A new theta-type thermosensitive replicon from Lactoccocus lactis as an integration vector for Enterococcus faecalis

We isolated a replication thermosensitive mutant of the theta-type lactococcal pUCL22 replicon. An improved version of this thermosensitive replicon was obtained by fusioning the replication repA gene with the downstream repB gene. The resulting plasmid was named pUCB3522Ts. It is highly instable at 42°C in Enterococcus faecalis. Integration into the chromosome via homologous recombination was monitored using the npr gene of E. faecalis JH2-2 as a target. A 513 bp PCR amplification product from an internal region of this npr gene was cloned into pUCB3522Ts. Integration of this construction into the JH2-2 npr gene was selected by shift temperature, from 30°C to 42°C. 85% of the analysed clones showed integration into the npr gene, demonstrating the practicality of this thermosensitive replicon as a genetic integrative tool for E. faecali

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

International audienceObjectives: 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

The Extracytoplasmic Function Sigma Factor SigV Plays a Key Role in the Original Model of Lysozyme Resistance and Virulence of Enterococcus faecalis

Background: Enterococcus faecalis is one of the leading agents of nosocomial infections. To cause diseases, pathogens or opportunistic bacteria have to adapt and survive to the defense systems encountered in the host. One of the most important compounds of the host innate defense response against invading microorganisms is lysozyme. It is found in a wide variety of body fluids, as well as in cells of the innate immune system. Lysozyme could act either as a muramidase and/ or as a cationic antimicrobial peptide. Like Staphylococcus aureus, E. faecalis is one of the few bacteria that are completely lysozyme resistant. Results: This study revealed that oatA (O-acetyl transferase) and dlt (D-Alanylation of lipoteicoic acids) genes contribute only partly to the lysozyme resistance of E. faecalis and that a specific transcriptional regulator, the extracytoplasmic function SigV sigma factor plays a key role in this event. Indeed, the sigV single mutant is as sensitive as the oatA/dltA double mutant, and the sigV/oatA/dltA triple mutant displays the highest level of lysozyme sensitivity suggesting synergistic effects of these genes. In S. aureus, mutation of both oatA and dlt genes abolishes completely the lysozyme resistance, whereas this is not the case in E. faecalis. Interestingly SigV does not control neither oatA nor dlt genes. Moreover, the sigV mutants clearly showed a reduced capacity to colonize host tissues, as they are significantly less recovered than the parental JH2-2 strain from organs of mice subjected to intravenous or urinary tract infections

A novel genomic island harbouring lsa(E) and lnu(B) genes and a defective prophage in a Streptococcus pyogenes isolate resistant to lincosamide, streptogramin A and pleuromutilin antibiotics

A lincosamide-resistant and macrolide-susceptible phenotype has not been described to date in Streptococcus pyogenes [group A streptococcus (GAS)]. The aim of this study was to characterize a GAS isolate susceptible to macrolides but resistant to lincosamide, streptogramin A and pleuromutilin antibiotics. Antimicrobial susceptibility was tested using the microdilution broth method and the resistance phenotype was tested by D-test. The GAS2887HUB isolate was subjected to whole-genome sequencing. The isolate showed a positive Gots' test (clindamycin inactivation). Whole-genome sequencing revealed that the strain was ST10 and emm93, and had five resistance genes [lnu(B), ant(6)-Ia, aph(3')-III, tet(M) and dfrG]. The tet(M) gene was located in a Tn916-like transposon. The lsa(E)-lnu(B)-containing sequence (inserted downstream of the rumA gene) was formed by a 39.6-kb prophage, followed by a gene cluster encoding aminoglycoside-streptothricin resistance [ant(6)Ia-sat4-aph(3')III] and lsa(E)-lnu(B) genes. This structure was not transferred by conjugation. This study identified a new genetic element carrying a determinant of lincosamide resistance in a GAS. Further molecular epidemiological surveys are needed to determine the prevalence of this mechanism of resistance in GAS

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 \u3b2-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

Genome-Wide Identification of Small RNAs in the Opportunistic Pathogen Enterococcus faecalis V583

Small RNA molecules (sRNAs) are key mediators of virulence and stress inducible gene expressions in some pathogens. In this work we identify sRNAs in the Gram positive opportunistic pathogen Enterococcus faecalis. We characterized 11 sRNAs by tiling microarray analysis, 5′ and 3′ RACE-PCR, and Northern blot analysis. Six sRNAs were specifically expressed at exponential phase, two sRNAs were observed at stationary phase, and three were detected during both phases. Searches of putative functions revealed that three of them (EFA0080_EFA0081 and EFB0062_EFB0063 on pTF1 and pTF2 plasmids, respectively, and EF0408_EF04092 located on the chromosome) are similar to antisense RNA involved in plasmid addiction modules. Moreover, EF1097_EF1098 shares strong homologies with tmRNA (bi-functional RNA acting as both a tRNA and an mRNA) and EF2205_EF2206 appears homologous to 4.5S RNA member of the Signal Recognition Particle (SRP) ribonucleoprotein complex. In addition, proteomic analysis of the ΔEF3314_EF3315 sRNA mutant suggests that it may be involved in the turnover of some abundant proteins. The expression patterns of these transcripts were evaluated by tiling array hybridizations performed with samples from cells grown under eleven different conditions some of which may be encountered during infection. Finally, distribution of these sRNAs among genome sequences of 54 E. faecalis strains was assessed. This is the first experimental genome-wide identification of sRNAs in E. faecalis and provides impetus to the understanding of gene regulation in this important human pathogen

Occurrence of Bacterial Pathogens and Human Noroviruses in Shellfish-Harvesting Areas and Their Catchments in France

During a 2-year study, the presence of human pathogenic bacteria and noroviruses was investigated in shellfish, seawater and/or surface sediments collected from three French coastal shellfish-harvesting areas as well as in freshwaters from the corresponding upstream catchments. Bacteria isolated from these samples were further analyzed. Escherichia coli isolates classified into the phylogenetic groups B2, or D and enterococci from Enterococcus faecalis and E. faecium species were tested for the presence of virulence genes and for antimicrobial susceptibility. Salmonella members were serotyped and the most abundant serovars (Typhimurium and its monophasic variants and Mbandaka) were genetically characterized by high discriminative subtyping methods. Campylobacter and Vibrio were identified at the species level, and haemolysin-producing Vibrio parahaemolyticus were searched by tdh- and trh- gene detection. Main results showed a low prevalence of Salmonella in shellfish samples where only members of S. Mbandaka were found. Campylobacter were more frequently isolated than Salmonella and a different distribution of Campylobacter species was observed in shellfish compared to rivers, strongly suggesting possible additional inputs of bacteria. Statistical associations between enteric bacteria, human noroviruses (HuNoVs) and concentration of fecal indicator bacteria revealed that the presence of Salmonella was correlated with that of Campylobacter jejuni and/or C. coli as well as to E. coli concentration. A positive correlation was also found between the presence of C. lari and the detection of HuNoVs. This study highlights the importance of simultaneous detection and characterization of enteric and marine pathogenic bacteria and human noroviruses not only in shellfish but also in catchment waters for a hazard assessment associated with microbial contamination of shellfish

Detection chain and electronic readout of the QUBIC instrument

The Q and U Bolometric Interferometer for Cosmology (QUBIC) Technical Demonstrator (TD) aiming to shows the feasibility of the combination of interferometry and bolometric detection. The electronic readout system is based on an array of 128 NbSi Transition Edge Sensors cooled at 350mK readout with 128 SQUIDs at 1K controlled and amplified by an Application Specific Integrated Circuit at 40K. This readout design allows a 128:1 Time Domain Multiplexing. We report the design and the performance of the detection chain in this paper. The technological demonstrator unwent a campaign of test in the lab. Evaluation of the QUBIC bolometers and readout electronics includes the measurement of I-V curves, time constant and the Noise Equivalent Power. Currently the mean Noise Equivalent Power is ~ 2 x 10⁻¹⁶ W/√Hz

Detection chain and electronic readout of the QUBIC instrument

The Q and U Bolometric Interferometer for Cosmology (QUBIC) Technical Demonstrator (TD) aiming to shows the feasibility of the combination of interferometry and bolometric detection. The electronic readout system is based on an array of 128 NbSi Transition Edge Sensors cooled at 350mK readout with 128 SQUIDs at 1K controlled and amplified by an Application Specific Integrated Circuit at 40K. This readout design allows a 128:1 Time Domain Multiplexing. We report the design and the performance of the detection chain in this paper. The technological demonstrator unwent a campaign of test in the lab. Evaluation of the QUBIC bolometers and readout electronics includes the measurement of I-V curves, time constant and the Noise Equivalent Power. Currently the mean Noise Equivalent Power is ~ 2 x 10⁻¹⁶ W/√Hz

Identification et caractérisation d'Ers, régulateur transcriptionnel impliqué dans la virulence d'Enterocccus faecalis

Ers a été identifié comme un régulateur transcriptionnel chez Enterococcus faecalis, une bactérie lactique responsable d infections nosocomiales. Cette protéine fait partie de la branche PrfA des régulateurs de la famille Crp/Fnr et serait, chez E. faecalis, l homologue de PrfA, régulateur majeur de la virulence chez Listeria monocytogenes. Elle possède en effet des similitudes structurales avec les protéines de cette famille et en particulier avec PrfA. De plus, on retrouve une séquence similaire de la boîte PrfA dans la région promotrice d ers. Celle-ci a été montrée comme étant incluse dans le segment d ADN reconnu par Ers. D autre part, cette protéine est impliquée dans la survie de la bactérie au stress oxydatif et dans les macrophages. Un mutant deltaers est, de plus, moins virulent que la souche sauvage dans un modèle murin de péritonite. Ers joue également un rôle dans le métabolisme de l arginine, du citrate et du glycérol. Les membres actuellement connus du régulon Ers sont les gènes ef0082 (codant un transporteur du glycérol), arcABC (codant des enzymes du métabolisme de l arginine), ef1459 (codant une protéine hypothétique), citF (codant la sous-unité alpha de la citrate lyase), ace (codant une adhésine impliquée dans la virulence) et enfin glpKOF (codant des protéines jouant un rôle dans le catabolisme du glycérol). L ensemble de ces résultats révèle qu Ers est un régulateur pléiotrope impliqué dans la virulence et le métabolisme cellulaire chez E. faecalis.Ers is a transcriptional regulator of Enterococcus faecalis, a lactic acid bacterium more and more involved in hospital acquired infection. It is part of the PrfA branch of the Crp/Fnr family regulators and may exert a PrfA-like activity in E. faecalis. This protein shares strong structural homologies with proteins of this family including PrfA, the major regulator of virulence in Listeria monocytogenes. Moreover, a sequence similar to the PrfA-box is found in the promoter regions of ers and it has been shown to be included in the DNA segment recognized by Ers. Furthermore, this protein is important for the survival of this opportunistic pathogen towards oxidative stress and within macrophages. In addition, in a mouse peritonitis model of virulence, the deltaers mutant appeared significantly less lethal than the JH2-2 wild type strain. Ers is also involved in the arginine, citrate and glycerol metabolism. At the present time, the Ers regulon is composed of ef0082 encoding a glycerol transporter, arcABC, encoding enzymes of the arginine deiminase system, ef1459 encoding a hypothetical protein, citF, encoding the alpha subunit of the citrate lyase involved in the citrate catabolism, ace, an adhesin involved in virulence and finally glpKOF, encoding two glycerol degradation enzymes, GlpK and GlpO, and a glycerol uptake facilitator, GplF. Taken together, these results indicate that the regulator Ers has a pleiotropic effect, especially in the cellular metabolism and in the virulence of E. faecalisCAEN-BU Sciences et STAPS (141182103) / SudocSudocFranceF