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

• Main Authors: Jie Chin, 秦傑
• Other Authors: Kuei-hsiang Lin
• Format: Others
• Language: zh-TW
• Published: 2012
• Online Access: http://ndltd.ncl.edu.tw/handle/13558812171306354047

碩士 === 高雄醫學大學 === 醫學研究所 === 100 === 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.