Comparison of in vitro static and dynamic assays to evaluate the efficacy of an antimicrobial drug combination against Staphylococcus aureus

An easily implementable strategy to reduce treatment failures in severe bacterial infections is to combine already available antibiotics. However, most in vitro combination assays are performed by exposing standard bacterial inocula to constant concentrations of antibiotics over less than 24h, which can be poorly representative of clinical situations. The aim of this study was to assess the ability of static and dynamic in vitro Time-Kill Studies (TKS) to identify the potential benefits of an antibiotic combination (here, amikacin and vancomycin) on two different inoculum sizes of two S. aureus strains. In the static TKS (sTKS), performed by exposing both strains over 24h to constant antibiotic concentrations, the activity of the two drugs combined was not significantly different the better drug used alone. However, the dynamic TKS (dTKS) performed over 5 days by exposing one strain to fluctuating concentrations representative of those observed in patients showed that, with the large inoculum, the activities of the drugs, used alone or in combination, significantly differed over time. Vancomycin did not kill bacteria, amikacin led to bacterial regrowth whereas the combination progressively decreased the bacterial load. Thus, dTKS revealed an enhanced effect of the combination on a large inoculum not observed in sTKS. The discrepancy between the sTKS and dTKS results highlights that the assessment of the efficacy of a combination for severe infections associated with a high bacterial load could be demanding. These situations probably require the implementation of dynamic assays over the entire expected treatment duration rather than the sole static assays performed with steady drug concentrations over 24h

Understanding the impact of antibiotic therapies on the respiratory tract resistome: A novel pooled-template metagenomic sequencing strategy

Determining the effects of antimicrobial therapies on airway microbiology at a population-level is essential. Such analysis allows, for example, surveillance of antibiotic-induced changes in pathogen prevalence, the emergence and spread of antibiotic resistance, and the transmission of multi-resistant organisms. However, current analytical strategies for understanding these processes are limited. Culture- and PCR-based assays for specific microbes require the a priori selection of targets, while antibiotic sensitivity testing typically provides no insight into either the molecular basis of resistance, or the carriage of resistance determinants by the wider commensal microbiota. Shotgun metagenomic sequencing provides an alternative approach that allows the microbial composition of clinical samples to be described in detail, including the prevalence of resistance genes and virulence traits. While highly informative, the application of metagenomics to large patient cohorts can be prohibitively expensive. Using sputum samples from a randomised placebo-controlled trial of erythromycin in adults with bronchiectasis, we describe a novel, cost-effective strategy for screening patient cohorts for changes in resistance gene prevalence. By combining metagenomic screening of pooled DNA extracts with validatory quantitative PCR-based analysis of candidate markers in individual samples, we identify population-level changes in the relative abundance of specific macrolide resistance genes. This approach has the potential to provide an important adjunct to current analytical strategies, particularly within the context of antimicrobial clinical trials

[Two efflux systems expressed simultaneously in clinical Pseudomonas aeruginosa].

International audienceActive efflux systems MexAB-OprM and MexXY were found to be overexpressed simultaneously in 12 multiresistant clinical isolates of Pseudomonas aeruginosa. Nine of these strains (agrZ mutants) harbored mutations in gene mexZ, the product of which down-regulates expression of operon mexXY. Eight of the 12 strains exhibited mutations in genes known to control transcription of operon mexAB-oprM, such as mexR (four nalB mutants) or PA3721 (three nalC mutants). One strain was a nalB/nalC double mutant. For MexAB-OprM as well as for MexXY, no clear correlation could be established between (i) the types of mutations, (ii) the over-expression levels of genes mexA or mexX, and (iii) the resistance levels to effluxed antibiotics. Finally, three and four isolates overproduced MexXY (agrW mutants) or MexAB-OprM (nalD mutants), respectively, without any mutation in the known regulator genes. These data show that clinical isolates are able to broaden their drug resistance profiles by coexpressing two Mex efflux pumps and suggest the existence of additional regulators for MexAB-OprM and MexXY

mcr-1 is borne by highly diverse Escherichia coli isolates since 2004 in food-producing animals in Europe.

International audienceIn November 2015, a plasmid-mediated colistin resistance, MCR-1, was described in animals, food and humans in China, and it was considered as a potential emerging threat to public health. Therefore, we screened for the mcr-1 gene a European collection of colistin-resistant Escherichia coli (n=218) and Salmonella spp. (n=74) isolated from diseased food-producing animals between 2004 and 2014 and characterized the mcr-1-positive clones.Screening for mcr-1 gene was performed by PCR on isolates for which inhibition diameter was 2 mg/L. Most of the mcr-1-positive E. coli belonged to the phylogroup A with two prevalent clonal complexes, CC10 and CC165, in which sequence type 10 and sequence type 100 were overrepresented and pulsed-field gel electrophoresis typing revealed a high diversity of pulsotypes.MCR-1 was detected yearly in European food-producing animal since 2004 with a high diversity of pulsotypes supporting the dissemination of mcr-1 via plasmids

Genetic analysis of a multiresistant strain of Pseudomonas aeruginosa producing PER-1 beta-lactamase.

International audienceA multiresistant strain of Pseudomonas aeruginosa, PA2345, belonging to serotype O:1, was isolated at the Teaching Hospital of Besançon, France. Resistance to beta-lactams, including third-generation cephalosporins, depended upon a chromosomally-located composite transposon carrying the bla(PER-1) gene encoding extended-spectrum beta-lactamase PER-1. PA2345 was unrelated genotypically to two previous PER-1-producing isolates of P. aeruginosa. Sequence analysis of the transposon in PA2345 revealed the presence of two insertion sequences (ISPa23 and ISPa24) with very different predicted transposases (TnpA1, TnpA2), which were both bordered by closely related 16-bp inverted repeats. High resistance of PA2345 to aminoglycosides was caused, in part, by a chromosomal class-I integron containing gene cassettes aadB, encoding an ANT(2'') enzyme, and aadA11, encoding a new ANT(3'') enzyme with 281 amino-acids that conferred elevated resistance to streptomycin and spectinomycin. Stable overproduction of efflux system MexXY contributed to resistance to amikacin, while mutations in the quinolone resistance-determining regions of gyrA and parC accounted for the high resistance of PA2345 to fluoroquinolones. The study indicates that multidrug resistance in P. aeruginosa might arise from sequential acquisition of a variety of mechanisms provided by both horizontal gene transfers and mutations in chromosomal genes

Time-Course Analysis of Main Markers of Primary Infection in Cats with the Feline Immunodeficiency Virus

Studies of the response of the immune system to feline immunodeficiency virus (FIV) during primary infection have shown that a subpopulation of CD8+ T-cells with an activated phenotype and reduced expression of the CD8β chain (denoted CD8βlow T cells) expands to reach up to 80% of the total CD8+ T cell count. The expansion of this subpopulation is considered to be a signature of FIV and an indicator of immune system alteration. We use a simple mathematical formalism to study the relationships over time between the dose of infection, the size of the CD8βlow population, and the circulating viral load in cats infected with FIV. Viremia profiles are described using a combination of two exponential laws, whereas the CD8βlow percentage (out of the total CD8+ population) is represented by a Gompertz law including an expansion phase and a saturation phase. Model parameters are estimated with a population approach using data from 102 experimentally infected cats. We examine the dose of infection as a potential covariate of parameters. We find that the rates of increase of viral load and of CD8βlow percentage are both correlated with the dose of infection. Cats that develop strong acute viremia also show the largest degree of CD8βlow expansion. The two simple models are robust tools for analysing the time course of CD8βlow percentage and circulating viral load in FIV-infected cats and may be useful for generating new insights on the disease and on the design of therapeutic strategies, potentially applicable to HIV infection