兩種指標性細菌對氟奎諾酮類與氯黴素類抗菌劑抗藥性機制之研究

Abstract

近幾十年來，國內外獸醫學界除均早已開始由疾病中或康復後的動物分離病原性細菌、指標性細菌(大腸桿菌與腸球菌)與/或人畜共通傳染性細菌(沙門氏菌與彎曲桿菌)外，並同時對臨床上常用抗菌劑的感受性進行調查與監控。但在上述調查及監控中，多忽視了健康動物腸道中上述細菌的抗藥性。因此，本論文主要針對健康產食性經濟動物腸道的指標性細菌對於獸醫與人醫臨床上經常被使用抗菌劑(包括：氟喹諾酮類與氯黴素類)的抗藥性現況與產生抗藥性的可能機制進行研究。研究樣本均是採自台灣地區健康豬隻與雞隻，進行大腸桿菌分離與其對氟喹諾酮類抗菌劑(Fluoroquinolones)抗藥性機制的研究。結果顯示：收集的大腸桿菌分離株並不具有qnrA、qnrB與qepA等基因，但3.33 %豬源菌株(12/360)與2 %雞源菌株(6/300)帶有qnrS基因。以南方轉漬法(Southern blot)分析帶有qnrS基因的質體，其大小介於50~165 kb；其中11株大腸桿菌同時於染色體的DNA gyrase(gyrA)或/與拓樸異構酶IV (topoisomerase IV) (parC)發生突變，因此，能對西普氟奎林羧酸(Ciprofloxacin；CIP)具有高程度的抗藥性。帶有qnrS基因的大腸桿菌菌株(11.1%；2/18)，如同時具有aminoglycoside acetyltransferase- aac(6’)-ib-cr時，除了對胺基醣苷類抗菌劑產生抗藥性外，亦可對CIP等氟喹諾酮類抗菌劑產生不同程度的抗藥性。此外，15株帶有qnrS基因的大腸桿菌(83.3%；15/18)，因同時具有超廣效乙內醯胺酶(Extended-spectrum β-lactamases, ESBL) CTX-M-1，故能對畜福(ceftiofur)與/或cefotaxime等頭孢子素類抗菌劑產生抗藥性。總結本試驗結果，推測當健康動物預防性投予喹諾酮類(Quinolones)與頭孢子素類(Cephalosporines)抗菌劑時，均可能產生篩選性壓力，造成大腸桿菌同時帶有blaCTX-M與qnr等基因，而此抗藥因子的存在亦明顯影響其抗藥性的表現。在革蘭氏陽性菌指標菌–糞腸球菌(Enterococcus faecalis；EF)的部分，樣本則收集自即將屠宰的健康豬隻與雞隻，上述分離菌株主用來研究EF對喹諾酮類抗菌劑之抗藥性表現型與基因型間的關係。結果顯示：豬源與雞源EF對於CIP抗藥性比例分別為54%(162/300)與53.5%(107/200)，這269株對CIP具抗藥性的糞腸球菌對於CIP、恩氟奎林羧酸(Enrofloxacin)與moxifloxacin的最小抑制濃度範圍分別為4–512 mg/L、8–512 mg/L與0.5–512 mg/L。分離株若在GyrA (Ser83–Arg/Ile；Glu87–Lys/Gly)與ParC(Ser80–Ile)產生突變，則會對CIP造成高程度的抗藥性，但若僅在ParC則不見明顯的影響，此外，本文為首次報告在EF的拓樸異構酶胺基酸發現三點突變。對36株具有抗藥性但於GyrA與ParC卻無胺基酸突變的菌株而言，由蛇根鹼(reserpine)抑制藥物輸出幫浦(efflux pump)的試驗結果證實：這些分離株所帶有的藥物輸出幫浦基因(emeA)表現量，除均較標準糞腸球菌(E. faecalis ATCC 29212)為高外，蛇根鹼可顯著下降EF對於CIP的最小抑制濃度。歸納上述結果得知，健康畜禽腸道的EF對於CIP產生抗藥性的機制包括：隨胺基酸突變數增加，而抗藥性愈強的拓樸異構酶突變外，細菌所帶有的藥物輸出幫浦亦可明顯造成抗藥性的上升。 另一方面，本研究於氯黴素(Chloramphenicol；CHL)停用後(2003年至2007年)為了瞭解豬隻大腸桿菌對氯黴素類抗菌劑抗藥性，故針對 50個不同豬場，共收集600個健康豬隻糞便樣本，調查大腸桿菌對於動物仍在使用的氟甲磺氯黴素(Florfenicol；FLO)與已禁用的CHL之抗藥性及其相關基因表現。研究結果顯示：豬源大腸桿菌對於FLO抗藥性比例由2003年的39.2%上升至2007年的78.3%。本次試驗分別由保育(61.5%)、肥育(62.5%)與母豬(51.5%)分離出351株具抗藥性的菌株，抗藥株帶有藥物輸出幫浦基因floR、cmlA與氯黴素乙醯轉移酶基因cat-1, cat-2與cat-3比例分別為82.9、61.3、10.8、3.7與0%，而同時攜有floR及cmlA基因菌株的比例為52.4%。抗藥性菌株在藥物輸出幫浦抑制劑Phe-Arg-β-naphthylamide存在時，FLO對於大腸桿菌最小抑制濃度下降4至64倍。上述結果指出：台灣豬隻大腸桿菌分離株，大部分均藉由藥物輸出幫浦對於FLO與CHL產生抗藥性。 綜合上述實驗結果得知，健康畜禽腸道菌叢亦可藉由各種機制產生與/或維持抗藥性，故當大量使用相關抗菌劑後仍會持續造成健康的宿主之分離菌表現不同程度的抗藥性與/或多重抗藥性，進而對公共衛生學、預防醫學與臨床治療造成直/間接的影響。至於停用部分抗生素或使用方法後，是否會造成不同細菌族群對抗菌劑感受性的改變，則仍需更一步適當的研究才可得知。

In recent years, international and domestic surveys to assess antimicrobial susceptibility among indicator bacteria (Escherichia coli and Enterococcus spp.) and zoonotic bacteria (Salmonella spp. and Campylobacter spp.) isolated from sick food-producing animals had been addressed. However, the background information about the bacterial resistance in healthy animals is totally neglected. The major focus of this dissertation is to investigate the levels and mechanisms of resistance of fluoroquinolones (FQ) and chloramphenicols in two indicator bacteria (Escherichia coli and Enterococcus faecalis) from two major food-producing animals, pig and chicken, in Taiwan. Our results indicated that the qnrS genes, but not qnrA, qnrB, and qepA were detected in 12/360 pig E. coli isolates (3.33%) and in 6/300 chicken E. coli isolates (2%). Southern blot hybridization analysis indicated that qnrS was located on plasmids ranging in size from 50-165 kb. Eleven of the 18 qnrS positive isolates which showed high ciprofloxacin resistance phenotype (MIC ≥ 8 mg/L) also had amino acid sequence variations in chromosomal quinolone resistance-determining regions of gyrA and parC. Besides, only two qnrS-positive isolates carried the aac(6’)-Ib-cr variant that mediating FQ acetylation. For the high percentage resistance of cephalosporins, the blaCTX-M gene was also examined from qnrS-positive isolates. The blaCTX-M gene was detected in fifteen isolates (15/18, 83.3%) in which 12 isolates were blaCTX-M-1 and 3 isolates were blaCTX-M-15. These results concluded that a close linkage between qnrS gene and blaCTX-M-1. The CTX-M and Qnr-based mechanisms might be co-emerging in E. coli strains isolated from healthy chickens and pigs under a selective pressure of quinolones and cephalosporines administration. The percentage of E. faecalis isolates resistant to ciprofloxacin was 54.0% (162/300) in pigs and 53.5% (107/200) in chickens. Two hundred sixty-nine ciprofloxacin-resistant isolates showed different levels of resistance to ciprofloxacin (MIC 4-512 mg/L), enrofloxacin (MIC 8-512 mg/L) and moxifloxacin (MIC 0.5-512 mg/L). Two mutations associated with resistance were detected in GyrA at Ser83 (to Arg/Ile) and Glu87 (to Lys/Gly) and one mutation was found in ParC at position 80 (Ser to Ile). In addition, triple-point mutation in DNA gyrase (GyrA) and topoisomerase IV (ParC) of E. faecalis was firstly reported in this paper. In the presence of reserpine, the level of resistance to ciprofloxacin for these 36 strains was decreased. The effect of reserpine on ciprofloxacin resistance was correlated with the level of expression of the emeA gene. In E. faecalis, our results demonstrate that not only point mutations in topoisomerase IV and DNA gyrase but also the efflux pump is the main mechanism for quinolones resistance. A similar approach but different sets of procine samples were used to elucidate the possible genetic determinants of resistance to florfenicol and chloramphenicol in porcine Escherichia coli. Six hundread fecal samples of healthy pigs from 50 different farms were collected from 2003 to 2007. The florfenicol resistance in the isolated E. coli strains doubled from 39.2% in 2003 to 78.3% in 2007. A total of 351 florfenicol-resistant E. coli isolates were isolated from nursery pigs (61.5%), grower-finisher pigs (62.5%), and sows (51.5%). The prevalence of resistance genes, floR, cmlA, cat-1, cat-2 and cat-3, was 82.9, 61.3, 10.8, 3.7, and 0%, respectively. Of the 351 florfenicol-resistant isolates, 184 (52.4%) were positive for both floR and cmlA. Furthermore, the results of efflux inhibitor studies with Phe-Arg-β-naphthylamide showed a 4- to 64-fold decrease in the florfenicol MIC levels. The FloR efflux pump may play a role in phenicol resistance among porcine E. coli isolates in Taiwan. In conclusions, our data clearly indicated that bacteria can continuously use various mechanisms to produce and/or maintain their antimicrobial resistance in healthy food-producing animals. The added risk to animal’s helath from resistance among E. coli and Enterococcus faecalis selected by florfenicol and enrofloxacin use is large. This reservoir phenomenon may directly enhance the expression and/or the levels of resistance/co-resistance as well as indirectly affect the public health, preventive medicine and clinical therapy. Furthermore, other microorganisms that might be expected to have become more susceptible as a consequence of withdrawing of chloramphenicol and water medication of enrofloxacin. Thus, the further adequate studies need to be done.