Proteomic analyses of a robust versus a poor chicken gastrointestinal colonizing isolate of Campylobacter jejuni

Campylobacter spp. are a significant contributor to the bacterial etiology of acute gastroenteritis in humans. Epidemiological evidence implicates poultry as a major source of the organism for human illness. However, the factors involved in colonization of poultry with Campylobacter spp. remain unclear. Determining colonization-associated factors at the proteome level should facilitate our understanding of Campylobacter spp. contamination of poultry. Therefore, proteomic analyses were utilized to identify expression differences between two Campylobacter jejuni isolates, a robust colonizer A74/C and a poor colonizing strain of the chicken gastrointestinal system designated NCTC 11168-PMSRU. Proteomic analyses by two-dimensional gel electrophoresis revealed the specific expression of an outer membrane-fibronectin binding protein, serine protease, and a putative aminopeptidase in the soluble portion of the robust colonizer A74C. Several proteins including a cysteine synthase and aconitate hydratase were detected specifically in the poor colonizer C. jejuni NCTC 11168-PMSRU isolate. Variation in the amino acid sequences resulting in different isoelectric points and relative mobility of the flagellin and C. jejuni major outer membrane (MOMP) protein were also detected between the two isolates. Western blotting of the bacterial proteins revealed the presence of two flagellin proteins in the poor colonizer versus one in the robust colonizing isolate, but no differences in MOMP. The results demonstrated that proteomics is useful for characterizing phenotypic variation among Campylobacter spp. isolates. Interestingly, different gene products potentially involved in robust colonization of chickens by Campylobacter spp. appear to conform to recently identified expression patterns in Biofilm or agar-adapted isolates.     

Cellular response of Campylobacter jejuni to trisodium phosphate

The highly alkaline compound trisodium phosphate (TSP) is used as an intervention to reduce the load of Campylobacter on poultry meat in U.S. poultry slaughter plants. The aim of the present study was to investigate the cellular responses of Campylobacter jejuni NCTC11168 when exposed to sublethal concentrations of TSP. Preexposure of C. jejuni to TSP resulted in a significant increase in heat sensitivity, suggesting that a combined heat and TSP treatment may increase reduction of C. jejuni. A microarray analysis identified a limited number of genes that were differently expressed after sublethal TSP exposure; however, the response was mainly associated with ion transport processes. C. jejuni NCTC11168 nhaA1 (Cj1655c) and nhaA2 (Cj1654c), which encode orthologues to the Escherichia coli NhaA cation/proton antiporter, were able to partially restore TSP, alkaline, and sodium resistance phenotypes to an E. coli cation/proton antiporter mutant. In addition, inhibition of resistance-nodulation-cell division (RND) multidrug efflux pumps by the inhibitor PaβN (Phe-Arg β-naphthylamide dihydrochloride) decreased tolerance to sublethal TSP. Therefore, we propose that NhaA1/NhaA2 cation/proton antiporters and RND multidrug efflux pumps function in tolerance to sublethal TSP exposure in C. jejuni.     

Bacteriophage F336 recognizes the capsular phosphoramidate modification of Campylobacter jejuni NCTC11168

Bacteriophages infecting the food-borne human pathogen Campylobacter jejuni could potentially be exploited to reduce bacterial counts in poultry prior to slaughter. This bacterium colonizes the intestinal tract of poultry in high numbers, and contaminated poultry meat is regarded as the major source of human campylobacteriosis. In this study, we used phage F336 belonging to the Myoviridae family to select a C. jejuni NCTC11168 phage-resistant strain, called 11168R, with the aim of investigating the mechanisms of phage resistance. We found that phage F336 has reduced adsorption to 11168R, thus indicating that the receptor is altered. While proteinase K-treated C. jejuni cells did not affect adsorption, periodate treatment resulted in reduced adsorption, suggesting that the phage binds to a carbohydrate moiety. Using high-resolution magic angle spinning nuclear magnetic resonance (NMR) spectroscopy, we found that 11168R lacks an O-methyl phosphoramidate (MeOPN) moiety attached to the GalfNAc on the capsular polysaccharide (CPS), which was further confirmed by mass spectroscopy. Sequence analysis of 11168R showed that the potentially hypervariable gene cj1421, which encodes the GalfNAc MeOPN transferase, contains a tract of 10 Gs, resulting in a nonfunctional gene product. However, when 11168R reverted back to phage sensitive, cj1421 contained 9 Gs, and the GalfNAc MeOPN was regained in this strain. In summary, we have identified the phase-variable MeOPN moiety, a common component of the diverse capsular polysaccharides of C. jejuni, as a novel receptor of phages infecting this bacterium.     

空肠弯曲菌cheA基因插入突变及其对小鼠空肠定植能力的影响

摘要：【目的】构建空肠弯曲菌（Campylobacter jejuni）cheA基因插入突变株,了解CheA与空肠弯曲菌小鼠体内定植的相关性。【方法】运用同源重组的原理构建空肠弯曲菌cheA基因突变株,采用PCR技术检测cheA突变株的构建情况。通过基因回补试验构建cheA基因回补株。空肠弯曲菌感染小鼠,运用小鼠空肠内容物涂板计数的方法检测cheA突变株、cheA基因回补株和野生株定植小鼠能力的差异。【结果】PCR检测显示成功构建cheA基因突变株。空肠弯曲菌cheA基因突变株定植小鼠空肠的数量明显减少（P<0.05）;cheA基因回补株定植小鼠空肠的数量跟野生株相比无明显差异（P>0.05）。【结论】本研究成功构建cheA基因突变株及其回补株。cheA基因可能参与空肠弯曲菌在小鼠体内定植的过程。     

Survival of cold-stressed Campylobacter jejuni on ground chicken and chicken skin during frozen storage

Campylobacter jejuni is prevalent in poultry, but the effect of combined refrigerated and frozen storage on its survival, conditions relevant to poultry processing and storage, has not been evaluated. Therefore, the effects of refrigeration at 4 degrees C, freezing at -20 degrees C, and a combination of refrigeration and freezing on the survival of C. jejuni in ground chicken and on chicken skin were examined. Samples were enumerated using tryptic soy agar containing sheep's blood and modified cefoperazone charcoal deoxycholate agar. Refrigerated storage alone for 3 to 7 days produced a reduction in cell counts of 0.34 to 0.81 log10 CFU/g in ground chicken and a reduction in cell counts of 0.31 to 0.63 log10 CFU/g on chicken skin. Declines were comparable for each sample type using either plating medium. Frozen storage, alone and with prerefrigeration, produced a reduction in cell counts of 0.56 to 1.57 log10 CFU/g in ground chicken and a reduction in cell counts of 1.38 to 3.39 log10 CFU/g on chicken skin over a 2-week period. The recovery of C. jejuni following freezing was similar on both plating media. The survival following frozen storage was greater in ground chicken than on chicken skin with or without prerefrigeration. Cell counts after freezing were lower on chicken skin samples that had been prerefrigerated for 7 days than in those that had been prerefrigerated for 0, 1, or 3 days. This was not observed for ground chicken samples, possibly due to their composition. C. jejuni survived storage at 4 and -20 degrees C with either sample type. This study indicates that, individually or in combination, refrigeration and freezing are not a substitute for safe handling and proper cooking of poultry.     

Reference isolates for the clonal complexes of Campylobacter jejuni

AIMS: To identify and make available through the National Collection of Type Cultures (NCTC) a set of reference isolates for the clonal complexes of Campylobacter jejuni. METHODS AND RESULTS: The development of a multilocus sequence typing scheme for C. jejuni enabled the genetic characterization of a large number of isolates (n = 814) from cases of human disease, animals, birds and their food products. The nucleotide sequence data were used to assign each isolate an allelic profile or sequence type (ST) and examine the C. jejuni population structure in terms of clonal complexes. The clonal complexes consisted of an abundant central or founder genotype (ST), after which the complex was named, together with very closely related, generally less abundant genotypes differing from the founder at one, two or three loci. The clonal complex is an informative unit for the study C. jejuni epidemiology. It provides data which enabled the choice of 13 C. jejuni founder isolates for submission to the NCTC as a representative cross-section of the C. jejuni population. CONCLUSIONS: These 13 isolates provide a defined resource for further research into aspects of C. jejuni biology such as genomic diversity, virulence and adaptation to particular hosts or environmental survival. SIGNIFICANCE AND IMPACT OF STUDY: This isolate collection is available through the NCTC and provides a resource for further research.     

Multiple N-acetyl neuraminic acid synthetase (neuB) genes in Campylobacter jejuni: identification and characterization of the gene involved in sialylation of lipo-oligosaccharide

N-acetyl neuraminic acid (NANA) is a common constituent of Campylobacter jejuni lipo-oligosaccharide (LOS). Such structures often mimic human gangliosides and are thought to be involved in the triggering of Guillain-Barré syndrome (GBS) and Miller-Fisher syndrome (MFS) following C. jejuni infection. Analysis of the C. jejuni NCTC 11168 genome sequence identified three putative NANA synthetase genes termed neuB1, neuB2 and neuB3. The NANA synthetase activity of all three C. jejuni neuB gene products was confirmed by complementation experiments in an Escherichia coli neuB-deficient strain. Isogenic mutants were created in all three neuB genes, and for one such mutant (neuB1) LOS was shown to have increased mobility. C. jejuni NCTC 11168 wild-type LOS bound cholera toxin, indicating the presence of NANA in a LOS structure mimicking the ganglioside GM1. This property was lost in the neuB1 mutant. Gas chromatography-mass spectrometry and fast atom bombardment-mass spectrometry analysis of LOS from wild-type and the neuB1 mutant strain demonstrated the lack of NANA in the latter. Expression of the neuB1 gene in E. coli confirmed that NeuB1 was capable of in vitro NANA biosynthesis through condensation of N-acetyl-D-mannosamine and phosphoenolpyruvate. Southern analysis demonstrated that the neuB1 gene was confined to strains of C. jejuni with LOS containing a single NANA residue. Mutagenesis of neuB2 and neuB3 did not affect LOS, but neuB3 mutants were aflagellate and non-motile. No phenotype was evident for neuB2 mutants in strain NCTC 11168, but for strain G1 the flagellin protein from the neuB2 mutant showed an apparent reduction in molecular size relative to the wild type. Thus, the neuB genes of C. jejuni appear to be involved in the biosynthesis of at least two distinct surface structures: LOS and flagella.     

Survival of Campylobacter jejuni in chicken meat at frozen storage temperatures

The aim of this study was to determine the survival of Campylobacter jejuni in chicken meat samples at frozen temperatures and given length of incubation and to determine the impact of aerobic bacteria on the survival of C. jejuni. The chicken meat samples were inoculated with C. jejuni NCTC 11351 suspensions and stored in bags at temperatures of -20°C and -70°C. The mean value of C. jejuni from meat samples decreased from 7.52 log10 CFU/g after 30 minutes of incubation at ambient temperature, to 3.87 log10 CFU/g on the eighth week of incubation at -20°C, and to 3.78 log10 CFU/g at incubation at -70°C after the same incubation period. Both freezing temperatures, -20°C and -70°C, decreased the number of campylobacters. The presence of aerobic mesophilic bacteria did not influence the survival of C. jejuni in chicken meet samples. Keeping poultry meat at freezing temperatures is important for the reduction of C. jejuni, which has a strong influence on the prevention of occurrence of campylobacteriosis in humans.     

The chemical structure and genetic locus of Campylobacter jejuni CG8486 (serotype HS:4) capsular polysaccharide: the identification of 6-deoxy-D-ido-heptopyranose

In line with our on-going efforts to create a multivalent anti-Campylobacter jejuni vaccine based on its capsule polysaccharides (CPSs), we report here the chemical structure and the genetic locus of the CPS produced by C. jejuni strain CG8486, which belongs to the serotype HS:4 CPS complex. C. jejuni CG8486 CPS was observed to be composed of approximately 17 disaccharide repeating blocks of 4-substituted N-acetyl-beta-D-glucopyranosamine and 3-substituted 6-deoxy-beta-D-ido-heptopyranose. A small number of 6-deoxy-beta-D-ido-heptopyranose units were observed to carry O-methyl phosphoramidate moieties at the O-2 or O-7 position. The gene content and organization of the CPS locus of C. jejuni CG8486 were comparable to those of C. jejuni strains NCTC 11168 and 81-176, but several CG8486 CPS genes were observed to be more divergent from those present in the CPS loci of NCTC 11168 and 81-176 CPS, which indicated that there are genetic characteristics specific to the C. jejuni HS:4 CPS complex. The efficacy of a glycoconjugate vaccine based on C. jejuni CG8486 CPS is presently being tested in an animal model, the results of which will be presented in future communications.     

Phase variation of a beta-1,3 galactosyltransferase involved in generation of the ganglioside GM1-like lipo-oligosaccharide of Campylobacter jejuni

Ganglioside mimicry by Campylobacter jejuni lipo-oligosaccharide (LOS) is thought to be a critical factor in the triggering of the Guillain-Barré and Miller-Fisher syndrome neuropathies after C. jejuni infection. The combination of a completed genome sequence and a ganglioside GM1-like LOS structure makes C. jejuni NCTC 11168 a useful model strain for the identification and characterization of the genes involved in the biosynthesis of ganglioside-mimicking LOS. Genome analysis identified a putative LOS biosynthetic cluster and, from this, we describe a putative gene (ORF Cj1139c), which we have termed wlaN, with a significant level of similarity to a number of bacterial glycosyltransferases. Mutation of this gene in C. jejuni NCTC 11168 resulted in a LOS molecule of increased electrophoretic mobility, which also failed to bind cholera toxin. Comparison of LOS structural data from wild type and the mutant strain indicated lack of a terminal beta-1,3-linked galactose residue in the latter. The wlaN gene product was demonstrated unambiguously as a beta-1,3 galactosyltransferase responsible for converting GM2-like LOS structures to GM1-like by in vitro expression. We also show that the presence of an intragenic homopolymeric tract renders the expression of a functional wlaN gene product phase variable, resulting in distinct C. jejuni NCTC 11168 cell populations with alternate GM1 or GM2 ganglioside-mimicking LOS structures. The distribution of wlaN among a number of C. jejuni strains with known LOS structure was determined and, for C. jejuni NCTC 12500, similar wlaN gene phase variation was shown to occur, so that this strain has the potential to synthesize a GM1-like LOS structure as well as the ganglioside GM2-like LOS structure proposed in the literature.     

Survival of Campylobacter jejuni strains of different origin in drinking water

AIMS: The aim of the study was to measure the survival of 19 Campylobacter jejuni strains of different origins, including two reference strains, four poultry-derived isolates, nine human isolates and four water isolates, in sterilized drinking water. METHODS AND RESULTS: Pure cultures of 19 C. jejuni strains were inoculated in sterile drinking water and incubated at 4 degrees C for 64 days. Survival was determined by culturability on both selective (Karmali agar) and non-selective [Columbia blood agar (CBA)] media. Culturability was shown to be strain and origin-dependent. Campylobacter jejuni showed prolonged survival on a non-selective than on a selective medium. CONCLUSIONS: The origin of the strain is a determining factor for the survival of C. jejuni in drinking water at 4 degrees C. Poultry isolates showed a prolonged survival, which could be an indication that these strains could play an important role in the transmission of campylobacteriosis through water. In addition, culture conditions are an important factor for evaluating the survival of C. jejuni in drinking water at 4 degrees C. The non-selective agar (CBA) allowed growth of C. jejuni over a longer period of time than the selective agar (Karmali). Furthermore, an enrichment broth (Bolton) allowed the recovery of all 19 C. jejuni strains during the 64 days of incubation at 4 degrees C. SIGNIFICANCE AND IMPACT OF THE STUDY: This study highlighted differences in culturability depending on culture conditions and on strain origin.     

Determination of toxicity of Campylobacter jejuni isolated from humans and from poultry carcasses acquired at various stages of production

AIM: The research focused on the determination of the toxicity variation associated with Campylobacter jejuni isolated from humans and chickens. METHODS AND RESULTS: Campylobacter jejuni isolates were obtained from chicken carcasses and from humans exhibiting symptoms of campylobacteriosis. Using HeLa cells as the in vitro model, toxicity was determined for each isolate. The mean toxicity level of the chicken isolates was significantly lower than that of the human isolates (P < 0.001). There was a wide range of toxicity in C. jejuni isolated from chickens and the percentage of isolates exhibiting low toxicity remaining relatively constant. All C. jejuni isolates from humans possessed either medium or high levels of toxicity. CONCLUSIONS: All wildtype C. jejuni isolates obtained from poultry carcasses may not be equally important as a human foodborne pathogen. SIGNIFICANCE AND IMPACT OF STUDY: Campylobacter jejuni remains a primary foodborne pathogen and increased efforts are needed to determine the impact of wildtype isolates in causing human illness. The present research indicates that all isolates may not be equally important in regards to disease potential. The information found should be included in efforts to develop C. jejuni detection, control and infection modelling.     

Detection and survival of Campylobacter in chicken eggs

AIMS: Campylobacter jejuni, a food-borne human pathogen, is widespread in poultry; however, the sources of infection and modes of transmission of this organism on chicken farms are not well understood. The objective of this study was to determine if vertical transmission of C. jejuni occurs via eggs. METHODS AND RESULTS: Using a temperature differential method, it was shown that Campylobacter had limited ability to penetrate the eggshell. When C. jejuni was directly inoculated into the egg yolk and the eggs were stored at 18 degrees C, the organism was able to survive for up to 14 days. However, viability of C. jejuni was dramatically shortened when injected into the albumen or the air sac. When freshly laid eggs from Campylobacter-inoculated specific pathogen-free (SPF) layers were tested, C. jejuni-contamination was detected in three of 65 pooled whole eggs (5-10 eggs in each pool) via culture and PCR. However, the organism was not detected from any of the 800 eggs (80 pools), collected from the same SPF flock, but kept at 18 degrees C for 7 days before testing. Likewise, Campylobacter was not recovered from any of 500 fresh eggs obtained from commercial broiler-breeder flocks that were actively shedding Campylobacter in faeces. Also, none of the 1000 eggs from broiler breeders obtained from a commercial hatchery were positive for Campylobacter. CONCLUSIONS: These results suggest that vertical transmission of C. jejuni through the egg is probably a rare event and does not play a major role in the introduction of Campylobacter to chicken flocks. SIGNIFICANCE AND IMPACT OF THE STUDY: Control of Campylobacter transmission to chicken flocks should focus on sources of infection that are not related to eggs.     

Identification and molecular characterisation of CmeB, a Campylobacter jejuni multidrug efflux pump

A multidrug efflux pump gene (cmeB) was identified from the published Campylobacter jejuni genome sequence. Secondary structural analysis showed that the gene encoded a protein belonging to the resistance nodulation cell division (RND) family of efflux transporters. The gene was inactivated by insertional mutagenesis. Compared with the wild-type strain (NCTC 11168), the resultant knockout strain (NCTC 11168-cmeB::kan(r)) displayed increased susceptibility to a range of antibiotics including beta-lactams, fluoroquinolones, macrolides, chloramphenicol, tetracycline, ethidium bromide, the dye acridine orange and the detergent sodium dodecyl sulfate. Accumulation of ciprofloxacin was increased in the knockout mutant, but carbonyl cyanide m-chlorophenyl hydrazone, a proton motive force inhibitor, had less effect upon ciprofloxacin accumulation in the knockout mutant compared with NCTC 11168. These data show that the identified gene encodes an RND-type multi-substrate efflux transporter, which contributes to intrinsic resistance to a range of structurally unrelated compounds in C. jejuni. This efflux pump has been named CmeB (for Campylobacter multidrug efflux).     

Correlation Between In Vitro Secretion of Virulence-Associated Proteins of <Emphasis Type="Italic">Campylobacter jejuni</Emphasis> and Colonization of Chickens

The mechanisms used by Campylobacter jejuni to colonize the (chicken) intestinal tract have not been defined. In this study, we obtained evidence that in the presence of chicken serum and mucus, C. jejuni secreted proteins that may play a role in the colonization of chicken gut (Campylobacter invasion antigen = Cia). C. jejuni strains NCTC11168V1 and 81-176, as well as an NCTC11168V1 flaA mutant, were found to colonize intestinal tract and secrete proteins in the presence of chicken mucus, chicken serum, or fetal bovine serum in cell culture–conditioned medium. C. jejuni strain NCTC11168V26, which was observed to be a poor colonizer compared with the other C. jejuni isolates, did not secrete Cia proteins. Secreted proteins were also recognized by Western immunoblot using sera from birds that had been colonized by C. jejuni. These data suggest that C. jejuni secretes Cia proteins during colonization of chicken gut and that these Cia proteins play an important role in colonization.     

The structures of the lipooligosaccharide and capsule polysaccharide of Campylobacter jejuni genome sequenced strain NCTC 11168.

Campylobacter jejuni infections are one of the leading causes of human gastroenteritis and are suspected of being a precursor to Guillain-Barré and Miller-Fisher syndromes. Recently, the complete genome sequence of C. jejuni NCTC 11168 was described. In this study, the molecular structure of the lipooligosaccharide and capsular polysaccharide of C. jejuni NCTC 11168 was investigated. The lipooligosaccharide was shown to exhibit carbohydrate structures analogous to the GM1a and GM2 carbohydrate epitopes of human gangliosides (shown below): The high Mr capsule polysaccharide was composed of beta-d-Ribp, beta-d-GalfNAc, alpha-d-GlcpA6(NGro), a uronic acid amidated with 2-amino-2-deoxyglycerol at C-6, and 6-O-methyl-d-glycero-alpha-l-gluco-heptopyranose as a side-branch (shown below): The structural information presented here will aid in the identification and characterization of specific enzymes that are involved in the biosynthesis of these structures and may lead to the discovery of potential therapeutic targets. In addition, the correlation of carbohydrate structure with gene complement will aid in the elucidation of the role of these surface carbohydrates in C. jejuni pathogenesis.     

Identification of Campylobacter jejuni ATCC 43431-specific genes by whole microbial genome comparisons

This study describes a novel approach to identify unique genomic DNA sequences from the unsequenced strain C. jejuni ATCC 43431 by comparison with the sequenced strain C. jejuni NCTC 11168. A shotgun DNA microarray was constructed by arraying 9,600 individual DNA fragments from a C. jejuni ATCC 43431 genomic library onto a glass slide. DNA fragments unique to C. jejuni ATCC 43431 were identified by competitive hybridization to the array with genomic DNA of C. jejuni NCTC 11168. The plasmids containing unique DNA fragments were sequenced, allowing the identification of up to 130 complete and incomplete genes. Potential biological roles were assigned to 66% of the unique open reading frames. The mean G+C content of these unique genes (26%) differs significantly from the G+C content of the entire C. jejuni genome (30.6%). This suggests that they may have been acquired through horizontal gene transfer from an organism with a G+C content lower than that of C. jejuni. Because the two C. jejuni strains differ by Penner serotype, a large proportion of the unique ATCC 43431 genes encode proteins involved in lipooligosaccharide and capsular biosynthesis, as expected. Several unique open reading frames encode enzymes which may contribute to genetic variability, i.e., restriction-modification systems and integrases. Interestingly, many of the unique C. jejuni ATCC 43431 genes show identity with a possible pathogenicity island from Helicobacter hepaticus and components of a potential type IV secretion system. In conclusion, this study provides a valuable resource to further investigate Campylobacter diversity and pathogenesis.