Proteomic characterization of persisters in Enterococcus faecium.

BACKGROUND: Enterococcus faecium is a Gram-positive bacterium, naturally present in the human intestinal microbiota, but is also an opportunistic pathogen responsible for healthcare-associated infections. Persisters are individuals of a subpopulation able to survive by arrest of growth coping with conditions that are lethal for the rest of the population. These persistent cells can grow again when the stress disappears from their environment and can cause relapses. RESULTS: In this study, we highlighted that ciprofloxacin (10-fold the MIC) led to the formation of persister cells of E. faecium. The kill curve was typically biphasic with an initial drop of survival (more than 2 orders of magnitude reduction) followed by a constant bacterial count. Growth curves and antimicrobial susceptibility tests of these persisters were similar to those of the original cells. In addition, by genomic analyses, we confirmed that the persisters were genotypically identical to the wild type. Comparative proteomic analysis revealed that 56 proteins have significantly different abundances in persisters compared to cells harvested before the addition of stressing agent. Most of them were related to energetic metabolisms, some polypeptides were involved in transcription regulation, and seven were stress proteins like CspA, PrsA, ClpX and particularly enzymes linked to the oxidative stress response. CONCLUSIONS: This work provided evidences that the pathogen E. faecium was able to enter a state of persister that may have an impact in chronic infections and relapses. Moreover, putative key effectors of this phenotypical behavior were identified by proteomic approach.

6-Polyaminosteroid Squalamine Analogues Display Antibacterial Activity against Resistant Pathogens.

A series of 6-polyaminosteroid analogues of squalamine were synthesized with moderate to good yields and evaluated for their in vitro antimicrobial properties against both susceptible and resistant Gram-positive (vancomycin-resistant Enterococcus faecium and methicillin-resistant Staphylococcus aureus) and Gram-negative (carbapenem-resistant Acinetobacter baumannii and Pseudomonas aeruginosa) bacterial strains. Minimum inhibitory concentrations against Gram-positive bacteria ranged from 4 to 16 µg/mL for the most effective compounds, 4k and 4n, and showed an additive or synergistic effect with vancomycin or oxacillin. On the other hand, the derivative 4f, which carries a spermine moiety like that of the natural trodusquemine molecule, was found to be the most active derivative against all the resistant Gram-negative bacteria tested, with an MIC value of 16 µg/mL. Our results suggest that 6-polyaminosteroid analogues of squalamine are interesting candidates for Gram-positive bacterial infection treatments, as well as potent adjuvants to fight Gram-negative bacterial resistance.

Identification of the iron-limitation stimulon in Staphylococcus lugdunensis.

During the infectious process, pathogens such as Staphylococcus lugdunensis have to cope with the condition of host-induced iron-limitation. Using the RNAseq approach, we performed the first global transcriptomic analysis of S. lugdunensis cells incubated in the absence and presence of iron chelator. One hundred and seventy-five genes were identified as members of the iron-limitation stimulon (127 up- and 48 downregulated). Six gene clusters known or likely required for the acquisition of iron have been identified. Among them, a novel Energy-Coupling Factor type transporter (ECF), homologous to the lhaSTA operon, has been found into a 13-gene putative operon and strongly overexpressed under iron-limitation condition. Moreover, the transcription of genes involved in resistance to oxidative stress (including catalase), virulence, transcriptional regulation, and hemin detoxification were also modified. These data provide some answers on the cellular response to the iron-limitation stress that is important for the opportunistic behavior of this pathogen.

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.

Phenotypic and proteomic approaches of the response to iron-limited condition in Staphylococcus lugdunensis.

BACKGROUND: Staphylococcus lugdunensis is a coagulase-negative Staphylococcus part of the commensal skin flora but emerge as an important opportunistic pathogen. Because iron limitation is a crucial stress during infectious process, we performed phenotypic study and compared proteomic profiles of this species incubated in absence and in presence of the iron chelator 2,2'-dipyridyl (DIP). RESULTS: No modification of cell morphology nor cell wall thickness were observed in presence of DIP. However iron-limitation condition promoted biofilm formation and reduced the ability to cope with oxidative stress (1 mM H2O2). In addition, S. lugdunensis N920143 cultured with DIP was significantly less virulent in the larvae of Galleria mellonella model of infection than that grown under standard conditions. We verified that these phenotypes were due to an iron limitation by complementation experiments with FeSO4. By mass spectrometry after trypsin digestion, we characterized the first iron-limitation stress proteome in S. lugdunensis. Among 1426 proteins identified, 349 polypeptides were differentially expressed. 222 were more and 127 less abundant in S. lugdunensis incubated in iron-limitation condition, and by RT-qPCR, some of the corresponding genes have been shown to be transcriptionally regulated. Our data revealed that proteins involved in iron metabolism and carriers were over-expressed, as well as several ABC transporters and polypeptides linked to cell wall metabolism. Conversely, enzymes playing a role in the oxidative stress response (especially catalase) were repressed. CONCLUSIONS: This phenotypic and global proteomic study allowed characterization of the response of S. lugdunensis to iron-limitation. We showed that iron-limitation promoted biofilm formation, but decrease the oxidative stress resistance that may, at least in part, explained the reduced virulence of S. lugdunensis observed under low iron condition.

Identification of the general stress stimulon related to colonization in Enterococcus faecalis.

Enterococcus faecalis has to cope with major stress conditions during colonization. To understand the effects of stress encountered during infection, the present study assessed the transcriptomic response of the bacteria facing exposure to serum, urine, bile salts, acid pH, or oxidative stress. Compared to non-stressed culture, 30% of the E. faecalis genes were differentially expressed. The transcriptome analysis reveals common but also specific responses, depending on stresses encountered: thus, urine exposure has the most important impact, and the highest number of genes with modified expression is involved in transport and metabolism. The results also pinpoint many stress-related sRNA or intergenic regions not yet characterized. This study identified the general stress stimulon related to infection: when the commensal bacterium initiates its response to stress related to infection, it increases its ability to survive to rough conditions for colonization, rather than promoting expression of virulence factors, and becomes this opportunistic pathogen that thrives in hospital settings.

The Transcriptional Repressor SmvR Is Important for Decreased Chlorhexidine Susceptibility in Enterobacter cloacae Complex.

Major facilitator superfamily (MFS) efflux pumps have been shown to be important for bacterial cells to cope with biocides such as chlorhexidine (CHX), a widely used molecule in hospital settings. In this work, we evaluated the role of two genes, smvA and smvR, in CHX resistance in Enterobacter cloacae complex (ECC). smvA encodes an MFS pump whereas smvR, located upstream of smvA, codes for a TetR-type transcriptional repressor. To this aim, we constructed corresponding deletion mutants from the ATCC 13047 strain (CHX MIC, 2 mg/liter) as well as strains overexpressing smvA or smvR in both ATCC 13047 and three clinical isolates exhibiting elevated CHX MICs (16 to 32 mg/liter). Determination of MICs revealed that smvA played a modest role in CHX resistance, in contrast to smvR that modulated the ability of ECC to survive in the presence of CHX. In clinical isolates, the overexpression of smvR significantly reduced MICs of CHX (2 to 8 mg/liter). Sequence analyses of smvR and promoter regions pointed out substitutions in conserved regions. Moreover, transcriptional studies revealed that SmvR acted as a repressor of smvA expression even if no quantitative correlation between the level of smvA mRNA and MICs of CHX could be observed. On the other hand, overproduction of smvA was able to complement the lack of the major resistance-nodulation-cell division (RND) superfamily efflux pump AcrB and restored resistance to ethidium bromide and acriflavine. Although SmvA could expel biocides such as CHX, other actors, whose expression is under SmvR control, should play a critical role in ECC.

Synthesis and evaluation of 1,3,4-oxadiazole derivatives for development as broad-spectrum antibiotics.

The reality and intensity of antibiotic resistance in pathogenic bacteria calls for the rapid development of new antimicrobial drugs. In bacteria, trans-translation is the primary quality control mechanism for rescuing ribosomes arrested during translation. Because trans-translation is absent in eukaryotes but necessary to avoid ribosomal stalling and therefore essential for bacterial survival, it is a promising target either for novel antibiotics or for improving the activities of the protein synthesis inhibitors already in use. Oxadiazole derivatives display strong bactericidal activity against a large number of bacteria, but their effects on trans-translation were recently questioned. In this work, a series of new 1,3,4-oxadiazole derivatives and analogs were synthesized and assessed for their efficiency as antimicrobial agents against a wide range of gram-positive and gram-negative pathogenic strains. Despite the strong antimicrobial activity observed in these molecules, it turns out that they do not target trans-translation in vivo, but they definitely act on other cellular pathways.

Genetic characterization of a VanG-type vancomycin-resistant Enterococcus faecium clinical isolate.

Objectives: To characterize, phenotypically and genotypically, the first Enterococcus faecium clinical isolate harbouring a vanG operon. Methods: The antibiotic resistance profile of E. faecium 16-346 was determined and its whole genome sequenced using PacBio technology. Attempts to transfer vancomycin resistance by filter mating were performed and the inducibility of expression of the vanG operon was studied by reverse-transcription quantitative PCR (RT-qPCR) in the presence or absence of subinhibitory concentrations of vancomycin. Results: E. faecium 16-346 was resistant to rifampicin (MIC >4 mg/L), erythromycin (MIC >4 mg/L), tetracycline (MIC >16 mg/L) and vancomycin (MIC 8 mg/L), but susceptible to teicoplanin (MIC 0.5 mg/L). The strain harboured the vanG operon in its chromosome, integrated in a 45.5 kb putative mobile genetic element, similar to that of Enterococcus faecalis BM4518. We were unable to transfer vancomycin resistance from E. faecium 16-346 to E. faecium BM4107 and E. faecalis JH2-2. Lastly, transcription of the vanG gene was inducible by vancomycin. Conclusions: This is, to the best of our knowledge, the first report of a VanG-type vancomycin-resistant strain of E. faecium. Despite the alarm pulled because of the therapeutic problems caused by VRE, our work shows that new resistant loci can still be found in E. faecium.

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.

Landscape of Resistance-Nodulation-Cell Division (RND)-Type Efflux Pumps in Enterobacter cloacae Complex.

In Gram-negative bacteria, the active efflux is an important mechanism of antimicrobial resistance, but little is known about theEnterobacter cloacaecomplex (ECC). It is mediated primarily by pumps belonging to the RND (resistance-nodulation-cell division) family, and only AcrB, part of the AcrAB-TolC tripartite system, was characterized in ECC. However, detailed genome sequence analysis of the strainE. cloacaesubsp.cloacaeATCC 13047 revealed to us that 10 other genes putatively coded for RND-type transporters. We then characterized the role of all of these candidates by construction of corresponding deletion mutants, which were tested for their antimicrobial susceptibility to 36 compounds, their virulence in the invertebrateGalleria mellonellamodel of infection, and their ability to form biofilm. Only the ΔacrBmutant displayed significantly different phenotypes compared to that of the wild-type strain: 4- to 32-fold decrease of MICs of several antibiotics, antiseptics, and dyes, increased production of biofilm, and attenuated virulence inG. mellonella In order to identify specific substrates of each pump, we individually expressed intransall operons containing an RND pump-encoding gene into the ΔacrBhypersusceptible strain. We showed that three other RND-type efflux systems (ECL_00053-00055, ECL_01758-01759, and ECL_02124-02125) were able to partially restore the wild-type phenotype and to superadd to and even enlarge the broad range of antimicrobial resistance. This is the first global study assessing the role of all RND efflux pumps chromosomally encoded by the ECC, which confirms the major role of AcrB in both pathogenicity and resistance and the potential involvement of other RND-type members in acquired resistance.

Sequential steps of daptomycin resistance in Enterococcus faecium and reversion to hypersusceptibility through IS-mediated inactivation of the liaFSR operon.

OBJECTIVES: To improve understanding of mechanisms of daptomycin resistance and to dissect the genetic basis of reversion to daptomycin hypersusceptibility in Enterococcus faecium. METHODS: Daptomycin-resistant mutants (Mut4, Mut8, Mut16, Mut32, Mut64 and Mut128 with MICs from 4 to 128 mg/L) were obtained in vitro from E. faecium strain Aus0004 (MIC at 2 mg/L). The entire genome sequences of Mut64 and Mut128 were determined as well as those of liaFSR and cls genes for other mutants and corresponding revertants (named Rev4 to Rev128). The study of daptomycin resistance stability was performed without any selective pressure. The expression of liaF, liaS and liaR genes was quantified by quantitative RT-PCR. RESULTS: By comparative genomic analysis, substitutions Asn13Ser in cls and Gly92Asp in liaS were identified in Mut64 and Mut128. Only the liaS mutation was found in Mut16 and Mut32 while Mut4 and Mut8 were devoid of any mutation. After 15 days, all mutants except Mut4 reverted to daptomycin hypersusceptibility (MICs from 0.12 to 0.25 mg/L). In all revertants (except Rev4 and Rev8), an IS was found in the liaFSR operon with a dramatic decrease of its expression: IS66 in the promoter region of liaF (Rev16 and Rev64), IS30 in liaR (Rev32) and IS982 in liaF (Rev128). CONCLUSIONS: We demonstrated the stepwise and sequential acquisition of mutations in liaS and in cls leading to daptomycin resistance in E. faecium, and the instability of daptomycin resistance as well as the role of liaFSR inactivation in reversion to daptomycin hypersusceptibility.

Cluster-dependent colistin hetero-resistance in Enterobacter cloacae complex.

OBJECTIVES: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Novel chromosome-encoded erm(47) determinant responsible for constitutive MLSB resistance in Helcococcus kunzii.

OBJECTIVES: The aim of the study was to identify the determinant responsible for erythromycin resistance in Helcococcus kunzii clinical isolate UCN99 and to characterize the genetic support and environment of this novel gene. METHODS: MICs were determined using the broth microdilution method according to EUCAST guidelines. The entire genome sequence of H. kunzii UCN99 was determined using a 454/Roche GS Junior sequencer. The fragment encompassing the new resistance gene and its own promoter was cloned into the pAT29 shuttle vector and the recombinant plasmid pAT29Ωerm(47) was expressed in both Staphylococcus aureus and Streptococcus agalactiae. The transcription start site (TSS) was experimentally determined by 5' RACE-PCR. RESULTS: UCN99 exhibited a constitutive macrolide/lincosamide/streptogramin B (MLSB) resistance phenotype, suggesting the presence of an Erm protein. WGS allowed the identification of a novel gene, named erm(47), encoding a protein sharing 44%-48% amino acid identity with known Erm methylases. In both S. aureus and S. agalactiae, the introduction of pAT29Ωerm(47) conferred a significant increase (≥16-fold) in MICs of all macrolides and lincosamides tested, as well as a 4-fold increase in MICs of quinupristin (streptogramin B), confirming the MLSB resistance. The TSS identification revealed the presence of a short leader peptide, potentially implicated in a translational attenuation mechanism. It was also demonstrated that erm(47) was harboured by a 81 kb genomic island integrated into a chromosomal gene. CONCLUSIONS: This is the first description of a novel MLSB resistance determinant, named erm(47). The prevalence of this gene among Gram-positive cocci must be further investigated to determine its clinical significance.

The polyamine N-acetyltransferase-like enzyme PmvE plays a role in the virulence of Enterococcus faecalis.

We previously showed that the mutant strain of Enterococcus faecalis lacking the transcriptional regulator SlyA is more virulent than the parental strain. We hypothesized that this phenotype was due to overexpression of the second gene of the slyA operon, ef_3001, renamed pmvE (for polyamine metabolism and virulence of E. faecalis). PmvE shares strong homologies with N(1)-spermidine/spermine acetyltransferase enzymes involved in the metabolism of polyamines. In this study, we used an E. faecalis strain carrying the recombinant plasmid pMSP3535-pmvE (V19/p3535-pmvE), which allows the induction of pmvE by addition of nisin. Thereby, we showed that the overexpression of PmvE increased the virulence of E. faecalis in the Galleria mellonella infection model, as well as the persistence within peritoneal macrophages. We were also able to show a direct interaction between the His-tagged recombinant PmvE (rPmvE) protein and putrescine by the surface plasmon resonance (SPR) technique on a Biacore instrument. Moreover, biochemical assays showed that PmvE possesses an N-acetyltransferase activity toward polyamine substrates. Our results suggest that PmvE contributes to the virulence of E. faecalis, likely through its involvement in the polyamine metabolism.

Complex Regulation Pathways of AmpC-Mediated ß-Lactam Resistance in Enterobacter cloacae Complex.

Enterobacter cloacae complex (ECC), an opportunistic pathogen causing numerous infections in hospitalized patients worldwide, is able to resist ß-lactams mainly by producing the AmpC ß-lactamase enzyme. AmpC expression is highly inducible in the presence of some ß-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 of ampC, ampR (which encodes the regulator protein of ampC), and ampG (encoding a permease) abolished ß-lactam resistance. Reverse transcription-quantitative PCR (qRT-PCR) experiments combined with phenotypic studies showed that cefotaxime (at high concentrations) and cefoxitin induced the expression of ampC in different ways: one involving NagZ (a N-acetyl-ß-D-glucosaminidase) and another independent of NagZ. Unlike the model established for Pseudomonas aeruginosa, inactivation of DacB (also known as PBP4) was not responsible for a constitutive ampC overexpression in ECC, whereas it caused AmpC-mediated high-level ß-lactam resistance, suggesting a post-transcriptional regulation mechanism. Global transcriptomic analysis by transcriptome sequencing (RNA-seq) of a dacB deletion 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 ß-lactam resistance in vivo as opposed to P. aeruginosa where dacB mutations have been commonly found. These findings bring new elements for a better understanding of ß-lactam resistance in ECC, which is essential for the identification of novel potential drug targets.

Genomic analysis of reduced susceptibility to tigecycline in Enterococcus faecium.

Tigecycline (TIG) is approved for use for the treatment of complicated intra-abdominal infections, skin and skin structure infections, as well as pneumonia. Acquired resistance or reduced susceptibility to TIG has been observed in Gram-negative rods, has seldom been reported in Gram-positive organisms, and has not yet been reported in Enterococcus faecium. Using the serial passage method, in vitro mutant AusTig and in vitro mutants HMtig1 and HMtig2 with decreased TIG susceptibility (MICs, 0.25 µg/ml) were obtained from strains E. faecium Aus0004 and HM1070 (MICs, 0.03 µg/ml), respectively. In addition, two vancomycin-resistant E. faecium clinical isolates (EF16 and EF22) with reduced susceptibility to TIG (MICs, 0.5 and 0.25 µg/ml, respectively) were studied. Compared to the wild-type strains, the in vitro mutants also showed an increase in the MICs of other tetracyclines. An efflux mechanism did not seem to be involved in the reduced TIG susceptibility, since the presence of efflux pump inhibitors (reserpine or pantoprazole) did not affect the MICs of TIG. Whole-genome sequencing of AusTig was carried out, and genomic comparison with the Aus0004 genome was performed. Four modifications leading to an amino acid substitution were found. These mutations affected the rpsJ gene (efau004_00094, coding for the S10 protein of the 30S ribosomal subunit), efau004_01228 (encoding a cation transporter), efau004_01636 (coding for a hypothetical protein), and efau004_02455 (encoding the l-lactate oxidase). The four other strains exhibiting reduced TIG susceptibility were screened for the candidate mutations. This analysis revealed that three of them showed an amino acid substitution in the same region of the RpsJ protein. In this study, we characterized for the first time genetic determinants linked to reduced TIG susceptibility in enterococci.

Involvement of Enterococcus faecalis small RNAs in stress response and virulence.

Candidate small RNAs (sRNAs) have recently been identified in Enterococcus faecalis, a Gram-positive opportunistic pathogen, and six of these candidate sRNAs with unknown functions were selected for a functional study. Deletion mutants and complemented strains were constructed, and their virulence was tested. We were unable to obtain the ef0869-0870 mutant, likely due to an essential role, and the ef0820-0821 sRNA seemed not to be involved in virulence. In contrast, the mutant lacking ef0408-0409 sRNA, homologous to the RNAII component of the toxin-antitoxin system, appeared more virulent and more able to colonize mouse organs. The three other mutants showed reduced virulence. In addition, we checked the responses of these mutant strains to several stresses encountered in the gastrointestinal tract or during the infection process. In parallel, the activities of the sRNA promoters were measured using transcriptional fusion constructions. To attempt to identify the regulons of these candidate sRNAs, proteomics profiles of the mutant strains were compared with that of the wild type. This showed that the selected sRNAs controlled the expression of proteins involved in diverse cellular processes and the stress response. The combined data highlight the roles of certain candidate sRNAs in the adaptation of E. faecalis to environmental changes and in the complex transition process from a commensal to a pathogen.

Physiological roles of small RNA molecules.

Unlike proteins, RNA molecules have emerged lately as key players in regulation in bacteria. Most reviews hitherto focused on the experimental and/or in silico methods 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.

AsrR is an oxidative stress sensing regulator modulating Enterococcus faecium opportunistic traits, antimicrobial resistance, and pathogenicity.

Oxidative stress serves as an important host/environmental signal that triggers a wide range of responses in microorganisms. Here, we identified an oxidative stress sensor and response regulator in the important multidrug-resistant nosocomial pathogen Enterococcus faecium belonging to the MarR family and called AsrR (antibiotic and stress response regulator). The AsrR regulator used cysteine oxidation to sense the hydrogen peroxide which results in its dissociation to promoter DNA. Transcriptome analysis showed that the AsrR regulon was composed of 181 genes, including representing functionally diverse groups involved in pathogenesis, antibiotic and antimicrobial peptide resistance, oxidative stress, and adaptive responses. Consistent with the upregulated expression of the pbp5 gene, encoding a low-affinity penicillin-binding protein, the asrR null mutant was found to be more resistant to ß-lactam antibiotics. Deletion of asrR markedly decreased the bactericidal activity of ampicillin and vancomycin, which are both commonly used to treat infections due to enterococci, and also led to over-expression of two major adhesins, acm and ecbA, which resulted in enhanced in vitro adhesion to human intestinal cells. Additional pathogenic traits were also reinforced in the asrR null mutant including greater capacity than the parental strain to form biofilm in vitro and greater persistance in Galleria mellonella colonization and mouse systemic infection models. Despite overexpression of oxidative stress-response genes, deletion of asrR was associated with a decreased oxidative stress resistance in vitro, which correlated with a reduced resistance to phagocytic killing by murine macrophages. Interestingly, both strains showed similar amounts of intracellular reactive oxygen species. Finally, we observed a mutator phenotype and enhanced DNA transfer frequencies in the asrR deleted strain. These data indicate that AsrR plays a major role in antimicrobial resistance and adaptation for survival within the host, thereby contributes importantly to the opportunistic traits of E. faecium.