台灣地區禽類鳥鼻氣管桿菌菌株之奎諾酮類抗藥性與gyrA基因之分析

Abstract

為了解台灣地區鳥鼻氣管桿菌(Ornithobacterium rhinotracheale)菌株對於奎諾酮類抗菌劑與gyrA基因之關係，所選用之試驗菌株來源為國立台灣大學獸醫學系預防醫學研究室所保存於2004 ~ 2006年間針對台灣地區屠宰場及禽鳥養殖場所採樣分離之O. rhinotracheale分離株，涵蓋物種包括雞、火雞、鴿、鴕鳥、鵪鶉、鳳頭蒼鷹等經濟動物及人工飼養寵物鳥共86株，以瓊脂稀釋法分別進行nalidixic acid、norfloxacin、ofloxacin、enrofloxacin與ciprofloxacin之奎諾酮類抗菌劑感受性試驗，結果O. rhinotracheale對上述抗菌劑的最小抑制濃度範圍分別為： 2~ > 128 μg/mL、< 0.125~ > 64μg/mL、< 0.125~ >32μg/mL、< 0.125~ > 8μg/mL及< 0.125~ > 16μg/mL。試驗同時亦針對分離株之DNA旋轉酶 (DNA gyrase- gyrA gene)進行序列分析，序列比對結果發現共有62株對奎諾酮類抗菌劑具有抗性，於GyrA第83或/與第87個胺基酸發生變異，其中85.5％ （53/62）及4.8％ （3/62）菌株於第83個胺基酸由絲胺酸（serine）分別突變為酪胺酸（tyrosin）及苯丙氨酸 (phenylalanine)； 9.8％（6/62）分別於第83及87個胺基酸由絲胺酸（serine）及天門冬氨酸（aspartic acid）突變為苯丙氨酸 (phenylalanine)及天門冬醯胺（aspargine）(5/6) 或丙氨酸 (alanine) (1/6)，且62株O. rhinotracheale之奎諾酮類抗菌劑感受性試驗結果同時對nalidixic acid （≧32 μg/mL； 96.8％）與norfloxacin （≧16 μg/mL； 90.3％）具有抗性，推測當O. rhinotracheale之GyrA第83或/與第87個胺基酸發生變異時與本菌對奎諾酮類抗菌劑產生之抗性有關（p＜0.01）。進一步利用高解析熱熔解分析方法成功的將四種不同型式之變異菌株以不同之熔解曲線區分開來，建立出快速鑑別O. rhinotracheale分離株之奎諾酮類抗藥性基因之診斷工具。另比較脈衝式電泳圖譜與抗菌劑感受性結果發現：於同時間地點與同動物來源所分離之菌株並非全然屬於同一基因型，推測可能因宿主之感染源複雜、禽場間之用藥方式不同、產生抗藥性的菌株因染色體DNA發生變異或菌株之基因體於Sal I限制酶切位上有些許差異而使PFGE圖譜有不同的基因型產生。

A total of 86 isolates of Ornithobacterium rhinotracheale were obtained from veterinary preventive medicine of National Taiwan University. Species include chickens, turkeys, pigeons, ostrichs, quails, and Accipiter trivirgatus respectively from 2004 to 2006. This study purpose is to analyze the quinolone resistant mutations of gyrA gene of O. rhinotracheale in Taiwan. Sixty-two (72.1 %, 62/86) quinolone resistant O. rhinotracheale strains were found by agar dilution method with the antibiotics nalidixic acid, norfloxacin, ofloxacin, enrofloxacin and ciprofloxacin ( the MIC range was 2 ~ > 128 μg/mL, < 0.125 ~ > 64 μg/mL, < 0.125~ > 32 μg/mL, <0.125 ~ > 8 μg/mL and < 0.125 ~ > 16 μg/mL, respectively) and sequencing of the polymerase chain reaction amplified products of the quinolone resistant determining regions (QRDRs) of gyrA showed that all isolates carrying single or double mutations in gyrA at codon 83 and 87. The mutation at 83 of GyrA from serine to tyrosine (S83T) and phenylalanine (S83P) was present in 85.5 %（53/62） and 4.8 %（3/62）, respectively. The double mutation at 83 and 87 of GyrA from serine and aspartic acid to phenylalanine and aspargine (5/6) or alanine (1/6) was present in 9.8 %（6/62）. The present study indicated that the presence of high-level resistance to quinolones in 62 O. rhinotracheale isolates (resistant to nalidixic acid and norfloxacin were ≧32 μg/mL; 96.8 % and ≧16 μg/mL; 90.3 %, respectively) was associated with mutations at codon 83 and 87 in GyrA (p <0.01). Sequence variants of four types were also revealed by high resolution melting (HRM) analytical scheme and established the rapid differentiation of gyrA mutations causing quinolone resistance in O. rhinotracheale. The compartment of pulsed-field gel electrophoresis (PFGE) results and susceptibility profile patterns showed that the strains isolated from the same location and species were not belonging to the same genotype. The resistance of the individual and the different antimicrobial disposal might cause the chromosomal variation on the Sal I restriction site of the quinolone resistant strains. And this resulted in the different genotypes in PFGE.