Pseudomonas aeruginosa菌株的ciprofloxacin 抗藥性與菌體藥物輸出幫浦系統調控基因nfxB, mexR, mexZ, mexT與mexOZ的變異之分子機轉研究

Molecular mechanism of ciprofloxacin resistance in Pseudomonas aeruginosa isolates associated with mutation in efflux pump systems regulatory gene of nfxB, mexR, mexZ, mexT and mexOZ gene

秦傑(Jie Chin)

指導教授 ： 林貴香

共同指導教授 ： 彭健芳(Chien-Fang Peng)

高雄醫學大學/醫學院/醫學研究所/碩士(2012年)

摘要

Pseudomonas aeruginosa一直是醫療院所當中造成院內感染的病菌之一，由於細菌本身內源性或是經由外界獲得抗藥性基因，抗藥性菌種比例持續不斷的增加使得醫師們遭受了臨床上治療的挑戰與困難。在治療受到P. aeruginosa感染的病患，臨床上能選擇及使用的藥物並不多，包括了fluoroquinolone 類的藥物，例如ciprofloxacin、 ofloxacin、levofloxacin等)；而細菌的抗藥機轉種類相當繁雜，其中一個為過度表現efflux pump systems，使得進到菌體內的抗生素不會累積而迅速排出菌體外而無法達到殺菌作用的效果。
本次研究是從2009年1月至2012年1月於高雄醫學大學附設中和紀念醫院收集共145株P. aeruginosa，其中對ciprofloxacin 有抗藥性的共有87株(60％)，介於中間值抗藥性的有28株(19.3％)，對ciprofloxacin 有感受性的有30株(20.7％)，針對其五種不同的efflux pump調控基因：nfxB、mexR、mexZ、mexOZ、mexT，利用PCR以及DNA sequencing的方法，於NCBI (National Center for Biotechnology Information) 網站進行相似性比對程式 BLAST ( Basic Local Alignment Search Tool )，將得到的DNA核酸定序結果與wild-type P. aeruginosa PAO1菌株序列相比對，判定序列突變的情形。結果發現nfxB基因突變的有13株(8.9％)，主要位置為胺基酸Arg21His及胺基酸Asp56Gly；mexR基因突變的有96株(66.2％)，主要位置為胺基酸Val126Glu；mexZ基因突變的有139株(95.9％)，主要位置為胺基酸Ser153Asn及胺基酸Arg105Leu；mexOZ基因突變的有144株(99.3％)，主要位置為核苷酸388 C→T 及核苷酸311 刪除A；mexT基因突變的有141株(97.2％)，主要位置為胺基酸Phe172Ile。
在87株抗藥性P. aeruginosa中，在mexZ及mexT這兩個基因都有突變的比例為100％，這結果分別導致胺基酸Ser153Asn及 Phe172Ile的改變，倘若再加上同時伴隨著mexR基因與mexOZ基因也有突變，共有61株(70.1％)的細菌是4個調控基因都有突變的情況。比較值得注意的是若就基因個別觀察時，發現到nfxB基因的突變會造成低程度(low-level)抗藥性的情形。而mexR基因突變的菌株若同時伴隨有胺基酸Cys30Arg、胺基酸Asn79Ser、胺基酸Arg114Cys，或是帶有胺基酸Gly51Val、胺基酸Arg70Gln、胺基酸Glu101Arg、胺基酸Val132Ala等這些位置的突變時，推測菌株對ciprofloxacin會產生較高的抗藥性。mexZ基因突變的菌株若是合併有多重基因點突變的話，例如突變點同時有胺基酸Asn43Arg、胺基酸Cys80Tyr、胺基酸Phe103Leu、胺基酸Gln107Leu、胺基酸Glu113Ala、胺基酸Ile128Val、胺基酸Ile137Val、胺基酸Asp149Asn、胺基酸Met158Leu、胺基酸Leu163Ile、胺基酸Ser168Gly、胺基酸Tyr172His、核苷酸661刪除A、核苷酸662-663插入C、核苷酸686-687插入G、核苷酸690刪除G、核苷酸867-868插入T、核苷酸869刪除A、核苷酸894-895插入A、核苷酸896刪除C、核苷酸950刪除G、核苷酸952-953 插入T等也是可能會使得菌株產生較高的抗藥性；另外在mexOZ基因則是觀察到若是在核苷酸449 A→G、核苷酸518 G→A、核苷酸444T→C這3個位置突變，以及mexT基因在胺基酸Trp 332Arg突變時，推測都有可能會造成P. aeruginosa對ciprofloxacin產生抗藥性的情況，以上都是比較特別的突變位置。
另外收集30株對ciprofloxacin有感受性的P. aeruginosa，結果發現在mexOZ基因位置對照下是完全都有突變的情況，比例是100％，其次則是mexT基因與mexZ基因，突變比例分別為93.3％與90％，再來則是mexR基因與nfxB基因，突變比例分別為46.7％與13.3％。結果顯示這30株菌株基因序列並非完全都與wild-type PAO1相同，推測會使細菌在MIC的結果表現上是呈現一種或數種低程度的抗藥性，但並未超出感受性所界定的濃度之外，特別是對於ciprofloxacin而言。因此若是P. aeruginosa的efflux pump調控基因的突變使得efflux pump systems的過度表現，對於欲被使用ciprofloxacin的病人來說，或許採取其它的治療方式如合併治療會較有幫助。

關鍵字

綠膿桿菌 ； 菌體藥物輸出幫浦系統調控基因

並列摘要

Pseudomonas aeruginosa is one of important nosocomial pathogens in hospital environments. Because of P. aeruginosa resistance due to their intrinsic and acquired resistant to a wide range of antimicrobial agents, that makes doctors suffer the challenges and difficulties for the treatment on clinical. There are many mechanisms to mediate P. aeruginosa isolates to become resistant strains. Efflux pump systems is one of them to be excessively display, which makes antibiotics cannot accumulate in bacterial cytoplasm, and unable to act their antibacterial activity on bacterial cells. Today, antibiotics used in the P. aeruginosa infectious disease are actually rarely. However the fluoroquinolones antibiotic, such as ciprofloxacin, is good choice for P. aeruginosa infections. In order to investigate molecular mechanism of ciprofloxacin resistance in P. aeruginosa isolates associated with efflux pump system, a total of 145 trains of P. aeruginosa isolates was obtained from patients clinical specimens during January 2009 to January 2012 admitted at Kaohsiung Medical University Hospital. Among these 145 trains of P. aeruginosa isolates, 87 (60%) isolates are resistance to the ciprofloxacin, and 28 (19.3%) isolates show intermediate resistance, and 30 (20.7%) isolates show susceptible to ciprofloxacin. By means of PCR technique and DNA sequencing of five different regulatory genes of nfxB、mexR、mexZ、mexOZ and mexT from each efflux pump system. Mutations of nfxB、mexR、mexZ、mexOZ and mexT were analyzed with the method of BLAST (Basic Local Alignment Search Tool), which is carried out from the NCBI (National Center for Biotechnology Information) for comparing the sequence of those genes with wild-type PAO1 to determine mutation of the DNA sequence tested. There are 8.9% of nfxB gene tested has been mutated, major mutation sites are amino acid Arg21His and Asp56Gly. And 66.2% of mexR gene have mutation occurred in main positions at amino acid Val126Glu. It were found that 95.9%, 99.3%, and 97.2% of mexZ, mexOZ, and mexOZ showed mutation with mostly appearing at amino acid Ser153Asn and Arg105Leu, nucleotide 388 C→T and 311 delete A, as well as amino acid Phe172Ile, individually. It was interesting to find that all 87 CIP-resistant of P. aeruginosa isolates have 100% mutations in mexZ and mexT leading amino acid change in Ser153Asn and Phe172Ile. While mexR and mexOZ genes have mutations simultaneously, 70.1% of CIP-resistant of P. aeruginosa isolates occurred mutation in those 5 regulatory genes tested. Our data showed that the mutation of nfxB gene will cause the low-level CIP-resistance presenting in P. aeruginosa isolates. In contrast, mutation of the mexR gene at sites of Cys30Arg, Asn79Ser, Arg114Cys, Gly51Val, Arg70Ser, Arg101Arg, and Val132Ala, which mediated high-level of ciprofloxacin resistance in P. aeruginosa isolates. In addition, it is also to find that high-level resistance of CIP-resistant P. aeruginosa isolates are caused by multiple sites mutation in mexZ gene as follows: Asn 43Arg, Cys80Tyr, Phe 103Leu, Gln107 Leu, Glu113Ala, Ile128Val, Ile137Val, Asp149Asn, Met158Leu, Leu163Ile, Ser168Gly, Tyr172His, nucleotide 661 delete A, nucleotide 662-663 insert G, nucleotide 690 delete G, nucleotide 867-868 insert T, nucleotide 869 delete A, nucleotide 894-895 insert A, nucleotide896 delete C, nucleotide 950 delete G, and nucleotide 952-953 insert T. However, mexOZ gene has 3 specific mutation sites in nucleotide 449 A→G, nucleotide 518G→A, nucleotide 444T→C. But, mexT gene has mutation at Try 332Arg that encoded P. aeruginosa resistance to ciprofloxaxin. In particularly, it was found that 30 CIP-susceptible P. aeruginosa did not show the complete nucleotide sequence of these five genes identical to that of wild-type PAO1 strain.
Therefore, there is 100% mutation occurring in mexOZ gene, and then the mexT gene (93.3%) and msxZ gene (90%), It was followed by mexR gene and nfxB gene, the mutation rate is 46.7% and 13.3% respectively. From our results, P. aeruginosa isolates have mutation more than 3 efflux pump system regulatory genes will leads efflux pump systems overexpression and encoded ciprofloxacin resistance. Our results showed that ciprofloxacin used for treatment against P. aeruginosa infectious disease should be considered carefully. Otherwise, it might be more suitable for combination of ciprofloxacin with other agents.

並列關鍵字

Pseudomonas aeruginosa ； efflux pump systems regulatory gene

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