Development of Host Immune Response to Bacteriophage in a Lung Transplant Recipient on Adjunctive Phage Therapy for a Multidrug-Resistant Pneumonia.

Bacteriophage therapy is the use of viruses to kill bacteria for the treatment of antibiotic-resistant infections. Little is known about the human immune response following phage therapy. We report the development of phage-specific CD4 T cells alongside rising phage-specific immunoglobulin G and neutralizing antibodies in response to adjunctive bacteriophage therapy used to treat a multidrug-resistant Pseudomonas aeruginosa pneumonia in a lung transplant recipient. Clinically, treatment was considered a success despite the development phage-specific immune responses.

Fine capsule variation affects bacteriophage susceptibility in Klebsiella pneumoniae ST258.

Multidrug resistant (MDR) carbapenemase-producing (CP) Klebsiella pneumoniae, belonging to clonal group CG258, is capable of causing severe disease in humans and is classified as an urgent threat by health agencies worldwide. Bacteriophages are being actively explored as therapeutic alternatives to antibiotics. In an effort to define a robust experimental approach for effective selection of lytic viruses for therapy, we have fully characterized the genomes of 18 Kumoniae target strains and tested them against novel lytic bacteriophages (n = 65). The genomes of K pneumoniae carrying blaNDM and blaKPC were sequenced and CG258 isolates selected for bacteriophage susceptibility testing. The local K pneumoniae CG258 population was dominated by sequence type ST258 clade 1 (86%) with variations in capsular locus (cps) and prophage content. CG258-specific bacteriophages primarily targeted the capsule, but successful infection is also likely blocked in some by immunity conferred by existing prophages. Five tailed bacteriophages against K pneumoniae ST258 clade 1 were selected for further characterization. Our findings show that effective control of K pneumoniae CG258 with bacteriophage will require mixes of diverse lytic viruses targeting relevant cps variants and allowing for variable prophage content. These insights will facilitate identification and selection of therapeutic bacteriophage candidates against this serious pathogen.

Variation in human genetic polymorphisms, their association with Helicobacter pylori acquisition and gastric cancer in a multi-ethnic country.

BACKGROUND AND AIM: The contribution of human genetic polymorphisms to Helicobacter pylori infection and gastric cancer (GC) development remains unclear due to geographic variation in the association between specific host genetic polymorphisms and GC. In the current study we investigated the association between polymorphisms related to immune and cancer-related pathways and H. pylori infection among the major ethnicities, Chinese, Malay and Indian, resident in Singapore and Malaysia as well as the association between these polymorphisms and GC development in ethnic Chinese patients. METHODS: Thirty-four polymorphisms in 26 genes were typed by mass spectrometry in 422 patients undergoing endoscopy (162 Chinese, 113 Indian and 87 Malay controls and 60 Chinese GC cases). Patients were assessed for evidence of H. pylori infection. Odds ratios (OR) and confidence intervals (CI) were obtained using logistic regression models. RESULT: The prevalence of 16 polymorphisms varied significantly among the ethnicities. In the Chinese subgroup, nominally significant associations were shown between (i) EBBR2+1963G (rs1801200) and H. pylori infection (per-allele OR: 0.48, 95% CI 0.23, 0.98, P = 0.04), (ii) PTGS2-1195G (rs689466) and an increased risk of GC on adjusting for H. pylori status (OR: 1.53, 95% CI 0.99, 2.37, P = 0.05), and (iii) IL1B-1473C (rs1143623) and a decreased risk of GC (OR: 0.64, 95% CI 0.41, 0.99, P = 0.05). Borderline significant associations were seen between IL2-330G (rs2069762) (OR 1.45, 95% CI 0.95, 2.15, P = 0.06) and IL13-1111T (rs1800925) (OR 0.65, 95% CI 0.42, 1.01, P = 0.06) and H. pylori infection. CONCLUSION: These findings contribute to the understanding of the genetic variation between ethnicities, which may influence H. pylori susceptibility and the outcome of infection.

A novel noninvasive genotyping method of Helicobacter pylori using stool specimens.

BACKGROUND & AIMS: The source(s) of the infection and the route(s) of transmission of Helicobacter pylori have not yet been clarified. This is to introduce a noninvasive protocol allowing molecular typing of H pylori using stool specimens. METHODS: The genotyping method is based on 2 H pylori-specific biprobe real-time polymerase chain reaction assays using fragments of the glmM and the recA genes as target sequences. Discrimination between strains results from differences in the melting temperature during melting curve analysis. In case of identical melting temperatures in both assays, sequence analysis of the glmM amplicon was performed to confirm strain identity. The method was validated using gastric biopsy specimens and stool specimens of 97 unrelated individuals suffering from abdominal pain and stool specimens of members of 10 families in Austria (infected index child and family members) and 8 African households. RESULTS: Of the 97 patients, 27 were infected as shown by culture, histology, and rapid urease test. The sensitivity of each of the assays was 100% in gastric biopsy specimens and 92.2% in stool specimens; the specificity was 100%. The discriminatory capacity of the method was 100%. Clonal identities were found in 9 of 10 (90%) European and 7 of 8 (87.5%) African households. In 2 African households, 2 different clonal lineages each were found. CONCLUSIONS: The genotyping protocol introduced allows for both accurate detection and discrimination of H pylori strains in stool samples. Large-scale studies using this protocol may contribute to the clarification of the transmission pathways of infection with H pylori.

Potential role of thiol:disulfide oxidoreductases in the pathogenesis of Helicobacter pylori.

Helicobacter pylori infections are responsible for a sequence of molecular events which ultimately result in the development of gastric diseases. The pathogenesis of H. pylori has been studied extensively with strong focus on the identification of virulence factors. In contrast, the involvement of thiol:disulfide oxidoreductases in bacterial pathogenesis is less well understood. This paper provides a review of the current knowledge of H. pylori putative thiol:disulfide oxidoreductases, and their potential role in promoting virulence and colonization. Several bioinformatic analyses served to complete the information on these oxidoreductases of H. pylori.

Immunoreactive proteins of Campylobacter concisus, an emergent intestinal pathogen.

Campylobacter concisus is an emerging pathogen of the human gastrointestinal tract. Recently, a significantly higher prevalence of C. concisus DNA and higher levels of antibodies specific to C. concisus was detected in children with Crohn's disease when compared with controls. The aim of this study was to identify C. concisus immunoreactive antigens. Proteins from C. concisus were separated using two-dimensional gel electrophoresis, and sera from 10 C. concisus-positive children with Crohn's disease were employed for immunoprobing. The patients' sera reacted with 69 spots, which corresponded to 31 proteins identified by mass spectrometry. The proteins were functionally classified as involved in chemotaxis, signal transduction, flagellar motility, surface binding and membrane protein assembly. Although the individual patients' sera reacted to different sets of proteins, common antigens that were recognized by all patients were flagellin B, ATP synthase F1 alpha subunit, and outer membrane protein 18. Cross-reactivity between proteins of the Campylobacter genus was tested using patients' sera absorbed with Campylobacter showae, Campylobacter jejuni and Campylobacter ureolyticus. Most of the C. concisus immunoreactive proteins identified in this study showed cross-reactivity with other species except for three antigens. In conclusion, this study has identified C. concisus proteins that are immunoreactive within patients with Crohn's disease.

The secretome of Campylobacter concisus.

A higher prevalence of Campylobacter concisus and higher levels of IgG antibodies specific to C. concisus in Crohn's disease patients than in controls were recently detected. In this study, 1D and 2D gel electrophoresis coupled with LTQ FT-MS and QStar tandem MS, respectively, were performed to characterize the secretome of a C. concisus strain isolated from a Crohn's disease patient. Two hundred and one secreted proteins were identified, of which 86 were bioinformatically predicted to be secreted. Searches were performed on the genome of C. concisus strain 13826, and 25 genes that have been associated with virulence or colonization in other organisms were identified. The zonula occludens toxin was found only in C. concisus among the Campylobacterales, although expanded searches revealed that this protein was present in two epsilon-proteobacterial species from extreme marine environments. Alignments and structural threading indicated that this toxin shared features with that of other virulent pathogens, including Neisseria meningitidis and Vibrio cholerae. Further comparative analyses identified several associations between the secretome of C. consisus and putative virulence factors of this bacterium. This study has identified several factors putatively associated with disease outcome, suggesting that C. concisus is a pathogen of the gastrointestinal tract.

Hepatocellular carcinoma: prevalence and molecular pathogenesis of Helicobacter spp.

Helicobacter pylori infection is one of the most common chronic bacterial infections in humans. The association of other Helicobacter spp. with extragastric diseases in animals is well established, and a role of these bacteria in human liver disease is becoming clearer. Several case-control studies have reported possible associations of Helicobacter spp. with various liver diseases, including hepatocellular carcinoma, which is the fifth most common type of carcinoma among men worldwide, and the eighth most common among women. Thus, it is important to understand molecular mechanisms that may lead to hepatotoxicity or hepatocellular dysfunction in which Helicobacter spp. may play a role in inducing malignant transformation of liver cells.

Wolinella succinogenes response to ox-bile stress.

The bacterium Wolinella succinogenes is the only known species of its genus. It was first isolated from cow ruminal fluid, and in cattle, it dwells in the reticulum and rumen compartments of the stomach. The global protein response of W. succinogenes to ox-bile was investigated with the aim to understand bile-tolerance mechanisms of the bacterium. Bacteria were grown in liquid media supplemented with different bile concentrations to determine its effects on growth and morphology. Proteomic analyses served to identify 14 proteins whose expression was modulated by the presence of 0.2% bile. Quantitative real-time PCR analyses of the expression of selected genes were employed to obtain independent confirmation of the proteomics data. Proteins differentially expressed revealed metabolic pathways involved in the adaptation of W. succinogenes to bile. The data suggested that bile stress elicited complex physiological responses rather than just specific pathways, and identified proteins previously unknown to be involved in the adaptation of bacteria to bile.

Novel real-time PCR assay for detection of Helicobacter pylori infection and simultaneous clarithromycin susceptibility testing of stool and biopsy specimens.

A biprobe real-time PCR protocol, followed by hybridization melting point analysis, to detect point mutations in the 23S rRNA gene of Helicobacter pylori associated with clarithromycin resistance was established and evaluated in a clinical study. Of 92 patients who underwent endoscopy, 45 were found to be H. pylori infected and invariably were also culture positive. Of the 45 isolates, 11 were shown to be resistant to clarithromycin by E-test. With respect to the detection of H. pylori infection, PCR showed sensitivities of 100% in biopsies and 98% in stool specimens and a specificity of 98% in both biopsy and stool samples. All clarithromycin-sensitive cases were identified as such by PCR in both biopsy and stool samples. Of the cases with a resistant strain, eight were identified as such in stool DNA and nine were identified in biopsy DNA. Failure of PCR to detect the resistant genotype in the biopsy DNA, stool DNA, or both (one case) was associated with mixed populations. In these cases, patients had not been treated for H. pylori infection before, and the sensitive population showed to be present in considerably higher numbers than the resistant population. In five of six cases in which infection with a resistant genotype only was identified by PCR, the patients had received clarithromycin-based eradication therapy in the past. Thus, the assay presented provides a highly accurate noninvasive method to detect H. pylori infection in stool and at the same time allows for culture-independent clarithromycin susceptibility testing.