Epidemiological cut-off values for non-O1/ non-O139 Vibrio cholerae disc diffusion data generated by standardised methods.

This work generates the data needed to set epidemiological cut-off values for disc-diffusion zone measurements of Vibrio cholerae. The susceptibility of 147 European isolates of non-O1/non-O139 V. cholerae to 19 antibiotics was established using a standardised disc diffusion method which specified incubation of Mueller Hinton agar plates at 35°C. Epidemiological cut-off values were calculated by analysis of the zone size data with the statistically based normalised resistance interpretation method. Cut-off values for 17 agents were calculated by analysis of the aggregated data from all 4 laboratories participating in this study. The cut-off values calculated were ≥18 mm for amoxicillin/clavulanate, ≥18 mm for amikacin, ≥19 mm for ampicillin, ≥27 mm for cefepime, ≥31 mm for cefotaxime, ≥24 mm for ceftazidime, ≥24 mm for chloramphenicol, ≥31 mm for ciprofloxacin, ≥16 mm for erythromycin, ≥ 27 mm for florfenicol, ≥16 mm for gentamicin, ≥23 mm for imipenem, ≥25 mm for meropenem, ≥29 mm for nalidixic acid, ≥28 mm for norfloxacin, ≥13 mm for streptomycin and ≥23 mm for tetracycline. For the other 2 agents the data from 1 laboratory was excluded from the censored aggregation because the data from that laboratory was considered excessively imprecise. The cut-off values for these 2 agents calculated for the aggregation of the data from 3 laboratories were ≥23 mm for trimethoprim and ≥24 mm for trimethoprim/sulfamethoxazole. These zone size data will be submitted to the Clinical Laboratory Standards Institute (CLSI) and European Committee for Antimicrobial Susceptibility Testing (EUCAST) for their consideration in setting international consensus epidemiological cut-off values for non O1/non-O139 V. cholerae.

Conformational maps of human 20S proteasomes reveal PA28- and immuno-dependent inter-ring crosstalks.

Hydrogen-Deuterium eXchange coupled to Mass Spectrometry (HDX-MS) is now common practice in structural biology. However, it is most of the time applied to rather small oligomeric complexes. Here, we report on the use of HDX-MS to investigate conformational differences between the human standard 20S (std20S) and immuno 20S (i20s) proteasomes alone or in complex with PA28αß or PA28γ activators. Their solvent accessibility is analyzed through a dedicated bioinformatic pipeline including stringent statistical analysis and 3D visualization. These data confirm the existence of allosteric differences between the std20S and i20S at the surface of the α-ring triggered from inside the catalytic ß-ring. Additionally, binding of the PA28 regulators to the 20S proteasomes modify solvent accessibility due to conformational changes of the ß-rings. This work is not only a proof-of-concept that HDX-MS can be used to get structural insights on large multi-protein complexes in solution, it also demonstrates that the binding of the std20S or i20S subtype to any of its PA28 activator triggers allosteric changes that are specific to this 20S/PA28 pair.

Top-Down and Intact Protein Mass Spectrometry Data Visualization for Proteoform Analysis Using VisioProt-MS.

The rise of intact protein analysis by mass spectrometry (MS) was accompanied by an increasing need for flexible tools allowing data visualization and analysis. These include inspection of the deconvoluted molecular weights of the proteoforms eluted alongside liquid chromatography (LC) through their representation in three-dimensional (3D) liquid chromatography coupled to mass spectrometry (LC-MS) maps (plots of deconvoluted molecular weights, retention times, and intensity of the MS signal). With this aim, we developed a free and open-source web application named VisioProt-MS (https://masstools.ipbs.fr/mstools/visioprot-ms/). VisioProt-MS is highly compatible with many algorithms and software developed by the community to integrate and deconvolute top-down and intact protein MS data. Its dynamic and user-friendly features greatly facilitate analysis through several graphical representations dedicated to MS and tandem mass spectrometry (MS/MS) analysis of proteoforms in complex samples. Here, we will illustrate the importance of LC-MS map visualization to optimize top-down acquisition/search parameters and analyze intact protein MS data. We will go through the main features of VisioProt-MS using the human proteasomal 20S core particle as a user-case.

HDX-Viewer: interactive 3D visualization of hydrogen-deuterium exchange data.

SUMMARY: With the advent of fully automated sample preparation robots for Hydrogen-Deuterium eXchange coupled to Mass Spectrometry (HDX-MS), this method has become paramount for ligand binding or epitope mapping screening, both in academic research and biopharmaceutical industries. However, bridging the gap between commercial HDX-MS software (for raw data interpretation) and molecular viewers (to map experiment results onto a 3D structure for biological interpretation) remains laborious and requires simple but sometimes limiting coding skills. We solved this bottleneck by developing HDX-Viewer, an open-source web-based application that facilitates and quickens HDX-MS data analysis. This user-friendly application automatically incorporates HDX-MS data from a custom template or commercial HDX-MS software in PDB files, and uploads them to an online 3D molecular viewer, thereby facilitating their visualization and biological interpretation. AVAILABILITY AND IMPLEMENTATION: The HDX-Viewer web application is released under the CeCILL (http://www.cecill.info) and GNU LGPL licenses and can be found at https://masstools.ipbs.fr/hdx-viewer. The source code is available at https://github.com/david-bouyssie/hdx-viewer.

Structural basis for chemically-induced homodimerization of a single domain antibody.

Chemically-induced dimerization (CID) systems are essential tools to interrogate and control biological systems. AcVHH is a single domain antibody homo-dimerizing upon caffeine binding. AcVHH has a strong potential for clinical applications through caffeine-mediated in vivo control of therapeutic gene networks. Here we provide the structural basis for caffeine-induced homo-dimerization of acVHH.

Antimicrobial Susceptibility among Urban Wastewater and Wild Shellfish Isolates of Non-O1/Non-O139 Vibrio cholerae from La Rance Estuary (Brittany, France).

The early 2000s marked the end of the Golden age of the antibiotics and the beginning of the awareness on the potential threat to human health due to the dissemination of antimicrobial resistance. As a base-line study, we investigated the antimicrobial susceptibility of 99 strains of non-O1/non-O139 Vibrio cholerae isolated from wastewater and shellfish in 2000/2001 within La Rance estuary (Brittany, France). All isolates were susceptible to amoxicillin-clavulanic acid, cefotaxime, imipenem, chloramphenicol, nalidixic acid, ciprofloxacin, norfloxacin, amikacin, gentamicin, tetracycline, doxycycline, trimethoprim-sulfamethoxazole, and erythromycin. The only resistances were to streptomycin, sulfonamides and ampicillin: 54.6% of the isolates had acquired resistance to at least one antimicrobial agent among them and only six isolates from cockles were multidrug resistant. On the basis of the distribution of a limited selection of resistance associated genes, our study shows that V. cholerae can constitute an environmental reservoir for these genes. However, none of our isolates harbored integron. This result casts doubt on the capacity of non-O1/non-O139 V. cholerae to acquire resistance-associated genes in such context, and on its potential role of indicator of the dissemination of antimicrobial resistance in the aquatic environment.

Antimicrobial Susceptibility of Autochthonous Aquatic Vibrio cholerae in Haiti.

We investigated the antimicrobial susceptibility of 50 environmental isolates of Vibrio cholerae non-O1/non-O139 collected in surface waters in Haiti in July 2012, during an active cholera outbreak. A panel of 16 antibiotics was tested on the isolates using the disk diffusion method and PCR detection of seven resistance-associated genes (strA/B, sul1/2, ermA/B, and mefA). All isolates were susceptible to amoxicillin-clavulanic acid, cefotaxime, imipenem, ciprofloxacin, norfloxacin, amikacin, and gentamicin. Nearly a quarter (22.0%) of the isolates were susceptible to all 16 antimicrobials tested and only 8.0% of the isolates (n = 4) were multidrug-resistant. The highest proportions of resistant isolates were observed for sulfonamide (70.0%), amoxicillin (12.0%), and trimethoprim-sulfamethoxazole (10.0%). One strain was resistant to erythromycin and one to doxycycline, two antibiotics used to treat cholera in Haiti. Among the 50 isolates, 78% possessed at least two resistance-associated genes, and the genes sul1, ermA, and strB were detected in all four multidrug-resistant isolates. Our results clearly indicate that the autochthonous population of V. cholerae non-O1/non-O139 found in surface waters in Haiti shows antimicrobial patterns different from that of the outbreak strain. The presence in the Haitian aquatic environment of V. cholerae non-O1/non-O139 with reduced susceptibility or resistance to antibiotics used in human medicine may constitute a mild public health threat.

No Evidence of Significant Levels of Toxigenic V. cholerae O1 in the Haitian Aquatic Environment During the 2012 Rainy Season.

BACKGROUND: On October 21, 2010, Haiti was struck by a cholera epidemic for the first time in over a century. Epidemiological and molecular genetic data have clearly demonstrated that the bacterium was imported. Nevertheless, the persistence of the epidemic for more than two years, the high incidence rates in some coastal areas and the seasonal exacerbations of the epidemic during the rainy seasons have prompted us to examine the levels of toxigenic Vibrio cholerae in the Haitian aquatic environment. METHODS: In July 2012, during the warm and rainy season, 36 aquatic stations were sampled to search for toxigenic V. cholerae. These stations included fresh, brackish and saline surface waters as well as waste water; the sampling sites were located in both rural and urban areas (around Port-au-Prince and Gonaïves) located in the West and Artibonite Departments. V. cholerae bacteria were detected in enrichment cultures of water samples (sample volumes included 1 L, 100 mL, 10 mL, 1 mL, 0.1 mL, 0.01 mL and 0.001 mL depending on the context). Detection methods included both culture on selective agar (for strain isolation) and PCR assays targeting the genes ompW (V. cholerae species), O1-rfb and O139-rfb (O1 and O139 V. cholerae serogroups, respectively), and the cholera toxin gene ctxA, which is present exclusively in toxigenic cholera strains. RESULTS: A total of 411 culturable V. cholerae isolates from 29 stations were obtained via selective culture; however, only one of these isolates displayed a late positive reaction with polyvalent anti-O1 serum. Positive V. cholerae PCR results were obtained from each of the 32 tested stations (a total of 77 enrichments out of 107 yielded a positive result); only one sample yielded a positive V. cholerae O1 PCR result. The cholera toxin gene ctxA was never detected via PCR with either primer pair, which includes samples derived from the two stations yielding positive O1 culture or positive O1 PCR results. Therefore, we could not demonstrate the presence of toxigenic V. cholerae O1 among the 36 stations sampled. This suggests that all water samples analyzed contained less than 10 toxigenic V. cholerae O1 bacteria per liter, a level 1000-fold below the dose that has been shown to provoke cholera in healthy adults. CONCLUSIONS: Currently, there is no evidence of a significant level of contamination of the aquatic environment in Haiti by the imported toxigenic V. cholerae O1 strain. The reemergence of cholera outbreaks in Haiti during rainy seasons is therefore more likely due to persisting outbreaks insufficiently tackled during the dry periods rather than the commonly suspected aquatic reservoir of toxigenic bacteria.

Influence of a nonfavorable environment, egg white, on resistance to heat and disinfectant, adhesion, and virulence of Salmonella enteritidis.

Although liquid egg white may be subjected to limited heat treatment when it is used in the fabrication of various foodstuffs, pathogenic bacteria such Salmonella Enteritidis could persist in this environment. Liquid egg white is not a favorable medium for Salmonella growth because of its alkaline pH and iron deficiency and the presence of ovotransferrin. Microorganisms adapted to a nonfavorable environment are often more resistant to stresses than are their laboratory-cultured counterparts. The objective of this study was to determine whether Salmonella exposed to an environment mimicking egg white conditions exhibited modified behavior that could have an impact on food safety. A medium resembling egg white (filtrate of egg white with added ovotransferrin) was used as an adaptation treatment to mimic the stress imparted by the egg white environment. There were no changes in resistance to heat and disinfection, in stainless steel adhesion, or in the virulence of Salmonella Enteritidis cultivated in the egg white medium. Egg white conditions do not appear to make Salmonella more virulent or more difficult to inactivate.

Occurrence of the tdh and trh genes in Vibrio parahaemolyticus isolates from waters and raw shellfish collected in two French coastal areas and from seafood imported into France.

The occurrence of the hemolysin genes, tdh and trh, in Vibrio parahaemolyticus strains isolated from environmental samples collected in two French coastal areas, clinical samples, and seafood products imported into France was studied. Polymerase chain reaction (PCR) with two sets of primers was used to detect the hemolysin genes. Most of the clinical isolates (91%) and 1.5% of the isolates from seafood possessed the hemolysin genes. Three and fifteen percent, respectively, of the two groups of environmental strains carried the hemolysin genes depending on the geographic site. The tdh and trh genes play important roles in virulence. Thus, our results indicate that pathogenic V. parahaemolyticus isolates are present in French coastal areas and in seafood imported into France. Furthermore, they may also be present in French seafood products.

Improved specific detection of Vibrio cholerae in environmental water samples by culture on selective medium and colony hybridization assay with an oligonucleotide probe.

We developed a rapid and efficient method based on culture on selective medium and colony hybridization assay for the detection of Vibrio cholerae in estuarine water samples. A 22-oligonucleotide sequence of the 16S-23S rDNA intergenic spacer region was labeled with digoxigenin and evaluated for specificity and sensitivity by dot blot and colony hybridization with collection strains and environmental and clinical isolates. No isolates of species other than V. cholerae hybridized with the oligonucleotide probe. Colony hybridization was then performed with mixed microbial populations from brackish and sea water samples isolated, after an enrichment step, on selective culture media. Plating on alkaline nutrient agar without added NaCl (modified alkaline nutrient agar, mANA) resulted in higher V. cholerae colony counts than did plating on other frequently used selective media, and favored direct detection by colony hybridization. The combined use of mANA agar and an oligonucleotide probe resulted in the specific recovery of V. cholerae and could be used for confirmation of the most-probable-number procedure usually used to count this bacterium in environmental samples.