Dynamac c/o US Environmental Protection Agency, 26 W Martin Luther King Dr, Cincinnati, OH, 45268, USA,
Microb Ecol. 2013 Oct;66(3):715-26. doi: 10.1007/s00248-013-0250-z. Epub 2013 Jun 14.
Hartmannella vermiformis and Acanthamoeba polyphaga are frequently isolated from drinking water and permissive to Legionella pneumophila parasitization. In this study, extracellular factor(s) produced by H. vermiformis and A. polyphaga were assessed for their effects on cultivability of L. pneumophila. Page's amoeba saline (PAS) was used as an encystment medium for H. vermiformis and A. polyphaga monolayers, and the culture supernatants (HvS and ApS, respectively) were assessed against L. pneumophila growth. Compared to PAS and ApS, HvS significantly inhibited L. pneumophila strain Philadelphia-1 (Ph-1) cultivability by 3 log(10) colony forming unit (CFU) mL(-1) after 3 days of exposure compared to <0.5 log(10) CFU mL(-1) reduction of strain Lp02 (P < 0.001). Flow cytometric analysis revealed changes in the percentage and cultivability of three bacterial subpopulations: intact/slightly damaged membrane (ISM), undefined membrane status (UD), and mixed type (MT). After 3 days of HvS exposure, the MT subpopulation decreased significantly (31.6 vs 67.2 %, respectively, P < 0.001), while the ISM and UD subpopulations increased (+26.7 and +6.9 %, respectively) with the ISM subpopulation appearing as viable but nonculturable (VBNC) cells. HvS was separated into two fractions based on molecular weight, with more than 99 % of the L. pneumophila inhibition arising from the <5 kDa fraction (P < 0.001). Liquid chromatography indicated the inhibitory molecule(s) are likely polar and elute from a Novapak C18 column between 6 and 15 min. These results demonstrate that H. vermiformis is capable of extracellular modulation of L. pneumophila cultivability and probably promote the VBNC state for this bacterium.
内变形虫和棘阿米巴常从饮用水中分离出来，并且允许嗜肺军团菌寄生。在这项研究中，评估了内变形虫和棘阿米巴产生的细胞外因子对嗜肺军团菌培养的影响。使用 Page 的变形虫生理盐水（PAS）作为内变形虫和棘阿米巴单层的包囊培养基，分别评估培养上清液（HvS 和 ApS）对嗜肺军团菌生长的影响。与 PAS 和 ApS 相比，HvS 在暴露 3 天后，对费城-1 （Ph-1）菌株的抑制作用显著，使嗜肺军团菌的培养物减少了 3 个对数（10）菌落形成单位（CFU）mL（-1），而对 Lp02 菌株的减少则小于 0.5 个对数（10）CFU mL（-1）（P < 0.001）。流式细胞术分析显示，三种细菌亚群的比例和可培养性发生了变化：完整/轻微受损的膜（ISM）、膜状态不明（UD）和混合类型（MT）。在 HvS 暴露 3 天后，MT 亚群显著减少（分别为 31.6%和 67.2%，P < 0.001），而 ISM 和 UD 亚群增加（分别增加+26.7%和+6.9%），ISM 亚群表现为活但不可培养（VBNC）细胞。根据分子量将 HvS 分离成两个部分，超过 99%的嗜肺军团菌抑制作用来自于 <5 kDa 部分（P < 0.001）。液相色谱表明，抑制分子可能是极性的，在 Novapak C18 柱上洗脱时间为 6-15 分钟。这些结果表明，内变形虫能够调节嗜肺军团菌的可培养性，并可能促进该细菌进入 VBNC 状态。
