Genomic variations leading to alterations in cell morphology of Campylobacter spp.

Campylobacter jejuni, the most common cause of bacterial diarrhoeal disease, is normally helical. However, it can also adopt straight rod, elongated helical and coccoid forms. Studying how helical morphology is generated, and how it switches between its different forms, is an important objective for understanding this pathogen. Here, we aimed to determine the genetic factors involved in generating the helical shape of Campylobacter. A C. jejuni transposon (Tn) mutant library was screened for non-helical mutants with inconsistent results. Whole genome sequence variation and morphological trends within this Tn library, and in various C. jejuni wild type strains, were compared and correlated to detect genomic elements associated with helical and rod morphologies. All rod-shaped C. jejuni Tn mutants and all rod-shaped laboratory, clinical and environmental C. jejuni and Campylobacter coli contained genetic changes within the pgp1 or pgp2 genes, which encode peptidoglycan modifying enzymes. We therefore confirm the importance of Pgp1 and Pgp2 in the maintenance of helical shape and extended this to a wide range of C. jejuni and C. coli isolates. Genome sequence analysis revealed variation in the sequence and length of homopolymeric tracts found within these genes, providing a potential mechanism of phase variation of cell shape.

Bacteriophage receptor binding protein based assays for the simultaneous detection of Campylobacter jejuni and Campylobacter coli.

Campylobacter jejuni and Campylobacter coli are the most common bacterial causes of foodborne gastroenteritis which is occasionally followed by a debilitating neuropathy known as Guillain-Barré syndrome. Rapid and specific detection of these pathogens is very important for effective control and quick treatment of infection. Most of the diagnostics available for these organisms are time consuming and require technical expertise with expensive instruments and reagents to perform. Bacteriophages bind to their host specifically through their receptor binding proteins (RBPs), which can be exploited for pathogen detection. We recently sequenced the genome of C. jejuni phage NCTC12673 and identified its putative host receptor binding protein, Gp047. In the current study, we localized the receptor binding domain to the C-terminal quarter of Gp047. CC-Gp047 could be produced recombinantly and was capable of agglutinating both C. jejuni and C. coli cells unlike the host range of the parent phage which is limited to a subset of C. jejuni isolates. The agglutination procedure could be performed within minutes on a glass slide at room temperature and was not hindered by the presence of buffers or nutrient media. This agglutination assay showed 100% specificity and the sensitivity was 95% for C. jejuni (n = 40) and 90% for C. coli (n = 19). CC-Gp047 was also expressed as a fusion with enhanced green fluorescent protein (EGFP). Chimeric EGFP_CC-Gp047 was able to specifically label C. jejuni and C. coli cells in mixed cultures allowing for the detection of these pathogens by fluorescent microscopy. This study describes a simple and rapid method for the detection of C. jejuni and C. coli using engineered phage RBPs and offers a promising new diagnostics platform for healthcare and surveillance laboratories.

Development of antimicrobial resistance in Campylobacter jejuni and Campylobacter coli adapted to biocides.

The potential for adaptive resistance of Campylobacter jejuni and Campylobacter coli after step-wise exposure to increasing sub-inhibitory concentrations of five biocides as triclosan, benzalkonium chloride, cetylpyridinium chloride, chlorhexidine diacetate and trisodium phosphate, was investigated, to identify the mechanisms underlying resistance. The biocide resistance and cross-resistance to the antimicrobials erythromycin and ciprofloxacin, and to sodium dodecyl sulphate, were examined according to the broth microdilution method. The presence of active efflux was studied on the basis of restored sensitivity in the presence of the efflux pump inhibitors phenylalanine-arginine beta-naphthylamide, 1-(1-naphthylmethyl)-piperazine, cyanide 3-chlorophenylhydrazone, verapamil and reserpine. Changes in the outer membrane protein profiles and morphological changes in adapted strains were studied, as compared with the parent strains. Repeated exposure of C. jejuni and C. coli to biocides resulted in partial increases in tolerance to biocides itself, to other biocides and antimicrobial compounds. The developed resistance was stable for up to 10 passages in biocide-free medium. More than one type of active efflux was identified in adapted strains. These adapted strains showed different alterations to their outer membrane protein profiles, along with morphological changes. The data presented here suggest that different mechanisms are involved in adaptation to biocides and that this adaptation is unique to each strain of Campylobacter and does not result from a single species-specific mechanism.

In vitro susceptibility to antimicrobial agents and ultrastructural characteristics related to swimming motility and drug action in Campylobacter jejuni and C. coli.

Campylobacter jejuni has recently been noted as the most common cause of bacterial food-borne diseases in Japan. In this study, we examined in vitro susceptibility to 36 antimicrobial agents of 109 strains of C. jejuni and C. coli isolated from chickens and patients with enteritis or Guillain-Barré syndrome from 1996 to 2009. Among these agents, carbapenems (imipenem, meropenem, panipenem, and biapenem) showed the greatest activity [minimal inhibitory concentration (MIC)(90), 0.03-0.125 microg/ml]. This was followed by sitafloxacin (MIC(90), 0.25 microg/ml), furazolidone and azithromycin (MIC(90), 0.5 microg/ml), gentamicin and clindamycin (MIC(90), 1 microg/ml), and clavulanic acid (beta-lactamase inhibitor; MIC(90), 2 microg/ml). All or most strains were resistant to aztreonam, sulfamethoxazole, and trimethoprim. Marked resistance was also observed for levofloxacin and tetracyclines. Resistance was not present for macrolides and rare for clindamycin. C. jejuni (and C. coli) exhibited high swimming motility and possessed a unique end-side (cup-like) structure at both ends, in contrast to Helicobacter pylori and Vibrio cholerae O1 and O139. The morphology of C. jejuni (and C. coli) changed drastically after exposure to imipenem (coccoid formation), meropenem (bulking and slight elongation), and sitafloxacin (marked elongation), and exhibited reduced motility. In the HEp-2 cell adherence model, unusually elongated bacteria were also observed for sitafloxacin. The data suggest that although resistance to antimicrobial agents (e.g., levofloxacin) has continuously been noted, carbapenems, sitafloxacin, and others such as beta-lactamase inhibitors alone showed good in vitro activity and that C. jejuni (and C. coli) demonstrated a unique ultrastructural nature related to high swimming motility and drug action.

Phenotypic characterisation of flagellin and flagella of urease-positive thermophilic campylobacters.

In this study, flagellin is purified biochemically from eight urease-positive thermophilic camplylobacters (UPTC) isolated from river water, sea water and mussels, and purified also from two isolates of Campylobacter jejuni and C. coli and fractionated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Results showed that no flagellin components were detected in the two Japanese UPTC isolates (CF89-12 and CF89-14) and the two UPTC NCTC strains (NCTC12893 and NCTC12894). Flagellin components, each consisting of a single peptide, with a heterogeneous molecular mass of approximately 52-63 kDa were demonstrated in the other four UPTC isolates (NCTC12892, NCTC12895, NCTC12896 and NI15F [from Northern Ireland]) and the two Japanese isolates of C. jejuni (JCM2013 and C. coli 27). The approximate molecular mass of flagellin from the flagellin-positive UPTC isolates was smaller than those of C. jejuni and C. coli. Flagella were not detected by electron microscopy in the four flagellin-negative UPTC isolates but they were detected in the four flagellin-positive UPTC isolates and the two isolates of C. jejuni and C. coli. Thus, significant phenotypic diversity for flagellin, which must be due to genotypic variations, was demonstrated among the UPTC isolates.

Pseudaminic acid, the major modification on Campylobacter flagellin, is synthesized via the Cj1293 gene.

Flagellins from Campylobacter jejuni 81-176 and Campylobacter coli VC167 are heavily glycosylated. The major modifications on both flagellins are pseudaminic acid (Pse5Ac7Ac), a nine carbon sugar that is similar to sialic acid, and an acetamidino-substituted analogue of pseudaminic acid (PseAm). Previous data have indicated that PseAm is synthesized via Pse5Ac7Ac in C. jejuni 81-176, but that the two sugars are synthesized using independent pathways in C. coli VC167. The Cj1293 gene of C. jejuni encodes a putative UDP-GlcNAc C6-dehydratase/C4-reductase that is similar to a protein required for glycosylation of Caulobacter crescentus flagellin. The Cj1293 gene is expressed either under the control of a sigma 54 promoter that overlaps the coding region of Cj1292 or as a polycistronic message under the control of a sigma 70 promoter upstream of Cj1292. A mutant in gene Cj1293 in C. jejuni 81-176 was non-motile and non-flagellated and accumulated unglycosylated flagellin intracellularly. This mutant was complemented in trans with the homologous C. jejuni gene, as well as the Helicobacter pylori homologue, HP0840, which has been shown to encode a protein with UDP-GlcNAc C6-dehydratase/C4-reductase activity. Mutation of Cj1293 in C. coli VC167 resulted in a fully motile strain that synthesized a flagella filament composed of flagellin in which Pse5Ac7Ac was replaced by PseAm. The filament from the C. coli Cj1293 mutant displayed increased solubility in SDS compared with the wild-type filament. A double mutant in C. coli VC167, defective in both Cj1293 and ptmD, encoding part of the independent PseAm pathway, was also non-motile and non-flagellated and accumulated unglycosylated flagellin intracellularly. Collectively, the data indicate that Cj1293 is essential for Pse5Ac7Ac biosynthesis from UDP-GlcNAc, and that glycosylation is required for flagella biogenesis in campylobacters.

Double-staining method for differentiation of morphological changes and membrane integrity of Campylobacter coli cells.

We developed a double-staining procedure involving NanoOrange dye (Molecular Probes, Eugene, Oreg.) and membrane integrity stains (LIVE/DEAD BacLight kit; Molecular Probes) to show the morphological and membrane integrity changes of Campylobacter coli cells during growth. The conversion from a spiral to a coccoid morphology via intermediary forms and the membrane integrity changes of the C. coli cells can be detected with the double-staining procedure. Our data indicate that young or actively growing cells are mainly spiral shaped (green-stained cells), but older cells undergo a degenerative change to coccoid forms (red-stained cells). Club-shaped transition cell forms were observed with NanoOrange stain. Chlorinated drinking water affected the viability but not the morphology of C. coli cells.

In vitro study on the effect of organic acids on Campylobacter jejuni/coli populations in mixtures of water and feed.

Gastroenteritis caused by Campylobacter spp. infection has been recognized as one of the important public health problems in the developed countries. Outbreaks mostly originate from the consumption of contaminated poultry or infected water. The aim of this study was to determine the bactericidal activity on Campylobacter spp. of organic acids individually and in combinations at different pH levels and times and to compare bactericidal activities with activities of commercially available products. Ten strains of Campylobacter spp. were added in a mixture of water with commercial broiler feed, separately adjusted by four acids: formic, acetic, propionic, and hydrochloric acids, into pH 4.0, 4.5, 5.0, and 5.5. A combination of three organic acids was used in two different formulation ratios: formic:acetic:propionic at 1:2:3 and 1:2:5, at pH 4.0, 4.5, 5.0, and 5.5. All organic acids showed the strongest bactericidal effect on Campylobacter at pH 4.0. In contrast, at pH 5.0 and 5.5, the bactericidal activity of the four acids was low. The combination of organic acids showed a synergistic bactericidal activity at pH 4.5. Interestingly, the effect of the combined organic acids was stronger than the commercial products. Morphological cell changes were studied by transmission electron microscopy to determine the effect of the organic acids on the cell structure of Campylobacter. Some loss of outer membranes of the bacteria could be found in treated groups. Therefore, it can be concluded that organic acids, individually or in combination, have a strong bactericidal effect on Campylobacter spp. Routine application of organic acids to the water supply on poultry farms could prevent or diminish Campylobacter transmission.

Effect of low temperatures on growth, structure, and metabolism of Campylobacter coli SP10.

The effect of low temperatures on the survival, structure, and metabolism of Campylobacter coli SP10, a virulent strain, was investigated. C. coli became nonculturable rapidly at 20 and 10 degree C and slightly later at 4 degrees C. Incubation in a microaerobic atmosphere improved survival, but after day 8, campylobacters were detectable by direct-count procedures only. The increase in the number of coccoid cells was most pronounced at 37 degrees C but also was noticeable at 20 and 10 degrees C. Two forms of coccoid cells were seen electron microscopically, but only one (20 and 10 degrees C) seemed to be a degenerative form. The flagella were shorter at 20 and 10 degrees C, a result which correlates well with the observed slight changes in the 62-kDa protein band. The fatty acid composition of bacterial cells was influenced significantly by low temperatures. An increase in the short-chain and unsaturated acids was noted; above all, a drastic increase in C19:0 cyc at 20 degrees C with a concomitant decrease in C18:1 trans9,cis11 was seen. The concentrations of excreted metabolites were analyzed to obtain information on metabolic activity. Depending on the magnitude of the temperature downshift, the production of organic acids decreased, but it was always observable after a temperature-specific lag phase and regardless of ability to be cultured. Under optimal conditions, succinate, lactate, and acetate were the main metabolites, other acids being of less importance. The pattern changed significantly at lower temperatures. Succinate was never detected at 20 degrees C and was only occasionally detected at 10 and 4 degrees C. At the same time, fumarate concentrations, which are normally not detectable at 37 degrees C, were highest at 20 degrees C and reduced at 10 and 4 degrees C. Inactivation of fumarate reductase was considered to be a possible explanation.

Interaction of Helicobacter pylori (strain 151) and Campylobacter coli with human peripheral polymorphonuclear granulocytes.

Helicobacter pylori associated gastritis is characterized by dense mucosal inflammatory infiltrations with predominantly neutrophilic granulocytes, together with a local and systemic immune response. Nevertheless, the natural course of the infection is chronic in nature, and active phagocytosis of H. pylori by mucosal granulocytes was only rarely observed. The aim of the present study was to investigate with electronmicroscopic methods the interaction of H. pylori with freshly harvested human peripheral granulocytes, with Campylobacter coli as control organism. Bacteria, either untreated or opsonized with complement or antiserum, were coincubated with phagocytes for up to 120 min. After defined time periods the following parameters were electronmicroscopically evaluated: i) internalization of bacteria., ii) morphological characteristics of bacteria and phagocytes, iii) decrease of lysosomes, and iv) by use of myeloperoxidase staining, the characteristics of phagolysosomal fusion. In the absence of complement, both organisms were internalized to a comparable extent. However, in contrast to C. coli, remarkable amounts of H. pylori cells remained extracellularly attached even after 120 min of coincubation, as well as internalized bacteria remained morphologically largely unimpaired. If complement was present, internalization and morphological destruction of H. pylori cells were significantly enhanced. The latter was characterized by rounding and swelling of H. pylori cells. It was already apparent in the extracellular space, and therefore probably induced by a complement effect, rather than by tee phagocytic action. Decrease of lysosomes, in general paralleled the degree of microbial uptake. Myeloperoxidase staining experiments furthermore showed an obviously regular consumption of lysosomal granules. However, if complement opsonization was excluded, lysosomal degranulation was not accompanied by a corresponding degradation of H. pylori cells, the latter indicating an at least partial resistance to phagocyte caused microbicidal mechanisms. In most of those cases ingested H. pylori cells were, in contrast to C. coli, surrounded by a rather "tight" phagosome. A possible explanation for this phenomenon could be a "leakage" of the phagosomal membrane, possibly caused by membranotoxic ammonia produced by the organism. If such an impairment of the phagocytic action would occur in vivo, it could lead to an impaired cellular defense, and therefore contribute to the chronic course of H. pylori infections.

Characterization of viable but nonculturable stage of C. coli, characterized with respect to electron microscopic findings, whole cell protein and lipooligosaccharide (LOS) patterns.

Campylobacter coli CK 205, isolated from swine feces, was examined for changes in cell morphology, protein and lipooligosaccharide (LOS) patterns during starvation-survival experiments. Bottles filled with sterile filtered A. dest. were seeded with campylobacters and incubated at 4 degrees C and 37 degrees C. Transition to the nonculturable stage occurred within 48 hours (37 degrees C) and 2 weeks (4 degrees C), respectively. In contrast to the culturability the electrophoretic studies showed no changes in whole cell protein or LOS patterns. The electron microscopic pictures revealed spiral and coccoid forms, partly with a slightly enlarged periplasmatic space or budding of the membrane. Totally intact non culturable spiral or coccoid forms might be regarded as dormancy forms that cannot be detected by conventional microbiological methods in water examination.

Variation in antigenicity and molecular weight of Campylobacter coli VC167 flagellin in different genetic backgrounds.

Campylobacter coli VC167 has been shown to undergo a reversible flagellar antigenic variation between antigenic type 1 (T1) and antigenic type 2 (T2). VC167 contains two flagellin genes, and the products of both genes are incorporated into a complex flagellar filament in both antigenic types. Although there are only minor amino acid changes in the flagellins expressed by T1 and T2 cells, the two antigenic types of flagellins can be distinguished by differences in apparent M(r) on sodium dodecyl sulfate-polyacrylamide gels and by immunoreactivity with T1-specific (LAH1) or T2-specific (LAH2) antiserum. The isolation of stable variants of T1 and T2 has allowed for the transfer via natural transformation of the flagellin structural genes from the T1 background into the T2 background and from the T2 background into the T1 background. In addition, the flagellin genes from VC167 T1 and T2 have been transferred into strains of Campylobacter jejuni. The results indicate that the observed antigenic variations of VC167 flagellins are dependent on the host genetic background and independent of the primary amino acid sequence. These data provide evidence that posttranslational modifications are responsible for the antigenic variation seen in VC167 flagellins.

Analysis of the role of flagella in the heat-labile Lior serotyping scheme of thermophilic Campylobacters by mutant allele exchange.

Flagellin mutations originally constructed in Campylobacter coli VC167 (serotype LIO8) by a gene replacement mutagenesis technique (P. Guerry, S. M. Logan, S. Thornton, and T. J. Trust, J. Bacteriol. 172:1853-1860, 1990) were moved from the original host into Campylobacter strains of a number of other Lior serogroups by a natural transformation procedure. This is the first report of the use of this transformation method to transfer a mutated locus among Campylobacter strains. Flagellin mutants were constructed in a number of heat-labile LIO serotypes and were serotyped and analyzed by immunoelectron microscopy with LIO typing antisera. In six cases, isogenic nonflagellated mutants were able to be serotyped in the same serogroup as their parent, and immunogold electron microscopy confirmed that antibodies in the typing antisera bound to components on the surface of both parent and mutant cells. However, in only one case, a strain belonging to serogroup LIO4, was a nonflagellated mutant untypeable, and immunogold electron microscopy showed that antibodies bound to the flagella filament of the parent but not to the cell surface. Furthermore, after introduction and expression as a flagellar filament of a LIO8 flagellin gene in this mutant, the strain could not be serotyped. These results indicate that a nonflagellar antigen is often the serodeterminant in the heat-labile Lior serotyping scheme.

[Morphological changes of Campylobacter jejuni and Campylobacter coli under various culture conditions and the accompanied changes in the cell composition].

The morphological transformation from the spiral form to the coccoidal form Campylobacter jejuni and Campylobacter coli was studied under various conditions by such techniques as electron microscopy, SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and chemical analyses. The conversion from the spiral form to the coccoidal form of Campylobacter in microaerophilic cultivation occurred in about 50% of the cells in 48 h and in about 90% of the cells in 72 h. At higher temperatures (37 C and 42 C) under aerophilic conditions, the spiral-form cells converted easily to the coccoidal form in phosphate-buffered saline. Electron-microscopic studies revealed that the envelopes of the coccoidal-form cells were soft in comparison with those of the spiral-form cells. The LPS and protein contents of the cells reached the highest levels after cultivation for 48 h under microaerophilic condition. The 33 K and 28 K polypeptide contents of 24-h and 48-h cultures were higher than those of 72-h and 96-h cultures.