Study On Molecular Antimicrobial Resistance Mechanisms Of Extended-spectrum β-lactamases Producing Gram-negative Bacteria

Antimicrobial resistance has become one of the most serious public health problems in the 21st century. The failure of clinical treatment and the break of nosocomial infection caused by antibiotic resistance has threatened human health. For the powerful antibacterial activity, broad antimicrobial spectrum and low toxicity, beta-lactam antibiotics, especially the third generation cephalosporins, play an important role in the treatment of gram-negative bacteria infection and has been the most popular antibiotics in clinical. But, resently, with the widespread use of numerous new beta-lactam antibiotics, the resistant strains increased quickly. One of the most important reason is that a new kind ofβ-lactamase appeared under the antibiotics selective pressure, called extended-spectrumβ-lactamases(ESBLs). ESBLs have spread substrates, and have the ability to hydrolyse penicillins, cephalosporins and monobactams. Since first described in 1983, ESBLs have been reported at almost everywhere in the world. There are many reports about the breaks of nosocomial infection induced by ESBLs-producing bateria. ESBLs-producing bateria, together with methicillin resistant Staphylococcus aureus(MRSA), vancomycin resistant enterococcus(VRE) and multiple resistant non-fermentative gram-negative bacteria, have become a worldwide tough problem in the field of anti-infection.Based on the above, investigating the infection condition of ESBLs-producing gram-negative bateria, monitoring their resistance, revealing the molecular epidemiological characteristics, inquiring into the molecular mechanisms of the emergence and dissemination of their multiple resistance, will provide significant theory support to clinical surveillance, prevention and therapy of ESBLs-producing strains. Thus, aim at the most popular ESBLs-producing gram-negative bacteria in hospital, we carried out the following research.1. Investigation of phenotypes and genotypes of antimicrobial resistance in extended-spectrumβ-lactamases -producing strains A total of 237 clinical isolates of nonrepetitive gram-negative bacteria were collected. Antibiotics susceptibilities were determined by K-B method. ESBLs-producing isolates were detected by phenotypic screening and confirmatory test according to the methods recommended by CLSI/NCCLS. Three-dimensional hydrolysis test, PCR amplification and nucleotide sequencing were used to analyze the genotypes of ESBLs. At the same time, aim at those strains producing ESBLs and plasmid-mediated AmpCβ-lactamases together, PCR method was used to detect the AmpC genes. The repetitive consensus sequence PCR(rep-PCR) was carried out to analyze the chromosome DNA homology of ESBLs-producing strains.The results showed, 96 isolates were identified as ESBLs -producing strains by phenotypic confirmatory test and the incidence was 40.5%. Detected rate of E.coli was 37.8% and Klebsiella was 40.5%. The ESBLs-producing isolates were more resistant to most antimicrobials than non-ESBLs-producers. The results of three-dimensional test indicated that among 123 ESBLs-producing doubtful isolates, 91 strains produced ESBLs alone and 10 strains produced ESBLs and AmpC together. The results ofβ-lactamases genotyping show that 71 isolates carried blaTEM(74.0%),41 isolates carried blaSHV(42.7%), 18 isolates carried blaCTX-M-1 group(18.8%) and 49 isolates carried blaCTX-M-9 group(51.0%) , the total detection rate of blaCTX-M was 69.8%. The results of PCR amplification using blaCTX-M-2 group,blaPER-1 and blaVEB-1 primers were negative. Sequencing analysis showed that the most frequently identified ESBLs genes in this group were blaTEM-1, blaSHV-12, blaSHV-2a blaCTX-M-14 and blaCTX-M-15. DHA-1 plasmid-mediated AmpC gene was detected in 7 strains among 10 phenotype positive strains and coexisted with many different ESBLs genes. The homology analysis indicated that most ESBLs-producing bacteria were different clones. But there were three types including more strains, they were from the same wards, same hospital and in the same period. So they were proposed to be the same clone or have very close relations and may prevailed in some hospital before.2. Study on mechanisms of transferable multiple resistance in ESBLs producing clinical isolatesAim at those 96 ESBLs-producing strains, plasmid conjugation test, resistant plasmids profile analysis were carried out. Detection of class 1 and 2 integron integrases, variable regions, and related antimicrobial resistance genes were amplified by PCR. Restriction fragment length polymorphism(PCR-RFLP), PCR-mapping, nested-PCR, and cloning, sequencing and expressing methods were used to investigate the epidemical conditions and characteristics of class 1 integron in ESBLs-producing bacteria and reveal the mechanisms of plasmid and gene cassette-integron system in the emergence and dissemination of multiple antimicrobial resistance.The results showed that ESBLs-producing bacteria generally carried 1~5 different numbers and 1.0~50kb different size natural plasmids and could transfer their resistance by conjugation. The transconjugant could get some part or whole resistance phenotypes and ESBLs genotypes of donator bacteria. Class 1 integrons were identified among 45 isolates and the positive rate was 46.9%. The class 1 integrons were broadly distributed in Escherichaia coli, Klebsiella pneumonia, Enterobacter cloacae, Enterobacter agglomerans and Serratia marcescens. The size of variable regions were always between 0.5 kb to 2.5kb and the most popular was 1.7 kb amplicon. More than one integrons could be detected in one strain. PCR-RFLP analysis revealed that all these strains carried 10 different types of integrons. If the integron is positive in donators, their transconjugants also carried integrons. Aminoglycoside modifying enzymes located in integron structure were detected by nested PCR. The positive rates of aac(3)-II, aac(6')-I and ant(3'')-I were 61.2%, 30.6% and 18.4% respectively。PCR-mapping and cloning and sequencing analysis revealed that most integrons harbored one or two antibiotic resistant gene cassettes, which encoding resistances to trimethoprim (dfrA1 and dfrA17), anmioglycosides (aadA5) and rifampin (arr-3). The assembly way of gene cassettes were as follows: dfrA17-aadA5,dfrA1-orfC,arr3-orf. In this study, an unknown open reading frame (orf) was found. Through the construction of prokaryotic expression vector, induced expression and antibiotics sensitivity test, we proposed that it may be a new dihydrofolate reductase gene cassette and encoding resistance to trimethoprim. Genes encodingβ-lactamases didn't locate in class 1 integrons in this study. Integrons and their associated gene cassettes couldn't always account for the total phenotypic resistance exhibited by the ESBLs producing bacteria. However, they played a significant role in the emergence and dissemination of multiple resistance.3. Molecular epidemiology study ofβ-lactamases producing multiple resistant Pseudomonas aeruginosaIn order to investigate the Molecular epidemiology characteristics ofβ-lactamases producing multiple resistant Pseudomonas aeruginosa, following experiments were carried out. Three-dimensional hydrolysis test and double disk synergy test were performed to analyze the phenotype ofβ-lactamases including ESBLs, AmpC and metalβ-lactamases (MBLs). PCR and sequencing methods were used to detect the genotypes ofβ-lactamase. Complete variable region amplification of Class 1 integron and PCR-RFLP analysis were performed to investigate the presence of class 1 integron and the way of resistance gene transferred. ERIC2-PCR was carried out for DNA typing of clonal strains.The resistance rate of 85 Pseudomonas aeruginosa clinical isolates against the third and fourth generation cephalosporins, aztreonam, aminoglycosides and quinolones were all more than 60.0%, the resistance rate to imipenem was up to 23.6% and 17 pan-resistant strains were found. Three-dimensional hydrolysis test showed that 14 strains appeared ESBLs-producing phenotypes in 17 pan-resistant strains. Double disk synergy test showed that 13 strains appeared MBL-producing phenotypes in 20 imipenem resistant strins. But the PCR results of IMP and VIM metalloβ-lactamase gene were negative. PCR amplification and sequencing results showed that among 85 PA, the positive rate of blaTEM was 77.6%, blaPER-1 was as high as 48.2% and blaSHV was 5.9% PER-1 type ESBLs-producing PA was reported for the first time in Hainan province. The homology analysis by ERIC-PCR confirmed that PER-1 type ESBLs-producing Pseudomonas aeruginosa were epidemic strains in nosocomial infection.Class 1 integrons were identified among 31 isolates and the positive rate was 36.5%.