Shaun Cawthraw

The genome-sequenced variant of Campylobacter jejuni NCTC 11168 and the original clonal clinical isolate differ markedly in colonization, gene expression, and virulence-associated phenotypes.

The genome sequence of the enteric bacterial pathogen Campylobacter jejuni NCTC 11168 (11168-GS) was published in 2000, providing a valuable resource for the identification of C. jejuni-specific colonization and virulence factors. Surprisingly, the 11168-GS clone was subsequently found to colonize 1-day-old chicks following oral challenge very poorly compared to other strains. In contrast, we have found that the original clinical isolate from which 11168-GS was derived, 11168-O, is an excellent colonizer of chicks. Other marked phenotypic differences were also identified: 11168-O invaded and translocated through tissue culture cells far more efficiently and rapidly than 11168-GS, was significantly more motile, and displayed a different morphology. Serotyping, multiple high-resolution molecular genotyping procedures, and subtractive hybridization did not yield observable genetic differences between the variants, suggesting that they are clonal. However, microarray transcriptional profiling of these strains under microaerobic and severely oxygen-limited conditions revealed dramatic expression differences for several gene families. Many of the differences were in respiration and metabolism genes and operons, suggesting that adaptation to different oxygen tensions may influence colonization potential. This correlates biologically with our observation that anaerobically priming 11168-GS or aerobically passaging 11168-O caused an increase or decrease, respectively, in colonization compared to the parent strain. Expression differences were also observed for several flagellar genes and other less well-characterized genes that may participate in motility. Targeted sequencing of the sigma factors revealed specific DNA differences undetected by the other genomic methods [corrected].

Host-Pathogen Interactions in Campylobacter Infections: the Host Perspective

SUMMARY Campylobacter is a major cause of acute bacterial diarrhea in humans worldwide. This study was aimed at summarizing the current understanding of host mechanisms involved in the defense against Campylobacter by evaluating data available from three sources: (i) epidemiological observations, (ii) observations of patients, and (iii) experimental observations including observations of animal models and human volunteer studies. Analysis of available data clearly indicates that an effective immune system is crucial for the host defense against Campylobacter infection. Innate, cell-mediated, and humoral immune responses are induced during Campylobacter infection, but the relative importance of these mechanisms in conferring protective immunity against reinfection is unclear. Frequent exposure to Campylobacter does lead to the induction of short-term protection against disease but most probably not against colonization. Recent progress in the development of more suitable animal models for studying Campylobacter infection has opened up possibilities to study the importance of innate and adaptive immunity during infection and in protection against reinfection. In addition, advances in genomics and proteomics technologies will enable more detailed molecular studies. Such studies combined with better integration of host and pathogen research driven by epidemiological findings may truly advance our understanding of Campylobacter infection in humans.

Identification of genetic differences between two Campylobacter jejuni strains with different colonization potentials

The consumption of poultry meat contaminated with Campylobacter jejuni is considered to be a risk factor for human campylobacteriosis. The development of targeted strategies to control campylobacters in broilers would benefit from knowledge of those bacterial factors important in colonization of the avian gut. During preliminary studies it was noted that C. jejuni NCTC 11168 was a poorer colonizer of chickens than strain 81116. This poor colonization could not be fully restored by in vivo passage, suggesting that it was a genetically endowed property of strain 11168. As the genome sequence is available for this strain, the technique of subtractive hybridization was used to identify gene fragments of strain 81116 not present in strain 11168. After two screening cycles, 24 out of 42 clones were identified as having DNA inserts specific for strain 81116. Six of these 24 clones contained gene fragment inserts with similarities to restriction-modification enzymes found in other bacteria. Two inserts had similarity to arsenic-resistance genes, whereas four others had similarities to cytochrome c oxidase III, dTDP-glucose 4,6-dehydratase, gamma-glutamyl transpeptidase and an abortive phage-resistance protein. At least some of these genes may be involved with colonization. A further six inserts had weak similarities to hypothetical proteins or to proteins with assigned functions from strain 11168. The remaining six clones had gene-fragment inserts with no database matches. Southern-blot analysis confirmed that strain-dependent variation existed for each of these DNA inserts. These results indicate that subtractive hybridization can successfully identify genes that are absent from the only C. jejuni strain for which the genome sequence is currently available.

Cytolethal Distending Toxin (CDT)-Negative Campylobacter jejuni Strains and Anti-CDT Neutralizing Antibodies Are Induced during Human Infection but Not during Colonization in Chickens

ABSTRACT The cytolethal distending toxin (CDT) of Campylobacter jejuni was detectable, using an in vitro assay, in most but not all of 24 strains tested. The reason for the absence of toxin activity in these naturally occurring CDT-negative C. jejuni strains was then investigated at the genetic level. CDT is encoded by three highly conserved genes, cdtA, -B, and -C. In the CDT-negative strains, two types of mutation were identified. The CDT activities of C. jejuni strains possessing both types of mutation were successfully complemented with the functional genes of C. jejuni 11168. The first type of mutation comprised a 667-bp deletion across cdtA and cdtB and considerable degeneration in the remainder of the cdt locus. Using a PCR technique to screen for this deletion, this mutation occurred in fewer than 3% of 147 human, veterinary, and environmental strains tested. The second type of mutation involved at least four nonsynonymous nucleotide changes, but only the replacement of proline with serine at CdtB position 95 was considered important for CDT activity. This was confirmed by site-directed mutagenesis. This type of mutation also occurred in fewer than 3% of strains as determined using a LightCycler biprobe assay. The detection of two CDT-negative clinical isolates raised questions about the role of CDT in some cases of human campylobacteriosis. To determine if anti-CDT antibodies are produced in human infection, a toxin neutralization assay was developed and validated using rabbit antisera. Pooled human sera from infected patients neutralized the toxin, indicating expression and immunogenicity during infection. However, no neutralizing antibodies were detected in colonized chickens despite the expression of CDT in the avian gut as indicated by reverse transcription-PCR.

The Campylobacter jejuni dccRS two‐component system is required for optimal in vivo colonization but is dispensable for in vitro growth

A Campylobacter jejuni two‐component signal transduction system (TCSTS), designated dccR‐dccS (diminished capacity to colonize; Cj1223c‐Cj1222c), has been found to be important for in vivo colonization but dispensable for in vitro growth. A ΔdccR response regulator mutant generated using the virulent strain 81–176 background exhibited significantly reduced colonization of immunocompetent limited flora (I‐LF) mice, severe combined immunodeficient limited flora (SCID‐LF) mice, and 1‐day‐old chicks. A ΔdccS sensor kinase mutant was likewise defective for colonization in the I‐LF mouse model. ΔdccR‐infected SCID‐LF mice also exhibited dramatically reduced inflammation relative to wild type‐infected SCID‐LF mice. Despite this diminished colonization capacity, the ΔdccRS mutants were indistinguishable from wild type for growth under numerous in vitro conditions as well as for various phenotypes. Microarray analysis identified several genes encoding putative periplasmic and membrane proteins as being regulated by this two‐component system; binding of purified His‐tagged DccR to the promoter region of two of these genes supports a direct protein–DNA interaction. A conserved repeat sequence was identified in the promoter regions of these genes and in three other promoter regions in the genome, including that of an operon encoding a putative type I secretion system. Two of the regulated target genes were found to be essential for optimal colonization. Both the two‐component system and the putative regulated genes have uncharacterized homologues in other Campylobacter and Helicobacter spp., suggesting that they may perform an important function in colonization among a variety of related pathogenic species.

Role of Intimin-Tir Interactions and the Tir-Cytoskeleton Coupling Protein in the Colonization of Calves and Lambs by Escherichia coli O157:H7

ABSTRACT Intimin facilitates intestinal colonization by enterohemorrhagic Escherichia coli O157:H7; however, the importance of intimin binding to its translocated receptor (Tir) as opposed to cellular coreceptors is unknown. The intimin-Tir interaction is needed for optimal actin assembly under adherent bacteria in vitro, a process which requires the Tir-cytoskeleton coupling protein (TccP/EspFU) in E. coli O157:H7. Here we report that E. coli O157:H7 tir mutants are at least as attenuated as isogenic eae mutants in calves and lambs, implying that the role of intimin in the colonization of reservoir hosts can be explained largely by its binding to Tir. Mutation of tccP uncoupled actin assembly from the intimin-Tir-mediated adherence of E. coli O157:H7 in vitro but did not impair intestinal colonization in calves and lambs, implying that pedestal formation may not be necessary for persistence. However, an E. coli O157:H7 tccP mutant induced typical attaching and effacing lesions in a bovine ligated ileal loop model of infection, suggesting that TccP-independent mechanisms of actin assembly may operate in vivo.

Campylobacter colonization of the chicken induces a proinflammatory response in mucosal tissues

Campylobacter jejuni is a major cause of human inflammatory enteritis, but colonizes the gastrointestinal tract of poultry to a high level in a commensal manner. In vitro, C. jejuni induces the production of cytokines from both human and avian-model epithelial cell and macrophage infections. This suggests that, in vivo, Campylobacter could induce proinflammatory signals in both hosts. We investigated whether a proinflammatory cytokine response can be measured in both day-of-hatch and 2-week-old Light Sussex chickens during infection with C. jejuni. A significant induction of proinflammatory chemokine transcript was observed in birds of both ages, compared with levels in mock-infected controls. This correlated with an influx of heterophils but was not associated with any pathology. These results suggest that in poultry there may be a controlled inflammatory process during colonization.

Genetic instability is associated with changes in the colonization potential of Campylobacter jejuni in the avian intestine

Aims:  A panel of pulsed field gel electrophoresis (PFGE) type variants of Campylobacter jejuni, previously identified as of clonal origin, were investigated to determine whether genomic instability could be observed during competitive growth.

A temperature‐regulated Campylobacter jejuni gluconate dehydrogenase is involved in respiration‐dependent energy conservation and chicken colonization

Campylobacter jejuni is a gastrointestinal pathogen of humans but can asymptomatically colonize the avian gut. C. jejuni therefore grows at both 37°C and 42°C, the internal temperatures of humans and birds respectively. Microarray and proteomic studies on temperature regulation in C. jejuni strain 81–176 revealed the upregulation at 42°C of two proteins, Cj0414 and Cj0415, orthologous to gluconate dehydrogenase (GADH) from Pectobacterium cypripedii. 81–176 demonstrated GADH activity, converting d‐gluconate to 2‐keto‐d‐gluconate, that was higher at 42°C than at 37°C. In contrast, cj0414 and cj0415 mutants lacked GADH activity. Wild‐type but not cj0415 mutant bacteria exhibited gluconate‐dependent respiration. Neither strain grew in defined media with d‐gluconate or 2‐keto‐d‐gluconate as a sole carbon source, revealing that gluconate was used as an electron donor rather than as a carbon source. When administered to chicks individually or in competition with wild‐type, the cj0415 mutant was impaired in establishing colonization. In contrast, there were few significant differences in colonization of BALB/c‐ByJ mice in single or mixed infections. These results suggest that the ability of C. jejuni to use gluconate as an electron donor via GADH activity is an important metabolic characteristic that is required for full colonization of avian but not mammalian hosts.

Longitudinal Molecular Epidemiological Study of Thermophilic Campylobacters on One Conventional Broiler Chicken Farm

ABSTRACT Improved understanding of the ecology and epidemiology of Campylobacter in the poultry farm environment is key to developing appropriate farm-based strategies for preventing flock colonization. The sources of Campylobacter causing broiler flock colonization were investigated on one poultry farm and its environment, from which samples were obtained on three occasions during each of 15 crop cycles. The farm was adjacent to a dairy farm, with which there was a shared concreted area and secondary entrance. There was considerable variation in the Campylobacter status of flocks at the various sampling times, at median ages of 20, 26, and 35 days, with 3 of the 15 flocks remaining negative at slaughter. Campylobacters were recoverable from various locations around the farm, even while the flock was Campylobacter negative, but the degree of environmental contamination increased substantially once the flock was positive. Molecular typing showed that strains from house surroundings and the dairy farm were similar to those subsequently detected in the flock and that several strains intermittently persisted through multiple crop cycles. The longitudinal nature of the study suggested that bovine fecal Campylobacter strains, initially recovered from the dairy yard, may subsequently colonize poultry. One such strain, despite being repeatedly recovered from the dairy areas, failed to colonize the concomitant flock during later crop cycles. The possibility of host adaptation of this strain was investigated with 16-day-old chickens experimentally exposed to this strain naturally present in, or spiked into, bovine feces. Although the birds became colonized by this infection model, the strain may preferentially infect cattle. The presence of Campylobacter genotypes in the external environment of the poultry farm, prior to their detection in broiler chickens, confirms the horizontal transmission of these bacteria into the flock and highlights the risk from multispecies farms.