RESUMO
Methicillin-resistant Staphylococcus aureus (MRSA) is an important cause of both healthcare-associated MRSA (HA-MRSA) and community-associated MRSA (CA-MRSA) infections. Severe MRSA infections have been associated with the virulence factor Panton-Valentine leukocidin (PVL). The aim of this study was to investigate susceptibility patterns, the presence of toxin genes, including that encoding PVL, and clonality among MRSA isolates collected from patients in Greece over a 12-year period. MRSA isolates were collected from January 2001 to December 2012 from six different hospitals. Antibiotic susceptibility was determined with the disk diffusion method and the Etest. The presence of the toxic shock syndrome toxin-1 gene (tst), the enterotoxin gene cluster (egc) and the PVL gene was tested with PCR. The genotypic characteristics of the strains were analysed by SCCmec and agr typing, and clonality was determined with pulsed-field gel electrophoresis and multilocus sequence typing. An increasing rate of MRSA among S. aureus infections was detected up to 2008. The majority of PVL-positive MRSA isolates belonged to a single clone, sequence type (ST)80-IV, which was disseminated both in the community and in hospitals, especially during the warmest months of the year. Carriage of tst was associated with ST30-IV, whereas egc was distributed in different clones. CA-MRSA isolates were recovered mainly from skin and soft tissue infections, whereas HA-MRSA isolates were associated with surgical and wound infections. During the period 2001-2012, ST80-IV predominated in the community and infiltrated the hospital settings in Greece, successfully replacing other PVL-positive clones. The predominance of ST239-III in HA-MRSA infections was constant, whereas new clones have also emerged. Polyclonality was statistically significantly higher among CA-MRSA isolates and isolates from adult patients.
Assuntos
Epidemias , Staphylococcus aureus Resistente à Meticilina/classificação , Staphylococcus aureus Resistente à Meticilina/isolamento & purificação , Infecções Estafilocócicas/epidemiologia , Infecções Estafilocócicas/microbiologia , Adulto , Antibacterianos/farmacologia , Toxinas Bacterianas/genética , Criança , Pré-Escolar , Infecções Comunitárias Adquiridas/epidemiologia , Infecções Comunitárias Adquiridas/microbiologia , Infecção Hospitalar/epidemiologia , Infecção Hospitalar/microbiologia , Feminino , Genótipo , Grécia/epidemiologia , Humanos , Masculino , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/genética , Testes de Sensibilidade Microbiana , Epidemiologia Molecular , Tipagem Molecular , Reação em Cadeia da Polimerase , Fatores de Virulência/genéticaRESUMO
An increasing number of infections caused by community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) carrying the Panton-Valentine leukocidin (PVL) genes was recently identified in Greece. In the present study, 170 patients with S. aureus infections and 123 uninfected children (<15 years old) who had been tested for nasal carriage were evaluated during a 2-year period. The MecA, PVL and superantigen family genes, and MRSA clones, were investigated by molecular methods. Sites of infection and laboratory findings for patients were recorded. The results were compared and statistically analysed. Among 123 uninfected children 73 (59%) carried S. aureus, including four MRSA strains. Of these, three MRSA and three methicillin-sensitive S. aureus (MSSA) strains were PVL-positive (p <0.0001). Ninety-six patients (96/170) exhibited skin and soft-tissue infections (SSTIs), and 74 exhibited invasive infections. The incidence of staphylococcal infections increased during July to September each year. In total, 110 S. aureus isolates were PVL-positive (81 from SSTIs and 29 from invasive infections, p <0.0001). Ninety-nine out of 106 MRSA (93%) isolates from 170 patients carried the PVL genes (p <0.0001); 97 belonged to the clonal complex CC80. Leukocyte and polymorphonuclear cell counts were higher among children with MRSA infections (p <0.005). MSSA predominated among patients with invasive infections (43/74), and carried mainly genes of the superantigen family. Children <5 years of age showed a higher risk of MRSA infection. The present study demonstrates that infections due to PVL-positive CA-MRSA spread easily among children, and SSTIs can lead to invasive infections. Nasal colonization may be an additional factor contributing to the emergence of CA-MRSA.
Assuntos
Portador Sadio/epidemiologia , Infecções Comunitárias Adquiridas/epidemiologia , Nariz/microbiologia , Infecções Estafilocócicas/epidemiologia , Staphylococcus aureus/genética , Staphylococcus aureus/isolamento & purificação , Proteínas de Bactérias/genética , Toxinas Bacterianas/genética , Portador Sadio/microbiologia , Criança , Pré-Escolar , Infecções Comunitárias Adquiridas/microbiologia , Infecções Comunitárias Adquiridas/fisiopatologia , Exotoxinas/genética , Feminino , Grécia/epidemiologia , Humanos , Incidência , Lactente , Leucocidinas/genética , Masculino , Resistência a Meticilina , Proteínas de Ligação às Penicilinas , Estações do Ano , Infecções Estafilocócicas/fisiopatologia , Superantígenos/genéticaRESUMO
AIM: Absolute and relative quantitative real-time reverse transcriptase polymerase chain reaction (RT-PCR) by the use of two mathematical models were applied in order to study the expression of tst gene encoding the toxic shock syndrome toxin-1 (TSST-1), among methicillin-resistant Staphylococcus aureus (MRSA). METHODS AND RESULTS: Thirteen epidemic MRSA belonging to different clones and carrying a variety of toxin genes were selected. tst gene expression was achieved by using absolute and relative quantitative real-time RT-PCR and the SYBR Green I. Absolute RT-PCR showed a statistically significant higher level of tst expression among strains isolated from soft tissue infections. Relative quantification was performed in relation to 23S rRNA expression by the application of two mathematical models, the 2(-DeltaDeltaCt) and the Pfaffl analysis methods. CONCLUSIONS: tst gene expression was best calculated by the relative real-time RT-PCR analysis applying the Pfaffl analysis method, taking into account the reactions' efficiencies. Level of tst expression was related to patients' infection and did not depend on the MRSA genetic profile. SIGNIFICANCE AND IMPACT OF THE STUDY: The results indicate that the application of the Pfaffl analysis method in the evaluation of relative real-time RT-PCR is more adequate.
Assuntos
Toxinas Bacterianas/genética , Enterotoxinas/genética , Resistência a Meticilina/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Infecções Estafilocócicas/diagnóstico , Staphylococcus aureus/isolamento & purificação , Superantígenos/genética , Expressão Gênica , Genótipo , Humanos , Modelos Teóricos , RNA Bacteriano/análise , RNA Mensageiro/análise , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/genéticaRESUMO
Staphylococcus aureus was identified as the cause of acute childhood osteomyelitis in 19 patients. A single clone of community-acquired methicillin-resistant S. aureus (MRSA) carrying the type IV mecA staphylococcal cassette chromosome and the Panton-Valentine leukocidin (PVL) genes was isolated from five patients. Among the remaining 14 patients, two methicillin-sensitive S. aureus (MSSA) isolates were PVL-positive. The maximal erythrocyte sedimentation rate and C-reactive protein values, and the time required for normalisation, were significantly different in patients with PVL-positive strains (MRSA and MSSA), suggesting that the production of PVL is an important factor that contributes to the course of the disease.
Assuntos
Toxinas Bacterianas/genética , Exotoxinas/genética , Leucocidinas/genética , Resistência a Meticilina/genética , Osteomielite/microbiologia , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/metabolismo , Adolescente , Sedimentação Sanguínea , Proteína C-Reativa/análise , Criança , Pré-Escolar , Infecções Comunitárias Adquiridas/microbiologia , Feminino , Humanos , Masculino , Staphylococcus aureus/isolamento & purificação , Fatores de TempoRESUMO
Methicillin-resistant coagulase-negative staphylococci (MR-CNS) (n = 132), isolated from pre-term neonates, were analysed to determine their antibiotic resistance patterns, clonal distribution, biofilm production and the presence of the ica operon. All MR-CNS were multiresistant, and 89% produced slime. A major clone was identified (77 isolates) among 115 Staphylococcus epidermidis isolates. Ten of 16 Staphylococcus haemolyticus isolates also belonged to a single clone. Most (80%) slime-positive isolates possessed all the ica genes tested, while the remaining 23 (20%) had a variety of gene combinations. The entire ica cluster was detected in three of 15 slime-negative isolates. One major and two minor slime-positive, multiresistant MR-CNS clones had disseminated among hospitalised pre-term neonates.
Assuntos
Adesinas Bacterianas/genética , Infecção Hospitalar/microbiologia , Resistência a Meticilina , Infecções Estafilocócicas/microbiologia , Staphylococcus/genética , Coagulase/análise , Hospitais Universitários , Humanos , Recém-Nascido , Unidades de Terapia Intensiva Neonatal , Testes de Sensibilidade Microbiana , Óperon/genética , Polissacarídeos Bacterianos/análise , Polissacarídeos Bacterianos/biossíntese , Sepse/microbiologia , Staphylococcus/efeitos dos fármacos , Staphylococcus/patogenicidadeRESUMO
Three collections of Staphylococcus aureus isolates (n = 1,058) were investigated to assess the spread of Panton-Valentine leukocidin (PVL)-producing strains in Greece and their association with skin and soft-tissue infections (SSTIs). The isolates were collected during 2001-2003 from inpatients and outpatients with invasive infections in two distinct geographical areas. Clonal types were identified according to their ClaI-mecA::ClaI-Tn554::pulsed-field gel electrophoresis patterns, and the presence of the lukS-PV and lukF-PV genes was assessed by PCR. In total, 287 (27%) S. aureus isolates carried the PVL genes: 45% of methicillin-resistant S. aureus (MRSA) and 12% of methicillin-sensitive S. aureus (MSSA). All the PVL-positive MRSA isolates belonged to a single clone that was disseminated in the community and hospitals. The PVL-positive MSSA isolates were polyclonal, with 14 of 65 isolates being associated with hospital-acquired infections. The community-acquired isolates were from SSTIs, while the hospital-acquired isolates were associated with surgical wound infections, especially those involving prosthetic devices. Thus, a unique clone of PVL-positive MRSA has spread in both the community and the hospital setting in Greece, and has replaced older clonal types.
