Baffone W, Citterio B, Vittoria E, Casaroli A, Campana R, Falzano L, Donelli G
Istituto di Scienze Tossicologiche, Igienistiche ed Ambientali, Università degli Studi di Urbino, Via Santa Chiara, 27, 61029 Urbino (PU), Rome, Italy.
Int J Food Microbiol. 2003 Dec 15;89(1):31-9. doi: 10.1016/s0168-1605(03)00102-8.
The viable but non-culturable (VBNC) forms of two environmental strains of Vibrio alginolyticus 1 and Vibrio parahaemolyticus 66 and one strain of V. parahaemolyticus ATCC 43996 showing virulence characteristics (hemolysin production, adhesive and/or cytotoxic ability, in vivo enteropathogenicity) were obtained by culturing bacteria in a microcosm consisting of artificial sea water (ASW) and incubating at 5 degrees C with shaking. Every 2 days, culturability of the cells in the microcosm was monitored by spread plates on BHI agar and total count and the percentage of viable cells were determined by double staining with DAPI and CTC. When cell growth was not detectable (<0.1 CFU/ml), the population was considered non-culturable and, then, the VBNC forms were resuscitated in a murine model. For each strain, eight male Balb/C mice were intragastrically inoculated with 0.1 ml of concentrated ASW bacterial culture. Two mice from each group were sacrificed at 2, 4, 8, and 12 days after challenge for autopsy and re-isolation of the microorganisms from the intestinal tissue cultures. Isolation was obtained in 25% of the animals challenged with the VBNC V. alginolyticus strain, in 37.5% of those challenged with the VBNC V. parahaemolyticus strain of environmental origin and in 50% of the animals infected with VBNC V. parahaemolyticus ATCC 43996. The strains thus isolated were again subjected to biological assays to determine the retention of pathogenicity. The virulence characteristics that seemed to disappear after resuscitation in the mouse were subsequently reactivated by means of two consecutive passages of the strains in the rat ileal loop model. The results obtained indicate that VBNC forms of the strains examined can be resuscitated and retain their virulence properties.
通过在由人工海水（ASW）组成的微宇宙中培养溶藻弧菌1和副溶血性弧菌66的两种环境菌株以及一株具有毒力特征（产生溶血素、具有黏附性和/或细胞毒性能力、体内肠致病性）的副溶血性弧菌ATCC 43996，获得了其活的但不可培养（VBNC）形式。将细菌在5℃振荡培养于人工海水中。每2天，通过在脑心浸液（BHI）琼脂平板上涂布计数来监测微宇宙中细胞的可培养性，并通过用4′,6-二脒基-2-苯基吲哚（DAPI）和羧基荧光素二乙酸盐琥珀酰亚胺酯（CTC）双重染色来测定活细胞总数和百分比。当检测不到细胞生长（<0.1 CFU/ml）时，该群体被认为不可培养，然后，在小鼠模型中复苏VBNC形式。对于每种菌株，给8只雄性Balb/C小鼠经胃内接种0.1 ml浓缩的人工海水细菌培养物。在攻击后2、4、8和12天，每组处死2只小鼠进行尸检，并从肠道组织培养物中重新分离微生物。在用VBNC溶藻弧菌菌株攻击的动物中，25%获得了分离；在用环境来源的VBNC副溶血性弧菌菌株攻击的动物中，37.5%获得了分离；在用VBNC副溶血性弧菌ATCC 43996感染的动物中，50%获得了分离。如此分离出的菌株再次进行生物学测定以确定致病性的保留情况。在小鼠中复苏后似乎消失的毒力特征随后通过在大鼠回肠袢模型中对菌株进行连续两代传代而重新激活。所获得的结果表明，所检测菌株的VBNC形式可以复苏并保留其毒力特性。
