Detection of Mycobacterium chelonae, Mycobacterium abscessus Group, and Mycobacterium fortuitum Complex by a Multiplex Real-Time PCR Directly from Clinical Samples Using the BD MAX System.

A new multiplex PCR test was designed to detect Mycobacterium chelonae, Mycobacterium abscessus group, and Mycobacterium fortuitum complex on the BD MAX System. A total of 197 clinical samples previously submitted for mycobacterial culture were tested using the new protocol. Samples were first treated with proteinase K, and then each sample was inoculated into the BD MAX Sample Buffer Tube. Extraction and multiplex PCR were performed by the BD MAX System, using the BD MAX ExK TNA-3 extraction kit and BD TNA Master Mix, along with specific in-house designed primers and probes for each target. The limit of detection of each target, as well as specificity, was evaluated. Of 197 clinical samples included in this study, 133 were positive and 60 were negative for mycobacteria by culture, and another 4 negative samples were spiked with M. chelonae ATCC 35752. The new multiplex PCR on the BD MAX had 97% concordant results with culture for M. abscessus group detection, 99% for M. chelonae, and 100% for M. fortuitum complex. The new multiplex PCR test performed on the BD MAX System proved to be a sensitive and specific test to detect M. chelonae, M. abscessus group, and M. fortuitum complex by real-time PCR on an automated sample-in results-out platform.

Differentiation of Klebsiella pneumoniae carbapenemase (KPC) variants by pyrosequencing.

A fast and reliable protocol using the pyrosequencing technique was developed to identify 11 different types of the KPC enzyme. A total of 65 blaKPC positive bacterial isolates were tested and characterized. In the end, the pyrosequencing proved to be a powerful tool for epidemiological studies of KPC producer isolates.

SPM-1-producing Pseudomonas aeruginosa: analysis of the ancestor relationship using multilocus sequence typing, pulsed-field gel electrophoresis, and automated ribotyping.

In Brazil, the spread of an endemic clone of SPM-1-producing Pseudomonas aeruginosa has been reported. Recently, a higher genomic variety has been observed among the SPM-1-producing P. aeruginosa isolates. The principal aim of this study was to analyze through multilocus sequence typing (MLST) analysis whether the recently isolated SPM-1-producing P. aeruginosa descend or not from a common ancestor. A total of 50 SPM-1-producing P. aeruginosa exhibiting 11 distinct ribotyping genotypes collected from 11 different Brazilian cities were studied. Three IMP-1-producing P. aeruginosa and two non-metallo-beta-lactamase-producing P. aeruginosa isolates were included in the study as controls. For assignment of allelic numbers and subsequent determination of sequence type (ST), the obtained sequences were compared to existing sequences in the MLST database (www.pubmlst.org/paeruginosa). The eBURSTv3 software was used in this study for establishing the evolutionary relationship and phylogenetic analysis. A total of 5 different STs were identified among 55 P. aeruginosa isolates. All of the SPM-1-producing P. aeruginosa presented an identical allelic profile (ST277), except for one strain. The three IMP-1-producing P. aeruginosa strains were classified as belonging to the ST593, whereas the non-metallo-beta-lactamase-producing P. aeruginosa showed two new distinct STs, ST594 and ST595. Our study shows that SPM-1-producing P. aeruginosa isolates as well as the IMP producers evaluated in this study descend from a common ancestor.

Molecular characterization of vancomycin-resistant Enterococci strains eight years apart from its first isolation in São Paulo, Brazil.

E. faecium was the first reported VRE species, carrying the vanA gene in Brazil. In spite of this, vancomycin-resistant E. faecalis has become the predominant species in Brazilian hospitals. The aim of this study was to evaluate the genetic relatedness of VREs isolated in a Brazilian teaching hospital eight years apart from its first isolation. We analyzed 38 VRE strains obtained from 81 surveillance cultures of patients admitted to the four largest intensive care units in Hospital São Paulo in February, 2006. Presence of the vanA gene was assayed by PCR and PFGE analysis was used for molecular characterization. All VRE strains carried the vanA gene. Two distinct clonal groups were observed among vancomycin-resistant E. faecalis. Vancomycin-resistant E. faecium belonged to five distinct clones were demonstrated by molecular typing. All of these clones were different from the first vancomycin-resistant enterococci clone isolated eight years ago in our hospital.

Molecular characterization of vancomycin-resistant Enterococci strains eight years apart from its first isolation in São Paulo, Brazil / Caracterização molecular de espécies de Enterococci resistentes à vancomicina oito anos após seu primeiro isolamento em São Paulo, Brasil

E. faecium was the first reported VRE species, carrying the vanA gene in Brazil. In spite of this, vancomycin-resistant E. faecalis has become the predominant species in Brazilian hospitals. The aim of this study was to evaluate the genetic relatedness of VREs isolated in a Brazilian teaching hospital eight years apart from its first isolation. We analyzed 38 VRE strains obtained from 81 surveillance cultures of patients admitted to the four largest intensive care units in Hospital São Paulo in February, 2006. Presence of the vanA gene was assayed by PCR and PFGE analysis was used for molecular characterization. All VRE strains carried the vanA gene. Two distinct clonal groups were observed among vancomycin-resistant E. faecalis. Vancomycin-resistant E. faecium belonged to five distinct clones were demonstrated by molecular typing. All of these clones were different from the first vancomycin-resistant enterococci clone isolated eight years ago in our hospital.

E. faecium contendo o gene vanA foi a primeira espécie de VRE descrita, no Brasil. Apesar disto, E. faecalis resistente a vancomicina tem se tornado a espécie predominante nos hospitais brasileiros.O objetivo desse estudo foi avaliar a relação genética de VREs isolados em um hospital de ensino brasileiro após oito anos de seu primeiro isolamento. Analisamos 37 isolados de VRE obtidos de 81 culturas de vigilância de pacientes admitidos nas quatro maiores Unidades de Tratamento Intensivo em Fevereiro de 2006. A presença do gene vanA foi analisada por PCR e a caracterização molecular por PFGE. Todas as amostras VRE carreavam o gene vanA. Entre os E. faecalis vancomicina-resistentes, dois distintos grupos clonais foram observados. E. faecium resistente a vancomicina pertencentes a cinco clones distintos foram demonstrados por tipagem molecular. Todos esses clones foram diferentes do primeiro clone de enterococo resistente a vancomicina isolado oito anos atrás em nosso hospital.

Genetic relatedness among clinical strains of Stenotrophomonas maltophilia in tertiary care hospital settings in São Paulo State, Brazil

Stenotrophomonas maltophilia is a Gram-negative bacillus, which is becoming widely recognized as an important nosocomial pathogen. The main objective of this study was to evaluate the genetic relatedness, by random amplified polymorphic DNA (RAPD) and pulsed-field gel electrophoresis (PFGE) of 86 clinical isolates of S. maltophilia (colonization 22, infection 64) obtained from 79 hospitalized patients, from different geographic regions of São Paulo State. The genotypic analysis performed by RAPD and PFGE was used in 24 isolates for genetic identity confirmation. The results were congruent between the two methods but it was not possible to link genetic profiles with the studied variables, clinical state and geographic area, probably due to the great variability among the strains. The analyses by PFGE confirmed identity in 5 pairs of microorganisms and RAPD, in this study, showed to be a useful tool for investigation of diversity leading the identification of 85 genetic profiles. The genetic diversity shown may be due to re-infection by different strains or co-infection by multiple strains which suggests multiple entry sources of the bacterium in the hospital setting or of acquisition by patient. In this setting, colonization, infection and re-infection occur with unknown frequency, raising the need for the establishment of specific control measures.

Stenotrophomonas maltophilia é um bacilo Gram-negativo, conhecido como importante patógeno nosocomial. O principal objetivo desse estudo foi avaliar a relação genética, através da análise randômica do polimorfismo de DNA (RAPD) e eletroforese em gel de campo pulsado (PFGE), de 86 isolados clínicos de S. maltophilia (22 de colonização, 64 de infecção) obtidos de 79 pacientes hospitalizados em diferentes regiões geográficas do estado de São Paulo. A análise genotípica foi realizada através da técnica RAPD e o PFGE foi usado em 24 isolados para confirmar a identidade genética dos mesmos. Os resultados foram coerentes entre os dois métodos, mas não foi possível correlacionar um perfil genético com as variáveis estudadas, estado clínico e área geográfica, provavelmente pela ampla variabilidade entre as linhagens. A análise por PFGE confirmou a identidade genética em 5 pares de microrganismos e o RAPD, neste estudo, mostrou ser uma ferramenta útil para investigação da diversidade, possibilitando identificar 85 perfis genéticos. A diversidade genética observada através do RAPD pode ser devido à re-infecção por diferentes linhagens ou co-infecção por linhagens distintas, sugerindo múltiplas fontes de entrada da bactéria no hospital ou de aquisição pelo paciente. Nesse ambiente, a colonização, infecção e re-infecção ocorrem com freqüência, o que leva à necessidade do estabelecimento de medidas de controle específicas.

Genetic relatedness among clinical strains of Stenotrophomonas maltophilia in tertiary care hospital settings in São Paulo State, Brazil

Stenotrophomonas maltophilia is a Gram-negative bacillus, which is becoming widely recognized as an important nosocomial pathogen. The main objective of this study was to evaluate the genetic relatedness, by random amplified polymorphic DNA (RAPD) and pulsed-field gel electrophoresis (PFGE) of 86 clinical isolates of S. maltophilia (colonization 22, infection 64) obtained from 79 hospitalized patients, from different geographic regions of São Paulo State. The genotypic analysis performed by RAPD and PFGE was used in 24 isolates for genetic identity confirmation. The results were congruent between the two methods but it was not possible to link genetic profiles with the studied variables, clinical state and geographic area, probably due to the great variability among the strains. The analyses by PFGE confirmed identity in 5 pairs of microorganisms and RAPD, in this study, showed to be a useful tool for investigation of diversity leading the identification of 85 genetic profiles. The genetic diversity shown may be due to re-infection by different strains or co-infection by multiple strains which suggests multiple entry sources of the bacterium in the hospital setting or of acquisition by patient. In this setting, colonization, infection and re-infection occur with unknown frequency, raising the need for the establishment of specific control measures.

Stenotrophomonas maltophilia é um bacilo Gram-negativo, conhecido como importante patógeno nosocomial. O principal objetivo desse estudo foi avaliar a relação genética, através da análise randômica do polimorfismo de DNA (RAPD) e eletroforese em gel de campo pulsado (PFGE), de 86 isolados clínicos de S. maltophilia (22 de colonização, 64 de infecção) obtidos de 79 pacientes hospitalizados em diferentes regiões geográficas do estado de São Paulo. A análise genotípica foi realizada através da técnica RAPD e o PFGE foi usado em 24 isolados para confirmar a identidade genética dos mesmos. Os resultados foram coerentes entre os dois métodos, mas não foi possível correlacionar um perfil genético com as variáveis estudadas, estado clínico e área geográfica, provavelmente pela ampla variabilidade entre as linhagens. A análise por PFGE confirmou a identidade genética em 5 pares de microrganismos e o RAPD, neste estudo, mostrou ser uma ferramenta útil para investigação da diversidade, possibilitando identificar 85 perfis genéticos. A diversidade genética observada através do RAPD pode ser devido à re-infecção por diferentes linhagens ou co-infecção por linhagens distintas, sugerindo múltiplas fontes de entrada da bactéria no hospital ou de aquisição pelo paciente. Nesse ambiente, a colonização, infecção e re-infecção ocorrem com freqüência, o que leva à necessidade do estabelecimento de medidas de controle específicas.

Prevalence of vancomycin-resistant Enterococcus fecal colonization among kidney transplant patients.

BACKGROUND: End stage renal disease patients are at risk of Vancomycin-Resistant Enterococcus (VRE) infections. The first reports of VRE isolation were from hemodialysis patients. However, to date, VRE fecal colonization rates as well as associated risk factors in kidney transplant patients have not yet been established in prospective studies. METHODS: We collected one or two stool samples from 280 kidney transplant patients and analysed the prevalence of VRE and its associated risk factors. Patients were evaluated according to the post-transplant period: group 1, less than 30 days after transplantation (102 patients), group 2, one to 6 months after transplantation (73 patients) and group 3, more than 6 months after transplantation (105 patients). RESULTS: The overall prevalence rate of fecal VRE colonization was 13.6% (38/280), respectively 13.7% for Group 1, 15.1% for group 2 and 12.4% for group 3. E. faecium and E. faecalis comprised 50% of all VRE isolates. No immunologic variables were clearly correlated with VRE colonization and no infections related to VRE colonization were reported. CONCLUSION: Fecal VRE colonization rates in kidney transplant patients were as high as those reported for other high-risk groups, such as critical care and hemodialysis patients. This high rate of VRE colonization observed in kidney transplant recipients may have clinical relevance in infectious complications.

Dissemination of IMP-1 metallo- beta -lactamase-producing Acinetobacter species in a Brazilian teaching hospital.

OBJECTIVE: To evaluate the emergence and dissemination of metallo- beta -lactamase (MBL)-producing Acinetobacter species. DESIGN: All carbapenem-resistant Acinetobacter strains (1 strain per patient) collected during the period 1993-2001 were evaluated. SETTING: A Brazilian tertiary care teaching hospital (Hospital Sao Paulo, Sao Paulo). METHODS: Seventy-three strains of carbapenem-resistant Acinetobacter species were recovered from the organism bank of the hospital. All isolates were tested for antimicrobial susceptibility by broth microdilution methods, and the production of MBL was initially assessed by phenotypic tests (MBL Etest strip and a disk approximation test). The MBL enzymes were identified by polymerase chain reaction using primers for bla(IMP), bla(VIM), and bla(SPM), followed by gene sequencing. Genetic similarity among the carbapenem-resistant strains was evaluated by automated ribotyping. RESULTS: Only colistin and ampicillin-sulbactam showed reasonable in vitro activity against carbapenem-resistant isolates (97% and 74% of isolates susceptible, respectively). More than half of the isolates (55%) had a positive MBL phenotypic test result and a positive polymerase chain reaction result for bla(IMP-1). The proportion of IMP-1-producing Acinetobacter isolates among carbapenem-resistant strains increased from 0% in the 1993-1997 period to 29% in 1998 and 100% in the 1999-2001 period. No carbapenem-resistant Acinetobacter isolates that harbored bla(VIM) or bla(SPM) were detected. Molecular typing results revealed 20 ribogroups among carbapenem-resistant isolates. During the study period of 1994-2001, we identified 2 major ribogroups, 52-1 (MBL-negative and MBL-positive strains) and 60-7 (MBL-positive strains), that had a coefficient of similarity of 0.85 or higher. CONCLUSIONS: Our results indicate that IMP-1-producing strains of Acinetobacter emerged in our institution in 1998. Since then, production of this MBL was detected not only in the major ribogroups of carbapenem-resistant Acinetobacter species but also among isolates that belonged to 17 distinct ribogroups, indicating that this important mechanism of antimicrobial resistance was disseminated among distinct clones.

Evaluation of three molecular typing techniques for nonfermentative Gram-negative bacilli.

OBJECTIVE: To evaluate three different DNA techniques for typing nonfermentative gram-negative bacilli isolated from Latin American hospitals. DESIGN: One hundred twenty-six nonfermentative gram-negative bacilli were typed. PARTICIPANTS: Pseudomonas aeruginosa (n = 64) and Acinetobacter baumannii (n = 42) samples were obtained from blood cultures of patients admitted to 10 medical centers in Latin America during 1998 and Stenotrophomonas maltophilia (n = 20) samples were obtained from patients admitted to the Hospital São Paulo between 1999 and 2001. METHODS: All samples were typed using automated ribotyping, PFGE, and ERIC-PCR. The discriminatory power for each technique was calculated using Hunter's generalized formula. RESULTS: All strains could be typed by automated ribotyping and ERIC-PCR, but two strains (1.6%) were not typeable by PFGE. All three techniques showed 100% reproducibility. The time to obtain the results was shorter for automated ribotyping and ERIC-PCR compared with PFGE. Likewise, the costs for ERIC-PCR and PFGE were lower than those for automated ribotyping. The interpretation of results was more complicated and more difficult with ERIC-PCR than with both PFGE and automated ribotyping. All techniques presented excellent discriminatory power for P. aeruginosa (0.98). PFGE presented the highest discriminatory power (0.94) for A. baumannii, and both PFGE and ERIC-PCR showed higher discriminatory power (0.90 for both) than automated ribotyping (0.82) for S. maltophilia. CONCLUSIONS: PFGE showed the highest discriminatory power for typing these nonfermentative gram-negative bacilli. However, automated ribotyping and ERIC-PCR can provide results in a shorter time period with similar discriminatory power.

Resistance trends of Acinetobacter spp. in Latin America and characterization of international dissemination of multi-drug resistant strains: five-year report of the SENTRY Antimicrobial Surveillance Program.

OBJECTIVES: To analyze the antimicrobial susceptibility of Acinetobacter spp. isolates collected from Latin American medical centers as part of the SENTRY Antimicrobial Surveillance Program and also to evaluate the dissemination of multi-drug resistant Acinetobacter spp. strains in the region. METHODS: A total of 826 isolates of Acinetobacter spp. from multiple infection sites were collected from January 1997 to December 2001 in ten medical centers and susceptibility tested to >25 selected agents by broth microdilution. Multi-drug resistant Acinetobacter spp. isolates were molecular typed. RESULTS: Resistance rates to carbapenems varied significantly among countries. A continued annual increase occurred in the Argentinean medical centers. In contrast, carbapenem resistance was rare in Chilean centers, and decreased significantly in the Brazilian institutions. Acinetobacter spp. isolates recovered from lower respiratory tract and bloodstream infections were associated with lower antimicrobial susceptibility rates. Resistance rates to imipenem were higher among isolates collected from intensive care units (13.5%) than among isolates from other units. A major ribogroup pattern (521-1) was detected among eight Acinetobacter spp. strains isolated from three distinct Latin American countries. CONCLUSIONS: This study found that antimicrobial resistance is still a major issue among Acinetobacter spp. isolates collected from some Latin American countries. The dissemination of a major bacterial cluster in different regions reinforces the importance of longitudinal surveillance programs, such as SENTRY, as valuable tools for monitoring antimicrobial susceptibility rates and guiding local interventions.

Evaluation of the prevalence and risk factors for colonization by vancomycin-resistant Enterococcus among patients on dialysis.

BACKGROUND: Vancomycin-resistant Enterococcus (VRE) has been reported among long-term dialysis patients, although risk factors for VRE colonization are not well defined. This study aims to appraise the prevalence and risk factors for VRE colonization among patients on long-term dialysis therapy, as well as the mechanisms for dissemination of vancomycin resistance. METHODS: This is a cross-sectional survey of 320 patients on long-term dialysis therapy at 2 hospitals of the Federal University of São Paulo from June 2001 to March 2003. Fecal samples were collected from each patient once a week for 1 month. Samples with positive test results for VRE were submitted to molecular typing through automated ribotyping. RESULTS: VRE prevalence was 14.4%. There were significant associations between VRE and dialysis type (hemodialysis, P = 0.04), number of hospitalizations (P = 0.03), low hemoglobin level (P = 0.03), and leukocytosis (P = 0.05). Among samples with VRE (n = 56), 25% were Enterococcus faecium; 10.7%, Enterococcus casseliflavus; 57.1%, Enterococcus gallinarum; and 3.6%, Enterococcus faecalis. All samples isolated were sensitive to teicoplanin, except for E faecium samples, which were strongly resistant, although 9 of 14 patients with this isolate presented the same ribogroup (111-S-4). Typing of 6 samples from 8 dialysis patients with E gallinarum was performed, showing a predominant ribogroup (112-S-4). CONCLUSION: Hospital environment, hemodialysis, anemia, and leukocytosis appear to be associated with VRE colonization. These results suggest that dissemination of these bacteria among patients on long-term dialysis therapy may be taking place.

Tipagem molecular de bacilos grem-negativos não-fermentadores / Molecular typing of non-fermentative gram-negative bacilli: comparison and application of different techniques

OBJETIVOS: (a) Avaliar três técnicas para a tipagem molecular de bacilos Gram-negativos não-fermentadores (BGN-NF) isolados na América Latina (AL); (b) propor modificações na técnica PFGE para bactérias sensíveis a degradação do DNA; e (c) avaliar a aplicabilidade destas técnicas de tipagem molecular em estudos longos de epidemiologia hospitalar. MÉTODOS: Três coleções diferentes de bactérias foram avaliadas. Primeiro foi estudado um grupo de 126 BGN-NF (64 P. aeruginosa, 42 A. baumannii e 20 S. maltophifa) isolados do sangue de pacientes atendidos em 10 centros médicos localizados na AL no ano de 1998 e. de pacientes atendidos no Hospital São Paulo (HSP) entre os anos de 1999 e 2001. Todos estes isolados clínicos foram submetidos à tipagem molecular pelas técnicas ribotipagem automatizada, PFGE e ERIC-PCR. O poder discriminatório (PD) de cada técnica foi calculado utilizando a fórmula de Hunter. Um segundo grupo de amostras bacterianas foi composto por 66 isolados clínicos sensíveis a degradação do DNA pela técnica PFGE e incluiu as seguintes espécies: E. coli (22 amostras), K. pneumoniae (4 amostras), E. cloacae (2 amostras), S. marcescens (12 amostras), P. aeruginosa (17 amostras), Acinetobacter spp. (5 amostras) e Salmonella spp. (4 amostras). Todas as 66 amostras foram submetidas à tipagem molecular pela técnica PFGE, sendo que a eletroforese foi realizada com e sem a adição de tiourea no tampão de corrida TBE. Um terceiro grupo incluiu 26 isolados clínicos de P. aeruginosa resistentes ao imipenem (PARI), isolados de pacientes atendidos no HSP durante um período de 6 anos (1997 a 2002). Todas as amostras eram representantes de feridas cirúrgicas e foram submetidas à tipagem molecular utilizando as técnicas PFGE e ribotipagem automatizada. RESULTADOS: Todas as amostras pertencentes ao primeiro grupo foram tipáveis pelas técnicas ERIC-PCR e ribotipagem automatizada e duas (1,6 por cento) amostras não foram tipáveis pela técnica PFGE. As três técnicas apresentaram 100 por cento de reprodutibilidade. O tempo necessário para realização da ribotipagem automatizada e ERIC-PCR foram menores quando comparados com o tempo necessário para realização do PFGE. O ERIC-PCR e o PFGE apresentaram custo mais baixo que a ribotipagem automatizada; no entanto, a interpretação dós resultados pelo ERIC-PCR foi mais difícil. As três técnicas…

The use of molecular typing to evaluate the dissemination of antimicrobial resistance among gram-negative rods in Brazilian hospitals

Antimicrobial resistance has increased rapidly in Brazil and worldwide during the past few years, giving rise to a growing necessity for antimicrobial resistance surveillance programs. These programs have been instituted in order to monitor bacterial resistance in various regions, and to guide empirical antimicrobial therapy. We evaluated the use of molecular typing in multicenter surveillance programs. We also studied the dissemination modes of selected resistance profiles. Antimicrobial susceptibility to various antimicrobial agents was evaluated by the reference broth microdilution method. Bacterial isolates with selected susceptibility patterns were characterized by pulsed field-gel electrophoresis (PFGE). A total of 119 Gram-negative bacteria were molecularly typed, including 22 imipenem-resistant Pseudomonas aeruginosa, 26 ESBL-producing Escherichia coli, 27 cefoxitin-resistant-ESBL-producing Klebsiella pneumoniae, 33 Enterobacter spp., 8 Citrobacter spp., and 3 S. marcescens isolates resistant to ceftazidime. The isolates were from clinically apparent bacteremia of patients hospitalized in medical centers located in 13 cities of 11 Brazilian states. Our molecular typing results revealed a great genetic diversity among isolates of the same species. However, some major PFGE patterns were found in more than one isolate. All repeated PFGE patterns were detected in only 2 isolates, which were isolated within the same institutions or in different medical centers. We conclude that the ability to characterize organisms phenotypically and genotypically is a powerful epidemiologic tool and it provides unique information that is very important for multicenter surveillance programs.

The use of molecular typing to evaluate the dissemination of antimicrobial resistance among Gram-negative rods in Brazilian hospitals.

Antimicrobial resistance has increased rapidly in Brazil and worldwide during the past few years, giving rise to a growing necessity for antimicrobial resistance surveillance programs. These programs have been instituted in order to monitor bacterial resistance in various regions, and to guide empirical antimicrobial therapy. We evaluated the use of molecular typing in multicenter surveillance programs. We also studied the dissemination modes of selected resistance profiles. Antimicrobial susceptibility to various antimicrobial agents was evaluated by the reference broth microdilution method. Bacterial isolates with selected susceptibility patterns were characterized by pulsed field-gel electrophoresis (PFGE). A total of 119 Gram-negative bacteria were molecularly typed, including 22 imipenem-resistant Pseudomonas aeruginosa, 26 ESBL-producing Escherichia coli, 27 cefoxitin-resistant-ESBL-producing Klebsiella pneumoniae, 33 Enterobacter spp., 8 Citrobacter spp., and 3 S. marcescens isolates resistant to ceftazidime. The isolates were from clinically apparent bacteremia of patients hospitalized in medical centers located in 13 cities of 11 Brazilian states. Our molecular typing results revealed a great genetic diversity among isolates of the same species. However, some major PFGE patterns were found in more than one isolate. All repeated PFGE patterns were detected in only 2 isolates, which were isolated within the same institutions or in different medical centers. We conclude that the ability to characterize organisms phenotypically and genotypically is a powerful epidemiologic tool and it provides unique information that is very important for multicenter surveillance programs.

Dissemination in distinct Brazilian regions of an epidemic carbapenem-resistant Pseudomonas aeruginosa producing SPM metallo-beta-lactamase.

BACKGROUND: In Brazil, carbapenem use has been limited by high carbapenem-resistance rates among Pseudomonas aeruginosa isolates. OBJECTIVE: The main objective of this study was to evaluate the presence of an epidemic P. aeruginosa strain in unrelated Brazilian hospitals. We also aimed to search for the gene blaSPM, which encodes production of SPM, a novel metallo-beta-lactamase (MBL). METHODS: A reference broth microdilution method was used for antimicrobial susceptibility testing. The isolates were typed by ribotyping and pulsed-field gel electrophoresis (PFGE). A disc-approximation test using MBL inhibitors was employed to screen isolates for MBL production. PCR was used to search for the gene blaSPM. RESULTS: A total of 43 clinical isolates of carbapenem-resistant P. aeruginosa were collected from 12 hospitals. Colistin retained greatest activity in vitro. A single ribogroup included 17 P. aeruginosa isolates (39.5%) collected from seven unrelated hospitals located in five Brazilian states. Sixteen of these isolates showed an identical PFGE pattern, and 15 produced an SPM-1-like MBL. The remaining 26 isolates were grouped into 25 diverse ribogroups; none were MBL producers. CONCLUSIONS: The emergence and dissemination of an epidemic clone has contributed to the high carbapenem resistance rates among P. aeruginosa isolates in Brazil. In addition, the production of SPM MBL has an important role in carbapenem resistance in this region. This is the first report of dissemination of an SPM-1-like-MBL-producing strain of P. aeruginosa among unrelated Brazilian hospitals.

Avaliaçäo da qualidade dos discos com antimicrobianos para testes de disco-difusäo disponíveis comercialmente no Brasil / Evaluation of the quality of the antimicrobial agents disks used in disk-diffusion tests comercially available in Brazil

Introduçäo: O teste de suscetibilidade a antimicrobianos representa um dos testes de maior importância clínica realizados pelo laboratório de microbiologia. Devido ao grande número de antimicrobianos e à complexidade dos mecanismos de resistência desenvolvidos pelas bactérias, fica muito difícil hoje a detecçäo de problemas nos testes de suscetibilidade pela simples avaliaçäo dos resultados obtidos. Sendo assim, é extremamente importante que haja uma avaliaçäo constante da qualidade destes testes. Objetivo: O objetivo do presente estudo foi avaliar a qualidade dos discos de antimicrobianos comercializados no Brasil. Material e métodos: Foram avaliados discos de 18 antimicrobianos obtidos de cinco diferentes fontes comerciais, os quais foram testados frente a quatro cepas bacterianas oriundas da ATCC, pelo método de difusäo em ágar, seguindo as recomendações do National Committee for Clinical Laboratory Standards (NCCLS). Cada teste foi repetido 20 vezes. Resultados: Nenhuma das marcas apresentou desempenho satisfatório para o uso na rotina de um laboratório de microbiologia. O melhor desempenho foi apresentado pela marca Cecon, com 89,6 por cento de concordância. A marca Sensifar apresentou taxa de concordância geral semelhante (90,8%). A marca com o pior desempenho foi a Pimenta Abreu, com apenas 58,6% de concordância. Conclusäo: Os resultados do presente estudo indicam que os discos de antimicrobianos comercializados no Brasil são de baixa qualidade, possivelmente refletindo a falta de controle de qualidade na produçäo e/ou estocagem dos produtos antes da sua distribuiçäo. Esses dados chamam a atençäo para a necessidade de implantaçäo de sistemas efetivos de fiscalizaçäo da comercializaçäo desses produtos e de programas criteriosos de controle de qualidade por parte dos laboratórios que os utilizam

Typing and molecular characterization of Streptococcus pneumoniae with reduced susceptibility to cefotaxime isolated in Latin America.

The treatment of systemic infections, especially meningitis, caused by Streptococcus pneumoniae nonsusceptible to third-generation cephalosporins, is extremely difficult due to the paucity of therapeutic options. The main objective of this study was to characterize isolates of S. pneumoniae with reduced susceptibility to cefotaxime (MICs, > or = 1 microg/ml) by different typing methods and to evaluate whether clonal dissemination of this pathogen had occurred among Latin American medical centers. A total of 46 isolates collected from respiratory tract specimens, blood cultures, cerebrospinal fluid, eye, and other sources were analyzed. The isolates were collected from Latin American medical centers located in Argentina, Brazil, Chile, Colombia, Mexico, and Uruguay through two multicenter surveillance programs, in 1997 and 1998. Isolates were serotyped and molecular typed by pulsed-field gel electrophoresis (PFGE) and automated ribotyping. Antimicrobial susceptibilities were determined to 19 drugs by reference broth microdilution methods. Ten isolates (21.7%) had cefotaxime MICs > or = 2 microg/ml, whereas 36 (78.3%) had cefotaxime MIC results at 1 microg/ml. All isolates were susceptible to gatifloxacin, levofloxacin, and vancomycin. The isolates were distributed among five major serotypes (%): 23F (39.1%), 14 (32.6%), 19F (23.9%), 9V (2.2%), and 6B (2.2%). However, distinct molecular patterns were detected among isolates with a unique serotype. Six and four PFGE patterns were identified among isolates with serotype 23F and 19F, respectively. When PFGE and automated ribotyping analyses were combined, four clusters were identified. The largest cluster (10 isolates) was represented by isolates with ribotype 18-2, major PFGE pattern I, and serotype 14. ATCC 700671 (international clone Spain 9V-3) also showed ribotype 18-2. This clone was detected in four countries: Argentina, Brazil, Chile, and Uruguay. A second cluster (8 isolates) were characterized by isolates with ribotype 17-4, PFGE type D, and serotype 23F, similar to ATCC 700669 (international clone Spain23F-1). Isolates from this cluster were identified in three countries: Brazil, Chile, and Mexico. Our results indicated that clonal dissemination of S. pneumoniae with reduced susceptibility to cefotaxime has occurred in Latin America mainly among serogroups 14, 19F, and 23F.

Improving typeability of multiple bacterial species using pulsed-field gel electrophoresis and thiourea.

Although pulsed-field gel electrophoresis is considered the "gold standard" technique for molecular typing, typeability may not be excellent for some bacterial species because of DNA degradation. Previous reports suggest that the addition of thiourea in the gel buffer can improved the typeability for some species. In the present study, 66 Gram-negative strains (seven species) known to be affected by DNA degradation and four control strains were evaluated by PFGE with and without the addition of 50 microg/M of thiourea to the buffer used in the electrophoresis. Macrorestriction patterns were obtained for all K. pneumoniae, S. marcescens, P. aeruginosa, and Salmonella spp., for 95.4% of E. coli, and for 50% of E. cloacae strains from the gels performed in the buffer with throurea. However, typeability was not improved for Acinetobacter spp. The range of non-typeable species for which thiourea can limit the problem of DNA degradation is considerably wider than described in previous publications.