鴨、鵝雷氏桿菌抗菌劑感受性與對氯黴素類與奎諾酮類藥物抗藥性機制之研究

Antimicrobial Susceptibility of Riemerella anatipestifer Isolates from Ducks and Geese and the Resistant Mechanisms of Chloramphenicol and Quinolones

陳燕萍(Yen-Ping Chen)

指導教授 ： 蔡向榮

國立臺灣大學/獸醫專業學院/獸醫學研究所/博士(2012年)

https://doi.org/10.6342/NTU.2012.01038

摘要

雷氏桿菌可以導致水禽的嚴重疾病，而由於血清型別複雜，且各血清型之間只有有限的交叉反應，使得疫苗不能完全保護水禽抵抗田間雷氏桿菌的感染。因此，抗菌劑治療與抗菌劑感受性的調查對於雷氏桿菌的控制非常重要。而且，雷氏桿菌的抗藥性機制尚未被報導，因此有必要進行調查。本研究以瓊膠紙錠擴散法調查1999年至2009年在臺灣分離之222株水禽雷氏桿菌對22種抗菌劑之感受性。結果顯示，菌株對colistin、nalidixic acid與kanamycin等較具抗藥性，對nitrofuratoin （已於2004年被禁止使用於動物）、doxycycline與amoxicillin/ clavulanic acid（clavulanic acid 未被核准用於動物）等較具感受性。此外，多種抗菌劑的抗藥性和多重抗藥性菌株的比率在2009年皆有明顯增加。另外，由最小抑菌濃度測試結果得知，雖然台灣已於2003年禁止於食用動物使用氯黴素，但2005年至2009年間分離的菌株仍有86.4％呈現氯黴素中等感受性或抗藥性。本研究結果顯示此氯黴素抗藥性基因為catB基因，且78.4％的菌株具有此基因，並且此catB基因位置是位於一個經命名為pRA0511的新質體上。質體pRA0511亦可轉譯其他的抗藥性相關蛋白，包括 TetX2和multi-drug ABC transporter permease/ATPase。然而，氯黴素乙醯轉移酶（CAT）無法不活化氟甲磺氯黴素。因此，本研究亦調查66株於1999年至2009年間分離的雷氏桿菌菌株對氯黴素與氟甲磺氯黴素之感受性，以及是否存在floR基因。結果顯示，9株具氟甲磺氯黴素中等感受性與抗藥性的菌株皆具有floR基因。floR基因為位於質體或染色體上，而且是以輸出幫浦的型式導致雷氏桿菌同時對氯黴素與氟甲磺氯黴素產生抗藥性。我們進一步將2個帶有floR基因的質體，pRA0726與pRA0846進行全長序列分析。pRA0726由11,706個鹼基組成，具有10個可能之open reading frames，包括floR、catB與一個新的blaOXA-209等抗藥性基因。pRA0846與pRA0726序列上最大不同在於前者缺少blaOXA-209抗藥性基因。質體消除試驗結果證實pRA0726具有抗氯黴素類與乙內醯胺類藥物之功能。另一方面，本研究亦調查田間分離與實驗室誘發突變之雷氏桿菌對喹諾酮類藥物抗藥性相關之拓樸異構酶突變。大部分對喹諾酮類藥物感受性降低的田間分離菌株在GyrA上具Ser10→Pro和/或Ser83→Arg/Ile突變，且在GyrB上具Thr140→Ile、Ile503→Val和/或Gln155→Lys突變。在實驗室誘發之突變株中，所有的突變皆發生於GyrA與GyrB，而在ParC與ParE並未發現任何突變，最常出現的突變為GyrA之Ser83→Ile/Asn，此現象與在田間分離株的發現相似，其次為Asp87→Ala/Gly/Tyr與Ser84→ Pro。而且，於GyrA之第183個胺基酸與於GyrB之第452個與第471個胺基酸之突變為首次被發現。大部分由較高濃度奎諾酮類藥物誘發之突變株以及具有較多突變點之突變株可顯現出較高之奎諾酮最小抑制濃度，但是一些具有相同突變之突變株卻表現出不同之最小抑制濃度，推測可能是因為過度表現幫浦系統或是其他可能之機制亦在本研究中被誘發。總結而言，本研究完成臺灣雷氏桿菌菌株之抗菌劑感受性變化趨勢調查，並且首次調查雷氏桿菌對氯黴素類與奎諾酮類藥物之抗藥性機制，同時亦完成3個新的多重抗藥性質體的特性分析，特別是著重在氯黴素類藥物的抗藥性上面。

關鍵字

雷氏桿菌 ； 抗菌劑感受性 ； 氯黴素 ； 氟甲磺氯黴素 ； 奎諾酮類藥物 ； 抗藥性

並列摘要

Riemerella anatipestifer is an etiological agent that can cause serious disease especially in waterfowl. Due to the existence of complex serotypes and limited cross reaction between serotypes, the vaccine cannot protect completely from the attack by wild R. anatipestifer. Therefore, chemotherapy and assessment of antimicrobial susceptibility are very important in the treatment of R. anatipestifer infection. In addition, the antimicrobial-resistance mechanisms in R. anatipestifer have not yet been reported, indicating the need for investigation. This study evaluated the susceptibility of 222 Taiwanese R. anatipestifer isolated from domestic waterfowl between 1999 and 2009 to 22 antimicrobials by agar disk diffusion method. Results showed that isolates were most resistant to colistin, nalidixic acid and kanamycin and most susceptible to nitrofurantoin which has been banned for use in domestic animals since 2004, doxycycline and amoxicillin/clavulanic acid (clavulanic acid is not approved for animals). Furthermore, the resistance rates to several antimicrobials and multi-resistance were increased obviously in 2009. In addition, from the minimum inhibitory concentration (MIC) test, 86.4% of isolates collected between 2005 and 2009 showed intermediate or resistance to chloramphenicol which has been prohibited for use in food-producing animals in Taiwan since 2003. The resistance gene was identified as catB gene with the prevalence of 78.4%. The position of the catB gene was then determined within a novel plasmid, designated pRA0511. pRA0511 also encoded other putative drug-resistance-associated proteins which were a TetX2 and a multi-drug ABC transporter permease/ATPase. CATs, however, are unable to inactivate florfenicol. Thus, 66 R. anatipestifer isolates collected between 1999 and 2009 were investigated for their susceptibility to chloramphenicol and florfenicol and the presence of floR gene. Results showed nine florfenicol intermediate or resistant isolates were all floR positive. The floR was located either in plasmid or chromosomal DNA and was as an efflux pump conferring resistance to both chloramphenicol and florfenicol. Furthermore, two novel floR-carrying plasmids designated pRA0726 and pRA0846 were sequenced completely. pRA0726 was 11,704 bp in size with 10 putative ORFs including a floR, a catB and a novel blaOXA-209 resistance genes. To our knowledge, this is the first report of presence of the floR and blaOXA-209 resistance genes in R. anatipestifer. The most differences between sequences of pRA0846 and pRA0726 were the absence of a blaOXA-209 gene in pRA0846. Plasmid curing tests demonstrated that pRA0726 carried functional coding proteins for resistance to phenicol and ß-lactam antimicrobials. On the other hand, this study also investigated the mutations of topoisomerases associated with quinolone resistance in clinical or laboratory-mutated R. anatipestifer. Most clinical isolates with reduced susceptibility to quinolones possessed amino acid alterations at Ser10 to Pro and/or Ser83 to Arg or Ile in GyrA and at Thr140 to Ile, Ile503 to Val and/or Gln155 to Val in GyrB. In laboratory mutants, all of the mutations were found in GyrA and GyrB but none in ParC and ParE. The most frequent mutation in GyrA was alterations at Ser83 to Ile or Asn which was concurred with aforementioned clinical strains, followed by alterations at Asp87 to Ala, Gly or Tyr and alterations at Ser84 to Pro. In addition, the mutations at positions 183 in GyrA and 452 and 471 in GyrB were newly described. Most mutants induced by higher concentration of quinolones and with more mutation points showed higher MIC values, although some mutants with identical mutation type showed different MIC profiles, suggesting that overexpression of the efflux system or other potential mechanisms might have also been induced. Conclusively, this study has described the trends in antimicrobial susceptibility of R. anatipestifer in Taiwan and the mechanisms of resistance to phenicols and quinolones for the first time in R. anatipestifer. In addition, three novel multidrug-resistance plasmids were characterized, more specifically associated with phenicol resistance.

並列關鍵字

Riemerella antipestifer ； antimicrobial susceptibility ； chloramphenicol ； florfenicol ； quinolones ； antimicrobial resistance

參考文獻

Chen, Y.P., Lee, S.H., Chen, C.L., Shien, J.H., Chang, T.C., Tsai, H.J. Antimicrobial susceptibility of Riemerella anatipestifer isolates from ducks and geese and the mutations of Gyrase associated with quinolone resistance. Taiwan Vet. J. (in press).

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