Genome sequence analysis of Helicobacter pylori strains associated with gastric ulceration and gastric cancer

<p>Abstract</p> <p>Background</p> <p>Persistent colonization of the human stomach by <it>Helicobacter pylori </it>is associated with asymptomatic gastric inflammation (gastritis) and an increased risk of duodenal ulceration, gastric ulceration, and non-cardia gastric cancer. In previous studies, the genome sequences of <it>H. pylori </it>strains from patients with gastritis or duodenal ulcer disease have been analyzed. In this study, we analyzed the genome sequences of an <it>H. pylori </it>strain (98-10) isolated from a patient with gastric cancer and an <it>H. pylori </it>strain (B128) isolated from a patient with gastric ulcer disease.</p> <p>Results</p> <p>Based on multilocus sequence typing, strain 98-10 was most closely related to <it>H. pylori </it>strains of East Asian origin and strain B128 was most closely related to strains of European origin. Strain 98-10 contained multiple features characteristic of East Asian strains, including a type s1c <it>vacA </it>allele and a <it>cagA </it>allele encoding an EPIYA-D tyrosine phosphorylation motif. A core genome of 1237 genes was present in all five strains for which genome sequences were available. Among the 1237 core genes, a subset of alleles was highly divergent in the East Asian strain 98-10, encoding proteins that exhibited <90% amino acid sequence identity compared to corresponding proteins in the other four strains. Unique strain-specific genes were identified in each of the newly sequenced strains, and a set of strain-specific genes was shared among <it>H. pylori </it>strains associated with gastric cancer or premalignant gastric lesions.</p> <p>Conclusion</p> <p>These data provide insight into the diversity that exists among <it>H. pylori </it>strains from diverse clinical and geographic origins. Highly divergent alleles and strain-specific genes identified in this study may represent useful biomarkers for analyzing geographic partitioning of <it>H. pylori </it>and for identifying strains capable of inducing malignant or premalignant gastric lesions.</p

Helicobacter pylori Perturbs Iron Trafficking in the Epithelium to Grow on the Cell Surface

Helicobacter pylori (Hp) injects the CagA effector protein into host epithelial cells and induces growth factor-like signaling, perturbs cell-cell junctions, and alters host cell polarity. This enables Hp to grow as microcolonies adhered to the host cell surface even in conditions that do not support growth of free-swimming bacteria. We hypothesized that CagA alters host cell physiology to allow Hp to obtain specific nutrients from or across the epithelial barrier. Using a polarized epithelium model system, we find that isogenic ΔcagA mutants are defective in cell surface microcolony formation, but exogenous addition of iron to the apical medium partially rescues this defect, suggesting that one of CagA's effects on host cells is to facilitate iron acquisition from the host. Hp adhered to the apical epithelial surface increase basolateral uptake of transferrin and induce its transcytosis in a CagA-dependent manner. Both CagA and VacA contribute to the perturbation of transferrin recycling, since VacA is involved in apical mislocalization of the transferrin receptor to sites of bacterial attachment. To determine if the transferrin recycling pathway is involved in Hp colonization of the cell surface, we silenced transferrin receptor expression during infection. This resulted in a reduced ability of Hp to colonize the polarized epithelium. To test whether CagA is important in promoting iron acquisition in vivo, we compared colonization of Hp in iron-replete vs. iron-deficient Mongolian gerbils. While wild type Hp and ΔcagA mutants colonized iron-replete gerbils at similar levels, ΔcagA mutants are markedly impaired in colonizing iron-deficient gerbils. Our study indicates that CagA and VacA act in concert to usurp the polarized process of host cell iron uptake, allowing Hp to use the cell surface as a replicative niche

The James Webb Space Telescope Mission

Twenty-six years ago a small committee report, building on earlier studies, expounded a compelling and poetic vision for the future of astronomy, calling for an infrared-optimized space telescope with an aperture of at least $4m$. With the support of their governments in the US, Europe, and Canada, 20,000 people realized that vision as the $6.5m$ James Webb Space Telescope. A generation of astronomers will celebrate their accomplishments for the life of the mission, potentially as long as 20 years, and beyond. This report and the scientific discoveries that follow are extended thank-you notes to the 20,000 team members. The telescope is working perfectly, with much better image quality than expected. In this and accompanying papers, we give a brief history, describe the observatory, outline its objectives and current observing program, and discuss the inventions and people who made it possible. We cite detailed reports on the design and the measured performance on orbit.Comment: Accepted by PASP for the special issue on The James Webb Space Telescope Overview, 29 pages, 4 figure

Surviving Sepsis Campaign: international guidelines for management of severe sepsis and septic shock, 2012

OBJECTIVE: To provide an update to the "Surviving Sepsis Campaign Guidelines for Management of Severe Sepsis and Septic Shock," last published in 2008. DESIGN: A consensus committee of 68 international experts representing 30 international organizations was convened. Nominal groups were assembled at key international meetings (for those committee members attending the conference). A formal conflict of interest policy was developed at the onset of the process and enforced throughout. The entire guidelines process was conducted independent of any industry funding. A stand-alone meeting was held for all subgroup heads, co- and vice-chairs, and selected individuals. Teleconferences and electronic-based discussion among subgroups and among the entire committee served as an integral part of the development. METHODS: The authors were advised to follow the principles of the Grading of Recommendations Assessment, Development and Evaluation (GRADE) system to guide assessment of quality of evidence from high (A) to very low (D) and to determine the strength of recommendations as strong (1) or weak (2). The potential drawbacks of making strong recommendations in the presence of low-quality evidence were emphasized. Recommendations were classified into three groups: (1) those directly targeting severe sepsis; (2) those targeting general care of the critically ill patient and considered high priority in severe sepsis; and (3) pediatric considerations. RESULTS: Key recommendations and suggestions, listed by category, include: early quantitative resuscitation of the septic patient during the first 6 h after recognition (1C); blood cultures before antibiotic therapy (1C); imaging studies performed promptly to confirm a potential source of infection (UG); administration of broad-spectrum antimicrobials therapy within 1 h of the recognition of septic shock (1B) and severe sepsis without septic shock (1C) as the goal of therapy; reassessment of antimicrobial therapy daily for de-escalation, when appropriate (1B); infection source control with attention to the balance of risks and benefits of the chosen method within 12 h of diagnosis (1C); initial fluid resuscitation with crystalloid (1B) and consideration of the addition of albumin in patients who continue to require substantial amounts of crystalloid to maintain adequate mean arterial pressure (2C) and the avoidance of hetastarch formulations (1B); initial fluid challenge in patients with sepsis-induced tissue hypoperfusion and suspicion of hypovolemia to achieve a minimum of 30 mL/kg of crystalloids (more rapid administration and greater amounts of fluid may be needed in some patients (1C); fluid challenge technique continued as long as hemodynamic improvement is based on either dynamic or static variables (UG); norepinephrine as the first-choice vasopressor to maintain mean arterial pressure ≥65 mmHg (1B); epinephrine when an additional agent is needed to maintain adequate blood pressure (2B); vasopressin (0.03 U/min) can be added to norepinephrine to either raise mean arterial pressure to target or to decrease norepinephrine dose but should not be used as the initial vasopressor (UG); dopamine is not recommended except in highly selected circumstances (2C); dobutamine infusion administered or added to vasopressor in the presence of (a) myocardial dysfunction as suggested by elevated cardiac filling pressures and low cardiac output, or (b) ongoing signs of hypoperfusion despite achieving adequate intravascular volume and adequate mean arterial pressure (1C); avoiding use of intravenous hydrocortisone in adult septic shock patients if adequate fluid resuscitation and vasopressor therapy are able to restore hemodynamic stability (2C); hemoglobin target of 7-9 g/dL in the absence of tissue hypoperfusion, ischemic coronary artery disease, or acute hemorrhage (1B); low tidal volume (1A) and limitation of inspiratory plateau pressure (1B) for acute respiratory distress syndrome (ARDS); application of at least a minimal amount of positive end-expiratory pressure (PEEP) in ARDS (1B); higher rather than lower level of PEEP for patients with sepsis-induced moderate or severe ARDS (2C); recruitment maneuvers in sepsis patients with severe refractory hypoxemia due to ARDS (2C); prone positioning in sepsis-induced ARDS patients with a PaO (2)/FiO (2) ratio of ≤100 mm Hg in facilities that have experience with such practices (2C); head-of-bed elevation in mechanically ventilated patients unless contraindicated (1B); a conservative fluid strategy for patients with established ARDS who do not have evidence of tissue hypoperfusion (1C); protocols for weaning and sedation (1A); minimizing use of either intermittent bolus sedation or continuous infusion sedation targeting specific titration endpoints (1B); avoidance of neuromuscular blockers if possible in the septic patient without ARDS (1C); a short course of neuromuscular blocker (no longer than 48 h) for patients with early ARDS and a PaO (2)/FI O (2) 180 mg/dL, targeting an upper blood glucose ≤180 mg/dL (1A); equivalency of continuous veno-venous hemofiltration or intermittent hemodialysis (2B); prophylaxis for deep vein thrombosis (1B); use of stress ulcer prophylaxis to prevent upper gastrointestinal bleeding in patients with bleeding risk factors (1B); oral or enteral (if necessary) feedings, as tolerated, rather than either complete fasting or provision of only intravenous glucose within the first 48 h after a diagnosis of severe sepsis/septic shock (2C); and addressing goals of care, including treatment plans and end-of-life planning (as appropriate) (1B), as early as feasible, but within 72 h of intensive care unit admission (2C). Recommendations specific to pediatric severe sepsis include: therapy with face mask oxygen, high flow nasal cannula oxygen, or nasopharyngeal continuous PEEP in the presence of respiratory distress and hypoxemia (2C), use of physical examination therapeutic endpoints such as capillary refill (2C); for septic shock associated with hypovolemia, the use of crystalloids or albumin to deliver a bolus of 20 mL/kg of crystalloids (or albumin equivalent) over 5-10 min (2C); more common use of inotropes and vasodilators for low cardiac output septic shock associated with elevated systemic vascular resistance (2C); and use of hydrocortisone only in children with suspected or proven "absolute"' adrenal insufficiency (2C). CONCLUSIONS: Strong agreement existed among a large cohort of international experts regarding many level 1 recommendations for the best care of patients with severe sepsis. Although a significant number of aspects of care have relatively weak support, evidence-based recommendations regarding the acute management of sepsis and septic shock are the foundation of improved outcomes for this important group of critically ill patients

<i>In Vitro</i> Characterization of the Anti-Bacterial Activity of SQ109 against <i>Helicobacter pylori</i>

<div><p>The most evident challenge to treatment of <i>Helicobacter pylori</i>, a bacterium responsible for gastritis, peptic ulcers and gastric cancer, is the increasing rate of resistance to all currently used therapeutic antibiotics. Thus, the development of novel therapies is urgently required. <i>N</i>-geranyl-N'-(2-adamantyl) ethane-1, 2-diamine (SQ109) is an ethylene diamine-based antitubercular drug that is currently in clinical trials for the treatment of tuberculosis (TB). Previous pharmacokinetic studies of SQ109 revealed that persistently high concentrations of SQ109 remain in the stomach 4 hours post oral administration in rats. This finding, combined with the need for new anti-<i>Helicobacter</i> therapies, prompted us to define the <i>in vitro</i> efficacy of SQ109 against <i>H. pylori</i>. Liquid broth micro-dilution was used for susceptibility studies to determine the antimicrobial activity of SQ109 against a total of 6 laboratory strains and 20 clinical isolates of <i>H. pylori</i>; the clinical isolates included a multi-drug resistant strain. All strains tested were susceptible to SQ109 with MIC and MBC ranges of 6-10 µM and 50-60 µM, respectively. SQ109 killing kinetics were concentration- and time-dependent. SQ109 killed <i>H. pylori</i> in 8-10 h at 140 µM (2MBCs) or 4-6 h at 200 µM (~3MBCs). Importantly, though the kinetics of killing were altered, SQ109 retained potent bactericidal activity against <i>H. pylori</i> at low pH. Additionally, SQ109 demonstrated robust thermal stability and was effective at killing slow growing or static bacteria. In fact, pretreatment of cultures with a bacteriostatic concentration of chloramphenicol (Cm) synergized the effects of typically bacteriostatic concentrations of SQ109 to the level of five-logs of bacterial killing. A molar-to-molar comparison of the efficacy of SQ109 as compared to metronidazole (MTZ), amoxicillin (AMX), rifampicin (RIF) and clarithromycin (CLR), revealed that SQ109 was superior to MTZ, AMX and RIF but not to CLR. Finally, the frequency of resistance to SQ109 was low and electron microscopy studies revealed that SQ109 interacted with bacterial inner membrane and cytoplasmic content(s). Collectively, our <i>in vitro</i> data demonstrate that SQ109 is an effective monotherapy against susceptible and multi-drug resistant strains of <i>H. pylori</i> and may be useful alone or in combination with other antibiotics for development as a new class of anti-<i>Helicobacter</i> drugs.</p> </div

Visualization of SQ109-induced morphological changes of <i>H. pylori</i> using transmission electron microscopy (TEM).

<p>Approximately 6 x 10⁷<i>H. pylori</i> cells were sampled following 8 h of exposure to PBS (<b>A</b>), 100 µM amoxicillin (<b>B</b>), 20 µM C ₁₂K-2β₁₂ peptide (<b>C</b>) or 140 µM SQ109 (<b>D</b>). Cells were examined at low magnification (6,500x) on a TEM and images were acquired and processed with AMT XR60B digital camera and AMTV600 software, respectively. (<b>A</b>) demonstrates normal spiral- or comma-shaped rod morphology of <i>H. pylori</i> cells as well as intact cell membranes following treatment with PBS as a negative control drug. Amoxicillin-treated cells (<b>B</b>) show characteristically swollen cells as well as detachment of the inner membrane from the outer membrane (white arrows). C₁₂K-2β₁₂-treated cells (<b>C</b>) show cell lysis, numerous ghost cells (C, white arrowheads) as well as the formation of electron dense structures within the extracellular medium and inside the cells. Like AMX-treated cells, SQ109-treated cells (<b>D</b>) present with inner membrane detachment from the outer membrane (white arrows), but are not significantly enlarged. Some ghost cells are visible (white arrowheads). Scale bars = 1500 nm. The data are representative images from two independent experiments.</p

TEM analysis of SQ109-induced morphological and ultra-structural changes in <i>H. pylori</i>-cell wall, membranes, and cytoplasm.

<p>Approximately 6 x 10⁷<i>H. pylori</i> cells were sampled following 2 h (top panels) or 8 h- (bottom panels) of culture in the presence of PBS (<b>A</b> and <b>B</b>) as a negative control or 140 µM SQ109 (C, D, <b>E</b>, <b>F</b>, <b>G</b>, and <b>H</b>). Bacterial cells cultured in the presence of 100 µM AMX or 20 µM C ₁₂K-2β₁₂ were included as positive controls (data shown in Figure S3). PBS-treated negative control cells showed evidence of normal spiral-comma rod shaped morphology in addition to smooth homogenous cytoplasm (<b><i>sc</i></b>) and intact cell walls (<b>A</b>, <b>B</b>). In contrast, after 2 h of culture, SQ109 induced the formation of spindle actin-like cytoskeleton structures (<b><i>a</i></b>) in some cells (<b>C</b>) that appeared to condense cytoplasmic contents and lead to detachment of the IM from OM (arrows; <b>C</b>, <b>E</b>, <b>F</b>, <b>G</b>, and <b>H</b>) of nearly all cells resulting in complete disappearance of the periplasmic region; 95-99% of these cells also showed a deformed coccoid morphology (<b>E</b>, <b>G</b>, and <b>H</b>). After 2h and 8 h treatment with SQ109, the cells further showed blebs (<b><i>b</i></b>), electron-dense structures (<b><i>e</i></b>), evidence of complete loss of the IM and part of the cell wall with only the OM remaining intact (<b>D</b>), formation of two aberrant cytoplasmic bodies from a single cell (arrowheads) (<b>G</b> and <b>H</b>), and formation of outer membrane vesicles (omv) and inner membrane vesicles (imv) (<b>C</b>, <b>F</b>, <b>G</b>, and <b>H</b>). All scale bars are of 200 nm except <b>B</b> and <b>E</b> which measure 1500 nm. Representative images from two independent experiments are presented.</p

Molar-to-molar comparison of the antibacterial activity of SQ109, amoxicillin, metronidazole, and clarithromycin.

<p>Time-kill assays for two drug concentrations, 70 µM (<b>A</b>) and 140 µM (<b>B</b>), were used to compare the bactericidal activity of SQ109 to those of conventional antibiotics currently used for the treatment of <i>H. pylori</i> infection. The data are representative results from three independent experiments. The horizontal dashed line on each graph indicates the limit of detection (500 bacteria).</p