Pesquisa de pátogenos oportunistas em medicamentos tópicos: padronização e análise comparativa de metodologias

Abstract

With the ample dissemination of the AIDS, a wide range microorganisms has blunted as emergent pathogens, causing important infection in immunocompromised patients. For example, Rhodococcus equi, an unusual cause of infection in humans, has been isolated from HIV-infected patients. The presence of these opportunistic microorganisms in pharmaceutical products has not receiv considerable attention. In the present work PCR assay were compared with standard microbiological methods for rapid detection of three bacterial species from artificially contaminated sample of topical creams. Artificially contaminated samples were incubated for 24 horas at 37º C. After incubation in broth with 10% tween 20, samples were streaked on seletive growth media. Biochemical identification of Bacillus cereus and R. equi was performed using API 20E® and API Coryne® identification systems, respectivelly. For Micrococcus luteus identification, colony an Gram-stained morphology of the bacterium and the biochemical tests of catalase and oxidase were used.DNA was extracted using phenol-chloroform method. DNA primers containing the specific sequences of the BC1 and BC2 for B. cereus, COX-R and COX-F for R. equi and ML-ISR-F and ML-ISR-F for M. luteus were used for detection in the PCR reaction. In order to validate this methodology, DNA sequences of the products were determined. The sequences of the cloned PCR products were analysed using Blastn and Blastx programs. It was observed a marked similarity (E.value 0,0) and a high identity (> 99%) to the bacteria used in the study. Both the PCR and the standard enrichment tests method detected B. cereus, R. equi and M. luteus in all of the deliberately contaminated samples. The time to complete the PCR assay including sample preparation and PCR amplification of the specific DNA bacterial targets was 27 h. Standard plating methods required 72-96 h for the bacteria to be isolated, purified and biochemecally identified. Rapid PCR detection of B. cereus, R. equi and M. luteus showed in this work suggests the use of this methodology in the microbiology control of non-sterile topical products, specially intended for use with immunocompromised patients.