Viable but nonculturable Vibrio cholerae 01 in biofilms in the aquatic environment and their role in cholera transmission
Viable but nonculturable Vibrio cholerae 01 in biofilms in the aquatic environment and their role in cholera transmission
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DOI：
10.1073/pnas.0705599104
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发表时间：
2007-11-06
影响因子：
11.1
通讯作者：
Colwell, Rita R.
中科院分区：
文献类型：
--
作者：
Alam, Munirul;Sultana, Marzia;Colwell, Rita R.
霍乱弧菌在水环境中持续存在，主要处于不可培养的状态。在这项研究中，在孟加拉国Mathbaria的生物膜中保持了495天的非培养球状霍乱弧菌01，池塘水在动物传代后成为可培养的。通过培养、直接荧光抗体(DFA)和多重聚合酶链式反应(PCR)监测细菌的可培养能力、生物被膜形成以及wbe、ctxA和rstR2基因。生物膜形成后，不可能进行DFA计数。此外，即使在室温(约25摄氏度)和4摄氏度(未检测到生长)下分别孵育54天和68天后，WBE仍可扩增，但ctKA不能扩增。与大约25℃相比，在4℃下培养的培养物的生物膜形成和延长的可培养能力较慢，这表明生物膜的产生与温度有关，并与培养能力的丧失有关。在小宇宙中孵育54天和68天后，分别在接近25℃和4℃的温度下出现小菌落，WBE阳性，ctxA和rstR2阴性，表明CTX phi失去了噬菌体。霍乱弧菌O1在微宇宙中被观察为游离细胞，孵育495天后不能培养，但同一微宇宙中的生物膜产生了可培养细胞。结论：生物膜可作为O1霍乱弧菌在两次流行之间的蓄水池，因为它在生物膜中具有长期的生存能力。与在Mathbaria池塘水中产生的生物膜相反，霍乱弧菌O1在霍乱粪便中存在的生物膜中，在与Mathbaria池塘水生物膜相同的条件下培养2个月后不能培养，这表明新排放到环境中的霍乱弧菌细胞对环境条件的适应能力明显低于细胞。
Vibrio cholerae persists in aquatic environments predominantly in a nonculturable state. In this study coccoid, nonculturable V. cholerae 01 in biofilms maintained for 495 days in Mathbaria, Bangladesh, pond water became culturable upon animal passage. Culturability, biofilm formation, and the wbe, ctxA, and rstR2 genes were monitored by culture, direct fluorescent antibody (DFA), and multiplex PCR. DFA counts were not possible after formation of biofilm. Furthermore, wbe, but not ctKA, were amplifiable, even after incubation for 54 and 68 days at room temperature (approximate to 25 degrees C) and 4 degrees C, respectively, when no growth was detectable. Slower biofilm formation and extended culturability were observed for cultures incubated at 4 degrees C, compared with approximate to 25 degrees C, suggesting biofilm production to be temperature dependent and linked to loss of culturability. Small colonies appearing after incubation in microcosms for 54 and 68 days at approximate to 25 degrees C and 4 degrees C, respectively, were wbe positive and ctxA and rstR2 negative, indicating loss of bacteriophage CTX Phi. The coccoid V. cholerae O1 observed as free cells in microcosms incubated for 495 days could not be cultured, but biofilms in the same microcosms yielded culturable cells. It is concluded that biofilms can act as a reservoir for V. cholerae O1 between epidemics because of its long-term viability in biofilms. In contrast to biofilms produced in Mathbaria pond water, V. cholerae O1 in biofilms present in cholera stools and incubated under identical conditions as the Mathbaria pond water biofilms could not be cultured after 2 months, indicating that those V. cholerae cells freshly discharged into the environment are significantly less robust than cells adapted to environmental conditions.