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The presence of Panton-Valentine leukocidin expressing Staphylococcus aureus colonization was investigated with a qualitative nucleic acid hybridization assay among 122 children and 19 staff in a child care center. Genotyping of 5 Panton-Valentine leukocidin-positive isolates by pulsed-field gel electrophoresis revealed that one child and a teacher from the same class were colonized with the clonally related strains. This finding allowed us to suggest that close contact with colonized people is a risk factor for being colonized.

We describe a rare case of multifocal necrotizing fasciitis (NF) complicating a single vaccine injection. Injection of hepatitis B vaccine of a 16-year-old immunocompetent woman developed into rapidly spreading multifocal NF of the right arm and the thighs, with septic shock. Treatment with antimicrobial therapy and surgical debridements allowed amputation to be avoided with a favourable outcome. The etiological agent was a methicillin-sensitive Staphylococcus aureus (MSSA) isolate harboring the Panton-Valentine leukocidin (PVL) and five enterotoxins. PVL has recently been reported in large series of methicillin-resistant SA cases and has been associated with necrotizing infections. Some strains of MSSA could harbor PVL and enterotoxins. PCR investigation is not frequent but could improve the understanding of the mechanisms of lesions. This case is in keeping with the increasing incidence of MSSA harboring PVL and enterotoxins with multifocal dissemination NF and emphasizes the necessary precautions for skin decontamination before vaccine injection.

Staphylococcus aureus is a major cause of skin and soft tissue infections, such as furuncles, carbuncles, and abscesses, but it also frequently colonizes the human skin and mucosa without causing clinical symptoms. Panton-Valentine leukocidin (PVL) is a pore-forming toxin that has been associated with soft tissue infections and necrotizing pneumonia. We have compared the genotypes, virulence gene repertoires, and phage patterns of 74 furunculosis isolates with those of 108 control strains from healthy nasal carriers. The large majority of furunculosis strains were methicillin sensitive. Clonal cluster (CC) 121 (CC121) and CC22 accounted for 70% of the furunculosis strains but for only 8% of the nasal isolates. The PVL-encoding genes luk-PV were detected in 85% of furunculosis strains, while their prevalence among colonizing S. aureus strains was below 1%. luk-PV genes were distributed over several lineages (CCs 5, 8, 22, 30, and 121 and sequence type 59). Even within the same lineages, luk-PV-positive phages characterized furunculosis strains, while their luk-PV-negative variants were frequent among nasal strains. The very tight epidemiological linkage between luk-PV and furunculosis, which could be separated from the genetic background of the S. aureus strain as well as from the gene makeup of the luk-PV-transducing phage, lends support to the notion of an important role for PVL in human furunculosis. These results make a case for the determination of luk-PV in recurrent soft tissue infections with methicillin-sensitive as well as methicillin-resistant S. aureus.

We describe a case of fatal pneumonia in a previously healthy 14-year-old boy. The patient was severely affected at the time of admission with high fever, tachypnea, tachycardia and peripheral cyanosis. The condition worsened despite treatment with antibiotics as well as respiratory and pressure support. Acidosis and critical leucopenia supervened and the patient died just short of 24 hours after admission. Subsequent bacterial cultivation showed Panton-Valentine Leucocidin-producing Staphylococcus aureus.

Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA), which often produces Panton-Valentine leucocidin (PVL), has emerged worldwide as a life-threatening pathogen. Herein, we describe molecular characteristics of MRSA isolated from abdominal cellulitis in a 7-year-old Japanese boy. This MRSA was PVL-positive and belonged to the Taiwanese multiple drug-resistant CA-MRSA clone with the genotype of ST59, staphylococcal cassette chromosome mec (SCCmec) VII (SCCmecV, according to recent reclassification), agr1a (a novel agr1 subtype), and SaPI (which carried seb1, a newly designated variant seb gene). This study demonstrates the first isolation of the Taiwanese PVL-positive ST59 MRSA clone in Japan. The data also demonstrate novel subtypes in agr1 and seb and suggest that a combination of agr1a, seb1, and PVL could contribute to cellulitis (and its recurrence). Recently, a variety of PVL-positive MRSA clones are accumulating in Japan.

Group A streptococci (GAS) may engage different sets of virulence strategies, depending on the site of infection and host context. We previously isolated 2 phenotypic variants of a globally disseminated M1T1 GAS clone: a virulent wild-type (WT) strain, characterized by a SpeB(+)/SpeA(-)/Sda1(low) phenotype, and a hypervirulent animal-passaged (AP) strain, better adapted to survive in vivo, with a SpeB(-)/SpeA(+)/Sda1(high) phenotype. This AP strain arises in vivo due to the selection of bacteria with mutations in covS, the sensor part of a key 2-component regulatory system, CovR/S. To determine whether covS mutations explain the hypervirulence of the AP strain, we deleted covS from WT bacteria (DeltaCovS) and were able to simulate the hypervirulence and gene expression phenotype of naturally selected AP bacteria. Correction of the covS mutation in AP bacteria reverted them back to the WT phenotype. Our data confirm that covS plays a direct role in regulating GAS virulence.

Panton-Valentine leukocidin toxin producing Staphylococcus aureus (PVLSA) is known to be responsible for recurrent soft tissue infections and more serious invasive infections including necrotising pneumonia, pyomyositis, and osteomyelitis. Most reported cases involving musculoskeletal infection in adults are associated with methicillin-resistant S. aureus (MRSA) PVL-producing strains. We present the case of an adult male with PVL toxin-producing methicillin-sensitive S. aureus (MSSA) osteomyelitis of the tibia which has not previously been described in adults and highlight issues of recognition, treatment, and surgical management of PVLSA osteomyelitis.

The hospital epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) has changed in the past few years due to the encroachment of community-associated MRSA (CA-MRSA) strains into health care settings. MRSA strains that were isolated during a 2-year period from patients of the Luzerner Kantonsspital were analyzed to elucidate their epidemiology. Moreover, extended surveillance of individuals who were contacts of those patients was carried out for 6 months to identify the routes of spread and to assess the quality of the infection control measures used in our setting. Patient data were collected to distinguish CA-MRSA strains from health care-associated MRSA (HA-MRSA) strains by epidemiological criteria, as defined by the Centers for Disease Control and Prevention (CDC). On the basis of the CDC definition, the majority of the strains were considered to be HA-MRSA. However, 87% of them belonged to staphylococcal cassette chromosome mec (SCCmec) types IV and V, which are traditionally associated with CA-MRSA. Surprisingly, classical nosocomial SCCmec types I and II represented a minority, whereas SCCmec type III was completely absent. By PFGE analysis, four predominant clonal lineages and 21 highly variable sporadic genotypes were detected. Twenty-eight percent of the MRSA strains studied carried the genes encoding the Panton-Valentine leukocidin (PVL), of which 21% and 83% were associated with SCCmec types IV and V, respectively. Among 289 contact individuals screened for MRSA carriage throughout the extended surveillance, a single secondary patient was discovered. The possibility of nosocomial transmission could be excluded. The high proportions of SCCmec type IV and V strains as well as PVL-positive strains suggest strong infiltration of CA-MRSA into our institution. Moreover, the low endemic prevalence of MRSA demonstrates that current infection control measures are sufficient to limit its spreading and the emergence of large epidemic outbreaks.

Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) expressing Panton-Valentine leukocidin (PVL) have been characterized worldwide, but it has not been done in Egypt. In this study, we analyzed the molecular characteristics of PVL+CA-MRSA in Egypt, compared their genetic patterns with that of PVL+ methicillin-susceptible S. aureus and PVL+CA-MRSA from different countries, and investigated the allelic variations among their lukS-PV and lukF-PV gene sequences. The prevalence of PVL+MRSA was 19.04%. They belonged to different genetic clones with multi-locus sequence types (MLST) 30, 80, and the novel type 1010. ST80 strains showed unique antibiotic resistance profile that was distinguishable from the European ST80 clone: they had no resistance to tetracycline and fusidic acid. Two nonsynonymous substitutions in the lukS-PV gene region and two haplotype variants among these isolates were detected. We revealed that PVL is a good marker for CA-MRSA infections and that PVL+ isolates belonged to different genotypes., (Copyright 2009 Elsevier GmbH. All rights reserved.)

Using strepavidin as a scaffold, we have assembled a composite immunotoxin that consists of recombinant Pseudomonas exotoxin A subunit (PE38) and recombinant 25-D1.16 Fab fragment which recognizes the SIINFEKL (pOV8) peptide from ovalbumin in association with H-2K(b) MHC class I protein. The composite immunotoxin exercises cytotoxicity against H-2K(b+) cells sensitized with pOV8 peptide but not with irrelevant peptide. Specific binding of the immunotoxin to H-2K(b+) cells infected with recombinant rabies virus (RV) expressing pOV8 epitope (RV-pOV8) resulted in the suppression of the production of virus particles by the infected cells. This strategy allows readily produce different immunotoxins with desired specificity by combining various targeting and toxin molecules. The results provide a proof of concept that composite immunotoxins can be utilized as novel immunotherapeutics to stop virus spread in the acute phase of the infection allowing winning time for the development of protective immune response., (2009 Elsevier B.V. All rights reserved.)

Background: Postinfluenza Staphylococcus aureus pneumonias are increasingly recognized as a major form of life-threatening infections., Methods: A mouse model of postinfluenza S. aureus pneumonia was developed. Mice were intranasally infected with bacteria alone or bacteria plus virus. Infection was assessed by mouse survival, lung histopathology, bacterial density in the lungs, and cellular response to infection., Results: Mice infected with both influenza virus and S. aureus showed higher mortality, greater lung parenchymal damage, and greater bacterial density at metastatic tissue sites than mice infected with only S. aureus. At 4 h, more polymorphonuclear leukocytes and fewer CD11c(+) cells were found in lung samples from mice infected with virus and bacteria than in those from mice infected with bacteria. alpha-Hemolysin and protein A were maximally expressed 4 h after infection, and Panton-Valentine leukocidin was maximally expressed 72 h after infection, with higher levels of alpha-hemolysin expression in mice infected with bacteria alone. Interferon gamma expression was higher in tissue collected from mice infected with virus plus bacteria than in those from bacteria-infected mice., Conclusions: The results from this model demonstrate diverse effects caused by antecedent influenza virus infection, which have a profound influence on the morbidity and mortality associated with S. aureus pneumonia.

Objectives: Community-associated methicillin-resistant Staphylococcus aureus (MRSA) including those encoding Panton-Valentine leucocidin (PVL) are often described as more susceptible to a range of antibiotics than their hospital-associated counterparts. Recent scattered reports of the emergence of multiresistant PVL-MRSA have highlighted the potential for resistance to emerge. Here we detail polyclonal multiply antibiotic-resistant PVL-MRSA occurring in England., Methods: PVL-MRSA from community-based and hospitalized patients located across England were identified by PCR. Isolates were characterized via MIC determinations, toxin gene profiling, PFGE, SCCmec, spa and agr typing. Multilocus sequence typing (MLST) was performed on selected isolates. Patient demographic and available disease data were retained for analysis., Results: Seventy-six PVL-MRSA isolates resistant to three further classes of antibiotic were identified between 2005 and 2008 from centres in each of the Health Protection Agency's geographic regions in England. Patient demographics were typical for PVL-MRSA, and some travel associations were identified along with clonal spread. One instance of familial transmission in the community was detected. PVL-MRSA belonging to MLST clonal complex (CC) 1 (sequence type 772) were consistently highly resistant; multiply antibiotic-resistant representatives of CCs 5, 8, 22, 59 and 80 were also identified. Ciprofloxacin resistance was common amongst the study isolates (51 of 76 isolates)., Conclusions: Genetically diverse multiply antibiotic-resistant PVL-MRSA were identified, and included representatives of a recently emerged multiresistant clone (dubbed the Bengal Bay clone). Risk factors and disease presentations were typical for PVL-MRSA infections. This work highlights the diminishing utility of ciprofloxacin susceptibility for putative identification of PVL-MRSA.

Panton-Valentine leukocidin (PVL)-negative, SCCmec type IVa strains are the most common strains of methicillin-resistant Staphylococcus aureus (MRSA) circulating in the community in South Korea. This report describes five elderly patients presenting in 2006 to 2007 with invasive community-associated MRSA infection caused by a PVL-negative, SCCmec type IVa strain with sequence type 72 and spa type t324.

Introduction: Very little is known of the diversity and expression of virulence factors of serotypes of Aggregatibacter actinomycetemcomitans. Toxic activity on Chinese hamster ovary (CHO) cells and cdt and ltx genotyping were evaluated in A. actinomycetemcomitans serotypes., Methods: Forty-one A. actinomycetemcomitans isolates were analysed for CHO cell growth inhibition. Genotyping was performed by polymerase chain reactions specific to the ltx promoter region, serotype-specific and cdt region and by sequencing of cdtB., Results: cdtABC was detected in 40 strains. Analysis of the cdtA upstream region revealed 10 cdt genotypes. Toxicity to CHO cells was detected for 92.7% of the isolates; however, no correlation between the toxic activity and the cdt genotype was detected. Serotype c was more prevalent among Brazilian samples (68.0%). Four serotype b isolates from subjects with aggressive periodontitis were associated with high leukotoxin production and exhibited moderate to strong toxic activity in CHO cells, but were classified in different cdt genotypes. High levels of toxicity in CHO cells were not associated with a particular serotype; 57.1% of serotype a isolates presented low toxicity to CHO cells whereas the highly toxic strains belonged to serotypes b and c. Sequencing of cdtB revealed a single nucleotide polymorphism of amino acid 281 but this was not related to the toxic activity in CHO cells., Conclusion: Differences in prevalence of the low and highly cytotoxic strains among serotypes reinforce the hypothesis that serotype b and c isolates of A. actinomycetemcomitans are more virulent than serotype a strains.

Methicillin-resistant Staphylococcus aureus (MRSA) arises when methicillin-susceptible S. aureus (MSSA) acquires the staphylococcal cassette chromosome mec (SCCmec). Most pvl-positive MRSA in Taiwan belong to ST59 lineage and carry SCCmec V. The genetic profiles of 51 MSSA were compared with those of 80 MRSA from the same hospitals. Nine pvl-positive MSSA (oxacillin MIC < or = 2 microg/mL) shared >80% similarity in pulsed field gel electrophoresis pattern with 17 pvl-positive SCCmec V MRSA. Further investigation found that 5 of these 9 isolates were MRSA by cefoxitin and carried SCCmec V. All 26 pvl-positive isolates had very similar genetic profile (ST59, protein A clonal complex [spa-CC] c2:441/437, and agr group I). The success of the ST59:SCCmec V MRSA may be due in part to its heterogeneous and borderline resistance to methicillin, which may be missed by testing only oxacillin, with subsequent exposure to beta-lactams causing the emergence of more resistant subpopulations.

The transcriptional regulator Rgg of Streptococcus pyogenes is essential for expression of the secreted cysteine protease SpeB. Although all isolates of S. pyogenes possess the speB gene, not all of them produce the protein in vitro. In a murine model of infection, the absence of SpeB production is associated with invasive disease. We speculated that naturally occurring mutations in rgg, which would also abrogate SpeB production, may be present in invasive isolates of S. pyogenes. Examination of the inferred Rgg sequences available in public databases revealed that the rgg gene in strain MGAS315 (a serotype M3 strain associated with invasive disease) encodes a proline at amino acid position 103 (Rgg(103P)); in contrast, all other strains encode a serine at this position (Rgg(103S)). A caseinolytic assay and Western blotting indicated that strain MGAS315 does not produce SpeB in vitro. Gene-swapping experiments showed that the rgg gene of MGAS315 is solely responsible for the lack of SpeB expression. In contrast to Rgg(103S), Rgg(103P) does not bind to the speB promoter in gel shift assays, which correlates with a lack of speB expression. Despite its inability to activate speB expression, Rgg(103P) retains the ability to bind to DNA upstream of norA and to influence its expression. Overall, this study illustrates how variation at the rgg locus may contribute to the phenotypic diversity of S. pyogenes.

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) has become a matter of concern worldwide, in particular in the USA. For the analysis of emergence and spread, clear definitions based on epidemiological origin are needed for discrimination between CA-MRSA, healthcare-associated community MRSA, and healthcare-associated MRSA (HA-MRSA). Although its role in pathogenesis is currently under debate, the capability for Panton-Valentine leukocidin formation is associated with the majority of CA-MRSA isolates from North America and from Europe. Most CA-MRSA isolates are attributed to clonal lineages different from HA-MRSA; there are, however, clonal lineages from which both HA-MRSA and CA-MRSA have been reported (e.g. ST1, ST5, ST8, and ST22); CA-MRSA ST8 (USA300), which is most frequent in the USA, has meanwhile been reported from Europe. CA-MRSA ST80 is widely disseminated in Europe; because of its pronounced oxacillin heteroresistance phenotype, cefoxitin-based assays are advisable for reliable detection. So far, CA-MRSA infections seem to be much less frequent in Europe than in the USA, where patients with particular predispositions and low social status are at especial risk.

We evaluated clinical outcomes and molecular epidemiology of methicillin-resistant Staphylococcus aureus carrying SCCmecIV recovered from patients who attended at a teaching hospital from Porto Alegre, Brazil. All Panton-Valentine leukocidin (PVL)-producer isolates belonged to clonal complex (CC) 30 (11 isolates, related to Oceania Southwest Pacific clone [OSPC]), and the PVL-negative isolates were typed as CC5 (2 isolates, related to the pediatric clone). Five patients had health care-associated infections (HCAIs) with hospital-onset, 5 HCAIs with community-onset, and 3 community-acquired infections without risks. A high overall mortality (30.8%) was found. This study show that OSPC isolates are not only causing community-associated infections but are also involved in HCAI in our country.

Many strains of community-acquired meticillin-resistant Staphylococcus aureus (MRSA) have a pore-forming leukotoxin, known as Panton-Valentine leukocidin (PVL), which can cause severe necrotising pneumonia. Linezolid (LZD) is a new antibacterial agent with potent antibacterial activity against MRSA. In this study, a mouse model of haematogenous pulmonary infection was used to compare the efficacies of LZD and vancomycin (VAN) against pulmonary infection caused by PVL-positive S. aureus. Following antibiotic administration for 3 days, the number of viable bacteria (mean+/-standard error of the mean) in the control, VAN and LZD groups was 6.77+/-0.14, 5.29+/-0.27 and 4.25+/-0.33 log colony-forming units/lung, respectively. LZD significantly decreased the number of viable bacteria in the lungs compared with the control and VAN groups (P<0.05). The survival rate at Day 7 post-inoculation was higher in the LZD group (100%) than in the VAN group (50%) or the control group (0%). Histopathological examination and cytokine analysis also showed the beneficial efficacy of LZD compared with VAN. In conclusion, LZD significantly reduced bacterial numbers and inflammation in a mouse model of PVL-positive S. aureus haematogenous infection and improved the survival rate of infected mice compared with VAN. LZD is clinically effective against PVL-positive S. aureus.

Streptococcus pyogenes (group A streptococcus [GAS]) is a versatile human pathogen, and emm1/sequence type 28 (ST28) is the most frequently isolated type from GAS infections. The emm1/ST28 strain is associated with necrotizing fasciitis and streptococcal toxic shock syndrome. Growth-phase regulation is one of the important regulatory mechanisms in GAS, which controls gene expression at restricted phases of growth. CovRS, a two-component regulatory system, is considered the regulator of streptococcal pyrogenic exotoxin B (SpeB) and is thought to be activated in the exponential phase of growth. In the present study, Northern hybridization analysis showed that 52% of the analyzed GAS strains expressed covR at the exponential phase, but 48% of the strains expressed covR at the early stationary phase of growth. Strains transcribing covR at the early stationary phase showed better growth and earlier SpeB expression than the other group of strains. Multilocus sequence typing and pulsed-field gel electrophoresis analysis showed only emm1/ST28 strains (which comprise a clonal cluster) were expressing covR at the early stationary phase of growth, indicating that emm1/ST28 strains have special characteristics which may be related to their worldwide distribution.

We report a patient who presented a severe Panton-Valentine-secreting methicillin-susceptible Staphylococcus aureus pneumonia with threatening multi-organ failure including acute respiratory distress syndrome, cardiac failure, renal failure and disseminated intravascular coagulation. Clinical and biological disease course using empiric therapy with treatment directed against toxin production (linezolid, clindamycin and intravenous immunoglobulins) was found to be quickly effective.

Several recent reports have suggested that community-associated meticillin-resistant Staphylococcus aureus (MRSA) clones, particularly those harbouring genes for Panton-Valentine leukocidin (PVL) or toxic shock syndrome toxin 1 (TSST-1), are increasingly responsible for infections in hospitals. Here, a retrospective study was carried out to investigate whether antimicrobial resistance patterns could be used to detect these pathogens in a French university hospital. Isolates were characterized by antimicrobial susceptibility testing, PCR profiling (PVL genes and tst), PFGE typing and multilocus sequence typing. Demographic and clinical data were collected from all patients. For PVL-positive MRSA, the typical antimicrobial resistance pattern (susceptible to fluoroquinolones, non-susceptible to fusidic acid, kanamycin resistant and susceptible to gentamicin and tobramycin) had a sensitivity of 77.8 % and a positive predictive value (PPV) of 100 %. For tst-positive MRSA, the antimicrobial resistance pattern (susceptible to fluoroquinolones and non-susceptible to fusidic acid) had a sensitivity of 100 % and a PPV of 72.4 %. These results suggest that phenotypic rules based on antimicrobial resistance patterns are potentially useful for the detection of PVL- and tst-positive MRSA isolates.

sThe role of Panton-Valentine leukocidin (PVL) in Staphylococcus aureus pathogenesis is controversial. Here, we show that an unintended point mutation in the agr P2 promoter of S. aureus caused the phenotypes in gene regulation and murine pneumonia attributed to PVL by earlier investigators. In agreement with other studies that failed to detect similar effects of PVL using community-associated methicillin-resistant S. aureus strains, we found no significant effect of PVL on gene expression or pathogenesis after we repaired the mutation. These findings provide further evidence that PVL does not have a major impact on S. aureus pathogenesis. Moreover, our results demonstrate that a single nucleotide polymorphism in an intergenic region can dramatically affect bacterial physiology and virulence. Finally, our work emphasizes the need to frequently evaluate the integrity of the S. aureus agr locus.

We aimed to enhance our case ascertainment of meticillin-resistant Staphylococcus aureus encoding Panton-Valentine leucocidin (PVL-MRSA), determine the patient demographic, risk factor and disease associations, and define the clonal diversity amongst isolates referred to the UK Health Protection Agency's Staphylococcus Reference Unit. PVL-MRSA collected during 2005-6 from community-based and hospitalised patients located across England and Wales were identified by polymerase chain reaction (PCR). Representative geographically and temporally unrelated isolates were characterised via toxin gene profiling, SCCmec, spa and agr typing, multilocus sequence typing (MLST) and minimum inhibitory concentration (MIC) determinations. PVL-MRSA were identified from 275 patients. Affected individuals were <1 to 95 years of age (mean 30, median 27 years). Forty-five isolates were from 18 household or community-based clusters and 23 isolates were from outbreaks in healthcare settings. Overall, 58% (n = 161) had skin and soft tissue infections and 9% (n = 25) presented with or developed more serious disease, including eight patients (3%) with necrotising pneumonia, five of whom subsequently died. PVL-MRSA were genetically diverse and harboured SCCmecIV or V(T)/VII. Representatives of MLST clonal complexes (CCs) 8, 30 and 80 were identified the most often. The 275 PVL-MRSA included internationally disseminated community-associated MRSA (CA-MRSA) strains, as well as other minor lineages, and were associated with typical risk factors and disease presentations.

Musculoskeletal infections caused by Panton-Valentine Leukocidin (PVL) secreting Stapylococcus aureus in children and adolescents have previously been reported. We report the first adult case in a 26 year-old British Army soldier who presented with a S. aureus septic arthritis. He was treated by surgical washout and antibiotics and discharged but was readmitted five months later with an ipsilateral femoral osteomyelitis requiring debridement. The causative S. aureus grown from tissue biopsy taken at time of surgery was found to encode the PVL gene. Whilst there is evidence that soldiers in Iraq have a greater rate of S. aureus colonisation on their skin, the proportion that encode the PVL gene is similar to that observed in the UK. Soldiers are however, subject to the known risk factors that increase vulnerability to PVL secreting S. aureus infection. Military clinicians need to be aware of PVL secreting S. aureus and have a low threshold for requesting specific testing in aggressive musculoskeletal S. aureus infections.

Panton-Valentine leukocidin (PVL) is a cytotoxin produced by Staphylococcus aureus that exhibits highly specific lytic activity against polymorphonuclear cells, monocytes, and macrophages. A 34-year-old man admitted for right parietal brain abscess and thickened dura mater in close proximity to a lytic bone lesion is presented. The abscess culture yielded methicillin-sensitive S. aureus that produced PVL. The patient did well after surgery and antibiotic treatment. A hematogenous infection, route of bone infection with progression to dura mater and brain parenchyma was hypothesized. To our knowledge this is the first reported case of a brain abscess due to PVL-positive S. aureus.

Pseudomonas aeruginosa exotoxin A (ETA) production depends on the virulence-factor regulator Vfr. Recent evidence indicates that the P. aeruginosa iron-starvation sigma factor PvdS also enhances ETA production through the ETA-regulatory gene regA. Mutants defective in vfr, regA and pvdS, plasmids that overexpress these genes individually and lacZ transcriptional/translational fusion plasmids were utilized to examine the relationship between vfr, regA and pvdS in regulating P. aeruginosa ETA production. ETA concentration and regA expression were reduced significantly in PAODeltavfr, but pvdS expression was not affected. Overexpression of Vfr produced a limited increase in ETA production in PAODeltapvdS, but not PAODeltaregA. Additionally, overexpression of either RegA or PvdS did not enhance ETA production in PAODeltavfr. RT-PCR analysis showed that iron did not affect the accumulation of vfr mRNA in PAO1. These results suggest that: (i) Vfr enhances toxA expression in PAO1 both directly and indirectly through regA, but not through pvdS; (ii) vfr expression is not regulated by iron; and (iii) both Vfr and PvdS cooperate in the presence of RegA to achieve a maximum level of toxA expression.

A gene unique to Streptococcus pyogenes, called vfr, that negatively regulates speB, an important extracellular proteinase, has been identified. Disruption of vfr markedly increased SpeB production in a clinical strain of S. pyogenes and relieved its growth phase dependency. These findings may provide important insights into the pathogenesis of invasive S. pyogenes infections.

Community-Acquired Methicillin-Resistant Staphylococcus aureus (CA-MRSA) is a worldwide emerging pathogen that is able to produce serious skin and soft- tissue infections such as necrotizing fasciitis, as well as pneumonia and osteomyelitis. We present a 14 month child with necrotizing fasciitis, confirmed by magnetic resonance imaging, produced by CA-MRSA Panton-Valentine leukocidin producer. The clinical outcome was good after early surgical treatment and the administration of intravenous clindamycin for two weeks. We review microbiological aspects and treatment guidelines of these infections.

Objectives: The rapid emergence of methicillin-resistant Staphylococcus aureus from the community (CA-MRSA) presents difficulties in making treatment choices. We evaluated whether combining another orally available agent commonly used to treat CA-MRSA with gemifloxacin would enhance gemifloxacin activity against CA-MRSA., Methods: Fifty strains of SCCmec IV, agr group 1, Panton-Valentine leucocidin-positive CA-MRSA were evaluated for susceptibilities to gemifloxacin, trimethoprim/sulfamethoxazole, doxycycline, levofloxacin, rifampicin, clindamycin and erythromycin. Twenty of these strains were evaluated for the potential for synergy between gemifloxacin and trimethoprim/sulfamethoxazole, clindamycin and rifampicin by time-kill analysis. Two strains were further evaluated in an in vitro pharmacokinetic/pharmacodynamic (PK/PD) model., Results: In time-kill analyses, gemifloxacin combined with trimethoprim/sulfamethoxazole produced additivity (6/20) or synergy (11/20) in 85% of the isolates tested. The addition of clindamycin to gemifloxacin showed additivity (3/20) or synergy (2/20) in 25% of the isolates. All isolates displayed indifference to the combination of gemifloxacin and rifampicin. In the PK/PD model, combining gemifloxacin and trimethoprim/sulfamethoxazole provided potent and sustained bactericidal activity to detection limits of 2 log(10) cfu/mL by 48 h; gemifloxacin combined with clindamycin or with rifampicin killed to detection limits by 56 h or later. One isolate developed efflux-mediated resistance to gemifloxacin at 96 h with gemifloxacin monotherapy. All combinations prevented the emergence of this resistance., Conclusions: Synergy or additivity was demonstrated by time-kill analysis between gemifloxacin and trimethoprim/sulfamethoxazole in most isolates tested. In the PK/PD model, the addition of trimethoprim/sulfamethoxazole, clindamycin and rifampicin enhanced the activity of gemifloxacin against CA-MRSA and suppressed the emergence of resistance to gemifloxacin.

Objectives: Panton-Valentine leucocidin (PVL)-positive Staphylococcus aureus are responsible for causing skin and soft tissue infections, with the potential to cause severe invasive disease. Recently, methicillin-resistant Staphylococcus aureus (MRSA) strains that produce PVL have emerged in the community. As residents of care homes are a key group at risk of MRSA colonization and infection, we have examined the epidemiology of MRSA in three large cohorts of residents in urban care homes to establish whether PVL-positive MRSA strains are present in this setting., Methods: Nasal swabs (n = 3037) collected from consenting residents of 69 care homes in Leeds, UK, were screened for MRSA using chromogenic agar over three periods (June-August 2005, November-December 2006 and October-November 2007). PCR amplification was used to detect genes encoding PVL. Antibiogram profile and PFGE were also used to characterize MRSA isolates (n = 601)., Results: MRSA prevalence was 21%, 20% and 19% in each cohort, respectively. The majority of the isolates were related epidemiologically to the predominant local nosocomial epidemic MRSA strain, EMRSA-15 (78%). No isolate carried the genes encoding PVL. Twelve percent of the isolates (n = 74) had increased susceptibility to non-beta-lactam agents and were distributed across 31 care homes., Conclusions: MRSA strains that produced PVL were not found to be colonizing residents of care homes between 2005 and 2007. Continued surveillance is, however, necessary to understand the interaction between MRSA in care homes and hospitals, especially to reduce the chance that the former may amplify community-associated MRSA strains.

The proportion of methicillin-resistant Staphylococcus aureus (MRSA) in Denmark has been below 1% for more than 30 years. However, a marked increase in community-onset MRSA (CO-MRSA) started in 2002. To identify possible risk factors for CO-MRSA infections, a nationwide case-control study was conducted in 2004. Cases (34) were patients with CO-MRSA infections; controls (87) were patients with community-onset methicillin-sensitive S. aureus infections (CO-MSSA). Demographic and clinical data and exposures to possible risk factors during the last 24 months were collected with a structured telephone-administered questionnaire. Skin and soft tissue were the predominant sites of infection, both for cases (68%) and for controls (60%). A large proportion of cases (26%) and controls (38%) had an underlying skin disease. The majority of cases (76%) and controls (61%) had received antibiotics within the last 6 months, and 51% and 31%, respectively, had been hospitalized within the previous year. In a multivariate analysis, non-Danish origin, defined as being from or having parents from outside Denmark, was the only independent risk factor for CO-MRSA infection (OR 30.5, 95% CI 3.6-257.3). Prior hospitalization for >7 days within the previous 6 months tended to be associated with CO-MRSA infection (OR 5.7, 95% CI 0.9-36.4). The predominant MRSA clones found in this study were CC80 (26%), CC8 (24%) and CC5 (18%). Resistance to three or more antimicrobial drug classes was seen in 47% of CO-MRSA isolates. Panton-Valentine leukocidin was found in 47% of CO-MRSA isolates. Apart from a non-Danish origin, CO-MRSA shared the same risk factors as CO-MSSA, which makes control a challenge.

We determined the entire nucleotide sequence of phiSa2958-carrying Panton-Valentine leukocidin (PVL) gene, which was lysogenized in a sequence type 5 staphylococcal cassette chromosome mec (SCCmec) type II strain of methicillin-resistant Staphylococcus aureus (MRSA). Based on the nucleotide sequences of PVL phages, we developed PCRs to discriminate among five PVL phages, with a preliminary classification into two morphological groups (elongated-head type and icosahedral-head type) with four PCRs, including two PCRs for identifying the gene lineage between lukS-PV and the tail gene. The phages were then classified into five types by four PCRs identifying each phage-specific structure. With these PCRs, we examined the PVL phage types of 67 MRSA strains isolated in Japan from 1979 through 1985 and since 2000 and found that two morphologically distinct phages were predominant in Japan. The icosahedral-head-type phage, represented by the phi108PVL type, was identified for 39 of 53 strains isolated from 1979 through 1985. Of 26 other Japanese isolates, 25 belonged either definitively or presumably to elongated-head types as follows: 3 belonged to the phiSa2958 type; 8 were determined to belong to an elongated-head type, but a determination of greater specificity was not made; and 14 belonged to a phiSa2958-like phage of unknown type. We induced prophages by treatment with mitomycin C from six strains of the phiSa2958 type or of phiSa2958-like unknown-type phages; five of six strains carried intact PVL-carrying phages, which can infect other S. aureus strains and might generate novel PVL-positive strains of S. aureus. That various SCCmec elements were carried by different strains of the same phage type suggests that S. aureus strains might independently acquire PVL phages before they acquire various SCCmec elements.

Background: Community-acquired pneumonia due to Panton-Valentine producing S.aureus is a serious infection recently described. Many cases have been reported worldwide., Aim: We report here the first case in Tunisia., Observation: Our patient is a previously healthy fourteen-year-old girl hospitalized for bilateral hypoxemic pneumonia. The clinical course had violently deteriorated two hours later, marked by massive hemoptysis that lead to rapid degradation of her hemodynamic state and death. Toxicologic research and bloodcultures were negatives. A post-mortem pleural specimen culture yielded a meticillin-resistant Staphylococcus aureus strain that carried the Panton-Valentine leucocidin genes., Conclusion: Community-acquired pneumonia due to Panton-Valentine producing Staphylococcus aureus is a serious affection unrecognized in our country. Thus, this pathogen must imperatively be included in the spectrum of those responsibles for pulmonary infections in children and young adults

Background: The predominant genetic background of community-associated methicillin-resistant Staphylococcus aureus has transitioned from USA400 to USA300 in most US communities. The explanation for this shift is unclear. We hypothesized that USA300 must be more pathogenic--specifically, that USA300 would have increased virulence when compared with USA400 in an animal model., Methods: Rats were inoculated intratracheally with 1 of 6 S. aureus isolates from the USA300 and USA400 backgrounds. We assessed mortality, in vivo bacterial growth, and histopathology. We assessed the in vitro expression of capsule and of selected genes believed to be important in virulence in S. aureus, including agr, saeRS, sarA, alpha-toxin (hla), and Panton-Valentine leukocidin (pvl)., Results: USA300 isolates were more lethal, produced more severe pneumonia, and had higher in vivo bacterial density in the lung than did USA400 isolates. In vitro expression of agr, saeRS, sarA, hla, and pvl were greater in USA300 isolates. USA300 isolates were unencapsulated, whereas 2 of 3 USA400 isolates produced capsule., Conclusions: USA300 isolates were more virulent than USA400 isolates in a model of necrotizing pneumonia. The explanation for this is unclear, but it likely results from increased expression of S. aureus regulatory systems (e.g., agr, saeRS, and sarA) and the resultant upregulation of key virulence factors including alpha-toxin and PVL.

The recent emergence of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) marked a quantum change in the biology and epidemiology of a major human pathogen. Various virulence determinants unique to CA-MRSA have been uncovered recently, which shed light on how these strains spread easily and sustainably among humans and frequently cause severe disease. The role of the Panton Valentine leukocidin (PVL) in CA-MRSA pathogenesis is a matter of much debate. Although epidemiological data have indicated a role for PVL in the CA-MRSA disease process, recent data from relevant animal models indicate that PVL does not impact virulence of prevalent CA-MRSA strains. Identifying specialized pathogenic traits of CA-MRSA remains a challenge that will yield new diagnostic tools and therapeutic targets for drug and vaccine development. Here, we discuss the roles of PVL, the arginine catabolic mobile element and phenol-soluble modulins in the pathogenesis of prevalent CA-MRSA strains.

We report two cases of invasive infections caused by Panton-Valentine leukocidin-positive, community-associated, methicillin-resistant Staphylococcus aureus (CA-MRSA) after kidney transplantation. This report emphasizes the clinical importance of considering CA-MRSA as a causative agent in the differential diagnosis of infections of the skin and soft tissues in organ transplant recipients.

Aggregatibacter (formerly Actinobacillus) actinomycetemcomitans is a pathogen that causes localized aggressive periodontitis and extraoral infections including infective endocarditis. Recently, we reported that A. actinomycetemcomitans is beta-hemolytic on certain growth media due to the production of leukotoxin (LtxA). Based on this observation and our ability to generate random transposon insertions in A. actinomycetemcomitans, we developed and carried out a rapid screen for LtxA mutants. Using PCR, we mapped several of the mutations to genes that are known or predicted to be required for LtxA production, including ltxA, ltxB, ltxD, and tdeA. In addition, we identified an insertion in a gene previously not recognized to be involved in LtxA biosynthesis, ptsH. ptsH encodes the protein HPr, a phosphocarrier protein that is part of the sugar phosphotransferase system. HPr results in the phosphorylation of other proteins and ultimately in the activation of adenylate cyclase and cyclic AMP (cAMP) production. The ptsH mutant showed only partial hemolysis on blood agar and did not produce LtxA. The phenotype was complemented by supplying wild-type ptsH in trans, and real-time PCR analysis showed that the ptsH mutant produced approximately 10-fold less ltxA mRNA than the wild-type strain. The levels of cAMP in the ptsH mutant were significantly lower than in the wild-type strain, and LtxA production could be restored by adding exogenous cAMP to the culture.

Unlabelled: Panton-Valentine leucocidin (PVL) toxin-producing strains of Staphylococcus aureus (S. aureus) are associated with skin abscesses and furunculosis, with necrotizing pneumonia being a relatively rare problem. Here, we describe a fatal case of necrotizing pneumonia in a 14-year-old child who presented initially with sore throat and pyrexia. He deteriorated rapidly, developing hypotension, multiple organ failure and purpura fulminans. S. aureus was isolated from the tracheal aspirate, which was found to be positive for PVL, toxic shock syndrome toxins (TSST) 1 and 2 and staphylococcal enterotoxin C (SEC). It was postulated that purpura fulminans and toxic shock syndrome were a result of the abovementioned exotoxins., Conclusion: This case highlights the emergence of PVL-positive community-acquired S. aureus infection and association of purpura fulminans with superantigens. Practitioners should be aware of this illness in order to initiate appropriate treatment.