In Vitro Contraction Of Resistant Pseudomonas Aeruginosa And Investigation Of Its Resistance Mechanism

Background: Bacterial resistance is one of the most pressing public health problems facing the world today.It is the main factors for increased bacterial resistance to use a large number of applications of antibacterial drug. On March 27, 2015, The United States announced the national plan of action against drug resistant bacteria. Pseudomonas bacteria is the main pathogenic bacteria for those patients who were infected by surgical implantation, such as stent implantation and pacemaker implantation,in Internal Medicine Department. PA, known is the representative strains of Pseudomonas. It belongs to the opportunistic pathogen exist in human skin, respiratory tract and intestines, often inducing nosocomial infection. PA tooks 1st rank in pathogenic bacteria in the respiratory tract from Chinese Ministry of Health antimicrobial resistance monitoring report in 2011. PA became to important pathogens on a series of serious pyogenic infections, sepsis, lung abscess, chronic bronchitis,within the plant operation infected such as heart intervention operation and artificial joint implantation.Along with extensive use of antibacterial drugs, PA’s drug resistance rapidly increased in multi-drug resistant and pan-resistant Pseudomonas aeruginosa isolation rate gradually increased, which is a huge challenge for infections treatment. Quinolones as the rapidly developed antibacterial medication in the past 20 years, the character is broad spectrum antibiotic, antimicrobial activity, and easy to use, vivid dosage form as the main application in the domestic and international clinical control of infectious diseases, second to β-lactam antibiotics. At the meantime, it became a serious issue acompany bacterial resistance to these drugs into the spread of the trend, the growing number of drug-resistant pathogens, the resistance level of increasingly heavier. The mechanisms of Pseudomonas aeruginosa resistant to quinolone mainly are mutations in topoisomerase II, IV; controlling bacterial active efflux systems of regulation of gene overexpression Erzhi active efflux enhanced and biofilm formation and cell membrane through permeability changes. Alone or in combination of several mechanisms of resistance caused by the PA decreased susceptibility to quinolone drugs.However, Pseudomonas aeruginosa resistance to quinolones and fluoroquinolones time, the correlation between doses, and drug resistance of PA resistance to quinolones and clinical application within the period quinolone sensitive to quinolone P. aeruginosa changes its resistance mechanism is not yet clear. Objective: In-depth understanding of my hospital system PA infection and resistance epidemic of quinolone situation to provide experience for local area PA infection of antibacterial drugs.It is important to have dose recommended for this hospital quinolone drug treatment PA infection and life cycle, via depth understanding the correlation between the use of PA quinolone and its casue of hospital infection strains resistant characteristics and mechanism. Methods: 1.All pathogens raw data will be entered into WHONET 5.4 provided by the WHO bacterial resistance monitoring network which is used for statistics of the sample distribution and resistance rates; World Health Organization(WHO) recommended defined daily dose method a variety of antimicrobial agents of DDDs, the DDD value of the various drugs, according to the commonly used daily dose for adults 16 edition of the "Chinese Pharmacopoeia 2005 edition of two new pharmacy", DDDs = a antimicrobial drug consumption(m) / drug DDD value, the study of antibacterial drug use frequency(DBD) said DDDs/100bed-days; using the Spearman correlation statistical methods to study the relationship between the use of antimicrobial agents and their resistance. Analysis of Pseudomonas aeruginosa isolated in the different samples the difference between the resistance rates of antibiotics commonly used in the ANOVA(analysis of variance), LSD(least significant difference method). 2. using the agar dilution method the MIC values; II topoisomerase(in gyr A, gyr B, par C and par E) gene was amplified by PCR, purification, sequencing; connection real-time quantitative PCR analysis of four kinds of efflux system membrane protein Mex A Mex C, Mex E, and Mex X gene expression level; using the χ2 test to analyze the relationship between gene mutation and efflux pump expression and drug resistance. 3.the use of ciprofloxacin, levofloxacin, and norfloxacin the gradient concentration(0.5MIC, 1MIC, 2MIC and 4MIC) of MH broth with clinical isolates,7PA, respectively, were cultured in vitro; and E-test method and agar dilution Determination of induction after strain the MIC value; PCR amplification of type â…¡ topoisomerase gene, purification, sequencing; real-time quantitative PCR analysis of four kinds of efflux system membrane to connect the protein Mex A Mex C the, Mex E, and Mex X gene expression level,; Statistics science software SPSS16.0 before induction and after induction of MIC values were compared using the t test, and inspection level α = 0.05, p <0.05 for the difference was statistically significant. Single factor analysis of variance(LSD) test three different concentrations of antimicrobial drug-induced, Pseudomonas aeruginosa MIC values difference. Standard p <0.05 significant difference as difference, p <0.01 as significant difference. 4. using the crystal violet staining and laser confocal microscopy study co-cultured in vitro with three different concentrations of quinolone drugs(0.5MIC, 1MIC, 2MIC and 4MIC) the difference of 9 days of P. aeruginosa generated biofilm capabilities, single factor analysis of variance the differences in the ability of PA with three different concentrations of antimicrobial drug-induced biofilm formation. Standard p <0.05 significant difference as difference, p <0.01 as significant difference. Results: 1.The patients who were infected pseudomonas aeruginosa in medical with respiratory infections as main, accounting for 55.2%, and respiratory infection of pseudomonas aeruginosa and other parts of the fungus infection, there were significant differences in the respectively, p<0.05; Our hospital pseudomonas aeruginosa in overall resistant higher level, antimicrobial resistance to the lowest for amelia card star, 2010 to 6.3% respectively, nine years pseudomonas aeruginosa cefotaxime resistant all remain below 30%, followed by ciprofloxacin; The correlation analysis found that found that three generation cephalosporin to the use of cefoperazone and cefotaxime of pseudomonas aeruginosa resistant is negatively related to the present, Levofloxacin, ciprofloxacin, beauty and e.faecalis imipenem dosage and pseudomonas aeruginosa resistant to the positive correlation between, and a statistical significance, p < 0.05. 2.All of the 150 strains of pseudomonas aeruginosa, the resistance rate to ciprofloxacin is the lowest the lower to 28.8%, Moxifloxacin is the most resistance to PA, rate as high as 96.3%, levofloxacin and levofloxacin take second place, 38 of 150 strains showed all antibiotics tested. 3.It is found that 26 strains exist gyr A Thr83(ACC)-Ile(ATC) mutations, no strains exist par C Ser80(TCG)-Leu(TTG) mutations, gyr B, par E gene mutations.But there were 4 strains with bases a missing in the same position,which lead to translation error of 46-59 amino acids correspondingly. Ciprofloxacin, levofloxacin was different in statistics(P < 0.05) between the group with Gyr A gene mutations and the group without, comparatly the sensitivity nofloxacin was not(P > 0.05). 4.We selected efflux pump overexpression positive phenotype through the MIC comparision in the condition of adding PA β N and not. efflux pump overexpression phenotype positive rate was 23.7%(9/38). Real-time PCR amplification quantitative fluorescence, discharge gene expression mex A high strain of six strains, the Numbers were 25, 31, 53, 71, 81 and 82 number. Mex E gene expression of discharging high strain have two strains, the Numbers were and use. In this trial not found in discharging mex C and mex X high expression gene strains of bacteria. 5.7strains of pseudomonas aeruginosa were cultured with ciprofloxacin, levofloxacin or nofloxacin, in that order, in different concentration(0.5 MIC, 1 MIC, 2 MIC, and 4 MIC). The MIC value of ciprofloxacin changed as 2.5-160 times as the original strains and nofloxacin’s is 4-32 times than the original strains, and the MIC value was 1-64 times compared with the original strains for levofloxacin.According to statistical analysis of LSD, it has significant different effect for three fluoroquinolones drug’s MIC value between drug concentration as 4 MIC and 0.5 MIC, 1 MIC, 2 MIC(p < 0.05). The other three concentrations have non- significant difference on influencing the MIC value; nofloxacin and levofloxacin in drug concentration of 2 MIC and 4 MIC have differences influence to the MIC value of induced strains, p < 0.05; ciprofloxacin and levofloxacin have no obvious difference with nofloxacin, p > 0.05. 6.Pseudomonas aeruginosas,which were inducted by three kinds of fluoroquinolone drug in different concentrations, were amplificated and purified by the PCR. The sequencing result, compared with the corresponding sequence of PAO1 model in Gen Bank, shows that 15 bacteria strains appeared the subunit gyr A and par C of mutation, the rate of mutation is 17.85%; Gry A subunit of mutations is 83 Thr-Gla(ten strains), 72 Asp-Gly(3 strains), 87Asp-Asn(1 strain); 4 strains have mutated at 80 th amino acids anticodon on QRDR area in par C genes Ser80(TCG)-Ieu(TTG); the other mutations in Gry A and par C QRDR have not been found; 98.8% of all strains were discharge genes Mex ACgenes overexpression, 45.23% are the overexpression strains of mex X, and mex E genes were overexpressed by 26.19% strains. The plant which had mex A genes overexpression was not found. 7.After dyed by Crystal violet, the OD value of these strains was inspected by SM-Mk3 microplate reader in the 570 nm single-wavelength. 39.29% of the strains appeared that A570 < 0.3; 48.21% were 0.3 < A570 < 1.0; 12.50% were A570 > 1.0. Analyzing the difference effect on generation amount of different strains(p > 0.05), it shows that there were no significant difference among seven strains of pseudomonas aeruginosa on generative capacity for biomembrane with different concentration of different fluoroquinolones; on contrary, significant differences were found with different concentration of fluoroquinolone p=0.000. According to the Least significant difference(LSD), after inducted by 1/2 MIC, clinical separated strains’ ability to produce biomembrane had significant differences with strains inducted by 1 MIC, 2 MIC and 4 MIC p = 0.000. And the rest three concentrations had no obvious differences(p > 0.05). 8. Strains of the experiment of membrane respectively in 24 h, observed 48 h, 72 h use CLSM observe and collecting images, not after drugs induced strains on the control of the biological membrane formation in the same observing time points late in after drugs induced strains, and control in the 72 h strains microscopically biological membrane structure than after drugs induced strains of osteoporosis, after LEV induction strains in 24 h colonies had gathered into a larger slice of biological membrane, and biological membrane formation faster than CIP induction strains. 0.5MIC after induction strains of biological membrane formation is stronger than the four MIC after induction strains. Conclusions: 1.Fluoroquinolone drugs, increased use of hydrocarbon of mycophenolate vinyl drugs and Pseudomonas aeruginosa resistance rate of change is positively correlated; the overall resistance rate of P. aeruginosa at a higher level. Amikacin, ceftazidime, ciprofloxacin may experience medication for the treatment of Pseudomonas aeruginosa hospital. 2.Fluoroquinolone drug resistance of P. aeruginosa gyr A subunit Thr83(ACC) â†' Ile(ATC) the mutations and efflux gene mex A overexpression. This study found first that the base deletion at the position of38 of Par C submit caused wrong translation and with that the PA’s resistance to FNQs. 3.Sensitive PA was susceptible to resistance after dealing with antibiotics and the overexpression of Par C was the main mechanism of drug resistance.it’s easy to develop multiple resistance of PA while the concentration of FNQs were fourfold of its MIC. 4.0.5MIC-induced strain biofilm formation is faster than the strain induced in the Ciprofloxacin. 5.It advised that combining with PPIs and enough(short?)course of treatment could postpone the development of PA’s resistance while treatingmoderate and severe infection coursed by sensitive PA.