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

Macrolide-Resistant Shigella sonnei

Shigella sonnei UCN59, isolated during an outbreak of S. sonnei in January 2007, was resistant to azithromycin (MIC 64 mg/L). The isolate contained a plasmid-borne mph(A) gene encoding a macrolide 2′-phosphotransferase that inactivates macrolides. Emergence of the mph(A) gene in S. sonnei may limit usefulness of azithromycin for treatment of shigellosis

Deciphering Normal Blood Gene Expression Variation—The NOWAC Postgenome Study

There is growing evidence that gene expression profiling of peripheral blood cells is a valuable tool for assessing gene signatures related to exposure, drug-response, or disease. However, the true promise of this approach can not be estimated until the scientific community has robust baseline data describing variation in gene expression patterns in normal individuals. Using a large representative sample set of postmenopausal women (N = 286) in the Norwegian Women and Cancer (NOWAC) postgenome study, we investigated variability of whole blood gene expression in the general population. In particular, we examined changes in blood gene expression caused by technical variability, normal inter-individual differences, and exposure variables at proportions and levels relevant to real-life situations. We observe that the overall changes in gene expression are subtle, implying the need for careful analytic approaches of the data. In particular, technical variability may not be ignored and subsequent adjustments must be considered in any analysis. Many new candidate genes were identified that are differentially expressed according to inter-individual (i.e. fasting, BMI) and exposure (i.e. smoking) factors, thus establishing that these effects are mirrored in blood. By focusing on the biological implications instead of directly comparing gene lists from several related studies in the literature, our analytic approach was able to identify significant similarities and effects consistent across these reports. This establishes the feasibility of blood gene expression profiling, if they are predicated upon careful experimental design and analysis in order to minimize confounding signals, artifacts of sample preparation and processing, and inter-individual differences

Etude de deux gènes spécifiques d'espèces de bactéries du genre Clostridium (le gène domestique de la triosephosphate isomerase et un gène d'autolysine de C. difficile)

L'objectif de ce travail était d'étudier deux gènes spécifiques d'espèces chez les bactéries du genre Clostridium, le gène tpi (codant la triosephospahte isomérase) et un gène d'autolysine de C. difficile. Le gène domestique tpi s'est révélé un marqueur taxonomique et phylogénétique alternatif au gène ADN ribosomal 16S. En effet, il nous a permis d'élaborer une démarche d'identification de douze espèces d'intérêt clinique au sein du genre Clostridium par restriction de l'amplicon tpi par l'enzyme Alul, qui génère des profils de restriction spécifique d'espèce. Nous avons également analysé un groupe de dix-neuf espèces incluant C. hastiforme, espèce chez laquelle certains isolats cliniques présentent une résistance de bas niveau à la vancomycine. Nous avons pu préciser la position taxonomique de ces espèces par analyse des séquences des gènes ADNr 16S et tpi. Le gène tpi a également pu être exploité comme cible diagnostique de C. difficile, entéropathogène nosocomial majeur. Nous avons en effet pu l'intégrer comme marqueur spécifique d'espèce dans une PCR multiplex ciblant les gènes tpi, tcdA et tcdB, permettant de façon combinée l'identification et la caractérisation du profil toxinique des souches de C. difficile isolées de prélèvement d'origine humaine ou animale, dès leur détection. Nous nous sommes ensuite intéressés à la caractérisation d'un gène d'autolysine chez C. difficile. En effet, les autolysines, enzymes hydrolysant le peptidoglycane bactérien, possèdent un rôle essentiel dans la physiologie des bactéries, pourraient présenter un intérêt taxonomique potentiel et pourraient contribuer à la virulence de certaines espèces pathogènes. Nous avons initialement amplifé par PCR un fragment de 230 pb environ chez cinq espèces du genre Clostridium. Chez C. difficile, le génome de la souche 630 étant entièrement séquencé, nous avons ensuite caractérisé in silico la séquence du gène acd codant une autolysine putative. Nous avons alors cloné ce gène et exprimé la protéine correspondante afin d'étudier les propriétés et fonctions de cette autolysine putative. La protéine Acd est une autolysine à activé N-acétyl-glucoseminidase. Le domaine catalytique de cette protéine apparaît conservé et implique probablement cette autolysine dans la physiologie de C. difficile.ROUEN-BU Médecine-Pharmacie (765402102) / SudocPARIS-BIUM (751062103) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF

Entretien alcoologique aux urgences (quel impact ?)

LILLE2-BU Santé-Recherche (593502101) / SudocPARIS-BIUM (751062103) / SudocSudocFranceF

Identification génotypique de bactéries du genre Clostridium par analyse du polymorphisme de gènes d'enzymes domestiques

Le polymorphisme de gènes domestiques de bactéries du genre Clostridium a été étudié afin de développer un marqueur taxonomique et phylogénétique alternatif à l'ADNr16S. Après avoir évalué plusieurs gènes domestiques candidats chez douze espèces de Clostridium (gène sod codant la superoxyde dismutase, gap, la glycéraldéhyde phosphate déshydrogénase, rpoB, l'ARN polymérase (sous-unité B)...), nous avons retenu le gène tpi codant une triosephosphate isomérase. Un fragment interne de 501 pb de ce gène a pu être amplifié chez les douze souches-types par PCR touch dolvn en utilisant un couple d'amorces dégénérées de 23 nucléotides chacune. Le séquençage des amplicons et l'alignement des séquences nous ont permis de visualiser plusieurs sites de restriction de l'enzyme AIuI, possiblement spécifiques d'espèces. Les amplicons digérés par AIuI ont alors généré des profils distincts, qui se sont avérés spécifiques d'espèce après analyse d'un plus grand nombre de souches. L'analyse phylogénétique sur la base des séquences du gène tpi est bien corrélée à celle basée sur les séquences d'ADNr 16S.ROUEN-BU Médecine-Pharmacie (765402102) / SudocSudocFranceF

Influence of Recombination on Development of Mutational Resistance to Linezolid in Enterococcus faecalis JH2-2

International audienc

Differences in Potential for Selection of Clindamycin-Resistant Mutants Between Inducible erm(A) and erm(C) Staphylococcus aureus Genes▿

In staphylococci, inducible macrolide-lincosamide-streptogramin B (MLSB) resistance is conferred by the erm(C) or erm(A) gene. This phenotype is characterized by the erythromycin-clindamycin “D-zone” test. Although clindamycin appears active in vitro, exposure of MLSB-inducible Staphylococcus aureus to this antibiotic may result in the selection of clindamycin-resistant mutants, either in vitro or in vivo. We have compared the frequencies of mutation to clindamycin resistance for 28 isolates of S. aureus inducibly resistant to erythromycin and bearing the erm(C) (n = 18) or erm(A) (n = 10) gene. Seven isolates susceptible to erythromycin or bearing the msr(A) gene (efflux) were used as controls. The frequencies of mutation to clindamycin resistance for the erm(A) isolates (mean ± standard deviation, 3.4 × 10−8 ± 2.4 × 10−8) were only slightly higher than those for the controls (1.1 × 10−8 ± 6.4 × 10−9). By contrast, erm(C) isolates displayed a mean frequency of mutation to clindamycin resistance (4.7 × 10−7 ± 5.5 × 10−7) 14-fold higher than that of the S. aureus isolates with erm(A). The difference was also observed, although to a lower extent, when erm(C) and erm(A) were cloned into S. aureus RN4220. We conclude that erm(C) and erm(A) have different genetic potentials for selection of clindamycin-resistant mutants. By the disk diffusion method, erm(C) and erm(A) isolates could be distinguished on the basis of high- and low-level resistance to oleandomycin, respectively

Resistance to macrolides by ribosomal mutation in clinical isolates of Turicella otitidis

International audienceThe genetic basis of erythromycin resistance in , a coryneform bacteria associated with otitis, was studied in five macrolide-resistant clinical isolates. Macrolide resistance genes were searched for by polymerase chain reaction (PCR). Genes for domain V of 23S rRNA () as well as (L4 protein) and (L22 protein) genes were characterised, amplified by PCR from total genomic DNA and sequenced. In the resistant isolates, cross-resistance to macrolides and clindamycin was associated with mutations at positions 2058 and/or 2059 ( numbering). Three isolates displayed A2058 mutations, one isolate had an A2059G mutation whereas another one contained mutations at positions 2058 and 2059. Southern blot experiments revealed that had three copies of the gene. In conclusion, resistance to macrolides in is due, at least in part, to mutations in the gene