| Clean rooms are essential in aseptic pharmaceutical or food production. Monitoring microbial distribution and identifying the predominant isolates is part of good manufacturing practices. The commonly used protocol for monitoring involves the use of media such as SCD agar and incubation at 30°C for 4 days. According to the European Union's good manufacturing practice directive, the permissible number of CFU on surface contact plates for grade A and B is 1 and 5 respectively. The number permitted by USP (U.S. Pharmacopeia) for class 100 and 10000 is 3 and 5 respectively (US general services administration 1992). To meet such requirements, all the surfaces within the clean room, air, floor and personnel hands, are disinfected routinely using a variety of disinfectants. Ultraviolet (UV) irradiation, Lysol (phenol) solution swabbing and Maskin (chlorhexidine gluconate, CHG) solution immergence are three widely used ways for disinfecting clean room air, furniture surface and personnel skins in many pharmaceutical factories.Maintaining the integrity of a clean room is a constant battle. To decide which method, or combination of methods, to be employed in disinfecting aseptic workshop, there is a need to understand the kind of bacteria that are the prime sources of contamination. Therefore, knowledge of the microbial diversity of clean rooms, as well as any extreme characteristics these microbes might possess, is essential to the development of disinfection technologies.The aim of this study was to isolate and identify the predominant bacteria strains distributed in clean rooms of a pharmaceutical workshop, In on-going investigations to determine and document possible microbial contamination in clean rooms, bacteria strains were isolated and their colony and cell morphology characteristics were compared. And the following process measures the biochemical and physiological characterization of predominant bacteria. To analyze the phylogenetic relationships of the predominant isolates, the 16S rDNA genes were amplified and their partial sequences were determined.The biochemical and physiological tests also showed that 3 of isolates are similar in most of the properties, and 16S rDNA sequence analyses further demonstrated that these 3 isolates share more than 99% similarities. Blasting analyses of partial 16S rDNA sequences confirmed that these three isolates belong to the genus Staphylococcus. However, molecular characterizations were at the limits of resolution for the differentiation of species in this genus. Combined with the results of biochemical and physiological tests, it can be preliminary concluded that one and rest one of the 3 isolates closely match 5. saprophyticus and S. cohnii subsp urealyticus, respectively, and one may be S. warneri or S. pasteuri. With the same methods, the others of the 5 isolates were identified as a Firmicutes Bacillus fusiformis ,and an Actinobacteria Microbacterium oleivorans respectively.To determine the extreme characteristics of these isolates, their resistance levels to 3 disinfectants (UV, phenol and CHG) were tested. In order to judge whether these isolates are just the normal bacteria in the environment, and the resisting abilities are the inherent characteristics of these strains, or plasmid hold or DNA changed mutants, Staphylococcus aureus was to be comparison, and comparing the different of resistance between Sa and isolated bacterium . The result showed: Although the 5 isolates have different resisting abilities to 3 disinfectants, two-factors variance analyses showed no significant differences between these 5 isolates and Staphylococcus aureus, suggesting that these isolates are just the normal bacteria in the environment, and the resisting abilities are the inherent characteristics of these strains, rather than plasmid hold or DNA changed mutants.
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