Williams Margaret M, Braun-Howland Ellen B
Wadsworth Center, New York State Department of Health, Albany, New York 12201, USA.
Appl Environ Microbiol. 2003 Sep;69(9):5463-71. doi: 10.1128/AEM.69.9.5463-5471.2003.
Bacteria indigenous to water distribution systems were used to grow multispecies biofilms within continuous-flow slide chambers. Six flow chambers were also inoculated with an Escherichia coli isolate obtained from potable water. The effect of disinfectants on bacterial populations was determined after exposure of established biofilms to 1 ppm of hypochlorous acid (ClOH) for 67 min or 4 ppm of monochloramine (NH(2)Cl) for 155 min. To test the ability of bacterial populations to initiate biofilm formation in the presence of disinfectants, we assessed the biofilms after 2 weeks of exposure to residual concentrations of 0.2 ppm of ClOH or 4 ppm of NH(2)Cl. Lastly, to determine the effect of recommended residual concentrations on newly established biofilms, we treated systems with 0.2 ppm of ClOH after 5 days of growth in the absence of disinfectant. Whole-cell in situ hybridizations using fluorescently tagged, 16S rRNA-targeted oligonucleotide probes performed on cryosectioned biofilms permitted the direct observation of metabolically active bacterial populations, including certain phylogenetic groups and species. The results of these studies confirmed the resistance of established bacterial biofilms to treatment with recommended levels of disinfectants. Specifically, Legionella pneumophila, E. coli, and beta and delta proteobacteria were identified within biofilms both before and after treatment. Furthermore, although it was undetected using routine monitoring techniques, the observation of rRNA-containing E. coli within biofilms demonstrated not only survival but also metabolic activity of this organism within the model distribution systems. The persistence of diverse bacterial species within disinfectant-treated biofilms suggests that current testing practices underestimate the risk to immunocompromised individuals of contracting waterborne disease.
利用水分配系统中的本土细菌在连续流动的载玻片腔室中培养多物种生物膜。六个流动腔室还用从饮用水中分离出的大肠杆菌进行了接种。在已形成的生物膜暴露于1 ppm的次氯酸（ClOH）67分钟或4 ppm的一氯胺（NH₂Cl）155分钟后，测定消毒剂对细菌种群的影响。为了测试细菌种群在消毒剂存在下启动生物膜形成的能力，我们在暴露于0.2 ppm的ClOH或4 ppm的NH₂Cl残留浓度2周后评估生物膜。最后，为了确定推荐残留浓度对新形成生物膜的影响，我们在无消毒剂的情况下生长5天后，用0.2 ppm的ClOH处理系统。使用荧光标记的、靶向16S rRNA的寡核苷酸探针在冷冻切片生物膜上进行全细胞原位杂交，从而可以直接观察代谢活跃的细菌种群，包括某些系统发育组和物种。这些研究结果证实了已形成的细菌生物膜对推荐水平消毒剂处理具有抗性。具体而言，在处理前后的生物膜中均鉴定出嗜肺军团菌、大肠杆菌以及β和δ变形菌。此外，尽管使用常规监测技术未检测到，但在生物膜中观察到含rRNA的大肠杆菌，这不仅证明了该生物体在模型分配系统中的存活，还证明了其代谢活性。消毒剂处理过的生物膜中多种细菌物种的持续存在表明，当前的检测方法低估了免疫功能低下个体感染水传播疾病的风险。
