Deepanjali A, Kumar H Sanath, Karunasagar I, Karunasagar I
Department of Fishery Microbiology, University of Agricultural Sciences, College of Fisheries, Mangalore 575 002, India.
Appl Environ Microbiol. 2005 Jul;71(7):3575-80. doi: 10.1128/AEM.71.7.3575-3580.2005.
The seasonal abundance of Vibrio parahaemolyticus in oysters from two estuaries along the southwest coast of India was studied by colony hybridization using nonradioactive labeled oligonucleotide probes. The density of total V. parahaemolyticus bacteria was determined using a probe binding to the tlh (thermolabile hemolysin) gene, and the density of pathogenic V. parahaemolyticus bacteria was determined by using a probe binding to the tdh (thermostable direct hemolysin) gene. Furthermore, the prevalence of V. parahaemolyticus was studied by PCR amplification of the toxR, tdh, and trh genes. PCR was performed directly with oyster homogenates and also following enrichment in alkaline peptone water for 6 and 18 h. V. parahaemolyticus was detected in 93.87% of the samples, and the densities ranged from <10 to 10(4) organisms per g. Pathogenic V. parahaemolyticus could be detected in 5 of 49 samples (10.2%) by colony hybridization using the tdh probe and in 3 of 49 samples (6.1%) by PCR. Isolates from one of the samples belonged to the pandemic serotype O3:K6. Twenty-nine of the 49 samples analyzed (59.3%) were positive as determined by PCR for the presence of the trh gene in the enrichment broth media. trh-positive V. parahaemolyticus was frequently found in oysters from India.
采用非放射性标记寡核苷酸探针通过菌落杂交法,研究了印度西南海岸两个河口牡蛎中副溶血性弧菌的季节性丰度。使用与tlh(不耐热溶血素)基因结合的探针测定副溶血性弧菌总菌密度,使用与tdh(耐热直接溶血素)基因结合的探针测定致病性副溶血性弧菌密度。此外,通过对toxR、tdh和trh基因进行PCR扩增研究副溶血性弧菌的流行情况。PCR直接用牡蛎匀浆进行,也在碱性蛋白胨水中富集6小时和18小时后进行。在93.87%的样本中检测到副溶血性弧菌,密度范围为每克<10至10⁴个菌体。通过使用tdh探针的菌落杂交法在49个样本中的5个(10.2%)中检测到致病性副溶血性弧菌,通过PCR在49个样本中的3个(6.1%)中检测到。从其中一个样本分离出的菌株属于大流行血清型O3:K6。在49个分析样本中,有29个(59.3%)通过PCR检测在富集肉汤培养基中存在trh基因呈阳性。trh阳性的副溶血性弧菌在印度牡蛎中经常被发现。