Targeting the rpoB gene using nested PCR-restriction fragment length polymorphism for identification of nontuberculous mycobacteria in hospital tap water.
Nontuberculous mycobacteria (NTM) are ubiquitous in the environment and can cause nosocomial infections in immunocompromised patients. Recently the presence of NTM in public drinking water and hospital water distribution systems has been reported. Their ability to form biofilms and their resistance to chlorine both contribute to their survival and colonization in water distribution systems. Here we analyzed thirty-two hospital tap water samples that were collected from different locations in three hospitals so as to evaluate the prevalence of NTM species. The water samples were concentrated by membrane filtration and then eluted with sterilized water following sonication. Two-step direct PCR targeting the rpoB gene, restriction fragment length polymorphism (RFLP) using the MspI restriction enzyme, and sequence analysis were performed for identification of NTM to the species level. The sequences of each PCR product were analyzed using BLASTN. Seven samples (7/32, 21.9%) were positive for NTM as determined by nested-PCR. The PCR-RFLP results indicated five different patterns among the seven positive PCR samples. The water-born NTM were identified, including M. peregrinum, M. chelonae (2 cases), M. abscessus, M. gordonae (2 cases), and Mycobacterium sp. JLS. The direct two-step PCR-RFLP method targeting the rpoB gene was effective for the detection and the differentiation of NTM species from hospital tap water.
Shin
Ji-Hyun
JH
Division of Bacterial Respiratory Infection, Center for Infectious Diseases, National Institute of Health, Centers for Disease Control and Prevention, Seoul, 122-701, Republic of Korea.
Lee
Hae-Kyung
HK
Cho
Eun-Jin
EJ
Yu
Jae-Yon
JY
Kang
Yeon-Ho
YH
D015373:Bacterial Typing Techniques; D012321:DNA-Directed RNA Polymerases / Q000235:genetics*; D019003:Deoxyribonuclease HpaII; D005618:Fresh Water / Q000382:microbiology*; D006761:Hospitals; D007723:Korea; D009161:Mycobacterium / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification; D016133:Polymerase Chain Reaction / Q000379:methods*; D012150:Polymorphism, Restriction Fragment Length; D013045:Species Specificity; D014881:Water Supply
D012321:DNA-Directed RNA Polymerases; C105078:RNA polymerase beta subunit; D019003:Deoxyribonuclease HpaII