Biofilm formation of Brazilian meticillin-resistant Staphylococcus aureus strains: prevalence of biofilm determinants and clonal profiles

Biofilms plays an important role in medical-device-related infections. This study aimed to determine the factors that influence adherence and biofilm production, as well as the relationship between strong biofilm production and genetic determinants in clinical isolates of meticillin-resistant Staphylococcus aureus (MRSA). Fifteen strains carrying different chromosomal cassettes recovered from hospitalized patients were selected; five SCCmecII, five SCCmecIII and five SCCmecIV. The SCCmec type, agr group and the presence of the virulence genes (bbp, clfA, icaA, icaD, fnbB, bap, sasC and IS256) were assessed by PCR. PFGE and multilocus sequence typing (MLST) techniques were also performed. The initial adhesion and biofilm formation were examined by quantitative assays. The surface tension and hydrophobicity of the strains were measured by the contact angle technique to evaluate the association between these parameters and adhesion ability. SCCmecIII and IV strains were less hydrophilic, with a high value for the electron acceptor parameter and higher adhesion in comparison with SCCmecII strains. Only SCCmecIII strains could be characterized as strong biofilm producers. The PFGE showed five major pulsotypes (AE); however, biofilm production was related to the dissemination of one specific PFGE clone (C) belonging to MLST ST239 (Brazilian epidemic clonal complex). The genes agrI, fnbB and IS256 in SCCmecIII strains were considered as genetic determinants associated with strong biofilm-formation by an ica-independent biofilm pathway. This study contributes to the understanding of biofilm production as an aggravating factor potentially involved in the persistence and severity of infections caused by multidrug-resistant MRSA belonging to this genotype.We thank FAPEMIG (Fundação de Amparo à Pesquisa de Minas Gerais, proceeding APQ 01398-11) and CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, PDSE proceeding 8952/11-6) for the financial support and scholarships. We also thank Dr Teruyo Ito, Juntendo University, Japan, and Dr Elsa Masae Mamizuka, Universidade de São Paulo, Brazil, for kindly providing the control strains used in this study.info:eu-repo/semantics/publishedVersio

Epidemiologia e fatores de risco associados à colonização por VRE e MRSA em uma unidade de terapia intensiva de adultos

This investigation included a total of 78 VRE-colonized patients and 17 MRSA-colonized patients through study of the incidence in the period April 2009 to January 2010. We evaluated the rates of infection/colonization with these phenotypes, risk factors for colonization, antimicrobial susceptibility profile and characterization of vanA gene in enterococci strains with high level vancomycin resistance. The identification of S. aureus and enterococci species was performed by conventional biochemical tests. The vancomycin minimal inhibitory concentration (MIC) was evaluated by E-test method. The antimicrobial susceptibility profile and high level aminoglycoside resistance were carried out by discdiffusion. To assess the genotype enterococcal strains expressing high-level vancomycin resistance, we used the polymerase chain reaction. Epidemiological data were recorded for all patients included in the study and were used for the risk factor analysis. A case-control study was then performed. The cases were defined as only patients with VRE colonization (n=21), and the controls were those without VRE colonization or infection from any organism (n=143). A total of 333 patients hospitalized were enrolled in this investigation. Of the 90 patients colonized with Enterococcus spp., 92 samples were isolated. Seventy seven patients (23.1%) were colonized with VanC VRE and only one patient (0.3%) with VanA-type. Four of 92 samples were identified as Enterococcus faecium, 11 as Enterococcus faecalis, 26 as Enterococcus gallinarum and 51 as Enterococcus casseliflavus. The risk factors that were determined through univariate analysis to be significantly associated with VRE colonization included nephropathy, diabetes mellitus, prior ICU antibiotic use, vancomycin and carbapenem use in the ICU. In the multivariate analysis, significant independent risk factors for VRE colonization were the nephropathy (P < 0.001), prior ICU antibiotic use (P = 0.03) and carbapenem use (P < 0.001). Our investigation revealed a low frequency of MRSA colonization (5.1%) with 23.5% of colonized patients progressed to infection by this organism (P <0.001, OR = 32.1), especially for cases of sepsis (P = 0.01, OR = 20.9). VRE colonization, particularly the VanC phenotype, was frequent in the ICU and although of little clinical importance, these microorganisms are considered reservoirs of resistance genes. There was a correlation between the vancomycin and carbapenems use and VRE colonization, although the results of multivariate analysis did not demonstrate vancomycin as an independent risk factor for VRE colonization. We found a low incidence of MRSA in the ICU and observed a significant relationship between colonization and the development of sepsis by this microorganism.Conselho Nacional de Desenvolvimento Científico e TecnológicoMestre em Imunologia e Parasitologia AplicadasFoi analisado um total de 78 pacientes colonizados por Enterococcus resistente à vancomicina (VRE) e 17 pacientes colonizados por Staphylococcus aureus resistente a meticilina (MRSA) através de estudo de incidência no período de abril de 2009 a janeiro de 2010. Foram avaliadas as taxas de infecção/colonização por esses fenótipos, fatores de risco para colonização, perfil de sensibilidade aos antimicrobianos e caracterização do gene vanA em amostras de VRE com alto nível de resistência à vancomicina. A identificação dos S. aureus e das espécies de enterococos foi realizada por testes bioquímicos convencionais. A concentração inibitória mínima (MIC) para vancomicina foi avaliada pelo método de E-test. O perfil de sensibilidade aos antimicrobianos e a resistência em nível elevado aos aminoglicosídeos foi realizada por disco difusão. Para avaliar o genótipo das cepas de VRE foi utilizado o PCR. Dados epidemiológicos foram registrados de todos os pacientes incluídos nesse estudo e para análise dos fatores de risco para colonização por VRE foi realizado um estudo caso x controle sendo definidos como caso os pacientes colonizados por VRE sem infecção por qualquer microrganismo (n=21) e como controle os pacientes sem colonização e infecção por VRE (n=143). Um total de 333 pacientes hospitalizados foi incluído nessa investigação. Dos 90 pacientes colonizados por Enterococcus spp., 92 amostras foram isoladas. Setenta e sete pacientes (23,1%) estavam colonizados com Enterococcus resistentes à vancomicina do fenótipo VanC e apenas um paciente (0,3%) com VanA. Quatro das 92 amostras foram identificadas como Enterococcus faecium, 11 como Enterococcus faecalis, 26 como Enterococcus gallinarum e 51 como Enterococcus casseliflavus. Os fatores de risco que foram significativamente associados com a colonização por VRE incluíram nefropatia, diabetes mellitus, uso de antibiótico prévio à UTI, uso de vancomicina e carbapenêmicos na unidade sendo o uso de antibiótico prévio à UTI (P = 0,03), uso de carbapenêmicos na unidade (P < 0,001) e nefropatia (P < 0,001) fatores de risco independentes para colonização. Nossa investigação evidenciou baixa frequência de colonização por MRSA (5,1%) durante o período estudado com 23,5% dos pacientes colonizados evoluindo com infecção por esse microrganismo (P < 0,001; OR = 32,1), com destaque para os casos de sepse (P = 0,01; OR = 20,9). A colonização por VRE, predominantemente do fenótipo VanC, foi frequente na UTI e embora de pouca importância clínica, esses microrganismos são considerados reservatórios de genes de resistência. Houve correlação positiva entre o uso de vancomicina e carbapenêmicos e a colonização por VRE. Apesar da baixa colonização por MRSA, observou-se uma relação entre a colonização e o desenvolvimento de sepse por esse microrganismo

Marcadores genéticos de risco para forte produção de biofilme em cepas clínicas de Staphylococcus aureus resistentes à meticilina e sua associação com o perfil clonal

Methicillin-resistant Staphylococcus aureus (MRSA) is one of the major human pathogens worldwide and its epidemiology has been the focus of numerous single and multicenter surveillance studies over the past years. In this study, a phenotypic and genotypic approach were used to determine the factors that influence adherence and biofilm production of the most common MRSA SCCmec types, and its relationship with antimicrobial resistance, virulence genes and the genetic background of S. aureus isolates. The strains used in this study were selected from a collection of clinical MRSA strains recovered from patients hospitalized in the Teaching Hospital of the Federal University of Uberlandia, isolated from infections at various anatomical sites and evaluated for SCCmec type. Fifteen strains carrying different chromosomal cassettes were selected, five SCCmec II, five SCCmec III and five SCCmec IV, recovered predominantly from blood (67%), surgical site infections (27%) and pneumonia (6.0%). The SCCmec type, agr group and the presence of the virulence genes (bbp, clfA, icaA, icaD, fnbB, bap, sasC and IS256) were assessed by PCR. The genetic relationship between the isolates and a possible association with the ability to form biofilm were investigated by pulsed field gel electrophoresis (PFGE). The initial adhesion and biofilm formation were examined by quantitative assays. To evaluate the association between the hydrophobicity and the ability of MRSA cell to adhere to an unmodified polystyrene surface, the surface tension and hydrophobicity of the strains were measured by contact angle technique. There were association among the values of the electron acceptor parameter, the degree of hydrophobicity and adhesion ability. SCCmec III and IV strains were less hydrophilic, showed higher values of the electron acceptor parameter and adhered better than SCCmec II strains. The analysis of biofilm production showed that SCCmec III strains were characterized as strong biofilm producers; with the average biomass of biofilm from 0.53 ± 0.12 compared with 0.04 ± 0.04 those non-producers/weak producers (SCCmec II e IV). The analysis of this study showed five major pulsotypes according to the PFGE (A-E) with a large genomic diversity observed by the number of subtypes in each pulsotype. However, biofilm production was related to the dissemination of one specific PFGE clone (Clone C). The presence of the genes agrI, fnbB and IS256 in clinical MRSA SCCmec III strains, were considered as genetic risk markers for strong biofilm-formation by an icaindependent biofilm pathway. This study contributes for the understanding of biofilm production as a virulence factor potentially involved in the persistence and severity of infections caused by Staphylococcus aureus belonging to this genotype.Coordenação de Aperfeiçoamento de Pessoal de Nível SuperiorDoutor em Imunologia e Parasitologia AplicadasStaphylococcus aureus resistente à meticilina (MRSA) é um dos principais patógenos humanos em todo o mundo e sua epidemiologia tem sido foco de numerosos estudos de vigilância unicêntricos e multicêntricos ao longo dos últimos anos. Neste estudo utilizamos abordagens fenotípicas e genotípicas para determinar os fatores que influenciam a adesão inicial e produção de biofilme em cepas clínicas de MRSA carreando os tipos de cassete cromossômico estafilocócico (SCCmec) mais frequentes nos hospitais brasileiros, e sua relação com resistência, genes de virulência e perfil clonal. As cepas de MRSA utilizadas neste estudo foram selecionadas a partir de uma coleção de amostras clínicas recuperadas de pacientes internados no Hospital de Clínicas da Universidade Federal de Uberlândia, isoladas de infecções em diversos sítios anatômicos. Foram selecionadas para o estudo quinze cepas carreando diferentes cassetes cromossômicos, cinco SCCmec II, cinco SCCmec III e cinco SCCmec IV, recuperadas predominantemente de sangue (67%), sítio cirúrgico (27%) e pneumonia (6,0%). O tipo de SCCmec, o grupo agr e a presença de genes de virulência (bbp, clfA, icaA, icaD, fnbB, bap, sasC e IS256) foram avaliados por PCR. A relação genética entre as amostras e a possível associação com a capacidade de formação de biofilme foram investigadas por eletroforese em gel de campo pulsado (PFGE). A adesão inicial e a capacidade de formação de biofilme foram examinadas por ensaios quantitativos. Adicionalmente, a associação entre a hidrofobicidade e a capacidade da célula de MRSA aderir a uma superfície de poliestireno não modificada, foi avaliada através da medida dos ângulos de contato. Houve associação entre o grau de hidrofobicidade e a capacidade de adesão. Cepas clínicas de MRSA carreando SCCmec III e IV foram menos hidrofílicas, apresentaram valores mais altos de tensão interfacial do componente aceptor de elétrons e aderiram melhor do que as cepas SCCmecII. As análises da produção de biofilme mostraram que cepas carreando SCCmec III foram caracterizadas como fortemente produtoras de biofilme, com a média da biomassa do biofilme de 0,53 ± 0,12 em comparação com 0,04 ± 0,04 daquelas nãoprodutoras/ fraco produtoras (SCCmec II e SCCmec IV). A tipagem molecular por PFGE evidenciou cinco pulsotipos (A-E) com uma grande diversidade clonal observada pelo número de subtipos em cada pulsotipo. Entretanto, a produção de biofilme esteve relacionada a disseminação de um clone específico (Clone C). Os genes agrI, fnbB e IS256 em cepas clínicas de MRSA carreando SCCmec III, foram considerados marcadores genéticos de risco para forte produção de biofilme por uma via independente de ica. Nosso estudo contribui para a compreensão da produção de biofilme como um fator de virulência, potencialmente envolvido na severidade e persistência de infecções causadas por S. aureus pertencentes a este genótipo

The nares as a CA-MRSA reservoir in the healthy elderly

Abstract:INTRODUCTION:The frequency of methicillin-resistant Staphylococcus aureus (MRSA) has increased in the community. This study evaluated the prevalence of MRSA and community-acquired (CA)-MRSA in 120 healthy elderly.METHODS:The MRSA were evaluated for the presence of the IS256, mecA, agr, icaA, icaD, fnbB , and pvl genes with PCR. Results: Frequency of S. aureus and MRSA colonization was 17.8% and 19%, respectively. CA-MRSA isolate showed SCC mec IV, fnbB+ , and icaD+ .CONCLUSIONS:CA-MRSA was detected, with genotype determined as SCC mec type IV/IS256/ fnbB+ / icaA / icaD+ / bbp-/agr2 / bap / pvl, characterizing this population as a possible reservoir of this organism in the community

Spread of multidrug-resistant Acinetobacter baumannii and Pseudomonas aeruginosa clones in patients with ventilator-associated pneumonia in an adult intensive care unit at a university hospital

Background:In Brazil, ventilator-associated pneumonia (VAP) caused by carbapenem resis- tant Acinetobacter baumanniiand Pseudomonas aeruginosaisolates are associated with signi&#64257;cant mortality, morbidity and costs. Studies on the clonal relatedness of these isolates could lay the foundation for effective infection prevention and control programs.Objectives: We sought to study the epidemiological and molecular characteristics of A. baumannii vs. P. aeruginosaVAP in an adult intensive care unit (ICU).Methods: It was conducted a cohort study of patients with VAP caused by carbapenem resistant A. baumanniiand P'. aeruginosaduring 14 months in an adult ICU. Genomic studies were used to investigate the clonal relatedness of carbapenem resistant OXA-23-producing A. baumanniiand P. aeruginosaclinical isolates. The risk factors for acquisition of VAP were also evaluated. Clinical isolates were collected for analysis as were samples from the environment and were typed using pulsed &#64257;eld gel electrophoresis.Results: Multivariate logistic regression analysis identi&#64257;ed trauma diagnosed at admission and inappropriate antimicrobial therapy as independent variables associated with the development of A. baumanniiVAP and hemodialysis as independent variable associated with P. aeruginosaVAP. All carbapenem resistant clinical and environmental isolates of A. baumanniiwere OXA-23 producers. No MBL-producer P. aeruginosawas detected. Molecular typing revealed a polyclonal pattern; however, clone A (clinical) and H (surface) were the most frequent among isolates of A. baumanniitested, with a greater pattern of resistance than other isolates. In P. aeruginosathe most frequent clone I was multi-sensitive.Conclusion: These &#64257;ndings suggest the requirement of constant monitoring of these microor- ganisms in order to control the spread of these clones in the hospital environment