title: Enhancement of Legionella pneumophila culture isolation from microenvironments by macrophage infectivity potentiator (mip) gene-specific nested polymerase chain reaction.
abstract: The combination of a Legionella pneumophila culture isolation technique and macrophage infectivity potentiator (mip) gene-specific nested polymerase chain reaction (PCR) is pivotal for effective routine use in an environmental water system laboratory. Detection of Legionella organisms in 169 environmental samples was performed by using modified buffered charcoal yeast extract (MBCYE) agar for conventional culture. Nested PCR specific for L. pneumophila was performed using boiled genomic DNA extracts from filtered and Chelex 100-treated water samples, or by using silica-gel membrane spin column-eluted DNA from concentrated pond, canal and river samples. Overall, the nested PCR was twelvefold more sensitive than the culture method. The target amplicons (471 basepairs) of all 4 biochemically characterized L. pneumophila isolates were sequenced. They had homology at the DNA and protein levels to 3' proximity of the mip-coding gene of L. pneumophila deposited in genome databases. EcoRI- or KpnI-digested PCR fragments with expected sizes were also confirmed in all 52 PCR-positive samples that were isolated from cooling towers and condenser drains. Viable but nonculturable L. pneumophila might have been present in 48 PCR-positive samples. This study demonstrates that detection of the genetically stable mip gene by nested PCR with a modified process of water sample preparation can be rapidly and effectively used to enhance isolation of the L. pneumophila taxon from microenvironments.
authors: Department of Microbiology, Faculty of Public Health, Mahidol University, 420/1 Rajvithi Road, Rajthewee, Bangkok 10400, Thailand.
authors: Rungrat
authors: R
authors: Nintasen
authors: Fuangfa
authors: F
authors: Utrarachkij
authors: Kanokrat
authors: K
authors: Siripanichgon
authors: Adisak
authors: A
authors: Bhumiratana
authors: Yasuhiko
authors: Y
authors: Suzuki
authors: Orasa
authors: O
authors: Suthienkul
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D003470:Culture Media; D004587:Electrophoresis, Agar Gel / Q000379:methods; D004783:Environmental Microbiology; D016952:Legionella pneumophila / Q000235:genetics / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D019696:Peptidylprolyl Isomerase / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D016415:Sequence Alignment; D014871:Water Microbiology
chemical_list: D001426:Bacterial Proteins; D003470:Culture Media; C512922:Mip protein, Legionella pneumophila; D019696:Peptidylprolyl Isomerase