Comparison of MICE and Etest with CLSI Agar Dilution for Antimicrobial Susceptibility Testing against Oxacillin-Resistant Staphylococcus spp.

Objective: the main objective of this study was to comparatively evaluate the performance of M. I. C. E. and Etest methodologies to that of agar dilution for determining the antimicrobial susceptibility profile of oxacillin-resistant Staphylococcus spp.Methods: A total of 100 oxacillin-resistant Staphylococcus spp. isolates were collected from hospitalized patients at a teaching hospital. Antimicrobial susceptibility testing for vancomycin, teicoplanin and linezolid was performed using the reference CLSI agar dilution method (2009), Etest and M. I. C. E. methodologies. the MIC values were interpreted according to CLSI susceptibility breakpoints and compared by regression analysis.Results: in general, the essential agreement (+/- 1-log(2)) between M. I. C. E. and CLSI agar dilution was 93.0%, 84.0% and 77.0% for linezolid, teicoplanin and vancomycin, respectively. Essential agreement rates between M. I. C. E. and Etest were excellent (>90.0%) for all antibiotics tested. Both strips (M. I. C. E. and Etest) yielded two very major errors for linezolid. Unacceptable minor rates were observed for teicoplanin against CoNS and for vancomycin against S. aureus.Conclusions: According to our results, linezolid and teicoplanin MICs against all staphylococci and S. aureus, respectively, were more accurately predicted by M. I. C. E. strips. However, the Etest showed better performance than M. I. C. E. for predicting vancomycin MICs against all staphylococci. Thus, microbiologists must be aware of the different performance of commercially available gradient strips against staphylococci.Thermo Fisher Scientific, São Paulo, BrazilConselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Universidade Federal de São Paulo, Lab Alerta, Disciplina Infectol, São Paulo, BrazilUniversidade Federal de São Paulo, Hosp São Paulo, Lab Cent, São Paulo, BrazilUniversidade Federal de São Paulo, Lab Alerta, Disciplina Infectol, São Paulo, BrazilUniversidade Federal de São Paulo, Hosp São Paulo, Lab Cent, São Paulo, BrazilCNPq: 307816/2009-5Web of Scienc

The route of antimicrobial resistance from the hospital effluent to the environment: focus on the occurrence of KPC-producing Aeromonas spp. and Enterobacteriaceae in sewage

We investigated the antimicrobial resistance profile and the occurrence of Klebsiella pneumoniae carbapenemase (KPC)-producing Gram-negative rods in sewage samples obtained from a Brazilian teaching hospital and from the wastewater treatment plant (WWTP) that receives it for treatment. We identified multidrug-resistant bacteria as well as KPC-2-producing Aeromonas spp. and several Enterobacteriaceae species, including Kluyvera spp., in the hospital effluent and in different sites of the WWTP. Most isolates showed the bla(KPC-2) gene harbored on a transposon that was carried by conjugative plasmids. the presence of KPC production among Aeromonas spp., Kluyvera spp., and other Enterobacteriaceae indicates the adaptability of such isolates to aquatic environments, not only in the hospital effluent but also throughout the WWTP. Although secondary treatment seems to decrease the amount of KPC producers in sewage, multidrug-resistant isolates are continually disposed in the urban river. Thus, sewage treatment regulations are urgently needed to decelerate the evolution of antimicrobial resistance beyond hospitals. (C) 2013 Elsevier Inc. All rights reserved.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Sanofi-AventisThermo FisherbioMerieuxUniversidade Federal de São Paulo, Dept Med, Div Infectol, Lab ALERTA, São Paulo, BrazilUniv Fed Rio de Janeiro, Inst Microbiol Paulo de Goes, Rio de Janeiro, BrazilUniversidade Federal de São Paulo, Dept Biofis, São Paulo, BrazilFundacao Parque Zool São Paulo, Lab Microbiol Aplicada, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Med, Div Infectol, Lab ALERTA, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biofis, São Paulo, BrazilFAPESP: 2009/11051-0CNPq: 307816/2009-5CNPq: 302981/2011-0Web of Scienc

Staphylococcus saprophyticus Recovered from Humans, Food, and Recreational Waters in Rio de Janeiro, Brazil.

Staphylococcus saprophyticus is an important agent of urinary tract infection (UTI) in young women, but information about this pathogen in human microbiota and in common environment is lacking. The aim of this study was to characterize S. saprophyticus isolates from genitoanal microbiota of 621 pregnant women, 10 minas cheese packs, and five beaches in Rio de Janeiro city and compare PFGE profiles of these isolates with five UTI PFGE clusters described in this city. We investigated 65 S. saprophyticus isolates from microbiota, 13 from minas cheese, and 30 from beaches and 32 UTI isolates. Antimicrobial resistance was determined by disk diffusion, MIC by agar dilution, and PCR. Erythromycin-resistance genes erm(C), msr(A), msr(B), mph(C), and lin(A) were found in 93% of isolates. Trimethoprim-sulfamethoxazole resistance correlated with dfrG or dfrA genes. Three cefoxitin-resistant isolates carried the mecA gene. All isolates obtained from cheese were susceptible to all antimicrobial agents. Six of 10 pregnant women with >1 isolate had monoclonal colonization. Isolates from pregnant women shared 100% similarity with UTI PFGE cluster types A and E obtained almost 10 years previously, suggesting temporal persistence of S. saprophyticus. Antimicrobial resistance of beach isolates reflected the profiles of human isolates. Taken together, results indicate a shared source for human and environmental isolates

Carbapenen resistance and susceptible to broad spectrum cephalosporins in Pseudomonas aeruginosa clinical isolates: evaluation of mechanisms of resistance involved

Objetivo: Esse estudo teve como objetivo avaliar os mecanismos de resistencia em isolados clinicos de P. aeruginosa que apresentavam fenotipo de resistencia aos carbapenens e sensibilidade as cefalosporinas de amplo espectro. Material e Metodos: Foram estudados 25 isolados de P. aeruginosa recuperadas de diferentes sitios infecciosos em tres distintos hospitais da cidade de São Paulo. Os isolados foram submetidos ao teste de sensibilidade antimicrobiana por microdiluicao em caldo (CLSI-2013). A similaridade genetica foi avaliada pela tecnica de PFGE, e a atividade enzimatica das carbapenemases foi avaliada pelo metodo de hidrolise enzimatica e MALDI-TOF. A pesquisa de genes que codificam as β-Lactamases foi realizada pela tecnica da PCR, seguido por sequenciamento. A desrrepressao de ampC e a hiperexpressao dos sistemas de efluxo foram avaliados fenotipicamente pela adicao de seus inibidores (cloxacilina e PAβN, respectivamente). A transcricao dos genes mexB, mexD, mexF e mexY, da β-lactamase cromossomal ampC e da porina oprD foi avaliada pela tecnica de qRT-PCR. As proteinas de membrana externa foram avaliadas pela tecnica de SDS-PAGE e em seguida foi realizada a tecnica da PCR. A resistencia aos aminoglicosideos e ao ciprofloxacino foi avaliada pela a tecnica da PCR, seguida por sequenciamento dos respectivos genes responsaveis por conferirem resistencia a estes antimicrobianos. Resultados: O fenotipo de resistencia a pelo menos um dos carbapenens (imipenem e meropenem) e sensibilidade as cefalosporinas de amplo espectro (cefepima e ceftazidima) foi confirmado em todos isolados clinicos estudados. A resistencia a ciprofloxacina e aos aminoglicosideos foi observada em cinco (20%) e dois (8%) desses isolados, respectivamente. A analise do perfil eletroforetico por PFGE permitiu o agrupamento dos isolados estudados em 17 padroes distintos sem a predominancia de um perfil clonal. Nenhum isolado apresentou atividade carbapenemase no teste de hidrolise, no entanto, no MALDI-TOF quatro isolados apresentaram resultados duvidosos. Porem, a pesquisa de genes codificadores de carbapenemases conhecidas foi negativa. Os genes cromossomais, blaampC e blaOXA-50 foram amplificados entre as amostras estudadas e seu sequenciamento revelou diversas mutacoes no gene blaOXA-50. O gene blaOXA-56 estava presente em um isolado. A inibicao in vitro da AmpC cromossomal e da expressao dos sistemas de efluxo produziu uma variacao de ≥ 2 diluicoes na CIMs dos antimicrobianos testados para a maioria dos isolados testados. A media da expressao relativa dos genes mexB, mexD, mexF e mexY nas 25 amostras de P. aeruginosa foram 1,92; 2,69; 6,04 e 1,56, respectivamente, em relacao a cepa referencia PAO1. A β-lactamase AmpC estava hiperexpressa em 40% dos isolados de P. aeruginosa, enquanto todos os isolados tiveram reducao da expressao de OprD. Uma alteracao na banda de 46 kDa foi identificada pelo SDS-PAGE em todos isolados clinicos estudados. Os cinco isolados resistentes a ciprofloxacina apresentaram mutacao na QRDR dos genes gyrA e parC e apenas um dos isolados resistentes aos aminoglicosideos carregava o gene de resistencia rmtD. Conclusao: Nossos resultados sugerem o envolvimento de multiplos mecanismos cromossomais, como a hiperexpressao da β-lactamase cromossomal AmpC e a reducao da expressao de OprD no fenotipo de resistencia aos carbapenens e sensibilidade as cefalosporinas de amplo espectro entre isolados clinicos de P. aeruginosaBV UNIFESP: Teses e dissertaçõe

Avaliação das metodologias M.I.C.E.®, Etest® e microdiluição em caldo para determinação da CIM em isolados clínicos Evaluation of M.I.C.E.TM, Etest® and CLSI broth microdilution methods for antimicrobial susceptibility testing of nosocomial bacterial isolates

INTRODUÇÃO: As fitas Oxoid® M.I.C.Evaluator® (M.I.C.E., Thermo Fisher Scientific, Basingstoke, UK), recém-lançadas no mercado brasileiro, representam uma alternativa rápida para a realização de testes de sensibilidade a antimicrobianos (TSA). OBJETIVO: Avaliar o desempenho da metodologia M.I.C.E. em relação à microdiluição em caldo (teste de referência) e ao Etest® (BioMérieux, Marcy l'Étoile, France). Material e métodos: Foram selecionados 160 isolados bacterianos, sendo P. aeruginosa (20), Acinetobacter spp. (20), K. pneumoniae (20), E. coli (20), S. aureus (20), Staphylococcus coagulase-negativa (20), E. faecalis (20) e E. faecium (20). Os TSAs foram realizados por microdiluição em caldo, Etest e M.I.C.E., seguindo-se as recomendações do Clinical Laboratory Standards Institute (CLSI, 2009) e dos respectivos fabricantes. Os resultados foram interpretados segundo os critérios estabelecidos pelo CLSI e comparados por análise de regressão. RESULTADOS: Avaliando-se todas as combinações de antimicrobianos vs. a espécie bacteriana, o desempenho da metodologia M.I.C.E. foi muito bom, apresentando uma concordância geral (variação na concentração inibitória mínima [CIM] ± 1-log2) > 90%, exceto para cefotaxima (85%) e vancomicina (76,3%), quando em comparação com os resultados da metodologia de referência. Quando comparado com o Etest, a metodologia M.I.C.E. apresentou concordância geral > 96%, com exceção para a combinação amoxicilina/ácido clavulânico (67,5%). CONCLUSÃO: Os resultados do TSA obtidos pela metodologia M.I.C.E. apresentaram boa correlação com aqueles obtidos pela microdiluição em caldo e pelo Etest, indicando que essa metodologia é uma alternativa rápida para a determinação da CIM pelos laboratórios de microbiologia clínica. Atenção especial deve ser dada á determinação da CIM para a combinação amoxicilina/ácido clavulânico.<br>INTRODUCTION: The Oxoid® M.I.C.EvaluatorTM methodology (M.I.C.E., Thermo Fisher Scientific, Basingstoke, UK), recently released into the market, represents a rapid alternative to antimicrobial susceptibility testing. OBJECTIVE: The objective of this study was to evaluate the performance of M.I.C.E. methodology in relation to broth microdilution (reference test) and Etest® (BioMérieux, Marcy l'Étoile, France). Material and method: A total of 160 bacterial isolates were collected comprising the following species: P. aeruginosa (20), Acinetobacter spp. (20), K. pneumoniae (20), E. coli (20), S. aureus (20), coagulase-negative Staphylococcus (20), E. faecalis (20) and E. faecium (20). Following Clinical Laboratory Standands Institute (CLSI) standards (2009) and the manufacturers' recommendations, antimicrobial susceptibility testing was performed using broth microdilution method, Etest and M.I.C.E. The results were interpreted according to the criteria established by CLSI and compared through regression analysis. RESULTS: All antimicrobial combinations vs. bacterial species were evaluated and M.I.C.E. methodology yielded good results with general correlation (MIC variation ± 1-log2) > 90%, except for cefotaxime (85%) and vancomycin (76.3%) when compared with the reference method. The M.I.C.E. results compared to Etest showed general correlation (> 96%), except for amoxicillin/clavulanic acid (67.5%) combination. CONCLUSION: AST results obtained from M.I.C.E. methodology showed a good correlation with those from broth microdilution and Etest, which corroborates its time effectiveness in the determination of MIC. However, the combination of amoxicillin/clavulanic acid requires further attention

Carbapenem-resistant and cephalosporin-susceptible: a worrisome phenotype among Pseudomonas aeruginosa clinical isolates in Brazil

The mechanisms involved in the uncommon resistance phenotype, carbapenem resistance and broad-spectrum cephalosporin susceptibility, were investigated in 25 Pseudomonas aeruginosa clinical isolates that exhibited this phenotype, which were recovered from three different hospitals located in São Paulo, Brazil. The antimicrobial susceptibility profile was determined by CLSI broth microdilution. β-lactamase-encoding genes were investigated by PCR followed by DNA sequencing. Carbapenem hydrolysis activity was investigated by spectrophotometer and MALDI-TOF assays. The mRNA transcription level of oprD was assessed by qRT-PCR and the outer membrane proteins profile was evaluated by SDS-PAGE. Genetic relationship among P. aeruginosa isolates was assessed by PFGE. Carbapenems hydrolysis was not detected by carbapenemase assay in the carbapenem-resistant and cephalosporin-susceptible P. aueruginosa clinical isolates. OprD decreased expression was observed in all P. aeruginosa isolates by qRT-PCR. The outer membrane protein profile by SDS-PAGE suggested a change in the expression of the 46 kDa porin that could correspond to OprD porin. The isolates were clustered into 17 genotypes without predominance of a specific PFGE pattern. These results emphasize the involvement of multiple chromosomal mechanisms in carbapenem-resistance among clinical isolates of P. aeruginosa, alert for adaptation of P. aeruginosa clinical isolates under antimicrobial selective pressure and make aware of the emergence of an uncommon phenotype among P. aeruginosa clinical isolates. Keywords: Gram-negative bacilli, Carbapenem resistance, OprD pori