Prevalence estimates of Helicobacter species infection in pancreatic and biliary tract cancers.

BACKGROUND: Helicobacter pylori infection is a well-established risk factor for gastric cancer and has been linked to other gastrointestinal diseases, including pancreatic and biliary tract cancers; however, the relevance of enterohepatic non-H. pylori helicobacters to the pathophysiology of these diseases remains unclear. MATERIALS AND METHODS: We estimated the prevalence of two enterohepatic non-H. pylori helicobacters (Helicobacter hepaticus and Helicobacter bilis) in the framework of a hospital-based case-control study involving 121 patients with biliary tract cancer, pancreatic cancer, or other gastrointestinal diseases. Bile and blood samples were collected from the patients undergoing endoscopic retrograde cholangiopancreatography. The presence of H. bilis, H. hepaticus, and other Helicobacter spp. was examined using bacterial culture, PCR-based detection, and serological tests. RESULTS: Culture of Helicobacter spp. from biliary brush samples was unsuccessful. Approximately 13.0% (15/115) of the bile samples collected from patients with a variety of gastrointestinal cancers, including pancreatic and biliary tract cancers, tested positive for one of the enterohepatic non-H. pylori helicobacter species as determined by PCR. Specifically, H. bilis and H. hepaticus DNA were detected in 11 and 4 bile samples, respectively. Approximately 20%-40% of the patients tested positive for serum non-H. pylori helicobacter IgG antibodies. The seroprevalence of H. bilis and H. hepaticus in the patients without evidence of H. pylori infection appeared to be higher in the pancreatic cancer group than in the control group. CONCLUSION: Our findings suggest a role for Helicobacter spp., especially H. bilis and H. hepaticus, in the etiology of pancreatic and biliary tract cancers.

Isolation and characterization of Helicobacter suis from human stomach.

Helicobacter suis, a bacterial species naturally hosted by pigs, can colonize the human stomach in the context of gastric diseases such as gastric mucosa-associated lymphoid tissue (MALT) lymphoma. Because H. suis has been successfully isolated from pigs, but not from humans, evidence linking human H. suis infection to gastric diseases has remained incomplete. In this study, we successfully in vitro cultured H. suis directly from human stomachs. Unlike Helicobacter pylori, the viability of H. suis decreases significantly on neutral pH; therefore, we achieved this using a low-pH medium for transport of gastric biopsies. Ultimately, we isolated H. suis from three patients with gastric diseases, including gastric MALT lymphoma. Successful eradication of H. suis yielded significant improvements in endoscopic and histopathological findings. Oral infection of mice with H. suis clinical isolates elicited gastric and systemic inflammatory responses; in addition, progression of gastric mucosal metaplasia was observed 4 mo postinfection. Because H. suis could be isolated from the stomachs of infected mice, our findings satisfied Koch's postulates. Although further prospective clinical studies are needed, H. suis, like H. pylori, is likely a gastric pathogen in humans. Furthermore, comparative genomic analysis of H. suis using complete genomes of clinical isolates revealed that the genome of each H. suis isolate contained highly plastic genomic regions encoding putative strain-specific virulence factors, including type IV secretion system-associated genes, and that H. suis isolates from humans and pigs were genetically very similar, suggesting possible pig-to-human transmission.

Genome-wide association meta-analysis identifies GP2 gene risk variants for pancreatic cancer.

Pancreatic cancer is the fourth leading cause of cancer-related deaths in Japan. To identify risk loci, we perform a meta-analysis of three genome-wide association studies comprising 2,039 pancreatic cancer patients and 32,592 controls in the Japanese population. Here, we identify 3 (13q12.2, 13q22.1, and 16p12.3) genome-wide significant loci (P < 5.0 × 10-8), of which 16p12.3 has not been reported in the Western population. The lead single nucleotide polymorphism (SNP) at 16p12.3 is rs78193826 (odds ratio = 1.46, 95% confidence interval = 1.29-1.66, P = 4.28 × 10-9), an Asian-specific, nonsynonymous glycoprotein 2 (GP2) gene variant. Associations between selected GP2 gene variants and pancreatic cancer are replicated in 10,822 additional cases and controls of East Asian origin. Functional analyses using cell lines provide supporting evidence of the effect of rs78193826 on KRAS activity. These findings suggest that GP2 gene variants are probably associated with pancreatic cancer susceptibility in populations of East Asian ancestry.

Analysis of the colonic mucosa associated microbiota (MAM) using brushing samples during colonic endoscopic procedures.

The mucosa-associated microbiota is an important component in human microbiota. The aim was to investigate mucosa-associated microbiota using brush samples during endoscopic procedures and compare with fecal microbiota. Seven patients who were planning to undergo a routine colonoscopy were recruited. Mucosal brushing samples were taken from 3 sites (terminal ileum, ascending and sigmoid colon), and a fecal sample was taken on the morning of colonoscopy. The samples were immediately placed in microcentrifuge tubes containing DNA stabilization reagent and analyzed using the next generation sequencer. The individual differences in microbiota were more evident than the differences of the sampling sites. Actinobacteria was more abundant and Bacteroidetes was less in the brush samples than those in the fecal samples. Taxonomic composition at the genus level and the proportion of genes responsible for some functions in the brushing samples tended to be different from those in the fecal samples. Bulleidia and Oribacteriumi were significantly more abundant and the proportions of genes responsible for transcription factors and phosphotransferase system were higher in ileal mucous than those in fecal samples. Brushing during colonoscopic procedure instead of using feces samples might be useful to analyze mucosa-associated microbiota.

Community-acquired fulminant colitis caused by binary toxin-producing Clostridium difficile in Japan.

We report a case of community-acquired fulminant colitis caused by Clostridium difficile in Japan. A 46-year-old woman was diagnosed with severe infectious enterocolitis and was admitted at another hospital. The stool culture was positive for toxigenic C. difficile. Since the patient presented with fulminant C. difficile infection (CDI) with toxic megacolon, respiratory insufficiency, and circulatory failure, she was transferred to Kyorin University Hospital for intensive care. Intubation and antibiotic therapy were performed. The general condition improved with conservative treatment, and she was discharged without sequelae. While the recovered isolate was toxin A and B-positive and binary toxin-positive, it was identified as polymerase chain reaction (PCR) ribotype ts0592 and slpA sequence type ts0592. The isolate was different from PCR ribotype 027 epidemic in Europe and North America. In Japan, binary toxin-producing strains are rare and have not caused an epidemic to date. Furthermore, there are few data on community-acquired CDI in Japan. In this case, a non-elderly woman with no major risk factors such as antibiotic use, administration of proton pump inhibitor and history of gastrointestinal surgery developed community-acquired fulminant CDI caused by the binary toxin-positive strain, and ICU treatment was required. Further studies focusing on the role of binary toxin-positive C. difficile in the severity of community-acquired CDI are necessary.

Enhanced infectivity of strains of Helicobacter pylori isolated from children compared with parental strains.

PURPOSE: Intra-familial infection, mother-to-child infection, is considered to be one of the main routes of transmission for Helicobacter pylori, in developed countries such as Japan. A major role for intra-familial spread in the pathogenicity of H. pylori is now beyond controversy, although the major route of transmission remains poorly understood. We performed this study to clarify the factors determining intra-familial transmission. METHODOLOGY: We used several H. pylori strains isolated from family members to compare infectivity. H. pylori K21 and K22 strains were isolated from the father and mother, and the K25 strain was isolated from the third child of the family. Mongolian gerbils were inoculated with H. pylori strains and the infectivity of three strains was compared in each experiment. In addition, the whole genome sequence, adhesion to gastric epithelial cells and the growth of static condition or continuous flow culture among three strains of H. pylori were analysed.Results/Key findings. Most of the colonies were determined as the same molecular type K25 in all of the four grouped animals and H. pylori K25 was observed as the dominant strain. The stronger adhesion capacity of the K25 strain was observed in comparison with the other two strains through in vitro analysis. By assessing the genomic profiles of H. pylori isolates from three strains, identified TnPZ regions were detected only in the K25 strain. CONCLUSION: The infectivity of H. pylori isolates intra-familial infection and animal infection were prescribed by the adhesion capacity and molecular type of each strain.

Evaluation of Risk Factors for Clostridium difficile Infection Based on Immunochromatography Testing and Toxigenic Culture Assay.

In recent years, the diagnostic method of choice for Clostridium difficile infection (CDI) is a rapid enzyme immunoassay in which glutamate dehydrogenase (GDH) antigen and C. difficile toxin can be detected (C. diff Quik Chek Complete; Alere Inc.) (Quik Chek). However, the clinical significance remains unclear in cases that demonstrate a positive result for GDH antigen and are negative for toxin. In this study, we used the Quik Chek test kit on fecal samples, with an additional toxin detection step using a toxigenic culture assay for the aforementioned cases. CDI risk factors were assessed among the 3 groups divided by the Quik Chek test results. The study involved 1,565 fecal samples from patients suspected to have CDI who were hospitalized during the period of April 2012 to March 2014. The 3 groups were defined as follows: both GDH antigen positive and toxin positive (by Quik Chek test) (toxin-positive [TP] group, n = 109), both GDH antigen and toxin negative (toxin-negative [TN] group, n = 111), and positive only for GDH antigen but toxin positive with subsequent toxigenic culture (toxigenic culture [TC] group, n = 72). The gender, age, number of hospitalization days, white blood cell (WBC) counts, serum albumin levels, body mass index (BMI), fecal consistency, and use of antibacterials and proton pump inhibiters (PPIs) were analyzed. The positive rate for the fecal direct Quik Chek test was 7.0% (109/1,565 cases). However, toxigenic culture assays using the Quik Chek test for only the GDH-antigen-positive/toxin-negative samples were 35.3% positive (72/204 cases). As a result, the true positive rate for C. difficile toxin detection was estimated to be 11.6% (181/1,565 cases). Moreover, significant differences (P < 0.05) in the number of hospitalization days (>50 days), WBC counts (>10,000 WBCs/µl), and use of PPIs comparing the TN, TP, and TC groups, were observed. The odds ratios (ORs) for the development of CDI were 1.61 (95% confidence interval [CI], 0.94 to 2.74) and 2.98 (95% CI, 1.59 to 5.58) for numbers of hospitalization days, 2.16 (95% CI, 1.24 to 3.75) and 2.24 (95% CI, 1.21 to 4.14) for WBC counts, and 9.03 (95% CI, 4.9 to 16.6) and 9.15 (95% CI, 4.59 to 18.2) for use of PPIs in the TP and TC groups, respectively. These findings demonstrated that the use of PPIs was a significant risk factor for CDI development. Moreover, antibacterials such as carbapenems, cephalosporins, and fluoroquinolones were demonstrated to be risk factors. In conclusion, identification of the TC group of patients is thought to be important, as this study demonstrates that this group bears the same high risk of developing CDI as the TP group.

Influence of Intestinal Indigenous Microbiota on Intrafamilial Infection by Helicobacter pylori in Japan.

Helicobacter pylori is a causative pathogen of chronic gastritis, gastric ulcer disease, and gastric cancer. Humans are known to be a natural host for H. pylori and tend to acquire the pathogen before the age of 5 years. The infection may then persist lifelong if eradication therapy is not applied. One of the modes of transmission of H. pylori is between family members, and therefore, the presence of infected family members is an important risk factor in children. However, other environmental factors have not been fully analyzed. The present study was performed to clarify whether and to what extent intestinal microbiota affect H. pylori intrafamilial infection. The fecal specimens from H. pylori-infected infants and H. pylori-infected and non-infected family members were collected in cohort studies conducted by Sasayama City, Hyogo Prefecture from 2010 to 2013. In total, 18 fecal DNA from 5 families were analyzed. Samples were amplified using 16S rRNA universal primers, and the amplicons were sequenced using the Ion PGM system. Principal-coordinate analysis demonstrated that there was no difference in intestinal microbiota between H. pylori-positive and H. pylori-negative groups. In intrafamilial comparison tests, the Manhattan distance of intestinal microbiota between the H. pylori-infected infant proband and H. pylori-negative mother was nearest in the family with low intestinal microbial diversity. However, in the family with the highest intestinal microbial diversity, the nearest Manhattan distance was shown between the H. pylori-infected infant proband and H. pylori-infected mother. The results in this study showed that the composition of the intestinal microbiota was very similar between members of the same family, and as such, colonization with organisms highly similar to the infected parent(s) may be a risk factor for H. pylori infection in children.

Helicobacter pylori sabA gene is associated with iron deficiency anemia in childhood and adolescence.

BACKGROUND: Gastric Helicobacter pylori colonization leads to iron deficiency anemia (IDA), especially in children and adolescents. However the pathogenesis is poorly understood. OBJECTIVE: We sought to identify specific H. pylori genes involved in IDA development, by comparing bacterial genome-wide expression profiling in patients affected or not. METHODS: H. pylori were isolated from four children with IDA and four from matched controls without IDA. Based on these isolates, cDNA microarrays under iron-replete or depleted conditions were systematically performed to compare gene expression profiles at the whole genome level. Real-time reverse-transcription (RT-) PCR and protein assays were performed for further assessing the profile differentiation of the identified H. pylori IDA-associated genes. RESULTS: We identified 29 and 11 genes with significantly higher or lower expression in the IDA isolates compared to non-IDA isolates, respectively. Especially notable were higher expression of sabA gene encoding sialic acid-binding adhesin in the IDA isolates, which was confirmed by real-time RT-PCR study. Moreover, iron-depletion in vitro led to up-regulation of fecA1 and frpB1 genes and down-regulation of pfr, as predicted. Known iron-regulated genes such as fur, pfr, fecA, and feoB did not significantly differ between both groups. The IDA isolates had significantly higher expression of vacuolating cytotoxin gene vacA than non-IDA isolates, consistent with the results of VacA protein assays. There were no significant differences in bacterial growth value between IDA and non-IDA isolates. CONCLUSIONS: It is likely that H. pylori carrying high expression of sabA causes IDA, especially in children and adolescents who have increased daily iron demand. In addition, it is possible that several host-interactive genes, including vacA, may play a synergistic role for sabA in IDA development.

Usefulness of detection of clarithromycin-resistant Helicobacter pylori from fecal specimens for young adults treated with eradication therapy.

BACKGROUND: To prevent Helicobacter pylori infection in the younger generation, it is necessary to investigate the prevalence of antibiotic-resistant H. pylori. OBJECTIVE: The aim of this study was to evaluate the method of PCR-based sequencing to detect clarithromycin (CAM) resistance-associated mutations using fecal samples as a noninvasive method. METHODS: DNA extracted from fecal specimens and isolates from gastric biopsy specimens were collected from patients with H. pylori infection. Antibiotic resistance to CAM was analyzed by molecular and culture methods. The detection rates of CAM resistance-associated mutations (A2142C or A2143G) were compared before and after eradication therapy. RESULTS: With CAM resistance of H. pylori evaluated by antibiotic susceptibility test as a gold standard, the sensitivity and the specificity of gene mutation detection from fecal DNA were 80% and 84.8%, respectively. In contrast, using DNA of isolated strains, the sensitivity and the specificity were 80% and 100%. Of the seven cases in which eradication was unsuccessful by triple therapy including CAM, CAM-resistant H. pylori, and resistance-associated mutations were detected in three cases, CAM-resistant H. pylori without the mutation was detected in two patients, and resistance-associated mutation was only detected in one patient. CONCLUSION: PCR-based sequencing to detect CAM resistance-associated mutations using isolates or fecal samples was useful for finding antibiotic-resistant H. pylori infection. Although the specificity of the detection from fecal samples compared with antibiotic susceptibility testing was lower than that from isolates, this fecal detection method is suitable especially for asymptomatic subjects including children. Further improvement is needed before clinical application.

Nationwide survey of Helicobacter pylori treatment for children and adolescents in Japan.

BACKGROUND: To prevent gastric cancer, a test-and-treat strategy for Helicobacter pylori has been proposed. This retrospective study assessed the clinical features, efficacy and safety of treatment for H. pylori infection in children and adolescents. METHODS: Questionnaires concerning the clinical features and treatment of H. pylori in children and adolescents were sent to doctors in 2013. It included questions on patient background, H. pylori-associated disease, first- and second-line treatment, success or failure of eradication, resistance to antibiotics, and occurrence of adverse events. In 2014, serious adverse events associated with treatment were analyzed. RESULTS: Invitation letters and questionnaires were sent to 1097 doctors, of whom 409 (37.3%) participated. Finally, 332 patients (mean age, 11.6 ± 3.4 years; male, n = 200) treated from 1997 to 2013 were analyzed. H. pylori-associated gastritis, iron deficiency anemia, and duodenal ulcer occurred most frequently. Success rates for first- and second-line treatments were 73.1% and 79.6%, respectively. Seventy-six H. pylori strains were analyzed for resistance to amoxicillin (AMPC) and clarithromycin (CAM), and 64 were analyzed for resistance to metronidazole (MNZ). CAM resistance was most frequent, occurring in 43.4% of patients; that of MNZ was 21.9%. Adverse events were observed in 13.8% of cases. In total, 587 cases of H. pylori infection were analyzed and no serious adverse events were observed. CONCLUSIONS: Treatment for H. pylori in children and adolescents is safe, but further studies on treatment regimens should be conducted to improve eradication rates and monitor increasing CAM resistance.

Microevolution of Virulence-Related Genes in Helicobacter pylori Familial Infection.

Helicobacter pylori, a bacterial pathogen that can infect human stomach causing gastritis, ulcers and cancer, is known to have a high degree of genome/epigenome diversity as the result of mutation and recombination. The bacteria often infect in childhood and persist for the life of the host. One of the reasons of the rapid evolution of H. pylori is that it changes its genome drastically for adaptation to a new host. To investigate microevolution and adaptation of the H. pylori genome, we undertook whole genome sequencing of the same or very similar sequence type in multi-locus sequence typing (MLST) with seven genes in members of the same family consisting of parents and children in Japan. Detection of nucleotide substitutions revealed likely transmission pathways involving children. Nonsynonymous (amino acid changing) mutations were found in virulence-related genes (cag genes, vacA, hcpDX, tnfα, ggt, htrA and the collagenase gene), outer membrane protein (OMP) genes and other cell surface-related protein genes, signal transduction genes and restriction-modification genes. We reconstructed various pathways by which H. pylori can adapt to a new human host, and our results raised the possibility that the mutational changes in virulence-related genes have a role in adaptation to a child host. Changes in restriction-modification genes might remodel the methylome and transcriptome to help adaptation. This study has provided insights into H. pylori transmission and virulence and has implications for basic research as well as clinical practice.

Low prevalence and incidence of Helicobacter pylori infection in children: a population-based study in Japan.

BACKGROUND: Infection of Helicobacter pylori mainly occurs in childhood. In Japan, incidence of gastric cancer is still high in the senior citizen population, but little is known about the current H. pylori infection status among children or their family members. METHODS: As a population-based study, the prevalence of H. pylori infection and change in infection status over a 1-year interval in children were determined. Family members of some participants were also invited to participate in the study to determine their infection status. All children of specific ages attending 16 schools in Sasayama, Hyogo Prefecture, were invited to participate. H. pylori infection was determined by the stool antigen test and diagnosis confirmed by polymerase chain reaction and the urea breath test. RESULTS: Helicobacter pylori prevalence was 1.9% among 689 children aged 0-8 years in 2010 and 1.8% among 835 children aged 0-11 in 2011. No feco-conversion was observed in 430 children aged 0-8 years (170 were aged 0-4 years) who provided follow-up stool samples after 1 year. The prevalence of infection was 6% (2 of 33) and 38% (6 of 16) in mothers of negative and positive probands (p = .04), respectively, and 12% (3 of 25) and 50% (8 of 16) (p = .01), respectively, in fathers. CONCLUSION: Helicobacter pylori prevalence in Japanese children is approximately 1.8%, which is much lower than that reported in Japanese adults. New infection may be rare. Parent-to-child infection is thought to be the main infection route of the infrequent infection for children in Japan.

Development of new PCR primers by comparative genomics for the detection of Helicobacter suis in gastric biopsy specimens.

BACKGROUND: Although the infection rate of Helicobacter suis is significantly lower than that of Helicobacter pylori, the H. suis infection is associated with a high rate of gastric mucosa-associated lymphoid tissue (MALT) lymphoma. In addition, in vitro cultivation of H. suis remains difficult, and some H. suis-infected patients show negative results on the urea breath test (UBT). MATERIALS AND METHODS: Female C57BL/6J mice were orally inoculated with mouse gastric mucosal homogenates containing H. suis strains TKY or SNTW101 isolated from a cynomolgus monkey or a patient suffering from nodular gastritis, respectively. The high-purity chromosomal DNA samples of H. suis strains TKY and SNTW101 were prepared from the infected mouse gastric mucosa. The SOLiD sequencing of two H. suis genomes enabled comparative genomics of 20 Helicobacter and 11 Campylobacter strains for the identification of the H. suis-specific nucleotide sequences. RESULTS: Oral inoculation with mouse gastric mucosal homogenates containing H. suis strains TKY and SNTW101 induced gastric MALT lymphoma and the formation of gastric lymphoid follicles, respectively, in C57BL/6J mice. Two conserved nucleotide sequences among six H. suis strains were identified and were used to design diagnostic PCR primers for the detection of H. suis. CONCLUSIONS: There was a strong association between the H. suis infection and gastric diseases in the C57BL/6 mouse model. PCR diagnosis using an H. suis-specific primer pair is a valuable method for detecting H. suis in gastric biopsy specimens.

Role of (p)ppGpp in biofilm formation and expression of filamentous structures in Bordetella pertussis.

Bordetella pertussis, the causative agent of whooping cough, is highly adapted to cause human infection. The production of virulence factors, such as adhesins and toxins, is just part of an array of mechanisms by which B. pertussis causes infection. The stringent response is a global bacterial response to nutritional limitation that is mediated by the accumulation of cellular ppGpp and pppGpp [termed together as (p)ppGpp]. Here, we demonstrate that production of (p)ppGpp was controlled by RelA and SpoT proteins in B. pertussis, and that mutation-induced loss of both proteins together caused deficiencies in (p)ppGpp production. The (p)ppGpp-deficient mutants also exhibited defects in growth regulation, decreases in viability under nutritionally limited conditions, increases in susceptibility to oxidative stress and defects in biofilm formation. Analysis of the secreted proteins and the respective transcripts showed that lack of (p)ppGpp led to decreased expression of fim3 and bsp22, which encode a fimbrial subunit and the self-polymerizing type III secretion system tip protein, respectively. Moreover, electron microscopic analysis also indicated that (p)ppGpp regulated the formation of filamentous structures. Most virulence genes - including fim3 and bsp22 - were expressed in the Bvg(+) phase during which the BvgAS two-component system was activated. Although fim3 and bsp22 were downregulated in a (p)ppGpp-deficient mutant, normal expression of fhaB, cyaA and ptxA persisted. Lack of coherence between virulence gene expression and (p)ppGpp production indicated that (p)ppGpp did not modulate the Bvg phase. Taken together, our data indicate that (p)ppGpp may govern an as-yet-unrecognized system that influences B. pertussis pathogenicity.

Effect of the steroid receptor antagonist RU486 (mifepristone) on an IFNγ-induced persistent Chlamydophila pneumoniae infection model in epithelial HEp-2 cells.

We have previously demonstrated that the steroid receptor antagonist mifepristone (RU486) causes growth inhibition of Chlamydophila pneumoniae by binding to and subsequently destroying the bacteria during their normal developmental cycle in epithelial HEp-2 cells. In the present study, we assessed the efficacy of treatment with RU486 against persistent C. pneumoniae infection in interferon (IFN)γ-treated HEp-2 cells. Assessment of bacterial growth modification, the number of infectious progenies, the formation of inclusions, and the expressions of the C. pneumoniae genes 16S rRNA and hsp60 were investigated in cells with or without IFNγ stimulation in the presence of RU486, using an inclusion-forming unit (IFU) assay, fluorescence microscopic analysis, and reverse transcription polymerase chain reaction (RT-PCR), respectively. Our results indicated that RU486 treatment produced growth inhibition and an absence of C. pneumoniae gene expression in normal HEp-2 cells and that this treatment failed to inhibit C. pneumoniae growth in HEp-2 cells stimulated with IFNγ. These results indicate that treatment with RU486 had a limited effect on C. pneumoniae growth only during the active developmental stage of the bacteria, suggesting that the bacterial target molecule of RU486 is not expressed sufficiently during persistent infection in which there is an aberrant developmental cycle. Thus, our findings provide valuable insight into the complicated chlamydial biological processes involved in the recurrent cycling between normal and persistent infections.

Analysis of outer membrane vesicle protein involved in biofilm formation of Helicobacter pylori.

Helicobacter pylori is one of the most common causes of bacterial infection in humans. Infection with H. pylori is closely associated with gastritis and peptic ulcers and is a risk factor for gastric cancer and mucosa-associated lymphoid tissue lymphoma. H. pylori forms biofilms on glass surfaces at the air-liquid interface in in-vitro batch cultures. We previously reported that strain TK1402 showed a strong biofilm-forming ability in vitro. We also suggested the outer membrane vesicles (OMV) produced by strain TK1402 might be related to its biofilm forming ability. In the present study, we analyzed the protein profile of the OMV produced by strain TK1402 and found a unique 22-kDa protein in TK1402 OMV cultured for 2-3 days. In addition, this protein could not be detected in the OMVs produced by other H. pylori strains. These results suggest that the 22-kDa protein is involved in effective biofilm formation by strain TK1402.

Urease-positive bacteria in the stomach induce a false-positive reaction in a urea breath test for diagnosis of Helicobacter pylori infection.

This study investigated the influence of urease-positive non-Helicobacter pylori bacteria on the results of a urea breath test (UBT) to evaluate the diagnostic utility of a UBT using film-coated [(13)C]urea tablets. The UBT was performed in 102 patients treated with a proton pump inhibitor and antibiotics for the eradication of H. pylori. Urease-producing bacteria other than H. pylori were isolated and identified from the oral cavity and stomach. In 4/102 patients, the UBT gave false-positive results. These false-positive results were found to be caused by the presence of urease-positive bacteria in the oral cavity and stomach. Five bacterial species with urease activity (Proteus mirabilis, Citrobacter freundii, Klebsiella pneumoniae, Enterobacter cloacae and Staphylococcus aureus) were subsequently isolated from the oral cavity and/or stomach. As there was no correlation between the in vitro urease activity of urease-positive non-H. pylori bacteria and the UBT value, and all of the patients with a false-positive UBT result were suffering from atrophic gastritis, it is possible that the false-positive results in the UBT were a result of colonization of urease-positive bacteria and gastric hypochlorhydric conditions. Thus, for the diagnosis of H. pylori infection using a UBT, the influence of stomach bacteria must be considered when interpreting the results.

Chlamydia pneumoniae growth inhibition in cells by the steroid receptor antagonist RU486 (mifepristone).

Since steroids are powerful anti-inflammatory agents and increase susceptibility to a variety of infections, including Chlamydia (Chlamydophila) pneumoniae respiratory tract infections, the effect of the steroid receptor antagonist RU486 (mifepristone) on C. pneumoniae growth in epithelial HEp-2 cells was examined. Treatment of HEp-2 cells with RU486 significantly inhibited the growth of C. pneumoniae in a dose-dependent manner. Electron microscopic studies also revealed that the treatment of infected cells with RU486 resulted in a marked destruction of infecting organisms. The addition of the host cell protein synthesis inhibitor cycloheximide to the infected cells did not alter the inhibition of C. pneumoniae growth by RU486. Pretreatment of C. pneumoniae organisms with RU486 before addition to culture also did not result in any modulation of bacterial growth in the cells. However, the binding of RU486 to C. pneumoniae organisms in cells at 24 h after infection was demonstrated by immune electron microscopy with anti-RU486 antibody. Incubation of cells with anti-RU486 antibody completely diminished the inhibition of C. pneumoniae growth by RU486. These results indicate that RU486 may directly bind to the bacteria within cells and cause the destruction of C. pneumoniae. This novel mode of regulation of C. pneumoniae growth in cells by RU486 might provide a new approach to understanding complicated aspects of C. pneumoniae infection.

Development of diabetes in non-obese diabetic mice promotes Chlamydia pneumoniae dissemination from lung to peripheral blood.

We examined a possible association between development of diabetes in non-obese diabetic (NOD) mice and dissemination of Chlamydia (Chlamydophila) pneumoniae from lung to peripheral blood. By real-time reverse transcription-polymerase chain reaction (RT-PCR) with primers for C. pneumoniae 16S rRNA, following multiple intranasal inoculations, we detected bacteria in lung in NOD mice with diabetes (38.5%) as well as Institute of Cancer Research, USA (ICR) mice (40%), but prevalence of bacteria in NOD mice without diabetes (pre-diabetic NOD mice and non-diabetic retired NOD mice) was very low (4.8%). The bacteria were only detected in peripheral blood mononuclear cells (PBMCs) cultured with hydrocortisone of the NOD mice with diabetes (53.8%). Results of immunostaining with fluorescein isothiocyanate-conjugated antichlamydia monoclonal antibody also showed the presence of bacterial antigens in the lungs and the PBMCs judged as positive by the RT-PCR. However, C. pneumoniae from cultured PBMCs of all NOD mice was undetected by cultivation method with inclusion-forming units assay. In addition, no influence of C. pneumoniae intranasal inoculation on development of diabetes in NOD mice was confirmed. Thus, the development of diabetes in NOD mouse appears to be one of critical factors for promoting the dissemination of C. pneumoniae from lung to peripheral blood.