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

Autor: Yung-Ting Wang, 王筠婷
Rok vydání: 2012
Druh dokumentu: 學位論文 ; thesis
Popis: 100
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.
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