【摘要】目的研究青海省血液透析用水中可培养微生物的多样性和生长特性，识别微生物污染风险，为改善透析用水水质提供数据支持。方法采集18 所医院血液透析中心的反渗机组出水口和输水管路回水口水样，经薄膜过滤法富集，在21℃下分别用胰化蛋白胨葡萄糖培养基(tryptone glucose extract agar,TGEA)和R2A 营养琼脂培养基(R2A agar medium,R2A)培养7 天，动态观察计数并用质谱仪和PCR 技术菌种鉴定。结果共检出12 种革兰氏阴性(G-)菌和7 种革兰氏阳性(G+)菌；回水口和出水口检出细菌中，回水口和出水口的G-菌(79.78%，95.76%)占比均大于G+菌(20.22%，4.24%)；出水口中检出的藤泽甲基杆菌(31.32CFU/ml)多于回水口(0.36 CFU/ml)，差异有统计学意义(U=5026.500，P＜0.001)。皮氏罗尔斯顿菌、棘皮鞘氨醇单胞菌和藤泽甲基杆菌TGEA(10.64CFU/ml、7.10CFU/ml和62.16CFU/ml)上生长数目多于R2A(0.82CFU/ml、6.10CFU/ml和29.36CFU/ml)，差异有统计学意义(U分别为29721.000,
127168.000,1808717.500，均P＜0.001)；大部分细菌在培养至第5 天进入稳定期，藤泽甲基杆菌和棘皮鞘氨醇单胞菌在第6 天进入生长对数期。结论透析用水回水口与出水口可培养微生物以G-菌为主，其种、属分布存在较大差异；个别细菌生长高峰期滞后，提示168h(7d)培养周期，可能会低估微生物污染水平；菌落生长过程中仅根据细菌的菌落形态特征区分其种类，可能使结果产生偏差。掌握透析用水中的微生物多样性、菌种分布及其生长特性，可为识别微生物风险环节和优化透析用水微生物监测标准提供依据。
【Abstract】Objective To study the diversity and growth characteristics of culturable microorganisms in hemodialysis water in Qinghai Province and to identify their contamination risk, in order to provide data support for improving the quality of dialysis water. Methods Hemodialysis water samples were collected from the delivery port of the reverse osmosis unit and the return port of the water pipeline in the hemodialysis centers of 18 hospitals. The bacteria in water were enriched by a membrane filtration method and then cultured in tryptone glucose extract agar (TGEA) and R2A agar medium (R2A) at 21℃ for 7 days. Colonies were dynamically observed and counted. The culturable bacteria were identified by mass spectrometry and PCR methods. Results Twelve Gram- negative bacteria and 7 Gram- positive bacteria were detected. The proportions of Gram-negative bacteria were 79.78% and 95.76%, higher than the proportions of Gram-positive bacteria of 20.22% and 4.24%, in water from return port and delivery port respectively. The number of Methylobacterium fujisawasense was 31.32 CFU/mL in water from delivery port, higher than that of 0.36 CFU/mL in water from return port (U=5026.500, P<0.001). The numbers of Ralstonia pickettii, Sphingomonas echinoides and Methylobacterium fujisawasense growing on TGEA were 10.64, 7.10 and 62.16 CFU/ml respectively, more than the
numbers growing on R2A of 0.82, 6.10 and 29.36 CFU/mL respectively (U=29721.000, 127168.000 and 1808717.5 respectively, P<0.001). Most bacteria entered the stabilization stage at the 5th day of incubation, but Methylobacterium fujisawasense and Sphingomonas echinoides progressed into the logarithmic growth stage at the 6th day of incubation. Conclusions The culturable microorganisms in dialysis water from delivery port and return port are mainly G- bacteria with substantial differences in the distribution of genus and species. The delayed growth peak in some bacteria suggests that incubation for 168 hours (7 days) may underestimate the level of microbial contamination in water. If the determination of bacterial species were solely based on morphological characteristics of the colonies in the processes of colony growth, the results would be biased. The knowledge about the diversity, species distribution and growth characteristics of bacteria in hemodialysis water is useful for the detection of bacterial contamination risk and the improvement of surveillance standards for dialysis water quality.
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