RESUMEN
We report the molecular characterization of methicillin-resistant Staphylococcus aureus (MRSA) with resistance to tigecycline and to daptomycin isolated from intensive-care-unit patients in Brazil over a 6-month period. Thirty-six isolates (25 from infection sites, 11 from nasal sites) recovered from 23 patients who presented with MRSA infection during this period were characterized by pulsed-field gel electrophoresis, multilocus sequence typing, staphylococcal cassette chromosome mec (SCCmec) typing, and antimicrobial susceptibility profiling. Ten isolates from six patients and two isolates from different patients were resistant to tigecycline and daptomycin, respectively. Eight pulsotypes were identified and one, type A, accounted for 21 isolates from 12 patients; type A isolates were SCCmecII as were a further nine isolates of other pulsotypes. All but four of the total isolates were sequence type (ST) 5 or ST105 and classified as clonal complex (CC) 5; the historically prevalent lineage in Brazil, ST239-SCCmecIII, was identified in only three patients. Tigecycline-resistant strains were all ST105-SCCmecII and two patients were nasally colonized by strains of the same pulsotype found in infection sites. Two ST5-SCCmecII were daptomycin resistant after 48 h incubation. The origin and mechanism of these resistant strains remains unknown and further studies are warranted to determine whether such clones are becoming endemic in Brazilian hospitals and to assess their impact on infection control practice.
Asunto(s)
Antibacterianos/farmacología , Daptomicina/farmacología , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Minociclina/análogos & derivados , Infecciones Estafilocócicas/tratamiento farmacológico , Proteínas Bacterianas/genética , Técnicas de Tipificación Bacteriana , Brasil/epidemiología , ADN Bacteriano/genética , Farmacorresistencia Bacteriana Múltiple , Electroforesis en Gel de Campo Pulsado , Hospitales , Humanos , Unidades de Cuidados Intensivos , Staphylococcus aureus Resistente a Meticilina/aislamiento & purificación , Pruebas de Sensibilidad Microbiana , Minociclina/farmacología , Epidemiología Molecular , Proteínas de Unión a las Penicilinas , Infecciones Estafilocócicas/epidemiología , Infecciones Estafilocócicas/genética , TigeciclinaRESUMEN
Enterococci are members of commensal flora of animals and insects, but are also important opportunistic pathogens. Our objective was to observe if there was any difference of virulence in several groups of E. faecalis, mainly between vancomycin-resistant E. faecalis (VREFS) of colonization and infection. VREFS and vancomycin-sensitive E. faecalis from Brazil were screened for the presence of virulence factor genes. Phenotypic assays were used to assess in vitro expression, to understand the pathogenic potential of these isolates and to determine whether a correlation exists between virulence and antibiotic resistance. Different virulence profiles were found suggesting that the disseminating clone may have generated several variations. However, our study showed that one constellation of traits appeared most commonly: gelatinase, aggregation substance and esp (GEA). These factors are important because they have been implicated in cell aggregation and biofilm formation. Biofilm formation may promote the conjugation of plasmids harboring resistance and virulence genes, enhancing the probability of entry of new resistance genes into species. Curiously, the profile GEA was not exclusive to VREFS, it was the second most observed in VSEFS isolates from colonization and infection in hospitalized patients and also from rectal swabs of healthy volunteers. Such strains appear to represent the entry gateway to new resistance genes into E. faecalis and may contribute to the spreading of E. faecalis mainly in hospitals.
Enterococci são membros da microbiota comensal de animais e insetos, mas também são importantes patógenos oportunistas. Nosso objetivo foi observar se há qualquer diferença na virulência nos diversos grupos de Enterococcus faecalis, principalmente nos E. faecalis resistente à vancomicina (VREFS) isolados de colonização e infecção. VREFS e E. faecalis sensíveis à vancomicina (VSEFS) do Brasil foram pesquisadas quanto a presença de fatores de virulência. Ensaios fenotípicos foram usados para obter a expressão in vivo, entender o potencial patogênico destas amostras e determinar se existe correlação entre virulência e resistência a antibióticos. Diferentes perfis de virulência foram encontrados sugerindo que o clone que está se disseminado pode ter gerado diversas variações. No entanto, nosso estudo mostrou que um conjunto de fatores parece ser mais comum entre as amostras: gelatinase, substância de agregação e esp (GEA). Estes fatores tem sido correlacionados com a agregação de células e formação de biofilmes. A formação de biofilme pode promover a conjugação de plasmídeos contendo genes de resistência entre as espécies. Curiosamente, o perfil GAE não foi exclusivo para VREFS, foi o segundo mais observado em amostras VSEFS provenientes de colonização e infecção em pacientes hospitalizados e também de swabs retais de voluntários saudáveis. Tais linhagens pacerem representar a "porta de entrada" para novos genes de resistência em E. faecalis e podem contribuir para a disseminação de E. faecalis principalmente nos hospitais.
Asunto(s)
Animales , Biopelículas , Pruebas Enzimáticas Clínicas , Enterococcus faecalis/aislamiento & purificación , Gelatinasas , Resistencia a la Vancomicina , Vancomicina/análisis , Vancomicina/aislamiento & purificación , Medios de Cultivo , Métodos , VirulenciaRESUMEN
Enterococci are members of commensal flora of animals and insects, but are also important opportunistic pathogens. Our objective was to observe if there was any difference of virulence in several groups of E. faecalis, mainly between vancomycin-resistant E. faecalis (VREFS) of colonization and infection. VREFS and vancomycin-sensitive E. faecalis from Brazil were screened for the presence of virulence factor genes. Phenotypic assays were used to assess in vitro expression, to understand the pathogenic potential of these isolates and to determine whether a correlation exists between virulence and antibiotic resistance. Different virulence profiles were found suggesting that the disseminating clone may have generated several variations. However, our study showed that one constellation of traits appeared most commonly: gelatinase, aggregation substance and esp (GEA). These factors are important because they have been implicated in cell aggregation and biofilm formation. Biofilm formation may promote the conjugation of plasmids harboring resistance and virulence genes, enhancing the probability of entry of new resistance genes into species. Curiously, the profile GEA was not exclusive to VREFS, it was the second most observed in VSEFS isolates from colonization and infection in hospitalized patients and also from rectal swabs of healthy volunteers. Such strains appear to represent the entry gateway to new resistance genes into E. faecalis and may contribute to the spreading of E. faecalis mainly in hospitals.
RESUMEN
Enterococci are leading causes of hospital-acquired infections that are often difficult to treat because of high-level aminoglycoside and glycopeptide resistance. Vancomycin-resistant enterococci are a global problem, and have been isolated with increasing frequency in hospitals in Brazil. The objective of this study was to determine the genetic relatedness of vancomycin-resistant Enterococcus faecium (VREFM) and vancomycin-sensitive E. faecium (VSEFM) isolated from human infections and faecal sources in Brazil, and to compare these isolates with those from domesticated animals. Isolates (n = 56) were classified by multilocus sequence typing (MLST) and assessed for putative virulence traits. The acm gene was detected in 98% of all isolates. The 56 isolates studied comprised 26 different MLST types. VSEFM isolates from the faeces of pigs were found to be distinct from all human isolates characterised previously by MLST, and were assigned new sequence type (ST) numbers. VREFM isolates were represented by four different STs (ST-114, ST-17, ST-281, ST-50). Among the 26 STs identified in this study, eBURST detected three groups of STs with related allelic profiles, and 19 unrelated STs. Among E. faecium isolates from Brazil, the esp gene was restricted to vancomycin-resistant isolates. Furthermore, isolates classified as ST-17 by MLST, an epidemic strain type isolated internationally with the purK-1 gene, were found among VREFM isolates from Brazil that also harboured the esp and hyl genes.
Asunto(s)
Enterococcus faecium/clasificación , Infecciones por Bacterias Grampositivas/microbiología , Resistencia a la Vancomicina/genética , Adhesinas Bacterianas/genética , Animales , Antibacterianos/farmacología , Proteínas Bacterianas/genética , Brasil/epidemiología , Portador Sano/microbiología , Infección Hospitalaria/microbiología , Enterococcus faecium/efectos de los fármacos , Enterococcus faecium/genética , Heces/microbiología , Infecciones por Bacterias Grampositivas/prevención & control , Hospitales , Humanos , Hialuronoglucosaminidasa/genética , Proteínas de la Membrana/genética , Pruebas de Sensibilidad Microbiana , Especificidad de la Especie , Porcinos/microbiología , Vancomicina/farmacología , Factores de Virulencia/genéticaRESUMEN
In 2000, Enterococcus faecalis resistant to vancomycin was first reported at a tertiary hospital in Porto Alegre, southern Brazil. The resistance spread to other hospitals and surveillance programs were established by hospital infection committees to prevent the spread of vancomycin-resistant enterococci. In February 2002, an isolate initially identified at the genus level as Enterococcus was obtained by surveillance culture (rectal swab) from a patient admitted to a hospital for treatment of septic arthritis in the shoulder. The isolate proved to be resistant to vancomycin by the disc diffusion method and confirmed by an E-test resulting in a minimal inhibitory concentration of > or = 256 microg/ml. This isolate was sent to a reference laboratory (Laboratorio Especial de Bacteriologia e Epidemiologia Molecular, Faculdade de Ciencias Farmaceuticas de Ribeirao Preto, USP) for further study and proved to be an E. gallinarum by the polymerase chain reaction (PCR) using specific primers for the species. Due to the phenotype of unusually high vancomycin resistance, the isolate presumably had the resistance genes (vanA and vanB) and this was confirmed by PCR, which indicated the presence of the vanA gene. A 10.8-kb Tn1546-related transposon was also identified by long-PCR. Interspecies transfer of the vancomycin-resistance gene from the donor E. gallinarum was performed in a successful conjugation experiment in vitro, using E. faecium GE-1 and E. faecalis JH22 as receptors. This is the first report of the detection of a vanA determinant naturally acquired by E. gallinarum in Brazil, indicating the importance of characterizing VRE by both phenotype and genotype methods.
Asunto(s)
Proteínas Bacterianas/genética , Ligasas de Carbono-Oxígeno/genética , Enterococcus/genética , Resistencia a la Vancomicina/genética , Proteínas Bacterianas/efectos de los fármacos , Brasil , Ligasas de Carbono-Oxígeno/efectos de los fármacos , Enterococcus/clasificación , Enterococcus/efectos de los fármacos , Genotipo , Humanos , Fenotipo , Reacción en Cadena de la PolimerasaRESUMEN
In 2000, Enterococcus faecalis resistant to vancomycin was first reported at a tertiary hospital in Porto Alegre, southern Brazil. The resistance spread to other hospitals and surveillance programs were established by hospital infection committees to prevent the spread of vancomycin-resistant enterococci. In February 2002, an isolate initially identified at the genus level as Enterococcus was obtained by surveillance culture (rectal swab) from a patient admitted to a hospital for treatment of septic arthritis in the shoulder. The isolate proved to be resistant to vancomycin by the disc diffusion method and confirmed by an E-test resulting in a minimal inhibitory concentration of > ou = 256 µg/ml. This isolate was sent to a reference laboratory (Laboratório Especial de Bacteriologia e Epidemiologia Molecular, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, USP) for further study and proved to be an E. gallinarum by the polymerase chain reaction (PCR) using specific primers for the species. Due to the phenotype of unusually high vancomycin resistance, the isolate presumably had the resistance genes (vanA and vanB) and this was confirmed by PCR, which indicated the presence of the vanA gene. A 10.8-kb Tn1546-related transposon was also identified by long-PCR. Interspecies transfer of the vancomycin-resistance gene from the donor E. gallinarum was performed in a successful conjugation experiment in vitro, using E. faecium GE-1 and E. faecalis JH22 as receptors. This is the first report of the detection of a vanA determinant naturally acquired by E. gallinarum in Brazil, indicating the importance of characterizing VRE by both phenotype and genotype methods.
Asunto(s)
Humanos , Proteínas Bacterianas/genética , Ligasas de Carbono-Oxígeno/genética , Enterococcus/genética , Resistencia a la Vancomicina/genética , Brasil , Proteínas Bacterianas/efectos de los fármacos , Ligasas de Carbono-Oxígeno/efectos de los fármacos , Enterococcus/clasificación , Enterococcus/efectos de los fármacos , Genotipo , Fenotipo , Reacción en Cadena de la PolimerasaRESUMEN
A vancomycin-resistant Enterococcus (VRE) was isolated from a blood culture of a patient in a Brazilian hospital who had a treatment history of a bone marrow transplant in the USA. The organism was identified as Enterococcus faecium, which exhibited an MIC (minimum inhibitory concentration) >or= 256 microg/mL for vancomycin. This was confirmed by E-test and the vanA gene was detected by PCR. Overlapping PCR revealed a left IR deletion and an additional 1.5 kb fragment between vanSH genes. DdeI digestion of vanRSHAX genes showed the determinant to be a T type variant, and the element was cloned and sequenced. These results revealed an IS1251 downstream of nucleotide 5820 of the VanA element. Insertions like this have not been reported previously in Brazil, but have been detected in the USA. The genotype and association with a patient previously treated in the USA suggest that this VRE was introduced from abroad, probably through inter-hospital strain spread.