A search for small noncoding RNAs in Staphylococcus aureus reveals a conserved sequence motif for regulation

Bioinformatic analysis of the intergenic regions of Staphylococcus aureus predicted multiple regulatory regions. From this analysis, we characterized 11 novel noncoding RNAs (RsaA‐K) that are expressed in several S. aureus strains under different experimental conditions. Many of them accumulate in the late-exponential phase of growth. All ncRNAs are stable and their expression is Hfq-independent. The transcription of several of them is regulated by the alternative sigma B factor (RsaA, D and F) while the expression of RsaE is agrA-dependent. Six of these ncRNAs are specific to S. aureus, four are conserved in other Staphylococci, and RsaE is also present in Bacillaceae. Transcriptomic and proteomic analysis indicated that RsaE regulates the synthesis of proteins involved in various metabolic pathways. Phylogenetic analysis combined with RNA structure probing, searches for RsaE‐mRNA base pairing, and toeprinting assays indicate that a conserved and unpaired UCCC sequence motif of RsaE binds to target mRNAs and prevents the formation of the ribosomal initiation complex. This study unexpectedly shows that most of the novel ncRNAs carry the conserved C−rich motif, suggesting that they are members of a class of ncRNAs that target mRNAs by a shared mechanis

Staphylococcus aureus Bloodstream Infection and Endocarditis―A Prospective Cohort Study

Equipe CHU UB (EA) Pôle MERS CT3 Hors Enjeu The VIRSTA Study Group : Clinical centres: Besançon: Catherine Chirouze, Elodie Curlier, Cécile Descottes-Genon, Bruno Hoen, Isabelle Patry, Lucie Vettoretti. Dijon: Pascal Chavanet, Jean-Christophe Eicher, Sandrine Gohier-Treuvelot, Marie-Christine Greusard, Catherine Neuwirth, André Péchinot, Lionel Piroth. Lyon: Marie Célard, Catherine Cornu, François Delahaye, Malika Hadid, Pascale Rausch. Montpellier: Audrey Coma, Florence Galtier, Philippe Géraud, Hélène Jean-Pierre, Vincent Le Moing, Catherine Sportouch, Jacques Reynes. Nancy: Nejla Aissa, Thanh Doco- Lecompte, François Goehringer, Nathalie Keil, Lorraine Letranchant, Hepher Malela, Thierry May, Christine Selton-Suty. Nîmes: Nathalie Bedos, Jean-Philippe Lavigne, Catherine Lechiche, Albert Sotto. Paris: Xavier Duval, Emila Ilic Habensus, Bernard Iung, Catherine Leport, Pascale Longuet, Raymond Ruimy. Rennes: Eric Bellissant, Pierre-Yves Donnio, Fabienne Le Gac, Christian Michelet, Matthieu Revest, Pierre Tattevin, Elise Thebault. Coordination and statistical analyses: François Alla, Pierre Braquet, Marie-Line Erpelding, Laetitia Minary, Sarah Tubiana. Centre National de Référence des staphylocoques: Michèle Bès, Jérôme Etienne, Anne Tristan, François Vandenesch. Sponsor CHU de Montpellier: Sandrine Barbas, Christine Delonca, Virginie Sussmuth, Anne Verchère. Alain Makinson reviewed the manuscript for English correctness.International audienceOBJECTIVES: To update the epidemiology of S. aureus bloodstream infection (SAB) in a high-income country and its link with infective endocarditis (IE).METHODS: All consecutive adult patients with incident SAB (n = 2008) were prospectively enrolled between 2009 and 2011 in 8 university hospitals in France. RESULTS: SAB was nosocomial in 54%, non-nosocomial healthcare related in 18% and community-acquired in 26%. Methicillin resistance was present in 19% of isolates. SAB Incidence of nosocomial SAB was 0.159/1000 patients-days of hospitalization (95% confidence interval [CI] 0.111-0.219). A deep focus of infection was detected in 37%, the two most frequent were IE (11%) and pneumonia (8%). The higher rates of IE were observed in injecting drug users (IE: 38%) and patients with prosthetic (IE: 33%) or native valve disease (IE: 20%) but 40% of IE occurred in patients without heart disease nor injecting drug use. IE was more frequent in case of community-acquired (IE: 21%, adjusted odds-ratio (aOR) = 2.9, CI = 2.0-4.3) or non-nosocomial healthcare-related SAB (IE: 12%, aOR = 2.3, CI = 1.4-3.5). S. aureus meningitis (IE: 59%), persistent bacteremia at 48 hours (IE: 25%) and C-reactive protein > 190 mg/L (IE: 15%) were also independently associated with IE. Criteria for severe sepsis or septic shock were met in 30% of SAB without IE (overall in hospital mortality rate 24%) and in 51% of IE (overall in hospital mortality rate 35%).CONCLUSION: SAB is still a severe disease, mostly related to healthcare in a high-income country. IE is the most frequent complication and occurs frequently in patients without known predisposing condition

Impact of sub-inhibitory antibiotics on fibronectin-mediated host cell adhesion and invasion by Staphylococcus aureus

<p>Abstract</p> <p>Background</p> <p><it>Staphylococcus aureus </it>is a well-armed pathogen prevalent in severe infections such as endocarditis and osteomyelitis. Fibronectin-binding proteins A and B, encoded by <it>fnb</it>A/B, are major pathogenesis determinants in these infections through their involvement in <it>S. aureus </it>adhesion to and invasion of host cells. Sub-minimum inhibitory concentrations (sub-MICs) of antibiotics, frequently occurring <it>in vivo </it>because of impaired drug diffusion at the infection site, can alter <it>S. aureus </it>phenotype. We therefore investigated their impact on <it>S. aureus </it>fibronectin-mediated adhesiveness and invasiveness.</p> <p>Methods</p> <p>After <it>in vitro </it>challenge of <it>S. aureus </it>8325-4 and clinical isolates with sub-MICs of major anti-staphylococcal agents, we explored <it>fnb</it>A/B transcription levels, bacterial adhesiveness to immobilised human fibronectin and human osteoblasts in culture, and bacterial invasion of human osteoblasts.</p> <p>Results</p> <p>Oxacillin, moxifloxacin and linezolid led to the development of a hyper-adhesive phenotype in the fibronectin adhesion assay that was consistent with an increase in <it>fnb</it>A/B transcription. Conversely, rifampin treatment decreased fibronectin binding in all strains tested without affecting <it>fnb</it>A/B transcription. Gentamicin and vancomycin had no impact on fibronectin binding or <it>fnb</it>A/B transcription levels. Only oxacillin-treated <it>S. aureus </it>displayed a significantly increased adhesion to cultured osteoblasts, but its invasiveness did not differ from that of untreated controls.</p> <p>Conclusion</p> <p>Our findings demonstrate that several antibiotics at sub-MICs modulate fibronectin binding in <it>S. aureus </it>in a drug-specific fashion. However, hyper- and hypo- adhesive phenotypes observed in controlled <it>in vitro </it>conditions were not fully confirmed in whole cell infection assays. The relevance of adhesion modulation during <it>in vivo </it>infections is thus still uncertain and requires further investigations.</p

RsaI, a multifaceted regulatory RNA, modulates the metabolism of the opportunistic pathogen Staphylococcus aureus RsaI, un ARN régulateur aux multiples facettes, module le métabolisme du pathogène opportuniste Staphylococcus aureus

Staphylococcus aureus est une bactérie commensale retrouvée chez environ 30 % des individus sains dont elle colonise la peau et la muqueuse nasale. Cependant, c’est également une bactérie pathogène opportuniste responsable d’infections diverses telles que orgelet, ostéomyélite, endocardite, ou encore septicémie en envahissant un grand nombre de tissus et d’organes. Cette bactérie est capable de s’adapter à des conditions hostiles et variées, telles que carence nutritive et stress osmotique, oxydant, ou thermique, ainsi qu’à la réponse immunitaire de l’hôte, car elle produit une grande diversité de facteurs de virulence. La synthèse de ces facteurs est finement régulée par des protéines et des ARN régulateurs majoritairement non codants, souvent désignés par l’abréviation sARN (dérivée de l’anglais, small RNA). Les facteurs de transcription et les systèmes à deux composants contrôlent l’expression des gènes impliqués non seulement dans le métabolisme, mais aussi dans la réponse au stress et la virulence [1]. Par exemple, la protéine du contrôle catabolique (carbon catabolite control protein A, CcpA) a un rôle essentiel dans le choix de la source carbonée en régulant le métabolisme central de la bactérie ainsi que la virulence [2, 3]. CcpA se fixe à une séquence promotrice spécifique appelée cre (catabolite-responsive element), qui est très conservée chez les bactéries à Gram positif [2]. Quant aux sARN, ils interagissent principalement avec leurs ARN messagers (ARNm) cibles. L’hybridation peut conduire à la stabilisation/déstabilisation de l’ARNm ou à l’activation/répression de sa traduction [4]. Nous avons montré que la transcription du sARN RsaI (RNA Staphylococcus aureus I) est réprimée par CcpA en présence de glucose [5]. L’induction de la synthèse de RsaI signale que la concentration en glucose diminue dans le milieu extracellulaire et que la croissance des bactéries est ralentie. En interagissant avec ses ARNm cibles ou d’autres sARN, il permet à la population bactérienne de modifier son métabolisme lorsque la source carbonée primaire est consommée

Staphylococcal entertotoxins of the enterotoxin gene cluster (egcSEs) induce nitrous oxide- and cytokine dependent tumor cell apoptosis in a broad panel of human tumor cells

International audienceThe egcSEs comprise five genetically linked staphylococcal enterotoxins, SEG, SEI, SElM, SElN, and SElO and two pseudotoxins which constitute an operon present in up to 80% of Staphylococcus aureus isolates. A preparation containing these proteins was recently used to treat advanced lung cancer with pleural effusion. We investigated the hypothesis that egcSEs induce nitrous oxide (NO) and associated cytokine production and that these agents may be involved in tumoricidal effects against a broad panel of clinically relevant human tumor cells. Preliminary studies showed that egcSEs and SEA activated T cells (range: 11-25%) in a concentration dependent manner. Peripheral blood mononuclear cells (PBMCs) stimulated with equimolar quantities of egcSEs expressed NO synthase and generated robust levels of nitrite (range: 200-250 μM), a breakdown product of NO; this reaction was inhibited by NG-monomethyl-L-arginine (L-NMMA) (0.3 mM), an NO synthase antagonist. Cell free supernatants (CSFs) of all egcSE-stimulated PBMCs were also equally effective in inducing concentration dependent tumor cell apoptosis in a broad panel of human tumor cells. The latter effect was due in part to the generation of NO and TNF-α since it was significantly abolished by L-NMMA, anti-TNF-α antibodies, respectively, and a combination thereof. A hierarchy of tumor cell sensitivity to these CFSs was as follows: lung carcinoma > osteogenic sarcoma > melanoma > breast carcinoma >neuroblastoma. Notably, SEG induced robust activation of NO/TNFα-dependent tumor cell apoptosis comparable to the other egcSEs and SEA despite TNF-α and IFN-γ levels that were 2 and 8 fold lower, respectively, than the other egcSEs and SEA. Thus, egcSEs produced by S. aureus induce NO synthase and the increased NO formation together with TNF-α appear to contribute to egcSE-mediated apoptosis against a broad panel of human tumor cells

Risk factors for treatment failure in orthopedic device-related methicillin-resistant Staphylococcus aureus infection

The purpose of this study was to determine the clinical and microbiological risk factors for treatment failure of methicillin-resistant Staphylococcus aureus (MRSA) orthopedic device-related infection (ODRI). A retrospective cohort study of patients with MRSA ODRI who were treated at Geneva University Hospitals between 2000 and 2008 was undertaken. Stored MRSA isolates were retrieved for genetic characterization and determination of the vancomycin minimum inhibitory concentration (MIC). Fifty-two patients were included, of whom 23 (44%) had joint arthroplasty and 29 (56%) had osteosynthesis. All 41 of the retrieved MRSA isolates were susceptible to vancomycin (MIC ≤ 2mg/L) and 35 (85%) shared genetic characteristics of the South German clone (ST228). During a median follow-up of 391days (range, 4-2,922days), 18 patients (35%) experienced treatment failure involving MRSA persistence or recurrence. Microbiological factors such as infection with the predominant clone and a vancomycin MIC of 2mg/L were not associated with treatment failure. Using a Cox proportional hazards model, implant retention (hazard ratio [HR], 4.9; 95% confidence interval [CI], 1.3-18.2; P = 0.017) and single-agent antimicrobial therapy (HR, 4.4; 95% CI, 1.2-16.3; P = 0.025) were independent predictors of treatment failure after debridement. Therapy using a combination of antimicrobials should be considered for patients with MRSA ODRI, especially when implant removal is not feasibl

Community-associated Methicillin-resistant Staphylococcus aureus, Switzerland

Two case-control studies evaluated the prevalence of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) carriage at hospital admission and characteristics of patients with CA-MRSA. Among 14,253 patients, CA-MRSA prevalence was 0.9/1,000 admissions. Although 5 CA-MRSA isolates contained Panton-Valentine leukocidin, only 1 patient had a previous skin infection. No easily modifiable risk factor for CA-MRSA was identified

Targeted proteomics links virulence factor expression with clinical severity in staphylococcal pneumonia

IntroductionThe bacterial pathogen Staphylococcus aureus harbors numerous virulence factors that impact infection severity. Beyond virulence gene presence or absence, the expression level of virulence proteins is known to vary across S. aureus lineages and isolates. However, the impact of expression level on severity is poorly understood due to the lack of high-throughput quantification methods of virulence proteins.MethodsWe present a targeted proteomic approach able to monitor 42 staphylococcal proteins in a single experiment. Using this approach, we compared the quantitative virulomes of 136 S. aureus isolates from a nationwide cohort of French patients with severe community-acquired staphylococcal pneumonia, all requiring intensive care. We used multivariable regression models adjusted for patient baseline health (Charlson comorbidity score) to identify the virulence factors whose in vitro expression level predicted pneumonia severity markers, namely leukopenia and hemoptysis, as well as patient survival.ResultsWe found that leukopenia was predicted by higher expression of HlgB, Nuc, and Tsst-1 and lower expression of BlaI and HlgC, while hemoptysis was predicted by higher expression of BlaZ and HlgB and lower expression of HlgC. Strikingly, mortality was independently predicted in a dose-dependent fashion by a single phage-encoded virulence factor, the Panton-Valentine leucocidin (PVL), both in logistic (OR 1.28; 95%CI[1.02;1.60]) and survival (HR 1.15; 95%CI[1.02;1.30]) regression models.DiscussionThese findings demonstrate that the in vitro expression level of virulence factors can be correlated with infection severity using targeted proteomics, a method that may be adapted to other bacterial pathogens

Pneumonia and New Methicillin-resistant Staphylococcus aureus Clone

Necrotizing pneumonia caused by Staphylococcus aureus strains carrying the Panton-Valentin leukocidin gene is a newly described disease entity. We report a new fatal case of necrotizing pneumonia. An S. aureus strain with an agr1 allele and of a new sequence type 377 was recovered, representing a new, emerging, community-acquired methicillin-resistant clone