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.

Aerosol route enhances the contamination of intact eggs and muscle of experimentally infected laying hens by Salmonella typhimurium DT104.

Commercial laying hens were infected with Salmonella typhimurium DT104 strain 16 alternatively via the crop (10(7) cfu per bird) or by an aerosol delivered directly to the beaks using a Collison nebuliser and Henderson apparatus (2 x 10(2) or 2 x 10(4) cfu per bird). Infection by both routes caused systemic infection and prolonged contamination of faeces. Contamination rates of eggs and muscle were much higher following the aerosol challenges despite the much lower doses given by this route. The frequency of Salmonella isolation from eggs rose from 1.7% following oral challenge to 14% and 25%, for each of the aerosol challenges respectively, and the frequency of isolation from muscle rose from 0% following the oral challenge to 27% following each of the aerosol challenges.

Experimental campylobacter infection and diarrhoea in immunodeficient mice.

The responses of previously untested immunodeficient mouse strains to campylobacter infection are described. Three strains of adult immunodeficient mice (SCID-Beige, C.B-17-SCID-Beige and RAG-2) were inoculated intragastrically with Campylobacter jejuni NCTC 11168. All mice became heavily colonised, but none developed clinical signs of disease. Immunocompetent BALB/c mice inoculated similarly had much lower colonisation levels. The co-administration of iron dextran had no effect on colonisation levels nor the development of clinical signs of disease. In contrast, C.B-17-SCID-Beige mice, when inoculated with one of a series of 10 clinical isolates of C. jejuni, were more heavily colonised for extended periods (up to 5 months) and approximately 10-20% of the mice became ill with diarrhoea. C. jejuni was detected in mouse faeces throughout at levels of 10(7)-10(9) cfu/g. All mice killed whilst ill with diarrhoea displayed histopathological lesions typical of human campylobacteriosis. Severe pathology was limited to the large intestine and was suggestive of an acute, bacteria-induced inflammation. Although blood was detected in the diarrhoeal stools, no evidence of mucosal epithelial cell invasion was found by immunohistology. No pathology was detected in tissue sections from any of the animals that had not developed signs of disease following C. jejuni inoculation. These immunodeficient mouse strains are readily, and heavily, colonised as adults by C. jejuni. The diarrhoea, although sporadic, was reproducibly produced, and could provide the basis for pathogenicity studies.

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.

Endogenous N-nitrosation and cancer of the biliary tract.

Evidence is presented that N-nitroso compounds occur in bile from patients who have undergone surgery for gallstones or had gastrectomy and from unoperated persons. It is unlikely, therefore, that local formation of nitrosamines can account for the excess risk for gallbladder cancer in the first two groups. Gastric formation remains the likeliest hypothesis.

The N-nitrosoproline test as a measure of cancer risk in geographical comparison studies: results from Italy and an overall comparison.

The N-nitrosoproline (NPRO) test has been used in studies in which populations at high risk of cancer have been compared with equivalent populations at lower risk, to examine whether the geographical variation in cancer risk correlates with propensity for endogenous nitrosation, as assessed by the NPRO test. The usual method employed has been to determine NPRO in 12- or 24-h urine samples, after ingestion of L-proline, in a representative sample of the general population. We present results from one such geographical study conducted in two regions of Italy (Florence and Cagliari) with an approximately three-fold variation in gastric cancer mortality. The nonsignificant difference in mean NPRO excretion between the two populations was insufficient to explain the difference in cancer risk. The fact that there are appreciable international differences in formation of NPRO suggests, firstly, that nitrosation may be of relevance to cancer risk in some countries but not in others and, secondly, that variations within one country may not be large enough for significant geographical differences to be evident. Multivariate analysis of individual, rather than grouped, results from our Italian study made it possible to quantify the relevance of different factors to NPRO formation: a major factor is exposure to nitrate. Important relationships may be missed by analysing only grouped data.

Inhibition of bacterially mediated N-nitrosation by ascorbate: therapeutic and mechanistic considerations.

Ascorbate is known to inhibit the acid-catalysed N-nitrosation reactions of nitrite in the normally acid stomach, suggesting a useful therapeutic application of this compound to reduce exposure to the carcinogenic products of such reactions. However, in the achlorhydric stomach, which is particularly predisposed to cancer, increased exposure to endogenous N-nitroso compounds may result from bacterially catalysed reactions. The mechanism of these bacterially mediated reactions is only just beginning to be understood, and, indeed, more than one such mechanism may exist. Despite its usual lack of reactivity towards nitrite at neutral pH, ascorbate proved to be a potent inhibitor of the bacterially mediated (Pseudomonas aeruginosa) nitrosation of morpholine, competing with morpholine for the nitrosating agent elaborated by the bacteria from nitrite (the kinetics of the inhibition were classically competitive). This and other data, particularly with regard to the dependence of the bacterially mediated reaction on amine pKa, are discussed in relation to the potential mechanisms of these bacterially mediated reactions.

The effect of gastric achlorhydria on the urinary recovery of nitrate in man: relevance to urinary nitrate as a measure of dietary nitrate exposure.

It is important to have a reliable method of assessing the dietary nitrate exposure of populations for a proper understanding of the potential health effects of the endogenous metabolites of this ion to be gained from epidemiological studies. Recently we strongly advocated the use of the nitrate analysis of 24 h urine samples as being a superior method for such studies. Our previous observations and those of others relating to nitrate pharmacology in healthy human volunteers formed the basis of this judgement. The purpose of this study was to determine whether gastric achlorhydria or hypochlorhydria has any significant gross effects on the urinary recovery of dietary nitrate and to what extent the inclusion of such individuals would compromise the results of potential epidemiological studies. The results demonstrated a significantly greater loss of dietary nitrate as measured by urinary recovery in achlorhydrics than in normochlorhydrics, presumably as a consequence of bacterial metabolism in the colonized stomach. Thus the average urinary nitrate recovery of a 1.5 mmol challenge was 33% in individuals with reduced stomach acidity as compared to 56% in normal controls. This significant further loss of nitrate (average 23%) when intragastric conditions favour bacterial colonization clearly indicates that for valid assessments and comparisons of nitrate exposure between populations it would be wise to exclude individuals with low levels of stomach acid where this is likely to lead to significant gastric colonization by nitrate-reducing bacteria (i.e. pH less than 4-5).

The effect of different sources of nitrate exposure on urinary nitrate recovery in humans and its relevance to the methods of estimating nitrate exposure in epidemiological studies.

It was demonstrated that nitrate concentration fluctuates dramatically in both urine and saliva throughout the study period and hence single time point ('spot') samples of either fluid give a poor measure of previous nitrate exposure. In contrast, the nitrate recovery in complete 24 h urine collections showed a strong positive correlation with previous nitrate exposure which was independent of the nature of the food matrix in which the nitrate was ingested. Regression analysis of the data showed that the apparent urinary recovery of 70% appears to consist of 55% arising from the original challenge and a background of approximately 0.22 mmol/day which is independent of the challenge and which may reflect endogenous mammalian synthesis of nitrate. It is therefore important that in future epidemiological studies, in which dietary nitrate exposures are to be determined, that analyses are conducted only on 24 h urine collections. A large inter-individual variation was found in the conversion of salivary nitrate to nitrite presumably mediated by the oral flora. The resulting differences in nitrite formation may be one of the factors determining the level of endogenous N-nitroso compound formation in an individual.

Remineralization of artificial caries-like lesions in human enamel in situ by chewing sorbitol gum.

The objective of the study was to determine quantitatively the effect on the potential for in situ remineralization of artificial caries-like lesions in human enamel when sugar-free gum containing mainly sorbitol as sweetener was chewed after meals and snacks. Artificial white-spot lesions were created in extracted human premolars and divided into three parts. One part was used as reference and the other two worn consecutively for two 21-day periods by 10 volunteers in a cast silver band cemented on lower molar teeth and covered with gauze to promote plaque formation. During the experimental periods, the subjects used fluoridated toothpaste twice daily, and consumed three meals (breakfast, lunch, and dinner) and two snacks (selected from chocolate bar, raisins, chocolate wafer, and iced cupcake). Sorbitol gum was chewed for 20 min immediately after each meal or snack during one of the experimental periods. The three parts of the enamel lesions were then sectioned (congruent to 80 microns) and examined together by means of quantitative microradiography and by polarized light microscopy. All estimates of mineral content indicated that significant remineralization occurred and was approximately doubled with gum-chewing. It is suggested that sorbitol gum stimulates salivation, which is responsible for the significantly enhanced remineralization, thus contributing to a therapeutic, caries-preventive effect. Because the gum was chewed immediately after meals and snacks, inhibition of demineralization may also have occurred.

The inhibition of bacterially mediated N-nitrosation by vitamin C: relevance to the inhibition of endogenous N-nitrosation in the achlorhydric stomach.

It has been suggested that endogenously formed N-nitroso compounds are involved in the aetiology of gastric cancer. In the model of gastric carcinogenesis postulated by Correa, gastric atrophy is an important early stage in the progression to carcinoma which results in the loss of stomach acidity, and colonization of the stomach by bacteria. As a consequence of the metabolic activity of these bacteria intragastric nitrite (a precursor to N-nitroso compounds) and possibly carcinogenic N-nitroso compounds become elevated, which may hasten the progression to carcinoma. Vitamin C has been shown to be an effective inhibitor of acid-catalysed N-nitroso compound formation, in vivo and in vitro, and this has been attributed to its relatively rapid reaction with nitrite in contrast to the slower rates of reaction of nitrite with secondary amines. However, N-nitroso compound formation in the achlorhydric stomach must proceed by mechanisms which operate at neutral pH values. One potential mechanism involves the enzymatic catalysis of N-nitrosation by a subpopulation of the bacteria colonizing the achlorhydric stomach which catalyse these reactions and in particular denitrifying organisms. In this study, we examined the effect of vitamin C on the formation of N-nitrosomorpholine from morpholine and nitrite when mediated by cells of an actively N-nitrosating denitrifying bacterium (Pseudomonas aeruginosa, BM1030) at neutral pH. Despite the fact that vitamin C ordinarily shows little reactivity towards nitrite at neutral pH it did prove to be a potent inhibitor of bacterial N-nitrosamine formation. This study provides some justification for the use of vitamin C as an inhibitor of endogenous N-nitrosation regardless of gastric pH.

Bacterially catalysed N-nitrosation reactions and their relative importance in the human stomach.

Human exposure to endogenously formed N-nitroso compounds has frequently been suggested as a causative factor in carcinogenesis where this is related to chronic bacterial infection such as is seen in gastric achlorhydria. At least two distinct mechanisms of endogenous formation have been identified. The first, a direct chemical reaction between secondary amino compounds and nitrite, is strongly pH dependent and does not proceed rapidly at neutral pH even in the presence of chemical catalysts. The second depends on the direct bacterial catalysis of N-nitrosation. The data presented demonstrate that the bacterially mediated reaction is catalysed by bacterial enzyme systems and proceeds much more rapidly at neutral pH than the chemical reaction. This suggests a particular relevance to the in vivo situation where neutral pH, bacteria and elevated nitrite concentrations are found. Drawing on the kinetic information presented regarding the bacterially mediated nitrosation reaction, the known kinetics of the chemical reaction and the published values for the relevant substrate concentrations in both the colonised and the normal acid stomach the bacterial and chemical reactions have been compared. Using these criteria, and assuming the presence of bacteria with the appropriate metabolic activity, it may be predicted that N-nitroso compounds may be formed in the colonized stomach at much higher concentrations than in the normal acid stomach. The difference in yield may be by two to four orders of magnitude. Different bacterial species and different isolates of the same species show considerable variation in their abilities to catalyse N-nitrosation reactions. The most rapid catalysis is associated with those bacteria capable of reducing nitrate and nitrite by the process of denitrification. The most significant clinical corollary of these studies is that although bacterial catalysis of N-nitrosation has been demonstrated unequivocally, bacterial colonization of the stomach may not itself necessarily result in elevated endogenous N-nitroso compound exposure despite the elevated nitrite concentrations normally associated with such colonization. An increase in exposure to endogenously formed N-nitroso compounds would only be predicted in those individuals where a significant proportion of the colonizing bacteria expressed significant N-nitrosation activity. As a consequence the carcinogenic risk may be restricted to only a small proportion of colonized individuals depending on the prevalence of sustained infection by bacteria with significant N-nitrosation activity, particularly denitrifiers.

Bacterially mediated N-nitrosation reactions and endogenous formation of N-nitroso compounds.

Results are presented demonstrating some factors that affect the kinetics of bacterially mediated N-nitrosation reactions. Two groups of bacteria, differing in their nitrate/nitrite metabolism, are contrasted. These findings are discussed in relation to a role for bacteria in endogenous N-nitrosation reactions.

Gastric hypoacidity as a risk factor for gastric and other cancers.

Evidence is presented and discussed to test the hypothesis that the excess risk of gastric cancer observed in patients with decreased gastric acidity is caused by metabolites of nitrite, possibly N-nitroso compounds.

An ultrastructural investigation of the filamentous surface appendages of suspensions of plaque bacteria as revealed by negative staining.

The filamentous surface appendages of freshly-collected aqueous suspensions of plaque bacteria, obtained from 1 day old supragingival plaque have been examined in the electron microscope by the technique of negative staining with methylamine tungstate. Approximately half of the bacteria revealed surface appendages as either fimbriae (45%), flagella (13%) or both (3%). The appendages were distributed either polarly, intermittently or peri-trichously around the bacteria and varied in length from 0.2 micron to more than 20 micron. It was not possible from these observations alone to determine either the topology of the appendages or their role as they existed originally in dental plaque.