric G: C tracts have been identified in all of the NJC genomes. Several genes within the NJC genomes are frameshifted or have other defects and are considered putative pseudogenes. In Escherichia coli, the conjugative F episome cointegrates into the chromosome via homologous recombination between insertion sequences (IS) elements common to both the chromosome and the F plasmid, resulting in an Hfr strain. Helicobacter strains that possess the Entner-Doudoroff (ED) pathway contain a phosphoglucose isomerase (PGI) distinct from those found in the other Epsilonproteobacteria. A complete oxidative tricarboxylic acid (TCA) cycle is present in C. jejuni subsp. jejuni, as evidenced by the presence of all three subunits of succinate dehydrogenase (encoded by sdhABC). The chapter talks about restriction/modification systems, and virulence/pathogenicity loci. Genomic data for the NJC genomes will lead to new typing methods and perhaps culture methods.

This chapter describes the clinical aspects of infection with Campylobacter jejuni (C. jejuni subsp. jejuni) and Campylobacter coli, which are the main causes of Campylobacter enteritis in humans. The prodrome can be highly misleading in the absence of abdominal symptoms, which may not appear for two or even three days. Patients with prodromal symptoms tend to have more severe illness than those whose illness starts with diarrhea. The chapter discusses special aspects of infection, intestinal complications, and extraintestinal infection. The urinary tract seems an unlikely place to find campylobacters because they do not tolerate acid conditions well, but there are two reports of apparent C. jejuni urinary infection. In the first, the main site of infection was thought to be the prostate, but in the other, there appeared to be cystitis in a 6-year-old girl. Infections will be missed unless cultures specific for campylobacters are set up when morphologically suspect bacteria are seen in urine samples. The association of Campylobacter enteritis with Guillain-Barré syndrome, which emerged during the mid-1980s, greatly improved one's understanding of the morbidity of the disease. The chapter also describes the role of endoscopy and rectal biopsy in the management of patients. No specific treatment is required for most patients with Campylobacter enteritis, other than the oral replacement of fluid and electrolytes lost through diarrhea and vomiting. Erythromycin was the first antimicrobial agent to be used. Serious systemic infection should be treated with an aminoglycoside, such as gentamicin, or imipenem.

This chapter describes the microbiology, epidemiology, and clinical features of infection with Campylobacter species other than C. jejuni subsp. jejuni and C. coli that are associated with human disease. The major habitat of C. fetus is the intestine, and it is commonly isolated from healthy sheep and cattle. C. fetus infections are often prolonged and result in relapse, but most patients will recover with appropriate antibiotic treatment and medical procedures. C. upsaliensis is a recognized human pathogen in both healthy and immunocompromised patients. C. hyointestinalis was identified and suggested as a possible cause of proliferative enteritis in pigs. Six Campylobacter species have an essential growth requirement for hydrogen or formate. A membrane-bound hemolytic phospholipase was detected and characterized in clinical strains of C. concisus isolated from Australian children with gastroenteritis. The presence of this potential virulence factor suggests C. concisus is an opportunistic pathogen. Two recent reviews summarize clinical presentation, pathogenicity, and other aspects of Arcobacter infection. The chapter also describes other Helicobacter and Campylobacter species. The clinical relevance of the newly recognized Campylobacter species has yet to be determined. PCR assays for the simultaneous detection and differentiation of the genera and individual species of Campylobacter, Helicobacter, and Arcobacter may make this task easier. Appreciation and application of an efficient protocol is essential for the isolation of non-jejuni, non-coli Campylobacter species for surveillance, epidemiological, and other studies.

The burden of disease can be defined as the summary of morbidity and mortality associated with different acute and chronic manifestations of Campylobacter infections. Burden of disease studies include elements of epidemiology, disease modeling, risk assessment, and burden projections. This chapter discusses challenges in the measurement of burden of illness. The major limitation of seroepidemiology is that it measures the incidence of Campylobacter-related seroreactions, and some of these may be reactions to previous exposures that took place long ago. To assess the burden of disease, the most important or relevant disease outcomes need to be defined. This can be illustrated by an outcome tree, which is a qualitative representation of the progression of a disease in time. One of the challenges in the measurement of burden of illness is to identify methods to include contribution from immunity. The costs of campylobacteriosis are somewhat lower than the viruses but still significant. When evaluating an intervention or policy, decision makers need to determine its ability to positively impact public health (effectiveness) at a reasonable cost (efficiency) in a fair manner for all affected parties. In industrialized countries and countries in the transition phase, recent studies have shed light on the burden of illness.

Knowledge of the epidemiology of the infections of the Campylobacter organism causes has been steadily accumulating. The majority of these infections are sporadic; Campylobacter jejuni is far less often recognized as a cause of outbreaks than is Salmonella. In the first comprehensive epidemiological report of the European Centre of Disease Prevention and Control (ECDC) covering 2005, campylobacteriosis was the most frequently occurring enteric bacterial infection, second only to Chlamydia infection among all diseases reported. Much of the difference in incidence rates between the otherwise comparable countries Denmark and The Netherlands is explained by the fact that all Danish laboratories report Campylobacter diagnoses to the national level, compared with 51% of the Dutch laboratories. As the efficacy of serological methods for diagnosis of Campylobacter infection are further validated, including appropriate control sera, and as mathematical models advance, the usefulness of seroepidemiology to study the incidence of Campylobacter infections will become clearer. The vast majority of Campylobacter infections are sporadic individual infections and often affect young adults and young children. Consumption of untreated water, raw milk, or milk products and contact with pets are important sources of infection. Routine treatment of drinking water and pasteurization of milk are core prevention strategies. Veterinary use of fluoroquinolones in poultry explains the recent rise in ciprofloxacin-resistant Campylobacter infections in humans in the United States and may explain much of the geographic variation in resistance around the world.

This chapter summarizes the various modern genotypic methods available for subtyping the various Campylobacter species, the use of genetic methods for investigating outbreaks of disease, the application and interpretation of large-scale techniques in broader epidemiological studies, and a perspective of future developments in the field. Discussions of three methods (pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and amplified fragment length polymorphism (AFLP)) used to compare relatively large numbers of strains follow in a section aimed at summarizing our knowledge of Campylobacter molecular epidemiology in a broader perspective. Limitations of MLST include cost and the degree of resolution that is possible with the quantity of data provided by seven highly conserved gene fragments. The molecular epidemiology of Campylobacter coli is less well studied, but more recent work offers some insights into host association. Current results suggest that the attribution of C. jejuni and C. coli isolates from human cases to their source will be best undertaken by means of a phylogenetic approach that uses analysis based on allele frequencies, or that follows robust definition of host associated sublineages within the main clonal groups. Molecular epidemiological studies of Campylobacter species have matured considerably since the early applications of plasmid profiling and restriction enzyme analysis in homogeneous electric fields.

This chapter presents an overview of the historic classifications and nomenclature of Campylobacter fetus. The current recognized nomenclature for Campylobacteraceae was officially accepted in 1980, published in the approved list of bacterial names, and in accordance with that used in Bergey’s Manual of Systematic Bacteriology. During 1950s to 1970s, identification, subspecies differentiation, and typing results were based on phenotypic methods that are limited in both reliability and interpretation. Screening programs aiming to control bovine genital campylobacteriosis and limit the economic impact are performed by veterinary health services. The majority of the molecular methods are sensitive and can be used to identify C. fetus. However, one study has investigated the use of animal models for subspecies differentiation, on the premise that C. fetus subsp. venerealis and C. fetus subsp. fetus have differences in their preference for host and niche. The use of animals for subspecies differentiation purposes is not practical and has never been publicly evaluated. Phenotyping of C. fetus has been confined to serotyping. The currently accepted serotyping scheme is based on the heat-stable O antigens and consists of only two serotypes, A and B. Multilocus sequence typing (MLST) results showed that C. fetus has a more clonal structure compared with most other species within the genusCampylobacter. MLST in combination with a C. fetus subsp. venerealis–specific PCR may be applied. Currently, the best method for typing purposes is pulsed-field gel electrophoresis (PFGE).

Patients with Campylobacter infection may manifest signs and symptoms of acute appendicitis and result in unnecessary surgery. Bacteremia, endocarditis, meningitis, urinary tract infection, and other extraintestinal diseases may result from Campylobacter infections. Fecal samples should routinely be submitted to the laboratory for isolation of Campylobacter species from patients with diarrheal illness. Commercial blood culture systems such as the BACTEC system (aerobic bottles) and Septi-Chek system support the growth of the common Campylobacter species. Advantages of molecular approaches over culture include same-day detection, additional data regarding mixed infections, and uncommonCampylobacter species that are often missed when routine culture and procedures that are amenable to automation and high-throughput capabilities are used. A microaerobic atmosphere containing approximately 5% O2, 10% CO2, and 85% N2 is required for optimal recovery of most Campylobacter species. Campylobacter species are difficult to identify phenotypically because of relative biochemical inactivity, special growth requirements, and complex taxonomy. The 16S rRNA gene and 23S rRNA gene are two widely used targets for the design of species-specific tests; PCR assays based on these targets have been described for 12 different Campylobacter species. Fluoroquinolones had good in vitro activity for all Campylobacter species as well as for members of the family of Enterobacteriaceae. Little information is available on the antimicrobial susceptibility of C. concisus. Multidrug resistance in Campylobacter appears to be occurring more frequently and poses the risk that an effective antimicrobial regimen to treat infection may be lacking.

Guillain-Barré Syndrome (GBS) is currently considered to be a true case of molecular mimicry mediated disease, at least in those patients with a preceding Campylobacter jejuni infection. There is convincing evidence from extensive histology, serology, and animal model studies that GBS is caused by an autoimmune response. This parallels the failure of natural immune tolerance in other disorders such as rheumatoid arthritis, systemic lupus erythematosus, and multiple sclerosis, which are generally classified as autoimmune diseases. GBS is therefore frequently classified as a typical postinfectious disease. Studies based on stool culture alone will therefore underestimate the frequency of C. jejuni infections in GBS. The current trend toward direct DNA sequence-based typing has also penetrated the Campylobacter research field. There appears to be an association between the specific clinical symptoms, GBS variants, and particular characteristics of C. jejuni strains that primarily resides in the lipooligosaccharides (LOS) biosynthesis genes. Two conditions need to be fulfilled to induce a cross-reactive immune response against autologous antigens by molecular mimicry. First, the microbial and autologous antigens need to be sufficiently similar to induce antibodies that can cross-react. Second, the microbial antigen needs to be sufficiently different from the autoantigen to break the tolerance of the immune system and induce a specific immune response. Many studies have reported an association between preceding C. jejuni infections and axonal forms of GBS. Considering the crucial role of antiganglioside antibodies in the pathogenesis of these forms of GBS, patients may profit from selective depletion of antibodies.

This chapter discusses the mechanisms involved in Campylobacter resistance to various antibiotics, with particular emphasis on new information obtained during the last few years. One's understanding of the resistance mechanisms is limited to Campylobacter jejuni and Campylobacter coli, and little information is available on antimicrobial resistance in other Campylobacter spp. Fluoroquinolone (FQ) antimicrobials are important in the treatment of enteric infections including campylobacteriosis. Macrolide antibiotics, such as erythromycin, clarithromycin, azithromycin, and telithromycin, are important drugs for the treatment of respiratory tract infections and gastric diseases caused by Helicobacter pylori and Campylobacter in humans. To date, two mechanisms have been described for Campylobacter resistance to tetracyclines. The first mechanism involves a ribosomal protection protein termed Tet(O). The second mechanism of tetracycline resistance in Campylobacter involves the efflux system. Several studies reported that the majority of C. jejuni and C. coli isolates produce β-lactamases. The multidrug efflux pump CmeABC has been shown to contribute to both intrinsic and acquired resistance to tetracycline. The contribution of efflux pumps to antimicrobial resistance in Campylobacter is increasingly recognized. Inhibition of the efflux systems may not only control antibiotic resistance but also prevent Campylobacter colonization in vivo. Future efforts should be directed for understanding the interplay of various mechanisms in conferring antimicrobial resistance and the molecular basis underlying the emergence and transmission of antibiotic-resistant Campylobacter.

PulseNet USA is a network of public health laboratories from all 50 states, federal food regulatory agency laboratories, and a few state agricultural laboratories in the United States dedicated to molecular surveillance of food-borne bacterial infections. Regional and national PulseNet networks inspired by PulseNet USA have been established in different parts of the world dedicated to molecular surveillance of food-borne infections. PulseNets work together in investigations of international outbreaks in building capacity for molecular surveillance all over the world and in the development and validation of new PulseNet methods to ensure that data generated in all participating networks are comparable. As Campylobacter species are a common cause of diarrheal illness, active case finding during an outbreak can inevitably turn up additional Campylobacter infections that may or may not be related to the outbreak. Multilocus sequencing typing (MLST) has recently emerged as the current method of choice for subtyping of Campylobacters. A new priority area for PulseNet USA is to identify the sources of sporadic food-borne infections. The use and the content of the PulseNet Campylobacter database is therefore different from the other PulseNet databases, which are primarily aimed at detection of disease clusters by pulsedfield gel electrophoresis (PFGE) . However, the introduction of multilocus variable number tandem repeat analysis and single nucleotide polymorphism analysis will open the door to reliable phylogenetic analysis and attribution analysis for the non-Campylobacter organisms in PulseNet tool.

This chapter discuss three critical aspects of the interaction of Campylobacter jejuni with host cells: (i) its ability to mediate its own uptake into nonphagocytic cells, (ii) its ability to modulate vesicular trafficking pathways to avoid delivery into lysosomes, and (iii) its ability to reprogram host cell gene expression to stimulate the production of proinflammatory cytokines. The strong phenotype associated with capsular mutants in different animal models is likely to be due to reasons other than their rather minor effect in bacterial internalization. Therefore, it is likely that the capsular polysaccharide may contribute to internalization indirectly, perhaps by promoting bacterial attachment to host cells. Bacterial internalization does not require dynamin, an essential component of the endocytic machinery associated with cavaealoe. Like other intracellular pathogens, C. jejuni must have evolved specific adaptations to survive within host cells. Intestinal epithelial cells are equipped to mount innate immune responses upon detection of microbial pathogens. The last few years have seen advances in the understanding of the cell biology of infection, although more studies will be required to gain an understanding of these issues on par with that of other pathogenic bacteria. Although many C. jejuni mutants apparently defective in some of these process have been identified (e.g., bacterial entry), the direct involvement of these determinants in C. jejuni– host cell interactions has not been demonstrated. The availability of powerful genetic tools, coupled with a better understanding of the cell biology of infection, can help to identify those bacterial determinants.

This chapter presents an overview of the interaction of Campylobacter jejuni with intestinal host cells and focuses on bacterial adherence and invasion into the intestinal epithelium, transcytosis across the epithelial mucosa, and ensuing damage to host cells. Campylobacter invasion into the epithelial mucosa appears to be an essential process leading to colitis. Although many researchers would agree with this general summary of Campylobacter invasion events, there still remains considerable confusion regarding how Campylobacter enter and cross the intestinal mucosa. In fact, researchers concluded, after treating T-84 cells with EGTA, that CadF-dependent invasion of epithelial cells occurs preferentially at the basolateral surface, which normally interacts with fibronectin. In the above study, the number of internalized C. jejuni F38011 increased approximately threefold after EGTA treatment and was then reduced ~80% by treating with anti-fibronectin antibody. A common theme among pathogenic invasive microorganisms is their ability to usurp the eukaryotic cell signaling systems both to allow for invasion and to trigger disease pathogenesis. The current data on signal transduction events involved in C. jejuni invasion suggest that host cell ‘’invasion receptors’’ reside in filipin III-sensitive membrane microdomains (i.e., lipid rafts). Clinical infections, experimental infections in humans and animals, and in vitro analyses in cultured human cells have now clearly demonstrated that cell adherence and invasiveness are necessary steps in Campylobacter-induced inflammatory diarrhea. Much progress has been made in the past 10 years in the understanding of the cell biology of these events.

This chapter is divided into three major sections. In the first section, a model of Campylobacter jejuni-mediated enteritis is presented. The second section presents a general overview of the organism's pathogenic mechanisms and virulence determinants. Finally, in the third section, various aspects of C. jejuni-host cell invasion and protein secretion are discussed. Specifically, in the third section C. jejuni protein export via the flagellar type III secretion system (T3SS), the development of an assay to identify C. jejuni secreted proteins, the evolutionary relatedness of the flagellum and virulence T3SS, and the putative roles of C. jejuni secreted proteins in disease, are discussed. Although much remains unknown regarding the identity and functional characteristics of the proteins exported via the flagellar apparatus, the chapter highlights evidence supporting the proposal that these proteins contribute to C. jejuni-mediated enteritis. Motility, adherence, invasion, protein secretion, intracellular survival, and toxin production may contribute to the pathogenicity of a given C. jejuni strain. C. jejuni nonmotile strain can be either secretion positive (i.e., the flaA flaB+ mutant) or secretion negative (i.e., the flaA flaB mutant), and the ability of the bacterium to secrete proteins can result in an increase in its invasive potential. Although these data helped clarify the relationship between C. jejuni motility, secretion, and host cell invasion, the significance of protein secretion and host cell invasion in C. jejuni-mediated gastroenteritis was not known.

This chapter addresses the current knowledge pertaining to innate immunity and Campylobacter, and highlights important areas of future research. With a better understanding of the intricacies of immunity comes the realization that our host defenses cannot be strictly divided into innate and adaptive systems. Host defense mechanisms relevant to protection against a gastrointestinal infection such as Campylobacter include the multiple cellular and soluble factors. Although the interplay between many of these components and Campylobacter are not fully understood, the chapter reviews the current knowledge and outlines areas in need of further study. Innate immune defenses found in the gastrointestinal tract appear extremely effective in limiting C. jejuni to the gut. The direct antimicrobial activities of these phagocytes is attributable to production of antimicrobial peptides/proteins, ROS, and RNS (the latter mainly by mononuclear phagocytes and/or macrophages). The sequencing of several Campylobacter genomes and improved mutagenesis techniques can facilitate molecular studies of particular Campylobacter genes with specific innate immune components. From the point of view of the host, genomewide association studies of genetic polymorphisms associated with alterations in host defense functions may provide insight into those genes that are important for defense against Campylobacter infection.

This chapter describes the composition of the Campylobacter jejuni chemotaxis system in the context of pathways found in other bacterial species and derives models of the mechanism of signal transduction in the C. jejuni chemotaxis pathway. The six C. jejuni chemotaxis signal transduction pathway genes are located in three separate regions of the genome. In two regions, the che genes are located with genes of apparently unrelated function, and the distances between open reading frames and the strand-specific grouping of open reading frames suggests an operon arrangement. The cheY gene is located adjacent to the Pgl protein glycosylation gene cluster and possibly at the start of an operon, where no other gene is likely to be involved in chemotaxis. The second region includes the genes cheV, cheA, and cheW, which are located next to one another, and the genes flanking the three che genes in the operon do not appear to be associated with chemotaxis. The third region of the C. jejuni chromosome in which che genes are located contains the genes cheR and cheB, which are likely to be cistronic and not cotranscribed with the flanking genes, rpiB and pebC. There is evidence that methylation- and demethylation- based adaptation of the chemoreceptors also occurs in C. jejuni. Clearly, this model for the most part is speculative, being largely based on the exploitation of genomic sequence data, but ongoing investigation of chemotaxis in C. jejuni is providing experimental support.

Early studies have been conducted to test various inbred and outbred strains of mice as colonization and disease models. The infecting strain of Campylobacter jejuni will influence the outcome of infection even in inbred mice. Therefore, C. jejuni strain and history in the laboratory should be taken into consideration. Murine models of colonization and enteritis induced by primary oral C. jejuni challenge have long been needed to explore the genetics of resistance to this pathogen and the genetics of virulence of the pathogen; however, the basis for why most immunocompetent mice are refractory to C. jejuni induced disease is still unknown. Typhlocolitis is the main pathological lesion that can be observed by either gross or histopathological examination, although inflammation is observed in other GI tissues, including the stomach and liver. Severe combined immunodeficient mice have been tested for colonization and enteritis induced by C. jejuni. Researchers showed that wild-type C3H mice with normal enteric flora were colonized inconsistently and inefficiently by C. jejuni 81-176. Models for C. jejuni vaccine development are dependent on animals capable of expressing disease after challenge and mounting an adaptive immune response after vaccination. The greater the volume of feces that is cultured, the higher the probability will be that an accurate identification of colonization status is made. Despite progress in development of tractable murine models for study of C. jejuni colonization and enteritis, gaps in model development still exist.

In acute motor axonal neuropathy (AMAN), immunoglobulin (Ig) G is deposited on the axolemma of the spinal anterior roots. This indicates that IgG, which binds effectively with complement, is an important factor in the development of AMAN. Patients who developed AMAN subsequent to Campylobacter jejuni enteritis had IgG antibodies against GM1, and their autoantibody titers decreased with the clinical course. Various ganglioside immunization protocols were examined to refine the procedure for establishing an animal model of AMAN. The most effective was subcutaneous injection of an emulsion of 2.5 mg of bovine brain ganglioside (BBG) mixtures, keyhole lympet hemocyanin (KLH) and complete Freund’s adjuvant (CFA), to Japanese white rabbits, with repeated injections at 3-week intervals. Under that protocol, all the rabbits developed marked flaccid paralysis associated with increased plasma anti-GM1 IgG antibody levels. Secondary breakdown of axons under severe demyelination in Guillain-Barré syndrome (GBS) patients has been reported. Electrophysiological evaluations were made for three rabbits inoculated with BBG, seven with GM1, eight with galactocerebroside (GalC), and seven control animals. In the AMAN rabbit model, despite marked limb weakness in the acute phase of the illness, neither compound muscle action potential amplitudes nor motor conduction velocities showed obvious changes, indicating that distal motor nerve conduction was preserved. Intravenous immunoglobulin (IVIG) is effective for GBS in shortening recovery time, but the mechanism of action has yet to be clarified. Anti-idiotypic antibodies in IVIG may affect antibody production by sending negative signals to B cells.

Campylobacter fetus has been recognized as a significant pathogen of livestock for nearly a century. The genome sequence of C. fetus subsp. fetus strain 82-40 has recently been determined. Analysis of this sequence has confirmed and extended previous observations and has also provided new insights into C. fetus metabolism, physiology, and pathogenesis. The majority of the C. fetus N-linked general glycosylation pathway genes are also found in C. fetus, although unlike in C. jejuni, the genes are arranged in multiple clusters. The major pathogenesis-related difference of C. fetus compared with C. jejuni is the presence of the C. fetus S-layer. Surface array protein, type A (SapA) lacked an amino-terminal signal sequence that would direct its secretion to the cell surface. Before this, the only surface-layer protein (SLP) that lacked a signal sequence was that of C. crescentus. Observations made during the cloning of the sapA genes were important toward understanding the mechanisms by which the expression and antigenic variation of the encoded proteins are controlled. To investigate the role of the S-layer proteins in ovine abortion, an in vivo model was developed that used pregnant ewes subcutaneously challenged with C. fetus subsp. fetus strain 23D. The outcome of the infection in terms of effects on the fetus is dependent on the interaction between the pathogen and the host response. This model also provides a context to understand the role of S-layer proteins as virulence factors in human infections.

Observational and experimental studies provide evidence of acquired immunity developing in humans exposed to Campylobacter jejuni, lending support for vaccine development. Campylobacter vaccine development strategy must integrate basic science knowledge of pathogenesis and immunity, as well as an appreciation of the antigens and host responses associated with the development of protective immunity. More practical concerns include the need to optimize vaccine production methodology, delivery methods, and regimen selection followed by an assessment of vaccine safety, immunogenicity, and efficacy in target populations. The major impact of diarrheal disease in industrialized countries such as the United States relates to short-term morbidity and economic burden. Surveys among travel medicine practitioners in the United States and the United Kingdom have provided estimates on the level of support and potential application for vaccines to prevent traveler’s diarrhea (TD), and specifically, Campylobacter-only vaccines. The survey was limited by low response rate but was able to assess providers providing care in high-volume practices. A major consideration in the development of any vaccine is safety. For Campylobacter, this includes symptoms that may be caused by a vaccine in the first 24 to 72 h, as well as the potential for postvaccination sequelae a few weeks after immunization. Campylobacter vaccine development at the U.S. Naval Medical Research Center recently completed clinical studies of subunit Campylobacter vaccine based on the flagellin protein. Increased understanding of the burden of campylobacteriosis combined with evidence of acquired immunity provides the need and rationale for disease prevention strategies that include vaccine approaches.

N-linked protein glycosylation is the most common type of protein modification in eukaryotes and is the topic of this chapter. The chapter demonstrates that the Campylobacter jejuni glycome is an excellent toolbox for glycobiologists to understand the fundamentals of this pathway, to develop new techniques for glycobiology, and to exploit this pathway for novel diagnostics and therapeutics. A section summarizes the N-linked proteins identified so far and provides further information on the roles for the posttranslational modification in Campylobacter which involves in cellular function. The importance of CjaA for the in vivo survival of Campylobacter has recently been shown in chicken colonization studies: birds immunized with an avirulent strain of Salmonella expressing plasmid-borne cjaA showed reduced C. jejuni colonization. In addition, gene clusters corresponding to the N-linked protein glycosylation pathway were shown to be present in various isolates of C. jejuni, C. lari RM2100, C. upsaliensis RM3195, C. jejuni subsp. doylei 269.97, C. coli RM2228, C. hominis ATCC BAA-381, C. curvus 525.92, C. concisus 13826, and C. fetus subsp. fetus 82-40, demonstrating that this pathway and potentially the bacillosamine-containing heptasaccharide are conserved among all Campylobacter species. C. jejuni provides researchers with an excellent model system because this organism has both well-characterized O-linked and N-linked protein glycosylation systems.

Protein glycosylation has long been recognized as an important posttranslational modification in eukaryotic systems and one that imparts unique and diverse biological functions to the respective proteins. Although there is a considerable gap in our knowledge on the process of O-linked glycosylation in prokaryotes as a result of the significant glycan diversity among prokaryotes, the O-linked flagellar glycosylation system of Campylobacter has received considerable attention and is one of the more detailed prokaryotic systems studied to date. The first evidence for posttranslational modification of Campylobacter flagellin came from Campylobacter coli VC167 flagellin by direct chemical analysis of purified flagellar peptides. Mapping of glycosylation sites of C. coli VC167 flagellin also confirmed a conservation in localization to the central region of the monomer. Comparative genomic analyses of Campylobacter isolates has revealed that the flagellar locus displays considerable genetic variability. The glycans on flagellin appear to play complex roles in the biology of Campylobacter. The role of glycan composition on flagellar filaments was examined in vivo. Flagellins from the Epsilonproteobacteria, including both Campylobacter and Helicobacter spp., are not recognized by TLR5 receptors. Significant progress has been made in defining at the molecular level the structural nature of the novel sialic acid-like nonulosonate sugars found to be decorating the flagellar filaments of Campylobacter, and it is clear that these types of studies will be integral to future work exploring the role of novel glycan moieties in biological interactions.

This chapter reviews the structure, biosynthesis, and genetic determination of Campylobacter jejuni lipooligosaccharides (LOSs), with particular reference to the core oligosaccharide region. In particular, the genetic bases for variation of the outer core are discussed, including gene content variation between LOS loci and mechanisms generating LOS core region variation. Although the chapter predominantly addresses the biosynthesis of the variable LOS outer core of C. jejuni, the biosynthesis and genetic determination of the inner core lipid A region is also examined. Variation in LOS structures is due to diversity of the constituent sugars (number of carbon atoms, ring form, isomeric form, anomeric configuration, etc.), the derivatization of the sugars with noncarbohydrate moieties, and the linkages between the individual sugars (or monosaccharides). The formation of these linkages is determined by the glycosyltransferases and other transferases encoded in the LOS biosynthesis locus. Sugar availability for incorporation into LOS is often determined by enzymes from the LOS biosynthesis locus that are involved in synthesis of sugar intermediates such as cytidine monophosphate-5-N-acetylneuraminic acid (CMPNeu5Ac). There is evidence of recombination occurring within the LOS locus to create mosaics of different classes. Functions can be assigned to 12 of the 13 open reading frames (ORFs) present in the LOS biosynthesis locus of OH4384, the only exception being ORF 12a. The variety of capsular polysaccharide (CPS) structures and biosynthesis loci observed in C. jejuni suggests that it would contribute as much as the LOS repertoire to an open pan-glycome.

This chapter provides a review of the biochemistry, genetics, and biological roles of Campylobacter capsular polysaccharides (CPSs). The chapter is limited to consideration of PS capsules that have been detected in Campylobacter jejuni and other closely related species such as Campylobacter coli. Differentiation between groups is not based on antigenic or structural differences of the polysaccharide (PS) chains, but on such features as the mechanisms of biosynthesis, assembly, genetic regulation, and sequence similarity. The first indirect evidence of CPS produced by C. jejuni was reported. The first one to be studied was the kpsM mutant of strain NCTC 11168. Importantly, the absence or presence of these modifying groups affects the stainability of the PSs with silver and/or its interaction with homologous antiserum, which may in part explain their previous lack of detection in some serotypes. Differentiation of CPS-producing strains could be based on the probes corresponding to the genes involved in particular biosynthetic pathways. It would also be interesting to study a role of phosphoramidate modification of CPS in colonization and interaction with the host immune system. Initial attempts to develop a vaccine against C. jejuni infection, particularly in chickens, have not been very successful. Initial studies in the development of a human vaccine have focused on the use of heat- and formalin-attenuated whole-cell vaccines. In another study, a live attenuated Salmonella strain vectoring the PEB1 antigen of C. jejuni, which is implicated in colonization ability was evaluated as a vaccine against Campylobacter infection, but no protection was observed.

The ultimate goal in metabolomics is to achieve unbiased identification and quantification of all the metabolites in a defined biological system. Much of the work in bacterial metabolomics has involved the study of well-established metabolic pathways such as the tricarboxylic acid cycle, glycolysis, and specific metabolic pathways of microorganisms used in industrial applications. In contrast, the field of Campylobacter metabolomics is very much in its infancy, and considering the lack of information on many of the novel glycoconjugate biosynthesis pathways in Campylobacter, there is much scope to use targeted metabolomics approaches to further define the substrates and genes involved in these metabolic pathways. The main challenges associated with the study of sugar nucleotide metabolites by nuclear magnetic resonance (NMR) have been the instability of the sugar nucleotides and their presence at low concentrations within the bacterial cells. UDP-α-D-QuiNAc4NAc is an important metabolite in the 2,4-diacetamido-bacillosamine biosynthesis pathway, and its accumulation in pseC had not been expected because it has been thought that the inactivation of pseC would lead to an accumulation of a novel precursor directly related to Pse5Ac7Ac biosynthesis. The focused metabolomics studies of flagellin glycosylation in Campylobacter jejuni 81-176 and Campylobacter coli VC167 were extensive and examined unknown gene functions, characterized novel biosynthetic substrates and novel flagellar glycans, and elucidated poorly understood metabolic pathways.

Campylobacters produce proteins required for motility that are absent in other well studied motile bacteria, and have portions of conserved pathways mixed with unique mechanisms of promoting flagellar gene regulation, biosynthesis, and motility. Chemotaxis is an essential property of flagellar motility that influences the movement of bacteria toward appropriate environmental and host niches that support ideal bacterial growth and away from components that are less beneficial for growth or harmful to the organism. Although much information regarding flagellar motility has been gleaned by analyzing predictions from genomic sequences, the field of flagellar motility in campylobacters was moved forward by the development of new genetic tools and strategies for studying these bacteria. Seminal works for understanding regulatory pathways for flagellar gene expression and assembly of proteins into a flagellum largely focused on those of Salmonella species followed by Vibrio and Pseudomonas species. Early studies focusing on antigens of Campylobacter jejuni that are recognized by convalescent human antisera after infection revealed that the major flagellin FlaA is the foremost immunodominant antigen. Thus, much early work regarding flagellar motility in campylobacters largely centered on the genetic organization and expression of the flagellin genes of C. jejuni and C. coli. Flagellar motility in campylobacters is also affected by phase variation. Much progress has been made in the last decade in identifying proteins of campylobacters required for flagellar motility and understanding the roles of these proteins in flagellar gene regulation, biosynthesis of the organelle, and chemotaxis.

Over 40 naturally competent bacterial species are known, and that number continues to grow. Almost all competent bacteria studied to date use components of the type II secretion/type IV pilus biogenesis family of proteins for transformation, with slight differences between the transformation machinery in each system. The two known exceptions are Helicobacter pylori, which requires components of a type IV secretion/conjugation system for transformation, and Campylobacter jejuni, in which some strains use both type II-like and type IV-like secretion systems. Three different mechanisms may contribute to generating genetic diversity. The first is local sequence change such as nucleotide substitutions or small insertions or deletions of one or a few nucleotides. Another mechanism is DNA rearrangement, where related sequences in the genome undergo recombination to create novel fusion genes, and duplicate or delete DNA segments. Finally, diversity can be generated by horizontal acquisition of DNA through mechanisms such as natural transformation, conjugation, and transduction. Given the natural competence of most Campylobacter species, antibiotic resistance is probably spread in part through transformation. Several species of Campylobacter are naturally competent for transformation, including C. jejuni and C. coli. The candidate gene approach identified recombinase RecA as having a function in transformation. recA mutants were unable to be transformed to streptomycin resistance in three C. jejuni strains tested, 81-176, VC83, and 81-116. Modification of C. jejuni proteins by addition of an N-linked glycan is due to the activity of a number of genes found in the pgl locus.

This chapter is divided into three sections. The first covers specific stresses and responses, is organized by the type of stress encountered, and discusses specific gene products or global response pathways that participate in countering the stress condition. The second and third sections cover two different types of pleiotropic whole-population differentiation strategies or outcomes: the viable but nonculturable (VBNC) and coccoid forms of Campylobacter, and Campylobacter jejuni biofilms. Also incorporated into the latter two sections are the types of stresses that induce the differentiation, the genes involved in their formation, and the effect of differentiation on survivability or growth of C. jejuni. To date, only two regulatory factors directly affecting heat stress have been characterized, one of which is the RacR (reduced ability to colonize) response regulator. As such, nonculturable cells can be classified as both viable and nonviable, and not necessarily specifically correlated with shape. The majority of research to date on the contribution of biofilms to stress tolerance in C. jejuni has been in the context of survival in aquatic environments. It is important to point out that although this chapter specifically reviewed survival strategies and stress responses, many of the genes and pathways discussed affect multiple or all aspects of the pathogenesis cycle, encompassing colonization, transmission, and virulence.

This chapter discusses the wealth of knowledge about iron metabolism of Campylobacter by discussing mechanisms of iron transport, iron storage, and iron-responsive regulation of genes involved in iron metabolism. Most of the data discussed in the chapter have been obtained by using Campylobacter jejuni, but the author also discusses about the data obtained for Campylobacter coli; it is thought that the mechanisms involved in iron metabolism are essentially similar in both species. The availability of free iron inside mammalian and avian hosts is extremely limited as a result of the toxicity of iron in combination with oxygen. Ferrous iron is utilized by many bacteria, and in Escherichia coli, the high-affinity ferrous transport system expressed under anaerobic conditions involves two proteins, FeoA and FeoB, and a probable transcriptional regulator, FeoC. Enterobactin, which is produced by members of the mammalian and avian intestinal microbial flora, has the potential of being a significant source of iron to C. jejuni. Genes with homology to fhuABD, which encode the outer membrane receptor and part of the ABC transport system of the E. coli ferrichrome uptake system, have been identified in a set of C. jejuni strains. The other genes belonging to iron metabolism found to be upregulated in the rabbit intestine include those encoding Cj0236c, Cj0722c–Cj0723c, Cj1613c, PanBC, and RpmA. The transcriptomic analysis of genes involved in iron metabolism has highlighted some important connections between iron limitation and C. jejuni metabolism.

Transcription factors are grouped on the basis of the presence of conserved motifs and their modes of DNA binding. Genome-wide analysis of several Campylobacter jejuni strains indicates that this species contains between approximately 1,650 and 1,800 genes. The C. jejuni genome carries only three sigma factors: RpoD, FliA, and RpoN; the remaining 34 regulators belong to the specific transcription factors. The main essential sigma factor regulating almost all C. jejuni promoters is RpoD. Genome-wide analysis of C. jejuni that is based on this sequence (TGGCAC-N5-TTGC) indicated the existence of 17 putative RpoN promoters. The 17 identified RpoN promoters of C. jejuni control the transcription of 23 genes, of which 15 encode proteins that are involved in the assembly of the flagella. The majority of MarR homologs are transcriptional repressors that are autoregulated. Comparative genomic analysis of C. jejuni revealed the existence of seven major plasticity regions (PR), three of which (PR4, PR5, and PR6) contain genes involved in the production and modification of antigenic surface structures. One mechanism of C. jejuni gene regulation may involve direct modulation of the function of the RNA polymerase. DNA-binding proteins dictate the correct regulation of gene expression, so that the optimal amount and type of proteins are produced in response to specific internal and external stimuli. Although considerable progress has been made, the knowledge of the mechanisms that control C. jejuni gene regulation is still fragmentary.

Campylobacter infections in humans are considered to be mainly food-borne, in which foods of animal origin play an important role. The majority of Campylobacter infections are sporadic (single) cases or small family outbreaks, and the actual source of these types of infection is rarely microbiologically identified. This chapter describes the detection and prevalence of Campylobacter in a wide range of different types of food. Food products, however, may harbor only low numbers of campylobacters, and bacterial cells may be seriously injured by processing procedures such as freezing, cooling, heating, and salting. Survival of Campylobacter on eggshells, however, is considered to be poor because of the sensitivity of the organism to drying. Unpasteurized milk is a well-documented cause of a number of outbreaks of campylobacteriosis. Sufficient heating of red meat products, which are relatively infrequently contaminated with low numbers of Campylobacter, will eliminate this risk of human infection. Several investigations on the detection of Campylobacter in different types of seafood have been carried out. The majority of Campylobacter studies on growth characteristics and survival were carried out during the early 1980s, and summarizing reviews can be found in articles by Doyle and Stern and Kazmi. Reduction of the potential risk of contaminated poultry products has to be achieved by the application of good hygienic practices by both the producers of poultry meat products and the consumers of these products.

Food of animal origin is considered the most important sources of Campylobacter causing infections in humans. This chapter reviews the current knowledge on antimicrobial susceptibility, occurrence of antimicrobial resistance, and transmission of antimicrobial resistant Campylobacter from food animals. Surveillance of antimicrobial resistance in Campylobacter is important for assessing the association between antimicrobial use and occurrence of antibiotic resistance. A total of 4,953 isolates from humans received by the Minnesota Department of Health were tested for resistance to nalidixic acid. Because fluoroquinolones (FQs) use in humans can lead to resistance in Campylobacter during therapy, it can be difficult to determine whether increased resistance in human strains is mainly caused by veterinary or human drug use. There are no known biological reasons why resistant Campylobacter should not transmit from animals to humans as well as susceptible Campylobacter. Several studies have shown that infections with quinolone-resistant Campylobacter in humans are associated with adverse effects for human health, mainly measured by prolonged diarrhea. Campylobacteriosis is one of the most common causes of diarrhea in humans worldwide. Studies aimed at the human health consequences of infections with antimicrobialresistant strains should help guide interventions aimed at limiting the spread of the most important types of resistant strains. The effects of successful intervention measures can be used to direct similar in interventions in other countries and have the potential to considerably improve food safety worldwide.

The handling and consumption of poultry is an important source of human campylobacteriosis. It is widely assumed that the control of Campylobacter in meat-producing poultry, with the aim of reducing the numbers of Campylobacter on poultry meat at the retail level, will reduce the public health burden of human campylobacteriosis. This chapter updates the knowledge of poultry infections and their control at the farm level and extends it to include the interface between the farm and the slaughterhouse. The focus of the chapter remains on the control of Campylobacter in primary production because in this food chain, the gut of living poultry is the only amplification point for Campylobacter. Epidemiological and exposure studies have implicated the handling and consumption of poultry meat as an important source for human campylobacteriosis. Colonization is largely in the ceca and is primarily confined to the intestinal mucous layer over the intestinal crypts of the villi. Reducing human campylobacteriosis is an important public health goal in most industrialized countries. Multispecies farming is a clear risk factor for Campylobacter-positive poultry flocks on the same site. The separation of Campylobacter-positive and -negative flocks and decontamination of the meat from positive flocks is an alternative strategy to reducing the risk of campylobacteriosis in humans. One practical control strategy that can be implemented at the farm-slaughterhouse interface is to separate colonized and noncolonized flocks during processing, and to subsequently treat the meat from Campylobacter-positive flocks.

This chapter discusses efforts to exploit Campylobacter-specific bacteriophages to reduce the numbers of C. jejuni and C. coli colonizing poultry and contaminating poultry meat products. All the phages reported by investigators in two studies had icosahedral heads and long contractile tails that were classified as members of the Myoviridae. Two phages with head diameters of 140.6 and 143.8 nm and large genome sizes of 320 kb were classified as group I. Five phages classified into group II had average head diameters of 99 nm and average genome sizes of 184 kb. A fourth phage had an icosahedral head that was classified as morphotype B1 of the Siphoviridae, while a fifth phage had an icosahedral head with a short tail of morphotype C1 in the Podoviridae. Campylobacter phages on the skin of retail chicken portions have been recovered at levels of 2 X 103 PFU/10 cm2 and this study found that Campylobacter phage could be isolated from chicken skin only when detectable levels of their host were also present. The spontaneous production of CampMu bacteriophages after bacteriophage therapy is of concern because Mu bacteriophages are potential agents of mutation. Attempts to utilize bacteriophage, initially for typing purposes and more recently for their biocontrol potential, have led to a greater awareness of the role that phage play in the complex ecology of Campylobacter. It should be noted that bacteriophage can shape the evolution of Campylobacter genomes as they do in other bacterial genera.