Morales-Morales Hugo A, Vidal Guadalupe, Olszewski John, Rock Channah M, Dasgupta Debanjana, Oshima Kevin H, Smith Geoffrey B
Biology Department, New Mexico State University, Las Cruces, NM 88003, USA.
Appl Environ Microbiol. 2003 Jul;69(7):4098-102. doi: 10.1128/AEM.69.7.4098-4102.2003.
The detection and identification of pathogens from water samples remain challenging due to variations in recovery rates and the cost of procedures. Ultrafiltration offers the possibility to concentrate viral, bacterial, and protozoan organisms in a single process by using size-exclusion-based filtration. In this study, two hollow-fiber ultrafilters with 50,000-molecular-weight cutoffs were evaluated to concentrate microorganisms from 2- and 10-liter water samples. When known quantities (10(5) to 10(6) CFU/liter) of two species of enteric bacteria were introduced and concentrated from 2 liters of sterile water, the addition of 0.1% Tween 80 increased Escherichia coli strain K-12 recoveries from 70 to 84% and Salmonella enterica serovar Enteritidis recoveries from 36 to 72%. An E. coli antibiotic-resistant strain, XL1-Blue, was recovered at a level (87%) similar to that for strain K-12 (96%) from 10 liters of sterile water. When E. coli XL1-Blue was introduced into 10 liters of nonsterile Rio Grande water with higher turbidity levels (23 to 29 nephelometric turbidity units) at two inoculum levels (9 x 10(5) and 2.4 x 10(3) per liter), the recovery efficiencies were 89 and 92%, respectively. The simultaneous addition of E. coli XL1-Blue (9 x 10(5) CFU/liter), Cryptosporidium parvum oocysts (10 oocysts/liter), phage T1 (10(5) PFU/liter), and phage PP7 (10(5) PFU/liter) to 10 liters of Rio Grande surface water resulted in mean recoveries of 96, 54, 59, and 46%, respectively. Using a variety of surface waters from around the United States, we obtained recovery efficiencies for bacteria and viruses that were similar to those observed with the Rio Grande samples, but recovery of Cryptosporidium oocysts was decreased, averaging 32% (the site of collection of these samples had previously been identified as problematic for oocyst recovery). Results indicate that the use of ultrafiltration for simultaneous recovery of bacterial, viral, and protozoan pathogens from variable surface waters is ready for field deployment.
由于回收率的差异和操作成本，从水样中检测和鉴定病原体仍然具有挑战性。超滤提供了一种通过基于尺寸排阻的过滤在单一过程中浓缩病毒、细菌和原生动物的可能性。在本研究中，评估了两种截留分子量为50,000的中空纤维超滤器，用于从2升和10升水样中浓缩微生物。当将已知数量（10⁵至10⁶CFU/升）的两种肠道细菌引入并从2升无菌水中浓缩时，添加0.1%吐温80可使大肠杆菌K-12菌株的回收率从70%提高到84%，肠炎沙门氏菌肠炎血清型的回收率从36%提高到72%。从10升无菌水中回收的大肠杆菌抗生素抗性菌株XL1-Blue的回收率（87%）与菌株K-12的回收率（96%）相似。当将大肠杆菌XL1-Blue以两种接种水平（每升9×10⁵和2.4×10³）引入10升浊度较高（23至29散射浊度单位）的非无菌格兰德河水中时，回收率分别为89%和92%。将大肠杆菌XL1-Blue（9×10⁵CFU/升）、微小隐孢子虫卵囊（10个卵囊/升）、噬菌体T1（10⁵PFU/升）和噬菌体PP7（10⁵PFU/升）同时添加到10升格兰德河地表水中，平均回收率分别为96%、54%、59%和46%。使用来自美国各地的各种地表水，我们获得的细菌和病毒回收率与格兰德河样品中观察到的回收率相似，但隐孢子虫卵囊的回收率降低，平均为32%（这些样品的采集地点先前已被确定为卵囊回收存在问题的地点）。结果表明，使用超滤从不同地表水同时回收细菌、病毒和原生动物病原体已准备好进行现场部署。
