Caracterização genotípica de amostras de Staphylococcus aureus resistentes à meticilina isoladas de diferentes hospitais do estado do Rio de Janeiro

A resistência aos antimicrobianos observada em Staphylococcus aureus resistentes à meticilina (MRSA) vem sendo considerada um dos maiores desafios a serem enfrentados na saúde humana, devido à ampla disseminação desta bactéria. As técnicas de epidemiologia molecular tradicionais, por anos, colaboraram para o mapeamento da circulação de clones mais resistentes. Dentre essas técnicas podemos destacar a caracterização do SCCmec (do inglês, Staphylococcal Cassette Chromosome mec) e o teste RM (do inglês, restriction-modification system). No Brasil, amostras do clone epidêmico Brasileiro (CEB) de MRSA (CC8-ST239-SCCmecIII) vinham sendo as mais frequentemente isoladas até meados dos anos 2000. Contudo, nas últimas décadas, vem sendo observado um fenômeno de variação clonal, com um aumento na ocorrência de amostras pertencentes a outras linhagens (CC1-SCCmecIV, CC5-SCCmecIV e CC5-SCCmecII), suplantando a linhagem previamente estabelecida. O trabalho teve como objetivo revelar quais os clones de MRSA estão atualmente predominando em infecções no Rio de Janeiro, em um universo de 51 hospitais, bem como identificar sua distribuição nos diferentes sítios do hospedeiro. Para isso, um total de 600 amostras de MRSA isoladas de pacientes admitidos nestes hospitais, entre julho de 2014 e agosto de 2017, foram incluídas neste estudo. A coleção foi submetida aos testes de susceptibilidades frente aos antimicrobianos recomendados pelo CLSI (2018). Posteriormente, essas amostras foram submetidas a dois métodos de caracterização genotípica: o teste RM e a tipagem do SCCmec. O teste RM identificou a prevalência do complexo clonal CC5, enquanto que a tipagem do SCCmec identificou os tipos IV e II como sendo os mais prevalentes dentro do Estado. Com base nesses resultados preliminares, foram identificadas as linhagens CC5-SCCmecIV e II, como sendo as mais prevalentes em circulação dentro do Estado, independentemente dos grupos amostrais analisados (origem clínica e faixa etária). Os resultados obtidos através dos testes de susceptibilidade identificaram que as linhagens de MRSA CC5-SCCmecIV e II apresentaram menores índices de resistência, quando comparadas com o CEB, às drogas que deixaram de ser utilizadas como alternativa terapêutica. Além disso, foi observado um aumento na quantidade de amostras pertencentes ao CC30. Concluindo, com os resultados apresentados podemos demonstrar que as linhagens de MRSA CC5-SCCmecIV e II estão suplantando a linhagem CEB anterior, pelo menos, dentro do Estado do Rio do Janeiro

Local Diversification of Methicillin- Resistant Staphylococcus aureus ST239 in South America After Its Rapid Worldwide Dissemination

The global spread of specific clones of methicillin-resistant Staphylococcus aureus (MRSA) has become a major public health problem, and understanding the dynamics of geographical spread requires worldwide surveillance. Over the past 20 years, the ST239 lineage of MRSA has been recognized as an emerging clone across the globe, with detailed studies focusing on isolates from Europe and Asia. Less is known about this lineage in South America, and, particularly, Brazil where it was the predominant lineage of MRSA in the early 1990s to 2000s. To gain a better understanding about the introduction and spread of ST239 MRSA in Brazil we undertook a comparative phylogenomic analysis of ST239 genomes, adding seven completed, closed Brazilian genomes. Brazilian ST239 isolates grouped in a subtree with those from South American, and Western, romance-language-speaking, European countries, here designated the South American clade. After an initial worldwide radiation in the 1960s and 1970s, we estimate that ST239 began to spread in South America and Brazil in approximately 1988. This clone demonstrates specific genomic changes that are suggestive of local divergence and adaptational change including agrC single-nucleotide polymorphisms variants, and a distinct pattern of virulence-associated genes (mainly the presence of the chp and the absence of sea and sasX). A survey of a geographically and chronologically diverse set of 100 Brazilian ST239 isolates identified this virulence genotype as the predominant pattern in Brazil, and uncovered an unexpectedly high prevalence of agr-dysfunction (30%). ST239 isolates from Brazil also appear to have undergone transposon (IS256) insertions in or near global regulatory genes (agr and mgr) that likely led to rapid reprogramming of bacterial traits. In general, the overall pattern observed in phylogenomic analyses of ST239 is of a rapid initial global radiation, with subsequent local spread and adaptation in multiple different geographic locations. Most ST239 isolates harbor the ardA gene, which we show here to have in vivo anti-restriction activity. We hypothesize that this gene may have improved the ability of this lineage to acquire multiple resistance genes and distinct virulence-associated genes in each local context. The allopatric divergence pattern of ST239 also may suggest strong selective pressures for specific traits in different geographical locations

Beyond Domestic Cats: Environmental Detection of <i>Sporothrix brasiliensis</i> DNA in a Hyperendemic Area of Sporotrichosis in Rio de Janeiro State, Brazil

In Brazil, sporotrichosis has transitioned from a rural to urban disease, driven by a shift in the initiation of infection from the accidental inoculation of organic matter to the traumatic implantation of the fungus by cats. Since the emergence of zoonotic sporotrichosis caused by Sporothrix brasiliensis, investigations have largely ignored the environmental habitat of the pathogen due to its association with domestic cats. Therefore, we investigated 18 environmental samples collected from rural areas of two cities where zoonotic sporotrichosis is endemic, but where domestic cats are scarce. We utilized traditional culture methods, and samples were also examined with two molecular methods used for the clinical diagnosis of sporotrichosis: a nested-PCR targeting the ITS region and a species-specific PCR targeting the calmodulin gene. No Sporothrix colonies were identified by traditional culture methods. However, the nested-PCR and the species-specific PCR for S. brasiliensis were positive for 18 and 5 samples, respectively. Sequencing revealed that positive results with the nested-PCR were due to non-specific amplification of other Ophiostomatales DNA, rather than Sporothrix spp. Three of the five amplicons from the species-specific PCR were suitable for sequencing and confirmed the presence of S. brasiliensis DNA. Hence, we confirmed that S. brasiliensis, as with other Sporothrix species, has an environmental habitat. Our findings underscore the challenges of nested-PCR for Sporothrix environmental studies and highlight that sequencing must follow PCR protocols to definitively identify Sporothrix spp. in environmental samples