Characterization of RNA Helicase CshA and Its Role in Protecting mRNAs and Small RNAs of Staphylococcus aureus Strain Newman

The toxin MazFsa in Staphylococcus aureus is a sequence-specific endoribonuclease that cleaves the majority of the mRNAs in vivo but spares many essential mRNAs (e.g., secY mRNA) and, surprisingly, an mRNA encoding a regulatory protein (i.e., sarA mRNA). We hypothesize that some mRNAs may be protected by RNA-binding protein(s) from degradation by MazFsa. Using heparin-Sepharose-enriched fractions that hybridized to sarA mRNA on Northwestern blots, we identified among multiple proteins the DEAD box RNA helicase CshA (NWMN_1985 or SA1885) by mass spectroscopy. Purified CshA exhibits typical RNA helicase activities, as exemplified by RNA-dependent ATPase activity and unwinding of the DNA-RNA duplex. A severe growth defect was observed in the cshA mutant compared with the parent when grown at 25°C but not at 37°C. Activation of MazFsa in the cshA mutant resulted in lower CFU per milliliter accompanied by a precipitous drop in viability (∼40%) compared to those of the parent and complemented strains. NanoString analysis reveals diminished expression of a small number of mRNAs and 22 small RNAs (sRNAs) in the cshA mutant versus the parent upon MazFsa induction, thus implying protection of these RNAs by CshA. In the case of the sRNA teg049 within the sarA locus, we showed that the protective effect was likely due to transcript stability as revealed by reduced half-life in the cshA mutant versus the parent. Accordingly, CshA likely stabilizes selective mRNAs and sRNAs in vivo and as a result enhances S. aureus survival upon MazFsa induction during stress

Plasmid-mediated quinolone resistance in Aeromonas allosaccharophila recovered from a Swiss lake

Objectives To search for plasmid-mediated qnr genes among waterborne environmental Aeromonas spp. recovered from Switzerland. Methods Isolates presenting MICs of nalidixic acid or ciprofloxacin ≥1 mg/L were screened for qnr genes by a multiplex PCR approach followed by sequencing. Plasmids were transferred by transformation, and further analysis of the genetic structures surrounding the qnrS2 gene was carried out by PCR and sequencing. Results A qnrS2 gene was identified from a single Aeromonas allosaccharophila isolate (Lugano lake, Lugano), as part of a mobile insertion cassette located on a broad host range IncU-type plasmid. This plasmid co-harboured a class 1 integron containing the aac(6′)-Ib-cr, blaOXA-1, catB3 and arr-3 gene cassettes. Conclusions These findings strengthen further the role of Aeromonas spp. as a reservoir of antimicrobial resistance determinants in the environmen

Rôle des résidus d'antibiotiques dans l'environnement hydrique sur la sélection et la diffusion de bactéries résistantes des genres "Aeromonas", "Acinetobacter" et "Legionella"

Les résidus d'antibiotiques et les bactéries résistantes sont déversées en quantités variées dans l'environnement, en conséquence d'une utilisation massive et souvent indiscriminée de ces substances dans les domaines médicaux, vétérinaires et en agrochimie. Les eaux de surface sont le réceptacle majeur de ces polluants et représentent également les sources d'eau directement ou indirectement utilisées pour la consommation humaine et animale. L'objectif de ce travail de thèse, effectué dans le cadre du Programme National de Recherche 49 "Antibiotic Resistance", a été d'évaluer, à l'aide d'outils empruntés à la bactériologie classique et à la microbiologie moléculaire, le rôle des résidus d'antibiotiques présents dans l'environnement hydrique sur l'émergence et / ou la persistance de bactéries résistantes. La recherche a été concentrée sur trois genres bactériens pathogènes pour l'homme et l'animal, à savoir les Aeromonas, les Acinetobacter et les Legionella et trois familles d'antibiotiques, les bêta-lactames, les quinolones / fluoroquinolones et les macrolides, abondamment utilisées en médecine humaine. L'analyse phénotypique de la résistance dans les divers milieux hydriques a mis en évidence la présence de souches résistantes dans les eaux les plus contaminées. Des Aeromonas résistants aux quatre générations de céphalosporines ont pu être isolés des eaux usées des hôpitaux et des stations d'épuration, tandis que les souches d'origine clinique se sont avérées beaucoup plus sensibles. Au contraire, les Acinetobacter de l'environnement hydrique sont apparus moins résistants que ceux d'origine humaine. Pour les Legionella, les valeurs des CMI différaient en fonction du type génétique de la souche (Sequence-based Type) et non de l'origine. Une corrélation statistique significative entre concentrations de ciprofloxacine et souches résistantes isolées a pu être mise en évidence dans les eaux usées des hôpitaux. L'effet éventuel de faibles concentrations hydriques de ceftriaxone, de ciprofloxacine et d'érythromycine sur la sélection et l'induction d'Aeromonas, d'Acinetobacter et de Legionella résistants a été étudié in vitro et in situ, ce dernier, par l'utilisation d'un bio réacteur reproduisant la phase biologique d'épuration des eaux (boues activées). Dans certains cas, ces faibles concentrations d'inducteurs ont pu modifier, in vitro, le profil de résistance de souches d'Aeromonas et d'Acinetobacter. Dans d'autres cas, des concentrations supérieures d'antibiotiques ont été nécessaires pour induire une résistance. Les nouveaux profils induits restent stables dans le temps. Par la technique d'électrophorèse sur gel en gradient de dénaturation (DGGE) et par l'utilisation de sondes moléculaires fluorescente (FISH), les effets qualitatifs et quantitatifs des résidus d'antibiotiques ajoutés dans le bio réacteur sur les trois populations considérées et sur la communauté microbienne totale présentes dans les boues activées ont pu être évalués. Enfin, l'étude de la diffusion des intégrons de classe 1 chez les souches d'Aeromonas et d'Acinetobacter provenant des divers environnements hydriques a été effectuée et mise en relation avec leur lieux d'isolement. Certains gènes de résistance ont pu être déterminés par séquençage

Characterization of RNA Helicase CshA and Its Role in Protecting mRNAs and Small RNAs of Staphylococcus aureus Strain Newman

The toxin MazFsa in Staphylococcus aureus is a sequence-specific endoribonuclease that cleaves the majority of the mRNAs in vivo but spares many essential mRNAs (e.g., secY mRNA) and, surprisingly, an mRNA encoding a regulatory protein (i.e., sarA mRNA). We hypothesize that some mRNAs may be protected by RNA-binding protein(s) from degradation by MazFsa. Using heparin-Sepharose-enriched fractions that hybridized to sarA mRNA on Northwestern blots, we identified among multiple proteins the DEAD box RNA helicase CshA (NWMN_1985 or SA1885) by mass spectroscopy. Purified CshA exhibits typical RNA helicase activities, as exemplified by RNA-dependent ATPase activity and unwinding of the DNA-RNA duplex. A severe growth defect was observed in the cshA mutant compared with the parent when grown at 25°C but not at 37°C. Activation of MazFsa in the cshA mutant resulted in lower CFU per milliliter accompanied by a precipitous drop in viability (∼40%) compared to those of the parent and complemented strains. NanoString analysis reveals diminished expression of a small number of mRNAs and 22 small RNAs (sRNAs) in the cshA mutant versus the parent upon MazFsa induction, thus implying protection of these RNAs by CshA. In the case of the sRNA teg049 within the sarA locus, we showed that the protective effect was likely due to transcript stability as revealed by reduced half-life in the cshA mutant versus the parent. Accordingly, CshA likely stabilizes selective mRNAs and sRNAs in vivo and as a result enhances S. aureus survival upon MazFsa induction during stress

A type IV modification-dependent restriction enzyme SauUSI from Staphylococcus aureus subsp. aureus USA300

A gene encoding a putative DNA helicase from Staphylococcus aureus USA300 was cloned and expressed in Escherichia coli. The protein was purified to over 90% purity by chromatography. The purified enzyme, SauUSI, predominantly cleaves modified DNA containing 5mC and 5-hydroxymethylcytosine. Cleavage of 5mC-modified plasmids indicated that the sites S5mCNGS (S = C or G) are preferentially digested. The endonuclease activity requires the presence of adenosine triphosphate (ATP) or dATP whereas the non-hydrolyzable γ-S-ATP does not support activity. SauUSI activity was inhibited by ethylenediaminetetraacetic acid. It is most active in Mg(++) buffers. No companion methylase gene was found near the SauUSI restriction gene. The absence of a cognate methylase and cleavage of modified DNA indicate that SauUSI belongs to type IV restriction endonucleases, a group that includes EcoK McrBC and Mrr. SauUSI belongs to a family of highly similar homologs found in other sequenced S. aureus, S. epidermidis and S. carnosus genomes. More distant SauUSI orthologs can be found in over 150 sequenced bacterial/archaea genomes. Finally, we demonstrated the biological function of the type IV REase in restricting 5mC-modified plasmid DNA by transformation into clinical S. aureus strain SA564, and in restricting phage λ infection when the endonuclease is expressed in E. coli

The Staphylococcus aureus CC398 lineage: An evolution driven by the acquisition of prophages and other mobile genetic elements

International audienceAmong clinically relevant lineages of Staphylococcus aureus, the lineage or clonal complex 398 (CC398) is of particular interest. Strains from this lineage were only described as livestock colonizers until 2007. Progressively, cases of infection were reported in humans in contact with farm animals, and now, CC398 isolates are increasingly identified as the cause of severe infections even in patients without any contact with animals. These observations suggest that CC398 isolates have spread not only in the community but also in the hospital setting. In addition, several recent studies have reported that CC398 strains are evolving towards increased virulence and antibiotic resistance. Identification of the origin and emergence of this clonal complex could probably benefit future large-scale studies that aim to detect sources of contamination and infection. Current evidence indicates that the evolution of CC398 strains towards these phenotypes has been driven by the acquisition of prophages and other mobile genetic elements. In this short review, we summarize the main knowledge of this major lineage of S. aureus that has become predominant in the human clinic worldwide within a single decade

Bypassing the restriction system to improve transformation of Staphylococcus epidermidis

Staphylococcus epidermidis is the leading cause of infections on indwelling medical devices worldwide. Intrinsic antibiotic resistance and vigorous biofilm production have rendered these infections difficult to treat and, in some cases, require the removal of the offending medical prostheses. With the exception of two widely-passaged isolates RP62A and 1457, the pathogenesis of infections caused by clinical S. epidermidis strains is poorly understood due to the strong genetic barrier that precludes efficient transformation of foreign DNA into clinical isolates. The difficulty in transforming clinical S. epidermidis isolates is primarily due to the type I and IV restriction modification systems which act as genetic barriers. Here, we showed that efficient plasmid transformation of clinical S. epidermidis isolates from clonal complexes 2, 10 and 89 could be realized by employing a plasmid artificial modification (PAM) in E. coli DC10B containing a Δdcm mutation. This transformative technique should facilitate our ability to genetically modify clinical isolates of S. epidermidis and hence improve our understanding of its pathogenesis in human infections.ImportanceStaphylococcus epidermidis is a source of considerable morbidity worldwide. The underlying mechanisms contributing to the commensal and pathogenic lifestyles of S. epidermidis are poorly understood. Genetic manipulations of clinically relevant strains of S. epidermidis are largely prohibited due to the presence of a strong restriction barrier. With the introductions of the tools presented here, genetic manipulation has now become possible with clinically relevant S. epidermidis isolates, thus improving our understanding of S. epidermidis as a pathogen

Prophages and adaptation of Staphylococcus aureus ST398 to the human clinic

International audienceIt has been suggested that prophages in the ST398 S. aureus clone are responsible for expanding ST398's spectrum of action and increasing its ability to cause human infections. We carried out the first characterization of the various prophages carried by 76 ST398 bloodstream infection (BSI) isolates obtained over 9 years of observation.Whole-genome sequencing of 22 representative isolates showed (1) the presence of the φ3-prophage and diverse genetic features typical of animal-associated isolates (i.e., SCCmec XI element, Tn916 transposon and non φ3-prophages) in a majority of BSI isolates, (2) one BSI isolate devoid of the φ3-prophage but otherwise similar to an animal-infecting isolate, (3) 35 prophages carrying numerous genes previously associated with virulence or immune evasion in animal models of staphylococcal infections. The analysis of prophage content in all 76 BSI isolates showed an increasing prevalence of polylysogeny over time. Overall, over the course of the last 10 years, the BSI isolates appear to have acquired increasing numbers of genetic features previously shown to contribute to bacterial adaptation and virulence in animal models of staphylococcal infections.We hypothesize that lysogeny has played a significant role in increasing the ability of the ST398 clone to cause infections in humans. Our findings highlight the risk that the ST398 lineage will increase its threat to public health by continuing to acquire virulence and/or multiple antibiotic-resistance genes from hospital-associated clones of Staphylococcus aureus