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The two-component regulatory system CovRS is the main regulator of virulence gene expression in Group B Streptococcus (GBS), the leading cause of invasive infections in neonates. In this study we analyzed by mass spectrometry the GBS extracellular protein complex (i.e. the exoproteome) of NEM316 wild-type (WT) strain and its isogenic covRS deletion mutant (ΔcovRS). A total of 53 proteins, 49 of which had classical secretion signals, were identified: 12 were released by both strains while 21 and 20 were released exclusively by WT and ΔcovRS strains, respectively. In addition to known surface proteins, we detected here unstudied cell-wall associated proteins and/or orthologs of putative virulence factors present in other pathogenic streptococci. While the functional role of these proteins remains to be elucidated, our data suggest that the analysis of the exoproteome of bacterial pathogens under different gene expression conditions may be a powerful tool for the rapid identification of novel virulence factors and vaccine candidates. Biological significance We believe that this manuscript will be of interest to Journal of Proteomics readers since the paper describes the identification of several putative virulence factors and vaccine candidates of the group B streptococcus, an important pathogen, using a simple proteomics strategy involving LC–MS analysis of culture supernatants obtained from two strains with divergent gene expression patterns. This technique provided the most comprehensive inventory of extracellular proteins obtained from a single streptococcal species thus far. The approach described has the added benefit of being easily applicable to a large number of different strains, making it ideal for the identification of conserved vaccine candidates.

Group B Streptococcus (GBS), a common commensal of the female genital tract, is the leading cause of invasive infections in neonates. Expression of major GBS virulence factors, such as the hemolysin operon cyl, is regulated directly at the transcriptional level by the CovSR two-component system. Using a random genetic approach, we identified a multi-spanning transmembrane protein, Abx1, essential for the production of the GBS hemolysin. Despite its similarity to eukaryotic CaaX proteases, the Abx1 function is not involved in a post-translational modification of the GBS hemolysin. Instead, we demonstrate that Abx1 regulates transcription of several virulence genes, including those comprising the hemolysin operon, by a CovSR-dependent mechanism. By combining genetic analyses, transcriptome profiling, and site-directed mutagenesis, we showed that Abx1 is a regulator of the histidine kinase CovS. Overexpression of Abx1 is sufficient to activate virulence gene expression through CovS, overcoming the need for an additional signal. Conversely, the absence of Abx1 has the opposite effect on virulence gene expression consistent with CovS locked in a kinase-competent state. Using a bacterial two-hybrid system, direct interaction between Abx1 and CovS was mapped specifically to CovS domains involved in signal processing. We demonstrate that the CovSR two-component system is the core of a signaling pathway integrating the regulation of CovS by Abx1 in addition to the regulation of CovR by the serine/threonine kinase Stk1. In conclusion, our study reports a regulatory function for Abx1, a member of a large protein family with a characteristic Abi-domain, which forms a signaling complex with the histidine kinase CovS in GBS., Author Summary The gram-positive Streptococcus genus includes three major human pathogens that are members of the normal microflora: Streptococcus pneumoniae (also known as the pneumococcus), Streptococcus pyogenes (Group A Streptococcus), and Streptococcus agalactiae (Group B Streptococcus). Their carriage in the population is highly dynamic and mostly asymptomatic. However, each of these species can cause a wide spectrum of diseases, from local infections to systemic and fatal infections including septicemia and meningitis. Expression of streptococcal virulence-associated genes is tightly regulated at the transcriptional level. However, the signal(s) and the precise molecular events controlling the switch from commensalism to virulence are not yet understood. In this study, we identified and characterized a bacterial protein essential for virulence gene expression in Group B Streptococcus, the main pathogen of neonates. We show that this transmembrane protein, named Abx1, interacts with the histidine kinase CovS to modulate the activity of the major regulator of virulence CovR. We define how a core set of four proteins, Abx1, CovS, CovR, and the serine/threonine kinase Stk1, interact to control the expression of virulence genes in S. agalactiae. We propose that Abx1-like proteins, that are widespread in bacteria, might be part of a conserved mechanism of two-component system regulation.

Objectives To determine the activity of oritavancin against methicillin-resistant staphylococci, vancomycin-resistant enterococci (VRE) and β-haemolytic streptococci recently isolated from acute bacterial skin and skin structure infections or bacteraemia in western Europe. Methods Forty-one centres in Spain (8), Italy (9), Germany (8), France (8) and the UK (8) submitted 866 isolates [204 methicillin-resistant Staphylococcus aureus (MRSA), 177 methicillin-resistant coagulase-negative staphylococci (MRCoNS), 101 VRE, 193 Streptococcus agalactiae and 191 Streptococcus pyogenes] that were collected during the first 6 months of 2011. These were re-identified and susceptibilities to oritavancin and comparators were determined. Results Oritavancin was very active against MRSA (MIC50/MIC90 0.03/0.06 mg/L), MRCoNS (0.06/0.12 mg/L), VRE (0.03/0.06 mg/L), S. agalactiae (0.03/0.06 mg/L) and S. pyogenes (0.06/0.25 mg/L). The highest oritavancin MIC observed was 0.25 mg/L (species were S. aureus, Staphylococcus epidermidis, Staphylococcus hominis, S. agalactiae, S. pyogenes and Enterococcus faecalis). Conclusions These data from recently collected Gram-positive bacteria in western Europe confirm the potent in vitro activity of oritavancin against a wide range of resistant MRSA, MRCoNS and VRE isolates, including ones resistant to newer agents.

Seventy-four unrelated clinical isolates of Streptococcus pneumoniae harboring the tet (M) gene were studied. Seven strains with low tetracycline (Tc) MICs (0.25 to 0.5 μg/ml) were found to harbor truncated tet (M) alleles that were inactivated by different frameshift mutations. In contrast, five strains bore deletions in the tet (M) promoter region, among which four displayed increased Tc MICs (16 to 64 μg/ml). The same promoter mutations were detected in Tc-resistant mutants selected in vitro from various susceptible strains. Sequence analysis revealed that these deletions might impede the formation of the transcriptional attenuator located immediately upstream of tet (M). Expression in Enterococcus faecalis of a tet (M) reporter gene transcribed from these promoter mutants conferred a level of Tc resistance similar to that observed in the parental S. pneumoniae strains. These results show that different levels of Tc susceptibility found in clinical isolates of S. pneumoniae can be explained by frameshift mutations within tet (M) and by alterations of the upstream transcriptional attenuator.

Group A Streptococcus (GAS) has acquired an arsenal of virulence factors, promoting life-threatening invasive infections such as necrotizing fasciitis. Current therapeutic regimens for necrotizing fasciitis include surgical debridement and treatment with cell wall-active antibiotics. Addition of clindamycin (CLI) is recommended, although clinical evidence is lacking. Reflecting the current clinical dilemma, an observational study showed that only 63% of the patients with severe invasive GAS infection received CLI. This work thus aimed to address whether CLI improves necrotizing fasciitis outcome by modulating virulence factors of CLI-susceptible and CLI-resistant GAS in vitro and in vivo. Treatment with CLI reduced extracellular DNase Sda1 and streptolysin O (SLO) activity in vivo, whereas subinhibitory CLI concentrations induced expression and activity of SLO, DNase, and Streptococcus pyogenes cell envelope protease in vitro. Our in vivo results suggest that CLI should be administered as soon as possible to patients with necrotizing fasciitis, while our in vitro studies emphasize that a high dosage of CLI is essential.

Background The prevalence of methicillin-resistant Staphylococcus aureus (MRSA) in cystic fibrosis (CF) patients has increased and MRSA seems to be associated with a poorer prognosis. The aim of this study was to assess the prevalence and clinical consequences of MRSA and methicillin-susceptible Staphylococcus aureus (MSSA), associated or not associated with Pseudomonas aeruginosa (PA). Methods In a retrospective study on 419 sputum producer patients (293 adults and 126 children > 7 years of age), we recorded patient characteristics, lung function, nutritional status, IV antibiotics and hospitalisations, the presence of SA and/or PA and FEV1 decline over 2 years. Results SA was found in 72% of the patients: MSSA in 68.2% of children and 48.8% of adults; MRSA in 17.5% of children and 17.8% of adults. Sixty percent of MRSA patients and 60.4% of MSSA patients also harboured PA. The rate of deterioration of clinical status of the various groups, as assessed from respiratory function, IV antibiotic courses and hospitalisations, increased in the order: no SA/no PA, MSSA alone, MRSA alone, MSSA/PA, MRSA/PA, and PA alone. Nutritional status did not differ between groups. Results were roughly similar for children and adults. The yearly FEV1 decline was significantly higher only for MRSA/PA patients (p = 0.03) compared to no SA/no PA patients. Conclusion Clinical condition of CF patients with MSSA only or MRSA only appeared similar, whereas MRSA/PA patients had more severe respiratory function than MSSA/PA patients. In CF patients, MRSA might be more deleterious than MSSA only when associated with PA.

Due to the differences in the management of Mycobacterium kansasii disease and tuberculosis, an accurate diagnosis is required. This report, which describes what we believe to be the first documented case of M. kansasii infection in a patient suffering from anorexia nervosa, sheds light on the possible occurrence of a non-tuberculous mycobacterial infection that can mimic tuberculosis, on the risk of a misleading interpretation of interferon-gamma release assays, and on the temporal response to these tests.

International audience; BACKGROUND: Observational studies showed that the profile of infective endocarditis (IE) significantly changed over the past decades. However, most studies involved referral centers. We conducted a population-based study to control for this referral bias. The objective was to update the description of characteristics of IE in France and to compare the profile of community-acquired versus healthcare-associated IE. METHODS: A prospective population-based observational study conducted in all medical facilities from 7 French regions (32% of French individuals aged ≥18 years) identified 497 adults with Duke-Li-definite IE who were first admitted to the hospital in 2008. Main measures included age-standardized and sex-standardized incidence of IE and multivariate Cox regression analysis for risk factors of in-hospital death. RESULTS: The age-standardized and sex-standardized annual incidence of IE was 33.8 (95% confidence interval [CI], 30.8-36.9) cases per million inhabitants. The incidence was highest in men aged 75-79 years. A majority of patients had no previously known heart disease. Staphylococci were the most common causal agents, accounting for 36.2% of cases (Staphylococcus aureus, 26.6%; coagulase-negative staphylococci, 9.7%). Healthcare-associated IE represented 26.7% of all cases and exhibited a clinical pattern significantly different from that of community-acquired IE. S. aureus as the causal agent of IE was the most important factor associated with in-hospital death in community-acquired IE (hazard ratio [HR], 2.82 [95% CI, 1.72-4.61]) and the single factor in healthcare-associated IE (HR, 2.54 [95% CI, 1.33-4.85]). CONCLUSIONS: S. aureus became both the leading cause and the most important prognostic factor of IE, and healthcare-associated IE appeared as a major subgroup of the disease.

International audience; Chronic meningococcemia is a form of sepsis with frequent polymorphous skin lesions. Both in vivo and in vitro data suggest that, in these lesions, meningococci gain access from the capillary lumen to the peripheral extravascular compartment, in the absence of vascular dislocation, through a paraendothelial route.

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. The capsular polysaccharide (CPS) is an important virulence factor and a vaccine target of the major neonatal pathogen group B Streptococcus (GBS). Population studies revealed no strong correlation between CPS type and multilocus sequence typing (MLST) cluster, with the remarkable exception of the worldwide spread of hypervirulent GBS CC17, which were all until recently CPS type III. Methods. A total of 965 GBS strains from invasive infection isolated in France were CPS typed and the presence of the CC17-specific surface protein encoding gene hvgA gene was investigated. Three hvgA-positive GBS strains screened were surprisingly CPS type IV and thus further characterized by MLST typing, pulsed-field gel electrophoresis (PFGE), and whole genome sequencing. Results. MLST and PFGE demonstrated a capsular switching from CPS type III to IV within the highly homogeneous GBS CC17. Sequence analysis revealed that this capsular switch was due to the exchange of a 35.5-kb DNA fragment containing the entire cps operon. Conclusions. This work shows that GBS CC17 hypervirulent strains have switched one of their main vaccine targets. Thus, continued surveillance of GBS population remains of the utmost importance during clinical trials of conjugate GBS vaccines. Streptococcus agalactiae or group B Streptococcus (GBS) is a gram-positive encapsulated commensal bacterium of the human intestine that is also present in the vagina of 15%–30% of healthy women. In neonates, it may turn into a deadly pathogen and is the

Group A Streptococcus (GAS) infections present high morbidity and mortality rates and consequently remain a significant health problem. The emm3 isolates induce more severe pathologies than all others. In this study, we tested, on a collection of invasive and non-invasive emm3 clinical isolates, whether in that genotype the invasive status of the strains affects the innate immune response. We show that phagocytosis is dependent on the invasiveness of the isolates. Interestingly, all emm3 isolates compromise macrophage integrity, already noticeable 1 h after infection. Inflammatory modulators (IL-6, TNF-α and IFN-β) are nevertheless detected during at least 6 h post-infection. This is a likely consequence of the macrophages not being all infected. The efficient and rapid induction of macrophage death could explain the virulence of the emm3 strains.

A new Vibrio cholerae serogroup O139 strain of unknown origin recently emerged in India and Bangladesh, causing a major outbreak of cholera. The genetic relationship between this epidemic strain and the O1 strain responsible for the 7th pandemic of cholera was studied by analyzing the DNA polymorphism of V. cholerae by pulsed-field gel electrophoresis and arbitrarily primed polymerase chain reaction. The restriction patterns of the reference strain O139 Bengal and 10 wild O139 strains isolated early in the Indian outbreak strikingly resemble that of the pandemic O1 strain of V. cholerae El Tor, thus suggesting a close genetic relationship among these strains. This similarity contrasts with the genetic heterogeneity of sporadic non-O1 strains isolated in various parts of the world. Study results strongly suggest that the new epidemic O139 strain is closely related to and might be derived from the pandemic O1 strain of V. cholerae.

International audience; Streptococcus agalactiae (group B Streptococcus, GBS), a normal constituent of the intestinal microbiota is the major cause of human neonatal infections and a worldwide spread 'hypervirulent' clone, GBS ST-17, is strongly associated with neonatal meningitis. Adhesion to epithelial and endothelial cells constitutes a key step of the infectious process. Therefore GBS surface-anchored proteins are obvious potential adhesion mediators of barrier crossing and determinant of hypervirulence. This review addresses the most recent molecular insights gained from studies on GBS surface proteins proven to be involved in the crossing of the brain-blood barrier and emphasizes on the specificity of a hypervirulent clone that displays meningeal tropism.

The goal of this study was to evaluate temporal trends in infective endocarditis (IE) incidence and clinical characteristics after 2002 French IE prophylaxis guideline modifications.There are limited data on changes in the epidemiology of IE since recent guidelines recommended restricting the indications of antibiotic prophylaxis of IE.Three 1-year population-based surveys were conducted in 1991, 1999, and 2008 in 3 French regions totaling 11 million inhabitants age ≥20 years. We prospectively collected IE cases from all medical centers and analyzed age- and sex-standardized IE annual incidence trends.Overall, 993 expert-validated IE cases were analyzed (323 in 1991; 331 in 1999; and 339 in 2008). IE incidence remained stable over time (95% confidence intervals given in parentheses/brackets): 35 (31 to 39), 33 (30 to 37), and 32 (28 to 35) cases per million in 1991, 1999, and 2008, respectively. Oral streptococci IE incidence did not increase either in the whole patient population (8.1 [6.4 to 10.1], 6.3 [4.8 to 8.1], and 6.3 [4.9 to 8.0] in 1991, 1999, and 2008, respectively) or in patients with pre-existing native valve disease. The increased incidence of Staphylococcus aureus IE (5.2 [3.9 to 6.8], 6.8 [5.3 to 8.6], and 8.2 [6.6 to 10.2]) was not significant in the whole patient population (p = 0.228) but was significant in the subgroup of patients without previously known native valve disease (1.6 [0.9 to 2.7], 3.7 [2.6 to 5.1], and 4.1 [3.0 to 5.6]; p = 0.012).Scaling down antibiotic prophylaxis indications was not associated with an increased incidence of oral streptococcal IE. A focus on avoidance of S. aureus bacteremia in all patients, including those with no previously known valve disease, will be required to improve IE prevention.

Low-level methicillin-resistant Staphylococcus aureus may be difficult to detect with the VITEK® 2 system (VK2). Here, we suggest that S. aureus exhibiting VK2-oxacillin MIC of 1 or 2 mg/L and a negative cefoxitin screen should be tested for the presence of mecA or its gene product.

Streptococcus salivarius is a commensal species commonly found in the human oral cavity and digestive tract, although it is also associated with human infections such as meningitis, endocarditis, and bacteremia. Here, we report the complete sequence of S. salivarius strain CCHSS3, isolated from human blood.

Excision and integration of Tn1545 occur by reciprocal site-specific recombination between 6 (or 7) bp variable sequences present in the recombining attachment (att) sites and designated overlap regions. We devised an assay for Tn1545 transposition in which derivatives containing the cis-acting transposition sequences (attTn 1545) integrate into a target replicon when complemented in trans by the transposon-encoded integrase Int-Tn. This assay was used to determine the characteristics of the DNA sequence that influence target site selection. Characterization of several integration sites indicated that a 20 bp segment, designated attB, contains the sequences required for target activity. It also appeared that (i) the target activity depends upon the extent of homology between the 7bp segments flanking the overlap regions in attB and attTn 1545, and (ii) the degree of homology between the two recombining overlap regions does not affect the level of target activity and has no influence on integration orientation.

Tuberculosis (TB) is rarely observed in cystic fibrosis (CF) patients. We report the first case of mediastinal TB, associated with leg pain and skin rash, in an adult patient with CF, and discuss factors suggestive of TB in the course of CF.

Streptococcus agalactiae (or group B streptococcus; GBS) is a leading cause of neonatal morbidity and mortality in the developed countries. Several epidemiological typing tools have been developed for GBS to investigate the association between genotype and disease and to assess genetic variations within genogroups. This study compared the semi-automated repetitive sequence-based PCR Diversilab® system (DL) with MLST and pulsed field gel electrophoresis (PFGE) for determining the relatedness of invasive GBS strains. We analysed 179 unrelated GBS strains isolated from adult (n=108) and neonatal (n=71) invasive infections. By MLST, strains were resolved into 6 clonal complexes (CCs) including 23 sequence-types (STs), and 4 unique STs, whereas DL differentiated these isolates into 12 rep-PCR clusters (rPCs) and 9 unique rep-PCR types. The discriminatory power of both methods was similar, with Simpson's diversity indexes of 71.9% and 70.6%, respectively. However, their clustering concordance was low with Wallace concordance coefficients inferior to 0.4. PFGE was performed on 31 isolates representative of the most relevant DLrPCs clustered within the 3 major MLST CCs (CC-17, CC-23 and CC-1). As already observed with MLST, the concordance of DL with PFGE was low for all three CCs (Wallace coefficient

Lethal meningitis triggered by the hypervirulent group B streptococcus clone ST-17 is mediated by a novel surface protein called HvgA., Streptococcus agalactiae (group B streptococcus; GBS) is a normal constituent of the intestinal microflora and the major cause of human neonatal meningitis. A single clone, GBS ST-17, is strongly associated with a deadly form of the infection called late-onset disease (LOD), which is characterized by meningitis in infants after the first week of life. The pathophysiology of LOD remains poorly understood, but our epidemiological and histopathological results point to an oral route of infection. Here, we identify a novel ST-17–specific surface-anchored protein that we call hypervirulent GBS adhesin (HvgA), and demonstrate that its expression is required for GBS hypervirulence. GBS strains that express HvgA adhered more efficiently to intestinal epithelial cells, choroid plexus epithelial cells, and microvascular endothelial cells that constitute the blood–brain barrier (BBB), than did strains that do not express HvgA. Heterologous expression of HvgA in nonadhesive bacteria conferred the ability to adhere to intestinal barrier and BBB-constituting cells. In orally inoculated mice, HvgA was required for intestinal colonization and translocation across the intestinal barrier and the BBB, leading to meningitis. In conclusion, HvgA is a critical virulence trait of GBS in the neonatal context and stands as a promising target for the development of novel diagnostic and antibacterial strategies.

The diagnostic sensitivities of the BD GeneOhm and Cepheid Xpert assays were compared using culture on log-serial dilutions of well-characterized methicillin-resistant Staphylococcus aureus (MRSA) and non-MRSA strains and on nasal and groin swabs from patients with histories of MRSA carriage. The sensitivities of GeneOhm and Xpert were high at 10 3 -CFU/ml MRSA concentrations (92.3% and 96.3%, respectively) although decreased considerably (

International audience; We have studied 457 Streptococcus pneumoniae isolated in 2007 from adults and children. For all isolates, both latex agglutination and molecular capsular typing were performed. Antibiotic resistance patterns were determined. S. pneumoniae 19A was the most frequently isolated serotype (34.7%) both in children and adults. It represented 12.8% of the strains isolated from invasive infections in adults and 27.0% in children and 63.6% (110/173) of strains isolated from acute otitis media. Between children and adults, no difference concerning antibiotic susceptibility was observed for penicillin, amoxicillin, cefotaxime, and erythromycin in strains isolated from invasive diseases. Comparing antibiotic susceptibilities according to the serotype, the 19A isolates appeared to be the least susceptible to penicillin (3.2%) and erythromycin (4.5%), followed by serotypes 19F and 14. We confirm the predominance of serotype 19A among S. pneumoniae responsible for invasive and noninvasive diseases either in children or adults in France.

Bacteria belonging to the Enterobacter genus are frequently isolated from clinical samples but are unusual causative agents of orthopedic implant infections. Twelve genetic clusters (clusters I to XII) and one sequence crowd (sequence crowd xiii ) can be distinguished within the Enterobacter cloacae nomenspecies on the basis of hsp60 sequence analysis, and until now, none of these clusters could be specifically associated with a disease. In order to investigate if specific genetic clusters would be involved in infections of orthopedic material, two series of bacterial clinical isolates identified as E. cloacae by routine phenotypic identification methods were collected either from infected orthopedic implants ( n = 21) or from randomly selected samples of diverse anatomical origins (control; n = 52). Analysis of the hsp60 gene showed that genetic clusters III, VI, and VIII were the most frequent genetic clusters detected in the control group, whereas cluster III was poorly represented among the orthopedic implant isolates ( P = 0.006). On the other hand, E. hormaechei (clusters VI and VIII), but not cluster III, is predominantly associated with infections of orthopedic implants and, more specifically, with infected material in the hip ( P = 0.019). These results support the hypothesis that, among the isolates within the E. cloacae complex, E. hormaechei and hsp60 gene sequencing-based cluster III are involved in pathogenesis in different ways and highlight the need for more accurate routine Enterobacter identification methods.

The type II fatty acid synthesis (FASII) pathway has been suggested to be a promising antimicrobial target, and the antibiotics platensimycin and platencin that target this pathway are claimed to be potentially effective against multiresistant Gram-positive bacteria. But a new study casts doubt on the value of this approach. Clinical isolates of a series of Gram-positive pathogens, including staphylococci, streptococci, pneumococci, and enterococci, are shown to overcome drug-induced blockade of FASII pathway inhibition when supplied with exogenous fatty acids either in laboratory growth medium or when infecting animals. Importantly, human serum is a highly effective source of fatty acids. The type II fatty acid biosynthesis pathway has been suggested to be a promising antimicrobial target. It is shown that major Gram-positive pathogen groups may circumvent the action of these antimicrobials by using host derived fatty acids instead, leaving these antibiotics ineffective. Antimicrobial drugs targeting the reportedly essential type II fatty acid synthesis (FASII) pathway1,2,3,4,5 have been recently acclaimed for their efficacy against infections caused by multiresistant Gram-positive bacteria6,7,8. Our findings show that the strategy for antibiotic development based on FASII pathway targets is fundamentally flawed by the fact that exogenous fatty acids fully bypass inhibition of this pathway in both in vitro and in vivo conditions. We demonstrate that major Gram-positive pathogens—such as streptococci, pneumococci, enterococci and staphylococci—overcome drug-induced FASII pathway inhibition when supplied with exogenous fatty acids, and human serum proves to be a highly effective source of fatty acids. For opportunist pathogen Streptococcus agalactiae, growth in serum leads to an overall decrease of FASII gene expression. No antibiotic inhibitor could have a stronger effect than the inactivation of the target gene, so we challenged the role of FASII using deletion mutants. Our results unequivocally show that the FASII target enzymes are dispensable in vivo during S. agalactiae infection. The results of this study largely compromise the use of FASII-based antimicrobials for treating sepsis caused by Gram-positive pathogens.

We report the first case of infection due to Streptococcus oligofermentans , which is a recently described oral Streptococcus species. It was responsible for the endocarditis and left forearm abscess of a 43-year-old woman. Identification was made using molecular techniques performed directly from valvular and surgical samples.

In streptococci, the secA2 locus includes genes encoding the following: (i) the accessory Sec components (SecA2, SecY2, and at least three accessory secretion proteins), (ii) two essential glycosyltranferases (GTs) (GtfA and GtfB), (iii) a variable number of dispensable additional GTs, and (iv) a secreted serine-rich LPXTG protein which is glycosylated in the cytoplasm and transported to the cell surface by this accessory Sec system. The secA2 locus of Streptococcus agalactiae strain NEM316 is structurally related to those found in other streptococci and encodes the serine-rich surface protein Srr1. We demonstrated that expression of Srr1 but not that of the SecA2 components and the associated GTs is regulated by the standalone transcriptional regulator Rga. Srr1 is synthesized as a glycosylated precursor, secreted by the SecA2 system, and anchored to the cell wall by the housekeeping sortase A. Srr1 was localized preferentially at the old poles. GtfA and/or GtfB, but not the six additional GTs, is essential for the production of Srr1. These GTs are involved in the attachment of GlcNac and sialic acid to Srr1. Full glycosylation of Srr1 is associated with the cell surface display of a protein that is more resistant to proteolytic attack. Srr1 contributes to bacterial adherence to human epithelial cell lines and virulence in a neonatal rat model. The extent of Srr1 glycosylation by GtfC to -H modulates bacterial adherence and virulence.

We have constructed and used an integrative vector, pAT112, that takes advantage of the transposition properties (integration and excision) of transposon Tn1545. This 4.9-kb plasmid is composed of: (i) the replication origin of pACYC184; (ii) the attachment site (att) of Tn1545; (iii) erythromycin-and kanamycin-resistance-encoding genes for selection in Gram- and Gram+ bacteria; and (iv) the transfer origin of IncP plasmid RK2, which allows mobilization of the vector from Escherichia coli to various Gram+ recipients. Integration of pAT112 requires the presence of the transposon-encoded integrase, Int-Tn, in the new host. This vector retains the insertion specificity of the parental element Tn1545 and utilises it to carry out insertional mutagenesis, as evaluated in Enterococcus faecalis. Since pAT112 contains the pACYC184 replicon and lacks most of the restriction sites that are commonly used for molecular cloning, a gene from a Gram+ bacterium disrupted with this vector can be recovered in E. coli by cleavage of genomic DNA, intramolecular ligation and transformation. Regeneration of the gene, by excision of pAT112, can be obtained in an E. coli strain expressing the excisionase and integrase of Tn1545. The functionality of this system was illustrated by characterization of an IS30-like structure in the chromosome of En. faecalis. Derivatives pAT113 and pAT114 contain ten unique cloning sites that allow screening of recombinants having DNA inserts by alpha-complementation in E. coli carrying the delta M15 deletion of lacZ alpha. These vectors are useful to clone and introduce foreign genes into the genomes of Gram+ bacteria.

Transposon Tn916 is a 16.4-kb broad-host-range conjugative transposon originally detected in the chromosome of Enterococcus faecalis DS16. Transposition of Tn916 and related transposons involves excision of a free, nonreplicative, covalently closed circular intermediate that is substrate for integration. Excisive recombination requires two transposon-encoded proteins, Xis-Tn and Int-Tn, whereas the latter protein alone is sufficient for integration. Here we report that conjugative transposition of Tn916 requires the presence of a functional integrase in both donor and recipient strains. We have constructed a mutant, designated Tn916-int1, by replacing the gene directing synthesis of Int-Tn by an allele inactivated in vitro. In mating experiments, transfer of Tn916-int1 from Bacillus subtilis to E. faecalis was detected only when the transposon-encoded integrase was supplied by trans-complementation in both the donor and the recipient. These results suggest that conjugative transposition of Tn916 requires circularization of the element in the donor followed by transfer and integration of the nonreplicative intermediate in the recipient.

Bacterial populations are subject to complex processes of diversification that involve mutation and horizontal DNA transfer mediated by transformation, transduction, or conjugation. Tracing the evolutionary events leading to genetic changes allows us to infer the history of a microbe. Here, we combine experimental and in silico approaches to explore the forces that drive the genome dynamics of Streptococcus agalactiae , the leading cause of neonatal infections. We demonstrate that large DNA segments of up to 334 kb of the chromosome of S. agalactiae can be transferred through conjugation from multiple initiation sites. Consistently, a genome-wide map analysis of nucleotide polymorphisms among eight human isolates demonstrated that each chromosome is a mosaic of large chromosomal fragments from different ancestors suggesting that large DNA exchanges have contributed to the genome dynamics in the natural population. The analysis of the resulting genetic flux led us to propose a model for the evolutionary history of this species in which clonal complexes of clinical importance derived from a single clone that evolved by exchanging large chromosomal regions with more distantly related strains. The emergence of this clone could be linked to selective sweeps associated with the reduction of genetic diversity in three regions within a large panel of human isolates. Up to now sex in bacteria has been assumed to involve mainly small regions; our results define S. agalactiae as an alternative paradigm in the study of bacterial evolution.

Group B streptococcus (GBS) is the most important cause of neonatal sepsis, which is mediated in part by TLR2. However, GBS components that potently induce cytokines via TLR2 are largely unknown. We found that GBS strains of the same serotype differ in released factors that activate TLR2. Several lines of genetic and biochemical evidence indicated that lipoteichoic acid (LTA), the most widely studied TLR2 agonist in Gram-positive bacteria, was not essential for TLR2 activation. We thus examined the role of GBS lipoproteins in this process by inactivating two genes essential for bacterial lipoprotein (BLP) maturation: the prolipoprotein diacylglyceryl transferase gene (lgt) and the lipoprotein signal peptidase gene (lsp). We found that Lgt modification of the N-terminal sequence called lipobox was not critical for Lsp cleavage of BLPs. In the absence of lgt and lsp, lipoprotein signal peptides were processed by the type I signal peptidase. Importantly, both the Δlgt and the Δlsp mutant were impaired in TLR2 activation. In contrast to released factors, fixed Δlgt and Δlsp GBS cells exhibited normal inflammatory activity indicating that extracellular toxins and cell wall components activate phagocytes through independent pathways. In addition, the Δlgt mutant exhibited increased lethality in a model of neonatal GBS sepsis. Notably, LTA comprised little, if any, inflammatory potency when extracted from Δlgt GBS. In conclusion, mature BLPs, and not LTA, are the major TLR2 activating factors from GBS and significantly contribute to GBS sepsis.

Genetic characterization of non-K1 Escherichia coli strains isolated from a mother and her neonate allowed us to provide evidence of the maternal origin of a late-onset neonatal infection. The use of ante- and peripartum antimicrobial prophylaxis with amoxicillin may have promoted the vertical transmission of this amoxicillin-resistant E. coli from mother to newborn. It allowed us to clarify the natural history of the disease.

A strain of Listeria monocytogenes, isolated from a patient with meningoencephalitis, was resistant to chloramphenicol, erythromycin, streptomycin, and tetracycline. The genes conferring resistance to these antibiotics were carried by a 37-kb plasmid, pIP811, that was self-transferable to other L monocytogenes cells, to enterococci-streptococci, and to Staphylococcus aureus. The efficacy of transfer and the stability of pIP811 were higher in enterococci-streptococci than in the other gram-positive bacteria. As indicated by nucleic acid hybridisation, the genes in pIP811 conferring resistance to chloramphenicol, erythromycin, and streptomycin were closely related to plasmid-borne determinants that are common in enterococci-streptococci. Plasmid pIP811 shared extensive sequence homology with pAM beta 1, the prototype broad host range resistance plasmid in these two groups of gram-positive cocci. These results suggest that emergence of multiple antibiotic resistance in Listeria spp is due to acquisition of a replicon originating in enterococci-streptococci. The dissemination of resistance to other strains of L monocytogenes is likely.

False-negative findings of polymerase chain reaction (PCR) for genuine pertussis as well as the numerous atypical forms of whooping cough make it difficult to diagnose this disease in young babies.For two years, real-time PCR was performed to test for Bordetella pertussis in 86 infants younger than 6 months hospitalized for apnea or paroxysmal and/or vomiting cough and in 205 of their household contacts, whether or not they coughed.Group 1 included 30 infants for whom PCR detected B. pertussis (25 of whom were also RSV+). PCR was also positive for at least one household contact in 25/30 families. This group included 16 babies with apnea and 12 who developed a whooping cough during follow-up. Group 2 comprised 12 infants whose PCR was negative while at least one household contact had positive results. Five of these infants had severe apnea and 6 developed a whooping cough. Group 3 included 44 infants (28 RSV +) for whom PCR was negative in the index case and in the household contacts: none developed a whooping cough during follow-up. Only 3 of the 54 positive household contacts had a paroxysmal cough or a typical whooping cough and 12 had no cough at all.Positive PCR in a household contact, symptomatic or not, is helpful for the diagnosis of atypical whooping cough in young infants.

During the winter 2005-2006, all infants

Objectives: Intrapartum antibiotic prophylaxis is recommended to prevent neonatal group B strepto- coccal (GBS) disease in colonized women, and penicillin or aminopenicillin constitute the first-line antibiotics. Most isolates are resistant to tetracycline, and resistance to macrolide-lincosamide- streptogramin (MLS) antibiotics is increasing. Therefore, laboratory testing for MLS resistance in GBS is now recommended for penicillin-allergic patients. The aim of this study was to compare the antimicrobial susceptibility of GBS as determined by the VITEK 2 system (bioMerieux, Marcy l'Etoile, France), agar diffusion methods and PCR-genotypic detection of resistance genes. Methods: One hundred and ten unrelated selected GBS clinical isolates were studied. The antibiotics tested (VITEK 2 and agar diffusion method) were benzylpenicillin, ampicillin, erythromycin, clinda- mycin, co-trimoxazole, tetracycline, kanamycin, streptomycin and vancomycin. A standardized double- disc (DD) diffusion test was performed for MLS antibiotics. Genotypic characterization of tetracycline, MLS and aminoglycoside resistance genes was performed by PCR. Results: All strains were susceptible to benzylpenicillin, ampicillin and vancomycin (category agree- ment (CA) between VITEK 2 and the diffusion method was 100%). Ninety-five (86%) strains were resistant to tetracycline (CA was 98.9%). Eighty-one strains (73.6%) harboured an MLS resistance phenotype; 50 (61.8%) an MLSB-constitutive phenotype, 25 (30.8%) an MLSB-inducible phenotype and 6 (7.4%) an M phenotype. The agreement between data of VITEK 2 and the DD diffusion test for the detection of MLSB-constitutive, MLSB-inducible and M phenotype isolates was 76%, 36% and 100%, respectively. Almost all discrepancies were due to failure to detect erythromycin resistance by VITEK 2. Conclusions: VITEK 2 allows accurate determination of GBS susceptibility for the majority of antibiotics, but has to be improved for erythromycin. Thus, the DD diffusion test remains the most simple and reliable method for macrolide resistance detection among this streptococcal species.

We developed a simple, specific, and sensitive two-multiplex-PCR assay that enabled the detection of all known group B streptococcal (GBS) capsular polysaccharides. This test is well adapted for GBS capsular polysaccharide typing in large-scale epidemiological studies.

This study investigated 41 infants, aged

The phylogenetically closely related species Streptococcus salivarius and Streptococcus vestibularis are oral bacteria that are considered commensals, although they can also be found in human infections. The relationship between these two species and the relationship between strains isolated from carriers and strains responsible for invasive infections were investigated by multilocus sequence typing and additional sequence analysis. The clustering of several S. vestibularis alleles and the extent of genomic divergence at certain loci support the conclusion that S. salivarius and S. vestibularis are separate species. The level of sequence diversity in S. salivarius alleles is generally high, whereas that in S. vestibularis alleles is low at certain loci, indicating that the latter species might have evolved recently. Cluster analysis indicated that there has been genetic exchange between S. salivarius and S. vestibularis at three of the nine loci investigated. Horizontal gene transfer between streptococci belonging to the S. salivarius group and other oral streptococci was also detected at several loci. A high level of recombination in S. salivarius was revealed by allele index association and split decomposition sequence analyses. Commensal and infection-associated S. salivarius strains could not be distinguished by cluster analysis, suggesting that the pathogen isolates are opportunistic. Taken together, our results indicate that there is a high level of gene exchange that contributes to the evolution of two streptococcal species from the human oral cavity.

International audience; Most bacteria require iron for growth. However, as it may not be directly available under aerobic conditions, bacteria may use iron-sequestering molecules, such as bacterially encoded siderophores, or heme, which is the major iron source in the animal host. Bacteria may also assimilate heme for purposes other than as an iron source. Once internalised, heme can activate, for example, a heme-dependent catalase and a cytochrome oxidase. In bacterial pathogen Streptococcus agalactiae, heme, in association with exogenous menaquinone, activates a respiratory chain. Respiration has radical effects on carbon metabolism. GBS respiration-grown cells display improved survival in an aerobic environment and greater virulence in a murine septicemia model. GBS might benefit from its ecological niches to capture heme and menaquinone, i.e., from other bacteria when it colonizes host mucosa, or from blood-containing organs during septicemia.

Streptococcus agalactiae is a leading cause of invasive infections in neonates, and responsible for bovine mastitis. It is also a commensal bacterium adapted to asymptomatic colonization of the mammalian gut and of the genitourinary tract. Here, we report the analysis of a collection of 75 strains of human and animal origin by using serotyping, multilocus sequence typing, whole genome DNA-array hybridizations and sequence comparison of putatively virulence-associated loci. Although the most variable parts of the genome are the previously predicted genomic islands, significant genetic variations were present in the genome backbone. Evolution within genes encoding surface and secreted proteins and those involved in the biosynthesis of different capsular types is mainly due to recombination events leading to the replacement of a locus of several genes or to the allelic exchange of the internal part of a gene. These two processes, which led to a broad diversity of surface protein patterns, are probably involved in the diversity of interactions with the host and its immune system. According to gene content comparisons and phylogeny, recent gene replacements by horizontal gene transfer may occur but are rare events. Although specific gene patterns, with respect to the origin of the strains and the epidemiological characteristics, were not identified, we show that the recently described hypervirulent ST-17 lineage is a homogeneous group. The study highlights for the first time that this lineage contains a specific and conserved set of surface proteins, probably accounting for its high capacity to cause infections in newborns.

Numerous Streptococcaceae produce an H2O-forming NADH oxidase, Nox-2, which has been generally implicated in aerobic survival. We examined the roles of Nox-2 in Group B Streptococcus (GBS), a leading agent of neonatal infections. While nox2 inactivation caused an aerobic growth arrest, no improvement was seen by addition of antioxidants to cultures, suggesting that this defect was not due to accumulation of toxic oxygen species. Using several approaches, we show that the observed inability of the nox2 mutant to grow aerobically is mainly due to an underlying defect in fatty acid (FA) biosynthesis: (i) the nox2 aerobic growth defect is fully and rapidly complemented by adding oleic acid to culture medium, and (ii) direct assimilation of this unsaturated FA in both wild type (WT) and nox2 GBS membranes is demonstrated and correlated with mutant growth rescue. We propose that NAD+ depletion in the nox2 mutant results in reduced acetyl-CoA production, which perturbs FA biosynthesis and hence blocks growth in aerobiosis. The nox2 aerobic growth defect was also complemented when GBS respiration metabolism was activated by exogenous haem and menaquinone. The membrane NADH oxidase activity generated by the functional respiratory chain thus compensates the cytoplasmic NADH oxidase deficiency. The nox2 mutant was attenuated for virulence, as assessed in lung, intraperitoneal and intravenous murine infection models. As the nox2 defect seems only to affect aerobic growth of GBS, its reduced virulence supports the suggestion that aerobic conditions and NADH oxidase activities are relevant to the GBS infection process.

Carbapenem-hydrolyzing β-lactamases can be metallo-β-lactamases (7), expanded-spectrum oxacillinases (8), or Ambler class A enzymes (1, 2, 4, 6, 9-12). The class A KPC β-lactamases hydrolyze all β-lactams except for cephamycins and have been rarely reported in studies of enterobacterial species (Klebsiella pneumoniae, Klebsiella oxytoca, Salmonella enterica, Enterobacter sp.) found in New England and the mid-Atlantic region of the United States; when they are reported, they are found to come from New York City hospitals in particular (2, 4, 6, 9-12). We report here a K. pneumoniae isolate from Paris that produced β-lactamase KPC-2. An 80-year-old man with a 5-year history of prostatic carcinoma and metastasis was admitted in February 2005 at the Cochin hospital in Paris for acute urine retention in the right nephrostomy. The bilateral nephrostomy was performed in December 2004 at a New York City hospital. No hospitalization or antibiotic treatment is known to have taken place during the period between the patient’s stay in New York and his emergency hospitalization in Paris. K. pneumoniae YC was isolated from urine and blood cultures and, according to disk diffusion susceptibility testing, was resistant or of intermediate susceptibility to all antibiotics except colistin and fosfomycin (3). No other K. pneumoniae isolates with similar antibiotic resistance patterns were recovered from the Cochin hospital during this same period of time. The MICs of β-lactams (3) for K. pneumoniae YC confirmed the disk diffusion results, with the MICs of expanded-spectrum cephalosporins and carbapenems being only slightly modified after the addition of clavulanic acid (Table ​(Table1).1). A crude β-lactamase extract of that isolate had significant imipenem hydrolysis activity (0.37 μmol of imipenem/min/mg of total protein) (5, 8). TABLE 1. MICs of β-lactams for K. pneumoniae YC, the transconjugant strain, and the reference strain Plasmid analysis detected two plasmids of >100 kb and ca. 75 kb in isolate YC. Using ampicillin (100 μg/ml) and sodium azide (100 μg/ml; Sigma-Aldrich, Saint-Quentin-Fallavier, France) as selective agents and sodium azide-resistant Escherichia coli J53 as the recipient strain, the ca.-75-kb plasmid was self-conjugative and conferred β-lactam resistance patterns for E. coli transconjugants similar with respect to the MICs obtained, except for those for cefoxitin, cefotaxime, ceftazidime, and aztreonam, which were lower than those for the parental strain (Table ​(Table1).1). A β-lactamase extract from a transconjugant culture subjected to analytical isoelectric focusing (8) identified two β-lactamases with pI values of 5.4 and 6.8. Using primers for the detection of Ambler class A and class B β-lactamase genes (7, 8), PCR experiments followed by sequencing identified a β-lactamase gene coding for the carbapenemase KPC-2 (pI 6.8) and the narrow-spectrum TEM-1 (pI 5.4), which were also identified from K. pneumoniae YC. Then, by the use of a series of successive PCR primers, the 2.8-kb surrounding sequences of the blaKPC-2 gene were found to be identical to those surrounding the same blaKPC-2 gene from a Salmonella isolate from Maryland (6). This report identified a carbapenem-hydrolyzing β-lactamase KPC in France that likely resulted from an intercontinental transfer of the KPC producer from the United States. Up to the time of this discovery, plasmid-mediated KPC enzymes had been a unique feature of the United States, whereas most other broad-spectrum antibiotic resistance genes have been reported in the United States after their initial discoveries elsewhere. The identification of KPC enzymes is worrisome, since they hydrolyze expanded-spectrum cephalosporins and carbapenems and thus could jeopardize therapy for serious infections caused by major nosocomial pathogens. In addition, their detection, based on careful interpretation of reduced carbapenem susceptibility in Enterobacteriaceae, remains difficult.

Streptococcus agalactiae (group B streptococcus [GBS]) is the leading cause of neonatal pneumonia, sepsis, and meningitis. An in silico genome analysis indicated that GBS strain NEM316 encodes 35 proteins containing an LPXTG motif which are thought to be covalently linked to the peptidoglycan by an enzyme called sortase. The role of these cell wall-anchored proteins in GBS pathogenesis was evaluated on a global level by inactivating the srtA gene. This gene encodes the major sortase SrtA that anchors most of the LPXTG-containing proteins. We chose the C5a peptidase (ScpB) and Alp2, an abundant immunogenic protein, as prototypical LPXTG-containing proteins. As expected, the SrtA knockout mutant was unable to anchor the C5a peptidase (ScpB) and Alp2 to the cell wall. Complementation with plasmid-borne srtA inserted into the chromosome restored the correct surface localization of both ScpB and Alp2. Interestingly, the SrtA mutant was impaired for binding to the major extracellular matrix components fibronectin and fibrinogen and displayed a significant reduction in adherence to human (A549, HeLa, and Caco-2) and murine (L2) epithelial cells compared to the parental wild-type strain. Surprisingly, the inactivation of srtA had no effect on the virulence of the type III strain of GBS in a neonatal rat model (measured by the 50% lethal dose and lung colonization) but strongly impaired the capacity of the strain to colonize the intestines of gnotobiotic mice in a competition assay. These results demonstrate that LPXTG-containing proteins are involved in cell adhesion and GBS persistence in vivo.

a real-time PCR assay for the detection of GBS from vaginal samples. Their test is based on the amplification of cfb, the gene encoding the Christie-Atkins-Munch-Petterson (CAMP) factor, and has been shown to be highly specific and sensitive. In the study reported here, we prospectively

Among 128 Streptococcus gallolyticus ( Streptococcus bovis ) isolates, 77.7% were resistant to tetracyclines and contained tet (M) and/or tet (L) and/or tet (O). A total of 59.4% had macrolide resistance and contained erm (B) and, rarely, mef (A). Among the one-third of isolates highly resistant to kanamycin and streptomycin, most harbored aphA3 and aad-6 genes.

Le streptocoque β-hemolytique du groupe A ( Streptococcus pyogenes , SGA) est un pathogene strictement humain responsable, dans la majorite des cas, d’infections benignes cutaneo- muqueuses comme l’angine et l’impetigo. Cependant, dans de rares cas, cette bacterie peut etre responsable d’infections invasives gravissimes (comme la fasciite necrosante qui se traduit par la destruction complete des tissus mous) et de syndromes de choc toxique, deux maladies souvent mortelles . En France, des donnees epidemiologiques recentes montrent que les infections invasives a SGA sont en augmentation depuis 2000, l’incidence des septicemies a SGA etant estimee en 2002 a 1,7 pour 100 000 habitants.