ABSTRACT
The choice of a suitable preservation method is critical for long-term microorganisms' viability. The strains should be preserved for long periods using reliable and reproducible methods that minimize genotypic and phenotypic variations and viability losses. The methodologies are usually designed for a better performance in isolated microorganisms. However, atypical primary isolates of Cryptococcus neoformans or Cryptococcus gattii, such as mixed species or even different species of a species complex, are a challenge for long-term preservation and taxonomic review studies. The aim of this study was to evaluate which of the four preservation methods tested presented better performance in the preservation of simulated coexistence strains of C. neoformans and C. gattii. Two environmental strains, one C. gattii and one C. neoformans, were mixed in vitro to test four different preservation methods (freezing at -20°C, -80°C, -196°C, and freeze-drying). The colony-forming units from each preservation method were evaluated, and colonies were randomly selected and cultivated in canavanine glycine bromothymol blue (CGB) agar to evaluate the amounts of CGB-positive (C. gattii) and CGB-negative (C. neoformans) colonies resulting from each preservation method after 1 week, 15 days, 1 month, 6 months, and 1 year. According to our results, cryopreservation at -20°C demonstrated was preferable for C. neoformans species, and further studies after long-term storage are necessary. Recovery of yeast cells after freeze-dried preservation in skim milk is better for both species. Ultrafreezing methods evaluated (-80°C and -196°C) also showed better results in the maintenance of C. gattii. Freeze-drying should be preferred for the maintenance of multilineage isolates from the C. neoformans and C. gattii species complexes.
Subject(s)
Cryopreservation/methods , Cryptococcus gattii/growth & development , Cryptococcus neoformans/growth & development , DNA, Fungal/genetics , Trees/microbiology , Cryptococcus gattii/genetics , Cryptococcus gattii/isolation & purification , Cryptococcus neoformans/genetics , Cryptococcus neoformans/isolation & purification , Culture Media , Freeze Drying , Freezing , Genomic Instability , Microbial Viability , Phenotype , TemperatureABSTRACT
Em vista de um estudo desenvolvido no Setor de Saneantes do Instituto Nacional de Controle deQualidade em Saúde da Fundação Oswaldo Cruz, referente à avaliação da atividade antimicrobiana dedesinfetantes, no presente trabalho foram analisados produtos desinfetantes de uso geral disponíveis nomercado brasileiro. Os desinfetantes foram coletados aleatoriamente, dos quais três (produtos A, B e C),apresentaram contaminação microbiana. Para efetuar o isolamento e a identificação dos microrganismoscontaminantes foram utilizados o aparelho Vitek® 2, a amplificação e o sequenciamento do gene rRNA16S. A análise realizada por meio de Vitek® 2 revelou a presença das bactérias Serratia marcescens eAchromobacter xylosoxidans, respectivamente, nos produtos A e B. No produto C foram detectadasAeromonas salmonicida pelo Vitek® 2 e Burkholderia lata pela técnica de amplificação da reação em cadeiada polimerase.
Subject(s)
Environmental Pollution , Quality Control , Disinfectants/analysis , Microbiology , Health Surveillance of Products , BrazilABSTRACT
Mycobacterium abscessus subps. bolletii (clone BRA100), entre outras micobactérias de crescimento rápido (MCR), tem sido isolada como agente etiológico de infecções localizadas e sistêmicas no Brasil. Neste estudo, foi avaliada a suscetibilidade de MCR pelo método confirmatório para Avaliação da Atividade Micobactericida de Desinfetantes frente a produtos à base de glutaraldeído, ácido peracético e ortoftalaldeído. Todos os produtos foram utilizados nas concentrações recomendadas pelos fabricantes.Foram avaliadas cinco cepas clínicas pertencentes ao clone BRA100, juntamente com as cepas de referência M. abscessus ATCC 19977, M. chelonae ATCC 35752, M. fortuitum ATCC 6841 e M. abscessus subps.bolletii INCQS 00594. Os três desinfetantes à base de glutaraldeído não foram eficazes contra M. abscessuse M. abscessus subps. bolletii. Os desinfetantes à base de ácido peracético demonstraram eficácia para todasas micobactérias empregadas, embora as cepas do clone BRA100 tenham demonstrado suscetibilidade reduzida ao ácido peracético B. O produto à base de ortoftalaldeído foi eficaz apenas frente a M. chelonae.Visando a redução de infecções nosocomiais, sugere-se que seja suspenso o uso de produtos à base de glutaraldeído como desinfetante de alto nível para quaisquer finalidades.
Subject(s)
Disinfectants , Cross Infection , MycobacteriumABSTRACT
Recent changes in Brazilian legislation for commercial disinfectants have been published due to the recent epidemic of nosocomial infections caused by rapidly growing mycobacteria (RGM) in many states of Brazil over the last 8years. One of these documents requires that all the manufacturers provide evidence of efficacy of sterilizing and disinfectant products, used for semi critical medical devices, against the Mycobacterium bovis BCG Moreau and Mycobacterium abscessus subsp. bolletii INCQS 00594 strains by using the Confirmative in vitro Test for Determining Tuberculocidal Activity of Disinfectants recommended by the Association of Official Analytical Chemists. These changes have caused additional costs and increased problems for importation of enrichment products at national laboratories where disinfectant efficacy assay service is performed. Middlebrook ADC Enrichment (ADC) is provided by a unique manufacturer and used in the official protocol. The aim of the present study was to evaluate an alternative in house low-cost enrichment composed of fetal bovine serum and glucose (FBSG) with ADC for performance of disinfectant efficacy assay against mycobacteria. After obtaining the growth curves for M. abscessus ATCC 19977, M. abscessus subsp. bolletii INCQS 00594, Mycobacterium chelonae ATCC 35752, and Mycobacterium fortuitum ATCC 6841 by using ADC enrichment and FBSG in Kirchners and 7H9 culture media. Through statistical analysis via the Kruskal-Wallis test on the evaluation of microorganism growth rate, it was observed that there was no inhibition of RGM growth by any of the enrichments used. These results suggest that low-cost enrichment FBSG may be used as a potential substitute of ADC for composition of media for mycobacterial growth, including in disinfectant tests.
