Antimicrobial-resistant Helicobacter pylori in Japan: Report of nationwide surveillance for 2018-2020.

BACKGROUND: Antimicrobial therapy is necessary to eradicate Helicobacter pylori infection. The emergence of antimicrobial-resistant bacteria poses a threat to continued treatment with antimicrobial agents. For those who prescribe antimicrobial therapy, it is necessary to constantly monitor the emergence of antimicrobial-resistant bacteria. METHOD: H. pylori clinical isolates were collected in Japan from August 2018 to December 2020 for antimicrobial susceptibility testing. The agar dilution method was used for the determination of the minimum inhibitory concentration (MIC) of clarithromycin (CLR), amoxicillin (AMX), metronidazole (MNZ), and sitafloxacin (STX). RESULTS: MICs for 938 H. pylori isolates were examined. The primary resistance rates of H. pylori clinical isolates for CLR, AMX, MNZ, and STX in Japan were 35.5%, 2.7%, 4.2%, and 27.6%, respectively. The primary resistance rates for CLR, AMX, and MNZ were significantly higher than those of the 2002-2005 isolates. The resistance rate for CLR was significantly higher in females (males: 30.7%, females: 41.5%, p < 0.001) and higher in the ≤29 years age group (54.8%) than in the other age groups, although there were no significant differences (p = 0.104). The MNZ resistance rate was significantly higher in the ≤29 years age group than in the other age groups (p = 0.004). The resistance rate for STX increased with age, but a significant difference was only seen between the 30-49 years age group and the ≥70 years age group (p < 0.001), and the resistance rate was significantly higher in strains isolated in the Kyushu region than in the other regions (p < 0.001). CONCLUSIONS: The primary resistance rates for CLR, AMX, and MNZ of H. pylori clinical isolates in Japan were higher than those of the 2002-2005 isolates. Continuous surveillance is needed to monitor the trends in antimicrobial-resistant H. pylori.

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.

Establishment of a reference panel of Helicobacter pylori strains for antimicrobial susceptibility testing.

BACKGROUND: Eradication treatment for Helicobacter pylori gastritis is covered by national health insurance since 2013 in Japan. However, eradication failure due to the increase of antimicrobial resistance has become a serious problem. The present study aims to establish a reference panel of Japanese H. pylori strains for antimicrobial susceptibility testing. METHOD: A total of 28 strains were collected from 4 medical facilities in Japan. Antimicrobial susceptibility tests (ASTs) to clarithromycin (CLR), amoxicillin (AMX), and metronidazole (MNZ), were used to select standard reference strains. Complete genome sequences were also determined. RESULTS: Three H. pylori strains (JSHR3, JSHR6 and JSHR31) were selected as standard reference strains by the Japanese Society for Helicobacter Research (JSHR). The minimum inhibitory concentrations (MICs) of the antibiotics against these 3 strains by agar dilution method with Brucella-based horse-serum-containing agar medium were as follows: JSHR3 (CLR 16 µg/ml, AMX 0.032 µg/ml and MNZ 4 µg/ml), JSHR6 (CLR 0.016 µg/ml, AMX 0.032 µg/ml and MNZ 4 µg/ml), and JSHR31 (CLR 16 µg/ml, AMX 1 µg/ml and MNZ 64 µg/ml). CONCLUSIONS: A reference panel of H. pylori JSHR strains was established. The panel consisted of JSHR6, which was antibiotic-susceptible, JSHR3, which was CLR-resistant, and JSHR31, which was multi-resistant. This reference panel will be essential for standardized ASTs before the optimal drugs are selected for eradication treatment.

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.

Lantibiotics Produced by Oral Inhabitants as a Trigger for Dysbiosis of Human Intestinal Microbiota.

Lantibiotics are a type of bacteriocin produced by Gram-positive bacteria and have a wide spectrum of Gram-positive antimicrobial activity. In this study, we determined that Mutacin I/III and Smb (a dipeptide lantibiotic), which are mainly produced by the widespread cariogenic bacterium Streptococcus mutans, have strong antimicrobial activities against many of the Gram-positive bacteria which constitute the intestinal microbiota. These lantibiotics also demonstrate resistance to acid and temperature. Based on these features, we predicted that lantibiotics may be able to persist into the intestinal tract maintaining a strong antimicrobial activity, affecting the intestinal microbiota. Saliva and fecal samples from 69 subjects were collected to test this hypothesis and the presence of lantibiotics and the composition of the intestinal microbiota were examined. We demonstrate that subjects possessing lantibiotic-producing bacteria in their oral cavity exhibited a tendency of decreased species richness and have significantly reduced abundance of the phylum Firmicutes in their intestinal microbiota. Similar results were obtained in the fecal microbiota of mice fed with S. mutans culture supernatant containing the lantibiotic bacteriocin Mutacin I. These results showed that lantibiotic bacteriocins produced in the oral cavity perturb the intestinal microbiota and suggest that oral bacteria may be one of the causative factors of intestinal microbiota dysbiosis.

Serum Pepsinogen Values in Japanese Junior High School Students With Reference to Helicobacter Pylori Infection.

BACKGROUND: Distributions of serum pepsinogen (PG) values were assessed in Helicobacter pylori-infected and non-infected junior high school students (aged 12-15 years) in Japan. METHODS: All junior high school students (1,225 in total) in Sasayama city, who were basically healthy, were asked to provide urine and serum samples, which were used to measure urine and serum H. pylori antibodies using ELISA kits and PG values. The subjects, whose urine and serum antibodies were both positive, were considered H. pylori infected. RESULTS: Of the 187 subjects who provided urine and blood samples, 8 were infected, 4 had discrepant results, 4 had negative serum antibody titers no less than 3.0 U/ml, and 171 were non-infected. In the H. pylori non-infected subjects, the median PG I and PG II values and PG I to PG II ratio (PG I/II) were 40.8 ng/mL, 9.5 ng/mL, and 4.4, respectively, whereas in the infected subjects, these values were 55.4 ng/mL, 17.0 ng/mL, and 3.3, respectively (each P < 0.01). In the non-infected subjects, PG I and PG II were significantly higher in males than in females (P < 0.01). CONCLUSIONS: The PG I and PG II values were higher, and the PG I/II was lower in H. pylori infected students than in non-infected students. In H. pylori non-infected students, males showed higher PG I and PG II values than females. The distributions of PG values in junior high school students differed from those in adults.

Acanthamoeba castellanii supports extracellular survival of Helicobacter pylori in co-culture.

This study aimed to demonstrate whether Helicobacter pylori is able to survive in co-culture with a protozoan, Acanthamoeba castellanii, in order to further investigate a possible aqueous environmental mode of transmission. Numbers of H. pylori in co-culture with A castellanii were assessed by colony forming unit (CFU) assay and cell morphology was observed by electron microscopy. Viable and intact H. pylori in co-culture were detected and the number of H. pylori in co-culture with A. castellanii was significantly higher than in bacterial single culture. It was also shown that co-culture of H. pylori with A. castellanii physically separated by a filter membrane negated this survival effect, suggesting that adherence of H. pylori to A. castellanii affects its survival. Scanning electron microscopy revealed helical forms of H. pylori in co-culture with A. castellanii, but not in single culture. These results imply that mutual interaction between H. pylori and A. castellanii in the environment is critical for survival of H. pylori. In addition, the H. pylori gene expression profile was found to differ between single and co-cultured cells using RNA-sequence analysis.

Effect of Helicobacter pylori biofilm formation on susceptibility to amoxicillin, metronidazole and clarithromycin.

The human gastric pathogen Helicobacter pylori forms biofilms in vitro and in vivo. We previously demonstrated that H. pylori biofilm formation in vitro decreased its susceptibility to clarithromycin (CAM). The aim of this study was to evaluate the effects of biofilm formation on amoxicillin (AMPC) and metronidazole (MNZ) susceptibility. In addition, we assessed the influence of biofilms of CAM resistant H. pylori on CAM susceptibility. It was shown that high levels of efflux pump gene transcripts were detected in biofilm cells of all H. pylori strains used in this study. H. pylori biofilm biomass was significantly decreased compared to initial biomass after treatment with the minimum inhibitory concentration (MIC) of AMPC. Similarly, the biofilm biomass of H. pylori decreased after treatment with MIC of MNZ, although the difference was not statistically significant. However, minimum bactericidal concentrations (MBCs) of AMPC or MNZ to biofilm cells were higher than those of planktonic cells. The biofilm biomasses of all of the CAM resistant strains were significantly decreased compared to initial biomass after treatment with 2x MIC of CAM. However, the viability of the CAM treated biofilm cells with 2x MIC of CAM was not significantly reduced compared to initial cell numbers with the exception of one strain. The viability of biofilm cells of all strains was higher than that of planktonic cells after treatment with various concentrations of CAM. These results indicate that biofilm cells were more resistant to these antibiotics than planktonic cells and that the assessment of the ability to form biofilms in H. pylori is important for eradication of this microorganism.

Role of Probiotics in Eradication Therapy for Helicobacter pylori Infection.

Probiotics are defined as, "Live microorganisms that, when administered in adequate amounts, confer a health benefit on the host", and have various effects including inhibitory capabilities on pathogens, stimulation of organ functions and activation of immune responses in the human. Probiotics were reported to inhibit Helicobacter pylori not only in vitro, but also in vivo studies. The mechanisms by which probiotics inhibit H. pylori infection include competition for nutrients, production of bactericidal substances, competitive inhibition of adherence and stimulation of host functions and immunity. In addition, probiotics are clinically used for eradication therapy of H. pylori infection, and the effects of probiotics as single treatment and combination use with other drugs including proton pump inhibitors and antibiotics against H. pylori are reported. It has been testified that probiotics increase the eradication rate and prevent adverse events including diarrhea, nausea, vomiting and taste disorder. In the Maastrich V/Florence Consensus Report 2017, it was stated that some probiotics may have a beneficial effect on H. pylori eradication and are effective in reducing side effects of eradication therapy, but more research is needed to answer several questions concerning the mechanisms of probiotics action. In addition, strain specificity, dosages and duration times of probiotics used for H. pylori eradication therapy need to be clarified in future studies.

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.

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.

Diversification of the AlpB Outer Membrane Protein of Helicobacter pylori Affects Biofilm Formation and Cellular Adhesion.

Helicobacter pylori is one of the most common causes of bacterial infection in humans, and it forms biofilms on human gastric mucosal epithelium as well as on in vitro abiotic surfaces. Bacterial biofilm is critical not only for environmental survival but also for successful infection. We previously demonstrated that strain TK1402, which was isolated from a Japanese patient with duodenal and gastric ulcers, has high biofilm-forming ability in vitro relative to other strains. In addition, we showed that outer membrane vesicles (OMV) play an important role in biofilm formation. The aim of this study was to analyze which protein(s) in the OMV contributes to biofilm formation in TK1402. We obtained a spontaneous mutant strain derived from TK1402 lacking biofilm-forming ability. The protein profiles of the OMV were compared between this mutant strain and the wild type, and it was found that AlpB, an outer membrane protein in the OMV of the mutant strain, was markedly decreased compared to that of the wild type. Restoration of TK1402 alpB to the mutant strain fully recovered the ability to form biofilm. However, restoration with alpB from other strains demonstrated incomplete recovery of biofilm-forming ability. We therefore inferred that the variable region of AlpB (amino acid positions 121 to 146) was involved in TK1402 biofilm formation. In addition, diversification of the AlpB sequence was shown to affect the ability to adhere to AGS cells. These results demonstrate a new insight into the molecular mechanisms of host colonization by H. pyloriIMPORTANCE Bacterial biofilm is critical not only for environmental survival but also for successful infection. The mechanism of Helicobacter pylori adherence to host cells mediated by cell surface adhesins has been the focus of many studies, but little is known regarding factors involved in H. pylori biofilm formation. Our study demonstrated that AlpB plays an important role in biofilm formation and that this property depends upon the specific sequence of alpB This in turn was shown to be important in the ability to adhere to gastric cells. We anticipate that these results will provide new insight into the molecular mechanisms of H. pylori colonization.

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.

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.

Rebamipide protects small intestinal mucosal injuries caused by indomethacin by modulating intestinal microbiota and the gene expression in intestinal mucosa in a rat model.

The effect of rebamipide, a mucosal protective drug, on small intestinal mucosal injury caused by indomethacin was examined using a rat model. Indomethacin administration (10 mg/kg, p.o.) induced intestinal mucosal injury was accompanied by an increase in the numbers of intestinal bacteria particularly Enterobacteriaceae in the jejunum and ileum. Rebamipide (30 and 100 mg/kg, p.o., given 5 times) was shown to inhibit the indomethacin-induced small intestinal mucosal injury and decreased the number of Enterococcaceae and Enterobacteriaceae in the jejunal mucosa to normal levels. It was also shown that the detection rate of segmented filamentous bacteria was increased by rebamipide. PCR array analysis of genes related to inflammation, oxidative stress and wound healing showed that indomethacin induced upregulation and downregulation of 14 and 3 genes, respectively in the rat jejunal mucosa by more than 5-fold compared to that of normal rats. Rebamipide suppressed the upregulated gene expression of TNFα and Duox2 in a dose-dependent manner. In conclusion, our study confirmed that disturbance of intestinal microbiota plays a crucial role in indomethacin-induced small intestinal mucosal injury, and suggests that rebamipide could be used as prophylaxis against non-steroidal anti-inflammatory drugs -induced gastrointestinal mucosal injury, by modulating microbiota and suppressing mucosal inflammation in the small intestine.

Role of IL-17A and IL-10 in the antigen induced inflammation model by Mycoplasma pneumoniae.

BACKGROUND: Mycoplasma pneumoniae is one of the causative organisms of community-acquired pneumonia which is found commonly in younger patients. Extrapulmonary complications similar to autoimmune disease are caused by M. pneumoniae following the initial infection. The mechanism and pathology of onset is not clear, but it is considered that excessive host immunoreactions play a part in the onset of mycoplasmal pneumonia and its extrapulmonary complications. In this study, we investigated the participation of the immune response, excluding the participation of Th1 and Th2 which has previously been investigated. RESULTS: In this study, the host immune response of an antigen induced inflammation model using SPF mice repeatedly sensitized with M. pneumoniae antigens was analyzed. The specificity of M. pneumoniae antigens in the Th17 response of murine lymphocytes in vitro was also examined. Frequent and concentrated sensitization induced exacerbation of lung inflammation immunologically and pathologically, and evoked intrapulmonary IL-17A and IL-10 production. M. pneumoniae antigen stimulation induced proliferation of mouse lymphocytes and caused production of IL-17A and IL-10. In addition, it was shown that IL-17A and IL-10 production was increased in the presence of IL-6 and TGF-ß1. CONCLUSIONS: It was shown that M. pneumoniae antigens induced potent immunoreaction and enhanced the Th17 cell response both in vivo and in vitro, and that both Treg and IL-10 are involved in the suppression of IL-17A production. This raises the possibility that breakdown of the immune balance may be part of the process leading to subsequent development of extrapulmonary mycoplasmal pneumonia.

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.

Differential impact of environmental factors on airborne live bacteria and inorganic particles in an underground walkway.

We previously reported that variations in the number and type of bacteria found in public spaces are influenced by environmental factors. However, based on field survey data alone, whether the dynamics of bacteria in the air change as a result of a single environmental factor or multiple factors working together remains unclear. To address this, mathematical modeling may be applied. We therefore conducted a reanalysis of the previously acquired data using principal component analysis (PCA) in conjunction with a generalized linear model (Glm2) and a statistical analysis of variance (ANOVA) test employing the χ2 distribution. The data used for the analysis were reused from a previous public environmental survey conducted at 8:00-20:00 on May 2, June 1, and July 5, 2016 (regular sampling) and at 5:50-7:50 and 20:15-24:15 on July 17, 2017 (baseline sampling) in the Sapporo underground walking space, a 520-meter-long underground walkway. The dataset consisted of 60 samples (22 samples for "bacterial flora"), including variables such as "temperature (T)," "humidity (H)," "atmospheric pressure (A)," "traffic pedestrians (TP)," "number of inorganic particles (Δ5: 1-5 µm)," "number of live airborne bacteria," and "bacterial flora." Our PCA with these environmental factors (T, H, A, and TP) revealed that the 60 samples could be categorized into four groups (G1 to G4), primarily based on variations in PC1 [Loadings: T(-0.62), H(-0.647), TP(0.399), A(0.196)] and PC2 [Loadings: A(-0.825), TP(0.501), H(0.209), T(-0.155)]. Notably, the number of inorganic particles significantly increased from G4 to G1, but the count of live bacteria was highest in G2, with no other clear pattern. Further analysis with Glm2 indicated that changes in inorganic particles could largely be explained by two variables (H/TP), while live bacteria levels were influenced by all explanatory variables (TP/A/H/T). ANOVA tests confirmed that inorganic particles and live bacteria were influenced by different factors. Moreover, there were minimal changes in bacterial flora observed among the groups (G1-G4). In conclusion, our findings suggest that the dynamics of live bacteria in the underground walkway differ from those of inorganic particles and are regulated in a complex manner by multiple environmental factors. This discovery may contribute to improving public health in urban settings.

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.