Pseudomonas aeruginosa 菌株Ciprofloxacin抗藥性與抗藥基因gyrA、gyrB、parC以及parE的變異之分子機轉研究

Molecular mechanism of Ciprofloxacin resistance in Pseudomonas aeruginosa isolates associated with target gene of gyrA, gyrB, parC and parE gene

王筠婷(Yung-Ting Wang)

指導教授 ： 彭健芳

高雄醫學大學/健康科學院/醫學檢驗生物技術學研究所/碩士(2012年)

摘要

Pseudomonas aeruginosa 為院內感染常見致病菌，對多種抗生素具有抗藥性。目前常用於治療P. aeruginosa感染的抗生素之一為fluoroquinolone類中ciprofloxacin (CIP)。P. aeruginosa對於CIP產生抗藥性主要的機制為基因突變 (gyrA、gyrB、parC以及parE基因) 或efflux pump 過度的表現。本篇的研究目的，分析目標基因突變與P. aeruginosa產生CIP抗藥性的分子機轉。主要針對DNA gyrase (gyrA與gyrB基因)以及topoisomerase IV (parC和parE基因)為研究對象。從2009年1月到2012年1月期間於高雄醫學大學附設醫院收集145株的P. aeruginosa，其中對於ciprofloxacin的最小抑菌濃度(MIC) ≥ 4 μg/ml共有87株(抗藥性)，MICs = 2 μg/ml共有28株(中等度抗藥性)，另外的30株為MICs ≤ 1μg/ml(感受性)，檢體來源以不重複病人為原則。測定MICs是使用VITEK 2系統(bioMerieux, France)。根據2012年CLSI所制定的P. aeruginosa 對於ciprofloxacin 的 MIC值判定標準，若MICs ≤ 1 μg/ml為敏感性(susceptible)、MICs = 2 μg/ml為intermediate susceptibility以及MICs ≥ 4 μg/ml為抗藥性(resistant)。本研究針對CIP抗藥性相關的2個酵素DNA gyrase (次單位為 gyrA 和gyrB基因) 和topoisomerase IV (次單位為parC和parE基因)的4基因的fluoroquinolone resistance-determining region (QRDR)，使用PCR技術來擴增，將所得的PCR產物定序，其所得序列與wild-type P. aeruginosa PAO1的序列作BLAST比對，判斷核甘酸序列是否有突變。結果之分析得知，在我們所偵測4個基因中並未觀察到有任何突變的現象存在於CIP-susceptible的菌株。CIP-resistant P. aeruginosa菌株中，發現DNA gyrase的gyrA基因產生突變的比率為83.9％。得知P. aeruginosa菌株在gyrA基因產生突變，會產生較高的抗藥性，主要的突變位置在Thr83Ile (98.6％)。其次為topoisomerase IV的parC基因突變的比率55.3％，主要的突變位置於Ser87Leu (83.3％)。其中有一株P. aeruginosa在parC基因產生雙突變於Ser87Leu和Gly85Leu。同時，觀察到gyrA與parC基因會一起產生突變的現象(52.9％)，而使得P. aeruginosa對於CIP產生較高抗藥性(MICs ≥ 4 μg/ml）。在gyrB與parE基因所觀察到的突變現象很少，在gyrB基因突變的比率為4.6％，均突變於Ser466Phe (100％)。parE基因突變的比率為10.3％，而不同的是突變位置沒有規律，但在本研究中觀察到新的突變位置共有Lys388Glu (10％)、Leu501Phe (10％)和Val460Phe (10％)。其中一株P. aeruginosa在parE基因產生雙突變Val460Phe與Ala473Val，並且使得P. aeruginosa菌株對CIP產生較高的抗藥性。至於有關CIP-intermediate susceptibility P. aeruginosa菌株， 在4個基因的突變比率(3.6％−46.4％) 都比CIP-resistant P. aeruginosa菌株較低(4.6％−83.9％)。CIP-intermediate susceptibility P. aeruginosa菌株，在gyrA基因突變的比率46.4％，突變位置為Thr83Ile (38.5％) 、Asp87Asn (23.1％) 、Asp87Tyr (15.4％) 、Asp87His (15.4％) 以及Asp87Gly (7.7％) ，主要突變位置於胺基酸87位置。CIP-intermediate susceptibility P. aeruginosa菌株突變於parC基因的比率為14.3％，突變位置Ser87Leu (50％) 、Gly85Cys (25％) 以及Glu91Lys (25％) 。在gyrB與parE基因有一株P. aeruginosa產生突變，突變的位置分別為Ser466Phe與Ala473Thr。由以上實驗結果得知，DNA gyrase (gyrA與gyrB基因)和topoisomerase IV (parC與parE 基因)產生突變會使得P. aeruginosa菌株對於ciprofloxacin產生抗藥性。而在gyrA基因突變Thr83Ile會使得P. aeruginosa菌株對CIP產生較高的抗藥性(MICs ≥ 4 μg/ml)，在gyrA基因突變於胺基酸87位置，是使P. aeruginosa對CIP產生較低的抗藥性(MICs = 2 μg/ml)。parC與parE基因產生突變會伴隨gyrA基因突變，所以使得P. aeruginosa菌株對於CIP產生較高的抗藥性。而本研究發現在parE基因觀察到新的突變位置於Lys388Glu、Leu501Phe和Val460Ala 也會導致P. aeruginosa菌株對CIP的抗藥性，此結果未見於其他的研究報告中。

關鍵字

綠膿桿菌 ； Ciprofloxacin抗藥性 ； 目標抗藥基因

並列摘要

Pseudomonas aeruginosa is an important pathogen in nosocomial infections and often shows multiple antibiotics resistance. Therapy for P. aeruginosa infections is currently limited to the use of a few antibiotics, including fluoroquinolones. In this study, ciprofloxacin (CIP) is one of fluoroquinolones class antibiotics used for elucidating molecular mechanism of resistance in P. aeruginosa isolates. Resistant to ciprofloxacin is associated with target gene mutations (such as gyrA, gyrB, parC and parE genes), and/or overexpression efflux pump systems. The aim of this study is to analyze the target gene mutations in P. aeruginosa isolates resistant to CIP. Of the target gene mutations, DNA gyrase (included gyrA and gyrB genes) and topoisomerase IV (included parC and pare genes) were investigated. Total of 145 strains of P. aeruginosa used in this study were collected from Kaohsiung Medical University hospital patients during January 2009 and January 2012. In our collected P. aeruginosa isolates, 87 strains show resistant to ciprofloxacin with the minimum inhibitory concentrations (MIC) ≥ 4 μg/ml, 28 strains show MICs = 2 μg/ml, and 30 strains show MICs ≤ 1 μg/ml. These P. aeruginosa strains isolated from different clinical specimens obtained from hospitalized patients. Antimicrobial susceptibility testing was determined by using the VITEK 2 system (bioMerieux, France). According to the CLSI in 2012, P. aeruginosa against ciprofloxacin MIC values was determined with the standard as follows: MICs ≤ 1 μg/ml is susceptible, and MICs = 2 μg/ml is intermediate susceptibility, and the MICs ≥ 4 μg/ml is resistant. This study is designed for mutational analysis of specific two enzymes of DNA gyrase (included gyrA and gyrB) and topoisomerase IV (included parC and parE) among CIP resistant P. aeruginosa isolates. Point mutations associated with the fluoroquinolone resistance-determining region (QRDR) of gyrA, gyrB, parC, parE genes were determined in this study for characterization of the CIP resistance in P. aeruginosa isolates. PCR technique was used for the analysis of point mutation. PCR products were subjected to sequence and analyze by the BLAST method compared with the reference strain of wild-type P. aeruginosa PAO1. From our data, we didn’t detect any nucleotide mutations in CIP-susceptible isolates among four genes tested. CIP-resistant P. aeruginosa isolates showing 83.9% mutation rate in DNA gyrase gyrA was observed. It was noted that gyrA gene mutations were associated with the high-level resistance to CIP. The major frequency of mutations site is occurring at Thr83Ile (98.6％), followed by topoisomerase IV parC gene mutation (55.3%) with the major mutation site at Ser87Leu (83.3%). In this study, one strain of P. aeruginosa showing double mutations at Ser87Leu and Gly85Leu in parC gene was detected. Moreover, gyrA gene mutational strains are also found to be associated with mutation in parC gene (52.9％). The double mutations occurring in gyrA and parC genes were associated with high-level resistant to CIP (MICs ≥ 4 μg/ml). It is interestingly found that gyrB and parE genes are commonly associated with the low frequency mutation. In the gyrB gene, only 4.6% mutation rate was detected, but the mutation sites are all detected at Ser466Phe (100％). In contrast, parE gene showed a variety of mutation sites at different codons. In this study, we found three novel mutation sites of parE gene at Lys388Glu, Leu501Phe, and Val460Ala. One strain of P. aeruginosa had a double mutations at Val460Ala and Ala473Val in parE gene mediated the high-level resistance to CIP. CIP-intermediate susceptibility P. aeruginosa isolates had mutation rate (3.6％−46.4％) lower than CIP-resistant isolates (4.6％−83.9％) among four genes tested. In gyrA gene, CIP-intermediate susceptibility P. aeruginosa isolates mutation rate is 46.4%. It was noteworthy that mutation sites at Thr83Ile (38.5%), Asp87Asn (23.1％), Asp87Tyr (15.4%) , Asp87His (15.4%) and Asp87Gly (7.7％) are occurred in codon 87 position of gyrA gene. Among CIP-intermediate susceptibility P. aeruginosa isolates, mutations rate is 14.3% in parC gene, and mutation site are detected at Ser87Leu (50%), Gly85Cys (25％) and Glu91Lys (25%). In particular, there are two CIP-intermediate susceptibility P. aeruginosa isolates, one strain showed mutation only in gyrB at Ser466Phe, the other one has mutation in parE gene at Ala473Thr site. In conclusion, mutations in DNA gyrase (gyrA and gyrB genes) and Topoisomerase IV (parC and parE gene) would mediated P. aeruginosa isolates resistant to ciprofloxacin. In gyrA Gene, mutation at Thr83Ile confers high-level resistant in P. aeruginosa (MICs ≥ 4 μg/ml). However, the gyrA gene mutation at codon 87 position contributed the low-level resistant to CIP (MICs = 2 μg/ml). ParC and parE gene mutations were associated simultaneously with gyrA gene mutation mediated P. aeruginosa showing high- level resistant to CIP. Also, three novel mutation sites at Lys388Glu, Leu501Phe and Val460Phe in parE gene were the first report in this study.

並列關鍵字

Pseudomonas aeruginosa ； Ciprofloxacin resistance ； target gene

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