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To screen the suitable monitoring method applicable to oligotrophs and review the bioburden and microbial species of oligotrophic environments in pharmaceutical water systems and cleanrooms.
The culture conditions and detection methods suitable for microorganisms under oligotrophic conditions were optimized by comparing the counting results of microorganisms in laboratory pure water with R2A and TSA media at different temperatures，media，and incubation times. In addition，monitoring of oligotrophs in pharmaceutical environments using both the traditional and oligotrophic assays were conducted，combining with the techniques of 16s rDNA sequencing and MALDI-TOF MS for multiphase microbial identification and analysis of the isolated microorganisms under oligotrophic conditions.
The colony counting methods，the type of culture media，and the incubation intervals significantly affected microbial enumeration. The isolation method using oligotrophic medium R2A was more effective than the TSA medium in this study. The profiles of microorganisms isolated from the R2A and TSA media were unique. The isolation rate of Gram-negative bacteria in the R2A medium reached 37.0%，however，that of Gram-negative bacteria in the TSA medium was only 14.0%. In addition，several strains of potential pathogens initially isolated from the R2A medium grew slowly in the TSA medium and were easily missed by the traditional monitoring method.
With the development of the pharmaceutical industry，the contamination proportion of human-associated Gram-positive cocci might gradually decrease，the oligotrophic culture method as a powerful supplement to traditional monitoring techniques can help to detect potential objectionable microorganism contamination risks at an early stage.
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| 科 Family | 属数 Number of genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) | 属 Genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) |
|---|---|---|---|---|---|---|
| 鹅膏菌科Amanitaceae | 2 | 11 | 5.26 | 鹅膏菌属 Amanita | 10 | 4.78 |
| 小菇科 Mycenaceae | 2 | 12 | 5.74 | 丝盖伞属 Inocybe | 5 | 2.39 |
| 多孔菌科 Polyporaceae | 8 | 14 | 6.70 | 蜡蘑属 Laccaria | 5 | 2.39 |
| 红菇科 Russulaceae | 3 | 23 | 11.00 | 小皮伞属 Marasmius | 6 | 2.87 |
| 小菇属 Mycena | 11 | 5.26 | ||||
| 光柄菇属 Pluteus | 5 | 2.39 | ||||
| 红菇属 Russula | 17 | 8.13 | ||||
| 栓菌属 Trametes | 5 | 2.39 |
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