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The Staphylococcus aureus enterotoxin gene cluster, egc , was detected in isolates from healthy individuals and in those from patients with bacteremia. The egc genes cooccur and are slightly enriched in strains from healthy carriers (present in 63.7% of carriage-associated isolates versus 52.9% of invasive isolates; P = 0.03). Multilocus sequence typing revealed that successful staphylococcal clones usually harbor the egc locus.

To define the clinical, microbiological, and therapeutic characteristics of haemophilus endocarditis, we reviewed the charts of 42 adults with haemophilus endocarditis (native valve disease, 37; prosthetic valve disease, five) who were followed up between 1983 and 1995 in France. The mean duration of symptoms before diagnosis was 34 days. The causative Haemophilus species were as follows: H. parainfluenzae (26 adults), H. aphrophilus (9), H. paraphrophilus (4), and H. influenzae (3). According to the Duke criteria, 38 cases of endocarditis were definitive and four were possible. Thirty-nine patients received combination antibacterial therapy and three received therapy with a beta-lactam agent alone (mean duration, 46 days). Arterial embolism occurred in 15 patients. Cardiac surgery was indicated for 18 patients; 16 of these surgeries were performed within 3 months. Two patients died of heart failure. In conclusion, haemophilus endocarditis is rare and is mainly due to H. parainfluenzae. Although surgery is often necessary, haemophilus endocarditis has a favorable prognosis.

(CA-MSSA) infection. The major difference between the 2 groups was the greater frequency of abnormal pulmonary imaging findings in the CA-MRSA group (67% of patients), compared with the CA-MSSA group (28% of patients) (

Background. Severe sepsis, combining acute osteomyelitis and lung involvement, has been described increasingly in healthy children with the spread of community-associated methicillin-resistant Staphylococcus aureus (CAMRSA). Methods. Outcomes (mortality, hematogenous spread, lung and bone involvements) of rabbit osteomyelitis caused by CA-MRSA LAC WT USA300 and its Panton-Valentine leukocidin (PVL)- and α-hemolysin (Hla)-negative isogenic derivatives (LACΔpvl and LACΔhla, respectively) were compared. Results. Three days after inoculation (D3), all LAC WT - and LACΔpvl-, and 72% of LACΔhla-infected rabbits had no hematogenous spread and similar lung and bone bacterial densities. LACΔpvl and LACΔhla caused less severe histological lung lesions than LAC WT (P ≤ .01). Between D3 and D9, 10 (53%) LAC WT -, 11 (55%) LACΔpvl-, but no LACΔhla-infected rabbits (P< .005) died of severe sepsis with disseminated infection. Unlike deceased animals, most LAC WT ,L ACΔpvl, and LACΔhla D14 survivors had no hematogenous spread (P< .001). LAC WT (88%) caused more bone abscesses than LACΔpvl (0, P= .001) or LACΔhla (30%, P= .01). Conclusion. In this model, both PVL and Hla seemed to be required for early lung involvement via hematogenous spread. Hla, but not PVL, significantly impacted severe sepsis-related mortality. PVL was the predominant factor determining late-stage bone abscesses.

International audience; Clonal complex 398 livestok-associated-MRSA (CC398 LA-MRSA) clone is described as a major animal pathogen that can also colonize and infect humans. CC398 methicillin susceptible Staphylococcus aureus (CC398 MSSA) is less described. We identified 126 CC398 MSSA strains of human origin within 6380 S. aureus isolates gathered between 2009 and 2011, from the French National Reference Centre for Staphylococci. They were characterized using antimicrobial susceptibility testing, spa typing, DNA microarrays (Identibac S. aureus Genotyping ®, Alere), CC398-specific sequence PCR, ermT (encoding macrolides résistance) PCR. Fifty-three CC398 LA-MRSA collected from French pigs and veal were used as comparators, and phylogenetic relations between human CC398 MSSA and animal CC398 MRSA populations were explored on the basis of spa-typing and DNA microarrays. CC398 MSSA were able to induce a large spectrum of infections (especially skin, bloodstream, and pneumonias). The prevalence rate of this clone was high in MSSA population, i.e., 24.7% in a local prospective study on nasal colonization, and 7.5% in a national prospective study on infective endocarditis. CC398 MSSA isolates were frequently (89%) erythromycin resistant, due to the presence of the ermT gene, a gene not detected in erythromycin resistant CC398 LA-MRSA strains. Expression of staphylococcal complement inhibitor (scn) and the chemotaxis inhibitory protein (chp), was also specific to this population. The CC398 MRSA signature included also a panel of antibiotic resistance genes, especially a type IV or V cassette mec and tetM. CC398 MSSA and CC398 LA-MRSA populations were closely related based on spa-typing and DNA microarrays, with the MRSA strains forming the most derived lineage in phylogenic trees. Both MSSA and MRSA populations may come from common ancestors, which would have evolved in the settings of different selective pressures, explaining the acquisition of ermT, chp and scn for MSSA, and antibiotic resistance genes for MRSA.

International audience; BACKGROUND: Staphylococcus epidermidis orthopedic device infections are caused by direct inoculation of commensal flora during surgery and remain rare, although S. epidermidis carriage is likely universal. We wondered whether S. epidermidis orthopedic device infection strains might constitute a sub-population of commensal isolates with specific virulence ability. Biofilm formation and invasion of osteoblasts by S. aureus contribute to bone and joint infection recurrence by protecting bacteria from the host-immune system and most antibiotics. We aimed to determine whether S. epidermidis orthopedic device infection isolates could be distinguished from commensal strains by their ability to invade osteoblasts and form biofilms. MATERIALS AND METHODS: Orthopedic device infection S. epidermidis strains (n?=?15) were compared to nasal carriage isolates (n?=?22). Osteoblast invasion was evaluated in an ex vivo infection model using MG63 osteoblastic cells co-cultured for 2 hours with bacteria. Adhesion of S. epidermidis to osteoblasts was explored by a flow cytometric approach, and internalized bacteria were quantified by plating cell lysates after selective killing of extra-cellular bacteria with gentamicin. Early and mature biofilm formations were evaluated by a crystal violet microtitration plate assay and the Biofilm Ring Test method. RESULTS: No difference was observed between commensal and infective strains in their ability to invade osteoblasts (internalization rate 308+/-631 and 347+/-431 CFU/well, respectively). This low internalization rate correlated with a low ability to adhere to osteoblasts. No difference was observed for biofilm formation between the two groups. CONCLUSION: Osteoblast invasion and biofilm formation levels failed to distinguish S. epidermidis orthopedic device infection strains from commensal isolates. This study provides the first assessment of the interaction between S. epidermidis strains isolated from orthopedic device infections and osteoblasts, and suggests that bone cell invasion is not a major pathophysiological mechanism in S. epidermidis orthopedic device infections, contrary to what is observed for S. aureus.

International audience; Microglial cells constitute the first line of defense of the central nervous system (CNS) against microbial invasion. Pathogens are detected thanks to an array of innate immune receptors termed pattern recognition receptors (PRRs). PRRs have been thoroughly characterized in bone marrow-derived macrophages, but the PRRs repertoire and functionality in microglial cells remain largely unknown. Microglial cells express various Toll-like Receptors and the Nod1/2 receptors. Recently, a novel innate immune signalling pathway, the inflammasome pathway has been uncovered. Inflammasome activation leads to caspase-1 activation, release of the proinflammatory cytokines, IL-1β and IL-18 and cell death in a process termed pyroptosis. One inflammasome receptor, NLRP3, has been characterized in microglial cells and associated with response to infections and in the initiation of neuro-degeneration in an Alzheimer's disease model. Legionella pneumophila (L.pneumophila) is a flagellated bacterium replicating within macrophages. In bone marrow-derived macrophages, L. pneumophila is detected in a flagellin-dependent manner by the Naip5-NLRC4 (Ipaf) inflammasome pathway. In this study, we decided to use L. pneumophila to investigate the presence and the functionality of this inflammasome in primary murine microglial cells. We show that microglial cells detect L. pneumophila infection in a flagellin-dependent manner leading to caspase-1-mediated bacterial growth restriction, infected cell death and secretion of the proinflammatory cytokines IL-1β and IL18. Overall, our data demonstrate that microglial cells have a functional Naip5-NLRC4 inflammasome likely to be important to monitor and clear CNS infections by flagellated bacteria.

Objectives: To examine the effect of subinhibitory concentrations (sub-MICs) of antistaphylococcal drugs on Panton-Valentine leucocidin (PVL), α-haemolysin (Hla) and protein A (SpA) expression by community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA). Methods: Five clinical isolates representing the main worldwide CA-MRSA clones were grown with sub-MICs (1/8, 1/4 and 1/2 MIC) of five antibiotics (clindamycin, daptomycin, linezolid, tigecycline and vancomycin). After 4 and 6 h of incubation, culture pellets were used for relative quantitative RT-PCR with primers specific for pvl, hla, spa and gyrB. The PVL, Hla and SpA concentrations were measured in the supernatant (for PVL and Hla) and in the cell pellet (for SpA) using specific ELISAs. Results: For all strains tested, clindamycin and linezolid dramatically reduced mRNA levels of PVL and SpA. Tigecycline also decreased the PVL and SpA mRNA levels of 3/5 and 4/5 strains tested, respectively, whereas daptomycin and vancomycin had no significant effect. PVL and SpA quantification confirmed the concentration-dependent inhibition of PVL and SpA production by clindamycin and, to a lesser extent, by linezolid and tigecycline. Only clindamycin decreased Hla mRNA expression, whereas linezolid, tigecycline and daptomycin showed heterogeneous strain-dependent results, and vancomycin had no significant effect. Analysis of the Hla level revealed a stronger concentration-dependent inhibition of Hla release by clindamycin than by linezolid. Conclusions: The effect of sub-MICs on virulence expression depended on the antibiotic and the virulence factor. Clindamycin and linezolid consistently suppressed the expression of different virulence factors by CA-MRSA, whereas tigecycline specifically suppressed PVL expression. Daptomycin and vancomycin seem to have no significant effects at these concentrations.

International audience; The 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.

International audience; Acquisition of nasal Staphylococcus aureus (S. aureus) colonization by contaminated hands is likely an important determinant of its nasal carriage rate in health care and lab setting. The objective of our cross-sectional study was to assess the prevalence of nasal methicillin-sensitive (MSSA) or -resistant Staphylococcus aureus (MRSA) carriage among health care professionals (HCPs) attending an international symposium and to study the association between compliance with hygiene rules, individual-related parameters, and medical conditions with nasal S. aureus carriage in this population. After obtaining consent, two nasal swabs were collected. Nasal MSSA and MRSA carriage was measured by the: i) molecular approach targeting spa, mecA and mecA-orfX junction sequences, and ii) culture on selective S. aureus media combined with mecA molecular detection of isolated strains. Information on compliance with hygiene rules, demographic variables, sector of activity and long-term medication was collected by anonymous questionnaire. The participation rate was 32.3%. In total, 176 subjects from 34 countries were included in the analysis. S. aureus was isolated from the nasal swabs of 57 (32.4%) subjects, of whom 3 (5.3%) harbored MRSA strains. Overall, 123 subjects reported working in microbiology laboratories with direct manipulation of S. aureus, and 29 acknowledged regular contacts with patients. In this exposed population, hydro-alcoholic solutions appeared to have a significant protective effect against nasal S. aureus carriage (OR?=?0.36; 95% CI: 0.15-0.85). Hospital work was associated with increased risk of nasal S. aureus carriage (OR?=?2.38; 95% CI: 1.07-5.29). The results of this study showed that compliance with basic rules of hygiene, such as the use of hydro-alcoholic solutions, could reduce the risk of nasal S. aureus colonization. Hydro-alcoholic solution could interrupt auto-transmission of the pathogen, consequently decreasing the overall nasal carriage rate, specifically in transient carriers.

International audience; BACKGROUND: Legionella is a water and soil bacterium that can infect humans, causing a pneumonia known as Legionnaires' disease. The pneumonia is almost exclusively caused by the species L. pneumophila, of which serogroup 1 is responsible for 90% of patients. Within serogroup 1, large differences in prevalence in clinical isolates have been described. A recent study, using a Dutch Legionella strain collection, identified five virulence associated markers. In our study, we verify whether these five Dutch markers can predict the patient or environmental origin of a French Legionella strain collection. In addition, we identify new potential virulence markers and verify whether these can predict better. A total of 219 French patient isolates and environmental strains were compared using a mixed-genome micro-array. The micro-array data were analysed to identify predictive markers, using a Random Forest algorithm combined with a logistic regression model. The sequences of the identified markers were compared with eleven known Legionella genomes, using BlastN and BlastX; the functionality for each of the predictive markers was checked in the literature. RESULTS: The five Dutch markers insufficiently predicted the patient or environmental origin of the French Legionella strains. Subsequent analyses identified four predictive markers for the French collection that were used for the logistic regression model. This model showed a negative predictive value of 91%. Three of the French markers differed from the Dutch markers, one showed considerable overlap and was found in one of the Legionella genomes (Lorraine strain). This marker encodes for a structural toxin protein RtxA, described for L. pneumophila as a factor involved in virulence and entry in both human cells and amoebae. CONCLUSIONS: The combination of a mixed-genome micro-array and statistical analysis using a Random Forest algorithm has identified virulence markers in a consistent way. The Lorraine strain and related Dutch and French Legionella strains contain a marker that encodes a RtxA protein which probably is involved in the increased prevalence in clinical isolates. The current set of predictive markers is insufficient to justify its use as a reliable test in the public health field in France. Our results suggest that genetic differences in Legionella strains exist between geographically distinct entities. It may be necessary to develop region-specific mixed-genome microarrays that are constantly adapted and updated.

International audience; Epidemic community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) is associated with more severe and acute forms of osteomyelitis than healthcare-associated (HA-) MRSA. Although S. aureus is now recognized as a facultative intracellular pathogen, the contribution of osteoblast invasion by CA-MRSA to the pathogenesis of osteomyelitis is unknown. Using an ex vivo model of intracellular infection of human osteoblasts, we demonstrated that CA-MRSA strains of diverse lineages share an enhanced ability to kill infected osteoblasts compared to HA-MRSA. Cytotoxicity comparisons of CA-MRSA isogenic deletion mutants revealed that phenol-soluble modulins (PSMs), a class of membrane-damaging exoproteins that are expressed at higher levels in CA-MRSA than in HA-MRSA, are involved in this osteoblast killing, whereas other major CA-MRSA virulence determinants, the Panton-Valentine leukocidin and alpha-toxin, are not involved. Similarly, functional agr and sarA regulators, which control the expression of PSMs and alpha-toxin, were required for the expression of the intracellular cytotoxic phenotype by CA-MRSA, whereas the saeRS regulator, which controls the expression of alpha-toxin but not PSMs, had no impact on cytotoxicity. Finally, PSM transcript levels determined by quantitative reverse-transcriptase PCR were significantly higher in CA-MRSA than in HA-MRSA strains and associated with cell damage in MRSA-infected osteoblasts. These findings provide new insights into the pathogenesis of severe CA-MRSA osteomyelitis and unravel a novel virulence strategy of CA-MRSA, based on the invasion and subsequent killing of osteoblasts by PSMs acting as intracellular toxins.

Tolerance of Staphylococcus lugdunensis to relatively high levels of cadmium is mediated by a 3117-bp plasmid, pLUG10. Sequencing reveals three major open reading frames (ORFs) in a single orientation. One ORF encompasses the origin of replication and its predicted product (RepL: 350 amino acids (aa)) shows 70% homology in its deduced aa sequence with Rep proteins of the pT181 family. A lagging strand conversion signal (palA) very similar to that of class 1 plasmids is present outside the rep-ori locus. The other two ORFs of 209 and 116 aa show 92.5% homology between their deduced aa sequences and the CadB and CadX peptides from the pOX6 plasmid of Staphylococcus aureus. The CadX-like peptide is 40% homologous to the S. aureus CadC product. Deletion of the C-terminal cadX gene by restriction enzyme digestion or frame-shift inactivation of the cadB gene reduced, but did not completely abolish, cadmium resistance. The two gene products may act cooperatively to confer cadmium resistance in S. lugdunensis.

In a consecutive series of patients with infective endocarditis, we compared the charts of 123 nonaddicted patients without previously known heart disease with those of 174 patients with native valve disease. The 2 groups were similar in age, sex, clinical findings, and mortality rates, but infective endocarditis was more often located on the aortic valve, more often due to Streptococcus bovis and enterococci in patients without previously known heart disease.

Resume Une activite bactericide a ete obtenue pour la cloxacilline, l'oxacilline, la gentamicine et la pefloxacine vis-a-vis de 5 souches de Staphylococcus aureus sensibles a la meticilline, en utilisant des concentrations d'antibiotiques correspondant a celles obtenues in vivo ; l'erythromycine, la pristinamycine et l'acide fusidique ne sont pas apparus comme bactericides. Aux concentrations obtenues par voie orale, la cloxacilline a ete la seule penicilline M bactericide en 6 heures sur les deux souches hyperproductrices de s-lactamases. L'etude de la bactericidie des associations avec la cloxacilline a mis en evidence soit des indifferences (avec la gentamicine et la pefloxacine), soit des antagonismes (avec l'acide fusidique).

Object To assess the relative risk of infective endocarditis associated with various procedures and the protective efficacy of antibiotic prophylaxis by a case-control study. Background Recommendations for the prevention of infective endocarditis are based on the hypothesis of a relationship between procedures and infective endocarditis which is supported by anecdotal reports and data from experimental models. Methods Cases met the Von Reyn's diagnostic criteria modified with echocardiographic and macroscopic findings, Controls were recruited from cardiology or medicinal wards. Cases (n=171) and controls were matched as regards sex, age and underlying cardiac condition, They were requested to indicate all the medical, surgical or dental procedures within the previous 3 months, Among potential confounding factors, infectious episodes and skin wounds in the previous 3 months were reported, Antibiotic prophylaxis administration was documented for type, dosage, duration and administration schedule. Results Cases significantly more frequently than controls had undergone at least one procedure (matched odds ratio, 1.6; 95% confidence interval, 1.01 to 2.53). Dental procedures considered as a whole were not associated with an increased risk, although scaling and root canal treatment showed a trend towards a higher risk of infective endocarditis ( P =0.065). Among non-dental procedures, only surgery appeared to be at risk (matched odds ratio, 4.7; 95% confidence interval, 1.02 to 22). Considering all procedures, the risk of infective endocarditis increased significantly with the number of procedures. While general co-morbid conditions did not differ between the two groups, cases significantly more frequently than controls had experienced an infectious episode or a skin wound In multivariate analysis, only infectious episodes and skin wounds significantly increased the risk of infective endocarditis. Scaling was the only independent risk factor for viridans streptococcal infective endocarditis. The 46% protective efficacy of antibiotic prophylaxis was not significant. Conclusions Procedures do increase the risk of infective endocarditis. The interpretation of the apparent low risk associated with dental procedures may be as a result of the current practice of antibiotic prophylaxis. Our data suggest that surgery should be more clearly mentioned in future guidelines, and reemphasize that a rigorous treatment of any focal infection in cardiac patients is mandatory. From the efficacy rate of antibiotic prophylaxis, it can be estimated that the overall incidence of infective endocarditis might be reduced by 5 to 10% in France by appropriate use of antibiotic prophylaxis in cardiac patients.

Fifty-three patients (42 men; 11 women) with Streptococcus bovis infective endocarditis attended a tertiary cardiology hospital between 1980 and 1991, and constituted 11% of the total number of infective endocarditis cases hospitalized there during that period. The mean age was 59 +/- 15 years; 15 had previously suffered valvular disease (12) or had a valvular prosthesis (3); one patient had had a previous infective endocarditis. The infective episode involved the aortic valve in 26 patients, both the aortic and mitral valves in 18 patients, the mitral valve only in six and other valves in three. Echocardiographic examination showed one or more vegetations in 44 patients. Cardiac failure was diagnosed in 35 patients and embolic episodes in 22, of whom 11 were cerebrovascular accidents. The patients became afebrile 19 +/- 39 days after starting antibiotic treatment. Valve replacement was performed in 37 patients during their initial hospitalization, and in four during follow-up. After a mean follow-up of 4.6 +/- 3.1 years with a 100% follow-up, 15 patients died: 1 preoperatively, one in the first 30 days after operation, 13 later (8141 operated patients and 5/12 non-operated patients). Actuarial survival was 73% at 5 years. Gastrointestinal signs were present in 12 patients; 43 patients (81%) had a full colonic examination which showed polyps in 20 patients and adenocarcinomas in seven. Of 11 late deaths four were related to a malignant colonic tumour. This study confirms that Streptococcus bovis infective endocarditis is 'relatively benign', but it stresses the frequency and potential severity of the associated colonic lesions, requiring colonoscopy and making the treatment of high risk lesions mandatory.

Bartonella (Rochalimaea) quintana is the agent of trench fever and is transmitted by the body louse. We searched for this organism in three alcoholic homeless men with endocarditis.Blood samples were cultured on a human endothelial cell line and on blood agar. Bacteria were identified by sequencing the amplified 16S ribosomal RNA gene. The presence of bartonella in tissue was assessed by Gram's staining, immunostaining, and polymerase-chain-reaction amplification. Serologic studies for antibodies to bartonella species were performed by indirect immunofluorescence and Western immunoblotting.B. quintana was isolated from one patient in the blood-agar culture and from the other two patients in the endothelial-cell culture. The organism was also identified by both immunostaining and molecular techniques in the valvular vegetations from the three patients and in a cervical lymph node from one patient. The 16S ribosomal RNA gene sequences of the three isolates were almost identical to that of the prototype strain of B. quintana. High titers of antibodies to B. quintana were detected in all three patients, but so were cross-reacting antibodies to chlamydia species. In all three patients studies were repeatedly negative for antibodies to the human immunodeficiency virus.B. quintana is a cause of endocarditis in homeless patients and may be serologically misdiagnosed as a chlamydial infection.

Resume • Objectif : Developper un moyen diagnostique pour reconnaitre l'origine bacterienne ou non bacterienne d'une meningite postoperatoire en neurochirurgie, en detectant dans le LCR l'ADN bacterien a l'aide de la technique de PCR (polymerase chain reaction). • Type d'etude : Etude de laboratoire. • Patients : Vingt-sept operes neurochirurgicaux traites en reanimation et satisfaisant aux criteres de meningite des CDC (Centers for Disease Control) et ayant plus de 100 polynucleaires neutrophiles par mm3 dans le LCR, ont ete repartis en deux groupes : le groupe MB+ (n = 7) en cas de culture de LCR positive ou le groupe MB− (n = 20) en cas de LCR sterile. Par ailleurs, le LCR de 43 patients neurochirurgicaux traites en reanimation, mais sans signes cliniques et biologiques de meningite, ont servi de controle. Seize echantillons sur les 43 ont ete ensemences avec des bacteries a des concentrations determinees. • Methodes : Dans l'ensemble des echantillons de LCR, l'ADN bacterien a ete recherche par amplification genique (PCR). Une etude prealable avait montre que la sensibilite de la PCR etait de 105 UFC·mL−1 pour 20 cycles et de 103 pour 25 cycles. • Resultats : Les 43 echantillons de LCR sterile, servant de controle, n'ont pas donne d'amplification en PCR dans tous les cas a 20 cycles et dans 42 cas sur 43 a 25 cycles. Les 16 echantillons controles qui avaient ete ensemences, ainsi que les 7 LCR du groupe MB+ avaient une amplification positive a 20 et 25 cycles. Les 20 LCR du groupe MB− n'avaient pas d'amplification a 20 cycles, mais celleci devenait positive a 25 cycles dans 19 cas sur 20. L'hybridation (southern blot) des produits d'amplification avec une amorce specifique des procaryotes a ete positive pour les LCR du groupe MB+ et du groupe MB− et negative avec les LCR controle et l'ADN humain. • Discussion : La presence de bacteries dans le LCR de patients atteints de meningite peut etre mise en evidence par l'intermediaire de la detection d'ADN bacterien. Les meningites aseptiques postoperatoires peuvent avoir une origine bacterienne. La PCR peut constituer une technique de routine permettant le diagnostic de meningite bacterienne en moins de 6 heures. De plus, le recours a des oligonucleotides specifiques permet d'identifier les bacteries en moins de 12 heures.

Background Descriptions of the epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) have seldom been produced in the Caribbean, which is a major tourism destination. Materials and Methods Using DNA microarrays and spa typing, we characterized 85 MRSA isolates from human skin and soft-tissue infections from five different islands. Results In the French West Indies (n = 72), the most frequently isolated clones were the same clones that are specifically isolated from mainland France [Lyon (n = 35) and Geraldine (n = 11) clones], whereas the clones that were most frequently isolated from the other islands (n = 13) corresponded with clones that have a worldwide endemic spread [Vienna/Hungarian/Brazilian (n = 5), Panton Valentine leukocidin-positive USA300 (n = 4), New York/Japan (n = 2), and pediatric (n = 1) clones]. Conclusion The distribution of the major MRSA clones in the French (Guadeloupe and Martinique) and non-French West Indies (Jamaica, Trinidad, and Tobago) is different, and the clones most closely resemble those found in the home countries of the travelers who visit the islands most frequently. The distribution might be affected by tourist migration, which is specific to each island.

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) can cause osteomyelitis with severe sepsis and/or local complications in which a Panton-Valentine leukocidin (PVL) role is suspected. In vitro sub-MIC antibiotic effects on growth and PVL production by 11 PVL + MRSA strains, including the major CA-MRSA clones (USA300, including the LAC strain; USA400; and USA1000), and 11 PVL + methicillin-susceptible S. aureus (MSSA) strains were tested in microplate culture. Time-kill analyses with ceftobiprole at its MIC were also run with LAC. Efficacies of ceftobiprole (40 mg/kg of body weight subcutaneously [s.c.] four times a day [q.i.d.]) or vancomycin (60 mg/kg intramuscularly [i.m.] twice a day [b.i.d.]) alone or combined with rifampin (10 mg/kg b.i.d.) against rabbit CA-MRSA osteomyelitis, induced by tibial injection of 3.4 × 10 7 CFU of LAC, were compared. Treatment, started 14 days postinoculation, lasted 14 days. In vitro , 6/11 strains cultured with sub-MICs of ceftobiprole produced 1.6- to 4.8-fold more PVL than did the controls, with no link to specific clones. Rifampin decreased PVL production by all tested strains. In time-kill analyses at the LAC MIC (0.75 mg/liter), PVL production rose transiently at 6 and 8 h and then declined 2-fold at 16 h, concomitant with a 2-log 10 -CFU-count decrease. In vivo , the mean log 10 CFU/g of bone for ceftobiprole (1.44 ± 0.40) was significantly lower than that for vancomycin (2.37 ± 1.22) ( P = 0.034), with 7/10 versus 5/11 bones sterilized, respectively. Combination with rifampin enhanced ceftobiprole (1.16 ± 0.04 CFU/g of bone [ P = 0.056], 11/11 sterile bones) and vancomycin (1.23 ± 0.06 CFU/g [ P = 0.011], 11/11 sterile bones) efficacies. Ceftobiprole bactericidal activity and the rifampin anti-PVL effect could play a role in these findings, which should be of interest for treating CA-MRSA osteomyelitis.

Currently, several methods are used for the detection of Legionella in clinical samples, and these methods constitute part of the criteria for defining legionellosis cases. Urinary antigen detection is the first-line diagnostic test, although this test is limited to L. pneumophila serogroup 1 (Lp1) (Helbig et al., J Clin Microbiol 41:838-840, 2003). The use of molecular techniques can improve Legionaire's disease (LD) diagnosis by detecting other serogroups and species (Diederen et al., J Clin Microbiol 46:671-677, 2008). The isolation of Legionella strains from pulmonary samples by axenic culture is still required to perform further epidemiological investigations (Blyth et al., N S W Public Health Bull 20:157-161, 2009; Fields et al., Clin Microbiol Rev 15:506-526, 2002) but demonstrates various sensitivities. Amoebic coculture has been described as a method to recover Legionella from clinical culture-negative specimens (La Scola et al., J Clin Microbiol 39:365-366, 2001; Rowbotham, J Clin Pathol 36:978-986, 1983) and can be proposed for optimizing Legionella strain isolation from samples contaminated by oropharyngeal flora. Identification of Legionella isolates is based on serological characterization, genotypic methods (with sequencing of the mip gene as the standard method) and, more recently, the Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) method.This chapter is limited to the identification of Legionella in clinical samples; antibody detection in human serum will not be discussed.

This thesis explores subglacial processes, glacier dynamics, and deglacial processes and patterns associated with the Cordilleran Ice Sheet (CIS) in Okanagan Valley and the neighbouring Thompson Plateau in southern British Columbia. Reconstructions of subglacial processes in an area of streamlined bedforms (drumlins) on Thompson Plateau reveal that sediments within drumlins and in intervening areas record evidence of lodgement, deformation, poreflow, conduit flow, debris flows, and suspension settling of fines within a network of subglacial cavities. These subglacial cavities developed within a regional bedrock basin. These sediments demonstrate that substrate deformation is not a dominant and pervasive process recorded within the drumlins. Based on i) drumlin morphology, including the presence of stoss-side crescentic troughs and en echelon arrangement, ii) drumlin composition consisting of sediment and bedrock, it is argued that drumlins on Thompson Plateau have an erosional origin. Further, regional spatial associations between drumlins and tunnel valleys (including some in bedrock) on Thompson Plateau and in Okanagan Valley, and arguments relying on the continuity of eroded sediments suggest that erosion by subglacial meltwater underburst(s) best explains the range of observations. Underbursts may have been associated with development and drainage of a subglacial ‘catch lake’ in Okanagan Valley. High geothermal heat flux in Okanagan Valley could have favoured subglacial lake development. Subglacial volcanic eruptions may have acted as triggers for lakedrainage. Lastly, deglaciation of the CIS led to development of a proglacial lake in Okanagan Valley (glacial Lake Penticton - gLP). Sediment delivery to gLP occurred via tributary valleys and possibly from an ice tongue along the valley axis. Regional delta correlations record a highstand of gLP at 500-525 m asl followed by a single drainage event which eroded a portion of the lacustrine valley fill to produce distinctive remnant valley-side benches (‘White Silt’ terraces) of lacustrine sediments. No evidence of glacioisostatic tilting could be discerned within the gLP basin, this is tentatively ascribed to lithospheric conditions favouring rapid crustal rebound, and to thin ice with uniform thickness in the gLP basin.

Currently, several methods are used for the detection of Legionella in clinical samples, and these methods constitute part of the criteria for defining legionellosis cases. Urinary antigen detection is the first-line diagnostic test, although this test is limited to L. pneumophila serogroup 1 (Lp1) (Helbig et al., J Clin Microbiol 41:838-840, 2003). The use of molecular techniques can improve Legionaire's disease (LD) diagnosis by detecting other serogroups and species (Diederen et al., J Clin Microbiol 46:671-677, 2008). The isolation of Legionella strains from pulmonary samples by axenic culture is still required to perform further epidemiological investigations (Blyth et al., N S W Public Health Bull 20:157-161, 2009; Fields et al., Clin Microbiol Rev 15:506-526, 2002) but demonstrates various sensitivities. Amoebic coculture has been described as a method to recover Legionella from clinical culture-negative specimens (La Scola et al., J Clin Microbiol 39:365-366, 2001; Rowbotham, J Clin Pathol 36:978-986, 1983) and can be proposed for optimizing Legionella strain isolation from samples contaminated by oropharyngeal flora. Identification of Legionella isolates is based on serological characterization, genotypic methods (with sequencing of the mip gene as the standard method) and, more recently, the Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) method.This chapter is limited to the identification of Legionella in clinical samples; antibody detection in human serum will not be discussed.

International audience; The aim of the present study was to detect the Staphylococcus aureus delta-toxin using Whole-Cell (WC) Matrix Assisted Laser Desorption Ionization - Time-of-Flight (MALDI-TOF) mass spectrometry (MS), correlate delta-toxin expression with accessory gene regulator (agr) status, and assess the prevalence of agr deficiency in clinical isolates with and without resistance to methicillin and glycopeptides. The position of the delta-toxin peak in the mass spectrum was identified using purified delta-toxin and isogenic wild type and mutant strains for agr-rnaIII, which encodes delta-toxin. Correlation between delta-toxin production and agr RNAIII expression was assessed by northern blotting. A series of 168 consecutive clinical isolates and 23 unrelated glycopeptide-intermediate S. aureus strains (GISA/heterogeneous GISA) were then tested by WC-MALDI-TOF MS. The delta-toxin peak was detected at 3005 +/- 5 Thomson, as expected for the naturally formylated delta toxin, or at 3035 +/- 5 Thomson for its G10S variant. Multivariate analysis showed that chronicity of S. aureus infection and glycopeptide resistance were significantly associated with delta-toxin deficiency (p = 0.048; CI 95%: 1.01-10.24; p = 0.023; CI 95%: 1.20-12.76, respectively). In conclusion, the S. aureus delta-toxin was identified in the WC-MALDI-TOF MS spectrum generated during routine identification procedures. Consequently, agr status can potentially predict infectious complications and rationalise application of novel virulence factor-based therapies.

A macrolide antimicrobial drug was administered to a newborn with cough. On day 23 of hospitalization, macrolide-resistant Bordetella pertussis was isolated from nasopharyngeal aspirates. DNA sequencing and PCR–restriction fragment length polymorphism showed a 2047 A-to-G mutation in the 3 copies of the 23S rRNA gene. Monitoring for macrolide resistance is essential in infants

Toxic shock syndrome (TSS) and septic shock (SS) share many clinical signs of an exacerbated inflammatory response. In this report, we investigated whether TSS presents similar features of delayed immunosuppression as described in SS. Five children with TSS from paediatric intensive care units in a university hospital were monitored. TSS cases were defined by the association of standardized clinical signs of TSS and confirmed by measurement of specific Vbeta expansions corresponding to toxin gene profile of the isolated strains. As in SS, an increased percentage of circulating regulatory T cells (Treg) was observed in patients with TSS. However, in contrast to SS, neither lymphopenia nor decreased HLA-DR expression on monocytes was measured. In conclusion, whereas SS and TSS exhibited similar clinical presentation, the present observation suggests that respective pathophysiological mechanisms induce different immune alterations. Future studies must isolate and better characterize the phenotypic and functional properties of Treg subsets during TSS to understand the mechanisms sustaining their increase, especially the putative role of superantigens.

Background Diseases from Staphylococcus aureus are a major problem in Indian hospitals and recent studies point to infiltration of community associated methicillin resistant S. aureus (CA-MRSA) into hospitals. Although CA-MRSA are genetically different from nosocomial MRSA, the distinction between the two groups is blurring as CA-MRSA are showing multidrug resistance and are endemic in many hospitals. Our survey of samples collected from Indian hospitals between 2004 and 2006 had shown mainly hospital associated methicillin resistant Staphylococcus aureus (HA-MRSA) carrying staphylococcal cassette chromosome mec (SCCmec) type III and IIIA. But S. aureus isolates collected from 2007 onwards from community and hospital settings in India have shown SCCmec type IV and V cassettes while several variations of type IV SCCmec cassettes from IVa to IVj have been found in other parts of the world. In the present study, we have collected nasal swabs from rural and urban healthy carriers and pus, blood etc from in patients from hospitals to study the distribution of SCCmec elements and sequence types (STs) in the community and hospital environment. We performed molecular characterization of all the isolates to determine their lineage and microarray of select isolates from each sequence type to analyze their toxins, virulence and immune-evasion factors. Results Molecular analyses of 68 S. aureus isolates from in and around Bengaluru and three other Indian cities have been carried out. The chosen isolates fall into fifteen STs with all major clonal complexes (CC) present along with some minor ones. The dominant MRSA clones are ST22 and ST772 among healthy carriers and patients. We are reporting three novel clones, two methicillin sensitive S. aureus (MSSA) isolates belonging to ST291 (related to ST398 which is live stock associated), and two MRSA clones, ST1208 (CC8), and ST672 as emerging clones in this study for the first time. Sixty nine percent of isolates carry Panton- Valentine Leucocidin genes (PVL) along with many other toxins. There is more diversity of STs among methicillin sensitive S. aureus than resistant ones. Microarray analysis of isolates belonging to different STs gives an insight into major toxins, virulence factors, adhesion and immune evasion factors present among the isolates in various parts of India. Conclusions S. aureus isolates reported in this study belong to a highly diverse group of STs and CC and we are reporting several new STs which have not been reported earlier along with factors influencing virulence and host pathogen interactions.

Cardiac surgery enters mainly into the class I of Altemeier (« clean surgery ). However, many factors may explain an intraoperative contamination : surgery of long duration, extra-corporeal circulation, aspiration of blood and air, immunodepression...). In fact, the infectious risk decreases from about 25 % with placebo to 5 % with prophylactic antibiotics. The staphylococcal infections are the most frequent (mediastinitis, endocarditis, parietal infections...). Cephalosporins, particularly of second-generation type (cefamandole, cefuroxime), perform better than antistaphylococcal penicillins. The combination with an aminoside may be used when Gram negative bacilli infection prevalence is high. Vancomycin is efficient but hypotension and renal impairment have been reported. Therefore, vancomycin is used in patients allergic to cephalosporins, when a high prevalence of methicillin-resistant Staphylococcus or enterococci infections is reported, or when the patient has recently received broad-spectrum antimicrobial therapy. The antibiotic doses must take into account the haemodilution due to extracorporeal circulation and the necessity to obtain sufficient serum concentrations throughout surgery. A prophylaxis of more than 48 hours is not associated with an improved outcome. In cardiac transplantation a prophylaxis is essential, but is still questionned during the insertion of pace-markers. In any case, the antibiotic prophylaxis must take into account the bacterial prevalence of each institution.

Twelve 16S-23S ribosomal DNA intergenic spacer (ITS-PCR) types were identified among 57 Staphylococcus intermedius isolates from humans and other animals. Six ITS-PCR types were host specific, and most human and canine strains belonged to the same types (A and J). Pigeon, horse, and mink strains appeared more heterogeneous.

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

A PMA (propidium monoazide) pretreatment protocol, in which PMA is applied directly to membrane filters, was developed for the PCR-based quantification (PMA-qPCR) of viable Legionella pneumophila. Using this method, the amplification of DNA from membrane-damaged L. pneumophila was strongly inhibited for samples containing a small number of dead bacteria.

BackgroundCoagulase-negative staphylococci, mainly Staphylococcus epidermidis, are the most frequent cause of late-onset sepsis (LOS) in the neonatal intensive care unit (NICU) setting. However, recent reports indicate that methicillin-resistant, vancomycin-heteroresistant Staphylococcus capitis could emerge as a significant pathogen in the NICU. We investigated the prevalence, clonality and vancomycin susceptibility of S. capitis isolated from the blood of NICU infants and compared these data to adult patients.Methodology/principal findingsWe conducted a retrospective laboratory-based survey of positive blood cultures in NICU infants ≥ 3 days of age (n = 527) and in adult ICU patients ≥ 18 years of age (n = 1473) who were hospitalized from 2004 to 2009 in two hospital centers in Lyon, France. S. capitis was the most frequent pathogen in NICU infants, ahead of S. epidermidis (39.1% vs. 23.5% of positive blood cultures, respectively). Conversely, S. capitis was rarely found in adult ICU patients (1.0%) compared to S. epidermidis (15.3%). S. capitis bloodstream isolates were more frequently resistant to methicillin when collected from NICU infants than from adult patients (95.6% vs. 53.3%, respectively). Furthermore, we collected and characterized 53 S. capitis bloodstream isolates from NICU infants and adult patients from six distant cities. All methicillin-resistant S. capitis isolates from NICU infants were clonally related as determined by pulsed-field gel electrophoresis. These isolates harbored a type V-related staphylococcal chromosomal cassette mec element, and constantly showed either vancomycin resistance (37.5%) or heteroresistance (62.5%). Conversely, the isolates that were collected outside of the NICU were genetically diverse and displayed much lower rates of vancomycin resistance and heteroresistance (7.7% and 23.1%, respectively).Conclusions/significanceA clonal population of methicillin-resistant S. capitis strains has spread into several French NICUs. These isolates exhibit reduced susceptibility to vancomycin, which is the most widely used antimicrobial agent in the NICU setting.

Panton–Valentine leukocidin (PVL)-producing Staphylococcus aureus is associated with a broad spectrum of diseases, ranging from common uncomplicated soft tissue infections to severe diseases such as complicated soft tissue infections, extensive bone and joint infections, and necrotising pneumonia. Specialised management of infection based on the presence of PVL may not be required for mild infections, whereas it could be lifesaving in other settings. Moreover, most severe PVL diseases are recently identified entities and a ‘gold standard’ treatment from comparatives studies of different therapeutic options is lacking. Thus, recommendations are based on expert opinions, which are elaborated based on theory, in vitro data and analogies with other toxin-mediated diseases. In this review, we consider the potential need for specialised PVL-based management and, if required, which tools should be used to achieve optimal management.

International audience; The epidemiology of methicillin-susceptible Staphylococcus aureus (MSSA) in Africa is poorly documented. From January 2007 to March 2008, 555 S. aureus isolates were collected from five African towns in Cameroon, Madagascar, Morocco, Niger, and Senegal; among these, 456 unique isolates were susceptible to methicillin. Approximately 50% of the MSSA isolates from each different participating centre were randomly selected for further molecular analysis. Of the 228 isolates investigated, 132 (58%) belonged to five major multilocus sequence typing (MLST) clonal complexes (CCs) (CC1, CC15, CC30, CC121 and CC152) that were not related to any successful methicillin-resistant S. aureus (MRSA) clones previously identified in the same study population. The luk-PV genes encoding Panton-Valentine leukocidin (PVL), present in 130 isolates overall (57%), were highly prevalent in isolates from Cameroon, Niger, and Senegal (West and Central Africa). This finding is of major concern, with regard to both a source of severe infections and a potential reservoir for PVL genes. This overrepresentation of PVL in MSSA could lead to the emergence and spread of successful, highly virulent PVL-positive MRSA clones, a phenomenon that has already started in Africa.

Staphylococcal species, notably, coagulase-negative staphylococci (CoNS), are frequently misidentified using phenotypic methods. The partial nucleotide sequences of the tuf and gap genes were determined in 47 reference strains to assess their suitability, practicability, and discriminatory power as target molecules for staphylococcal identification. The partial tuf gene sequence was selected and further assessed with a collection of 186 strains, including 35 species and subspecies. Then, to evaluate the efficacy of this genotyping method versus the technology of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS), the 186 strains were identified using MALDI-TOF-MS (Axima® Shimadzu) coupled to the SARAMIS® database (AnagnosTec). The French National Reference Center for Staphylococci identification method was used as a reference. One hundred and eighty-four strains (98.9%) were correctly identified by tuf gene sequencing. Only one strain was misidentified and one was unidentified. MALDI-TOF-MS identified correctly 138 isolates (74.2%). Four strains were misidentified, 39 were unidentified, five were identified at the group (hominis/warneri) level, and one strain was identified at the genus level. These results confirm the value of MALDI-TOF-MS identification for common species in clinical laboratory practice and the value of the partial tuf gene sequence for the identification of all staphylococcal species as required in a reference laboratory.

Flow cytometry is a powerful tool for analyzing the adhesion to and invasion of Staphylococcus aureus (S. aureus) to eukaryotic cells. Established techniques have used bacteria that have been genetically modified to express fluorescent proteins or directly labeled with fluorochromes prior to infection. Such approaches are appropriate in most cases; however, the use of genetically or chemically altered bacteria could introduce a bias when measuring fine differences in adhesion and invasiveness. Here, we describe a combined flow cytometry-based invasion and adhesion assay that does not require the processing of bacteria prior to internalization. This method was performed on osteoblastic MG-63 cells infected with S. aureus reference strain 8325-4 and its invasion-deficient isogenic mutant, which carries deletions in the genes encoding fibronectin-binding proteins A and B. The data from this assay were compared to those obtained using the standard gentamicin protection assay. The results obtained by the two methods were consistent. Moreover, quantification of internalized bacteria was more reproducible using the flow cytometry-based assay than the gentamicin protection assay, which allowed for the simultaneous quantification of host cell adhesion and invasion.

Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) isolates are widespread in many countries, with varying distribution and epidemiology. The aim of this study was to collect and characterise the CA-MRSA isolates circulating in Italy, since only some case reports have been published. Eighteen Panton-Valentine-positive CA-MRSA isolates were collected from different Italian hospitals during the period 2005-2009 from severe infections (skin and soft tissue infections, n = 10; necrotising pneumonia, n = 7; and sepsis, n = 1). Accessory gene regulator (agr) typing, staphylococcal cassette chromosome (SCC) mec typing, spa typing, multi-locus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE) and DNA microarray were applied to categorise isolates into clones and to compare the relevant genetic features of each clone. Six different clones were identified, the most common (7 out of 18 isolates, 38.8%) being agrI/ST8/SCCmecIV, corresponding to the USA300 clone. Six out of the seven USA300 isolates did not harbour the arginine catabolic mobile element (ACME). Four strains (22.2%) were agrIII/ST80/SCCmecIV, corresponding to the European clone. Two of the other clones, namely, agrIII/ST88/SCCmecV and agrIII/ST772/SCCmecV, corresponded to CA-MRSA clones rarely found in other countries and probably originating from Africa or the Indian subcontinent. The results of microarray hybridisations showed that the distribution of resistance genes and other virulence factors was specific to each clone. Some characteristics could be exploited as specific markers for a clone or a group of isolates, e.g. the mer operon, recovered only in ACME-negative USA300 strains. DNA microarray contributed to a more complete description of the variety of different CA-MRSA clones circulating in Italy.

Sequences related to the Staphylococcus aureus accessory gene regulator ( agr ) were demonstrated in S. lugdunensis by Southern blot analysis of 13 strains and sequencing of the S. lugdunensis agr -like locus ( agr-sl ). Northern blot analysis of cellular RNA revealed the presence of a transcript having homology with the agr -P3 transcript (RNAIII) for three of the six strains tested. The three strains containing this transcript produce a hemolysin with phenotypic properties similar to that of S. aureus δ-hemolysin. Nevertheless, unlike agr -P3 from S. aureus, agr-sl does not encode any potential peptides homologous to S. aureus δ-hemolysin, suggesting that the hemolytic activity detected in S. lugdunensis is encoded elsewhere and may be controlled by agr-sl .

DNA analysis using pulsed-field gel electrophoresis (PFGE) has emerged as one of the most sensitive epidemiological tools for the characterization of coagulase-negative staphylococci (CNST). The significance of some minor differences observed between the DNA restriction pulsed patterns of two CNST strains are difficult to interpret since they can theoretically be due to minor chromosomal rearrangements or to phage DNA integration. The latter possibility was investigated by comparing DNA restriction patterns of Staphylococcus epidermidis strains with those of their lysogenized derivatives. In vitro lysogenisation was obtained by exposing the strains to phage 118II. The pulsed patterns of the lysogenized strains were compared to those of their parental strains, revealing a shift in size of approximately 50 kb in a single band which was shown by Southern blotting to contain prophage. One strain was lysogenized ten times, revealing a potential preferred attachment site for phage 118II. These results confirm that chromosomal integration of a phage can be responsible for minor stable variations in DNA restriction patterns.

Introduction L’esmolol est un beta-bloquant de courte duree d’action. Une etude a montre un benefice sur la survie chez des patients en choc septique traite par esmolol [1] . L’effet microcirculatoire precoce est inconnu. Le but de notre etude est d’evaluer l’effet de l’esmolol sur la microcirculation sublinguale et digestive sur un modele porcin de sepsis. Materiel et methodes Douze porcelets ont ete anesthesies et ventiles mecaniquement. Apres une periode temoin (t0), ils ont recu a t1 une suspension de souches vivantes de Pseudomonas aeruginosa. A partir de t5, le groupe E (6 porcelets) a recu une perfusion d’esmolol (Frequence cardiaque (FC) cible Resultats L’esmolol a permis une baisse significative de la FC (En t11 FC groupe E = 89 (84–91) vs. FC groupe C = 119 (108 ; 130), p = 0,004). Aucune difference significative entre les 2 groupes n’a ete notee pour les volumes de fluides, les doses de noradrenaline, l’IC. L’hetDIG a augmente, le MFISL ( Fig. 1 ) et la PPVSL ont baissee de maniere significative dans le groupe C contrairement au groupe E. La PPVSL a differe significativement entre les 2 groupes en t11. Pour les autres parametres (MFIDIG, hetSL) une alteration plus marquee non significative a ete notee dans le groupe C. Le transport en oxygene (DO2) et la lactatemie etait inchange dans les 2 groupes, mais la consommation en oxygene (VO2) et l’extraction en oxygene (EO2) a augmente dans le groupe E ( Tableau 1 ). Discussion L’effet macrocirculatoire est coherent avec la litterature. L’esmolol, malgre la baisse de FC, n’a pas entraine d’alteration microcirculatoire, au contraire, cette derniere semble mieux preservee dans le groupe. On observe une augmentation de l’extraction en oxygene et une augmentation de la VO2 dans le groupe esmolol, peut-etre via un recrutement microcirculatoire dont le mecanisme reste a determiner.

Background. Panton-Valentine leukocidin (PVL)–positive methicillin-susceptible Staphylococcus aureus and methicillin-resistant S. aureus (MSSA and MRSA, respectively) are both associated with severe infections, such as necrotizing pneumonia. The epidemiological profile of PVL-positive community-acquired (CA) MRSA has been extensively studied, but few corresponding data on PVL-positive MSSA are available. Objectives. The objectives of the study were to investigate the global population structure of PVL-positive MSSA, to compare it with that reported for CA-MRSA, and thus to examine the phylogenetic relationship between these pathogens. Methods. We determined the agr types, multilocus sequence types, and toxin gene profiles of 211 PVL-positive MSSA clinical isolates collected in 19 countries throughout the world between 1981 and 2007. Results. The predominant lineages of PVL-positive MSSA were agr3/ST30, agr4/ST121, agr3/ST1, agr2/ST5, and agr3/ST80. Except for agr4/ST121, these lineages are also reported to be prevalent among CA-MRSA. PVLpositive MSSA lineages that are genetically related to CA-MRSA have gradually replaced other lineages (especially agr4/ST121) over the past 2 decades. Within a given sequence type, the toxin gene content of PVL-positive MSSA strains was very similar to that of PVL-positive CA-MRSA. Conclusions. The molecular epidemiological profiles of PVL-positive MSSA and CA-MRSA are dynamically interrelated, with the former appearing to constitute a reservoir for the latter.

Staphylococcus aureus strains producing Panton-Valentine leukocidin (PVL) have been epidemiologically linked to specific human infections. To evaluate immunological tests that may be used to diagnose infections with PVL-producing strains, we prospectively collected pus, respiratory tract specimens, and joint fluid specimens from which S. aureus had been isolated in clinical laboratories in six countries. An enzyme-linked immunosorbent assay (ELISA) and an immunochromatographic test (ICT) targeting LukS-PV were performed directly with clinical samples for the detection of PVL. The same tests were applied to S. aureus culture supernatants. The corresponding S. aureus isolates were characterized by PCR for the presence of the PVL locus ( lukS-PV and lukF-PV ) and the mec A gene. A total of 185 samples from 144 skin infections, 23 bone and joint infections, and 18 lower respiratory tract infections were analyzed. By PCR, 72/185 S. aureus isolates were PVL locus positive (PVL + ); 28 of these were also mecA positive. PVL was detected in the supernatants of all PVL + strains by both ELISA and an ICT, while no signal was observed with PVL-negative strains. The PVL concentrations in human clinical samples that grew PVL + strains ranged from 0 to 399 μg/ml by ELISA. By the use of 0.015 μg/ml of PVL as a cutoff value, PVL was detected in 65/72 (90%) of the clinical samples by ELISA. The sensitivity and specificity of the ELISA test were 90% and 100%, respectively. By the ICT, PVL was detected in 57/72 (79%) of the samples, and the sensitivity and specificity of ICT were 79% and 100%, respectively. PVL is expressed by S. aureus during human infection, and a PVL-specific ELISA and ICT could be reliable tests for the diagnosis of infections caused by PVL-producing strains.

Three patients with extensive necrotizing pneumonia due to Panton-Valentine leukocidin-positive Staphylococcus aureus strains and with aggravating factors (leukopenia count of less than 3 × 10 9 /liter in all three cases and hemoptysis in two cases) were successfully treated with toxin-suppressing agents introduced rapidly after hospital admission.

The glacial landscape of the Dobrzyn Plateau in central Poland contains a complex suite of enigmatic bedforms consisting of elongate and sinuous sediment ridges occurring within an anabranched network of tunnel channels terminating at a former ice margin marked by extensive glaciofluvial fans. The term “glacial curvilineations” is proposed to describe these enigmatic bedforms that occur as fields of parallel, sinuous ridges separated by troughs. The regional pattern of glacial curvilineations replicates the morphology and pattern of tunnel channel margins and suggests a common genesis. Tunnel channels and glacial curvilineations are eroded in the interbedded diamictons and glaciofluvial sediments of the plateau. We propose that tunnel channels and glacial curvilineations are the products of subglacial meltwater erosion. Curvilineations are erosional remnants produced by longitudinal vortices within the tunnel channel-forming flows. Parallelism of curvilineations reflects spacing of turbulent structures within the subglacial flow. Tunnel channel margins also control the sinuosity of curvilineations by dictating both the path and curvature of longitudinal vortices. Arguments for meltwater erosion are strengthened by the presence of extensive glaciofluvial fans at tunnel channel termini: they are the depositional counterparts to the extensive subglacial fluvial dissection of the plateau. Understanding curvilineation genesis may shed light on glacier hydrologic processes. Tunnel channels develop abruptly in the plateau surface suggesting localized point sources of meltwater, such as drainage of subglacial and/or supraglacial lakes. The extent of tunnel channels and lateral continuity of glacial curvilineations suggests broad sheet-like flows as wide as a few kilometres.

Legionella pneumophila and L. longbeachae are two species of a large genus of bacteria that are ubiquitous in nature. L. pneumophila is mainly found in natural and artificial water circuits while L. longbeachae is mainly present in soil. Under the appropriate conditions both species are human pathogens, capable of causing a severe form of pneumonia termed Legionnaires' disease. Here we report the sequencing and analysis of four L. longbeachae genomes, one complete genome sequence of L. longbeachae strain NSW150 serogroup (Sg) 1, and three draft genome sequences another belonging to Sg1 and two to Sg2. The genome organization and gene content of the four L. longbeachae genomes are highly conserved, indicating strong pressure for niche adaptation. Analysis and comparison of L. longbeachae strain NSW150 with L. pneumophila revealed common but also unexpected features specific to this pathogen. The interaction with host cells shows distinct features from L. pneumophila, as L. longbeachae possesses a unique repertoire of putative Dot/Icm type IV secretion system substrates, eukaryotic-like and eukaryotic domain proteins, and encodes additional secretion systems. However, analysis of the ability of a dotA mutant of L. longbeachae NSW150 to replicate in the Acanthamoeba castellanii and in a mouse lung infection model showed that the Dot/Icm type IV secretion system is also essential for the virulence of L. longbeachae. In contrast to L. pneumophila, L. longbeachae does not encode flagella, thereby providing a possible explanation for differences in mouse susceptibility to infection between the two pathogens. Furthermore, transcriptome analysis revealed that L. longbeachae has a less pronounced biphasic life cycle as compared to L. pneumophila, and genome analysis and electron microscopy suggested that L. longbeachae is encapsulated. These species-specific differences may account for the different environmental niches and disease epidemiology of these two Legionella species., Author Summary Legionella longbeachae, found in potting soil, and L. pneumophila, present in aquatic environments, are opportunistic human pathogens that cause Legionnaires' disease, a severe and often fatal pneumonia. The analysis and comparison of the genome sequences of four L. longbeachae genomes together with the study of its gene expression program and virulence pattern in different infection models provides important new insight on the organism's lifestyle and virulence strategies. L. longbeachae harbors a unique repertoire of secreted substrates, many of which encode eukaryotic like domains that may help the pathogen to subvert host functions and cause disease. Curiously, L. longbeachae may also be able to interact with plants. Several proteins present mainly in plants and phytopathogenic bacteria and several enzymes that might confer the ability to degrade plant material were identified in its genome. Interestingly, L. longbeachae encodes a chemotaxis system but no flagella, in contrast L. pneumophila encodes flagella but no chemotaxis system. It will be an interesting aspect of future research to understand these peculiarities. Finally, the genome sequence and analysis reported here will aid in understanding how L. longbeachae causes disease and will open new possibilities to develop tools for rapid identification and risk prediction of L. longbeachae infection.

Resume Une enquete visant a decrire l'incidence et les caracteristiques demographiques, cliniques, bacteriologiques, et evolutives de l'endocardite infectieuse en France a ete conduite entre le 1er novembre 1990 et le 31 octobre 1991, de facon exhaustive, dans trois regions francaises — Ile de France, Lorraine, et Rhone-Alpes —. Selon les criteres de Von Reyn modifies par l'echocardiographie, 386 cas d'EI certaine, probable, ou possible ont ete retenus, ainsi que 24 cas d'EI survenues moins d'un an apres la pose d'une prothese valvulaire. L'incidence etait de 23,6 cas / million d'habitants, plus elevee chez les hommes — 31,9 cas / million d'hommes — que chez les femmes — 15,7 cas / million de femmes. L'âge etait de 56,3 ± 19,0 ans; 65 % etaient des hommes; 57 % provenaient de la region Ile de France, 13 % de la region Lorraine, 30 % de la region Rhone-Alpes; 31 % etaient considerees comme certaines, 53 % comme probables, et 16 % comme possibles. Le cœur etait presume sain dans 36,5 % des cas; 10,9 % des patients etaient porteurs d'une cardiopathie non valvulaire, 52,6% d'une atteinte valvulaire, pour laquelle 65 sujets (16,8 % du total) ont recu une ou plusieurs protheses valvulaires. Le siege de l'EI etait mitral dans 39 %, aortique dans 37 %, mitro-aortique dans 13 %, tricuspide dans 6 %. Le micro-organisme a ete retrouve dans 89,0 %. Il s'agissait dans 20,6 % des cas d'un staphylocoque, Staphylococcus aureus dans plus de trois quarts des cas, et dans 58,7 % d'un streptocoque, surtout non groupable (48 % des streptocoques), ou du groupe D (42 % des streptocoques). La porte d'entree fut retrouvee ou presumee chez 66,9 %, dentaire (26,0 %), digestive (12,7 %), iatrogene (9,1 %), cutanee (5,0 %), urinaire (3,9 %). Dans 19 cas (5,2 %), il s'agissait d'un toxicomane. Sur le plan evolutif, 16,7 % ont ete operes au cours du premier mois, 24,7 % au cours des 2 premiers mois; 12,2 % sont decedes au cours du premier mois, 16,1 % au cours des 2 premiers mois.