| Summary: | 碩士 === 高雄醫學大學 === 公共衛生學研究所碩士班 === 93 === A total of 41 strains of Legionella species isolated from hospital environmental water supplying system and different hot spring water sources, and 5 reference strains of Legionella pneumophia [ATCC 43111 (serogroup 1), ATCC 33154 (serogroup 2), ATCC 33155 (serogroup 3), ATCC 33156 (serogroup 4), ATCC 33216(serogroup 5)] were used to study the mutational analysis of nucleotide sequences of virulence gene lvgA. In addition, phenotypic character of invasion assay in each reference strain was also investigated.
By using a specific pair of primer L711 (5’-TGTCAATTTTGGGCTAAT-3’) and primer U86 (5’-TATAATTTTCTCTTGGGGATT-3’), PCR testing was performed for detection of lvgA gene in Legionella strains tested. The results showed that 5 reference isolates of Legionella pneumophia and 25 isolates of Legionella pneumophia obtained from environmental sources harbored the lvgA gene. However 15 isolates of Legionella species and 1 isolate of L. micadadei originated from environmental sources were not presenting the lvgA gene. Moreover, a specific primer pair of LF2 (5’-ACGGCTTTTTTACCTTTTCTT-3’) and LR2 (5’-TTATGTGCTTTTGCATCAATG-3’) was designed for PCR testing to amplify the entire lvgA gene of Legionella pneumophia tested, and the PCR amplicons were subject to nucleotide sequencing.
Sequence analysis of nucleotide sequences of 30 strains of the lvgA gene-positive Legionella pneumophia tested was analyzed and compared with the original lvgA gene (accession no. AF181867). Nucleotide analysis after sequencing showed that Legionella pneumophia serogroup 1(ATCC 43111) shared a 98% identity with the original lvgA gene (accession no. AF181867) in Legionella pneumophia serogroup 1(ATCC BAA-74), and that the amino acid sequence had an S72P, and M131L substitutions. In particular, Legionella pneumophia serogroup 2(ATCC 33154) shared a 95% identity with the original lvgA gene(accession no. AF181867), and that the amino acid sequence had an S72P, H114Y, D122E, E133Q, E194K, N195I, K197M, A202G and M131L substitutions. However, Legionella pneumophia serogroup 3(ATCC 33155) shared a 97% identity with the original lvgA gene (accession no. AF181867) of , and that the amino acid sequence only had an S72P substitution. On the other hand, Legionella pneumophia serogroup 4(ATCC ATCC 33156 ) shared a 96% identity with the original lvgA gene (accession no. AF181867), and that the amino acid sequence had an V13A, D14E, S72P, S82P, and E133Q substitutions. But Legionella pneumophia serogroup 5 (ATCC 33216 ) shared a 96% identity with the original lvgA gene (accession no. AF181867), and that the amino acid sequence had S72P, H117Y, D122E, and E133Q substitutions.
Amplification products were separated, purified and used for PCR cloning into vector pGEM-T Easy (Promega). E. coli XL-1 blue was used as competent cells for recombinant DNA transformation. The transformants were further examined for the presence of lvgA gene and invasion assay with cell line A549. The transformant E. coli harboring the lvgA gene showed the same invasive ability as original Legionella pneumophia tested penetrating and multiplying the cell line A549. In contrast, E. coli XL-1 blue has no ability to invasive into cell line A549. In our results showed the lvgA gene is a virulence factor for Legionella pneumophia and can express the intracellular invasion into cell line A549 even E. coli strain harboring the lvgA gene. Meantime, primed PCR testing combined with nucleotide sequence mutational analysis for molecular typing of Legionella pneumophia isolates. In this study, it was found able to identify an epidemic strain of Legionella pneumophia serogroup 1 that was isolated from both patients (reference strains) and a hospital environmental isolate.
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