Characterisation of cytolethal distending toxin (CDT) mutants of Campylobacter jejuni.

In order to assess the contribution of cytolethal distending toxin (CDT) to the toxigenicity and pathogenicity of Campylobacter jejuni, the C. jejuni 81-176 and C. jejuni NCTC 11168 CDTs were inactivated by insertional mutation of the cdtB toxin subunit. Cell-free sonicates from isogenic C. jejuni 81-176 cdtB- strains were found to be greatly attenuated in HeLa cytotoxicity assays, whilst still retaining some toxigenicity. Sonicates from a C. jejuni NCTC 11168 cdtB- strain produced no detectable cytotoxicity. When orally administered to adult severe combined immunodeficient (SCID) mice, C. jejuni cdtB mutant strains were unaffected in enteric colonisation abilities but demonstrated impaired invasiveness into blood, spleen and liver tissues. These data suggest that CDT may be the principal toxin produced by this species and that some C. jejuni strains may generate additional toxigenic factor(s) distinct from CDT.

Coaggregation between aquatic bacteria is mediated by specific-growth-phase-dependent lectin-saccharide interactions.

Coaggregating strains of aquatic bacteria were identified by partial 16S rRNA gene sequencing. The coaggregation abilities of four strains of Blastomonas natatoria and one strain of Micrococcus luteus varied with culture age but were always maximum in the stationary phase of growth. Each member of a coaggregating pair carried either a heat- and protease-sensitive protein (lectin) adhesin or a saccharide receptor, as coaggregation was reversed by sugars.

Extended survival and persistence of Campylobacter spp. in water and aquatic biofilms and their detection by immunofluorescent-antibody and -rRNA staining.

In water microcosm experiments, the survival times of Campylobacter isolates differed by up to twofold, as determined by culturing; this difference increased to fourfold when particular combinations of temperature and oxygenation were used. The mean survival times were much longer at 4 and 10 degrees C (202 and 176 h, respectively) than at 22 and 37 degrees C (43 and 22 h, respectively). The influence of anaerobiosis on survival time was less dramatic and differed considerably between isolates. In a two-stage water distribution model preparation containing a biofilm consisting of standardized autochthonous water microflora, Campylobacter isolates continued to differ in survival time. However, the survival times of cultures were considerably longer in the presence of the autochthonous water microflora (strains CH1 and 9752 survived 700 and 360 h, respectively, at 4 degrees C) than in the sterile microcosms (strains CH1 and 9752 survived 230 and 157 h, respectively). Although increased temperature and oxygenation were generally detrimental to culturability, the interaction of these two factors influenced the two strains examined differently. When the organisms were grown aerobically at 30 degrees C, the survival of the two strains was reversed; aerobiosis decreased the survival time of strain CH1 by 30%, but unexpectedly improved the persistence time of strain 9752 by more than threefold. Persistence times within biofilms were much longer when they were determined by detection methods not involving culturing. Immunofluorescent-antibody staining demonstrated that the pathogen persisted up to the termination of the experiments after 28 and 42 days of incubation at 30 and 4 degrees C, respectively. The specificity of detection within intact biofilms was reduced because of high background fluorescence. However, preliminary studies with a Campylobacter-specific rRNA probe revealed the same extended persistence of the pathogen within the biofilms.

Carbon load in aquatic ecosystems affects the diversity and biomass of water biofilm consortia and the persistence of the pathogen Campylobacter jejuni within them.

The influence of carbon load on autochthonous water microflora population distribution and diversity, and on the persistence of Campylobacter jejuni, was examined with a two-stage aquatic biofilm model. Serine was chosen since it is a carbon source utilised by C. jejuni and concentrations were chosen to reflect upper limits of amino acid load reported in surface water. The total viable count of the autochthonous biofilm microflora increased with increasing serine concentration (10-fold and 20-fold with 5 nM and 5 µM serine, respectively), as did the counts of the microflora in the planktonic phase. Differences in biofilm species distribution as determined by culture were small with changes in temperature or the addition of serine; but was markedly affected by serine as determined by light microscopy, becoming more luxuriant and dominated by long filamentous cells. The addition of serine to the water significantly and progressively reduced the persistence of C. jejuni, which decreased by 25% and 50% with serine concentrations of 5 nM and 5 µM respectively. We have demonstrated that carbon load affects the species diversity and density of both the planktonic and biofilm phase of aquatic autochthonous microflora. Although the survival of C. jejuni in water in a culturable form was sufficient for this to be an important vehicle for its transmission, carbon load significantly influenced survival; an increase in serine concentration significantly reduced survival.