Common genomic features of <Emphasis Type="Italic">Campylobacter jejuni</Emphasis> subsp. <Emphasis Type="Italic">doylei</Emphasis> strains distinguish them from <Emphasis Type="Italic">C. jejuni</Emphasis> subsp. <Emphasis Type="Italic">jejuni</Emphasis>

Background  

Campylobacter jejuni has been divided into two subspecies: C. jejuni subsp. jejuni (Cjj) and C. jejuni subsp. doylei (Cjd). Nearly all of the C. jejuni strains isolated are Cjj; nevertheless, although Cjd strains are isolated infrequently, they differ from Cjj in two key aspects: they are obtained primarily from human clinical samples and are associated often with bacteremia, in addition to gastroenteritis. In this study, we utilized multilocus sequence typing (MLST) and a DNA microarray-based comparative genomic indexing (CGI) approach to examine the genomic diversity and gene content of Cjd strains.     

Colonization of broilers by <Emphasis Type="Italic">Campylobacter jejuni</Emphasis> internalized within <Emphasis Type="Italic">Acanthamoeba castellanii</Emphasis>

Although Campylobacter survives within amoeba in-vitro, it is unknown if intra-amoeba Campylobacter jejuni can colonize broilers. Five groups of 28 day-of-hatch chicks were placed into separate isolators. Groups (1) and (2) were challenged with page’s amoeba saline (PAS), and disinfected planktonic C. jejuni NCTC 11168, respectively. Groups (3), (4) and (5) were challenged with a C. jejuni positive control, C. jejuni in PAS, and intra-amoeba C. jejuni, respectively. After 1, 3, 7 and 14 days post challenge, seven birds from each unit were examined for C. jejuni colonization. For the first time we report that intra-amoeba C. jejuni colonized broilers.     

Performance of a 70-mer oligonucleotide microarray for genotyping of <Emphasis Type="Italic">Campylobacter jejuni</Emphasis>

Background  

Campylobacter jejuni is widespread in the environment and is the major cause of bacterial gastroenteritis in humans. In the present study we use microarray-based comparative genomic hybridizations (CGH), pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) to analyze closely related C. jejuni isolates from chicken and human infection.     

Multilocus sequence types of Finnish bovine <Emphasis Type="Italic">Campylobacter jejuni</Emphasis> isolates and their attribution to human infections

Background  

Campylobacter jejuni is the most common bacterial cause of human gastroenteritis worldwide. Due to the sporadic nature of infection, sources often remain unknown. Multilocus sequence typing (MLST) has been successfully applied to population genetics of Campylobacter jejuni and mathematical modelling can be applied to the sequence data. Here, we analysed the population structure of a total of 250 Finnish C. jejuni isolates from bovines, poultry meat and humans collected in 2003 using a combination of Bayesian clustering (BAPS software) and phylogenetic analysis.     

Antimicrobial activity of copper surfaces against suspensions of <Emphasis Type="Italic">Salmonella enterica</Emphasis> and <Emphasis Type="Italic">Campylobacter jejuni</Emphasis>

Background  

Salmonella enterica and Campylobacter jejuni are amongst the more prevalent bacterial pathogens that cause foodborne diseases. These microorganisms are common contaminants of poultry and poultry products. This study was aimed to evaluate the antibacterial activity of metallic copper surfaces on these important enteropathogens, and to determine the potential acquisition of copper by food exposed to this metal.     

Temperature-dependent phenotypic variation of <Emphasis Type="Italic">Campylobacter jejuni</Emphasis> lipooligosaccharides

Background  

Campylobacter jejuni is a major bacterial cause of food-borne enteritis, and its lipooligosaccharide (LOS) plays an initiating role in the development of the autoimmune neuropathy, Guillain-Barré syndrome, by induction of anti-neural cross-reactive antibodies through ganglioside molecular mimicry.     

Sequence variability of <Emphasis Type="Italic">Campylobacter</Emphasis>temperate bacteriophages

Background  

Prophages integrated within the chromosomes of Campylobacter jejuni isolates have been demonstrated very recently. Prior work with Campylobacter temperate bacteriophages, as well as evidence from prophages in other enteric bacteria, suggests these prophages might have a role in the biology and virulence of the organism. However, very little is known about the genetic variability of Campylobacter prophages which, if present, could lead to differential phenotypes in isolates carrying the phages versus those that do not. As a first step in the characterization of C. jejuni prophages, we investigated the distribution of prophage DNA within a C. jejuni population assessed the DNA and protein sequence variability within a subset of the putative prophages found.     

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

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

Characterization of <Emphasis Type="Italic">Campylobacter</Emphasis> phages including analysis of host range by selected <Emphasis Type="Italic">Campylobacter</Emphasis> Penner serotypes

Background  

The predominant food borne pathogen in the western world today is Campylobacter. Campylobacter specific bacteriophages (phages) have been proposed as an alternative agent for reducing the burden of Campylobacter in broilers. One concern in relation to phage biocontrol is the narrow host range often displayed by phages. To identify the potential of phages as a Campylobacter reducing agent we needed to determine their infectivity on a panel of isolates representing the Campylobacter strains found in broilers as well as humans.     

Serine phosphorylation of cortactin is required for maximal host cell invasion by <Emphasis Type="Italic">Campylobacter jejuni</Emphasis>

Background

Campylobacter jejuni causes acute disease characterized by severe diarrhea containing blood and leukocytes, fever, and abdominal cramping. Disease caused by C. jejuni is dependent on numerous bacterial and host factors. C. jejuni invasion of the intestinal epithelial cells is seen in both clinical samples and animal models indicating that host cell invasion is, in part, necessary for disease. C. jejuni utilizes a flagellar Type III Secretion System (T3SS) to deliver the Campylobacter invasion antigens (Cia) to host cells. The Cia proteins modulate host cell signaling leading to actin cytoskeleton rearrangement necessary for C. jejuni host cell invasion, and are required for the development of disease.

Results

This study was based on the hypothesis that the C. jejuni CiaD effector protein mediates Erk 1/2 dependent cytoskeleton rearrangement. We showed that CiaD was required for the maximal phosphorylation of Erk 1/2 by performing an immunoblot with a p-Erk 1/2 specific antibody and that Erk 1/2 participates in C. jejuni invasion of host cells by performing the gentamicin protection assay in the presence and absence of the PD98059 (a potent inhibitor of Erk 1/2 activation). CiaD was also found to be required for the maximal phosphorylation of cortactin S405 and S418, as judged by immunoblot analysis. The response of human INT 407 epithelial cells to infection with C. jejuni was evaluated by confocal microscopy and scanning electron microscopy to determine the extent of membrane ruffling. This analysis revealed that CiaD, Erk 1/2, and cortactin participate in C. jejuni-induced membrane ruffling. Finally, cortactin and N-WASP were found to be involved in C. jejuni invasion of host cells using siRNA to N-WASP, and siRNA to cortactin, coupled with the gentamicin protection assay.

Conclusion

We conclude that CiaD is involved in the activation of Erk 1/2 and that activated Erk 1/2 facilitates C. jejuni invasion by phosphorylation of cortactin on serine 405 and 418. This is the first time that cortactin and N-WASP have been shown to be involved in C. jejuni invasion of host cells. These data also provide a mechanistic basis for the requirement of Erk 1/2 in C. jejuni-mediated cytoskeletal rearrangement.

     

Production and characterization of anti-<Emphasis Type="Italic">Campylobacter jejuni</Emphasis> IgY derived from egg yolks

Background

Campylobacter jejuni is a major cause of foodborne disease having chickens as an important reservoir. Its control at the farm would lower the contamination of the final products and therefore also lower the risk of transmission to humans. At the farm, C. jejuni is rarely found in chickens before they reach 2 weeks of age. Past studies have shown that maternal antibodies could hamper C. jejuni gut colonization. The objective of this study was to compare protocols to use in order to produce anti-C. jejuni antibodies derived from egg yolks in the perspective to be used as feed additives for the control of chicken C. jejuni colonization. Laying hens were naturally contaminated with four well-characterized strains or injected with either outer membrane proteins or formalin-killed whole bacteria derived from these same strains. Eggs were collected and IgYs present in the yolks were extracted. The amount and the specificity of the recovered antibodies were characterized.

Results

It was observed that injection yielded eggs with superior concentrations of both total and anti-C. jejuni antibodies. Equivalent performances for antibodies recovered from all protocols were observed for the ability of the antibodies to agglutinate the live C. jejuni homologous strains, to hinder their motility or to lyse the bacteria. Western blot analyses showed that proteins from all strains could be recognized by all IgY extracts. All these characteristics were strain specific. The characterization assays were also made for heterologous strains and weaker results were observed when compared to the homologous strains.

Conclusions

Based on these results, only an IgY quantitative based selection can be made in regards to which protocol would give the best anti-C. jejuni IgY enriched egg-yolks as all tested protocols were equivalent in terms of the recovered antibody ability to recognized the tested C. jejuni strains.

     

A newly constructed primer pair for the PCR amplification,cloningand sequencing of the flagellin (<Emphasis Type="Italic">flaA</Emphasis>) gene from isolatesof urease-negative <Emphasis Type="Italic">Campylobacter lari</Emphasis>

A newly constructed primer pair (lari-Af/lari-Ar) designed to generate a product of the flagellin (flaA) gene for urease-negative Campylobacter lari produced a PCR amplicon of about 1700 bp for 16 isolates from 7 seagulls, 5 humans, 3 food animals and one mussel in Japan and Northern Ireland. Nucleotide sequencing and alignments of the flaA amplicons from these isolates demonstrated that the deduced amino acid sequences of the possible open reading frame were 564–572 amino acid residues in length with calculated molecular weights of 58,804 to 59,463. The deduced amino acid sequence similarity analysis strongly suggested that the ORF of the flaA from the 16 isolates showed 70–75% sequence similarities to those of Campylobacter jejuni isolates. The approximate Mr of the flagellin purified from some of the isolates of urease-negative C. lari was estimated to range from 59.6 to 61.8 kDa. Thus, flagellin from the isolates of urease-negative C. lari was shown for the first time to have a molecular size similar to those of C. jejuni and Campylobacter coli isolates, but to be different from the shorter flaA and smaller flagellin of urease-positive thermophilic Campylobacter (UPTC) isolates. Flagellins from C. lari spp., consisting of the two representative taxa of urease-negative C. lari and UPTC, thus show genotypic and phenotypic diversity.     

Role of Oxidative Stress in <Emphasis Type="Italic">C. jejuni</Emphasis> Inactivation During Freeze-Thaw Treatment

Campylobacter jejuni represents one of the leading causes of bacterial enteritis throughout the world. Poultry is an important source of C. jejuni. Despite hygiene measures taken in the production chain, C. jejuni is frequently isolated from poultry meat. C. jejuni is a microaerophilic pathogen, affected by oxidative stress. Freeze-thaw treatment induces cell death by several mechanisms, including oxidative stress. In this article, we investigate the role of oxidative stress in C. jejuni sensitivity during and after a freeze-thaw treatment. This treatment results in dead and sublethally injured cells. The latter population might have an increased sensitivity to oxidative stress. To test this, cells were stored for another 24 h at 4°C under aerobic conditions and compared to cells that were not treated. C. jejuni survival was measured in different media (water, BHI broth, chicken juice, and chicken fillets) to test the environment protective effect. Different strains were tested, including sodB (encoding the superoxide dismutase) and cj1371 (encoding a periplasmic protein) mutants. Cell death was particularly important in water but similar in BHI, chicken juice, and chicken fillets. The sodB mutant was more sensitive to freeze-thaw treatment, suggesting that the killing mechanism involves production of superoxide anions. On the contrary, the cj1371 mutant was more sensitive to storage at 4°C, suggesting that it does not play a role in the detoxification of reactive oxygen species. Storage at 4°C after freeze-thaw treatment increases cell death of oxidative stress-sensitive populations. Sensitization to oxidative stress, freeze-thaw treatment, and further storage at 4°C could be a way to reduce C. jejuni populations on carcasses.     

Antagonistic Activity of <Emphasis Type="Italic">Lactobacillus acidophilus</Emphasis> ATCC 4356 on the Growth and Adhesion/Invasion Characteristics of Human <Emphasis Type="Italic">Campylobacter jejuni</Emphasis>

The aim of this research was to determine the potential probiotic activity of Lactobacillus acidophilus ATCC 4356 against several human Campylobacter jejuni isolates. The ability to inhibit the pathogen’s growth was evaluated by co-culture experiments as well as by antimicrobial assays with cell-free culture supernatant (CFCS), while interference with adhesion/invasion to intestinal Caco-2 cells was studied by exclusion, competition, and displacement tests. In the co-culture experiments L. acidophilus ATCC 4356 strain reduced the growth of C. jejuni with variable percentages of inhibition related to the contact time. The CFCS showed inhibitory activity against C. jejuni strains, stability to low pH, and thermal treatment and sensitivity to proteinase K and trypsin. L. acidophilus ATCC 4356 was able to reduce the adhesion and invasion to Caco-2 cells by most of the human C. jejuni strains. Displacement and exclusion mechanisms seem to be the preferred modalities, which caused a significant reduction of adhesion/invasion of pathogens to intestinal cells. The observed inhibitory properties of L. acidophilus ATCC 4356 on growth ability and on cells adhesion/invasion of C. jejuni may offer potential use of this strain for the management of Campylobacter infections.     

Production of glycoprotein vaccines in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Background  

Conjugate vaccines in which polysaccharide antigens are covalently linked to carrier proteins belong to the most effective and safest vaccines against bacterial pathogens. State-of-the art production of conjugate vaccines using chemical methods is a laborious, multi-step process. In vivo enzymatic coupling using the general glycosylation pathway of Campylobacter jejuni in recombinant Escherichia coli has been suggested as a simpler method for producing conjugate vaccines. In this study we describe the in vivo biosynthesis of two novel conjugate vaccine candidates against Shigella dysenteriae type 1, an important bacterial pathogen causing severe gastro-intestinal disease states mainly in developing countries.     

Comparative genomic assessment of Multi-Locus Sequence Typing: rapid accumulation of genomic heterogeneity among clonal isolates of <Emphasis Type="Italic">Campylobacter jejuni</Emphasis>

Background  

Multi-Locus Sequence Typing (MLST) has emerged as a leading molecular typing method owing to its high ability to discriminate among bacterial isolates, the relative ease with which data acquisition and analysis can be standardized, and the high portability of the resulting sequence data. While MLST has been successfully applied to the study of the population structure for a number of different bacterial species, it has also provided compelling evidence for high rates of recombination in some species. We have analyzed a set of Campylobacter jejuni strains using MLST and Comparative Genomic Hybridization (CGH) on a full-genome microarray in order to determine whether recombination and high levels of genomic mosaicism adversely affect the inference of strain relationships based on the analysis of a restricted number of genetic loci.     

Unexpected differential metabolic responses of <Emphasis Type="Italic">Campylobacter jejuni</Emphasis> to the abundant presence of glutamate and fucose

Introduction

Campylobacter jejuni is the leading cause of foodborne bacterial enteritis in humans, and yet little is known in regard to how genetic diversity and metabolic capabilities among isolates affect their metabolic phenotype and pathogenicity.

Objectives

For instance, the C. jejuni 11168 strain can utilize both l-fucose and l-glutamate as a carbon source, which provides the strain with a competitive advantage in some environments and in this study we set out to assess the metabolic response of C. jejuni 11168 to the presence of l-fucose and l-glutamate in the growth medium.

Methods

To achieve this, untargeted hydrophilic liquid chromatography coupled to mass spectrometry was used to obtain metabolite profiles of supernatant extracts obtained at three different time points up to 24 h.

Results

This study identified both the depletion and the production and subsequent release of a multitude of expected and unexpected metabolites during the growth of C. jejuni 11168 under three different conditions. A large set of standards allowed identification of a number of metabolites. Further mass spectrometry fragmentation analysis allowed the additional annotation of substrate-specific metabolites. The results show that C. jejuni 11168 upon l-fucose addition indeed produces degradation products of the fucose pathway. Furthermore, methionine was faster depleted from the medium, consistent with previously-observed methionine auxotrophy.

Conclusions

Moreover, a multitude of not previously annotated metabolites in C. jejuni were found to be increased specifically upon l-fucose addition. These metabolites may well play a role in the pathogenicity of this C. jejuni strain.

     

Comparison of 16S rRNA gene sequences of genus <Emphasis Type="Italic">Methanobrevibacter</Emphasis>

Background  

The phylogeny of the genus Methanobrevibacter was established almost 25 years ago on the basis of the similarities of the 16S rRNA oligonucleotide catalogs. Since then, many 16S rRNA gene sequences of newly isolated strains or clones representing the genus Methanobrevibacter have been deposited. We tried to reorganize the 16S rRNA gene sequences of this genus and revise the taxonomic affiliation of the isolates and clones representing the genus Methanobrevibacter.     

Invasion of epithelial cells by <Emphasis Type="Italic">Campylobacter jejuni</Emphasis> is independent of caveolae

Caveolae are 25–100 nm flask-like membrane structures enriched in cholesterol and glycosphingolipids. Researchers have proposed that Campylobacter jejuni require caveolae for cell invasion based on the finding that treatment of cells with the cholesterol-depleting compounds filipin III or methyl-β-cyclodextrin (MβCD) block bacterial internalization in a dose-dependent manner. The purpose of this study was to determine the role of caveolae and caveolin-1, a principal component of caveolae, in C. jejuni internalization. Consistent with previous work, we found that the treatment of HeLa cells with MβCD inhibited C. jejuni internalization. However, we also found that the treatment of HeLa cells with caveolin-1 siRNA, which resulted in greater than a 90% knockdown in caveolin-1 protein levels, had no effect on C. jejuni internalization. Based on this observation we performed a series of experiments that demonstrate that MβCD acts broadly, disrupting host cell lipid rafts and C. jejuni- induced cell signaling. More specifically, we found that MβCD inhibits the cellular events necessary for C. jejuni internalization, including membrane ruffling and Rac1 GTPase activation. We also demonstrate that MβCD disrupted the association of the β₁ integrin and EGF receptor, which are required for the maximal invasion of epithelial cells. In agreement with these findings, C. jejuni were able to invade human Caco-2 cells, which are devoid of caveolae, at a level equal to that of HeLa cells. Taken together, the results of our study demonstrate that C. jejuni internalization occurs in a caveolae-independent manner.     

Bactericidal effect of hydrolysable and condensed tannin extracts on <Emphasis Type="Italic">Campylobacter jejuni</Emphasis> in vitro

Strategies are sought to reduce intestinal colonisation of food-producing animals by Campylobacter jejuni, a leading bacterial cause of human foodborne illness worldwide. Presently, we tested the antimicrobial activity of hydrolysable-rich blackberry, cranberry and chestnut tannin extracts and condensed tannin-rich mimosa, quebracho and sorghum tannins (each at 100 mg/mL) against C. jejuni via disc diffusion assay in the presence of supplemental casamino acids. We found that when compared to non-tannin-treated controls, all tested tannins inhibited the growth of C. jejuni and that inhibition by the condensed tannin-rich mimosa and quebracho extracts was mitigated in nutrient-limited medium supplemented with casamino acids. When tested in broth culture, both chestnut and mimosa extracts inhibited growth of C. jejuni and this inhibition was much greater in nutrient-limited than in full-strength medium. Consistent with observations from the disc diffusion assay, the inhibitory activity of the condensed tannin-rich mimosa extracts but not the hydrolysable tannin-rich chestnut extracts was mitigated by casamino acid supplementation to the nutrient-limited medium, likely because the added amino acids saturated the binding potential of the condensed tannins. These results demonstrate the antimicrobial activity of various hydrolysable and condensed tannin-rich extracts against C. jejuni and reveal that condensed tannins may be less efficient than hydrolysable tannins in controlling C. jejuni in gut environments containing high concentrations of amino acids and soluble proteins.