ABSTRACT: Enterococcus faecium has become a major opportunistic pathogen with the emergence of multidrug-resistant clones that are well-adapted to the hospital environment. As part of the vast diversity of gut microbiota, they are faced with different environmental stress, including antimicrobial pressure. By contrast, little is known about the effect of non-antibiotic molecules on bacterial physiology while numerous drugs are used in inpatients, especially those hospitalized in intensive care units (ICUs). The aim of this study was to investigate the impact of the most prescribed xenobiotics in ICUs on fitness, pathogenicity and antimicrobial resistance of E. faecium. Several phenotypic analysis was carried out and we rapidly brought to light that caspofungin, an antifungal agent belonging to the echinocandin family, seemed to have an important impact on E. faecium growth. Since the fungal target of caspofungin [beta-(1,3)-glucan synthase] is absent in enterococci, the mechanism of caspofungin action was investigated by several approaches. First, we decided to confirm this result by electronic microscopy and a peptidoglycan analysis by Ultra Performance Liquid Chromatography coupled with mass spectrometry (UPLC-MS/MS). Again, we highlighted that caspofungin even at subinhibitory concentrations (SICs) seemed to have an impact on cell wall organization especially in muropeptide precursors abundance. Then, a transcriptomic analysis was performed by RNA-seq (HiSeq 2500, Illumina) using the vanB-positive reference strain E. faecium Aus0004 in the presence or absence of caspofungin SIC (8 mg/L i.e., ¼ of the MIC). Transcriptomic analysis showed that the expression of 568 genes (19.9% of the genome) was significantly altered in the presence of caspofungin SIC, with 323 genes induced (fold change >2, p-value <0.1) and 245 genes repressed (fold change <-2, p-value <0.1). Regarding the repressed genes, the pdhABCD operon is largely downregulated (fold changes -4.3, -9.7, -6.9 and -6.4, respectively). This operon encoded components of the pyruvate deshydrogenase multienzyme complex involved in bacterial energetic pathway by the citrate cycle (i.e., TCA cycle). Moreover, it seemed that the glycerol metabolism pathway and in particular the glpOKF operon is downregulated too. The dramatic alteration of TCA seemed to have an drastic impact on bacterial cells viability indeed decrease of glycerol metabolism could explain the conformational modifications of peptidoglycan.
