Part I 台灣地區1993~1994及2002~2003年收集之大腸桿菌中，第一型與第二型integron之分析 Part II 台灣分離之大腸桿菌中，gyrA，parC及AcrAB efflux pump在fluoroquinolone抗藥性之角色分析

Part I Analysis of class 1 and class 2 integron in Escherichia coli isolated in 1993~1994 and 2002~2003 in Taiwan Part II The role of gyrA, parC, and AcrAB efflux pump in fluoroquinolone resistance in E. coli isolates from Taiwan

張淙銘(Tsung-Ming Chang)

指導教授 ： 張玲麗

高雄醫學大學/醫學院/醫學研究所/碩士(2004年)

摘要

Part I
抗菌藥物普遍用於治療細菌感染後，往往伴隨細菌抗藥性增加。而integron是近年來被認為繼抗藥性質體與轉位子外，另一個與細菌獲得抗藥性基因及與抗藥基因散佈有關的機制。目前發現，許多不同的抗藥基因是位在一種mobile element稱為基因片匣，而integron可主導基因片匣以特定部位重組作用的方式嵌入或移出integron，造成integron所攜帶之抗藥基因改變及抗藥基因的散佈，因此，integron及基因片匣在臨床菌株抗藥性上扮演著重要的角色。
本實驗針對1993~1994年及2002~2003年收集之大腸桿菌（Escherichia coli, E. coli），利用聚合酶連鎖反應、核苷酸定序、限制酶切割試驗及南方雜交等方法，分析第一型（class 1）與第二型（class 2）integron在E. coli的存在、傳遞情形及所攜帶之基因片匣種類，並評估比較integron及攜帶之基因片匣是否隨著菌種收集年代的不同及藥物使用情況而有所改變，以及不同型integron的存在對細菌多重抗藥性的影響。
結果顯示在2002~2003年收集之436株受測的E. coli菌株中，第一型及第二型 integron出現率分別為64﹪及6﹪，而所攜帶的基因片匣主要以trimethoprim類的抗藥基因：dfr；與aminoglycosides類抗藥基因：aadA、aadB和aacA為主。與10年前的結果相較之下，主要差異在於目前臨床E. coli菌株的第一型integron攜帶率由52﹪增加為64﹪，且基因片匣組合方式以dfr17-aadA5的組合最常見，由10年前的9﹪增加為29﹪，進一步以南方雜交法分析後，證實有一部份攜帶dfr17-aadA5的integron位於可轉移性質體上。而在第二型integron的攜帶率以及所攜帶的基因片匣上則無明顯變化。另外， integron陽性菌株比陰性菌株具有明顯多重抗藥性的情形。所以未來除了持續觀察integron在細菌抗藥性的影響之外，也必須一併探討細菌的多重抗藥機制，以作為未來控制抗藥性散佈的方向。

Part II
Fluoroquinolone（FQ）是一種具有廣效性抑制革蘭氏陽性及陰性菌生長的抗生素，且被臨床使用的頻率也相當的高，在抗菌藥物普遍用於治療感染性疾病的狀況下，也促使抗藥性菌株不斷的產生。由於目前在臨床FQ抗藥性菌株中所發現到的抗藥性機轉幾乎都是由DNA gyrase以及DNA topoisomerase IV之基因突變所造成，而且主要發生突變的基因為gyrA與parC的QRDR（quinolone resistant determining region），因此本研究以聚合酶連鎖反應-單股構形多形性與核酸自動定序來分析自臨床檢體分離出的74株對FQ有抗藥性之大腸桿菌中，其gyrA與parC的QRDR發生突變的情形，結果發現在74株大腸桿菌中皆有gyrA與parC QRDR的突變發生，在gyrA之QRDR發生突變的主要位置在Ser83與Asp87，且具有多個gyrA 之QRDR突變點（≧2個）者會顯著提高對ciprofloxacin、levofloxacin及moxifloxacin的抗藥性，而parC之QRDR發生突變的主要位置在Ser80與Glu84，具有多個parC之QRDR突變點（≧2個）者則無顯著提高對FQ的抗藥性。
另外有愈來愈多的研究顯示菌株過度表現multidrug efflux pump也是增加對FQ抗藥性與多重抗藥性的原因，因此吾人利用real-time PCR評估一個最常被提出與FQ抗藥性有關且存在於大腸桿菌之RND-type的acrAB multidrug efflux system的表現與否，結果有51﹪之菌株acrAB過度表現，造成顯著提高對ciprofloxacin、ofloxacin、levofloxacin與moxifloxacin的抗藥性，並且伴隨增加對cefalozin、piperacillin以及amoxicillin/clavulanic acid三種β-lactam類抗生素的抗藥性。
綜合本研究結果可知，臨床分離之大腸桿菌對FQ產生抗藥性的主要機轉為gyrA的QRDR發生突變，以及acrAB過度表現所造成。

關鍵字

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並列摘要

Part I
Increasing use of various drugs for the treatment of infectious diseases has resulted in an increase of drug-resistant clinical isolates. Integron has been considered a major factor in the development and dissemination of multiple drug resistance among bacteria in recent years. Many of the known resistance genes are contained in mobile elements called gene cassettes. Via site-specific recombination catalyzed by an integron-encoded integrase, cassettes can be integrated into or excised from their receptor elements. Because of the ability of integron to acquire gene cassettes, therefore, integron and antibiotic-resistant gene cassettes play an important role in the development and dessemination of multiple antibiotic-resistance in bacterial strains.
In this study, by using PCR, DNA sequencing, restriction enzyme digestion, Southern hybridization, the presence and transfer of class 1, 2 integrons and the numbers, kinds and combinations of gene cassettes within these integrons in E. coli isolated in 1993~1994 and 2002~2003 were analyzed. The change of integrons and gene cassettes between different time periods were compared. Furthermore, relationship between various classes of integrons and multiple drug resistance was also evaluated.
Among 436 clinical strain of E. coli isolated in 2002~2003, the prevalence rate of class 1, 2 integron were 64% and 6%, respectively. Predominant gene cassettes contain trimethoprim resistance gene, dfr and of three aminoglycosides resistance gene, aadA, aadB and aacA. Comparison the results in the present study with our previous study in 1993 showed that the presence of class I integron in clinical strains of E. coli was increased from 52% to 64% and predominant gene cassette array was dfr17-aadA5 which increased from 9% to 29%. Some of the dfr17-aadA5 contained class 1 integrons was located on transferable plasmid by Southern hybridization. However, the prevalence rate of class II integron was not significant change. Furthermore, the more multidrug resistance was observed for the strain in which integron was detectable. Further studies of the process of gene cassettes and its interaction with integron, drug resistance could provide new insight about resistant spread control.

Part II
Fluoroquinolones (FQ) have broad-spectrum activity against gram-positive and gram-negative bacteria, and have been commonly used in clinical setting. However, increasing use of these drugs for the treatment of infectious diseases has resulted in an increase of drug-resistant clinical isolates.
To date, FQ resistance was mainly due to presence of mutations in the quinolone resistant determining regions (QRDRs) of gyrA and parC. In this study, by using single strand conformational polymorphism (SSCP) and direct sequencing, 74 FQ-resistant E. coli isolates carrying at least one mutation in gyrA at codon 83 and/or 87 and in parC at codon 80 and/or 84 were observed. Strains carrying two or more gyrA mutations were increase resistance to ciprofloxacin, levofloxacin and moxifloxacin significantly.
Overexpression of efflux pumps has been demonstrated to play an important role in isolates displaying resistance to several antimicrobials including FQ. In this study, real-time PCR was performed to assess and quantify the expression of the AcrAB efflux pumps of E. coli. Results showed that 51% of the isolates overexpressed the acrAB gene. Extrusion of ciprofloxacin, ofloxacin, levofloxacin, moxifloxacin, cefalozin, piperacillin and amoxacillin/clavulanic acid was specific to the AcrAB pump.
In conclusion, gyrA mutations and AcrAB efflux pumps play important role in resistance of E. coli to FQ.

並列關鍵字

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參考文獻

Part I

1. Witte, W. (1998). Medical consequences of antibiotic use in agriculture. Science 279, 996-997.

2. Mazel, D. & Davies, J. (1999). Antibiotic resistance in microbes. Cell Mol Life Sci 56, 742-754.

3. Hsueh, P. R., Liu, C. Y. & Luh, K. T. (2002). Current status of antimicrobial resistance in Taiwan. Emerg Infect Dis 8, 132-137.

4. Hall, R. M. (1997). Mobile gene cassettes and integrons: moving antibiotic resistance genes in gram-negative bacteria. Ciba Found Symp 207, 192-202; discussion 202-195.