Aminoglycoside-resistant Pseudomonas Aeruginosa And The Molecular Mechanism Of Drug Resistance

Pseudomonas aeruginosa ( Pae) is one of the main pathogens of nosocomial infections. Intensive Care Unit in China separated from Gram-negative bacteria, Pseudomonas aeruginosa ranked the first. Because of the natural and acquired drug resistance, Pseudomonas aeruginosa resistants to many antibiotics, and has caused great difficulties to the clinical treatment.β-lactams, aminoglycosides and quinolones antibacterial drugs are common infection in clinical medicining Pae. In recent years, as new emerging drug resistance genes, the three types of antimicrobial agents are generally increasing, and even multi-drug resistant (MDR) and pan-drug resistance (PDR) strains. Due to a broad spectrum antibacterial aminoglycoside antibiotics, In clinical and animal husbandry and veterinary medicine widely used in industry. However, the problem of drug abuse and excessive use of aminoglycoside has become highlights. Bacterial resistance to the drugs is because of the bacteria producing aminoglycoside-modifying enzymes (AMEs), 16SrRNA methylase target site and the role of aminoglycoside antibiotics, 16S rRNA gene mutation. Aminoglycoside-modifying enzymes according to the functions can be divided into acetyltransferase (AAC), phosphate transferase (APH), nucleotide transferase (ANT), modificate the corresponding position of the - OH and - NH2 of aminoglycoside drugs respectively, and reduct or loss their affinity ribosomal target site. The genes encoding AMEs originated in the antibiotic-producing strains, Gene expression of enzymes such as antibiotic-producing strains play the role of self-protection, Many studies have shown that resistant bacteria generated by the existence of self-protection mechanisms is the same with mechanism of resistance of clinical drug-resistant.Integration through a self-coded integrase to get insurance outside the gene or gene fragment of free expression and make to express the genetic component system. Integration can located the bacterial chromosome, located on plasmids, also on the transposon.On the mechanisms of bacterial antibiotic resistance, resulting in, including transposons and plasmid conjugation, including the discovery of a number of mobile devices, through a comparative analysis of sequences of these components, the final discovery of the presence of integrons.ObjectivePseudomonas aeruginosa is a pathogen of nosocomial infection, and can cause severe nosocomial infections. To realize the antibacterial activity of clinical commonly used seven antibiotics to sixty Pseudomonas aeruginosa isolates (the thirsty strains from outpatients,the other thirsty strains from inpatients) , vitro combined antibacterial effect of polymyxin B with other six antibiotics (gentamicin,netilmicin,amikacin,tobramycin,ciprofloxacin,and imipenem), and the difference antibacterial effect of antibiotics to these isolates from different sources. To Understand the presence of aminoglycoside resistance-associated genes and 16SrRNA methylase genes among strains, as well as the affinity,By clinical isolates of Pseudomonas aeruginosa resistant features were analyzed to detect the relevant drug-resistant genes and integrons carrying case to have a preliminary clinical study of Pseudomonas aeruginosa to antibiotics commonly used in the molecular mechanisms to understand the different resistance mechanism of interaction patterns as well as drug resistance gene in the characteristics of horizontal transmission among strains, and provide laboratory basis for clinical medicine and help. Methods:1. Drug sensitivity test and Co-sensitivity test60 Pseudomonas aeruginosa strains are from the First Affiliated Hospital of Guangzhou Medical College of clinical specimens,agar dilution method determined the minimal inbititory concentration of single antibiotic and combined-use bntibiotics to Pseudomonas aeruginosas, and analysis susceptibility to these antibiotics. We detect drug susceptibility of polymyxin B and other six kinds and the joint, try to open the new ways to treat infections Pae, play a tremendous role in the drug selection, reducting dosage and toxicity.2. Multi-drug resistant Pseudomonas aeruginosa aminoglycoside-modifying enzymes and genetic analysis of 16SrRNA methylase,Against the strains in clinical isolates of Pae, aminoglycoside modifying enzyme genes, 16S rRNA methylases and other genes were detected by polymerase chain reaction (PCR) method, and Cluster analysis was applied to realise the consanguinity of sample stains.3. Identification and analysis of integronsTo use PCR methods, through the integration of sub-constant region amplification method, selection of strains carrying integrons and use of restriction fragment length polymorphism (RFLP) enzyme analysis method for sub-classification of these integration. Sequencing and analysising integron-positive strains amplified constant region. Integrons carrying resistance gene cassettes is mediated by the multidrug-resistant Pseudomonas aeruginosa or not.Results:1. In strains of Pseudomonas aeurginosa, the different drug-resistant rates were 70%(netilmicin), 63.3%(tobramycin), 63.3%(gentamicin), 53.3%(ciprofloxacin), 40%(im ipenem), 13.3%(amikacin) respectively,the drug resistance rate of polymycin B was zero. The muti-drug resistant rate and pan-drug resistant rate were 55%(33/60) and 31.7%(19/60). Polymycin B combined-use with imipenem and ciprofloxacin had obviously combined antibacterial effect. Polymycin B combined-use with netilmicin can reduce mutual MICG to each other obviously. The antibacterial effect (MICG) of all kinds of antibiotics to isolatesfrom inpatients and outpatients were obviously different.2. In 21 aminoglycoside-resistant strains(20 strains of them were multi-drug resistant),the positive rates of aminoglycoside modifying enzyme genes, such as aac(6,)-Ⅰ,aac(6,)-Ⅱ,ant(2,,)-Ⅰ,ant(3,,)-Ⅰand aac(3)-Ⅱ,were respectively 61.9%,61.9%,47.6%,42.9%,and 4.8%, the gene of oprD2 was not detected in only 1 strain, some of the gene types,such as aac(6,)-Ⅰae,aph(3,)-Ⅲ,aac(6,)-aph(2,,),ant(4,)-Ⅰwere not detected. The positive rates of 16SrRNA methylase genotypes , such as armA and rmtA were respectively 38.1% and 90.4%, genotypes rmtC and rmtD were not detected. Cluster analysis showed that the Pseudomonas aeurginosa isolates present clone spread.3. 30 clinical isolates of 16 strains of Pseudomonas aeruginosa integron were the positive amplifications on variable region constant region,the fragment length was between 0.7kb and 4kb. We detected five Gene cassettes of different combinations, which contains coding aminoglycosides,β-lactams and quinolones antibiotic resistance genes and two cases for the new gene cassette ,which contained aacA4- VIM,aadA2-OXA10-aacA4-blaIMP9-aatI1,the accession numbers were GQ89065 and GU122165 in Genbank respectively. Another three cases of sequence matches were consistent with Genbank accession numbers for the FJ917747, FJ817423, GU367339.4. Class I integron 3'-end of the qacEΔ1-sul1 and class I integrase gene positive rates were same with positive strains.Conclusion:1.Clinical P. aeruginosa to commonly used antibiotics showed multidrug resistance more. Polymyxin B alone have a good effect against P. aeruginosa, and ciprofloxacin (fluoroquinolone), imipenem (β-lactam) combined have significant antibacterial effects of combined. Multi-drug resistance and pan-resistant strains, in order to reduce the dosage and side effects, improve the antibacterial effect of polymyxin B which could be considered with the combination of these antibiotics.2. Most of the tests commonly used in clinical Pseudomonas aeruginosa, Pseudomonas aeruginosa has generated widespread anti-infective drug, especially aminoglycoside antibiotics. The common strain of aminoglycoside modifying enzyme genes detected rate of drug resistance, 16SrRNA methyltransferase genotype rmtA and armA had higher detection rate. 30 test strains had propagation.3. Pseudomonas aeruginosa resistance and multiple drug resistance, was closely related with the integron which mainedly carried aminoglycosides andβ-lactam antibiotic resistance genes, and two kinds of gene cassettes which carrying new combinations of integron were first detected .4. Class I integron 3'-end of the qacEΔ1-sul1 and class I integrase gene positive rates were same with positive strains, it Showed that the two genes were closely associated with intI1 exist, and could be used as Class I integron marker genes.