BACKGROUND Neisseria meningitidis strains belonging to clonal complex 11 is the cause of numerous outbreaks and epidemics in the United States, Canada and Europe, accounting for 49.5% of cases of meningococcal disease caused by serogroup C worldwide. In Brazil, it is the second most frequent clonal complex within this serogroup. The genetic characterisation of cc11/ET-15 variants is important for the epidemiological monitoring of meningococcal disease, through the identification of circulating epidemic clones, to support specific actions of Health Surveillance aiming outbreaks control. OBJECTIVES The objective of this study was to identify features in the genome of cc11/ET-15 clones through whole-genome sequencing (WGS), that differ from cc11/non-ET-15 strains that could explain their virulence. METHODS The whole genome of three cc11/ET-15 representative strains were sequenced with a minimum coverage of 100X with the MiSeq System and compared to the genome of cc11/non-ET-15 strains. RESULTS Genome analysis of cc11/ET-15 variants showed the presence of resistance factors, mobile genetic elements and virulence factors not found in cc11/non-ET-15 strains. MAIN CONCLUSIONS Our results show that these strains carry virulence factors not identified in cc11/non-ET-15 strains, which could explain the high lethality rates attributed to this clone worldwide.

Resumo Materiais de referência são ferramentas indispensáveis para a garantia da qualidade laboratorial. São amplamente utilizados no controle interno da qualidade de ensaios analíticos e na validação de metodologias, e como itens de ensaio em ensaios de proficiência. Materiais de referência microbiológicos são representados principalmente por cepas de referência, cuja utilização como controle interno de ensaios é inquestionável e recomendada por vários manuais microbiológicos. No Brasil, a prática de produção de materiais de referência microbiológicos avançou, nos últimos anos, no desenvolvimento de materiais específicos para ensaios de proficiência. O mesmo não ocorreu com estudos relacionados ao estabelecimento de cepas de referência, por falta de políticas de incentivo, mantendo a dependência do país no uso de cepas de referência internacionais. Este artigo tem como objetivos ressaltar a importância do uso de materiais de referência microbiológicos no controle da qualidade laboratorial e discutir a necessidade do desenvolvimento desses materiais no Brasil. O presente trabalho apresenta uma breve explicação sobre materiais de referência microbiológicos e aponta questões sobre a dependência do país na aquisição de materiais biológicos internacionais. Descreve os padrões de qualidade relacionados à produção desses materiais e a situação das coleções de cultura e dos laboratórios de referência brasileiros que fornecem cepas de referência. Este estudo menciona também recomendações práticas sobre subcultivos de cepas de referência.

Abstract Reference materials are crucial for laboratory quality assurance. They are widely used for the internal quality control of analytical tests, in the validation of methodologies and as test items in proficiency tests. Microbiological reference materials are represented mainly by reference strains, whose use in laboratory internal quality control is unquestionable and recommended by several microbiological manuals. In Brazil, the practice of producing microbiological reference materials has advanced in recent years in the development of specific materials for proficiency testing. However, the same did not occur with the establishment of reference strains due to a lack of incentive policies, maintaining the country dependant on the use of international reference strains. This article aims to emphasize the importance of the use of microbiological reference materials in laboratory quality control and discuss the need for the development of such materials in Brazil. The paper presents a brief explanation of microbiological reference materials and points out questions concerning the country’s dependence on the acquisition of international biological materials. It also describes quality standards related to the production of these materials and the situation of culture collections and Brazilian reference laboratories that supply reference strains. The study also mentions practical recommendations on the subcultures of reference strains.

Resumo Cronobacter é uma bactéria oportunista, associada a infecções em neonatos devido ao consumo de fórmulas infantis desidratadas. Atualmente, sabe-se que Cronobacter pode causar infecções em indivíduos de qualquer idade, sendo, em idosos, a sua maior prevalência, e a infecção urinária, a síndrome mais comum neste grupo. A identificação de fórmulas infantis desidratadas como veículo de contaminação em casos de meningite causada por Cronobacter em neonatos levou as agências de regulação a criarem medidas específicas para o controle destes patógenos nestes produtos. Uma revisão bibliográfica foi realizada sobre a epidemiologia das infecções por Cronobacter, a ocorrência do patógeno e a regulação na área de alimentos no Brasil. Constatou-se que casos de infecções foram reportados no país no período de 1997-2013, com maior ocorrência em neonatos. O número preciso de casos não pôde ser determinado e, em alguns dos relatos, a identificação fidedigna dos micro-organismos pode ser questionável. Na maioria dos casos relatados o veículo de contaminação não foi investigado ou não pôde ser determinado. Já ocorreram relatos da contaminação de amostras de fórmulas infantis desidratadas comercializadas no Brasil por Cronobacter, mas sua ocorrência parece ter sido reduzida após a adoção de medidas sanitárias mais rígidas pelos órgãos reguladores. Cronobacter também foi isolada de temperos e condimentos, farinhas, queijo e misturas de cereais para crianças; destas amostras, quatro espécies foram identificadas: C. sakazakii, C. malonaticus, C. dublinensis e C. muytjensii. A presença do patógeno em diferentes classes de alimentos é preocupante pois pode representar um risco caso sejam consumidos por indivíduos pertencentes a algum grupo de risco (neonatos e idosos). O incentivo à realização de pesquisas sobre a epidemiologia do gênero Cronobacter é importante para identificar as classes de alimentos que podem atuar como veículo de contaminação e a real prevalência das infecções no Brasil.

Abstract Cronobacter is an opportunistic bacterium associated with infections in newborns due to the consumption of powdered infant formulae. It is now known that Cronobacter can cause infections in individuals of any age with a higher prevalence in the elderly, urinary infections being the most common syndrome in this group. The identification of powdered infant formulae as the contamination vehicle in cases of meningitis caused by Cronobacter in neonates led the regulatory agencies to establish specific measures to control such pathogens in these products. Thus a literature review was carried out concerning the epidemiology of Cronobacter infections, the occurrence of the pathogen and food regulations in Brazil. It was found that cases of the infection were reported in Brazil from 1997-2013, with a higher occurrence in neonates. The precise number of cases could not be determined, and in some reports, identification of the microorganism was questionable. In most of the cases reported the contamination vehicle was not investigated or could not be determined. There have been reports of contamination by Cronobacter in samples of powdered infant formulae commercialized in Brazil, but their occurrence seems to have reduced after the adoption of stricter sanitary measures by the regulatory agencies. Cronobacter has also been isolated from spices and herbs, flours, cheese and from cereal mixes destined for children; and from these samples, four species were identified: C. sakazakii, C. malonaticus, C. dublinensis and C. muytjensii. The presence of the pathogen in different types of food is worrying because it may represent a risk if consumed by individuals belonging to one of the risk groups (neonates and elderly). It is important to encourage research about the epidemiology of the genus Cronobacter in order to identify the classes of food that can act as contamination vehicles and the real prevalence of infections in Brazil.

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.

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.

Abstract Background Several in-house PCR-based assays have been described for the detection of bacterial meningitis caused by Neisseria meningitidis, Streptococcus pneumoniae, and Haemophilus influenzae from clinical samples. PCR-based methods targeting different bacterial genes are frequently used by different laboratories worldwide, but no standard method has ever been established. The aim of our study was to compare different in-house and a commercial PCR-based tests for the detection of bacterial pathogens causing meningitis and invasive disease in humans. Methods A total of 110 isolates and 134 clinical samples (99 cerebrospinal fluid and 35 blood samples) collected from suspected cases of invasive disease were analyzed. Specific sets of primers frequently used for PCR-diagnosis of the three pathogens were used and compared with the results achieved using the multiplex approach described here. Several different gene targets were used for each microorganism, namely ctrA, crgA and nspA for N. meningitidis, ply for S. pneumoniae, P6 and bexA for H. influenzae. Results All used methods were fast, specific and sensitive, while some of the targets used for the in-house PCR assay detected lower concentrations of genomic DNA than the commercial method. An additional PCR reaction is described for the differentiation of capsulated and non-capsulated H. influenzae strains, the while commercial method only detects capsulated strains. Conclusions The in-house PCR methods here compared showed to be rapid, sensitive, highly specific, and cheaper than commercial methods. The in-house PCR methods could be easily adopted by public laboratories of developing countries for diagnostic purposes. The best results were achieved using primers targeting the genes nspA, ply, and P6 which were able to detect the lowest DNA concentrations for each specific target.

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.

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.

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.

Doenças transmitidas por alimentos (DTA), causadas por microrganismos, representam um importante problema de saúde pública. O queijo Minas Frescal é um produto nacional, de tecnologia simples e elevada aceitação no País, tornando sua produção uma importante atividade econômica. Muitos microrganismos podem estar presentes neste alimento e, entre eles, destaca-se a bactéria Escherichia coli (E. coli). De modo geral, E. coli é um comensal inofensivo, entretanto, algumas cepas podem apresentar potencial patogênico. Diversos surtos de DTA, associados ao consumo de queijos contaminados, têm sido relatados e a presença de cepas patogênicas de E. coli cada vez mais observada. Este estudo teve como objetivo isolar, avaliar a suscetibilidade antimicrobiana e caracterizar molecularmente, através da Multiplex PCR, as cepas patogênicas de E. coli, isoladas de queijo Minas Frescal comercializado no município do Rio de Janeiro. Trinta amostras foram analisadas e cinco cepas de E. coli enteropatogênica (EPEC) identificadas. A avaliação da suscetibilidade antimicrobiana revelou 40% de resistência à ampicilina e 40% de perfil de resistência intermediária à associação ampicilina-sulbactam. Tais resultados representam um alerta para as autoridades sanitárias, uma vez que este alimento é um produto de pronto consumo e, portanto, precisa ser inócuo para que a saúde da população não seja colocada em risco.

Foodborne disease caused by microorganisms is a problem of public health. Minas soft cheese is a national product manufactured using simple technology; it has high level of acceptance in the country making its production an important economic activity. Many microorganisms may be present in foods including the bacterium Escherichia coli (E. coli). Overall, E. coli is a harmless commensal bacterium; however, some strains may have a pathogenic potential. Several outbreaks of foodborne diseases associated with consumption of contaminated cheese have been reported, and the presence of pathogenic strains of E. coli has increased. The objective of this study was to isolate, evaluate the antimicrobial susceptibility, and characterize, by Multiplex PCR, the pathogenic E. coli strains isolated from Minas cheese commercialized in Rio de Janeiro. Thirty samples were analyzed and five strains of E. coli (EPEC) were identified. The assessment of antimicrobial susceptibility revealed 40% of the isolates resistant to ampicillin and 40% with intermediate resistance to ampicillin-sulbactam combination. These findings are a warning signal to health authorities since Minas cheese is a ready to eat food product, and therefore should not pose health risks to the population.

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.