Stress response and pathogenic potential of Campylobacter jejuni cells exposed to starvation

Campylobacter jejuni is a Gram-negative, fragile, spiral bacterium, known worldwide to be a major cause of acute human enteritis. Like many other food-borne bacteria, campylobacters must be able to survive under diverse conditions both inside the host and in the environment. Understanding stress response mechanisms provides information necessary for improving food processing and strategies that enhance food safety as well as clarifying the pathogenesis of campylobacteriosis. We investigated the relation between stress response to starvation and pathogenic potential in C. jejuni. Starvation changed the morphology and physiology of C. jejuni cells. However, the lower metabolic activity of 5-h-starved culture was not a dormant state, but probably a viable but non-culturable (VBNC) form of the cells, since starved C. jejuni induced heat stress resistance. The health hazard potential of starved cells is still unclear. We showed that, in spite of starvation, C. jejuni survived in vitro within Caco-2 enterocites up to 4 days and caused systemic campylobacteriosis in vivo in a mouse model. However, bacterial numbers in investigated organs were significantly lower and the infection was resolved sooner. Our results show that nutrient insufficiency is responsible for C. jejuni transformation, influencing but not abolishing its survival and virulence properties while in the VBNC state.     

Cloning of a Candida utilis gene which complements leu2 mutation in Saccharomyces cerevisiae

Summary DNA fragments containing the LEU2 gene of Candida utilis have been isolated, utilizing the genome library (constructed in YRp12) of this organism. Two recombinant plasmids pZR84 and pZR32, containing the cloned LEU2 gene, were 4.24 kb and 10.4 kb, respectively, and were shown to complement leu2 mutation in Saccharomyces cerevisiae and leuB mutation in Escherichia coli. The cloned fragment in pZR84 contained one restriction site each for EcoRI and PvuII, and two for HindIII, but none for SalI, BamHI or Pstl. This cloned fragment hybridized with the total DNA from C. utilis and from Leu⁺ transformants of S. cerevisiae, but not with that from untransformed S. cerevisiae. Subcloning analyses showed that a 2.34 kb BamHI HindIII fragment of the cloned C. utilis sequence contains the region essential for the expression of the LEU2 gene.Journal Article No. 11669 from the Michigan Agricultural Experiment Station     

A putative adhesin gene cloned from Campylobacter jejuni

Thirteen Campylobacter jejuni strains of human origin showed differing behaviours when analysed for their ability to bind the Caco-2 cell line in vitro, suggesting variations in genetic complements and/or regulation. We designed an oligonucleotide probe corresponding to a highly conserved part of adhesins from various Gram-negative bacteria. Among our laboratory collection, Southern hybridization has demonstrated that only a discrete number of strains harbour this sequence. The corresponding gene has been cloned from our prototype strain and sequence analysis has confirmed homology with Gram-negative bacterial adhesins. The ORF corresponded to 869 amino acids; we named this protein P95. Protein sequence similarity assessment demonstrated that this gene product belongs to the family of proteins including the filamentous haemagglutinin of Bordetella pertussis and the high-molecular-weight surface-exposed adhesins of Haemophilus influenzae. Comparison of adhesion and hybridization results emphasized the involvement of this gene in an essential pathogenic process of Campylobacter.     

Real-time multiplex PCR for simultaneous detection of Campylobacter jejuni, Salmonella, Shigella and Yersinia species in fecal samples

Diarrheal diseases due to notifiable bacterial infections require rapid diagnosis of the causative pathogens. To facilitate detection, a real-time multiplex PCR was developed that identifies common diarrhea-causing bacteria in fecal samples. On the basis of published sequence data, sets of primers and probes were designed that were specific for Campylobacter jejuni, Salmonella, Shigella/enteroinvasive Escherichia coli EIEC, and Yersinia species, suitable for use in a one-tube PCR assay. The assay was assessed using a list of 137 well-defined intestinal bacterial strains or isolates. Furthermore, 393 routine clinical stool samples were analyzed, and the results of real-time multiplex PCR were compared with those obtained by established microbiological methods. The PCR yielded results within 3 h including DNA purification. No false-positive signals or cross-reactions were observed. The analytical sensitivity was 10³ cfu mL⁻¹ for Campylobacter jejuni, 10⁴ cfu mL⁻¹ for Salmonella, and 10⁵ cfu mL⁻¹ for Shigella/EIEC and Yersinia, respectively. Compared with culture, PCR detected 79 out of 81 Campylobacter jejuni (97.5%), 71 out of 74 Salmonella (96%), 8 out of 8 Shigella (100%), and 10 out of 10 Yersinia-positive (100%) clinical samples. In culture-negative samples (n = 192), PCR additionally detected 2 Shigella, 1 Salmonella, and 5 Campylobacter jejuni infections. Thus, the new real-time multiplex PCR provides reliable results within a short time and might be useful as an additional diagnostic tool whenever time is important in the diagnosis of enteropathogenic bacteria.     

Comparative analysis of Campylobacter lari cytolethal distending toxin (CDT) effect on HeLa cells

We aimed to clarify if Campylobacter lari exerts a cytolethal distending toxin (CDT) effect on HeLa cells. Campylobacter cell lysates (CCLys) from C. jejuni 81–176 and urease‐positive thermophilic Campylobacter (UPTC) CF89‐12 and UPTC NCTC12893 isolates were shown to exert a CDT effect on HeLa cells with morphological changes examined by Giemsa staining and microscopy. However, Campylobacter lari JCM2530^T isolate showed no effect. In addition, Campylobacter cell culture supernatant wash gave low or absent toxic effects with both C. jejuni and C. lari organisms. When western blot analysis was carried out to clarify if there was a CDTB effect in the CCLys and soluble fractions from Campylobacter isolates, which had a CDT effect on HeLa cells or did not have any effect, anti‐recombinant CjCDTB antibodies identified an immunoreactively positive signal at around approximately 25 kDa on all the C. lari isolates examined, as well as the C. jejuni 81116 strain. Thus, all the Campylobacter isolates including those without any CDT effect were shown to express CDTB at the translational level. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Serological assessment of synthetic peptides of <Emphasis Type="Italic">Campylobacter jejuni</Emphasis> NCTC11168 FlaA protein using antibodies against multiple serotypes

The flagellum of Campylobacter jejuni is not only responsible for initiating colonization of the gastrointestinal tract of host animals but is also a major antigen that induces protective immune responses. However, protection is limited to the homologous strain and the ability to protect against multiple serotypes has yet to be determined. In this study, we have shown that FlaA is an immmunodominant protein on NCTC11168 CJ1 flagella and we mapped the immunoreactive epitopes on the protein by probing a series of overlapping synthetic peptides spanning the entire sequence with sera against multiple C. jejuni serotypes. Amino acid residues 176–205 (P8), 376–405 (P16) and 501–530 (P21) were immunodominant and cross-reactive. The mucosal IgA in the intestinal secretions of CJ1-infected birds reacted significantly with peptides P16 and P21 indicating that the specificity of the mucosal response is different from the systemic response. Antisera raised against formalin-killed CJ1 cells and purified flagellin showed positive reactivity with a subset of peptides identified by antisera against live C. jejuni. This study provides insight into the specificity of the host immune responses to the FlaA protein of C. jejuni and suggests that these sequences merit further testing for their immunogenicity and potential as subunit vaccine candidates for multiple serotypes.     

The role of WlaRG, WlaTB and WlaTC in lipooligosaccharide synthesis by Campylobacter jejuni strain 81116

Campylobacter jejuni is a major bacterial cause of gastroenteritis world-wide. C. jejuni produces a range of glycans including lipooligosaccharide (LOS), an important virulence factor. The genetic content of the LOS synthesis locus varies between C. jejuni strains and 19 classes have been described. Three LOS synthesis genes of C. jejuni strain 81116 (NCTC 11828), wlaRG, wlaTB and wlaTC were the focus of this study. WlaRG and the remaining two proteins of interest share sequence similarity to aminotransferases and glycosyltransferases, respectively. These genes were insertionally inactivated and phenotypically characterised. Each mutant produced truncated LOS. Mutants lacking WlaRG, WlaTB and WlaTC produced LOS with reduced immunogenicity. Both the wlaRG and wlaTC mutants were non-motile and aflagellate. In vitro invasion and adhesion assays revealed that the wlaRG, wlaTB and wlaTC mutants displayed reduced adherence to chicken embryo fibroblasts. All mutants were less invasive of human cells than 81116 confirming the role of intact LOS during invasion of human cells in vitro. Here we propose the general composition for the 81116 LOS core backbone based on capillary electrophoresis-mass spectrometry.     

Discrimination from other thermophilic Campylobacter and genometyping at the chromosomal genomic DNA level of Campylobacter lari

Since the respective undigested and intact chromosomal DNAs from C. coli JCM2529^T, from two strains of C. coli, C. jejuni JCM2013 and from two strains of C. jejuni migrated as fragments of around 1,900 kb and the chromosomal DNAs from C. lari JCM2530^T and from eleven strains of C. lari migrated as fragments of around 1,640 kb, pulsed-field gel electrophoresis (PFGE) was clearly demonstrated to be useful for the discrimination of C. lari from two other thermophilic species of Campylobacter (C. coli and C. jejuni). PFGE revealed that SmaI generated nine distinctly different and distinguishable banding profiles from the DNA of 18 strains of C. lari, which included the type strain. Three types of banding profile, namely, A, B and C, exemplified by the nine profiles included those from five, four and three strains of C. lari, respectively. Our present attempt to discriminate and to genome-type C. lari at the chromosomal genomic level using PFGE and SmaI may be a valuable step towards a discriminative and molecular-epidemiologic study of C. lari.     

Campylobacter spp among Children with acute diarrhea attending Mulago hospital in Kampala - Uganda

Background

Campylobacter infections occur worldwide. A recent study in Kampala, Uganda, found that 87% of broiler chickens had Campylobacter jejuni; these are potential source of human infection. Isolation rate in developing countries is between 5–35%. This study aimed at finding prevalence of children with campylobacter infection among children with acute diarrhea attending Mulago hospital.

Objective

The objective was to establish the proportion of children infected with Campylobacter spp among children with acute diarrhea at Mulago hospital.

Methods

A crossectional study from July to October 2005 was conducted involved 226 children with acute diarrhea. Serial sampling was done a total of 226 stool specimens were obtained and cultured on selective media. Identification was done using biochemical test and susceptibility using standard discs diffusion method.

Results

Campylobacter spp were isolated in 21 (9.3%) of 226 stool specimens analyzed. Campylobacter jejuni 17 (80.9%), Campylobacter lari 2 (9.5%), Campylobacter coli 1 (4.5%) and Campylobacter jejuni/coli 1(4.5%). All Campylobacter isolates were sensitive to erythromycin, and 20% had intermediate resistance to Ampicillin.

Conclusion

Campylobacter spp are prevalent among children with acute diarrhea in Kampala- Uganda. A large multicenter study should be undertaken so that the extent of campylobacter infection in our setting can be established.     

Use of a selective medium and a membrane filter method for isolation ofCampylobacter species from Spanish paediatric patients

A study was conducted to assess the value of a combination of two culture methods for isolation ofCampylobacter spp. from Spanish children. Seven hundred twenty-nine diarrhoea] stool specimens from 599 patients were examined forCampylobacter spp. by culturing them on charcoal cefoperazone deoxycholate agar and on blood agar with a membrane filter. One hundred sixteenCampylobacter strains were isolated from a total of 108 specimens; 75 (64.6%) wereCampylobacter jejuni, 32 (27.5%) wereCampylobacter coli, 8 (6.8%) were non-typeable, and one (0.9%) wasCampylobacter upsaliensis. Campylobacters were isolated from 99 positive samples using charcoal cefoperazone deoxycholate agar alone. The filtration technique alone yielded only 86 positive samples. Seven specimens yielded differentCampylobacter spp. with different media. The only catalase-negative strain was recovered using the filter method. The combination of the selective medium with the filter method increased the isolation rate ofCampylobacter strains by 14.1%. Isolation rates of campylobacters using the filter method were similar to those reported in European studies, in which a similar frequency ofCampylobacter upsaliensis was observed. The addition of a filter method for routine laboratory isolation of campylobacters should be considered in selected age groups (in children <10 years of age) or in areas where catalase-negative or weakly-positiveCampylobacter strains may be of epidemiological significance.     

Campylobacter jejuni: A brief overview on pathogenicity-associated factors and disease-mediating mechanisms

Campylobacter jejuni has long been recognized as a cause of bacterial food-borne illness, and surprisingly, it remains the most prevalent bacterial food-borne pathogen in the industrial world to date. Natural reservoirs for this Gram-negative, spiral-shaped bacterium are wild birds, whose intestines offer a suitable biological niche for the survival and dissemination of C. jejuni Chickens become colonized shortly after birth and are the most important source for human infection. In the last decade, effective intervention strategies to limit infections caused by this elusive pathogen were hindered mainly because of a paucity in understanding the virulence mechanisms of C. jejuni and in part, unavailability of an adequate animal model for the disease. However, recent developments in deciphering molecular mechanisms of virulence of C. jejuni made it clear that C. jejuni is a unique pathogen, being able to execute N-linked glycosylation of more than 30 proteins related to colonization, adherence, and invasion. Moreover, the flagellum is not only depicted to facilitate motility but as well secretion of Campylobacter invasive antigens (Cia). The only toxin of C. jejuni, the so-called cytolethal distending toxin (CdtA,B,C), seems to be important for cell cycle control and induction of host cell apoptosis and has been recognized as a major pathogenicity-associated factor. In contrast to other diarrhoea-causing bacteria, no other classical virulence factors have yet been identified in C. jejuni. Instead, host factors seem to play a major role for pathogenesis of campylobacteriosis of man. Indeed, several lines of evidence suggest exploitation of different adaptation strategies by this pathogen depending on its requirement, whether to establish itself in the natural avian reservoir or during the course of human infection.     

Investigation of infection with Campylobacter jejuni in a man with hypogammaglobulinaemia using PCR-single-stranded conformational polymorphism (PCR-SSCP) typing

This study investigated several episodes of infection of Campylobacter jejuni in an immunocompromised male with hypogammaglobulinaemia, presenting with diarrhoea and bacteraemia over a 16-month period, by employing three phenotyping and four genotyping schemes, including the single-stranded conformational polymorphism (SSCP) technique to establish if infection was reinfection or persistent infection. Four isolates from blood culture and two faecal isolates of Campylobacter jejuni were obtained from the patient by direct selective plating on Skirrow Selective agar. Isolates were characterised at the sub-species level by Penner serology, Preston biotyping, Preston phage-typing, as well as E3CJC2 restriction fragment length polymorphism (RFLP), 16S ribotyping, flagellin (flaA) RFLP and single-stranded conformational polymorphism (SSCP) analyses. Phenotyping and genotyping sub-species analyses demonstrated that the patient was infected with at least two different strains of Campylobacter jejuni, i. e. one strain that persisted throughout the 16-month period and another strain that was transient suggesting reinfection from a different source. SSCP analysis was the most discriminatory of all the typing schemes examined and demonstrated an altered genotype of the persistent strain, whereby there were subtle modifications to the hypervariable regions of the flaA gene. Overall, as SSCP examines the hypervariable region of the flaA gene and as this technique can detect point mutations, differences between SSCP banding patterns may represent markers and thus examine mutations that occur under immune selection, thereby permitting the C. jejuni to evade the host immune response. In conclusion, this study describes the novel use of SSCP genotyping of C. jejuni and demonstrated that this method is a highly discriminatory technique which may be beneficial in outbreak characterisation, but which is not suitable to examine the clonal patterns of C. jejuni over a long period of time.     

Homogeneity and diversity of genome polymorphism in a set of herpes simplex virus type 1 strains classified as the same genotypic group

Summary Restriction fragment length polymorphism (RFLP) of 327 strains of herpes simplex virus type 1 (HSV-1) isolated in Japan were analyzed using six restriction endonucleases (REs) recognizing 6 base pairs (BamHI,HindIII,KpnI,PvuII,SalI,XhoI). The presence of strains with the same RFLP profile became evident. Fifteen strains of each of the two predominant sets with the same RFLP profile were further analyzed using two different methods, that is analyses of RFLP using 3 restriction endonucleases recognizing 4 base pairs (4-bp REs) (HaeIII,HhaI,MboI) and analyses of the variation of 3 reiterated sequences (reiterations I, IV, VII). Most of the epidemiologically unrelated strains could be differentiated by variation of the reiterations. RFLPs differentiating the strains were detected with the 4-bp REs, and epidemiologically related strains shared a specific RFLP, thereby confirming the relationship. These results verified the utility of the two methods for use in molecular epidemiological studies. Homogeneity of RFLP among the strains suggested derivation from a common ancestor (making up a genotypic group) while diversity in strains of the same genotypic group was indicative of a separate replication.     

Binding of outer membrane preparations of Campylobacter jejuni to INT 457 cell membranes and extracellular matrix proteins

Binding of outer membrane (OM) preparations of the thermophilic Campylobacter species C. jejuni to epithelial cell membranes and extracellular matrix proteins were studied in an in vitro model system using enzyme-linked immunosorbent assay. The OM preparations exhibited significant binding to INT 407 intestinal cell membranes. The process of adhesion was modulated by enzymatic, chemical or immunological pretreatment of the bacteria. Following oxidation of the lipopolysaccharide (LPS) with sodium meta-periodate, the OM preparations essentially retained their binding properties. After pretreatment with proteinase K, the OM preparations lost their binding capacity and the apparent molecular mass of the major OM protein shifted from 42 to 24 kDa. Preincubation of C. jejuni bacteria with C. jejuni-specific antiserum reduced adhesion significantly; preincubation with LPS-specific monoclonal antibodies only to a minimal extent. The OM preparations also bound significantly to the extracellular matrix proteins collagen and fibronectin; however, they bound virtually no bovine serum albumin or horse serum.     

Construction of new yeast vectors and cloning of the nif (nitrogen fixation) gene cluster of Klebsiella pneumoniae in yeast

Summary Two vectors, termed pG63.11 (7.6 Kb) and pHCG3 (9.6 Kb), suitable for yeast transformation have been constructed. The pHCG3 vector has cosmid properties. Both vectors contain a single 3.3 Kb EcoRI-HindIII fragment of yeast origin which carries the yeast URA3 gene (1.1 Kb) and the origin of replication of the 2 µm plasmid (2.2 Kb). They confer ampicillin resistance and they contain 5 unique EcoRI,HpaI,HindIII,BamHI and SalI restriction sites. Cosmid pHCG3 was used to clone the nitrogen fixation (nif) gene cluster of Klebsiella pneumoniae carried by twoHindIII fragments of 17 and 26 Kb, respectively. The resulting cosmid, termed pGPC875 (53 Kb) which conferred a Nif⁺ phenotype to Escherichia coli, was introduced in yeast by transformation. No acetylene reduction activity was detectable in the transformants. However it was shown that the entire information for nitrogen fixation can be replicated and maintained intact in yeast for more than 50 generations of growth.     

Restriction site and genetic map of Cucurbita pepo chloroplast DNA

Summary A detailed restriction map of squash chloroplast DNA (cpDNA) was constructed with five restriction endonuclease, SalI, PvuII, BglI, SacII, and PstI. The cleavage sites were mapped by sequential digestion of cpDNA using low-gelling temperature agarose. The restriction map shows that squash cpDNA is an approximately 153 kilobase (kb) circle with a large inverted repeat sequence of 23.3 kb, separated by a large (83.7 kb) and a small (22.7 kb) single copy region. Genes for a number of chloroplast polypeptides were localized on the map by hybridizing the cpDNA restriction fragments to heterologous gene-specific probes from tobacco, pea, tomato, maize, and spinach chloroplasts. The gene locations and organization of squash cpDNA are highly conserved and similar to chloroplast genomes of tomato, pepper, and Ginkgo.Abbreviations cpDNA chloroplast DNA - kb kilobases - IR inverted repeat. Gene names follow the nomenclature recommendation of Hallick and Bottomley (1983)     

Rapid identification by PCR of the genus Campylobacter and of five Campylobacter species enteropathogenic for man and animals

The nucleotide sequences for the 16S ribosomal RNA gene were compared for 33 species comprising the ε subdivision of the Proteobacteria (genera: Campylobacter, Helicobacter, Arcobacter and Wolinella). Regions specific for the genus Campylobacter and for five Campylobacter species important in human and/or veterinary medicine were identified. From these regions, PCR primer pairs were designed for use in species-specific identification. Primer pairs were validated against strains representing all taxa of campylobacter-like organisms. They did not amplify products other than from their five target species (C. upsaliensis, C. helveticus, C. fetus, C. hyointestinalis and C. lari), and they generated amplicons of defined size from large numbers of strains of those species. A primer pair suitable for identification of the genus Campylobacter was also identified and validated. This generated amplicons from all species of Campylobacter as well as from unnamed groups known to be within the genus, but not from any species or unnamed strains of Helicobacter, Arcobacter or Wolinella.