Unexpected Cell Wall Alteration-Mediated Bactericidal Activity of the Antifungal Caspofungin against Vancomycin-Resistant Enterococcus faecium.

Enterococcus faecium has become a major opportunistic pathogen with the emergence of vancomycin-resistant enterococci (VRE). As part of the gut microbiota, they have to cope with numerous stresses, including effects of antibiotics and other xenobiotics, especially in patients hospitalized in intensive care units (ICUs) who receive many medications. The aim of this study was to investigate the impact of the most frequently prescribed xenobiotics for ICU patients on fitness, pathogenicity, and antimicrobial resistance of the vanB-positive E. faecium Aus0004 reference strain. Several phenotypic analyses were carried out, and we observed that caspofungin, an antifungal agent belonging to the family of echinocandins, had an important effect on E. faecium growth in vitro We confirmed this effect by electron microscopy and peptidoglycan analysis and showed that, even at a subinhibitory concentration (1/4× MIC, 8 mg/liter), caspofungin had an impact on cell wall organization, especially with respect to the abundance of some muropeptide precursors. By transcriptome sequencing (RNA-seq), it was also shown that around 20% of the transcriptome was altered in the presence of caspofungin, with 321 and 259 significantly upregulated and downregulated genes, respectively. Since the fungal target of caspofungin (i.e., ß-1,3-glucan synthase) was absent in bacteria, the mechanistic pathway of caspofungin activity was investigated. The repression of genes involved in the metabolism of pyruvate seemed to have a drastic impact on bacterial cell viability, while a decrease of glycerol metabolism could explain the conformational modifications of peptidoglycan. This is the first report of caspofungin antibacterial activity against E. faecium, highlighting the potential impact of nonantibiotic xenobiotics against bacterial pathogens.

Small RNAs in vancomycin-resistant Enterococcus faecium involved in daptomycin response and resistance.

Vancomycin-resistant Enterococcus faecium is a leading cause of hospital-acquired infections and outbreaks. Regulatory RNAs (sRNAs) are major players in adaptive responses, including antibiotic resistance. They were extensively studied in gram-negative bacteria, but less information is available for gram-positive pathogens. No sRNAs are described in E. faecium. We sought to identify a set of sRNAs expressed in vancomycin-resistant E. faecium Aus0004 strain to assess their roles in daptomycin response and resistance. Genomic and transcriptomic analyses revealed a set of 61 sRNA candidates, including 10 that were further tested and validated by Northern and qPCR. RNA-seq was performed with and without subinhibitory concentrations (SICs) of daptomycin, an antibiotic used to treat enterococcal infections. After daptomycin SIC exposure, the expression of 260 coding and srna genes was altered, with 80 upregulated and 180 downregulated, including 51% involved in carbohydrate and transport metabolisms. Daptomycin SIC exposure significantly affected the expression of seven sRNAs, including one experimentally confirmed, sRNA_0160. We studied sRNA expression in isogenic mutants with increasing levels of daptomycin resistance and observed that expression of several sRNAs, including sRNA_0160, was modified in the stepwise mutants. This first genome-wide sRNA identification in E. faecium suggests that some sRNAs are linked to antibiotic stress response and resistance.

Nationwide molecular epidemiology of methicillin-resistant Staphylococcus aureus responsible for horse infections in France.

BACKGROUND: The epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) isolated in horse infections is not well documented, especially in France. The aim of the study was to evaluate the prevalence of MRSA isolates in horse infections from 2007 to 2013 in France and to characterize phenotypically and genotypically this collection. RESULTS: Out of 1393 S. aureus horse isolates, 85 (6.1%) were confirmed to be MRSA. Interestingly, the prevalence of MRSA significantly increased from 2007-2009 to 2010-2013 (0.7 vs. 9.5%, P <0.0001). Resistance to methicillin was due to the presence of the mecA gene in 84 strains (98.8%) while one strain (1.2%) possessed the mecC gene. The vast majority of the strains (83/85, 97.6%) was resistant to at least three different classes of antibiotics. Multi-locus sequence typing (MLST) showed that MRSA strains belonged mainly since not all belong to two sequence types (STs): ST398 (53/85, 62.4%) and ST8 (28/85, 32.9%). It is worth to note that all ST398 MRSA isolates were detected in the period 2010-2013. Other molecular typing methods were also used, such SCC mec analysis, spa typing and rep-PCR (Diversilab, bioMérieux). All these four techniques were in good agreement, with spa typing and rep-PCR being more discriminative than MLST and SCC mec typing. CONCLUSIONS: This study is the first epidemiological study in France with extensive characterization of MRSA isolates associated with horse infections in stud farms. It shows that there is a significant increase of MRSA prevalence between 2007 and 2013, which mainly results from the spread of ST398 clones. It also highlights the importance of horses as a potential reservoir of important antimicrobial resistance genes.

Subinhibitory Concentrations of Ciprofloxacin Enhance Antimicrobial Resistance and Pathogenicity of Enterococcus faecium.

Enterococcus faecium has emerged as a major opportunistic pathogen for 2 decades with the spread of hospital-adapted multidrug-resistant clones. As members of the intestinal microbiota, they are subjected to numerous bacterial stresses, including antibiotics at subinhibitory concentrations (SICs). Since fluoroquinolones are extensively prescribed, SICs are very likely to occur in vivo, with potential effects on bacterial metabolism with subsequent modulation of opportunistic traits. The aim of this study was to evaluate globally the impact of SICs of ciprofloxacin on antimicrobial resistance and pathogenicity of E. faecium Transcriptomic analysis was performed by RNA sequencing (RNA-seq) (HiSeq 2500; Illumina) using the vanB-positive reference strain E. faecium Aus0004 in the absence or presence of ciprofloxacin SIC (0.38 mg/liter, i.e., 1/8 of the MIC). Several genetic and phenotypic tests were used for validation. In the presence of ciprofloxacin SIC, 196 genes were significantly induced, whereas 286 genes were significantly repressed, meaning that 16.8% of the E. faecium genome was altered. Among upregulated genes, EFAU004_02294 (fold change, 14.3) encoded a protein (Qnr of E. faecium [EfmQnr]) homologue of Qnr proteins involved in quinolone resistance in Gram-negative bacilli. Its implication in intrinsic and adaptive fluoroquinolone (FQ) resistance in E. faecium was experimentally ascertained. Moreover, EFAU004_02292, coding for the collagen adhesin Acm, was also induced by the SIC of ciprofloxacin (fold change, 8.2), and higher adhesion capabilities were demonstrated phenotypically. Both EfmQnr and Acm determinants may play an important role in the transition from a commensal to a pathogenic state of E. faecium that resides in the gut of patients receiving fluoroquinolone therapy.