Caracterização molecular de bastonetes Gram positivos irregulares e actinomicetos aeróbios obtidos de espécimes clínicos, de ensaios de esterilidade e de áreas limpas

Abstract

Irregular Gram positive rods (coryneform bacteria; IGPRs) comprise a group of species that has a wide phenotypic diversity and makes the conventional identification limited and that may be present in the environment, humans and animals hosts. In order to provide further accurate identification of these microorganisms in a genus and species terms it is recommended the use of molecular methods. In this study, we analyzed the 16S rRNA gene sequences and other conserved genes (housekeeping) in order to elucidate the identification of clinical isolates, pharmaceutical and clean room areas. We have documented a nosocomial outbreak caused by multidrug-resistant (MDR) C. striatum in Rio de Janeiro. C. striatum identification was confirmed by 16S rRNA and rpoB gene sequencing. C. striatum was mostly isolated in pure culture from tracheal aspirates of adults undergoing endotracheal intubation procedures. The analysis by pulsed-field gel electrophoresis (PFGE) indicated the presence of four PFGE profiles, including two related clones of MDR strains (PFGE I and II). The data demonstrated the predominance of PFGE I, mostly isolated from patients of intensive care units and surgical wards. A potential causal link between death and MDR C. striatum (PFGE I and II) infection was observed in five cases. We also performed the identification of Nocardia species of human infections by multilocus sequence analysis (MLSA) and characterized their antimicrobial and phenotypic profiles. An overview from 1970 to 2013 of the case reports on Nocardia species related to human infections, except mycetomas, in Brazil, was also accomplished. Molecular characterization by four-locus (gyrB-16S-secA1-hsp65) has provided the species identification N. nova, N. cyriacigeorgica, N. asiatica and N. exalbida/gamkensis. N. nova strains isolated from different clinical specimens of one patient showed identical antimicrobial susceptibility patterns. PFGE analysis performed to determine the genetic relatedness of these N. nova strains two distinct profiles, which were designated A and B. This is the first report on identification of Nocardia species by MLSA in Brazil. The IGPRs obtained in clean room environments that could not be identified by conventional criteria were studied by 16S rRNA gene sequence analysis that allowed the identification of 95.83% at genus level and of 35.42% at the species level. The most common genus found in the clean room environments were Microbacterium and Streptomyces. The analysis of rpoB and recA genes sequence of seventeen Microbacterium strains contributed to the identification of three strains at species level. MLSA of seven Streptomyces strains identified a single sample at the species level. Moreover, the rpoB gene sequence analysis was effective in identifying Corynebacterium strains at species level. A new genus and species were found among clean room environmental strains belonging to the Actinobacteria class. Based on the morphological, genotypic and biochemical data presented in this study the 3117BRRJ strain represent a novel specie of the genus Nocardioides, for which the name Nocardioides brasiliensis sp. nov. was proposed and the 3712BRRJ and 3371BRRJ represent a new genus and species for which the name Guaraldella brasiliensis nov. were proposed.