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International audience; Objectives - Time to blood culture positivity (TTP), a routinely available parameter in automated blood culture systems, may be a proxy for infectious burden in patients with bloodstream infections. We aimed to study the association between TTP and infective endocarditis (IE), or death, in patients with Staphylococcus aureus bacteraemia. Methods - VIRSTA is a multicenter prospective cohort study that included all adult patients with S. aureus bacteraemia in eight university hospitals in France (2009-2011). We analyzed data from four centers which collected data on TTP. Regression models were used to study the association between TTP and definite IE (Duke-Li criteria), and 30 day-mortality. Results - We included 587 patients with S. aureus bacteraemia: mean age was 65.3±16.3 years, 420/587 patients (71.6%) were male, 121/587 (20.6%) died, and 42/587 (7.2%) had definite IE. Median TTP of first positive blood culture was 13.7 h (interquartile range, 9.9-18). On multivariate analysis, 30-day mortality was associated with TTP≤13.7 h (74/295 (25.1%) vs 47/292 (16.1%), P=0.02), as well as old age, McCabe score, methicillin resistance, stroke, pneumonia, and C-Reactive Protein. TTP was also independently associated with IE, but with a U-shape curve: IE was more common in the first (TTP18 h, 8/146, 5.5%) quartiles of TTP, P=0.002. Conclusions - TTP provides reliable information in patients with S. aureus bacteraemia, on the risk of IE, and prognosis, with short TTP being an independent predictor of death. This data readily available at no cost may be used to identify patients who require specific attention.

Background The French government has set up a community-based learning programme on health promotion for undergraduate health students to involve them in key public health objectives. At the University of Lyon, students first underwent formal instruction, including e-learning, lectures, and interactive seminars, and then became health educators for school pupils. The main objective of the present study was to assess the process of implementing this programme during the 2018–2019 academic year. Methods The satisfaction and perception of medical and midwife students with community-based learning experiences were assessed by a questionnaire, semi-directive interviews, and observations. Replies to the questionnaire were described by median and interquartile range or by proportion. A paired Wilcoxon-Mann–Whitney test was used to compare self-evaluated students’ competence scores before and after the seminars (alpha risk of 5%). Thematic analyses using grounded theory were performed on recorded and transcribed interviews, and on transcribed notes taken during the observations. Results Over time the students have evolved from a negative perception of the community-based learning to a positive one. The students were mostly satisfied by interactive seminars that allowed them to gain confidence and competencies in health education. Their involvement in the programme increased their self-esteem. They became more aware of their educative responsibilities regarding public health issues as future professionals. Conclusions The students had a positive perception of the implementation of a community-based learning programme in our University, as it appeared a pertinent strategy to raise their awareness of prevention and health education issues.

Background. The French government set up a community-based service learning programme on health promotion for undergraduate health students to involve them in key public health objectives. At the University of Lyon, students first underwent formal instruction, including e-learning, lectures, and interactive seminars, and then became health educators for school pupils. The main objective of the present study was to assess the process of implementing this programme during the 2018-2019 academic year.Methods. Satisfaction and perception of medical and midwife students with service learning experiences were assessed by a questionnaire, semi-directive interviews, and observations.Results. Over time the students evolved from a negative perception of service learning to a positive one. Students were mostly satisfied by interactive seminars that allowed them to gain confidence and competence in health education. They felt valued by taking part in this service learning. They became more aware of their educative responsibilities on public health issues as future professionals.Conclusions. Students had a positive perception of the implementation of a community-based service learning programme in our University, as it seems a good way to raise their awareness of prevention and health education issues.

PurposeStaphylococcus aureus causes severe forms of community-acquired pneumonia (CAP), namely staphylococcal pleuropneumonia in young children and staphylococcal necrotising pneumonia in older patients. Methicillin resistance and the Panton–Valentine leukocidin (PVL) toxin, as well as less specific factors, have been associated with poor outcome in severe CAP, but their roles are unclear.MethodsA prospective multicentre cohort study of severe staphylococcal CAP was conducted in 77 paediatric and adult intensive care units in France between January 2011 and December 2016. After age-clustering, risk factors for mortality, including pre-existing conditions, clinical presentation, laboratory features, strain genetic lineage, PVL, other virulence factors and methicillin resistance were assessed using univariate and multivariable Cox and LASSO (least absolute shrinkage and selection operator) regressions.ResultsOut of 163 included patients, aged 1 month to 87 years, 85 (52.1%) had PVL-positive CAP; there were 20 (12.3%) patients aged S. aureus lineages and the presence of other determinants of virulence.ConclusionPVL was associated with staphylococcal pleuropneumonia in toddlers and was a risk factor for mortality in older patients with severe CAP, independently of methicillin resistance, S. aureus genetic background and other virulence factors.

BackgroundStaphylococcus aureus causes severe forms of community-acquired pneumonia (CAP), namely staphylococcal pleuropneumonia in young children and staphylococcal necrotizing pneumonia in older patients. Methicillin resistance and the Panton-Valentine leukocidin (PVL) toxin have both been associated with poor outcome in severe CAP, but their respective roles are unclear.MethodsProspective multicenter cohort study of severe staphylococcal CAP conducted in 77 pediatric and adult intensive care units in France between January 2011 and December 2016. Clinical features and outcomes were compared between toddlers (ResultsOf 163 included patients, aged one month to 87 years, 85 (52.1%) had PVL-positive CAP; there were 20 (12.3%) toddlers, among whom 19 (95%) had PVL-positive CAP. The features of PVL-positive CAP in toddlers matched with the historical description of staphylococcal pleuropneumonia, with a lower mortality (n = 3/19, 15%) compared to PVL-positive CAP in older patients (n=31/66, 47%). Mortality in older patients was independently predicted by PVL-positivity (hazard ratio 1.81, 95% CI, 1.03 to 3.17) and methicillin resistance (2.37, 95% CI 1.29 to 4.34). As genetic diversity was comparably high in PVL-positive and PVL-negative isolates, confounding by microbial population structure was unlikely.ConclusionPVL was associated with staphylococcal pleuropneumonia in toddlers and was a risk factor for mortality in older patients with severe CAP, independently of methicillin resistance. Funded by the French ministry of Health (PHRC 2010-A01132-37).

Background: The first characterisation of Panton-Valentine Leucocidin (PVL)-associated staphylococcal necrotizing pneumonia (SNP) was published in 2002. A major controversy regarding the role played by PVL in the severity of community-acquired pneumonia (CAP) ensued. To address this issue, all French intensive care units (ICU) were asked to prospectively include their cases of staphylococcal CAP between January 2011 and December 2016. Method: Patients with CAP who fulfilled the following criteria were included: clinical and radiological signs of lung infection, at least one assessment of S. aureus involvement (positive culture from blood/pleural fluid, respiratory sample), and admission to ICU. At the time of data collection, clinicians were not aware of PVL status. Patterns in patient age distribution were detected using a clustering procedure. Risk factors for mortality were identified using multivariate Cox regression. Findings: One-hundred and sixty-three patients, aged from one month to 87 years, were included; 52.1% were PVL-positive. Unexpectedly, PVL-positive cases distributed into two independent clusters, with a cut-off of around 3 years of age, whereas PVL-negative cases were only observed in adolescents and adults. Features of the

Contexte : Mise en place en France en septembre 2010, la Premiere Annee Commune des Etudes de Sante (PACES) est caracterisee par un enseignement de masse, un concours tres selectif et une grande quantite d’information a memoriser. Dans ce contexte tres particulier, nous avons, pour la premiere fois, determine les approches d’apprentissage specifiquement adoptees par les etudiants et etudie leur impact sur la reussite au concours. Materiel et methodes : Une version francaise du questionnaire R-SPQ-2F de Biggs a ete administree aux etudiants de PACES 2011-2012 avant les partiels en decembre. Les approches d’apprentissage specifiques ont ete determinees apres analyse factorielle exploratoire du R-SPQ-2F et selection des items pertinents dans ce contexte. L’association entre les approches et la bonne reussite aux partiels a ete etudiee par un modele de regression binomiale. Resultats : Au total, 1977 etudiants sont inclus dans l’analyse. Les etudiants qui ont le plus de chance de reussir sont i) ceux qui s’interessent a leurs etudes (risque relatif (RR) de reussite de 1,5) et ii) ceux qui se limitent au contenu du cours (approche de surface, RR = 1,4). Les etudiants qui apprennent par cœur (approche de surface) ont 1,6 plus de risque d’echouer que les autres. Conclusion : Ce concours de PACES tres selectif et tres stressant a pour effet d’encourager une certaine approche de surface tres forte en vue de la reussite au concours au detriment d’une approche plus en profondeur indispensable a une formation solide.

Staphylococcus aureus is a commensal and pathogen of both humans and bovines. While the epidemiology of both groups has been extensively studied individually, little is known about the potential zoonotic transfer from animal strains to human being and vice versa. To determine the S. aureus prevalence of bovine mastitis in Algeria and the zoonotic transfer of strains to human beings, mastitis milk samples were collected, and professionals in a close contact with bovines were nasal swabbed. S. aureus isolates were all characterized by methicillin resistance and spa-typing. DNA microarrays analysis was performed on a subset of strains in order to detect other virulence factors, including toxins, and to assign the isolates to theirs MLST clonal complexes. Overall, 116/222 (52.3%) cows suffered from mastitis, whose 38.8% (45/116) infected with S. aureus. Human nasal carriage was of 38% (49/129), with only 4 MRSA carriers (3.1%). A higher diversity of spa-types was observed in human (35/50) than in bovine (18/67) isolates, with a predominance of clonal complexes CC97 and CC22 in bovines. The typical animal clone CC97 was occasionally detected in human beings. Conversely, the CC22 S. aureus clone largely switched from humans to bovines. Our study highlights the potential dynamics of animal and human S. aureus strains in the farm environment in Algeria, which may represent a health threat in both populations.

In the past 20 to 30 years, methicillin-resistant Staphylococcus aureus (MRSA) strains have been present in hospitals and have become a major cause of hospital-acquired infection. Methicillin resistance rates of S. aureus vary considerably between countries, with a high prevalence in the United States, and southern Europe (>20%) and a low prevalence in northern Europe (< or =5%). Community-acquired MRSA emerged worldwide in the late 1990s. There has been great confusion in the literature between healthcare-associated MRSA infections occurring in the community in patients who are at risk of acquiring hospital MRSA (such as those with past history of hospital admission, immunocompromised status, etc.), and true CA-MRSA infections due to strains that are present in the community only.

International audience; OBJECTIVES: Legionella pneumophila serogroup 1 (Lp1) sequence type 47 is the leading cause of legionellosis in north-western Europe, but, surprisingly, it is rarely isolated from environmental samples. Comparative genomics was applied to develop a PCR assay and to better understand the evolution of this strain. METHODS: Comparative analysis of 36 genomes representative of the Lp species was used to identify specific PCR targets, which were then evaluated in silico on 545 sequenced genomes and in~vitro on 436 Legionella strains, 106 respiratory samples, and three environmental samples from proven ST47 sources. Phylogenetic analyses were performed to understand the evolution of ST47. RESULTS: The gene LPO\₁073 was characterized as being 100% conserved in all 129 ST47 genomes analysed. A real-time PCR designed to detect LPO\₁073 was positive for all 110 ST47 strains tested and agreed with culture and typing results previously obtained for 106 respiratory samples. The three environmental samples were also positive. Surprisingly, 26 of the 44 ST109 strains tested among 342 non-ST47 strains scored positive for LPO\₁073. SNP-based phylogenetic analysis was undertaken to understand this result: the PCR-positive ST109 genomes were almost identical to ST47 genomes, with the exception of a recombined region probably acquired by ST47 from a ST62(-like) strain. CONCLUSION: The genomic analysis allowed the design of a highly specific PCR assay for rapid detection of ST47 strains. Furthermore, it allowed us to uncover the evolution of ST47 strains from ST109 by homologous recombination with ST62. We hypothesize that this recombination generated the leading cause of legionellosis in north-western Europe.

Monitoring the emergence of antibiotic resistance is a recent issue in the treatment of Legionnaires' disease. Macrolides are recommended as first-line therapy, but resistance mechanisms have not been studied in Legionella species. Our aim was to determine the molecular basis of macrolide resistance in L. pneumophila . Twelve independent lineages from a common susceptible L. pneumophila ancestral strain were propagated under conditions of erythromycin or azithromycin pressure to produce high-level macrolide resistance. Whole-genome sequencing was performed on 12 selected clones, and we investigated mutations common to all lineages. We reconstructed the dynamics of mutation for each lineage and demonstrated their involvement in decreased susceptibility to macrolides. The resistant mutants were produced in a limited number of passages to obtain a 4,096-fold increase in erythromycin MICs. Mutations affected highly conserved 5-amino-acid regions of L4 and L22 ribosomal proteins and of domain V of 23S rRNA (G2057, A2058, A2059, and C2611 nucleotides). The early mechanisms mainly affected L4 and L22 proteins and induced a 32-fold increase in the MICs of the selector drug. Additional mutations related to 23S rRNA mostly occurred later and were responsible for a major increase of macrolide MICs, depending on the mutated nucleotide, the substitution, and the number of mutated genes among the three rrl copies. The major mechanisms of the decreased susceptibility to macrolides in L. pneumophila and their dynamics were determined. The results showed that macrolide resistance could be easily selected in L. pneumophila and warrant further investigations in both clinical and environmental settings.

Dobrzyn Plateau in central Poland contains a suite of enigmatic glacial bedforms termed ‘glacial curvilineations’ (GCL). These landforms occur within tunnel valleys forming a complex anabranched network. GCL consist of a series of parallel ridges extending for as much as 5 km. Over their length, GCL ridges are parallel to neighboring ridges and to tunnel valley margins. Internal GCL composition consists of sand, gravel and till distributed randomly within ridges and in intervening troughs. Electric resistivity tomography surveys confirm the sediment distribution seen in field exposures. Truncated beds near the ridge forms and the heterogeneous nature and distribution of sediments suggest an erosional (rather than accretionary) origin for GCL. In this case, internal GCL composition is not related to GCL genesis: it records proglacial and subglacial conditions prior to GCL shaping by erosion. The terminal zone of the anabranched tunnel valley network is characterized by a broad apron-like ice-marginal sedimentary complex. Sand and gravel dominate sedimentary facies within this zone. Sediments record evidence of meltwater sedimentation associated with supraglacial melting near the ice margin and high-energy subglacial outbursts emanating from tunnel valleys. This interpretation suggests that the GCL surface is a regional unconformity representing an erosional landscape where sediments within landforms offer limited insights into landform genesis (cf., Sharpe et al., 2004 ). Subglacial meltwater erosion is proposed as unifying model of landscape development as it accounts for both erosion of tunnel valleys and GCL, and meltwater-dominated sedimentation in the ice-marginal sedimentary complex.

The antibiotic susceptibility and molecular epidemiology of Panton-Valentine leukocidin (PVL)-positive meticillin-resistant Staphylococcus aureus (MRSA) isolates reported from 17 countries in the Americas, Europe and, Australia-Asia were analysed. Among a total of 3236 non-duplicate isolates, the lowest susceptibility was observed to erythromycin in all regions. Susceptibility to ciprofloxacin showed large variation (25%, 75% and 84% in the Americas, Europe and Australia-Asia, respectively). Two vancomycin-intermediate PVL-positive MRSA isolates were reported, one from Hong Kong and the other from The Netherlands. Resistance to trimethoprim/sulfamethoxazole and linezolid was

We have studied the nature of ciprofloxacin resistance in methicillin sensitive and resistant Staphylococcus aureus among patients in a tertiary care hospital in Bengaluru, South India. All the isolates were highly resistant to ciprofloxacin. Molecular characterization of these samples performed using Staphylococcal Cassette Chromosome typing and multilocus sequence typing showed that 37.5% of total isolates and 59% of MRSA were sequence type (ST)772 and the rest were other STs. This indicates high prevalence of CA-MRSA in this tertiary care hospital serving the Indian community. Mutations responsible for ciprofloxacin resistance among these isolates in DNA gyrase (gyrA and gyrB) and topoisomerase IV (grlA and grlB) were analyzed by PCR amplification of specific fragments and sequencing. We found that for ST772 and five other STs present in this collection, single mutation in the gyrA gene, Ser-84→Leu, was sufficient for the high resistance. In vitro generation of ciprofloxacin resistance in two sensitive ST772 isolates by exposure to increasing antibiotic concentrations also resulted in the same single mutation of gyrA. The factors responsible for high ciprofloxacin resistance are varied and are dependent on the genetic background of the isolates and the environment. This is the first report on the mechanism of ciprofloxacin resistance among the most prevalent Indian CA-MRSA.

The contamination of aerosols by washbasin water colonized by Legionella in a hospital was evaluated. Aerosol samples were collected by two impingement technologies. Legionella was never detected by culture in all the (aerosol) samples. However, 45% (18/40) of aerosol samples were positive for Legionella spp. by polymerase chain reaction, with measurable concentrations in 10% of samples (4/40). Moreover, immunoassay detected Legionella pneumophila serogroup 1 and L. anisa, and potentially viable bacteria were seen on viability testing. These data suggest that colonized hospital washbasins could represent risks of exposure to Legionella aerosol inhalation, especially by immunocompromised patients.

The Okanagan Centre section is the stratotype for marine oxygen isotope stage (MIS) 4 sediments (Okanagan Centre Drift) in the southern interior of British Columbia, Canada. Previous work suggested that these sediments record two glacial and two interglacial cycles. This study reports on detailed sedimentological and geochronological investigations of lithostratigraphic units comprising the Okanagan Centre sequence, revealing successive deposition of subaqueous and subaerial outwash, a subglacial till and glaciolacustrine sediments during MIS 4. A limiting optical age of 113 ± 8 ka defines the base of this sequence. Sedimentological, paleopedological, optical dating and tephrochronological data from sediments near the middle of the sequence reveal soil development (MIS 3) in eolian sediments deposited on a river terrace overlying a deglaciated surface. Within these sediments, identification of Mt. St. Helens set C tephra suggest sedimentation between 50 and 35k 14C a BP. Optical dating corroborates the tephrochronology and suggests that this surface formed after ∼52 ± 7 ka. The record of MIS 2 glaciation is restricted to deglaciation, and overlies MIS 3 sediments above an unconformity possibly related to regional subglacial meltwater erosion. Eolian sediments containing Mt Mazama set O tephra (∼7.62k cal a BP) cap the sequence.

Background: To characterize the methicillin-resistant Staphylococcus aureus (MRSA) clones present in Istanbul, 102 MRSA isolates collected during a 5-year period at the Istanbul Medical Faculty Hospital were characterized using microarray analysis and phenotypic resistance profiles. Methods: Resistance to methicillin was detected with a cefoxitin disk diffusion assay and confirmed with a MRSA-agar and MRSA detection kit. Antimicrobial susceptibility testing was performed by a disk diffusion assay and interpreted according to the 2012 guidelines of the Antibiogram Committee of the French Society for Microbiology. Decreased susceptibility to glycopeptides was confirmed using the population analysis profile-area under the curve (PAP-AUC) method. The presence of the mecA gene was detected by polymerase chain reaction. Bacterial DNA was extracted according to the manufacturer's recommended protocol using commercial extraction kits. Strains were extensively characterized using the DNA microarray. Results: Isolates were grouped into six clonal complexes. The most frequently detected clone was the Vienna/Hungarian/Brazilian clone (ST239-MRSA-III), which accounted for 53.9% of the isolates. These isolates were resistant to multiple antibiotics, particularly penicillin, tetracycline, rifampicin, kanamycin, tobramycin, gentamicin, levofloxacin, erythromycin, lincomycin and fosfomycin. Furthermore, three isolates were detected by population analysis profile as heterogeneous vancomycin-intermediate S. aureus (hVISA). The UK-EMRSA-15 clone (ST22-MRSA-IV PVL negative) was detected in 9.8% of the isolates and was mainly susceptible to all anti-staphylococcal antibiotics. Seven isolates (6.9%) were positive for PVL genes and were assigned to the CC80-MRSA-IV clone (European CA-MRSA clone, three isolates), ST8-MRSA-IV clone (USA300 clone, two isolates, one ACME-positive) or ST22-MRSA-IV clone (“Regensburg EMRSA” clone, two isolates). All other clones were detected in one to six isolates and corresponded to well-known clones (e.g., Pediatric clone, Dublin EMRSA clone, WA MRSA-54/63, WA MRSA-1/57). Conclusions: This work highlighted both the high prevalence of ST239-MRSA-III clone and the large diversity of the other MRSA clones detected in a university hospital in Istanbul.

Objectifs : Presenter un panorama general des MD-PhD programs , les doubles cursus americains permettant l’obtention d’un doctorat en science et d’un diplome d’exercice de la medecine, et proposer le developpement d’une filiere pilote au sein de l’Universite de Lyon. Exegese : Les MD-PhD programs permettent la formation de medecins chercheurs tout en reduisant la duree globale d’etudes. Ils ont atteint les objectifs initialement fixes aux Etats-Unis et dans les pays ou ils se sont exportes. L’Universite de Lyon dispose des structures d’enseignement et de recherche, notamment l’Ecole Normale Superieure de Lyon et les facultes de medecine, pour accueillir une filiere pilote.

Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) have emerged worldwide. These CA-MRSA are different from classical hospital-acquired MRSA. They share common characteristics: they affect mainly young subjects, without past medical history. The majority of strains produce the Panton-Valentine leukocidin. They are mainly responsible for suppurative skin infections and rarely for invasive infections such as necrotizing pneumonia. The situation in the US is alarming with a main circulating clone the USA300 clone, whereas in Europe, the diffusion of CA-MRSA strains remains limited. It is important to take advantage of the experience acquired from the US to limit the potential spread of such CA-MRSA strains.

Legionella is the causative agent of Legionnaires' disease, a severe form of pneumonia. Detection of Legionella pneumophila serogroup 1 antigen in urine samples has shortened the delay of diagnosis and subsequent treatment initiation resulting in decreased mortality. Improved surveillance of potable water system reinforces the community prevention. In France, the National Reference Center for Legionella collects the strains responsible for sporadic or epidemic cases and crosslinks the data including epidemiological pattern, clinical presentation, and genetic analysis of the strains. Regarding host-pathogen interactions, major advances have been made recently in the understanding of L. pneumophila ability to subvert the host intracellular trafficking and the innate immune response leading to infection control.

Endemic strains of Legionella pneumophila sequence type 1 (ST1), in particular the ST1/Paris pulsotype, are dispersed worldwide and represent about 10% of culture-proven clinical cases of Legionnaires' disease in France. The high rate of isolation of this strain from both clinical and environmental samples makes identification of the source of infection difficult during epidemiological investigations. The full-length genome sequence of this strain was recently determined, and it revealed the presence of a CRISPR/ cas complex. The aim of this study was to develop and evaluate a spoligotyping tool based on the diversity of this CRISPR locus that would allow the accurate subtyping of the L. pneumophila serogroup 1 ST1/Paris pulsotype. The CRISPR loci of 28 L. pneumophila ST1/Paris pulsotype isolates were sequenced, and 42 different spacers regions were characterized. A membrane-based spoligotyping method was developed and used to determine the subtypes of 406 L. pneumophila isolates, including 233 with the ST1/Paris pulsotype profile that were collected in France from 2000 to 2011. A total of 46 different spoligotypes were detected, and 41 of these were specifically identified in the ST1/Paris pulsotype isolates. In 27 of 33 epidemiological investigations, the environmental source of contamination was confirmed by comparing spoligotypes of clinical isolates with those of environmental isolates. With an index of discrimination of 79.72% (95% confidence interval, 75.82 to 83.63), spoligotyping of the L. pneumophila ST1/Paris pulsotype has the potential to be a useful complementary genotyping tool for discriminating isolates with undistinguishable pulsed-field gel electrophoresis (PFGE) and ST genotypes, which could help to identify environmental sources of infection.

Staphylococcus aureus is a pathogen and a skin commensal that is today also common in the infant gut flora. We examine the role of S. aureus virulence factors for gut colonization. S. aureus isolated from quantitative stool cultures of 49 Swedish infants followed from birth to 12 months of age were assessed for 30 virulence-associated genes, spa type, and agr allele by serial polymerase chain reaction (PCR) assays. Strains carrying genes encoding collagen-binding protein, and the superantigens S. aureus enterotoxin O/M (SEO/SEM) had higher stool counts than strains lacking these genes, whereas genes for S. aureus enterotoxin A (SEA) were associated with low counts. A cluster of strains belonging to agr allele I and the spa clonal cluster 630 (spa-CC 630) that carried genes encoding SEO/SEM, SEC, collagen-binding protein, and elastin-binding protein were all long-time colonizers. Thus, certain S. aureus virulence factors might promote gut colonization.

Menstrual toxic shock syndrome (MTSS) is a severe toxin-mediated disease associated with Staphylococcus aureus producing toxic shock syndrome toxin 1 (TSST-1), a superantigen that mediates a potent activation of Vβ-2 T cells. In animal models, superantigen treatment of responsive T cells induces their initial proliferation, followed by unresponsiveness upon further superantigen stimulation. To determine whether T cell unresponsiveness occurs in humans during the acute phase of MTSS, we collected T cells from a patient with MTSS and restimulated them ex vivo with recombinant TSST-1. The expansion of T cells collected during the acute phase of disease was compared with positive controls including basal-state T cells (collected 70 days after MTSS) restimulated with TSST-1, and T cells stimulated with enterotoxin B superantigen. We found that TSST-1-induced expansion of acute phase T cells was not inferior to that observed in positive controls. We conclude that T cells were still reactive to TSST-1 during the acute phase of MTSS in this patient. As the persistence of TSST-1 production could thus be associated with further expansion of TSST-1-reactive T cells and a rapid worsening of symptoms, this study adds further support to the need for immediate eradication of the focus of infection as soon as MTSS is suspected.

Previous articles reported that beta-lactam antibiotics increase the expression of Staphylococcus aureus Panton-Valentine leukocidin (PVL) by activating its transcription. We investigated the mechanisms underlying the inductor effect of beta-lactams on PVL expression by determining targets and regulatory pathways possibly implicated in this process. We measured PVL production in the presence of oxacillin (nonselective), imipenem (penicillin-binding protein 1 [PBP1] selective), cefotaxime (PBP2 selective), cefaclore (PBP3 selective), and cefoxitin (PBP4 selective). In vitro , we observed increased PVL production consistent with luk -PV mRNA levels that were 20 to 25 times higher for community-acquired methicillin-resistant S. aureus (CA-MRSA) cultures treated with PBP1-binding oxacillin and imipenem than for cultures treated with other beta-lactams or no antibiotic at all. This effect was also observed in vivo , with increased PVL mRNA levels in lung tissues from CA-MRSA-infected mice treated with imipenem but not cefoxitin. To confirm the involvement of PBP1 inhibition in this pathway, PBP1 depletion by use of an inducible pbp1 antisense RNA showed a dose-dependent relationship between the level of pbp1 antisense RNA and the luk -PV mRNA level. Upon imipenem treatment of exponential-phase cultures, we observed an increased sarA mRNA level after 30 min of incubation followed by a decreased rot mRNA level after 1 to 4 h of incubation. Unlike the agr and saeRS positive regulators, which were nonessential for PVL induction by beta-lactams, the sarA (positive) and rot (negative) PVL regulators were necessary for PVL induction by imipenem. Our results suggest that antibiotics binding to PBP1 increase PVL expression by modulating sarA and rot , which are essential mediators of the inductor effect of beta-lactams on PVL expression.

Legionella pneumophila , a bacterium that replicates within aquatic amoebae and persists in the environment as a free-living microbe, is the causative agent of Legionnaires' disease. Among the many Legionella species described, L. pneumophila is associated with 90% of human disease, and within the 15 serogroups (Sg), L. pneumophila Sg1 causes more than 84% of Legionnaires' disease worldwide. Thus, rapid and specific identification of L. pneumophila Sg1 is of the utmost importance for evaluation of the contamination of collective water systems and the risk posed. Previously we had shown that about 20 kb of the 33-kb locus carrying the genes coding for the proteins involved in lipopolysaccharide biosynthesis (LPS gene cluster) by L. pneumophila was highly specific for Sg1 strains and that three genes ( lpp0831 , wzm , and wzt ) may serve as genetic markers. Here we report the sequencing and comparative analyses of this specific region of the LPS gene cluster in L. pneumophila Sg6, -10, -12, -13, and -14. Indeed, the wzm and wzt genes were present only in the Sg1 LPS gene cluster, which showed a very specific gene content with respect to the other five serogroups investigated. Based on this observation, we designed primers and developed a classical and a real-time PCR method for the detection and simultaneous identification of L. pneumophila Sg1 in clinical and environmental isolates. Evaluation of the selected primers with 454 Legionella and 38 non- Legionella strains demonstrated 100% specificity. Sensitivity, specificity, and predictive values were further evaluated with 209 DNA extracts from water samples of hospital water supply systems and with 96 respiratory specimens. The results showed that the newly developed quantitative Sg1-specific PCR method is a highly specific and efficient tool for the surveillance and rapid detection of high-risk L. pneumophila Sg1 in water and clinical samples.