Department of Microbiology, All India Institute of Medical Sciences, New Delhi, India.
International Centre for Genetic Engineering & Biotechnology, New Delhi, India.
Indian J Med Res. 2017 Sep;146(3):392-400. doi: 10.4103/ijmr.IJMR_623_16.
BACKGROUND & OBJECTIVES: Genomic constitution of the bacterium Legionella pneumophila plays an important role in providing them a pathogenic potential. Here, we report the standardization and application of multiplex polymerase chain reaction (PCR) for the detection of molecular markers of pathogenic potential in L. pneumophila in hospital environment.
Culture of the standard strains of L. pneumophila was performed in buffered charcoal-yeast extract agar with L-cysteine at p H 6.9. Primers were designed for multiplex PCR, and standardization for the detection of five markers annotated to L. pneumophila plasmid pLPP (11A2), lipopolysaccharide synthesis (19H4), CMP-N-acetylneuraminic acid synthetase (10B12), conjugative coupling factor (24B1) and hypothetical protein (8D6) was done. A total of 195 water samples and 200 swabs were collected from the hospital environment. The bacterium was isolated from the hospital environment by culture and confirmed by 16S rRNA gene PCR and restriction enzyme analysis. A total of 45 L. pneumophila isolates were studied using the standardized multiplex PCR.
The PCR was sensitive to detect 0.1 ng/μl DNA and specific for the two standard strains used in the study. Of the 45 hospital isolates tested, 11 isolates had four markers, 12 isolates had three markers, 10 isolates had two markers, nine isolates had one marker and three isolates had none of the markers. None of the isolates had all the five markers.
INTERPRETATION & CONCLUSIONS: The findings of this study showed the presence of gene markers of pathogenic potential of the bacterium L. pneumophila. However, the genomic constitution of the environmental isolates should be correlated with clinical isolates to prove their pathogenic potential. Rapid diagnostic methods such as multiplex PCR reported here, for elucidating gene markers, could help in future epidemiological studies of bacterium L. pneumophila.
嗜肺军团菌的基因组构成在赋予其致病潜能方面发挥着重要作用。本研究报告了一种用于检测医院环境中嗜肺军团菌致病潜能分子标记物的多重聚合酶链反应（PCR）的标准化和应用。
在 pH 值为 6.9 的缓冲炭酵母提取物琼脂中培养嗜肺军团菌标准菌株。设计了用于多重 PCR 的引物，并对标记在嗜肺军团菌质粒 pLPP（11A2）、脂多糖合成（19H4）、CMP-N-乙酰神经氨酸合成酶（10B12）、共轭偶联因子（24B1）和假设蛋白（8D6）上的 5 个标记物的检测进行了标准化。从医院环境中采集了 195 份水样和 200 份拭子。通过培养从医院环境中分离出细菌，并通过 16S rRNA 基因 PCR 和限制性内切酶分析进行确认。使用标准化的多重 PCR 对 45 株嗜肺军团菌分离株进行了研究。
PCR 对 0.1 ng/μl DNA 的检测具有敏感性，且对研究中使用的两种标准菌株具有特异性。在所测试的 45 株医院分离株中，11 株有 4 个标记物，12 株有 3 个标记物，10 株有 2 个标记物，9 株有 1 个标记物，3 株没有标记物。没有分离株具有所有 5 个标记物。
本研究结果表明，存在嗜肺军团菌的致病潜能基因标记物。然而，环境分离株的基因组构成应与临床分离株相关联，以证明其致病潜能。这里报道的多重 PCR 等快速诊断方法可以帮助阐明嗜肺军团菌的基因标记物，有助于未来对嗜肺军团菌的流行病学研究。
