Summary: The opportunistic pathogen Enterococcus faecalis is well equipped with peroxidatic activities. It harbours three loci encoding a NADH peroxidase, an alkyl hydroperoxide reductase and a protein (EF2932) belonging to the AhpC/TSA family. We present results demonstrating that ef2932 does encode a thiol peroxidase (Tpx) and show that it is part of the regulon of the hydrogen peroxide regulator HypR. Characterization of unmarked deletion mutants showed that all three peroxidases are important for the defence against externally provided H₂O₂. Exposure to internal generated H₂O₂ by aerobic growth on glycerol, lactose, galactose or ribose showed that Npr was absolutely required for aerobic growth on glycerol and optimal growth on the other substrates. Growth on glycerol was also dependent on Ahp. Addition of catalase restored growth of the mutants, and therefore, extracellular H₂O₂ concentrations have been determined. This showed that the time point of growth arrest of the Δnpr mutant correlated with the highest H₂O₂ concentration measured. Analysis of the survival of the different strains inside peritoneal macrophages revealed that Tpx was the most important antioxidant activity for protecting the cells against the hostile phagocyte environment. Finally, the Δtpx and the triple mutant showed attenuated virulence in a mouse peritonitis model.

Unlike proteins, RNA molecules have emerged lately as key players in regulation in bacteria. Most reviews hitherto focused on the experimental and/orin silicomethods used to identify genes encoding small RNAs (sRNAs) or on the diverse mechanisms of these RNA regulators to modulate expression of their targets. However, less is known about their biological functions and their implications in various physiological responses. This review aims to compile what is known presently about the diverse roles of sRNA transcripts in the regulation of metabolic processes, in different growth conditions, in adaptation to stress and in microbial pathogenesis. Several recent studies revealed that sRNA molecules are implicated in carbon metabolism and transport, amino acid metabolism or metal sensing. Moreover, regulatory RNAs participate in cellular adaptation to environmental changes, e.g. through quorum sensing systems or development of biofilms, and analyses of several sRNAs under various physiological stresses and culture conditions have already been performed. In addition, recent experiments performed with Gram-positive and Gram-negative pathogens showed that regulatory RNAs play important roles in microbial virulence and during infection. The combined results show the diversity of regulation mechanisms and physiological processes in which sRNA molecules are key actors.

Enterobacter cloacaecomplex (ECC), an opportunistic pathogen causing numerous infections in hospitalized patients worldwide, is able to resist β-lactams mainly by producing the AmpC β-lactamase enzyme. AmpC expression is highly inducible in the presence of some β-lactams, but the underlying genetic regulation, which is intricately linked to peptidoglycan recycling, is still poorly understood. In this study, we constructed different mutant strains that were affected in genes encoding enzymes suspected to be involved in this pathway. As expected, the inactivation ofampC,ampR(which encodes the regulator protein ofampC), andampG(encoding a permease) abolished β-lactam resistance. Reverse transcription-quantitative PCR (qRT-PCR) experiments combined with phenotypic studies showed that cefotaxime (at high concentrations) and cefoxitin induced the expression ofampCin different ways: one involving NagZ (aN-acetyl-β-d-glucosaminidase) and another independent of NagZ. Unlike the model established forPseudomonas aeruginosa, inactivation of DacB (also known as PBP4) was not responsible for a constitutiveampCoverexpression in ECC, whereas it caused AmpC-mediated high-level β-lactam resistance, suggesting a post-transcriptional regulation mechanism. Global transcriptomic analysis by transcriptome sequencing (RNA-seq) of adacBdeletion mutant confirmed these results. Lastly, analysis of 37 ECC clinical isolates showed that amino acid changes in the AmpD sequence were likely the most crucial event involved in the development of high-level β-lactam resistancein vivoas opposed toP. aeruginosawheredacBmutations have been commonly found. These findings bring new elements for a better understanding of β-lactam resistance in ECC, which is essential for the identification of novel potential drug targets.

Enterococcus faecalis is an opportunistic pathogen that causes numerous infectious diseases in humans and is a major agent of nosocomial infections. In this work, we showed that the recently identified transcriptional regulator Ers (PrfA like), known to be involved in the cellular metabolism and the virulence of E. faecalis, acts as a repressor of ace, which encodes a collagen-binding protein. We characterized the promoter region of ace, and transcriptional analysis by reverse transcription-quantitative PCR and mobility shift protein-DNA binding assays revealed that Ers directly regulates the expression of ace. Transcription of ace appeared to be induced by the presence of bile salts, probably via the deregulation of ers. Moreover, with an ace deletion mutant and the complemented strain and by using an insect (Galleria mellonella) virulence model, as well as in vivo-in vitro murine macrophage models, we demonstrated for the first time that Ace can be considered a virulence factor for E. faecalis. Furthermore, animal experiments revealed that Ace is also involved in urinary tract infection by E. faecalis.

The worldwide ratio of Enterococcus faecalis-Enterococcus faecium infections is currently changing in favor of E. faecium. Intrinsic and acquired antimicrobial resistance traits of this latter species can explain this evolution as well as the diffusion of hospital-adapted strains belonging to the clonal complex CC17. Like other enterococci, E. faecium is naturally resistant to cephalosporins and aminoglycosides (at low level). Because of its high genome plasticity, it can also acquire numerous other resistances. It is noteworthy that most modern isolates of E. faecium are highly resistant to ampicillin while a non-negligible proportion of them (depending on geographical locations) are resistant to glycopeptides (especially in the USA). Even if resistance to newer antimicrobial agents (linezolid, daptomycin, tigecycline) is still uncommon, some clinical isolates with reduced susceptibility or resistance have already been reported and better understanding of resistance mechanisms is needed for prediction and prevention of their dissemination.

Aims of this study were to: (i) evaluate whether the cluster membership could have an impact on hetero-resistance phenotype to colistin in the Enterobacter cloacae complex (ECC); and (ii) determine the genetic mechanism of colistin hetero-resistance in ECC. A collection of 124 clinical isolates belonging to 13 clusters were used to analyse the hetero-resistance phenotype (MICs were determined using the broth microdilution method, Etest and population analysis profiling). Different mutants (ΔphoP, ΔphoQ, ΔphoPQ, ΔpmrA, ΔpmrB, ΔpmrAB, ΔarnE, ΔarnF and ΔarnBCADTEF) were constructed and tested for their colistin hetero-resistance phenotype. Based on broth microdilution and Etest results, it was shown that the hetero-resistance to colistin depended on the cluster: strains from clusters I, II, IV, VII, IX, X, XI and XII were usually hetero-resistant, whereas those from clusters III, V, VI, VIII and XIII were categorized as susceptible. However, for some cluster V and VIII strains, a small proportion (<10-7) of cells appeared resistant when tested by population analysis profiling. From a mechanistic point of view, analysis of mutants revealed that the mechanism of hetero-resistance was mainly due to the expression of the arn operon and the phoP/phoQ two-component regulatory system. Because the colistin hetero-resistance appeared cluster-dependent in the ECC, it should be advocated to determine the cluster of the strain associated with the infection in parallel with the MIC of colistin. The resistance mechanism may not be similar to other Enterobacteriaceae since only the two-component regulatory system PhoP/PhoQ (and not PmrA/PmrB) seemed to play a role in resistance regulation.