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We detected Helicobacter cinaedi in 4 of 10 patients with infected aortic aneurysms diagnosed using blood or tissue culture in Aichi, Japan, during September 2017-January 2021. Infected aortic aneurysms caused by H. cinaedi had a higher detection rate and better results after treatment than previously reported, without recurrent infection.

Helicobacter cinaediinfects the human gut and causes invasive infections such as bacteremia and cellulitis by bacterial translocation. However, howH. cinaediattaches to host cells and establishes infection has not been elucidated. In this study, we focused on a novel autotransporter protein, i.e.,H. cinaediautotransporter protein A (HcaA) to examine its relationship withH. cinaedipathogenicity. The cytotoxicity ofH. cinaediinfection in the colon epithelial cell lines (Caco-2 and HT-29) by lactate dehydrogenase assay showed a significant reduction in cytotoxicity by HcaA knockout. Adhesion assays further revealed that the HcaA-knockout strain showed significantly reduced attachment ofH. cinaedito the human epithelial colorectal adenocarcinoma cell line (Caco-2) compared to the wild-type strain. To determine the role of HcaA inH. cinaediinfectionin vivo, H. cinaediwild-type and HcaA-knockout strains were orally infected C57BL/6 mice. The colonized bacteria were then measured 7, 14, and 28-days post-infection. The number of colonizedH. cinaedicells was significantly lower in HcaA-knockout strain infections than in wild-type strain infections at 7 days post-infection. Recombinant HcaA protein showed strong adhesion characteristics to the human monocytic cell line (U937) by adhesion assay used recombinant purified HcaA protein. The adherent activity was diminished by the replacement of the RGD motif in HcaA with RAD, indicating the contribution of the RGD motif in HcaA to host cell adherence. These results suggest that HcaA, a novel autotransporter protein inH. cinaedi, plays a significant role in establishing infection as an adhesin.IMPORTANCEHelicobacterspecies are classified as gastric or enterohepatic according to their habitat. EnterohepaticHelicobacterspecies inhabit the intestine, colon and liver; among them,H. cinaedihas been most frequently isolated from humans.H. cinaedicommonly causes bacteremia and cellulitis in immunocompromised hosts. Here, we focused on theH. cinaediautotransporter protein A (HcaA), a novel virulence factor inH. cinaedi. We discovered that HcaA contributed to cell adhesion via its RGD motif. Furthermore, in animal experiments, the number of colonized bacteria was decreased in HcaA knockout strain infections, supporting the hypothesis that HcaA contributes to the adhesion ofH. cinaedito host cells. Our study provides a novel mechanism for the establishment ofH. cinaediinfections and is expected to provide new insights into the role of autotransporter proteins in the establishment ofHelicobacterspecies infection.

SUMMARYHelicobacter suishosted by hogs is the most prevalent gastric non-Helicobacter pylori Helicobacterspecies found in humans. Recent studies suggest that theH. suisinfection has already induced many cases of gastric disease. However, the infection period and route ofH. suisfrom hogs remain unclear. Because diagnostic methods based on the urease activity ofH. suisoften yield negative judgments, there is no reliable method for diagnosingH. suisinfection in clinical practice without gastric biopsy specimens. We developed the world’s first ELISA to simultaneously diagnoseH. suisandH. pyloriinfection in a single test. The area under the ROC curve was 0.9648 or 0.9200 for identifyingH. suisorH. pyloriinfection, respectively. The sensitivity, specificity, and positive and negative predictive values for identifyingH. suisinfection were 100%, 92.6%, 76.9%, and 100%, and those for identifyingH. pyloriinfection were 88.2%, 87.5%, 65.2%, and 96.6%, respectively. (150 words)

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.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.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).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.

Significance This study directly demonstrates virulence-associated features of Helicobacter suis infection using isolates obtained from human stomachs. H. suis is the second-most-prevalent Helicobacter species in the human stomach, but diagnostic tests for Helicobacter pylori often fail to diagnose H. suis. H. suis does not have orthologs of either cytotoxin-associated gene A (CagA) or vacuolating cytotoxin A (VacA), two major virulence factors in H. pylori, suggesting that the molecular pathogenicity of H. suis and clinical manifestations caused by H. suis infection are different from those caused by H. pylori. This study paves the way for epidemiological research aimed at identifying the causal relationships between H. suis and gastric diseases and developing diagnostic methods., 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.

Helicobacter suis strain SNTW101c, which was originally obtained from a patient with nodular gastritis, has been maintained in mouse stomach because of difficulty culturing it in vitro. Recently, we succeeded in culturing this strain in vitro. Here, we report the complete genome sequence of H. suis strain SNTW101c.

Background: 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. The aim of this study is to elucidate the pathogenicity of H. suis by genetic characterization and through animals infected with H. suis isolated from human stomachs. Methods: H. suis from human gastric biopsies was isolated as previously described in pigs, with modifications. Mice were orally infected with H. suis for 4 months, and then pathogenesis was analyzed. Whole-genome analysis of H. suis isolates was performed by short-read and long-read sequencing, and their genomes were subjected to comparative analysis. Findings: H. suis was successfully isolated from three patients with gastric diseases, including gastric MALT lymphoma. Successful eradication of H. suis yielded significant improvements in endoscopic and histopathological findings in all patients. H. suis infection in mice using clinical isolates elicited gastric and systemic inflammatory responses, and H. suis could be isolated from the stomachs of infected mice. Therefore, according to Koch’s postulates, these findings demonstrate that H. suis is a human gastric pathogen. Genomic analysis revealed high similarity between isolates from humans and pigs, suggesting possible horizonal transmission of H. suis. Interpretation: This is the first study to directly demonstrate virulence-associated features of H. suis infection using isolates obtained from human stomachs. Since H. suis would have been missed by routine diagnostic tests for Helicobacter pylori, large-scale epidemiological research on H. suis is needed to identify causal relationships with gastric diseases and develop novel diagnostic methods. Funding: MEXT/JSPS KAKENHI Grant numbers JP19H03474 for (HM) and JP20K08365 for (KT), and AMED Grant Numbers JP20fk0108148 (ER) and JP20fk0108133 (MS). Conflict of Interest: ER has received research funding from GlaxoSmithKline, outside the submitted work. All other authors declare no competing interests. Ethical Approval: Ethical approvals were obtained from all participating hospitals and the National Institute of Infectious Diseases (NIID). Written informed consent was obtained from all participants.

β-Lactams are often used to treat Helicobacter cinaedi infections; however, the mechanism underlying β-lactam resistance is unknown. In this study, we investigated β-lactam resistance in an H. cinaedi strain, MRY12-0051 (MICs of amoxicillin [AMX] and ceftriaxone [CRO], 32 and 128 μg/ml; obtained from human feces). Based on a comparative whole-genome analysis of MRY12-0051 and the CRO-susceptible H. cinaedi strain MRY08-1234 (MICs of AMX and CRO, 1 and 4 μg/ml; obtained from human blood), we identified five mutations in genes encoding penicillin-binding proteins (PBPs), including two in pbpA , one in pbp2 , and two in ftsI . Transformation and penicillin binding assays indicated that CRO resistance was mainly associated with mutations in pbpA ; mutations in ftsI also led to increased resistance to AMX. Knocking out cmeB and cmeD , which encode resistance-nodulation-division-type efflux pump components, in H. cinaedi type strain CCUG18818 (AMX MIC, 4 to 8 μg/ml) resulted in 8- and 64-fold decreases, respectively, in the AMX MIC. Hence, MICs of AMX in H. cinaedi become similar to those of Helicobacter pylori isolates in the absence of cmeD . In conclusion, the difference in susceptibility to β-lactams between H. pylori and H. cinaedi is explained by differences in efflux pump components. Mutations in pbpA are the primary determinant of high resistance to β-lactams in H. cinaedi .

We recently demonstrated that the Rv2613c protein from Mycobacterium tuberculosis H37Rv is a novel diadenosine 5′,5‴-P1,P4-tetraphosphate (Ap4A) phosphorylase (MtAPA) that forms a tetramer. Mycobacterium avium and Mycobacterium smegmatis express proteins named MAV_3489 and MSMEG_2932, respectively, that are homologous to MtAPA. Here we showed that the MAV_3489 and MSMEG_2932 proteins possess Ap4A phosphorylase activity and enzymatic properties similar to those of MtAPA. Furthermore, gel-filtration column chromatography revealed that MAV_3489 and MSMEG_2932 assembled into homotetramers in solution, indicating that they may also form unique Ap4A-binding sites composed of tetramers.

Molecular testing can rapidly detect Helicobacter pylori susceptibility using gastric biopsies. Allele-specific polymerase chain reaction (ASP-PCR) was used to identify H. pylori 23S rRNA and gyrA mutation using gastric biopsies from Colombian patients and confirmed by PCR and sequencing of the 23S rRNA and gyrA genes. The sensitivity and specificity of ASP-PCR were compared with susceptibilities measured by agar dilution. Samples included gastric biopsies from 107 biopsies with H. pylori infections and 20 H. pylori negative. The sensitivity and specificity of ASP-PCR for the 23S rRNA gene were both 100%. The sensitivity and specificity of ASP-PCR for the gyrA gene, published in 2007 by Nishizawa et al., were 52% and 92.7%, respectively; the lower sensitivity was due to the presence of mutation N87I in our samples, which were not detected by the test. In this study, we designed new primers to detect the mutation N87I in GyrA. The ASP-PCR was performed with the original primers plus the new primers. The molecular test with the new primers improved the sensitivity to 100%. In conclusion, ASP-PCR provides a specific and rapid means of predicting resistance to clarithromycin and levofloxacin in gastric biopsies.

A 56-year-old previously healthy man was hospitalized due to a 10-day history of neck pain and an elevated C-reactive protein level. Gram-negative spiral bacilli were isolated from his blood, and Helicobacter cinaedi was confirmed using 16S rRNA sequencing. The infectious focus was not identified by initial cervical magnetic resonance imaging (MRI); however, repeated MRI demonstrated prominent high signal intensity in the entire region of the C6-C7 vertebrae and C6/C7 disc space. Furthermore, fluorodeoxyglucose-positron emission tomography/computed tomography showed no significant uptake, other than in the C6-C7 region. The patient was successfully treated with ceftriaxone for six weeks without sequelae.

Diadenosine 5′,5′′′-P1,P4-tetraphosphate (Ap4A) phosphorylase from Mycobacterium tuberculosis H37Rv (MtAPA) belongs to the histidine triad motif (HIT) superfamily, but is the only member with an alanine residue at position 149 (Ala-149). Enzymatic analysis revealed that the Ala-149 deletion mutant displayed substrate specificity for diadenosine 5′,5′′′-P1,P5-pentaphosphate and was inactive on Ap4A and other substrates that are utilized by the wild-type enzyme.

γ-Glutamyltranspeptidase and asparaginase have been shown to play important roles in Helicobacter pylori colonization and cell death induced by H. pylori infection. In this study, the association of γ-glutamyltranspeptidase and asparaginase was elucidated by comparing activities of both deamidases in H. pylori strains from patients with chronic gastritis, gastric and duodenal ulcers, and gastric cancer. γ-Glutamyltranspeptidase activities in H. pylori strains from patients with gastric cancer were significantly higher than in those from patients with chronic gastritis or gastric ulcers. There was a wide range of asparaginase activities in H. pylori strains from patients with gastric cancer and these were not significantly than those from patients with other diseases. To identify the contributions of γ-glutamyltranspeptidase and asparaginase to gastric cell inflammation, human gastric epithelial cells (AGS line) were infected with H. pylori wild-type and knockout strains and inflammatory responses evaluated by induction of interleukin-8 (IL-8). IL-8 response was significantly decreased by knockout of the γ-glutamyltranspeptidase-encoding gene but not by knockout of the asparaginase-encoding gene. Additionally, IL-8 induction by infection with the H. pylori wild-type strain was significantly decreased by adding glutamine during infection. These findings indicate that IL-8 induction caused by γ-glutamyltranspeptidase activity in H. pylori is mainly attributable to depletion of glutamine. These data suggest that γ-glutamyltranspeptidase plays a significant role in the chronic inflammation caused by H. pylori infection.

Forty-six Helicobacter cinaedi isolates from the same hospital were analyzed by multilocus sequence typing. Most H. cinaedi isolates exhibited clonal complex 9 and were mainly isolated from immunocompromised patients in the same ward. Three Helicobacter fennelliae isolates were obtained from the same ward and exhibited the same pulsed-field gel electrophoresis patterns. All isolates were resistant to clarithromycin and ciprofloxacin. H. cinaedi and H. fennelliae must be carefully monitored to prevent nosocomial infection.

We present here the draft whole-genome shotgun sequence of an uncultivated strain SNTW101 of Helicobacter suis , which has been maintained in the stomachs of mice. This strain was originally isolated from gastric biopsy specimens of a urea breath test-negative Japanese patient suffering from nodular gastritis.

Volume 1, no. 4, [e00512-13][1], 2013. After publication, we detected the presence of a putative cytolethal distending toxin (CDT) gene cluster in Helicobacter fennelliae MRY12-0050. In the last paragraph before the accession numbers, the third sentence should read as follows: “ H. cinaedi and H.

Background and Aim: Helicobacter pylori eradication clearly decreases peptic ulcer recurrence rates. H. pylori eradication is achieved in 70–90% of cases, but treatment failures due to poor patient compliance and resistant organisms do occur. Lactobacillus gasseri can suppress both clarithromycin-susceptible and -resistant strains of H. pylori in vitro. The aim of this study was to determine the effect of pretreatment with L. gasseri- containing yogurt on H. pylori eradication. We conducted a randomized, controlled clinical trial in patients with H. pylori infection. Methods: A total of 229 patients were randomized into either a 1-week triple therapy of rabeprazole (10 mg bid), amoxicillin (750 mg bid), and clarithromycin (200 mg bid) or triple therapy plus L. gasseri-containing yogurt. In the yogurt-plus-triple therapy groups, yogurt containing L. gasseri OLL2716 (112 g) was given twice daily for 4 weeks (3 weeks pretreatment and also 1 week during eradication therapy). Clarithromycin resistance was determined by the detection of a mutation in 23S rRNA using nested polymerase chain reaction and the direct sequencing of DNA from pretreatment feces. H. pylori eradication was diagnosed based on the urea breath test and a stool antigen test after 8 weeks of eradication. Results: The status of H. pylori susceptibility to clarithromycin was successively determined in 188 out of 229 samples. The rate of infection with clarithromycin-resistant strains of H. pylori was 27.1%. Overall eradication (intention to treat/per protocol) was 69.3/74.5% for the triple-only group, and 82.6/85.6% for the yogurt-plus-triple group (P = 0.018/P = 0.041). Eradication of primary clarithromycin-resistant strains tended to be higher for yogurt-plus-triple therapy than triple-only therapy (38.5 vs 28.0%, respectively, P = 0.458). Conclusion: This study confirmed that the major cause of treatment failure is resistance to clarithromycin. A 4-week treatment with L. gasseri-containing yogurt improves the efficacy of triple therapy in patients with H. pylori infection.

Background and Aims: Fluoroquinolone-containing regimens have been suggested as an alternate to standard triple therapy for the treatment of Helicobacter pylori infections. To determine the relationship between fluoroquinolone resistance and mutations of GyrA and GyrB in H. pylori, we exchanged the mutations at positions 87and 91 of GyrA among fluoroquinolone-resistant clinical isolates. GyrB of a strain with no mutations in GyrA was also analyzed to identify mechanisms of resistance to norfloxacin. Materials & Methods: Natural transformation was performed using the amplified fragment of the gyrA and gyrB gene as donor DNA. The amino acid sequences of GyrA and GyrB were determined by DNA sequencing of the gyrA and gyrB genes. Results: Norfloxacin-resistant strains which had mutations at position 87 and 91 became susceptible when the mutations were converted to the wild type. When the mutation from Asp to Asn at position 91 was exchanged to the mutation from Asn to Lys at position 87, the MIC to levofloxacin, gatifloxacin, and sitafloxacin increased. Norfloxacin-resistant strain TS132 with no mutations in GyrA but had a mutation at position 463 in GyrB. Transformants obtained by natural transformation using gyrB DNA of TS132 had a mutation at position 463 of GyrB and revealed resistant to norfloxacin and levofloxacin. Conclusion: Mutation from Asn to Lys at position 87 of GyrA confers higher resistance to levofloxacin and gatifloxacin than does mutation from Asp to Asn at position 91. We propose that mutation at position 463 in GyrB as a novel mechanism of fluoroquinolone resistance in H. pylori.

OBJECTIVE: Traditional Helicobacter pylori (H. pylori) eradication therapy has been undermined by increasing antimicrobial, especially clarithromycin, resistance. Susceptibility testing in some areas is difficult to achieve or unavailable. We aimed to determine whether gastric biopsy specimens stored at room temperature for rapid urease test (RUT) were suitable for clarithromycin susceptibility testing of H. pylori. METHODS: After 30 days of storage at room temperature, DNA was extracted from gastric biopsies present in RUTs (Hpfast). H. pylori status and clarithromycin susceptibility were evaluated using H. pylori-specific polymerase chain reaction (PCR) for ureA, vacA, and allele-specific primer-PCR of the 23S rRNA genes. The PCR results were compared with histology, RUT, and culture results. H. pylori positive was defined as RUT and either culture or histology positive; H. pylori negative as RUT, culture and histology negative. RESULTS: Samples from 31 patients were evaluated; 11 were H. pylori positive including 9 by culture; seven of which had allele-specific primer-PCR results from the RUT specimen for the detection of mutations of the 23S rRNA gene. When both tests were available, culture and PCR results were concordant in 7 cases. In 15 of the 20 histology, RUT and culture negative patients, three PCR were negative. In one patient, all of the three tests were positive; and in three only the 23S rRNA was positive and in one only ureA was positive. CONCLUSION: Gastric biopsy specimens stored in the gel of RUT for 30 days can be used for molecular testing to confirm the diagnosis of H. pylori infection and test for clarithromycin susceptibility.

Objectives To elucidate the relationship between the mutations of penicillin-binding protein (PBP)1, PBP2 and PBP3 and amoxicillin resistance in Helicobacter pylori. Methods The mutations detected only in clinical amoxicillin-resistant strains were determined by comparison of the deduced amino acid sequences of PBP1(HP0597), PBP2(HP1556) and PBP3(HP1565) encoded by the pbp1, ftsI and pbp2 genes, respectively, in 13 clinical H. pylori strains and three ATCC strains. The contribution of the mutations in PBPs was analysed by the natural transformation of the amoxicillin-susceptible strain ATCC 700392 with various combinations of the pbp1, ftsI and pbp2 genes from the amoxicillin-resistant strain TH743 (MIC of amoxicillin: 8 mg/L). Results We initially identified six, four and two mutations of PBP1, PBP2 and PBP3, respectively, which were detected only in amoxicillin-resistant strains. By the natural transformation of an amoxicillin-susceptible strain ATCC 700392, we found that mutations in PBP1 and PBP3 conferred higher resistance to amoxicillin than mutations in PBP1 and PBP2, or mutations only in PBP1. Furthermore, mutations in PBP1, PBP2 and PBP3 conferred a 256-fold higher amoxicillin resistance when compared with ATCC 700392. Conclusions Multiple mutations in PBP2 and PBP3, in addition to mutations in PBP1, confer higher amoxicillin resistance in H. pylori.

The 23S rRNA gene in clinical isolates of Helicobacter pylori isolated between 1995 and 2004 from Japan was investigated and the relationship between mutations in this gene and clarithromycin susceptibility was studied. Among nine mutations that have previously been reported to confer clarithromycin resistance, an adenine→guanine transition at position 2142 (A2142G) or 2143 (A2143G) was detected in all clarithromycin-resistant strains ( n =67) but not in any clarithromycin-susceptible strains ( n =17). Mutations at positions 2182, 2223, 2244 and 2288 have previously been reported to confer clarithromycin resistance in H. pylori isolates from Bangladesh, China and Brazil. However, these mutations were not associated with clarithromycin resistance in H. pylori isolates from Japan in this study. Other mutations at positions 2115, 2144 and 2711, which have also been reported to confer clarithromycin resistance in H. pylori from Sweden and Italy, were not detected in the strains in this study. Our results suggest that susceptibility to clarithromycin is predicted by detection of mutations at positions 2142 and 2143 of the 23S rRNA gene in H. pylori isolates in Japan.

In the past year, a substantial number of (putative) novel Helicobacter species have been described, including Helicobacter himalayensis colonizing the Himalayan marmot and Helicobacter apodemus, colonizing the Korean striped field mouse. In addition, a putative novel gastric Helicobacter species was identified in wild gorillas and chimpanzees, for which the name "Candidatus H. homininae" was proposed. A high incidence of gastric non-H. pylori Helicobacter infection was described in China and multiple case reports have described the involvement of enterohepatic Helicobacter species, especially Helicobacter cinaedi, in a wide range of diseases. Several studies in rodent models further elucidated the mechanisms underlying the development of gastric mucosa-associated lymphoid tissue lymphoma during infection with gastric non-H. pylori Helicobacters. The effects of infection with gastric Helicobacters on the development of neuroinflammation were investigated and several enterohepatic Helicobacter species were shown to affect the composition of the gut microbiota, to influence vaccine efficiency as well as the progression of cancer in distant sites of the body.

A 43-year-old man was referred to our hospital for an acute-onset fever and left flank pain. He had been previously diagnosed with lymphangioma, and abdominal computed tomography showed pararenal cysts with fat stranding around the left kidney, of which infection was subsequently confirmed on magnetic resonance imaging. Gram-negative spiral bacilli were isolated from two sets of blood cultures, and Helicobacter cinaedi was identified using 16S rRNA sequencing. The patient was successfully treated with ceftriaxone therapy without recurrence. A multilocus sequence typing analysis indicated the current H. cinaedi strain differed from previous strains isolated in Japan.

Summary Background The only authorized second-line Helicobacter pylori regimen in Japan is proton pump inhibitor + amoxycillin + clarithromycin. However, it has been reported that this second-line regimen is not effective. In this study, we evaluated the efficacy of dual and triple eradication therapies using rabeprazole as second-line H. pylori eradication regimens. Aim To evaluate the efficacy of dual and triple eradication therapies using rabeprazole as second-line H. pylori eradication therapy. Methods Sixty-two H. pylori-positive patients with first-line eradication failure were randomly assigned to two groups. The RAM group was administered rabeprazole 20 mg + amoxycillin 1500 mg + metronidazole 500 mg daily for 1 week. The RA group was administered rabeprazole 40 mg + amoxycillin 2000 mg daily for 2 weeks. Eradication of H. pylori infection was determined by 13C-urea breath testing at 8 weeks after completion of treatment. Prior to treatment, amoxycillin, clarithromycin and metronidazole susceptibility, and CYP2C19 phenotype status were determined. Results Eradication rates for the RAM and RA groups were 97% and 74%, respectively. Eradication rates were not influenced by CYP2C19 phenotype in either group. Eradication rates for clarithromycin-resistant patients were 100% in the RAM group and 77% in the RA group. Conclusions One week with RAM therapy and 2 weeks with RA therapy were effective as second-line eradication therapy for H. pylori infection; moreover, RAM was more effective than RA therapy.

Summary Background Levels of pepsinogen have been reported to correlate with the degree of gastric atrophy in Helicobacter pylori-infected gastric mucosa. Aim To investigate the relationship between PG levels and histological atrophy before and after H. pylori eradication. Methods Eradication therapy was conducted on 180 H. pylori-positive patients with upper gastrointestinal conditions. Endoscopy was performed prior to and at 2, 12 and 24 months after successful eradication therapy. Histological findings were scored using the updated Sydney System in the antrum and the corpus of the stomach. Pepsinogen was measured. Results Pepsinogen I levels dropped significantly at 2 months after the eradication. By 12 months and 24 months post-eradication they had risen again. Histological improvement was seen in atrophy at all sites at 24 months after the eradication. A significant correlation between atrophy and pepsinogen I was seen from prior to eradication. The correlation coefficient was greater at 2 months post-eradication, decreased again at 12 months and was no longer significant at 24 months. Conclusions Our results suggest that pepsinogen levels correlate with the degree of gastric mucosal atrophy prior to and soon after successful eradication therapy, but that the degree of correlation subsequently declines over time.

Helicobacter pylori (H. pylori) is known to be associated with peptic ulcer, chronic gastritis, MALT lymphoma, and gastric cancer. In this study, we investigated the usefulness of a new E-plate serum antibody kit, developed in Japan using H. pylori strains isolated from Japanese patients, in the assessment of eradication, with an HM-CAP kit for comparison. The subjects were 100 H. pylori positive patients who underwent upper gastrointestinal endoscopy between October 1999 and June 2000. Eradication assessment performed using E-plate kit with the cut-off values for the delta value of the antibody titre set at −20%, −30%, and −40%, yielded sensitivity/specificity of 93.9%/85.7%, 92.4%/85.7%, and 72.7%/92.9%, respectively, for agreement rates with eradication assessments using endoscopic biopsy specimens at 2 months post-eradication therapy. Use of the HM-CAP kit with cut-off values of −5%, −10%, and −15% yielded sensitivity/specificity of 97.0%/28.6%, 83.3%/85.7%, and 53.0%/100%, respectively. These results indicated that the E-plate kit is more useful for eradication assessment in Japan.

Gastric infection of clarithromycin (CAM)-resistant Helicobacter pylori is one of the major causes of failure to eradicate this organism. A noninvasive and useful method for the detection of CAM-resistant H. pylori from human feces by restriction fragment length polymorphism (RFLP)-nested polymerase chain reaction (PCR) targeting the mutation of the 23S rRNA gene that confers CAM-resistance in H. pylori was developed in this study. Our nested PCR method detected DNA of H. pylori in feces with high sensitivity and specificity compared with both an enzyme-linked immunoadsorbent assay (ELISA) of H. pylori in feces and the isolation of H. pylori from gastric biopsy. Furthermore, the results of mutation analysis of the H. pylori 23S rRNA gene amplified from feces completely correlated with both that of the H. pylori 23S rRNA gene amplified from the isolates of gastric biopsy and the susceptibility of H. pylori isolates to CAM. Therefore, our results show that this RFLP/nested PCR method is useful for the accurate diagnosis of CAM-resistant H. pylori infection from feces.

The aim of this study was to determine the susceptibilities to clarithromycin, amoxycillin and metronidazole of Helicobacter pylori isolates from the antrum and corpus of Japanese patients examined during the period 1995–2001. There was an increase, from 6.2% in 1995 to 22.1% in 2000–2001, in the proportion of patients infected with clarithromycin-resistant H. pylori. Of patients infected with clarithromycin-resistant H. pylori, 39.1% were infected with both clarithromycin-susceptible and -resistant H. pylori. Furthermore, the MIC90 of clarithromycin for H. pylori rose from < 1 mg/L in 1995– 1998 to 8 mg/L in 1999. In contrast, the MIC90s of amoxycillin and metronidazole were ≤ 0.125 and 4 mg/L, respectively, throughout the study period. The results showed that, while most H. pylori isolates were susceptible to amoxycillin and metronidazole, resistance to clarithromycin among H. pylori isolates increased markedly in Japan during 1995–2001. The results also indicated a need to test the susceptibility of H. pylori isolates from more than two samples obtained from two different sites in the stomach of a single patient in order to diagnose the presence of clarithromycin-resistant H. pylori correctly.

Acinetobacter baumannii has emerged worldwide as an important nosocomial pathogen in medical institutions. Here, we present the draft genome sequences of A. baumannii strains MRY09-0642, MRY10-0558, and MRY12-0277 that were isolated from nosocomial outbreaks in Japan between 2008 and 2012 and that are resistant to antimicrobial agents, including carbapenems, fluoroquinolones, and aminoglycosides.

Helicobacter fennelliae , a human enterohepatic pathogen, causes bacteremia and colitis. We isolated H. fennelliae strain MRY12-0050 from a female patient; this strain was isolated from 2 other patients from the same hospital during the same period, suggesting human-to-human transmission. This is the first report of an H. fennelliae genome sequence.

γ-Glutamyltranspeptidase and asparaginase have been shown to play important roles in Helicobacter pylori colonization and cell death induced by H. pylori infection. In this study, the association of γ-glutamyltranspeptidase and asparaginase was elucidated by comparing activities of both deamidases in H. pylori strains from patients with chronic gastritis, gastric and duodenal ulcers, and gastric cancer. γ-Glutamyltranspeptidase activities in H. pylori strains from patients with gastric cancer were significantly higher than in those from patients with chronic gastritis or gastric ulcers. There was a wide range of asparaginase activities in H. pylori strains from patients with gastric cancer and these were not significantly than those from patients with other diseases. To identify the contributions of γ-glutamyltranspeptidase and asparaginase to gastric cell inflammation, human gastric epithelial cells (AGS line) were infected with H. pylori wild-type and knockout strains and inflammatory responses evaluated by induction of interleukin-8 (IL-8). IL-8 response was significantly decreased by knockout of the γ-glutamyltranspeptidase-encoding gene but not by knockout of the asparaginase-encoding gene. Additionally, IL-8 induction by infection with the H. pylori wild-type strain was significantly decreased by adding glutamine during infection. These findings indicate that IL-8 induction caused by γ-glutamyltranspeptidase activity in H. pylori is mainly attributable to depletion of glutamine. These data suggest that γ-glutamyltranspeptidase plays a significant role in the chronic inflammation caused by H. pylori infection.

Increased resistance of Helicobacter pylori to clarithromycin and metronidazole has resulted in recommendation to substitute fluoroquinolones for eradication therapy. The aims of the study were to determine the prevalence and changes in primary levofloxacin resistance related to H. pylori gyrA sequences. The study utilized H. pylori strains isolated from patients undergoing gastroscopy in Bogotá, Colombia from 2009 to 2014. Levofloxacin susceptibility was assessed by agar dilution. Mutations in gyrA sequences affecting the quinolone resistance-determining region (QRDR) were evaluated by direct sequencing. Overall, the mean prevalence of primary levofloxacin resistance was 18.2% (80 of 439 samples). Resistance increased from 11.8% (12/102) in 2009 to 27.3% (21/77) in 2014 (p = 0.001). gyrA mutations in levofloxacin resistant strains were present in QRDR positions 87 and 91. The most common mutation was N87I (43.8%, 35/80) followed by D91N (28.8%, 23/80) and N87K (11.3%, 9/80). Levofloxacin resistance increased markedly in Colombia during the six-year study period. Primary levofloxacin resistance was most often mediated by point mutations in gyrA, with N87I being the most common QRDR mutation related to levofloxacin resistance.

Helicobacter pylori is an important pathogen whose primary niche is the human stomach. H. pylori is etio-logically associated with gastric inflammation (gastritis), peptic ulcer disease, and gastric cancer. Both noninvasive (e.g., urea breath and stool antigen tests) and invasive (gastric biopsy for histology, culture, or PCR) tests are used for diagnosis. PCR detection of H. pylori has been reported using a variety of clinical samples including gastric biopsy, gastric juice, saliva, dental plaque, and stools as well as environmental samples. Whenever possibly, noninvasive tests are preferred over invasive tests. H. pylori are excreted in the stool. Culture from stool is variable whereas stool antigen testing is widely used. Stool consists of a complicated mixture of commensal bacteria and chemicals and often includes inhibitors of PCR. Nevertheless, simple extraction methods are available to efficiently extract DNA from human stools and nested-PCR targeting the 23S rRNA gene have proven to be highly sensitive for the detection of H. pylori. Detection of clarithromycin susceptibility/resistance is important clinically and the mutation of the 23S rRNA gene responsible for resistance can also be detected using stool. This described method can be modified for other clinical samples such as gastric juice or biopsy material.

Helicobacter cinaedi colonizes the colons of human and animals and can cause colitis, cellulitis, and sepsis in humans, with infections in immunocompromised patients being increasingly recognized. However, methods for analyzing the molecular epidemiology of H. cinaedi are not yet established. A genotyping method involving multilocus sequence typing (MLST) was developed and used to analyze 50 H. cinaedi isolates from Japanese hospitals in addition to 6 reference strains. Pulsed-field gel electrophoresis (PFGE) results were also compared with the MLST results. Based on the genomic information from strain CCUG18818, 21 housekeeping genes were selected as candidates for MLST and were observed to have high homology (96.5 to 100%) between isolates. Following a comparison of the 21 housekeeping genes from 8 H. cinaedi isolates, 7 genes were chosen for MLST, revealing 14 sequence types (STs). The isolates from 3 hospitals belonged to the same STs, but the isolates from the other 4 hospitals belonged to different STs. Isolates belonging to ST6 were analyzed by PFGE and showed similar, but not identical, patterns between isolates. Isolates belonging to ST9, ST10, and ST11, which belonged to the same clonal complex, had the same pattern. All isolates were found to contain mutations in GyrA and the 23S rRNA gene that confer ciprofloxacin and clarithromycin resistance, respectively, in H. cinaedi . These results raise concerns about the increase in H. cinaedi isolates resistant to clarithromycin and ciprofloxacin in Japan.

Fluoroquinolone-containing regimens have been suggested as an alternate to standard triple therapy for the treatment of Helicobacter pylori infections. To determine the relationship between fluoroquinolone resistance and mutations of GyrA and GyrB in H. pylori, we exchanged the mutations at positions 87 and 91 of GyrA among fluoroquinolone-resistant clinical isolates. GyrB of a strain with no mutations in GyrA was also analyzed to identify mechanisms of resistance to norfloxacin.Natural transformation was performed using the amplified fragment of the gyrA and gyrB gene as donor DNA. The amino acid sequences of GyrA and GyrB were determined by DNA sequencing of the gyrA and gyrB genes.Norfloxacin-resistant strains which had mutations at position 87 and 91 became susceptible when the mutations were converted to the wild type. When the mutation from Asp to Asn at position 91 was exchanged to the mutation from Asn to Lys at position 87, the MIC to levofloxacin, gatifloxacin, and sitafloxacin increased. Norfloxacin-resistant strain TS132 with no mutations in GyrA but had a mutation at position 463 in GyrB. Transformants obtained by natural transformation using gyrB DNA of TS132 had a mutation at position 463 of GyrB and revealed resistant to norfloxacin and levofloxacin.Mutation from Asn to Lys at position 87 of GyrA confers higher resistance to levofloxacin and gatifloxacin than does mutation from Asp to Asn at position 91. We propose that mutation at position 463 in GyrB as a novel mechanism of fluoroquinolone resistance in H. pylori.

Despite the fact that sequential therapy has been evaluated in more than 2500 patients and has been shown to on average provide Helicobacter pylori eradication in 90% to 94%, some authorities still question whether it should be a first-line anti-H. pylori regimen. Here, we discuss H. pylori eradication using experience and expectations with other common bacterial infections as a frame of reference. H. pylori is no exception and near 100% success is expected for optimized regimens treating susceptible infections. As such, the proper comparator would be the relation to 100% eradication. Superiority to another, often proven inferior, therapy per se provides little or no useful information. Treatment failures in infectious diseases are typically easily explainable and most often relate to the presence of antimicrobial resistance or failure to take the drugs. We provide a model for predicting the results of H. pylori combination therapies in relation to the pattern and prevalence of resistance. The results are consistent with clinical practice and explain why sequential is typically superior and essentially never inferior to triple therapy. We also show when meta-analysis is an inappropriate technique for the analysis of H. pylori clinical trials and discuss how to appropriately use the technique. Finally, we discuss why the location of studies (eg, Italy), is unimportant and explain why, from the standpoint of a therapy for an infectious disease, sequential therapy is a significant advance and should be considered one of the replacements for the outdated legacy triple therapy (proton pump inhibitor--clarithromycin--amoxicillin).