Research Of Active Efflux Mechanism Of AcrAB-TolC Multi-drug Efflux Pump Of Escherichia Coli

Objective:E. coli is one of the most common pathogens of community and nosocomial infections, is one of the important pathogens. As the antibiotics are widely used in clinic, resistance to antibiotics has become more and more serious. Multiple drug-resistant E. coli appeared and spread, seriously affecting the effects of therapy, increasing the cost of treatment. Treatment of multiple drug-resistant E. coli is one of important goals in clinic. Bacterial resistance mechanisms include production of inactivated enzyme,change drug target, change the outer membrane permeability, active efflux, biological membrane and so on. The latter three are the main mechanisms of multi-resistant. Active efflux system is one of the important methods of multiple drug-resistant. AcrAB-TolC efflux pumps is the dominant multiple drug efflux pump of E.coli,.The substrates are extensive.Studying of E. coli in tianjin region efflux pump AcrAB-TolC carrying situation, and relevance with drug resistance in clinical strains, and inhibitory of carbonyl cyanide chlorobenzene hydrazone (CCCP) to efflux pump exploring possible, new ideas for clinical multiple drug-resistant E. coli infection prevention and controlling.Methods:1、Strains collection and identification:A total of90strains of Escherichia coli were isolated from The Second Hospital of Tianjin Medical University from July.2008to September.2009. Excluding the repeated strain of same cases in the same place, all strains are identification by routine biochemical. Standard strain of E. coli ATCC25922as a quality control strains. staphylococcus aureus ATCC25923negative control strains for acra/acrb gene, the two kinds of standard strains were preserved strains of tianjin infectious disease research institute.2, the grouping resistance pattern:All90clinical strains of e. coli to6kinds of antimicrobial drugs by he K-B disc diffusion method. the sensitivity of the drug susceptibility results according to the clinical laboratory standardization institute (CLSI2011) criterion, the resistance pattern is divided into multiple drug-resistant group, two drug resistance group,one drug resistance group, all sensitive group. 3, Amplification of efflux pump gene acra/acrb:application of polymerase chain reaction (PCR) amplification of90clinical isolated strains of E. coli of acra/acrb gene fragments, PCR products send Beijing huada technology company, use the positive and negative two-way DNA sequence analysis.4, CCCP inhibitory experiment:select10multiple drug-resistant E. coli strains for CCCP inhibitory experiments, to trace the broth dilution method and pump inhibitors CCCP (final concentration of100umol/L) before and after multiple drug-resistant strains of five kinds of antimicrobial minimum bacteriostatic concentration (MIC).Results:1, The total collection of90strains of e. coli,37strains of isolated bacteria from urethra,36from intestinal,5from The respiratory tract,11from other secretions(blood, ascites, etc.).2, in vitro antibiotics sensitive experiment results:90strains of E. coli on6kinds of antimicrobial drugs showed different degrees of resistance, resistance rate from high to low:ciprofloxacin as67%,levofloxacin61%,gentamicin53%, cefotaxime50%, aztreonam20%, amikacin10%. Drug resistance pattern of30double drug-resistant strains (33.3%),26multiple drug-resistant strains (28.9%),22single drug resistant strains (24.4%),12whole sensitive strains12(13.3%).3, acra/acrb amplification and sequencing results:90clinical isolated E. coli strains,55(61.1%) are visible of567bp (acra) and516bp (acrb) fragments amplified. Staphylococcus aureus ATCC25923did not see the fragments amplified. Clinical strain246of acra gene and clinical strains b716of acrb gene amplification are seleted for positive and negative two-way DNA sequence analysis, the sequencing results hitches in GeneBank database rows homology comparison after confirmed:clinical strains246, acra gene homology of98%, the clinical strains b716,acrb gene homology of99%. The multi-resistant group of22strains of acra positive (84.6%),21strains of acrb positive (80.8%); Two drug resistant group of16strains of acra tested positive (53.3%),17strains of acrb tested positive (56.7%); Single drug resistant group of12strains of acra positive (54.5%),13strains of acrb positive(59.1%); All sensitive group of5strains of acra and4strains of acrb(33.3%) were positive (41.7%),.So acra/acrb genes of efflux pump in4kinds of resistance pattern is common in clinical strains of E. coli, the positive rate was highest in the multi-resistant group,84.6%and80.8%, respectively. Of multiple drug resistance and the sensitive group of acra gene and acrb positive rate, statistical analysis using chi-square test, acra gene andAcrb gene,the result is statistically significant.4, CCCP inhibit experimental results:10multiple drug-resistant strains of E. coli clinical strains after application of CCCP, the MICs of most strains of5kinds of antibiotic have a different degree of decline, can restore their sensitivity to antibiotics of most strains. MIC of strain1034to ceftriaxone decline highest in multiples of128times, strain A620, C636,1034to5kinds of antibiotics were from resistant to sensitive. But strain B1581and strain O MIC of5kinds of antibiotics, which are basicly has no changed.Conclusions:1,90clinical strains of E. coli to6kinds of antibiotics were showed different degrees of resistance, higher resistante rate prevalence of fluoroquinolones, but resistant rate of amikacin is low; Resistance pattern is given priority to with two drug resistance and multi drug resistance.2, acra/acrb efflux pump genes in4kinds of resistance pattern is common in clinical strains of E. coli, among them, the positive rate of multiple drug-resistant group are the highest of84.6%and80.8%, respectively, in all the sensitive group were the lowest of41.7%and33.3%, respectively. The difference has statistical significance (P<0.05).3, Most bacterias have active efflux effect, CCCP can inhibit the efflux system of bacteria to antibiotics effectively, MIC falling multiples,demonstates that active efflux of efflux pumpEeffect sizes, or active efflux mechanism with other mechanisms may lead to multiple drug-resistant of E. coli to antibacterial drugs.