Dysbiosis of Gut Microbiome Is Associated With Rupture of Cerebral Aneurysms.

BACKGROUND AND PURPOSE: Environmental factors are important with respect to the rupture of cerebral aneurysms. However, the relationship between the gut microbiome, an environmental factor, and aneurysm rupture is unclear. Therefore, we compared the gut microbiome in patients with unruptured intracranial aneurysms (UIAs) and ruptured aneurysms (RAs) to identify the specific bacteria causing the rupture of cerebral aneurysms. METHODS: A multicenter, prospective case-control study was conducted over one year from 2019 to 2020. The fecal samples of patients with stable UIAs and RAs immediately after onset were collected. Their gut microbiomes were analyzed using 16S rRNA sequencing. Subsequently, a phylogenetic tree was constructed, and polymerase chain reaction was performed to identify the specific species. RESULTS: A total of 28 RAs and 33 UIAs were included in this study. There was no difference in patient characteristics between RAs and UIAs: age, sex, hypertension, dyslipidemia, diabetes status, body mass index, and smoking. No difference was observed in alpha diversity; however, beta diversity was significantly different in the unweighted UniFrac distances. At the phylum level, the relative abundance of Campylobacter in the RA group was larger than that in the UIA group. Furthermore, the gut microbiome in the RA and UIA groups exhibited significantly different taxonomies. However, Campylobacter was focused on because it is widely known as pathogenic among these bacteria. Then, a phylogenetic tree of operational taxonomic units related to Campylobacter was constructed and 4 species were identified. Polymerase chain reaction for these species identified that the abundance of the genus Campylobacter and Campylobacter ureolyticus was significantly higher in the RA group. CONCLUSIONS: The gut microbiome profile of patients with stable UIAs and RAs were significantly different. The genus Campylobacter and Campylobacter ureolyticus may be associated with the rupture of cerebral aneurysms.

Enhancement of Campylobacter hepaticus culturing to facilitate downstream applications.

Campylobacter hepaticus causes Spotty Liver Disease (SLD) in chickens. C. hepaticus is fastidious and slow-growing, presenting difficulties when growing this bacterium for the preparation of bacterin vaccines and experimental disease challenge trials. This study applied genomic analysis and in vitro experiments to develop an enhanced C. hepaticus liquid culture method. In silico analysis of the anabolic pathways encoded by C. hepaticus revealed that the bacterium is unable to biosynthesise L-cysteine, L-lysine and L-arginine. It was found that L-cysteine added to Brucella broth, significantly enhanced the growth of C. hepaticus, but L-lysine or L-arginine addition did not enhance growth. Brucella broth supplemented with L-cysteine (0.4 mM), L-glutamine (4 mM), and sodium pyruvate (10 mM) gave high-density growth of C. hepaticus and resulted in an almost tenfold increase in culture density compared to the growth in Brucella broth alone (log10 = 9.3 vs 8.4 CFU/mL). The type of culture flask used also significantly affected C. hepaticus culture density. An SLD challenge trial demonstrated that C. hepaticus grown in the enhanced culture conditions retained full virulence. The enhanced liquid culture method developed in this study enables the efficient production of bacterial biomass and therefore facilitates further studies of SLD biology and vaccine development.

Improvement of Bolton broth by supplementation with tazobactam for the isolation of Campylobacter from chicken rinses.

Overgrowth of extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli (E. coli) on Campylobacter media prevents the latter's selective isolation, thereby making the improvement of Campylobacter-selective media necessary. We evaluated tazobactam (an ESBL inhibitor) to supplement Bolton enrichment broth (Tz-Bolton broth) for the selective isolation of Campylobacter in chicken carcass rinses. First, using 20 strains of ESBL-producing E. coli and 13 Campylobacter strains, we found 4 µg/mL of tazobactam to be optimal for inhibiting the ESBL-producing E. coli while allowing the growth of all tested Campylobacter strains. Next, 80 whole chicken carcasses were rinsed with buffered peptone water (BPW), and 25 mL of BPW rinse was mixed with 2 × blood-free Bolton broth (25 mL) with or without tazobactam followed by incubation at 42°C for 48 h under microaerobic conditions. A loopful of the incubated broth was inoculated on modified charcoal-cefoperazone-deoxycholate agar (mCCDA) and microaerobically incubated at 42°C for 48 h. The tazobactam supplemented Bolton broth showed a higher Campylobacter isolation rate (38.8%, p < 0.05) than normal Bolton broth (15%). Moreover, the number of mCCDA plates with non-Campylobacter was much lower (p < 0.05) after enrichment in Tz-Bolton broth (0%) than in the normal Bolton broth (80%), suggesting that selectivity of the modified broth was superior to normal Bolton broth.

Growth of Campylobacter incubated aerobically in fumarate-pyruvate media or media supplemented with dairy, meat, or soy extracts and peptones.

The ability of Campylobacter to grow aerobically in media supplemented with fumarate-pyruvate or with dairy, meat, or soy extracts or peptones was examined. Optical densities (OD) of Campylobacter cultured in basal media, media supplemented with fumarate-pyruvate or with 1.0, 2.5, 5.0, or 7.5% beef extract was measured. Growth was also compared in media supplemented with other extracts or peptones. Finally, cfu/mL of Campylobacter recovered from basal media or media supplemented with fumarate-pyruvate, casamino acids, beef extract, soytone, or beef extract and soytone was determined. Results indicated that OD of cultures grown in media supplemented with fumarate-pyruvate or with 5.0 or 7.5% beef extract were higher than OD of isolates grown in basal media or media supplemented with lower concentrations of beef extract. Highest OD were produced by isolates grown in media supplemented with beef extract, peptone from meat, polypeptone, proteose peptone, or soytone. Also, more cfu/mL were recovered from media with fumarate-pyruvate, beef extract, soytone, or beef extract-soytone than from basal media or media with casamino acids. Findings indicate that media supplemented with organic acids, vitamins, and minerals and media supplemented with extracts or peptones containing these metabolites can support aerobic growth of Campylobacter.

Survival after cryogenic freezing of Campylobacter species in ground Turkey patties treated with polyphosphates.

The use of polyphosphate-based marinades in the processing of poultry has been previously shown to increase the survival of Campylobacter species present in the exudates derived from these products. This study investigates the effects that some of the same polyphosphates have on the survival of Campylobacter species within a ground turkey product subjected to cryogenic freezing. Ground turkey patties with two different polyphosphate formulations added in two different concentrations were artificially contaminated with known concentrations of Campylobacter jejuni or Campylobacter coli. The patties were cryogenically frozen at -80°F (-62.2°C) with liquid nitrogen vapor and held at -20°C for 7 or 33 days, after which the number of Campylobacter surviving in the patties was determined. On average the cryogenic freezing resulted in a 2.5-log decrease in the survival of C. jejuni cells and a 2.9-log decrease in C. coli cells present in the turkey patties. Additionally, the presence of polyphosphates in the turkey patties had no effect on Campylobacter survival up to the maximum allowed concentration (0.5%) for polyphosphates in poultry marinades. Finally, it was determined that the added polyphosphates had little effect on the pH of the ground turkey meat; an effect which previously had been implicated in the enhancement of Campylobacter survival due to the presence of polyphosphates.

Effects of polyphosphate additives on the pH of processed chicken exudates and the survival of Campylobacter.

Campylobacter spp. are nutritionally fastidious organisms that are sensitive to normal atmospheric oxygen levels and lack homologues of common cold shock genes. At first glance, these bacteria seem ill equipped to persist within food products under processing and storage conditions; however, they survive in numbers sufficient to cause the largest number of foodborne bacterial disease annually. A mechanism proposed to play a role in Campylobacter survival is the addition of polyphosphate-containing marinades during poultry processing. Campylobacter jejuni and Campylobacter coli strains incubated in chicken exudates collected from poultry treated with a marinade demonstrated considerable survival advantages (1 to 4 log CFU/ml) over the same strains incubated in chicken exudate from untreated birds. Polyphosphates, which constitute a large portion of the commercial poultry marinades, were shown to account for a majority of the observed influence of the marinades on Campylobacter survival. When six different food grade polyphosphates (disodium pyrophosphate, tetrasodium pyrophosphate, pentasodium triphosphate, sodium polyphosphate, monosodium phosphate, and trisodium phosphate) were utilized to compare the survival of Campylobacter strains in chicken exudate, significant differences were observed with regard to Campylobacter survival between the different polyphosphates. It was then determined that the addition of polyphosphates to chicken exudate increased the pH of the exudate, with the more sodiated polyphosphates increasing the pH to a greater degree than the less sodiated polyphosphates. It was confirmed that the change in pH mediated by polyphosphates is responsible for the observed increases in Campylobacter survival.

Effect of growth phase on the adherence to and invasion of Caco-2 epithelial cells by Campylobacter.

The effect of growth phase on the adherence to and invasion of Caco-2 epithelial cells by five strains of Campylobacter was studied. No significant differences were observed between the behaviors in the exponential or stationary phases for the most stationary-phase tolerant strains (C. jejuni 118 and C. coli LP2), while the strains that produced a greater reduction in the viability in the stationary phase (C. jejuni 11351, C. jejuni 11168 and C. jejuni LP1), also presented reduced adherence to and invasion of Caco-2 cells. In order to find a possible explanation for the observed differences, the presence of putative virulence factors was studied in the analyzed strains. In spite of the fact that C. jejuni 118 and C. jejuni 11168 strains showed a different adherence to and invasion of Caco-2 cells behavior, they posses identical alleles for ciaB, cadF, and pldA loci. From the virulence factors analyzed, only the flaA locus was different among both strains.

Campylobacter and Arcobacter species sensitivity to commercial orange oil fractions.

Seven orange oil fractions were screened for their ability to inhibit the growth of selected Campylobacter and Arcobacter spp. using the standard agar-disk diffusion assay. Cold pressed (CP) terpeneless Valencia orange oil was found to be the most inhibitory to both Campylobacter jejuni and Campylobacter coli, exhibiting maximum zones of inhibition up to 80+/-0.0 mm. Five-fold concentrated Valencia oil and distilled d-limonene resulted in Campylobacter inhibition zones ranging from 11.0+/-1.4 to 44+/-1.4 mm against both C. jejuni and C. coli. No inhibition of Arcobacter spp. was detected by 6 out of 7 orange fractions except CP terpeneless Valencia orange oil which produced inhibition zones varying from 9.5+/-0.7 to 29+/-1.4 mm. Naturally occurring C. jejuni UAF 244 was isolated from a whole retail chicken, confirmed by hippuricase gene PCR assay, and used to determine antimicrobial capacities of the CP terpeneless Valencia orange oil and limonene when applied on chicken legs and thighs. The two types of chicken parts did not influence the antimicrobial strength of both orange fractions. While the observed reduction of C. jejuni cells attached to the skin varied approximately 1.5 to 2 logarithms compared to the control, the growth inhibition of the bacterial cells by limonene in the rinse increased by 6-fold and complete inhibition without recovery of detectable viable cells occurred when CP Valencia orange oil was applied. The study demonstrated the potential of the selected commercial orange oil fractions to serve as natural antimicrobials against C. jejuni, C. coli, and Arcobacter spp.

Efficacy of iron chelators on Campylobacter concentrations in turkey semen.

Campylobacter is a leading bacterial cause of human foodborne infections in the United States. Recent studies suggest that the organism is highly prevalent in poultry semen and may contribute to vertical transmission between the breeder hen and offspring. Because Campylobacter requires iron for its growth and survival, the objective of this study was to determine if the addition of natural and synthetic chelators such as ovotransferrin, desferrioxaime, EDTA, or 2,2'-dipyridyl could reduce or eliminate Campylobacter in turkey semen. In a preliminary study without semen, a commercial poultry semen extender was supplemented with various concentrations of ovotransferrin, desferrioxaime, EDTA, or 2,2'-dipyridyl and inoculated with an average of 10(8) cfu/mL of a wild-type Campylobacter coli turkey semen isolate. At 6 and 24 h of storage at 4 degrees C, a sample was taken from each treatment group and enumerated for Campylobacter. In all 3 trials, Campylobacter was undetectable (< 10(2)) in the commercial poultry semen extender supplemented with 20 mg/mL of 2,2'-dipyridyl. There were no differences observed in Campylobacter concentrations in the commercial poultry semen extender supplemented with ovotransferrin, desferrioxaime, or EDTA compared with unsupplemented controls. In a follow-up study, pooled semen samples were randomly collected from toms, diluted with a commercial poultry semen extender supplemented with 5, 10, or 20 mg/mL of 2,2'-dipyridyl and inoculated with an average of 10(8) cfu/mL of a wild-type C. coli turkey semen isolate. At 6 and 24 h of storage at 4 degrees C, samples were taken from each treatment group, enumerated for Campylobacter, and evaluated for sperm viability. In all 3 trials, supplementing the commercial poultry semen extender with 20 mg/mL of 2,2'-dipyryidyl significantly reduced (3 to 4 logs) Campylobacter concentrations when compared with the positive controls. Sperm viability was also reduced with this treatment, and, therefore, the use of 2,2'-dipyridyl may not be a practical treatment for reducing Campylobacter in poultry semen.

Thermophilic Campylobacter spp. in turkey samples: evaluation of two automated enzyme immunoassays and conventional microbiological techniques.

AIMS: To determine the sensitivity and specificity of two automated enzyme immunoassays (EIA), EiaFoss and Minividas, and a conventional microbiological culture technique for detecting thermophilic Campylobacter spp. in turkey samples. METHODS AND RESULTS: A total of 286 samples (faecal, meat, neckskin and environmental samples) were collected over a period of 4 months at a turkey slaughterhouse and meat-cutting plant in Denmark. Faecal and environmental samples were tested by the conventional culture method and by the two EIAs, whereas meat and neckskin samples were tested by the two EIAs only. Two enrichment broths were used, Campylobacter Enrichment Broth (CEB) and Preston Broth (PB). Verification of positive test results was carried out by conventional culture on selective solid media. The specificities of all methods were high. The sensitivities of the EIAs were higher than that of the conventional culture technique but varied depending on the type of sample and enrichment broth. For neckskin samples, the Minividas had a significantly higher sensitivity than the EiaFoss and using PB instead of CEB as the enrichment broth significantly improved the sensitivity for both EIAs. CONCLUSIONS: Both EIAs provided more accurate results than the conventional culture technique. Furthermore, neckskin samples enriched in PB resulted in more positive test results and Campylobacter growth than samples enriched in CEB. SIGNIFICANCE AND IMPACT OF THE STUDY: The Eiafoss and Minividas proved to be reliable methods for detecting Campylobacter spp. in various samples. However, the results emphasize the need for the development of specific enrichment protocols for specific samples.

Mutans streptococci in subgingival plaque of treated and untreated patients with periodontitis.

BACKGROUND: The etiology of root caries is thought to be identical to coronal caries, though root caries seem to be more complicated because of the higher susceptibility of exposed roots (dentin) by periodontal therapy to demineralization than intact enamel. This implies that mutans streptococci are the most likely pathogens in the development of root caries. Although it is known that both the numbers of mutans streptococci and the frequency of isolation in root caries lesions are negatively correlated with the distance from the gingival margin, the subgingival sulcus has not been considered a possible habitat for mutans streptococci. However, subgingival occurrence of mutans streptococci in both untreated and treated periodontal patients has not been documented well in the literature. OBJECTIVE: To investigate the presence and levels of mutans streptococci in the subgingival plaque of patients (n=154) in different stages of periodontal therapy. The subgingival sulcus may be a possible habitat for mutans streptococci. This localisation of mutans streptococci may be of importance in the development of root caries after periodontal surgery. MATERIALS AND METHODS: In this cross-sectional study, subgingival plaque samples from 154 consecutive adult periodontitis patients were tested for presence and levels of mutans streptococci and putative periodontal pathogens by anaerobic cultures. These patients were divided into 4 groups based on their stage of periodontal treatment: (1) untreated patients; (2) patients after initial periodontal therapy only; (3) patients in the maintenance phase who not underwent periodontal surgery; (4) patients after periodontal surgery. RESULTS: The prevalence of mutans streptococci in the 4 study groups varied from 82% in untreated patients to 94% in maintenance patients. The mean proportion of mutans streptococci was 6.65% in maintenance patients versus 1.86% in untreated patients (p=0.005) and 2.51% in patients after scaling and root planing (p=0.041). CONCLUSIONS: The subgingival area is a microbial habitat for mutans streptococci that may be of importance in the development of root caries in periodontitis patients.

"Checkerboard" assessments of periodontal microbiota and serum antibody responses: a case-control study.

BACKGROUND: We explored the association between subgingival microbial profiles and serum IgG responses to periodontal microbiota in relation to clinical periodontal status. METHODS: One hundred thirty-one (131) periodontitis patients aged 29 to 74 years (mean 51.8) were age- and gender-matched with 74 periodontally intact controls (range 26 to 77, mean 49.3). Smoking habits and health history were recorded and assessments of plaque, bleeding on probing, probing depth, and attachment level were performed at 6 sites per tooth on all present teeth, excluding third molars. Subgingival plaque samples were obtained from each tooth in one upper and one lower quadrant (maximum 14 samples/subject; 2,440 samples total) and analyzed with respect to 19 species by means of whole genomic DNA probes. Serum IgG antibodies against the same 19 species were assessed by an immunoassay. RESULTS: Cases displayed an average of 22.7 teeth, 20.3 sites with probing depth > or =6 mm, and 18.9 sites with attachment loss > or =6 mm. Corresponding figures for controls were 27.1, 0.1, and 1.0, respectively. Heavy smoking was 3 times more frequent among cases than controls (32.1% versus 9.6%). Higher levels of Porphyromonas gingivalis, Porphyromonas endodontalis, Prevotella intermedia, Prevotella nigrescens, Prevotella melaninogenica, Bacteroides forsythus, Fusobacterium nucleatum, Treponema denticola, Eubacterium nodatum, Peptostreptococcus micros, and Campylobacter rectus were found in cases and higher levels of Eikenella corrodens, Veillonella parvula, and Actinomyces naeslundii in controls. Cases displayed higher IgG levels against P. gingivalis and Actinobacillus actinomycetemcomitans, while controls displayed higher levels against F. nucleatum, T. denticola, E. nodatum, and Capnocytophaga ochracea. Positive correlations between bacterial colonization and antibody responses were identified for 9 species in controls. In cases, however, statistically significant correlations were observed for only 3 species out of which only one was positive (V. parvula). Both bacterial levels and antibody responses declined in ages over 55 years. A logistic regression employing selected elements of bacterial colonization and antibody responses as independent variables resulted in 81.1% correct diagnosis, with sensitivity of 83.1%, specificity of 77.8%, positive predictability of 86%, and negative predictability of 73.7%. Smoking did not reach statistical significance in this model. CONCLUSION: A combined microbial colonization/antibody response profile can effectively discriminate between periodontitis patients and periodontally intact controls.

The prevalence of pathogenic periodontal microflora in healthy young adult smokers.

BACKGROUND: Smoking is a major risk factor in periodontitis, although the mechanisms of its effects are not well understood. The overall goal of this clinical study was to determine if smoking enhances the colonization of the oral cavity by pathogenic bacteria in a periodontitis-free population. The prevalence of Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, Fusobacterium nucleatum, Campylobacter rectus, Eikenella corrodens, Bacteroides forsythus, and Treponema denticola was investigated in 25 smokers and 25 non-smokers by using DNA probes. METHODS: The subjects were 21 to 35 years of age with a healthy periodontium or slight gingivitis and were systemically healthy. The test group included subjects who had a minimum of a 1.5 pack-year history of smoking, while the control subjects never smoked. Subgingival plaque samples were taken by paper point following the assessment of multiple clinical parameters. RESULTS: This investigation showed: 1) no statistically significant differences were noted in any clinical parameter measured between the groups; 2) of the 8 subjects who were infected by at least 1 tested pathogen, seven were smokers (P= 0.02); 3) infected smokers had a 15.7+/-3.5 pack-year history and smoked a mean of 27+/-5 cigarettes/day versus 4.4+/-0.8 pack years and 15+/-1 cigarettes/day for the non-infected smokers (P = 0.0001 and P = 0.004); and 4) smokers were 18 times more likely to exhibit the presence of pathogens than non-smokers. CONCLUSIONS: These data indicate that the prevalence of colonization of the sulcus by pathogenic bacterial species in periodontitis-free individuals is related to the quantity and duration of cigarette smoking.

Clinical and microbiological characteristics of smokers with early onset periodontitis.

Cigarette smoking is a potential risk factor which has recently been associated with periodontal disease progression. The objective of this study was to compare the microbial profile of smokers and non-smokers in a group of patients with early onset periodontitis. The study population consisted of 60 healthy individuals, 40 males and 20 females aged 22 to 35 yr, exhibiting early onset periodontitis. Thirty patients were smokers (30.9 cigarettes/d) and 30 non-smokers. Smokers had a higher proportion of deep pockets (PD >5 mm), especially in the maxilla anterior and premolar regions (p < 0.001) and presented a significantly greater mean probing depth and attachment loss (p <0.05) in diseased sites and a significantly greater alveolar bone loss (p <0.01) compared to non-smokers. Two pooled bacterial samples were obtained from each patient. Samples were collected from the deepest periodontal pockets of each quadrant. The samples were cultured anaerobically and in 10% CO2 plus air for bacterial isolation using selective and non-selective media. Isolates were characterized to species level by conventional biochemical tests and various identification kits. Smokers harboured a greater number of bacteria in total. Analysis of bacterial counts using the ANOVA (Mann-Whitney U-test) showed that Staphylococcus aureus, Peptostreptococcus micros, Campylobacter concisus, Escherichia coli, Bacteroides forsythus, C. gracilis, C. rectus, Porphyromonas gingivalis, Selenomonas sputigena, Candida albicans and Aspergillus fumigatus were found in significantly higher numbers and more frequently in smokers while Streptococcus intermedius, A. naeslundii, A. israelii and Eubacterium lentum were detected more frequently and in significantly higher proportions in non-smokers. The isolation of bacteria belonging to the exogenous flora such as E. coli, C. albicans, A. fumigatus and S. aureus in smokers' microbiota underscores the importance of the host that is adversely affected by cigarette smoking.

Coronal leakage of mixed anaerobic bacteria after obturation and post space preparation.

OBJECTIVES: The purpose of this study was to assess bacterial leakage of a mixed anaerobic community of organisms in obturated canals after post space preparation. STUDY DESIGN: A mixed microbial community of strict anaerobic organisms (F. nucleatum, P. micros and C. rectus) was developed. With the use of an in vitro model system, coronal leakage was assessed in 40 anterior teeth after obturation and post space preparation. The specific leakage time in days for each organism to penetrate through the obturating material was determined. In addition, colonization of the apical canal space was assessed by scanning electron microscope after longitudinal splitting of randomly selected specimens. RESULTS: Eighty percent of the teeth demonstrated coronal leakage of F. nucleatum and C. rectus by the 90 day interval. Bacterial penetration occurred from 48 days to 84 days. Scanning electron microscope examination showed a heterogeneous biofilm of coccal and bacillary species colonizing the apical portion of the canal wall. CONCLUSIONS: This study demonstrated that coronal leakage phenomena do occur after loss of coronal seals. The model system developed using mixed, anaerobic bacterial cultures is more clinically relevant and may be used to assess bacterial penetration through gutta percha obturation.

Isolation and partial characterization of the Campylobacter rectus cytotoxin.

Previously, we reported the antigranulocytic activity of Campylobacter rectus media supernatants containing lipopolysaccharide (LPS) and a 104 kDa protein. Here, we monitored the release of protein and LPS through the growth cycle of C. rectus ATCC 33238 and identified the 104 kDa protein as the cytotoxin. LPS in media supernatants was quantitated by a KDO assay; the 104 kDa protein was detected on immunoblots with specific antibody (A104) and quantitated by amino acid analysis of membrane immobilized protein bands. C. rectus cell product release was independent of cell lysis. Over 24 h, the 104 kDa protein was released linearly while LPS was released in two plateaus; both increased in C. rectus culture supernatants 3 h after inoculation achieving maximum concentrations at 21 h of 3.1 micrograms/ml and 14.6 micrograms/ml, respectively. In 2 h, trypan blue viability assays, 37-47 micrograms of 12, 18 and 24 h supernatant protein killed 33-43% of HL-60 cells. Supernatant toxicity was heat sensitive and inhibited by A104. Sequencing the 16 N-terminal amino acids of the cytotoxin distinguished it from described C. rectus proteins. Similarities between epitopes and amino acid compositions of the Actinobacillus actinomycetemcomitans leukotoxin and C. rectus cytotoxin were observed. These data indicate that C. rectus secretes a 104 kDa cytotoxin.

Better preservation of Campylobacter jejuni/C. coli in a defined medium.

Campylobacter jejuni and C. coli strains were preserved at -10 degrees C in different stock media to determine their efficacy to preserve the organism for a longer period of time. An improved defined stock culture medium was developed for the organism by removal and effective neutralisation of the toxic metabolites. Comparative study revealed that phosphate buffer saline (PBS), pH 6.7 supplemented with 0.2 per cent charcoal, 0.025 per cent FBP (ferrous sulphate, sodium metabisulphate and sodium pyruvate), 0.1 per cent L-cystein and 10 per cent glycerol could support survival of C. jejuni coli strains for as long as 135 days at -10 degrees C followed by George's medium, brucella broth with 15 per cent glycerol, fetal calf serum with 50 per cent TSYB (tryplicase soy yeast-broth) and glycerol transport broth respectively.

Use of tri-gas incubator for routine culture of Campylobacter species from fecal specimens.

We evaluated a tri-gas incubator for Campylobacter isolation to be used instead of an anaerobic jar. Fecal specimens were cultured in duplicate onto charcoal selective medium and incubated at 43 degrees C for 48 h in two different environments: a tri-gas incubator (Forma Scientific) adjusted to provide an atmosphere of 10% CO2, 10% O2, and the balance N2; and evacuated anaerobic jars with a replacement gas mixture of 10% CO2, 5% O2, and 85% N2. A total of 106 Campylobacter jejuni and 8 Campylobacter coli isolates were obtained from 2,348 stool specimens. Of the positive specimens, 113 isolates came from the incubator and 111 isolates came from the anaerobic jars. An additional 32 previously positive specimens were replated onto charcoal selective medium and retested by both methods. We recovered 27 C. jejuni isolates, 26 isolates by each method. The isolates from the incubator typically produced discrete colonies, while the isolates from the anaerobic jar showed some degree of swarming in colony formation. The tri-gas incubator provided a cost-effective method for culturing Campylobacter spp.

Purification, characterization, and localization of a protein antigen shared by thermophilic campylobacters.

A protein antigen with an apparent molecular weight (Mr) of 31,000 was isolated from 0.2 M glycine hydrochloride (pH 2.2) extracts of a typical human fecal isolate, Campylobacter jejuni VC74. The protein was purified to homogeneity on a preparative scale by immunoaffinity chromatography followed by molecular sieving with a Superose 12 column. Isoelectric focusing under nondenaturing conditions indicated a pI of 9.3, and amino acid composition analysis showed that the protein was unusually rich in lysine, containing 14.9 mol% of this basic amino acid. Cysteine and tryptophan were absent. The protein also contained approximately 35% hydrophobic amino acid residues, and N-terminal amino acid analysis showed that 17 of the first 38 residues were hydrophobic. This amino-terminal sequence to residue 22 was virtually identical to that of an antigenically cross-reactive 31,000-Mr protein isolated from another C. jejuni strain belonging to a different heat-labile serogroup. Western blotting (immunoblotting) of glycine extracts of other C. jejuni, Campylobacter coli, and Campylobacter laridis strains belonging to different thermolabile and thermostable serotypes, as well as Campylobacter fetus, with a rabbit polyclonal antiserum raised against the purified C. jejuni VC74 protein showed that all C. jejuni, C. coli, and C. laridis strains tested contained a 31,000-Mr protein with epitopes which were antigenically cross-reactive with the C. jejuni VC74 protein. The antigenically cross-reactive epitopes of this protein were also readily detected by immunodot blot assay of glycine extracts of C. jejuni, C. coli, and C. laridis with monospecific polyclonal antisera to the 31,000-Mr protein, suggesting that this serological test could be a useful addition to those currently employed in the rapid identification of these important pathogens. Slide agglutination reactions, immunofluorescence assay, and immunogold electron microscopy with antisera to purified 31,000-Mr protein and trypsin treatment of whole cells indicated that the cross-reactive epitopes of the 31,000-Mr protein were not exposed on the cell surface. Cell fractionation analysis and immunogold electron microscopy located the protein on the outer surface of the cytoplasmic membrane. This finding suggests that the 31,000-Mr protein is not a good candidate for inclusion in a monovalent subunit Campylobacter vaccine.

[Semiquantitative culture of Campylobacter pylori in gastroduodenal disorder].

Results of semiquantitative culture of the gastric mucosa for Campylobacter pylori (C. pylori) are reported. The samples were obtained by biopsy at the pyloric antrum along the lesser curvature from 197 patients with various gastric or duodenal disorders. 1) C. pylori was found in most cases with duodenal ulcer (100%), gastric ulcer (87.7%) and acute gastric mucosal lesion (AGML) (87.5%). Relatively heavy infestation was usual in these diseases, while relatively small amounts of the organism were found in various frequencies in other gastroduodenal disorders. 2) Samples from gastric ulcer cases under H2-blocker treatment had reduced amount of C. pylori as compared with those from untreated cases (p less than 0.01). C. pylori decreased in quantity in each case of AGML after successful treatment with a H2-blocker but not in a case without favorable response. 3) C. pylori distributed evenly among the gastric mucosa sampled at the margin of the ulcer and that at a relatively healthy portion in the pyloric antrum of the same stomach. 4) There was a modest, positive correlation between the amount of C. pylori and that of inflammatory cells, especially of neutrophilic granulocyte, in the gastric mucosa (r = 0.679), while there was a weak, negative correlation between the former and the extent of intestinal metaplasia of the epithelial cells (r = -0.479). 5) Quantitative rather than qualitative observation of C. pylori seems mandatory for considering the relevance of the bacterium to gastric and duodenal disorders. Although C. pylori can be found in the mucosa of the stomach with any type of disorder, its quantity tends to parallel with the activity of duodenal as well as gastric ulcer.