痢疾桿菌Shigellaflexneri自發性抗藥性質體變異株與自發性毒性質體變異株的分析

• Main Author: 陳冠州
• Other Authors: 陳建華
• Format: Others
• Language: zh-TW
• Published: 2004
• Online Access: http://ndltd.ncl.edu.tw/handle/38866676204092335511

碩士 === 國立中興大學 === 分子生物學研究所 === 92 === Forty-two Taiwan isolates and 1 ATCC isolate of Shigella flexneri were collected and their minimum inhibitory concentrations (MIC) of ampicillin, streptomycin and nalidixic acid were determined. The results showed that MIC of ampicillin and streptomycin for the 42 Taiwan isolates were 64 - 512 μg/ml and 128 - 512 μg/ml, respectively, whereas MIC of ampicillin and streptomycin for the ATCC isolate were 2 and 8 μg/ml, respectively. On the other hand, only 1 Taiwan isolate had MIC of nalidixic acid to be 1024 μg/ml. Except for this Taiwan isolate, MIC of nalidixic acid for all other Taiwan isolates and the ATCC isolate were 1 μg/ml. In the previous study, one Taiwan isolate (SH595) was picked and a resistant plasmid (R plasmid) responsible for its resistance to ampicillin and streptomycin was identified. In addition to the R plasmid, SH595 carried 4 cryptic small plasmids and one large plasmid likely involved in its virulence. SH595 had MIC of streptomycin to be 128 μg/ml. Many plasmid mutants resistant to streptomycin of 200 μg/ml were selected. These plasmid mutants could be grouped into two types. The type 1 mutants had an insertion of either a 15-kb or 26-kb Tn21-like transposon in one of the four cryptic small plasmids. The Tn21-like transponson was likely originated from the R plasmid in SH595. The type II mutants had an extra 40-kb plasmid, with the 6 endogeneous plasmids seemingly unchanged in size (Wang, 2002; Chen, 1999). In this study, the 40-kb plasmid was transformed into E. coli and analyzed by PCR, Southern hybridization, sequencing and restriction analysis. The results indicated that the plasmid consists of a 36-kb Tn21-like transposon and a 4-kb sequence containing a ColE1 origin of replication. It seems that SH595 contained a cryptic low-copied or undetectable 4-kb plasmid in the bacteria. An insertion of the 36-kb Tn21-like transposon in this plasmid resulted in generation of the type II mutant which showed enhanced resistance to streptomycin. The relationship between the R plasmid and the three Tn21-like transposons (15-kb, 26-kb and 36-kb) awaits further study. In the second part of this study, another class of plasmid mutants of S. flexneri was studied. Plasmid mutants from two Taiwan isolates (SH2308 and SH2576) were selected by loss of the capabilities of congo red binding. One SH2308 plasmid mutant and 3 SH2576 plasmid mutants were selected for further PFGE and Southern analyses. The SH2308 mutant lost a large plasmid completely, whereas two SH2576 mutants had a large plasmid, similar in size to that in SH2308, integrated into the chromosome in different locations. The other SH2576 mutant had a further deletion after integration of the same large plasmid in the chromosome. SH2308 and SH2576 both showed multiplication after infection into human macrophage cells and had cytotoxicity toward the cells. But the four mutants lose the multiplication ability and cytotoxicity. The large plasmid in SH2308, and likely the large plasmid in SH2576, could be defined as the virulence plasmid.