Abstract: Abstract To determine whether Staphylococcus aureus Panton-Valentine leukocidin (PVL) is expressed during human infection, anti-PVL antibody titres were compared in patients with PVL-positive and PVL-negative staphylococcal infections, and in patients with no evidence of S. aureus infection. Patients with PVL-positive strains had higher levels of anti-PVL antibodies than individuals of both control groups. The median anti-PVL titre increased 8.6-fold during the course of PVL-positive infection and 1.4-fold during PVL-negative infection. These results indicate that only PVL-positive S. aureus strains elicit significant anti-PVL antibody production in humans, and demonstrate the production of PVL during PVL-positive S. aureus infection. The protective role of this immune response remains to be established.
Abstract: OBJECTIVE: To examine the effect of intratracheal heparin instillation on Legionella pneumophila-related acute lung injury (ALI) and systemic dissemination. DESIGN: Prospective, controlled experimental study. SETTING: University research laboratory. INTERVENTIONS: A/J mice received 5 microg of sulfated heparin intratracheally co-instilled with 10(6) or 10(8) colony-forming units (CFU) of a virulent isolate of L. pneumophila. MEASUREMENTS AND RESULTS: ALI was assessed in control groups (PBS and PBS-heparin) and on days 1, 2 and 3 post-infection, in terms of the lung wet-to-dry (W/D) weight ratios and of lung endothelial permeability to radio-labeled albumin (Perm-I(125)). Lung bacterial loads were measured and systemic spread was assessed by blood and target organ culture. The alveolar inflammatory response was evaluated by measuring the cytokine levels (TNF-alpha, IFN-gamma, IL-6 and IL-12p70) in bronchoalveolar lavage fluids (BALF). Co-instilled heparin improved mouse survival after the 10(8) CFU challenge (p < 0.01). On day 2, heparin co-instillation significantly reduced the W/D ratio and Perm-I(125) (p < 0.01 and p < 0.001 respectively), improved lung bacterial clearance (p < 0.001), prevented systemic dissemination (blood, liver, spleen, kidneys and brain cultures, all p < 0.05) and significantly increased IFN-gamma and IL-12p70 levels in BALF (p < 0.05). CONCLUSIONS: Heparin co-instillation during intratracheal L. pneumophila challenge has a protective effect on the alveolar-capillary barrier and prevents bacterial dissemination. These results tend to confirm the competitive inhibition by heparin of L. pneumophila attachment to lung epithelium in vivo, and point to the possible involvement of a heparan-sulfate adhesin in L. pneumophila binding to pneumocytes.
Abstract: Legionella pneumophila is the causative agent of Legionnaires' disease. This bacterium is ubiquitous in aqueous environments and uses amoebae as an intracellular replicative niche. Real-time PCR has been developed for rapid detection of Legionella DNA in water samples. In addition to culturable bacteria, this method may also detect dead and viable but noncultivable (VBNC) legionellae. In order to understand the significance of positive PCR results in this setting, we prepared water samples containing known concentrations of L. pneumophila and analyzed them comparatively by means of conventional culture, real-time PCR, viability labeling, and immunodetection (solid-phase cytometry). We also examined the influence of chlorination on the results of the four methods. The different techniques yielded similar results for nonchlorinated water samples but not for chlorinated samples. After treatment for 24 h with 0.5 and 1 ppm chlorine, all cultures were negative, PCR and immunodetection showed about 10(6) genome units and bacteria/ml, and total-viable-count (TVC) labeling detected 10(5) and 10(2) metabolically active bacteria/ml, respectively. Thus, PCR also detected bacteria that were VBNC. The recoverability of VBNC forms was confirmed by 5 days of coculture with Acanthamoeba polyphaga. Therefore, some TVC-positive bacteria were potentially infective. These data show that L. pneumophila PCR detects not only culturable bacteria but also VBNC forms and dead bacterial DNA at low chlorine concentrations.
Abstract: Clonal analysis and PCR screening for the presence of Panton-Valentine leukocidin (PVL) genes was performed among 694 methicillin-resistant Staphylococcus aureus (MRSA) cases collected during a 2-y period in Greece. The detection rate of PVL-positive MRSA is high, both in the community and in hospital. Clonal analysis revealed the predominance among the PVL-positive strains of the clonal complex CC80 (ST80-IV) and the emergence of ST377 clone carrying agr1 allele and SCCmec type V.
Abstract: We report the case of a 12-year-old boy who developed staphylococcal toxic shock syndrome associated with S. aureus pharyngeal colonization or infection. The diagnosis was rapidly confirmed by detecting the Vbeta signature of the toxic shock syndrome toxin-1 in peripheral blood, based on transient T cell depletion rapidly followed by massive expansion of Vbeta 2-positive T cells.
Abstract: The capacity of Staphylococcus aureus strain LUG855 to release Panton-Valentine leukocidin (PVL) in the presence of sub-inhibitory concentrations of anti-staphylococcal drugs was examined. Oxacillin enhanced PVL release 2.5-fold, while clindamycin, linezolid, fusidic acid and rifampicin were inhibitory, and vancomycin, pristinamycin, tetracycline, ofloxacin and co-trimoxazole had no effect. In combination with oxacillin, sub-inhibitory concentrations of clindamycin or rifampicin inhibited PVL induction significantly, linezolid was less inhibitory, and fusidic acid did not inhibit PVL induction by oxacillin. These data support the use of oxacillin in combination with clindamycin, rifampicin or linezolid for the treatment of PVL-positive S. aureus infections.
Abstract: Staphylococcus aureus superantigenic toxins are responsible for menstrual and non-menstrual toxic shock syndrome (TSS). We compared the clinical and biological characteristics of 21 cases of menstrual TSS (MTSS) with 34 cases of non-menstrual TSS (NMTSS) diagnosed in France from December 2003 to June 2006. All 55 S. aureus isolates had been spontaneously referred to the French National Staphylococcal Reference Center, where they were screened for superantigenic toxin gene sequences. Most of the patients had previously been in good health. The most striking differences between MTSS and NMTSS were the higher frequency in NMTSS of neurological disorders (p=0.028), of S. aureus isolation by blood culture (50% versus 0% in MTSS), and the higher mortality rate in NMTSS (22% versus 0% in MTSS). The tst and sea genes were less frequent in isolates causing NMTSS than in those causing MTSS (p<0.001 and 0.051, respectively). Higher mortality was significantly associated with the presence of the sed gene (p=0.041), but when considering NMTSS survivors and non-survivors, no clinical or bacteriological factors predictive of vital outcome were identified. Specific antitoxinic therapy was rarely prescribed, and never in fatal cases. Higher mortality was observed in NMTSS than in MTSS, and no definite factors could explain the higher severity of NMTSS. NMTSS would require more aggressive therapy, comprising systematic rapid wound debridement, antistaphylococcal agents, including an antitoxin antibiotics, and intravenous immunoglobulin.
Abstract: In staphylococci, inducible macrolide-lincosamide-streptogramin B (MLS(B)) resistance is conferred by the erm(C) or erm(A) gene. This phenotype is characterized by the erythromycin-clindamycin "D-zone" test. Although clindamycin appears active in vitro, exposure of MLS(B)-inducible Staphylococcus aureus to this antibiotic may result in the selection of clindamycin-resistant mutants, either in vitro or in vivo. We have compared the frequencies of mutation to clindamycin resistance for 28 isolates of S. aureus inducibly resistant to erythromycin and bearing the erm(C) (n = 18) or erm(A) (n = 10) gene. Seven isolates susceptible to erythromycin or bearing the msr(A) gene (efflux) were used as controls. The frequencies of mutation to clindamycin resistance for the erm(A) isolates (mean +/- standard deviation, 3.4 x 10(-8) +/- 2.4 x 10(-8)) were only slightly higher than those for the controls (1.1 x 10(-8) +/- 6.4 x 10(-9)). By contrast, erm(C) isolates displayed a mean frequency of mutation to clindamycin resistance (4.7 x 10(-7) +/- 5.5 x 10(-7)) 14-fold higher than that of the S. aureus isolates with erm(A). The difference was also observed, although to a lower extent, when erm(C) and erm(A) were cloned into S. aureus RN4220. We conclude that erm(C) and erm(A) have different genetic potentials for selection of clindamycin-resistant mutants. By the disk diffusion method, erm(C) and erm(A) isolates could be distinguished on the basis of high- and low-level resistance to oleandomycin, respectively.
Abstract: Legionella pneumophila is a common cause of hospital and community-acquired pneumonia, being transmitted by inhalation of aqueous aerosols. Most outbreaks are linked to contaminated hot water systems and cooling towers. Our study was about the molecular typing of 35 strains of L. pneumophila including four clinical isolates and 31 environmental strains isolated from the distribution systems of 14 hotels. Among the clinical strains, two have the same pattern, however, all were different from the studied environmental strains. For the 31 environmental strains, ten patterns were obtained. Among which, a same pulsotype was found for four strains isolated from four different establishments. In addition, two different pulsotypes were found for strains isolated from the same establishment. The pulsed-field gel electrophoresis showed the existence of various patterns. Although cases of legionellosis were declared in these hotels, there are no epidemiological links between the clinical and environmental strains.
Abstract: Genomics can provide the basis for understanding the evolution of emerging, lethal human pathogens such as Legionella pneumophila, the causative agent of Legionnaires' disease. This bacterium replicates within amoebae and persists in the environment as a free-living microbe. 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 over 84% of Legionnaires' disease worldwide. Why L. pneumophila Sg1 is so predominant is unknown. Here, we report the first comprehensive screen of the gene content of 217 L. pneumophila and 32 non-L. pneumophila strains isolated from humans and the environment using a Legionella DNA-array. Strikingly, we uncovered a high conservation of virulence- and eukaryotic-like genes, indicating strong environmental selection pressures for their preservation. No specific hybridization profile differentiated clinical and environmental strains or strains of different serogroups. Surprisingly, the gene cluster coding the determinants of the core and the O side-chain synthesis of the lipopolysaccaride (LPS cluster) determining Sg1 was present in diverse genomic backgrounds, strongly implicating the LPS of Sg1 itself as a principal cause of the high prevalence of Sg1 strains in human disease and suggesting that the LPS cluster can be transferred horizontally. Genomic analysis also revealed that L. pneumophila is a genetically diverse species, in part due to horizontal gene transfer of mobile genetic elements among L. pneumophila strains, but also between different Legionella species. However, the genomic background also plays a role in disease causation as demonstrated by the identification of a globally distributed epidemic strain exhibiting the genotype of the sequenced L. pneumophila strain Paris.
Abstract: We report the first case of intrafamily transmission of a C-MRSA clone harbouring toxic shock syndrome toxin-1 (TSST-1). Because of the risk of this clone to spread in the community, family members of these patients should be screened to detect and prevent the diffusion of recurrent or new infections.
Abstract: Most clinical isolates of Staphylococcus aureus harbour genes encoding superantigenic toxins that bind the Vbeta domain of T-cells, but little information is available concerning superantigenic toxin production during staphylococcal toxic shock syndrome (TSS) and septic shock. This prospective study investigated 14 patients with staphylococcal TSS or septic shock; the toxin gene profile of each isolate was determined and flow-cytometry was used to identify the discriminant Vbeta signature (DVbetaS) of each superantigenic toxin in vitro. Attempts were also made to identify in-vivo production of superantigenic toxin DVbetaS in patients' blood. The DVbetaS identified in vitro were: toxic shock syndrome toxin (TSST)-1, Vbeta 2; staphylococcal enterotoxin (SE), Vbeta 9, Vbeta 22; SEB, Vbeta 3, Vbeta 14, Vbeta 17; SED, Vbeta 1, Vbeta 8; egc, Vbeta 5.3, Vbeta 7.1, Vbeta 9, Vbeta 23; and SElK, Vbeta 5.1. The DVbetaS of TSST-1 and SEB were detected in patients with menstrual and non-menstrual TSS, respectively, whereas no Vbeta signature was detected during septic shock. All patients with septic shock (but only one patient with TSS) had lymphopenia and/or impaired cellular immunity. Detection of a superantigenic toxin DVbetaS may help to show which toxin is produced during staphylococcal TSS, thus confirming the diagnosis and hastening the administration of anti-toxin therapy. In contrast, this approach failed to demonstrate superantigenic toxin involvement in cases of septic shock. In this latter condition, a superantigenic toxin may not be produced by S. aureus, or its production may occur without expansion of targeted T-cells because of T-cell apoptosis and/or anergy.
Abstract: Pus samples were prospectively collected from patients with Staphylococcus aureus skin infections and tested for Panton-Valentine leukocidin (PVL). PVL was detected at concentrations that were toxic for rabbit skin in all specimens from patients infected with strains harbouring PVL genes.
Abstract: OBJECTIVE: The aim of this study was to estimate the frequency of methicillin-resistant Staphylococcus aureus (MRSA) strains in the French community and the proportion of PVL-MRSA. DESIGN: A cross-sectional study was made during a 3-month period in 2003 through a network of private-sector, community-based medical laboratories selected throughout France: the Labville network. Each MRSA isolate was included and characterized by the Staphylococci national reference centre. The total number of S. aureus isolates was also collected. RESULTS: Among the 283 patients infected or colonized by MRSA, 166 (59%) were considered as healthcare-associated, 14 (5%) as nursing-associated, and 39 (14%) as community-acquired. The proportion of methicillin resistance among S. aureus was 14%. Taking into account the sampling design, the incidence of MRSA cases in French outpatients was estimated to be 0.50 (CI 95%: 0.41-0.60) per 10,000 inhabitants. The molecular analysis confirmed that 80.6% belong to the Lyon clone, the most prevalent hospital MRSA clone spreading in France, and 10.6% to a closely related clone. An emerging MRSA clone containing the tst1 gene was detected in six patients and the PVL positive ST80 clone only in one, 22-year old, patient. CONCLUSION: Most of MRSA cases diagnosed in the community in France in 2003 were elderly with specific risk factors and harbored hospital strains. The prevalence of PVL-MRSA remained low.
Abstract: We conducted a prospective multicenter study of methicillin-resistant Staphylococcus aureus (MRSA) isolates, including the first five consecutive clinical isolates, collected between September 2006 and February 2007 in 23 hospitals located throughout France (Fig. 1). The 111 isolates were tested for their antibiotic susceptibility patterns and were extensively characterized by screening for drug resistance and agr alleles, multilocus sequence typing (ST), staphylococcal cassette chromosome mec (SCCmec) typing, spa typing, and PCR profiling of 21 toxin genes. Clones were designated by their ST followed by their SCCmec type (I to VI). The Lyon clone ST8-IV or ST8-IV(variant) (n = 77; 69.4%) was widely distributed. Four minor clones were also detected, namely, the "classical" Pediatric clone ST5-IV (n = 9; 8.1%), the "new" Pediatric clone ST5-VI (n = 8; 7.2%), the clone Geraldine ST5-I(truncated) (n = 7; 6.3%), and the European clone ST80-IV (n = 4; 3.6%). The six other isolates were related to five rare clones. Relative to that of other European countries, the situation in France is marked by the predominance of a specific major clone and the worrying emergence of minor clones with enhanced virulence and new antibiotic susceptibility profiles.
Abstract: Methicillin-resistant Staphylococcus aureus is increasingly responsible for staphylococcal infections in the community. A large percentage of the community-acquired methicillin-resistant (CA-MRSA) strains in the USA produce Panton-Valentine leukocidin (PVL), which is associated with severe infections. The virulence of the clinical CA-MRSA strain USA300 was compared to that of its isogenic pvl-deleted mutant, and it was shown that PVL contributes to lung and muscle tissue destruction, respectively, in murine necrotizing pneumonia and skin infection models. Mice infected with the USA300 strain developed a dominant anti-PVL response. The PVL subunits were therefore tested as vaccinogens against this isolate, and their vaccine efficacy correlated with both the route of vaccination and infection. These data suggest that PVL is a virulence factor in murine CA-MRSA infections.
Abstract: Healthcare-associated infections (HAIs) have been a hot topic for several decades. An understanding of HAIs should be based on an understanding of the organisms that cause infection and determine prevention. Although some improvements in control in hospitals have been recorded, the community setting is now implicated, and the role of microbiology in diagnosis, detection of carriers and strain typing of organisms is evident. As healthcare systems vary widely, prevention strategies must be designed accordingly. Hand hygiene, however, remains applicable in all settings, and the WHO is strongly promoting alcohol-based hand rubs to interrupt transmission. Some countries are only beginning to develop standards, whereas compliance is obligatory in others. Economics and cost factors are common to all countries, and litigation is increasingly a factor in some.
Abstract: The aim of this study was to examine the production of superantigenic toxins in vivo and in vitro in two patients with streptococcal toxic shock syndrome (TSS). In the first patient, a woman with puerperal fever and Streptococcus pyogenes peritonitis, flow cytometry of blood cells and in vitro studies of the isolate showed massive expansion of Vbeta 2-positive T cells corresponding to SpeC production. In the second case, involving a patient with streptococcal TSS and purpura fulminans following non-steroidal anti-inflammatory drug (NSAID) therapy, no Vbeta expansion of T cells was observed in vivo, but the SpeC Vbeta signature was also detected in vitro. In this latter patient, NSAID administration and/or severe disseminated infection might partly explain the absence of Vbeta T cell expansion in vivo. Combined in vivo and in vitro detection of a superantigenic toxin Vbeta signature may be useful to determine which superantigenic toxin is involved in individual cases of streptococcal TSS.
Abstract: OBJECTIVE: The purpose of this study was to assess the virulence potential of Staphylococcus aureus strains isolated from diabetic foot ulcers and to discriminate noninfected from infected ulcers. RESEARCH DESIGN AND METHODS: Diabetic patients hospitalized in a diabetic foot department with a foot ulcer were prospectively enrolled if they had been free of antibiotic treatment over the previous 6 months. At admission, ulcers were classified as infected or noninfected on the basis of clinical examination, according to the International Working Group on the Diabetic Foot system. Only patients carrying S. aureus as the sole pathogen were included. In individuals with a grade 1 ulcer, a second bacterial specimen was obtained 1 month later. Using virulence genotyping markers, clonality tools, and an in vivo Caenorhabditis elegans model, we correlated the virulence of 132 S. aureus strains with grade, time of collection, and ulcer outcome. RESULTS: Among virulence genes, the most relevant combination derived from the logistic regression was the association of cap8, sea, sei, lukE, and hlgv (area under the curve 0.958). These markers were useful to distinguish noninfected (grade 1) from infected (grades 2-4) ulcers and to predict wound status at the follow-up. With use of the nematode model, S. aureus strains isolated from grade 1 ulcers were found to be significantly less virulent than strains from ulcers at or above grade 2 (P < 0.001). CONCLUSIONS: This study highlights the coexistence of two S. aureus populations on diabetic foot ulcers. A combination of five genes that may help distinguish colonized grade 1 from infected grade >or=2 wounds, predict ulcer outcome, and contribute to more appropriate use of antibiotics was discovered.
Abstract: The virulence of SCCmec type IV hospital-acquired methicillin-resistant Staphylococcus aureus (MRSA) clinical isolates belonging to the major sequence type 8 (ST8 [Lyon clone]) and to a minor upcoming clone, ST5, was compared with that of methicillin-susceptible S. aureus (MSSA) isolates of matching sequence types. In vitro adhesion to human airway epithelial cells (HAECs) as an indicator of dissemination and mortality in a murine sepsis model as an indicator of virulence were evaluated. Ten MRSA isolates and 8 MSSA isolates of ST8 and 8 MRSA isolates and 8 MSSA isolates of ST5 were characterized with respect to multilocus sequence type; agr, spa, and capsule typing; in vitro doubling time; toxin and adhesin gene profiles; and adherence to HAECs. Adherence was significantly lower in the MRSA ST5 group than in the ST8 groups. Infections with MRSA and MSSA isolates ST8 and ST5 were compared. No change in virulence related to the presence of SCCmec was observed, since ST8 but not ST5 caused a significantly lower mortality in its presence. Despite their similar genetic backgrounds, individual clonal MRSA and MSSA isolates were heterogeneous in adherence and virulence. No one of these specific virulence factors determined in vitro was related to mouse mortality. In conclusion, in a bacteremic model, mortality was dependent on the ST and was differentially modulated by SCCmec; within an ST, clonality was not associated with a homogenous outcome.
Abstract: PURPOSE: To identify bacterial agents in the aqueous humor of patients with postoperative endophthalmitis using eubacterial polymerase chain reaction (PCR) and conventional culture. SETTING: University Hospital of Lyon E. Herriot, Lyon, France. METHODS: Broad-range eubacterial PCR amplification followed by direct sequencing was used to identify microbial pathogens in ocular samples from 30 patients with acute or delayed-onset endophthalmitis, mainly after cataract surgery. Ocular samples included aqueous humor collected before the first intravitreal injection of antibiotics and vitreous samples collected at the time of the therapeutic pars plana vitrectomy. RESULTS: Cultures were positive in 32% of cases and PCR in 61% of cases with aqueous humor samples. When associated, culture and PCR of aqueous humor samples allowed for a microbiological diagnosis in 71% of cases. Microorganisms cultured by conventional techniques matched those identified by PCR. When applied on vitreous pretreated with intravitreal antibiotics, PCR increased the identification rate from 18% to 62%. CONCLUSIONS: Polymerase chain reaction assay of initial aqueous humor samples contributed to the diagnosis of endophthalmitis in 30% of cases. Previous use of intravitreal antibiotics did not seem to affect the ability to PCR-amplify DNA in the short term. Polymerase chain reaction-based technology was a useful adjunct to conventional culture because when used with aqueous humor samples only, the association of both techniques allowed for a microbiological diagnosis in 71% of cases of postoperative acute and delayed-onset endophthalmitis.
Abstract: We characterized 34 methicillin-resistant Staphylococcus aureus strains isolated in Paraguay in 2005. The strains belonged to two clones. The major clone (sequence type 5 [ST5] or ST221, spa type t149, staphylococcal cassette chromosome mec [SCCmec] type I) was similar to the Cordobes/Chilean clone spreading through South America, and the minor clone (ST239 or ST889, spa type t037, SCCmec type IIIA) was related to the Brazilian clone.
Abstract: We report 2 outbreaks of Panton-Valentine leukocidin-positive, doxycycline-resistant, methicillin-susceptible Staphylococcus aureus infections in French soldiers operating in Côte d'Ivoire. In a transssectional survey, nasal carriage of this strain was found in 2.9% of 273 soldiers about to be sent to Côte d'Ivoire and was associated with prior malaria prophylaxis with doxycycline.
Abstract: We determined the agr type, multilocus sequence type, protein A gene type (spa typing), toxin gene profile, and antimicrobial drug resistance profile of 469 isolates of Panton-Valentine leukocidin-positive community-acquired methicillin-resistant Staphylococcus aureus isolates (PVL-positive CA-MRSA). The isolates had been collected from around the world from 1999 through 2005 by the French National Reference Center for Staphylococci. We found that some continent-specific clones described in 2003, such as clone ST8, have now spread all over the world. Likewise, some PVL-positive CA-MRSA have spread to several countries on various continents. New clones have emerged (e.g., ST377) on new genetic backgrounds. PVL-positive CA-MRSA that were usually susceptible to most antistaphylococcal antimicrobial agents have acquired new resistance determinants (e.g., to gentamicin) in certain countries. The major trait shared by all these clones is a short staphylococcal chromosomal cassette mec element of type IV or V.
Abstract: Legionnaires disease, more formally known as legionellosis, is a relatively common form of severe pneumonia caused by Legionella, a genus of waterborne bacteria. Legionellosis is acquired by inhalation of legionellae from contaminated environmental sources. Legionella pneumophila serogroup 1 is responsible for more than 80% of cases in most countries. More than 1500 cases were reported in France in 2005. Initial diagnosis is based on tests for urinary antigens. The mortality rate for legionellosis depends on the promptness of appropriate antibiotic therapy. Macrolides (erythromycin or intravenous azithromycin, which is preferred to erythromycin for its better pharmacodynamic properties) and fluoroquinolones (levofloxacin) are the antibiotics of choice for severe legionellosis.
Abstract: Staphylococcal cassette chromosome mec (SCCmec) typing, in combination with genotyping of the Staphylococcus aureus chromosome, has become essential for defining methicillin-resistant S. aureus (MRSA) clones in epidemiological studies. We have developed a convenient system for SCCmec type assignment. The system consists of six multiplex PCRs (M-PCRs) for identifying the ccr gene complex (ccr), the mec gene complex (mec), and specific structures in the junkyard (J) regions: M-PCR with primer set 1 (M-PCR 1) identified five types of ccr genes; M-PCR 2 identified class A to class C mec; M-PCRs 3 and 4 identified specific open reading frames in the J1 regions of type I and IV and of type II, III, and V SCCmec elements, respectively; M-PCR 5 identified the transposons Tn554 and PsiTn554 integrated into the J2 regions of type II and III SCCmec elements; and M-PCR 6 identified plasmids pT181 and pUB110 integrated into J3 regions. The system was validated with 99 MRSA strains carrying SCCmec elements of different types. The SCCmec types of 93 out of the 99 MRSA strains could be assigned. The SCCmec type assignments were identical to those made with a PCR system that uses numerous primer pairs to identify genes or gene alleles. Our system of six M-PCRs is thus a convenient and reliable method for typing SCCmec elements.
Abstract: We evaluated a ready-to-use real-time quantitative Legionella pneumophila PCR assay system by testing 136 hot-water-system samples collected from 55 sites as well as 49 cooling tower samples collected from 20 different sites, in parallel with the standard culture method. The PCR assay was reproducible and suitable for routine quantification of L. pneumophila. An acceptable correlation between PCR and culture results was obtained for sanitary hot-water samples but not for cooling tower samples. We also monitored the same L. pneumophila-contaminated cooling tower for 13 months by analyzing 104 serial samples. The culture and PCR results were extremely variable over time, but the curves were similar. The differences between the PCR and culture results did not change over time and were not affected by regular biocide treatment. This ready-to-use PCR assay for L. pneumophila quantification could permit more timely disinfection of cooling towers.
Abstract: Staphylococcus aureus is a major cause of nosocomial and community-acquired infections. The predominance of two capsular polysaccharides, types 5 and 8, on the surface of clinical isolates led to the development of a conjugate vaccine (StaphVAX) based on capsular polysaccharides types 5 and 8 conjugated to a carrier protein. We have studied the capsular phenotypes and genotypes of 195 isolates representative of all clinical syndromes that encompassed both hospital and community-acquired infections. These isolates were mainly detected in France between January 2001 and December 2004. In this population, most of clinical isolates (87%) expressed either capsular polysaccharide type 5 (42%) or 8 (45%), whereas 13% were nontypeable by the serotyping method with antibodies specific to capsular polysaccharide type 5 or 8. These 26 nontypeable strains were further serotyped and were demonstrated to express the cell wall surface antigen 336, a polyribitol phosphate N-acetylglucosamine, which resembles cell wall teichoic acid. Among methicillin-resistant Staphylococcus aureus (MRSA) strains, we found a predominance of serotype 5 for 64% of strains, whereas MSSA isolates were predominantly capsular serotype 8 (60%). All S. aureus clinical isolates included in the present study have been investigated by PCR method, demonstrating that all isolates carried either the cap5 or the cap8 locus.
Abstract: The Staphylococcus aureus Panton-Valentine leukocidin (PVL) is a pore-forming toxin secreted by strains epidemiologically associated with the current outbreak of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) and with the often-lethal necrotizing pneumonia. To investigate the role of PVL in pulmonary disease, we tested the pathogenicity of clinical isolates, isogenic PVL-negative and PVL-positive S. aureus strains, as well as purified PVL, in a mouse acute pneumonia model. Here we show that PVL is sufficient to cause pneumonia and that the expression of this leukotoxin induces global changes in transcriptional levels of genes encoding secreted and cell wall-anchored staphylococcal proteins, including the lung inflammatory factor staphylococcal protein A (Spa).
Abstract: We examined the capacity of Staphylococcus aureus strains to release Panton-Valentine leukocidin (PVL) in the presence of antibiotics. No PVL was detected when S. aureus was incubated at inhibitory concentrations, while subinhibitory concentrations of oxacillin enhanced the PVL level; clindamycin, linezolid, and fusidic acid were inhibitory; and vancomycin had roughly no effect.
Abstract: BACKGROUND: Necrotizing pneumonia due to Panton-Valentine leukocidin-producing strains of Staphylococcus aureus is associated with a high mortality rate. We sought factors associated with vital outcome in 50 cases occurring from 1986 through 2005. METHODS: We compared the clinical and biological characteristics of 50 patients according to their vital outcome and examined the characteristics of the corresponding S. aureus isolates. RESULTS: The overall mortality rate was 56%, and the median survival time was 10 days. All of the deaths were attributed to S. aureus infection and were secondary to refractory shock and/or respiratory failure. Fatal outcome was associated with classical severity factors, such as the need for mechanical ventilation or inotrope support, and with onset of the acute respiratory distress syndrome. Airway bleeding was strongly associated with fatal outcome (P=.002). Patients who had focal staphylococcal infection before the onset of pneumonia had a significantly lower mortality rate (P=.002). The main biological feature associated with death was leukopenia (P<.001). In multivariate analysis, leukopenia and erythroderma occurring within the first 24 h after admission to the hospital were independently associated with fatal outcome. Erythroderma was not associated with toxic shock syndrome toxin. CONCLUSIONS: Airway bleeding, erythroderma, and leukopenia are associated with fatal outcome from Panton-Valentine leukocidin-positive S. aureus necrotizing pneumonia. More work is needed to develop more efficacious therapy against this highly lethal disease.
Abstract: BACKGROUND: The precise role of Staphylococcus aureus toxins and nasal carriage in common skin infections remains unclear. OBJECTIVES: To seek correlations between toxin expression, S. aureus nasal carriage and clinical manifestations in patients with community-acquired furuncles and impetigo. METHODS: From November 2004 to August 2005, we studied clinical data and bacteriological samples prospectively collected from 121 patients presenting with furuncles or impetigo. RESULTS: Sixty-four patients (31 with furuncles and 33 with impetigo) had S. aureus-positive skin culture. Panton-Valentine leukocidin (PVL) genes were present in 13 of 31 (42%) isolates from furuncles and were associated with epidemic furunculosis. Exfoliative toxin genes were present in 10 of 10 (100%) and 12 of 21 (57%) bullous and nonbullous impetigo isolates, respectively. Nasal carriage of S. aureus was found in 58% of patients overall. It was strongly associated with chronic furunculosis but not with simple furuncles (88% vs. 29%, P < 0.007). Skin and nose isolates from a given patient always had identical characteristics. Methicillin-resistant S. aureus accounted for four of 64 (6%) positive skin cultures. CONCLUSIONS: PVL is not involved in all types of furuncles but is associated with epidemic furunculosis. Both bullous and nonbullous forms of impetigo are associated with exfoliative toxins. Staphylococcus aureus nasal carriage is associated with the chronicity of furuncles.
Abstract: Panton-Valentine Leucocidin (PVL) is associated in the USA with community-acquired meticillin resistant strains of Staphylococcus aureus (CA-MRSA). Bone and joint infection due to such strains appears to be more severe, necessiting longer antibiotic course and various surgical procedure. Our study of 14 PVL positive bone and joint infection, performed in France where PVL is rarely (2/14) associated with meticillin resistance, demonstrates that severity is linked with PVL secretion more than with resistance. Considering PVL associated bone and joint infections as a toxin-mediated disease, prompt diagnosis is needed in order to start specific therapeutic procedures. PVL mediated infection could be evoked in front of severe acute osteomyelitis or arthritis, with radiological abnormalities present in the first days of evolution and with pejorative evolution despite antibiotic treatment. Evolution toward multifocal osteomyelitis and/or multiple abscesses seems to be a major characteristic of such infection. Therapeutic approach should use an association of parenteral antibiotics with at least one molecule active against protein synthesis like Clindamycin, associated with betalactams or Vancomycin in area of high incidence of CA-MRSA. Surgical procedure should be considered whenever focal abscesses of bones or adjacent tissue is detected and should be repeated in most cases.
Abstract: Microbial adherence to host cells is an early key step in the establishment of infection. During the course of Legionnaire's disease, Legionella interactions with host cells are best documented for resident macrophages. However, L. pneumophila can also replicate within type I and type II pneumocytes, which cover almost the entire alveolar surface. In the presence of zinc, we observed a significant and concentration-dependent increase in L. pneumophila adherence to and invasion of type II pneumocytes. The zinc-dependent adherence mechanism seemed to be host-cell-independent, as a similar increase in cytoadherence was observed with macrophages. We also found that zinc-dependent adherence of L. pneumophila appears to involve recognition of zinc-binding pneumocyte receptors by a bacterial adhesin, and heparan-sulfated host cell receptors, but not type IV pili.
Abstract: BACKGROUND: Panton-Valentine leukocidin (PVL) is a necrotizing toxin secreted by Staphylococcus aureus. PVL-positive S. aureus osteomyelitis and arthritis have been described. METHODS: We analyzed demographic, clinical, laboratory, microbiologic, and imaging data in a study group of 14 pediatric cases with PVL-positive S. aureus osteomyelitis and arthritis diagnosed between 2001 and 2005 and compared results with a control group of 17 pediatric cases of PVL-negative S. aureus osteomyelitis and arthritis treated in our institution during the same period. Treatments and outcome were studied. RESULTS: The severity of PVL-positive S. aureus bone and joint infections was indicated by the presence of severe sepsis in all cases and of septic shock in 6 of the 14 patients. By comparison, severe sepsis was not noted in the control group (P = 0.004). On admission, the median C-reactive protein value was significantly higher in the study group (202.6 mg/L versus 83 mg/L in the control group; P = 0.001). Eleven patients with PVL-positive infection had local extension of the infection by magnetic resonance imaging and 7 patients had severe deep-seated infectious complications by computed tomography. By contrast only 1 patient in the control group presented with bone abscess without extension and none had deep-seated infection (P < 0.001). The median length of hospitalization was 45.5 days in the study group versus 13 days in the control group (P < 0.001). The median duration of intravenous antibacterial chemotherapy was 48 days versus 11.3 days in the control group (P < 0.001). Ten patients (71%) of the study group required surgical procedures with a mean of 3 procedures (range, 1-5) whereas 3 patients (17%) of the control group required 1 surgical drainage each (P = 0.002). All the patients survived, but only 2 patients of the study group were free of long-term complications, whereas there were no long-term complications noted in the control group. CONCLUSION: PVL-positive S. aureus bone and joint infection is severe and requires prolonged treatment. Local complications are more frequent and often need repeated surgical drainage.
Abstract: The epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) at an international level shows that most MRSA strains belong to a few pandemic clones. At the local level, a predominance of one or two clones was generally reported. However, the situation is evolving and new clones are emerging worldwide, some of them with specific biological characteristics, such as the presence of Panton-Valentine leucocidin (PVL). Understanding these changes at the local and international levels is of great importance. Our objective was to analyze the evolution of MRSA epidemiology at multiple sites on a local level (Western Switzerland) over a period of 8 years. Data were based on MRSA reports from seven sentinel laboratories and infection control programs covering different areas. Pulsed-field gel electrophoresis was used to type MRSA isolates. From 1997 to 2004, a total of 2,256 patients with MRSA were reported. Results showed the presence of four predominant clones (accounting for 86% of patients), which could be related to known international clones (Berlin, New York/Japan, Southern Germany, and Iberian clones). Within the small geographic region, the 8-year follow-up period in the different areas showed spacio-temporal differences in the relative proportions of the four clones. Other international MRSA clones, as well as clones showing genetic characteristics identical to those of community-acquired MRSA (SCCmec type IV and the presence of PVL genes), were also identified but presumably did not disseminate. Despite the worldwide predominance of a few MRSA clones, our data showed that at a local level, the epidemiology of MRSA might be different from one hospital to another. Moreover, MRSA clones were replaced by other emerging clones, suggesting a rapid change.
Abstract: Staphylococcus aureus produces many virulence factors, most of which act in a synergistic and coordinated fashion. Some appear to be specifically associated with certain severe infections and are produced by meticillin-resistant Staphylococcus aureus (MRSA) clones distributed worldwide. Superantigenic exotoxins appear to be major virulence factors in hospital MRSA clones (HA-MRSA), and staphylococcal enterotoxin A (SEA) may be involved in the physiopathology of septic shock. Panton Valentine Leucocidin (PVL) has emerged as a major virulence factor in community-acquired Staphylococcus aureus (CA-MRSA) infections. In particular, the leukotoxic action of PVL is responsible for the high mortality rate associated with necrotizing pneumonia. CA-MRSA can also harbour the toxic shock toxin 1 (TSST-1) and rarely the exfoliative toxin.
Abstract: We report the association of a generalized pustular psoriasis and infection by Staphylococcus aureus which produced Panton-Valentine leukocidin in a 5-year-old child. Another S. aureus strain with the same toxin gene content was also isolated among three family members presenting with cutaneous lesions. Although a methicillin-resistant staphylococcal strain has been reported in association with pustular psoriasis, this is the first report of a Panton-Valentine leukocidin strain associated with generalized pustular psoriasis. The causal relationship between S. aureus produced Panton-Valentine leukocidin and skin lesions is discussed.
Abstract: Methicillin-susceptible Staphylococcus aureus (MSSA) strains can produce superantigenic toxins that may trigger a massive release of pro-inflammatory cytokines, which are involved in the onset of septic shock. This 1-year prospective pilot study assessed the role of the production of superantigenic toxins in the outcome of immunocompetent patients hospitalised for community-acquired MSSA bacteraemia. Thirty-seven patients were enrolled, of whom 14 died in hospital. Fourteen patients had septic shock, and the mortality rate in this subgroup was 56%. Twenty-seven (73%) isolates produced at least one superantigenic toxin, but this did not influence the rate of occurrence of septic shock or death.
Abstract: Methicillin-resistant Staphylococcus aureus (MRSA) clones harboring the toxic shock syndrome toxin 1 (tst) gene have been detected in France and in Switzerland since 2002. During a passive survey conducted between 2002 and 2003, we collected 103 tst-positive S. aureus isolates from 42 towns in France, of which 27 were resistant to methicillin. The tst-positive MRSA belonged to two clones: a major clone comprising 25 isolates of sequence type (ST) 5 and agr group 2 and a minor clone comprising two isolates of ST30 and agr3. The tst-positive MRSA clones were associated with both hospital-acquired (12 cases) and community-acquired (8 cases) infections. The MRSA clones were mainly isolated from children (overall median age, 3 years). They caused a variety of clinical syndromes, including toxic shock syndrome and suppurative infections. Both clones were found to harbor a type IV staphylococcal chromosomal cassette mec (SCCmec) and to have similar antibiotic resistance profiles (usually resistant to oxacillin, kanamycin, and tobramycin and with intermediate resistance to fusidic acid). The origin of these clones is unclear. The tst-positive agr2 MRSA clone has the same sequence type (ST5) of two pandemic nosocomial MRSA clones, namely, the Pediatric clone and the New York/Japan clone. These findings suggest that all these clones are phylogenetically related. The pulsotype of the tst-positive MRSA clones differed from that of methicillin-sensitive S. aureus (MSSA) clones by a single band involving the SCCmec element. These findings suggest that the tst-positive MRSA clones may have emerged from their respective MSSA counterparts.
Abstract: Quantitative Legionella PCRs targeting the 16S rRNA gene (specific for the genus Legionella) and the mip gene (specific for the species Legionella pneumophila) were applied to a total of 223 hot water system samples (131 in one laboratory and 92 in another laboratory) and 37 cooling tower samples (all in the same laboratory). The PCR results were compared with those of conventional culture. 16S rRNA gene PCR results were nonquantifiable for 2.8% of cooling tower samples and up to 39.1% of hot water system samples, and this was highly predictive of Legionella CFU counts below 250/liter. PCR cutoff values for identifying hot water system samples containing >10(3) CFU/liter legionellae were determined separately in each laboratory. The cutoffs differed widely between the laboratories and had sensitivities from 87.7 to 92.9% and specificities from 77.3 to 96.5%. The best specificity was obtained with mip PCR. PCR cutoffs could not be determined for cooling tower samples, as the results were highly variable and often high for culture-negative samples. Thus, quantitative Legionella PCR appears to be applicable to samples from hot water systems, but the positivity cutoff has to be determined in each laboratory.
Abstract: We report 4 cases of community-acquired infections due to Staphylococcus aureus producing Panton-Valentin leukocidin (SA-PVL) with uncommon multivisceral localizations. These cases highlight the need to screen for PVL in patients with serious staphylococcal infections. All patients were cured. Two of them received intravenous immunoglobulins in addition to antibiotics.
Abstract: We analysed 38 French isolates of Legionella anisa by means of pulsed-field gel electrophoresis (PFGE) with single or double digestion. Double digestion was more discriminatory than single digestion, and can thus be useful for epidemiological studies of L. anisa. Several isolates from different parts of France clustered together on the basis of their PFGE patterns (similarity cutoff of 80%), suggesting that the L. anisa population structure is homogenous or that a few clones of L. anisa strains have spread widely in France.
Abstract: 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.
Abstract: A community-wide outbreak of legionnaires disease occurred in Pas-de-Calais, France, in November 2003-January 2004. Eighteen (21%) of 86 laboratory-confirmed cases were fatal. A case-control study identified smoking, silicosis, and spending >100 min outdoors daily as risk factors for acquiring the disease. Legionella pneumophila strain Lens was isolated from cooling towers, wastewater, and air samples from plant A. This unique strain matched all 23 clinical isolates, as assessed by pulsed-field gel electrophoresis subtyping. Modeling of atmospheric dispersion of aerosols emitted from plant A cooling towers showed good coverage of the communes where patients lived and showed that the dispersion extended over a distance of at least 6 km from plant A. No other aerosol-producing installation was identified as a plausible source, and no common source of indoor exposure was found. These findings implicate plant A as the most likely outbreak source and suggest that the distance of airborne transmission of L. pneumophila may be greater than previously reported.
Abstract: This study focuses on the interest of the hypervariable 23S-5S ribosomal intergenic spacer region (ISR) of the genus Legionella to analyze the phylogenic diversity of Legionella at the species and subspecies levels and to identify isolates directly from clinical specimens. The method, using a real-time PCR assay with a single primer pair followed by sequencing, was able to identify correctly 49 reference strains of Legionella belonging to 37 different species, including those implicated in human infections, and to clearly differentiate the three subspecies of L. pneumophila. Based on sequence similarities, the 23S-5S ISR sequences were much more variable than the rpoB and mip sequences (P<0.0001 by the Wilcoxon signed rank test). The 23S-5S ISR method was able to cluster Legionella species in accordance with phenotypic traits, such as autofluorescence or fatty acid membrane composition. Using maximum parsimony methods, the rpoB and 23S-5S ISR data sets were shown to be incongruent (P<0.001). In contrast, the 23S-5S ISR and the mip data sets were found to be congruent (P=0.313), suggesting the interest of combining these two regions to demonstrate phylogenetic links between Legionella species. This molecular assay was shown able to both detect Legionella DNA directly in respiratory specimens from patients exhibiting a Legionella infection and provide accurate identification of the bacterium at the species level in the tested specimens. These properties open a wide range of applications to the 23S-5S ISR sequencing method, from taxonomic analyses to clinical and epidemiological investigations.
Abstract: The accessory gene regulator (agr) is a crucial regulatory component of Staphylococcus aureus involved in the control of bacterial virulence factor expression. We developed a real-time multiplex quantitative PCR assay for the rapid determination of S. aureus agr type. This assay represents a rapid and affordable alternative to sequence-based strategies for assessing relevant epidemiological information.
Abstract: BACKGROUND: Community-acquired skin and soft-tissue infections due to methicillin-resistant Staphylococcus aureus (MRSA) are an emerging clinical and epidemiological problem. OBJECTIVES: To characterize community-acquired skin infections caused by S. aureus, and especially MRSA. METHODS: From November 1999 to December 2003, we conducted in a French hospital a prospective epidemiological, clinical and bacteriological study of skin infections acquired in the community, applying strict criteria for true community-acquired MRSA (CA-MRSA) and health-care-associated MRSA (HCA-MRSA). RESULTS: One hundred and ninety-seven patients had 207 skin infections (154 primary and 53 secondary infections). Twenty-two (11%) patients had skin infections caused by MRSA. The incidence of MRSA skin infections acquired in the community rose from 4% in 2000 to 17% in 2003, but the increase was not statistically significant. Six patients (3%) were infected by CA-MRSA and 15 (8%) by HCA-MRSA; one patient was lost to follow-up and could not be classified. CA-MRSA and HCA-MRSA had different epidemiological, clinical and biological characteristics. CA-MRSA infections were more severe than HCA-MRSA infections: all the CA-MRSA infections (six of six, 100%) required surgical treatment, compared with only two (15%) of 13 with HCA-MRSA infection (P < 0.001). CA-MRSA all belonged to the same clonal strain, harbouring an agr type 3 allele and the Panton-Valentine leucocidin genes (not detected in HCA-MRSA) and possessing a specific antibiotype. CONCLUSIONS: Two populations of MRSA causing skin infections are emerging in the French community, with distinct epidemiological, clinical and biological characteristics.
Abstract: Current DNA amplification-based typing methods for bacterial pathogens often lack interlaboratory reproducibility. In this international study, DNA sequence-based typing of the Staphylococcus aureus protein A gene (spa, 110 to 422 bp) showed 100% intra- and interlaboratory reproducibility without extensive harmonization of protocols for 30 blind-coded S. aureus DNA samples sent to 10 laboratories. Specialized software for automated sequence analysis ensured a common typing nomenclature.
Abstract: Forty-five Panton-Valentine leukocidin (PVL)-positive, methicillin-resistant Staphylococcus aureus strains were isolated in Algeria between 2003 and 2004; 18 isolates were isolated in the community and 27 in a hospital. Five PVL-positive hospital isolates were resistant to multiple antibiotics, including ofloxacin and gentamicin for three isolates.
Abstract: To probe encephalopathy pathogenesis during toxic shock syndrome (TSS), we investigated the fate of bloodborne TSS toxin-1 (TSST-1) as it moves through the choroid plexus epithelium that forms the main blood-cerebrospinal fluid (CSF) barrier and the effect that TSST-1 has on choroidal barrier properties and on cultured neuronal cell viability. TSST-1 showed a slow, diffusional movement across a cellular model of the blood-CSF barrier but did not compromise the integrity of the barrier. Relevant to the acute symptoms of TSS, a combination of human leukocytes and the toxin induced a decrease in CSF clearance of the pyrogenic prostaglandin E(2) (PGE(2)). The direct effects that TSST-1 had on primary cortical neuron cultures and a neuronal cell line involved elevated caspase 3/7 levels, which correlated with an increase in neuronal cell death. The results of the present study suggest that TSST-1 can affect the brain, by inducing both an intracerebral increase in PGE(2) concentration and caspase-dependent neuronal death, which are possibly relevant to long-term intoxication.
Abstract: The aims of the study presented here were to determine the prevalence of Staphylococcus aureus carriage and, specifically, community-acquired methicillin-resistant S. aureus (CA-MRSA) carriage in children and their parents in Israel and to determine the genetic relatedness of these isolates. S. aureus was isolated from 580 of 3,373 (17.2%) individuals screened. The predominant type identified by pulsed-field gel electrophoresis was strain ST45-MSSA (25%). Five MRSA isolates were detected, and two of these were classified as CA-MRSA, based on the following criteria: no previous contact with a healthcare facility, absence of a multidrug-resistant (MDR) phenotype, and presence of SCCmec type IV. Isolates were negative for pvl and were classified as ST-45-MRSA. Although CA-MRSA is still rare in Israel, the genetic relatedness of the strains found in this study to a successful MSSA clone warrants close follow up.
Abstract: OBJECTIVE: To better understand the role of indirect transmission in community-acquired infection with methicillin-resistant Staphylococcus aureus (MRSA). DESIGN: Prospective case-control study. SETTING: A French teaching hospital. PATIENTS: A total of 198 case patients and 198 control patients with MRSA or methicillin-susceptible S. aureus infection diagnosed between April 2002 and July 2003. RESULTS: Multivariate analysis showed a highly significant independent link between MRSA infection at admission and prior receipt of home nursing care (odds ratio [OR], 3.7; P<.001). Other independent risk factors were prior hospitalization (OR, 3.8; P<.001), transfer from another institution (OR, 2.3; P=.008), and age older than 65 years (OR, 1.6; P=.04). Prior home nursing care showed a frequency dose-response relationship. Eleven MRSA-infected patients had had home nursing procedures but no hospital stay in the previous 3 years. These patients' MRSA strains were related to the prevalent MRSA clone currently spreading in French hospitals. CONCLUSION: Home nursing care appears to be an independent risk factor for MRSA acquisition in the community. The reservoir probably consists of MRSA carriers discharged from the hospital. Community nurses seem to be a potential vector.
Abstract: Staphylococcal chromosome cassette mec (SCCmec) elements within major lineages of healthcare- and community-associated methicillin-resistant Staphylococcus aureus (MRSA) clones were characterised using intra-SCCmec multilocus sequencing. A strong correlation was observed between sequence- and PCR-based typing methods (p <0.001). However, phylogenetic analysis of the SCCmec locus using concatenated sequences evidenced few recombination events. Sequence type (ST)-SCCmec1 was found in SCCmec elements types I and IV, suggesting the evolution of an SCCmecI element into an SCCmecIV element. This coincided with the spread of the clone harbouring this SCCmec element into the community. No correlation was observed between ST-SCCmec lineage and MRSA lineage, confirming multiple acquisitions of SCCmec by S. aureus. This was exemplified by the SCCmecIV ST-SCCmec10 element, which was detected in all of the clonal complexes examined, including healthcare- and community-associated MRSA. The acquisition of this SCCmec element was five- to ten-fold more common than that of others. Models of MRSA clone evolution suggest that this SCCmec was first found in the paediatric clone.
Abstract: The lvh region of the Legionella pneumophila genome, which encodes a type IV secretion system, is located on a plasmid-like element in strains Paris (pP36) and Philadelphia (pLP45). The pP36 element has been described either integrated in the chromosome or excised as a multi-copy plasmid, in a similar manner to pLP45. In this paper, the chromosomal integration of pP36 in the Paris strain genome was described, occurring through site-specific recombination at the 3' end of a transfer-messenger RNA gene by recombination between attachment sites, in a similar manner to pathogenicity islands. This integration was growth-phase dependent, occurring during the exponential phase. Several pP36-borne genes were expressed during the lag phase of bacterial growth, coinciding with the peak amount of the episomal form of pP36. Expression of the same genes decreased during the exponential and stationary phases, owing to the integration phenomenon and a loss of episomal copies of pP36. A similar plasmid-like element was described in the Lens strain genome, suggesting that the mobility of the lvh region is a phenomenon widespread among Legionella sp.
Abstract: Exfoliative toxin D (ETD) was identified recently as a new exfoliative toxin serotype. Like other exfoliative toxins, ETD induces intra-epidermal cleavage through the granular layer of the epidermis of neonatal mice. The distribution of ETD production was investigated in Staphylococcus aureus isolates from infected and colonised patients in France. The etd gene was found in 55 (10.5%) of 522 isolates tested. Isolates responsible for bullous impetigo and generalised staphylococcal scalded skin syndrome did not harbour etd, but etd was significantly more frequent in isolates causing cutaneous abscesses and furuncles. Most etd- and Panton-Valentine leukocidin-positive strains belonged to the clone of community-acquired methicillin-resistant S. aureus spreading currently throughout France.
Abstract: There has been renewed interest in the superantigens as antitumor agents with the discovery of a group of bacterial superantigens known as the enterotoxin gene cluster (egc staphylococcal enterotoxins [SEs]). This article discusses the mechanisms by which egc SEs induce tumor killing and pleurodesis. The application of SE homolog and nucleic acid compositions as vaccines and for treatment of established tumors is reviewed. Finally, the use of native SEs ex vivo-intratumorally and intravesicularly administered superantigens against established tumors-is described and the interrelation between superantigen therapy and chemoradiotherapy.
Abstract: Necrotizing pneumonia caused by Staphylococcus aureus strains carrying the Panton-Valentin leukocidin gene is a newly described disease entity. We report a new fatal case of necrotizing pneumonia. An S. aureus strain with an agr1 allele and of a new sequence type 377 was recovered, representing a new, emerging, community-acquired methicillin-resistant clone.
Abstract: The methicillin-resistant Staphylococcus aureus (MRSA) Lyon clone, detected throughout France, contains the enterotoxin A gene (sea), like other pandemic clones of clonal complex 8 (CC8). The egc locus was detected in MRSA pandemic clones of CC5, CC22, and CC45, occasionally with the toxic shock syndrome toxin 1 gene. The representative strain of the EMRSA-16 clone (CC30) harbored both sea and the egc locus.
Abstract: To test the hypothesis that the Staphylococcus aureus enterotoxin gene cluster (egc) can generate new enterotoxin genes by recombination, we analyzed the egc locus in a broad panel of 666 clinical isolates of S. aureus. egc was present in 63% of isolates, confirming its high prevalence. The archetypal organization of the egc locus, consisting of five enterotoxin genes plus two pseudogenes, was found in 409 of 421 egc-positive strains. The egc locus was incomplete in a few strains and occasionally harbored an insertion sequence and transposase genes. These strains may represent evolutionary intermediates of the egc locus. One strain with an atypical egc locus produced two new enterotoxins, designated SElV and SElU2, generated by (i) recombination between selm and sei, producing selv, and (ii) a limited deletion in the varphient1-varphient2 pseudogenes, producing selu2. Recombinant SElV and SElU2 had superantigen activity, as they specifically activated the T-cell families Vbeta 6, Vbeta 18, and Vbeta 21 (SElV) and Vbeta 13.2 and Vbeta 14 (SElU2). Immunoscope analysis showed a Gaussian CDR3 size distribution of T-cell receptor Vbeta chain junctional transcripts of expanded Vbeta subsets in toxin-stimulated cultures, reflecting a high level of polyclonality. These data show that egc is indeed capable of generating new superantigen genes through recombination.
Abstract: Staphylococcus aureus is a major human pathogen responsible for a variety of toxin-mediated and suppurative diseases. About 50 staphylococcal virulence factors have been described to date. In this review, we examine the clinical implications of key staphylococcal virulence factors in toxin-mediated diseases, septic shock, and severe focal infections such as arthritis, infective endocarditis, pneumonia acquired during mechanical ventilation, and necrotizing pneumonia. Staphylococcal pathogenicity is sometimes due principally to a single virulence factor, as in toxic shock syndrome and necrotizing pneumonia. In contrast, several virulence factors are involved in other staphylococcal disease, such as septic shock. A better knowledge of the mechanism of action of each virulence factor involved in the different staphylococcal diseases could open the way to the use of specific inhibitors in the clinical setting.
Abstract: Reported here is what is believed to be the first case of community-acquired pneumonia caused by a Panton-Valentine leukocidin-producing strain of Staphylococcus aureus in Spain. Although lung infections caused by S. aureus strains carrying this powerful leukocidin gene are associated with necrotizing pneumonia and high mortality, the previously healthy 28-year-old male reported here experienced a good clinical course and recovery. It is thought that the hematogenous origin of the infection, the patient's lack of any previous respiratory viral infection and prompt initiation of therapy may explain the satisfactory evolution in this case.
Abstract: Panton-Valentine leukocidin (PVL) is a pore-forming toxin secreted by Staphylococcus aureus that has recently been associated with necrotizing pneumonia. In the present study, we report that in vitro, PVL induces polymorphonuclear cell death by necrosis or by apoptosis, depending on the PVL concentration. PVL-induced apoptosis was associated with a rapid disruption of mitochondrial homeostasis and activation of caspase-9 and caspase-3, suggesting that PVL-induced apoptosis is preferentially mediated by the mitochondrial pathway. Polymorphonuclear cell exposure to PVL leads to mitochondrial localization of the toxin, whereas Bax, 1 of the 2 essential proapoptotic members of the Bcl-2 family, was still localized in the cytosol. Addition of PVL to isolated mitochondria induced the release of the apoptogenic proteins cytochrome c and Smac/DIABLO. Therefore, we suggest that PVL, which belongs to the pore-forming toxin family, could act at the mitochondrion level by creating pores in the mitochondrial outer membrane. Furthermore, LukS-PV, 1 of the 2 components of PVL, was detected in lung sections of patients with necrotizing pneumonia together with DNA fragmentation, suggesting that PVL induces apoptosis in vivo and thereby is directly involved in the pathophysiology of necrotizing pneumonia.
