Protocol for detecting Helicobacter suis infection in gastric biopsies and serum by PCR and ELISA.

Infection with Helicobacter suis, which causes many cases of gastric disease, is not reliably diagnosed. Here, we present a protocol for detecting H. suis infection. We describe steps for collecting gastric biopsies and sera from patients, preparing DNA for PCR, and targeting the H. suis-specific gene. We then define procedures for inoculating biopsies onto primary agar plates and transferring colonies to secondary agar plates. Finally, we detail whole-genome sequencing of bacteria and assess H. suis infection in sera with ELISA. For complete details on the use and execution of these protocols, please refer to Matsui et al.1.

Development of serological assays to identify Helicobacter suis and H. pylori infections.

Helicobacter suis, hosted by hogs, is the most prevalent gastric non-Helicobacter pylori Helicobacter species found in humans. Recent studies have suggested that H. suis infection has caused many cases of gastric disease, but the transmission route from hogs remains unclear. Diagnostic methods based on H. suis urease activity often yield negative results, and there is no reliable method for diagnosing H. suis infection in clinical practice without gastric biopsy specimens. This study presents the world's first use of whole-bacterial cell ELISA to simultaneously assess H. suis and H. pylori infections. The ELISAs showed high accuracy, with an area under the ROC curve of 0.96, 100% sensitivity, 92.6% specificity, 76.9% positive predictive value, and 100% negative predictive value for the H. suis test, and an area under the ROC curve of 0.92, 88.2% sensitivity, 87.5% specificity, 65.2% positive predictive value, and 96.6% negative predictive value for the H. pylori test.

Association between the proportion of laparoscopic approaches for digestive surgeries and the incidence of consequent surgical site infections, 2009-2019: A retrospective observational study based on national surveillance data in Japan.

BACKGROUND: Surgical site infections (SSIs) are among the most common healthcare-associated infections. Laparoscopy is increasingly being used in various surgical procedures. However, no study has examined the association between the proportion of laparoscopic procedures and the incidence of SSIs in digestive surgery using nationwide surveillance data. METHODS: We retrospectively investigated national SSI surveillance data from the Japan Nosocomial Infections Surveillance between 2009 and 2019. The annual trend of the SSI rate and the proportion of laparoscopic procedures were assessed, focusing on five major digestive surgeries. This was based on data from 109,544 (appendix surgery), 206,459 (gallbladder surgery), 60,225 (small bowel surgery), 363,677 (colon surgery), and 134,695 (rectal surgery) procedures. The effect of a 10% increase in the proportion of laparoscopic procedures on the reduction of the SSI rate was estimated using mixed-effect logistic regression. FINDINGS: The average SSI rate of the five digestive surgeries decreased from 11.8% in 2009 to 8.1% in 2019. The proportion of laparoscopic procedures in each of the five digestive surgeries increased continuously (p<0.001). The SSI rate for laparoscopic procedures was always lower than that for open procedures. The results were consistent between all and core hospitals participating in the surveillance. The odds ratios of the 10% increase in the proportion of laparoscopic procedures for five digestive surgeries were always <0.950 (p<0.001). CONCLUSION: An increase in the proportion of laparoscopic procedures was associated with a reduction in the SSI rate in digestive surgeries.

Association between the frequency of surgeries for video-assisted thoracic surgery and the incidence of consequent surgical site infections: a retrospective observational study based on national surveillance data.

BACKGROUND: The association between the frequency of surgeries and the incidence of surgical site infections (SSIs) has been reported for various surgeries. However, no previous study has explored this association among video-assisted thoracic surgeries (VATS). Hence, we aimed to investigate the association between the frequency of surgeries and SSI in video-assisted thoracic surgeries. METHODS: We analyzed the data of 26,878 thoracic surgeries, including 21,154 VATS, which were collected during a national surveillance in Japan between 2014 and 2018. The frequency of surgeries per hospital department was categorized into low (< 50/year), moderate (50-100/ year), and high (> 100/year). Chi-squared test or Fisher's exact test was used for discrete explanatory variables, whereas Wilcoxon's rank-sum test or Kruskal-Wallis test was used for continuous explanatory variables. Univariate analysis of the department groups was conducted to explore confounding factors associated with both SSIs and the department groups. We used a multiple logistic regression model focusing on VATS and stratified by the National Nosocomial Infections Surveillance System (NNIS) risk index. RESULTS: The rates of SSIs in the hospital groups with low, moderate, and high frequency of surgeries were 1.39, 1.05, and 1.28%, respectively. In the NNIS risk index 1 stratum, the incidence of SSIs was significantly lower in the moderate-frequency of surgeries group than that in the other groups (odds ratio [OR]: vs. low-frequency of surgeries: 2.48 [95% confidence interval [CI]: 1.20-5.13], P = 0.0143; vs. high-frequency of surgeries: 2.43 [95% CI: 1.44-4.11], P = 0.0009). In the stratum of NNIS risk indices 2 and 3, the incidence of SSI was significantly higher in the low-frequency of surgeries group (OR: 4.83, 95% CI: 1.47-15.93; P = 0.0095). CONCLUSION: The result suggests that for departments with low-frequency of surgeries, an increase in the frequency of surgeries to > 50 per department annually potentially leads to a decrease in the incidence of SSIs. This occurs through an increase in the experience of the departmental surgeons and contributes to the improvement of VATS outcomes in thoracic surgeries.

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.

Corrigendum: Networking and Specificity-Changing DNA Methyltransferases in Helicobacter pylori.

[This corrects the article DOI: 10.3389/fmicb.2020.01628.].

Networking and Specificity-Changing DNA Methyltransferases in Helicobacter pylori.

Epigenetic DNA base methylation plays important roles in gene expression regulation. We here describe a gene expression regulation network consisting of many DNA methyltransferases each frequently changing its target sequence-specificity. Our object Helicobacter pylori, a bacterium responsible for most incidence of stomach cancer, carries a large and variable repertoire of sequence-specific DNA methyltransferases. By creating a dozen of single-gene knockout strains for the methyltransferases, we revealed that they form a network controlling methylome, transcriptome and adaptive phenotype sets. The methyltransferases interact with each other in a hierarchical way, sometimes regulated positively by one methyltransferase but negatively with another. Motility, oxidative stress tolerance and DNA damage repair are likewise regulated by multiple methyltransferases. Their regulation sometimes involves translation start and stop codons suggesting coupling of methylation, transcription and translation. The methyltransferases frequently change their sequence-specificity through gene conversion of their target recognition domain and switch their target sets to remodel the network. The emerging picture of a metamorphosing gene regulation network, or firework, consisting of epigenetic systems ever-changing their specificity in search for adaptation, provides a new paradigm in understanding global gene regulation and adaptive evolution.

A prolonged multispecies outbreak of IMP-6 carbapenemase-producing Enterobacterales due to horizontal transmission of the IncN plasmid.

A multispecies outbreak of IMP-6 carbapenemase-producing Enterobacterales (IMP-6-CPE) occurred at an acute care hospital in Japan. This study was conducted to understand the mechanisms of IMP-6-CPE transmission by pulsed-field gel electrophoresis (PFGE), multilocus sequence typing and whole-genome sequencing (WGS), and identify risk factors for IMP-6-CPE acquisition in patients who underwent abdominal surgery. Between July 2013 and March 2014, 22 hospitalized patients infected or colonized with IMP-6-CPE (Escherichia coli [n = 8], Klebsiella oxytoca [n = 5], Enterobacter cloacae [n = 5], Klebsiella pneumoniae [n = 3] and Klebsiella aerogenes [n = 1]) were identified. There were diverse PFGE profiles and sequence types (STs) in most of the species except for K. oxytoca. All isolates of K. oxytoca belonged to ST29 with similar PFGE profiles, suggesting their clonal transmission. Plasmid analysis by WGS revealed that all 22 isolates but one shared a ca. 50-kb IncN plasmid backbone with blaIMP-6 suggesting interspecies gene transmission, and typing of plasmids explained epidemiological links among cases. A case-control study showed pancreatoduodenectomy, changing drains in fluoroscopy room, continuous peritoneal lavage and enteric fistula were associated with IMP-6-CPE acquisition among the patients. Plasmid analysis of isolates in an outbreak of IMP-6-CPE suggested interspecies gene transmission and helped to clarify hidden epidemiological links between cases.

Injectable Polypeptide Hydrogel Depot System for Assessment of the Immune Response-Inducing Efficacy of Sustained Antigen Release Alone.

Vaccines typically contain an antigen, delivery system (vehicle), and adjuvant, all of which contribute to inducing a potent immune response. Consequently, design of new vaccines is difficult, because the contributions and interactions of these components are difficult to distinguish. Here, it is aimed to develop an easy-to-use, non-immunogenic, injectable depot system for sustained antigen release that will be suitable for assessing the efficacy of prolonged antigen exposure per se for inducing an immune response. This should mimic real-life infections. Recombinant elastin-like polypeptides with periodic cysteine residues (cELPs) are selected, which reportedly show little or no immunogenicity, as carriers and tetanus toxoid (Ttd) as an antigen. After subcutaneous injection of the mixture, cELP rapidly forms a disulfide cross-linked hydrogel in situ, within which Ttd is physically incorporated, affording a biodegradable antigen depot. A series of Ttd-containing hydrogels is examined. A single injection induces high levels of tetanus antibody with high avidity for at least 20 weeks in mice. The chain length of cELP proves critical, whereas differences in hydrophobicity has little effect, although hydrophilic cELPs are more rapidly biodegraded. This system's ability to distinguish the contribution of sustained antigen release to antibody induction should be helpful for rational design of next-generation vaccines.

Draft Genome Sequence of Helicobacter suis Strain SNTW101, Isolated from a Japanese Patient with Nodular Gastritis.

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.

Linezolid-resistant Staphylococcus epidermidis associated with long-term, repeated linezolid use in a pediatric patient.

We report an 8-year-old patient with catheter-related bacteremia caused by linezolid-resistant Staphylococcus epidermidis that was isolated after the long-term, repeated use of linezolid. Three S. epidermidis strains isolated from this patient were bacteriologically analyzed. While the strain isolated prior to linezolid initiation was susceptible to linezolid, two strains after linezolid therapy displayed low-level linezolid susceptibility (MIC, 4 mg/L) and linezolid resistance (MIC, 16 mg/L). T2500A mutation in two copies and G2575T mutations in three copies of 23S rRNA were detected in the low-susceptible strain and the resistant strain, respectively. Linezolid-resistant S. epidermidis infection is rare, but may occur with the long-term administration of linezolid.

Mycobacterium avium infection induces the resistance of the interferon-γ response in mouse spleen cells at late stages of infection.

BACKGROUND: Bacterial infections cause an increase in the population of hematopoietic stem cells (HSCs). To investigate the downstream factors associated with hematopoietic stem cells, mice are infected with Mycobacterium avium (M. avium). RESULTS: Mycobacterium avium (M. avium) infection induces the enlargement of the spleen and changes in histopathology, including changes to the lineage populations. A dramatic expansion of Lin-c-kit+Sca-1+ (KSL) cells in mouse bone marrow cells and spleen cells was detected 4 weeks after infection with M. avium; however, there was no difference in the engraft activity between infected and un-infected mouse bone marrow cells. We tested the cytokine and cytokine-related gene expression after M. avium infection and found that IFN-γ expression increased and peaked at 4 weeks in both bone marrow and spleen cells. The expression of Sca-1 gene peaked at 4 weeks in the bone marrow but peaked at 2 weeks in spleen cells, although the Sca-1 surface marker peaked at 4 weeks after infection in both bone marrow and spleen cells. Interferon regulatory factor-2 (IRF-2) expression did not change in the bone marrow cells, whereas it decreased in spleen cells at 4 weeks and IRF-1 expression was up-regulated in both bone marrow and spleen cells after infection. However, the up-regulation of IRF-1 was not correlated with IFN-γ expression in the M. avium-infected mouse spleen cells. CONCLUSIONS: This finding suggests that the IFN-γ production mediated by M. avium infection alters the population of KSL cells during host defense, and the down-regulation of the IFN-γ response in spleen cells occurs at the late stage after M. avium infection.

The Other Helicobacters.

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.

Pararenal Lymphatic Cyst Infection Caused by Helicobacter cinaedi.

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.

Inhibition of adhesion of Clostridium difficile to human intestinal cells after treatment with serum and intestinal fluid isolated from mice immunized with nontoxigenic C. difficile membrane fraction.

Diarrhea and pseudomembrane colitis caused by Clostridium difficile infection is a global health concern because of the high recurrence rate after standard antibiotic therapy. Vaccination presents a powerful countermeasure against disease recurrence. In this study, mice vaccinated with the nontoxigenic C. difficile membrane fraction generated a marked immune response to the antigen, as demonstrated by the serum IgG and intestinal fluid IgA levels. Significantly, pretreatment with harvested IgG- and IgA-containing fluids was sufficient to prevent in vitro adhesion of C. difficile to human Caco-2 intestinal cells. These results highlight the potential of nontoxigenic C. difficile membrane fraction as a vaccine candidate for C. difficile infection.

Emergence of third-generation cephalosporin-resistant Enterobacteriacae in a Japanese critical care setting.

AIM: Extended-spectrum beta-lactamases and AmpC beta-lactamases give resistance to Enterobacteriaceae against cephalosporins, which are important drugs in various clinical settings. Within 5 days, a total of eight strains of Citrobacter freundii and two strains of Escherichia coli, all of which were resistant to third-generation cephalosporins, were isolated from six different patients in a critical care center. We carried out epidemiological and genetic studies to confirm the situation to be an outbreak by a single strain or a beta-lactamase-producing gene. METHODS: A review of patients' clinical data, pulsed-field gel electrophoresis epidemiological study against the strains, and polymerase chain reaction analysis for the detection of extended-spectrum beta-lactamase genes (TEM, SHV, CTX-M-1, CTX-M-2, and CTX-M-9) and multiplex polymerase chain reaction analysis for plasmid-mediated AmpC-beta-lactamase genes were carried out. RESULTS: Pulsed-field gel electrophoresis showed none of the subjects were genetically identical organisms. However, all strains possessed either extended-spectrum beta-lactamases (TEM, CTX-M-2, and CTX-M-9) or AmpC-beta-lactamases. One patient carried both extended-spectrum beta-lactamase- and AmpC- beta-lactamase-producing organisms. CONCLUSION: This study revealed that various types of easily transmittable drug resistance genes exist in the critical care setting concurrently. This case was not an outbreak of such strains, but the present situation might indicate a more serious condition than a mere outbreak of a single resistant strain. Enterobacteriaceae that possess extended-spectrum beta-lactamases or AmpC-beta-lactamases are increasingly common in many medical institutions in Japan, and constitutive monitoring of drug-resistant organisms and proper contact precautions are essential to prevent possible outbreaks.

Role of γ-glutamyltranspeptidase in the pathogenesis of Helicobacter pylori infection.

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.

Structural insights into the novel diadenosine 5',5‴-P¹,P4-tetraphosphate phosphorylase from Mycobacterium tuberculosis H37Rv.

Rv2613c is a diadenosine 5',5‴-P(1),P(4)-tetraphosphate (Ap(4)A) phosphorylase from Mycobacterium tuberculosis H37Rv. Sequence analysis suggests that Rv2613c belongs to the histidine triad (HIT) motif superfamily, which includes HIT family diadenosine polyphosphate (Ap(n)A) hydrolases and Ap(4)A phosphorylases. However, the amino acid sequence of Rv2613c is more similar to that of HIT family Ap(n)A hydrolases than to that of typical Ap(4)A phosphorylases. Here, we report the crystal structure of Rv2613c, which is the first structure of a protein with Ap(n)A phosphorylase activity, and characterized the structural basis of its catalytic activity. Our results showed that the structure of Rv2613c is similar to those of other HIT superfamily proteins. However, Asn139, Gly146, and Ser147 in the active site of Rv2613c replace the corresponding Gln, Gln, and Thr residues that are normally found in HIT family Ap(n)A hydrolases. Furthermore, analyses of Rv2613c mutants revealed that Asn139, Gly146, and Ser147 are important active-site residues and that Asn139 has a critical role in catalysis. The position of Gly146 might influence the phosphorylase activity. In addition, the tetrameric structure of Rv2613c and the presence of Trp160 might be essential for the formation of the Ap(4)A binding site. These structural insights into Rv2613c may facilitate the development of novel structure-based inhibitors for treating tuberculosis.

Biochemical and pathophysiological characterization of Helicobacter pylori asparaginase.

Asparaginase was purified from Helicobacter pylori 26695 and its pathophysiological role explored. The K(m) value of asparagine was 9.75 ± 1.81 µM at pH 7.0, and the optimum pH range was broad and around a neutral pH. H. pylori asparaginase converted extracellular asparagine to aspartate. H. pylori cells were unable to take up extracellular asparagine directly. Instead, aspartate produced by the action of the asparaginase was transported into H. pylori cells, where it was partially converted to ß-alanine. Asparaginase exhibited striking cytotoxic activity against histiocytic lymphoma cell line U937 cells via asparagine deprivation. The cytotoxic activity of live H. pylori cells against U937 cells was significantly diminished by deletion of the asparaginase gene, indicating that asparaginase functions as a cytotoxic agent of the bacterium. The cytotoxic effect was negligible for gastric epithelial cell line AGS cells, suggesting that the effect differs across host cell types. An asparaginase-deficient mutant strain was significantly less capable of colonizing Mongolian gerbils. Since asparagine depletion by exogenous asparaginase has been shown to suppress lymphocyte proliferation in vivo, the present results suggest that H. pylori asparaginase may be involved in inhibition of normal lymphocyte function at the gastric niche, allowing H. pylori to evade the host immune system.

The nosocomial transmission of Helicobacter cinaedi infections in immunocompromised patients.

BACKGROUND: We encountered 15 cases of Helicobacter cinaedi (H. cinaedi) infection between March and July 2008. PATIENT, METHOD, AND RESULT: The underlying diseases were hematological malignancies in a majority of cases, many of which received chemotherapy. All patients had a fever. The fever was followed by cellulitis in three, a skin rash in six, pain in the lower limbs in three, and diarrhea in three cases. We analyzed the bacterial 23S rRNA genes. The fifteen strains were divided according to base sequence into Groups A, B, and C, respectively. All four cases in Group A were women and all ten in Group C were men, indicating that the gender of the patient corresponded precisely to the genotypes of the separated bacilli in these two groups. These findings also suggested the strong possibility of nosocomial spread. CONCLUSION: It is highly likely that H. cinaedi infections have been overlooked due to the difficulties encountered in culturing the bacterium. The possibility of septicemia caused by H. cinaedi should be suspected especially in immunocompromised patients such as those undergoing chemotherapy, with symptoms such as fever, rash, arthritis, cellulitis, leg pain, and other systemic or local symptoms.