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The Study of Resistance to Fluoroquinolones in Escherichia Coli

Author: JingPengWei
Tutor: LuoYu；MaoLiNa
School: Zhengzhou University
Course: Pathogen Biology
Keywords: Escherichia coli Fluoroquinolone Cross-resistance Related resistant Resistance mechanisms
CLC: R96
Type: Master's thesis
Year: 2009
Downloads: 86
Quote: 0
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Abstract


Background: The quinolones are a large class of synthetic antibacterial drug, the first time since 1962, successfully developed the first quinolones - nalidixic acid since the development of these drugs very quickly. The fluoroquinolones Is a new generation of broad-spectrum antibiotics, has become mainstream quinolones. Fluoroquinolones with broad spectrum antimicrobial, antibacterial activity, less toxicity, and widely distributed in the body, ease of administration, has been widely used in medical and veterinary clinical. E. coli infection is important hospital pathogens, has a variety of complex mechanisms of resistance. With the widespread use of fluoroquinolones, especially clinical irrational drug use, caused by the drug-resistant strains of Escherichia coli of these drugs has increased rapidly, increasing levels of drug resistance. Fluoroquinolone resistance become a serious problem in the clinical treatment. Escherichia coli of fluoroquinolones resistance mechanisms become widespread concern at home and abroad. Known mechanism of resistance of Escherichia coli to fluoroquinolones in three ways: (1) gene mutations lead to drug targets to promote enzyme-DNA gyrase and topoisomerase IV mutations; (2) gene mutations lead to drug The decline in the concentration of bacteria in vivo accumulation; (3) of the plasmid-mediated resistance mechanisms. The first mechanism is the main mechanism of Escherichia coli to fluoroquinolones resistance. Understanding of the region, the units of E. coli fluoroquinolone resistance status quo-depth study of Escherichia coli resistant to the generation and dissemination of accurate detection of drug-resistant phenotype and genotype for control of drug resistance and development and hospital infections, the rational use of antimicrobial drugs is important. For this reason, this topic by analyzing clinical Escherichia coli resistance monitoring data, and by in vitro induced experimentally induced Escherichia coli highly resistant to fluoroquinolones detect gyrA and parC genes induced resistant strains of quinolone resistance determinants District (quinolone resistance determining region, QRDR) mutations, in-depth study of Escherichia coli resistant to fluoroquinolones. Objective: To analyze clinical Escherichia coli resistance monitoring data to understand the clinical Escherichia coli of fluoroquinolone resistance characteristics; through in vitro multi-step induction experiments, testing before and after induction of E. coli to fluoroquinolones sensitivity and cross-resistance, the detection of E. coli ultra-broad-spectrum-β-lactamase (extendedspectrum beta lactamases, ESBLs) and ESBLs producing Escherichia coli fluoroquinolones resistance for clinical the fluoroquinolones to provide guidance; study of Escherichia coli type Ⅱ topoisomerase gene mutations and their relationship resistance to fluoroquinolones, to understand the mechanism of resistance of Escherichia coli to fluoroquinolones. Method: use whonet5.4 software statistics in our hospital in 2008 (First Affiliated Hospital of Henan College of Traditional Chinese Medicine) clinical Escherichia coli resistance monitoring data. Clinical isolates of Escherichia coli with the Phoenix-100 fully automated bacterial identification system will be identified to species. Determination of ciprofloxacin, levofloxacin, gatifloxacin gatifloxacin minimum inhibitory concentration (minimal inhibitory concentration, MIC) of the agar dilution method. 37 of Escherichia coli as an experimental strains selected based on the identification and susceptibility results and incorporated ciprofloxacin induced group (strain CE1-CE14 14), levofloxacin (strain induced group LE1-LE11 11) , plus gatifloxacin induced group (12 strains GE1-GE12) three experimental groups. And Escherichia coli ATCC25922 as quality control strains. Experimental strains footwork ciprofloxacin-induced resistance test (strain CE1-CE14), levofloxacin-induced resistance test (strain LE1-LE11) processing Gatifloxacin-induced drug test (strain GE1- GE12). The experimental procedure is as follows: Preparation of 1 × 10 ~ 8CFU/ml bacterial suspension with sterile saline, take 10 μl of the bacteria suspension was inoculated in 2ml of MH broth to containing 1/4MIC antibacterial drug concentration to a final amount of bacterial inoculum for 5 × 10 ~ 5CFU/ml, 35 ℃ for 24h. The selection of well grown 24h bacterial culture was diluted with sterile saline the bacterial suspension Preparation of 1 × 10 ~ 8CFU/ml, taken 10μl bacterial suspension was inoculated in 2ml of MH broth containing 1/2MIC antibacterial drug concentration incubated at 35 ℃ for 24h. The above steps through continuous turn kind of culture, the concentration of the drug since 1/4MIC, gradually increase the concentration of the drug in MH broth containing up to 128 times the MIC antibacterial drug concentration. Resistant vaccination induced antimicrobial agents in MH broth, 35 ° C train, of every 24h transferred species once, cultured for 5 consecutive days. Paper confirmatory test for the determination of the clinical isolates and laboratory strains ESBLs. Experimental strains the fluoroquinolone MIC agar dilution method; gyrA parC after the determination of DNA sequences amplified by PCR. Results: in 2008, the clinical total isolates 223 (excluding repeated strains), 94 (ESBLs positive strains positive rate of 42.2%). ESBLs positive strains to ciprofloxacin, levofloxacin, gatifloxacin gatifloxacin resistance rates were 87.8%, 84.9%, 84%, of ESBLs negative strains to ciprofloxacin, levofloxacin, plus Gatifloxacin resistance rates were 71.2%, 73% and 72.3%, respectively. Ciprofloxacin induced group of 14 experimental bacteria CE1-CE6, CE8-CE12, CE14 of 12 strains after subculture after increasing concentration of the drug inducing stable high ciprofloxacin-resistant strains. Ciprofloxacin MIC induced resistant strains 128-512μg/ml, with the original strains (MIC 0.016-2μg/ml) compared 128-32000 times. Induced group levofloxacin 11 experimental bacteria LE1, LE2, LE4, LE6, LE8, LE9, LE10 7 strains after increasing the concentration of the drug subculture induced a stable high-resistant strains of levofloxacin. Levofloxacin induction of drug-resistant strains of MIC 128-256μg/ml, with the original strains (MIC 2-4μg/ml) compared with an increase of 32-64 times. Induced group gatifloxacin 12 experimental bacteria and GE3, GE5, GE8, GE9, GE11 strains after increasing the concentration of the drug subculture after induction of stable high resistance to gatifloxacin strains. Gatifloxacin MIC for the induction of drug-resistant strains 128-512μg/ml, compared with the original strain (MIC 2-4μg/ml), an increase of 32-128 times. Highly resistant strains induced cross-resistance between fluoroquinolones. The results of experimental strains of ESBLs unchanged before and after induction. ESBLs-producing Escherichia coli related resistance to fluoroquinolones. Resistant strains of Escherichia coli ATCC25922, susceptible strains CE1 and induced CE1I CE6I LE1I LE2I LE8I GE3I GE5I GE8I by PCR amplification of gyrA and parC. Results All strains are on the electrophoretic pattern of amplification products the 648bp gyrA and 417bp parC bands. After gyrA and parC gene sequencing. The results 8 gyrA gene induction of drug-resistant strains of quinolone resistance-determining region (QRDR) within the first 83 serine (TCG) are leucine (TTG) instead, 87 aspartate (GAC) are asparagine (AAC) instead. The parC gene induction of drug-resistant strains QRDR 8 within the first 80 serine (AGC) are isoleucine (ATC) instead. Escherichia coli ATCC25922 and susceptible strains CE1 gyrA and parC gene QRDR amino acid sequence does not change. Conclusion: In the long-term antimicrobial selective pressure, allows Escherichia coli acquired drug resistance. Coli existence of cross-resistance to fluoroquinolones, and Escherichia coli producing ESBLs related resistance to fluoroquinolones. 3.DNA gyrase and topoisomerase IV change is the main mechanism of Escherichia coli fluoroquinolone resistance.

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