RESUMEN
Multiple locus sequence typing (MLST) was undertaken to extend the genetic characterization of 29 isolates of Bacillus cereus and Bacillus thuringiensis previously characterized in terms of presence/absence of sequences encoding virulence factors and via variable number tandem repeat (VNTR). Additional analysis involved polymerase chain reaction for the presence of sequences (be, cytK, inA, pag, lef, cya and cap), encoding putative virulence factors, not investigated in the earlier study. MLST analysis ascribed novel and unique sequence types to each of the isolates. A phylogenetic tree was constructed from a single sequence of 2,838 bp of concatenated loci sequences. The strains were not monophyletic by analysis of any specific housekeeping gene or virulence characteristic. No clear association in relation to source of isolation or to genotypic profile based on the presence or absence of putative virulence genes could be identified. Comparison of VNTR profiling with MLST data suggested a correlation between these two methods of genetic analysis. In common with the majority of previous studies, MLST was unable to provide clarification of the basis for pathogenicity among members of the B. cereus complex. Nevertheless, our application of MLST served to reinforce the notion that B. cereus and B. thuringiensis should be considered as the same species.
Asunto(s)
Bacillus cereus/genética , Bacillus thuringiensis/genética , Repeticiones de Minisatélite/genética , Tipificación de Secuencias Multilocus , Bacillus cereus/patogenicidad , Bacillus thuringiensis/patogenicidad , Brasil , Genotipo , Filogenia , Reacción en Cadena de la Polimerasa , Factores de Virulencia/genéticaRESUMEN
Neisseria meningitidis, Haemophilus influenzae and Streptococcus pneumoniae are leading causes of meningitis and acute invasive infections. PCR-based methods are widely used for the diagnosis and surveillance of bacterial pathogens because of their high sensitivity, specificity and high-throughput capabilities compared with conventional laboratory methods. This study evaluated a high-resolution melting qualitative PCR analysis method for the simultaneous detection of these three pathogens. The assay has been optimized to detect three species-specific genes of each organism isolated from clinical samples, enabling accurate identification of the etiological agent. The method proved to be highly sensitive and cheaper than the real-time PCR TaqMan® system because it is probe-free; it could be used for the diagnosis of invasive diseases in public health laboratories of developing countries.
Asunto(s)
Meningitis Bacterianas , Neisseria meningitidis , Humanos , Análisis Costo-Beneficio , Meningitis Bacterianas/diagnóstico , Meningitis Bacterianas/microbiología , Neisseria meningitidis/genética , Streptococcus pneumoniae/genética , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Sensibilidad y EspecificidadRESUMEN
Meningococcal strains belonging to clonal complex cc60 are not associated with hypervirulent lineages and were never reported as causing disease in Latin American countries. This is the first report of a fatal meningitis case caused by a cc60 clonal complex meningococcus in Brazil. Despite the immune-compromised state of the patient, the fatal outcome here described shows the potential pathogenic behavior of strains belonging to this clonal complex and how compromised hosts can be susceptible to meningococcal infections even if the strain is not particularly invasive.
Asunto(s)
Infecciones Oportunistas Relacionadas con el SIDA/microbiología , ADN Bacteriano/análisis , Meningitis Meningocócica/microbiología , Neisseria meningitidis Serogrupo C/genética , Adulto , Resultado Fatal , Humanos , Masculino , SerotipificaciónRESUMEN
ABSTRACT Introduction: The increasing incidence of multi-resistant microorganisms has been considered a public health problem. One of the routines included in hospital practice is the screening of colonized and/or infected patients. Objective: The aim of this study was to evaluate the genetic variability and clonal relationships of extended-spectrum beta-lactamase (ESBL)-producing K. pneumoniae, from surveillance cultures, at an intensive care unit, in Rio de Janeiro, Brazil. Material and methods: Seventy K. pneumoniae isolates were obtained from rectal swabs (March 2013 to March 2014). Antimicrobial susceptibility was assessed by VITEK 2 System. Resistant genes blaSHV, blaTEM, blaOXA-1, blaKPC, blaOXA-48, blaCTX-M-15, blaVIM, blaIMP and blaNDM were investigated by polymerase chain reaction (PCR); genetic diversity, by Enterobacterial Repetitive Intergenic Consensus-PCR (ERIC-PCR). Results: Strains showed high resistance rates to cefepime (94%), ceftazidime (96%), ertapenem (61%), imipenem (54%) meropenem (43%) and ciprofloxacin (69%). The most prevalent genes were blaSHV (69%), blaTEM (63%), blaOXA-1 (60%), blaKPC (57%), blaCTX-M-15 (47%), blaOXA-48 (16%). Genes blaVIM, blaIMP and blaNDM were not detected. Twenty nine profiles of resistance genes were observed, with 23% carrying at least five genes. A great genetic diversity (68 ERIC profiles) was also observed among the strains. Conclusion: Although no clonal relationship was observed within the isolates, this study revealed alarming data on the antimicrobial resistance deficiently monitored for preventive purposes in Brazil. Our data allow us to conclude that the inclusion of surveillance cultures in health facilities is a recommended strategy aiming particularly at preventing the spread of resistance genes in the hospital environment and, consequently, reducing morbidity and mortality.
RESUMO Introdução: O aumento da incidência de microrganismos multirresistentes é considerado um dos principais problemas de saúde pública. Uma das rotinas incluídas na prática hospitalar é a busca de pacientes colonizados e/ou infectados. Objetivo: Avaliar a variabilidade genética e as relações clonais de K. pneumoniae produtoras de betalactamases de espectro estendido (ESBL) em culturas de vigilância de unidade de terapia intensiva (UTI) no Rio de Janeiro, Brasil. Materiais e métodos: Setenta isolados obtidos a partir de swab retal, (março/2013 a março/2014). O perfil de suscetibilidade a antibióticos foi avaliado pelo sistema VITEK 2. Foram pesquisados os genes de resistência: blaSHV, blaTEM, blaOXA-1, blaKPC, blaOXA-48, blaCTX-M-15, blaVIM, blaIMP e blaNDM pela reação em cadeia da polimerase (PCR). A diversidade genética foi avaliada por Enterobacterial Repetitive Intergenic Consensus-PCR (ERIC-PCR). Resultados: Foram detectados altos percentuais de resistência a cefepime (94%), ceftazidima (96%), ertapenem (61%), imipenem (54%) meropenem (43%) e ciprofloxacino (69%). Os genes prevalentes foram: blaSHV (69%), blaTEM (63%), blaOXA-1 (60%), blaKPC (57%), blaCTX-M-15 (47%), blaOXA-48 (16%). Os genes blaVIM, blaIMP e blaNDM não foram detectados. Foram observados 29 perfis em relação aos genes de resistência, com 23% apresentando pelo menos cinco genes. Uma grande diversidade genética (68 perfis) foi observada entre as cepas. Conclusão: Embora não tenha sido observada relação clonal entre os isolados, este estudo revelou dados alarmantes quanto à resistência microbiana em monitoramento preventivo, abordagem ainda pouco adotada no Brasil. Nossos dados permitem concluir que a inclusão de culturas de vigilância nas unidades de saúde é uma estratégia recomendada, visando principalmente à prevenção da disseminação dos genes de resistência no ambiente hospitalar e, consequentemente, redução da morbimortalidade.
RESUMEN
Among the vaccines produced by Bio-Manguinhos, a major centre for manufacturingthe immunobiological products in Latin America, stands out the yellow fever (YF) vaccine.To guarantee the excellence and safety of the YF vaccine, the quality control tests has to be performedthroughout its production. The World Health Organization (WHO) demands the producersto guarantee the absence of Mycoplasma orale, M. pneumoniae, M. gallisepticum and M. synoviaein the biological products. Mycoplasma is a fastidious microorganism, requiring about 35 daysfor attaining the conclusive culturing test. In this study PCR methods were selected for amplifying16S rRNA gene fragments for detecting mycoplasma in the intermediate products of YF vaccine.This standardized methodology was specific and sensitive to detect the low concentrations of mycoplasma in spiked intermediary vaccine products; and the absence of unspecific amplification was also demonstrated. The detection rates ranged from 3.1 to 12.5 colony formingunits and showed 100 % of sensitivity and specificity in the tested samples. The PCR protocol for detecting mycoplasmal DNA in YF vaccine was validated by analysing 286 samples. Bio-Manguinhos produces annually 10,000,000 YF vaccine doses, and this method has been successfully employed, complementing the traditional approach in the mycoplasma detection since 2008.
Dentre as vacinas produzidas por Bio-Manguinhos, um importante centro de produção deimunobiológicos da América Latina, destaca-se a vacina de febre amarela (FA) que é produzidaem ovos embrionados. Para garantir a excelência e a segurança da vacina, testes de controlede qualidade são realizados durante a produção. A Organização Mundial de Saúde (OMS) exigedos produtores a ausência de Mycoplasma orale, M. pneumoniae, M. gallisepticum e M. synoviaeem produtos biológicos. Micoplasmas são micro-organismos fastidiosos, sendo necessários35 dias para que os testes de cultura sejam conclusivos. Neste estudo foram selecionadosmétodos de amplificação de fragmentos do gene 16S rRNA para detecção de micoplasmas emprodutos intermediários da vacina de FA. Esta metodologia padronizada foi capaz de detectarbaixas concentrações de micoplasmas nos produtos intermediários e a ausência de amplificaçãoinespecífica foi demonstrada. O limite de detecção variou entre 3,1 e 12,5 unidades formadorasde colônia; e nas amostras testadas a sensibilidade e a especificidade foram de 100 %. O protocolode PCR para detecção de micoplasmas na vacina foi validado pela análise de 286 amostras.Bio-Manguinhos produz 10.000.000 doses de vacina de febre amarela por ano e, desde 2008,este método tem sido empregado com sucesso, complementando-se a abordagem tradicional.
Asunto(s)
Humanos , Reacción en Cadena de la Polimerasa , Control de Calidad , Vacunas , Fiebre Amarilla , Vacuna contra la Fiebre AmarillaRESUMEN
ABSTRACT Introduction: Cross-contamination by Staphylococcus aureus among patients, professionals and medical supplies in health facilities is a constant concern, leading many researchers to study the prevalence of this pathogen in asymptomatic carriers. Objective: We investigated the colonization and the antimicrobial susceptibility profile of Staphylococcus spp. on surfaces of medical articles and in professionals from two basic health units in the city of Rio de Janeiro. Materials and methods: Seventy-nine samples resulted in 49 isolates which underwent phenotypic and molecular characterization by polymerase chain reaction (PCR) of coa, mecA and femA genes. Results: According to the phenotypes, the isolates were identified as S. aureus (n = 35, 71.42%) and coagulase-negative Staphylococcus (CoNS) (n = 14, 28.57%). Among these 14 isolates, 42.85% were methicillin-resistant coagulase negative Staphylococcus (MRCoNS). Among the 35 S. aureus, 31.42% were methicillin resistant (MRSA), and 2.8% were vancomycin resistant, characterized as VRSA. Sixty-eight percent were susceptible to methicillin (MSSA). Genes coa, femA and mecA were amplified from 75.51%, 71.42% and 30.61% of the isolates, respectively. After amplification of the mecA gene, 20.41% were characterized as MRSA, and 10.20% as MRCoNS. The vancomycin-resistant strain was characterized as VRSA after detection of the vanB gene. Conclusion: Our results show a higher frequency of MSSA and MRCoNS among S. aureus and CoNS respectively, colonizing devices and health professionals. However, the already described transfer of the staphylococcal cassette chromosome mec (SSCmec) from MRCoNS to MSSA may alter these results, increasing the frequency of MRSA strains. .
RESUMO Introdução: A contaminação cruzada por Staphylococcus aureus entre pacientes, profissionais e materiais de uso médico em unidades de saúde é uma preocupação constante, o que leva pesquisadores a estudar a prevalência desse patógeno em portadores assintomáticos. Objetivos: Investigamos a colonização e o perfil de suscetibilidade aos antimicrobianos de Staphylococcus spp. em superfícies de artigos médicos e em profissionais de duas unidades básicas de saúde no município do Rio de Janeiro. Materiais e métodos: Foram coletadas 79 amostras que resultaram em 49 isolados, submetidos à caracterização fenotípica e molecular por meio da reação em cadeia da polimerase (PCR) dos genes coa, femA e mecA. Resultados: De acordo com os fenótipos apresentados, os isolados foram identificados como S. aureus (n = 35; 71,42%) e Staphylococcus coagulase negativa (CoNS) (n = 14; 28,57%). Destes 14 isolados, 42,85% foram Staphylococcus coagulase negativa resistentes a meticilina (MRCoNS). Dos 35 S. aureus, 31,42% foram resistentes a meticilina (MRSA). Uma cepa foi resistente a vancomicina e identificada como S. aureus resistente a vancomicina (VRSA) após a detecção do gene vanB. Sessenta e oito por cento foram suscetíveis a meticilina (MSSA). Os genes coa, femA e mecA foram amplificados em 75,51%; 71,42% e 30,61% dos isolados, respectivamente. Após amplificação do gene mecA, 20,41% foram classificados como MRSA e 10,20% como MRCoNS. Conclusão: Nossos resultados mostraram frequência maior de MSSA e MRCoNS entre S. aureus e CoNS, respectivamente, colonizando equipamentos e profissionais de saúde. No entanto, a já descrita transferência do cassete cromossômico estafilocócico mec (SSCmec) de MRCoNS para MSSA poderia alterar esses resultados, aumentando a frequência de cepas MRSA. .
RESUMEN
Cronobacter spp. é uma bactéria oportunista associada a surtos de infecção em neonatos e criançasem virtude de consumo de fórmulas infantis desidratadas (FID). Neste contexto, o setor reguladortem criado normas específicas para o controle destes agentes patogênicos nas fórmulas infantis. Nesteestudo foi pesquisada a ocorrência de Cronobacter spp. em 60 amostras de FID comercializadas no Riode Janeiro, Brasil. Foram analisadas 30 amostras de fórmulas infantis para lactantes (0-6 meses) e 30 defórmulas infantis de seguimento para lactantes (> 6 meses) seguindo-se a metodologia de cultivo descritano Bacteriological Analytical Manual OnlineFDA (2012). A identificação das colônias característicasfoi realizada com uso de kits ID32E, API20E e do sistema Vitek 2.0; e pela reação da polimerase emcadeia (PCR) com alvo no gene gluA. Nenhuma amostra apresentou contaminação por Cronobacterspp. Concluiu-se que a ocorrência de Cronobacter spp. em FID parece ser baixa, o que indica que osprodutores estão cumprindo o disposto nas normas brasileiras vigentes de forma a evitar a contaminaçãodos produtos por este micro-organismo...
Asunto(s)
Conservación de Alimentos , Cronobacter , Fórmulas Infantiles , Microbiología de Alimentos , Reacción en Cadena de la PolimerasaRESUMEN
Multiple locus sequence typing (MLST) was undertaken to extend the genetic characterization of 29 isolates of Bacillus cereus and Bacillus thuringiensis previously characterized in terms of presence/absence of sequences encoding virulence factors and via variable number tandem repeat (VNTR). Additional analysis involved polymerase chain reaction for the presence of sequences (be, cytK, inA, pag, lef, cya and cap), encoding putative virulence factors, not investigated in the earlier study. MLST analysis ascribed novel and unique sequence types to each of the isolates. A phylogenetic tree was constructed from a single sequence of 2,838 bp of concatenated loci sequences. The strains were not monophyletic by analysis of any specific housekeeping gene or virulence characteristic. No clear association in relation to source of isolation or to genotypic profile based on the presence or absence of putative virulence genes could be identified. Comparison of VNTR profiling with MLST data suggested a correlation between these two methods of genetic analysis. In common with the majority of previous studies, MLST was unable to provide clarification of the basis for pathogenicity among members of the B. cereus complex. Nevertheless, our application of MLST served to reinforce the notion that B. cereus and B. thuringiensis should be considered as the same species.
Asunto(s)
Bacillus cereus/genética , Bacillus thuringiensis/genética , Tipificación de Secuencias Multilocus , Repeticiones de Minisatélite/genética , Brasil , Bacillus cereus/patogenicidad , Bacillus thuringiensis/patogenicidad , Genotipo , Filogenia , Reacción en Cadena de la Polimerasa , Factores de Virulencia/genéticaRESUMEN
Background: Neisseria meningitidis serogroup B has been predominant in Brazil, but no broadly effective vaccine is available to prevent endemic meningococcal disease. To understand genetic diversity among serogroup B strains in Brazil, we selected a nationally representative sample of clinical disease isolates from 2004, and a temporally representative sample for the state of SaËo Paulo (19882006) for study (n = 372). Methods: We performed multi-locus sequence typing (MLST) and sequence analysis of five outer membrane protein (OMP) genes, including novel vaccine targets fHbp and nadA. Results: In 2004, strain B:4:P1.15,19 clonal complex ST-32/ET-5 (cc32) predominated throughout Brazil; regional variation in MLST sequence type (ST), fetA, and porB was significant but diversity was limited for nadA and fHbp. Between 1988 and 1996, the SaËo Paulo isolates shifted from clonal complex ST-41/44/Lineage 3 (cc41/44) to cc32. OMP variation was associated with but not predicted by cc or ST. Overall, fHbp variant 1/subfamily B was present in 80% of isolates and showed little diversity. The majority of nadA were similar to reference allele 1. Conclusions: A predominant serogroup B lineage has circulated in Brazil for over a decade with significant regional and temporal diversity in ST, fetA, and porB, but not in nadA and fHbp.
Asunto(s)
Epidemiología , Epidemiología Molecular , MeningitisRESUMEN
Meningococcal strains belonging to clonal complex cc60 are not associated with hypervirulent lineages and were never reported as causing disease in Latin American countries. This is the first report of a fatal meningitis case caused by a cc60 clonal complex meningococcus in Brazil. Despite the immune-compromised state of the patient, the fatal outcome here described shows the potential pathogenic behavior of strains belonging to this clonal complex and how compromised hosts can be susceptible to meningococcal infections even if the strain is not particularly invasive.
Asunto(s)
Adulto , Humanos , Masculino , Infecciones Oportunistas Relacionadas con el SIDA/microbiología , ADN Bacteriano/análisis , Meningitis Meningocócica/microbiología , Neisseria meningitidis Serogrupo C/genética , Resultado Fatal , SerotipificaciónRESUMEN
A sensibilidade ao ciprofloxacin foi testada in vitro em 83 cepas de estafilococos coagulase-negativas isoladas de infecçöes humanas. De 83 cepas, 82 mostraram-se sensíveis ao antibiótico pelo método de difusäo com disco (5microng) e foram inibidas na concentraçäo de 1microng/ml da droga
Asunto(s)
Quinolinas/farmacología , Staphylococcus/efectos de los fármacos , Técnicas In VitroRESUMEN
Foi determinada a concentraçäo inibitória mínima in vitro do ciprofloxacin para cepas de enterobactérias, enterecocos, staphylococcus, Pseudomonas e bactérias anaeróbias estritas isoladas de diversos processos infecciosos de pacientes do Rio de Janeiro, RJ. A maioria das cepas de Escherichia coli e Klebsiella pneumoniae foi inibida com 0,25microng/ml do antimicrobiano, enquanto 1,0microng/ml foi necessário para inibir 90% das cepas de Proteus e Staphylococcus. A CIM90 para cepas de Pseudomonas aeruginosa e Streptococcus faecalis atingiu 2,0microng/ml; no entanto, para bactérias anaeróbias estritas (Bacteroides fragilis e Clostridium perfringens) elevou-se para 16 e 18micron/ml, respectivamente. A análise da curva de morte empregando amostra-padräo de Escherichia coli (ATCC-25922) revelou o efeito bactericida do ciprofloxacin