利用高解析熱熔解分析法偵測Campylobacter jejuni gyrA之基因突變並探討與奎諾酮類藥物感受性變化之關係

The Evaluation Between the gyrA Mutation Detected by HRM and Quinolone Susceptibility in Campylobacter jejuni

范揚棋(Yang-Chi Fan) ； 王嘉蘭(Chia-Lan Wang) ； 周崇熙(Chung-Hsi Chou) ； 蔡向榮(Hsiang-Jung Tsai)

《臺灣獸醫學雜誌》 40卷1期 (2014/03) Pp. 61-69

摘要

本研究中針對2009－2012年間採集自臺灣地區水禽養殖場之95株Campylobacter jejuni分離株進行奎諾酮類藥物抗藥性之研究。結果顯示本群菌株對nalidixic acid與ciprofloxacin產生抗藥性之比率分別為61.2%（58/95）及57.9%（55/95）。進一步針對C. jeijuni之gyrA奎諾酮類藥物抗藥性決定區（quinolone resistant determining regions，QRDRs）進行突變序列探測，其中33.7%分離株之gryA基因沒有任何突變點發生；22.1%分離株之gyrA基因的第86個胺基酸位由酥胺酸（threonine）突變為丙胺酸（alanine），9.5%分離株之基因的第90個胺基酸由天門冬氨酸（aspatic acid）突變為天門冬醯胺（asparagine）；34.7%分離株gyrA之基因的第81及86個胺基酸由組胺酸（histidine）與酥胺酸突變為丙胺酸及天門冬醯胺。本研究同時利用高解析熱熔解（high-resolution melting）法分析gyrA基因之突變，由四種不同熔點曲線可有效區分出不同之gyrA基因序列與抗藥性結果，此技術具有快速偵測、分析C. jejuni分離株對奎諾酮類藥物抗藥性特性之能力。

關鍵字

奎諾酮類藥物抗藥性 ； gyrA基因變異 ； 高解析熱熔解分析法

並列摘要

A total of 95 isolates of Campylobacter jejuni isolated from domestic waterfowl from 2009 to 2012 in Taiwan were subjected to quinolone resistance analysis. The resistant rates to nalidixic acid and ciprofloxacin were 61.2% (58/95) and 57.9% (55/95), respectively. All isolates were further screened for mutations in the quinolone resistant determining regions (QRDRs) gyrA genes. In the analysis of gyrA mutation, 33.7% of isolates showed no amino acid change, however, 22.1% of isolates were found with one-point mutation at codon 86, 9.5% of isolates at codon 90 and 34.7% of isolates were found with two-point mutations at codon 81 and 86. Four patterns of melting temperature curve were revealed in the high-resolution melting (HRM) analysis. The consistent changes in melting temperatures of different amplicon sequences allow the mutated-gyrA strains to be distinguished from wild-type strains. This rapid detection of gyrA mutations by HRM assay is feasible for extensive quinolone resistance monitoring program.

並列關鍵字

campylobacter jejuni ； Quinolone resistance ； gyrA Mutiation ； High-resolution melting assay