Blanch A R, García-Aljaro C, Muniesa M, Jofre J
Dept of Microbiology, University of Barcelona, Diagonal 645, 08028 Barcelona, Spain.
Water Sci Technol. 2003;47(3):109-16.
Verotoxigenic Escherichia coli strains have been related with waterborne outbreaks. Besides 0157:H7, several serotypes of E. coli and other enterobacteria have been implicated in outbreaks and reported to carry the shiga toxin genes. Shiga toxins, stx1 and stx2, are important virulence factors of these strains. These genes have been linked to bacteriophages and consequently are susceptible to lateral transmission. To better understand the ecology of these genes a study of the presence of the shiga toxin 2 gene (stx2) among coliform bacteria present in sewage samples was carried out. A procedure based on colony hybridisation was developed for the isolation of enterobacteria carrying this gene. Colony growth on Chromocult agar was transferred to a membrane and hybridised with a gene specific probe. The procedure allowed detection of about one colony carrying the gene among around 1,000 faecal coliform colonies. The numbers of bacteria carrying the gene in sewage were also estimated by PCR indicating that the numbers of bacteria carrying the stx2 gene were about 1/1,000 faecal coliforms. The detected numbers by both methods were similar. Positive colony hybridisation was detected in four sewage origins. Fifty-two colonies showing positive signal were isolated from the Chromocult agar plates, confirmed to be stx2 positive by PCR and phenotypically characterised. Results of the characterisation showed certain diversity among the isolates even in isolates from the same sample. Most of these isolates would not have been isolated with the methods regularly used for the isolation of E. coli 0157:H7 strains. The method will allow study of the numbers and characteristics of bacteria carrying the stx2 gene in different water environments and isolate them in order to determine their role in the spread of the gene.
产志贺毒素大肠杆菌菌株与水源性疾病暴发有关。除了O157:H7之外，几种大肠杆菌血清型及其他肠杆菌科细菌也与疾病暴发有关，并且据报道它们携带志贺毒素基因。志贺毒素stx1和stx2是这些菌株的重要毒力因子。这些基因与噬菌体有关，因此易于横向传播。为了更好地了解这些基因的生态学，对污水样本中大肠菌群中志贺毒素2基因（stx2）的存在情况进行了研究。开发了一种基于菌落杂交的方法来分离携带该基因的肠杆菌科细菌。在显色培养基上生长的菌落被转移到膜上，并与基因特异性探针杂交。该方法能够在大约1000个粪大肠菌群菌落中检测到约一个携带该基因的菌落。还通过PCR估计了污水中携带该基因的细菌数量，表明携带stx2基因的细菌数量约为每1000个粪大肠菌群中有1个。两种方法检测到的数量相似。在四个污水源中检测到阳性菌落杂交。从显色培养基平板上分离出52个显示阳性信号的菌落，通过PCR确认stx2呈阳性并进行了表型特征分析。特征分析结果表明，即使是来自同一样本的分离株之间也存在一定的多样性。这些分离株中的大多数用常规用于分离大肠杆菌O157:H7菌株的方法是无法分离出来的。该方法将有助于研究不同水环境中携带stx2基因的细菌数量和特征，并将它们分离出来，以确定它们在该基因传播中的作用。
