Gastric Helicobacter pylori infection perturbs human oral microbiota

Background We investigated the effects of gastric Helicobacter pylori infection on the daytime and overnight human oral microbiota. Methods Twenty four volunteers were recruited. Ten tested positive for H. pylori infection by the Carbon-14 Urea Breath Test, and the rest were negative. Two oral swabs were collected: one immediately after waking up in the morning and before brushing teeth, and another in the evening before teeth-brushing. DNA extract acquired from each swab was subjected to Illumina sequencing of 16S rRNA gene amplicons. The microbial abundance and composition were analysed in relation to H. pylori infection status. Results Helicobacter pylori-positive individuals had significant changes in the alpha and beta diversities in the daytime samples in comparison to those who were H. pylori negative. To identify which taxa could be significantly affected within the cohorts in the daytime, we employed the LEfSe method. When compared against UBT-negative samples, significantly higher abundances were detected in both Pseudomonas and Roseomonas, while Fusobacterium, Solobacterium, Haemophilus and Streptococcus were significantly decreased in the UBT-positive samples. Discussion Our data demonstrated that H. pylori infection affects the human daytime oral microbiota. The hitherto undocumented changes of several bacterial genera due to H. pylori infection require more studies to examine their potential health effects on affected individuals

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field

The effect of environmental conditions on biofilm formation of Burkholderia pseudomallei clinical isolates.

Burkholderia pseudomallei, a Gram-negative saprophytic bacterium, is the causative agent of the potentially fatal melioidosis disease in humans. In this study, environmental parameters including temperature, nutrient content, pH and the presence of glucose were shown to play a role in in vitro biofilm formation by 28 B. pseudomallei clinical isolates, including four isolates with large colony variants (LCVs) and small colony variants (SCVs) morphotypes. Enhanced biofilm formation was observed when the isolates were tested in LB medium, at 30 °C, at pH 7.2, and in the presence of as little as 2 mM glucose respectively. It was also shown that all SVCs displayed significantly greater capacity to form biofilms than the corresponding LCVs when cultured in LB at 37 °C. In addition, octanoyl-homoserine lactone (C(8)-HSL), a quorum sensing molecule, was identified by mass spectrometry analysis in bacterial isolates referred to as LCV CTH, LCV VIT, SCV TOM, SCV CTH, 1 and 3, and the presence of other AHL's with higher masses; decanoyl-homoserine lactone (C(10)-HSL) and dodecanoyl-homoserine lactone (C(12)-HSL) were also found in all tested strain in this study. Last but not least, we had successfully acquired two Bacillus sp. soil isolates, termed KW and SA respectively, which possessed strong AHLs degradation activity. Biofilm formation of B. pseudomallei isolates was significantly decreased after treated with culture supernatants of KW and SA strains, demonstrating that AHLs may play a role in B. pseudomallei biofilm formation

Phenotypic Detection of Metallo-β-Lactamase in Imipenem-Resistant Pseudomonas aeruginosa

Carbapenems are the primary choice of treatment for severe Pseudomonas aeruginosa infection. However, the emergence of carbapenem resistance due to the production of metallo-β-lactamases (MBLs) is of global concern. In this study, 90 imipenem- (IPM- or IP-) resistant P. aeruginosa (IRPA) isolates, including 32 previously tested positive and genotyped for MBL genes by PCR, were subjected to double-disk synergy test (DDST), combined disk test (CDT), and imipenem/imipenem-inhibitor (IP/IPI) E-test to evaluate their MBLs detection capability. All three methods were shown to have a sensitivity of 100%. However, DDST was the most specific of the three (96.6%), followed by IP/IPI E-test interpreted based on the single criteria of IP/IPI ≥8 as positive (62.1%), and CDT was the least specific (43.1%). Based on the data from this evaluation, we propose that only IRPA with IP MIC >16 μg/mL and IP/IPI ≥8 by IP/IPI E-test should be taken as positive for MBL activity. With the new dual interpretation criteria, the MBL IP/IPI E-test was shown to achieve 100% sensitivity as well as specificity for the IRPA in this study. Therefore, the IP/IPI E-test is a viable alternative phenotypic assay to detect MBL production in IRPA in our population in circumstances where PCR detection is not a feasible option

AHL identification of <i>B. pseudomallei</i> isolates using TLC and mass spectrometry analysis.

<p>AHL identification of <i>B. pseudomallei</i> isolates using TLC and mass spectrometry analysis.</p

AHL identification of <i>B. pseudomallei</i> wild type via analytical TLC assay.

<p>TLC analysis of short chain AHL production in wildtype and colony morphotypes of <i>B. pseudomallei</i> (A) and long chain AHL production in wildtype and colony morphotypes of <i>B. pseudomallei</i> (B). Lane 1: K96342, Lane 2: strain 1, Lane 3: strain 3, Lane 4: LCV TOM, Lane 5: LCV CTH, Lane 6: LCV OCY, Lane 7: LCV VL, Lane 8: SCV TOM, Lane 9: SCV CTH, Lane 10: SCV OCY and Lane 11: SCV VL.</p

<i>B. pseudomallei</i> AHLs treatment with <i>E. coli</i> culture supernatants.

<p>Detection of AHL molecules in <i>B. pseudomallei</i> bioactive compound extracts prepared after treatment with <i>E.coli</i> culture supernatants for short chain AHL production in wildtype and colony morphotypes of <i>B. pseudomallei</i> (A) and long chain AHL production in wildtype and colony morphotypes of <i>B. pseudomallei</i> (B). Lane 1: K96342, Lane 2: strain 1, Lane 3: strain 3, Lane 4: LCV TOM, Lane 5: LCV CTH, Lane 6: LCV OCY, Lane 7: LCV VL, Lane 8: SCV TOM, Lane 9: SCV CTH, Lane 10: SCV OCY and Lane 11: SCV VL.</p

Effect of glucose on biofilm formation by <i>B. pseudomallei</i> wild type and colony morphotypes.

<p>Biofilm formation by <i>B. pseudomallei</i> (A) wild type isolates at 30°C and 37°C, (B) colony morphotypes at 37°C and (C) colony morphotypes at 30°C, in LB supplemented with glucose of different concentrations.</p

Effect of growth medium on biofilm formation by <i>B. pseudomallei</i> wild type and colony morphotypes.

<p>Biofilm formation by <i>B. pseudomallei</i> (A) wild type isolates at 30°C and 37°C, (B) colony morphotypes at 37°C and (C) colony morphotypes at 30°C, in LB and MVBM. The results in Panel A are reported as the overall mean value for biofilm formation of 24 wild type isolates.</p