Genotypic Characterization And Antibiotic-resistance Of Acinetobacter Baumanii Isolated From Patients With Nosocomial Infections In Part Of Xiamen Area

objeetives:1. In order to understand the drug-resistance of Acinetobacter baumanii which were seleeted from hospital infections in Xiamen area, graped differences among the different time, then Provide a better clinical selection of appropriate antibiotics.2. To explore and optimize Pulsed-field gel eleetrophoresis method for Acinetobacter baumanii operating, understand their genetic polymorphism and clarity genetic relationship between strains.3. Aalysised the cluster outeomes of Acinetobacter baumanii gene map together with epidemiological data. Distinguished dominant clonal strains as well as their distribution in different time, different regions and different human, then diagnosised whether they were outbreak strains.4. Analysis the common drug resistant gene carried by extensively drug-resistant Acinetobacter baumanii.Methods:1.Strains collection:during January2009to December2010, selected samples including sputum,blood,Urine,faeces and other ones from nosocomial Infection Patients in three hosPitals(174th HosPital, Zhongshan HosPital,1st HosPital), isolation and identlfication were carrying out by using Walkaway96automatic ideniification system(Dering,USA) removed those repeated samples from the same patient.2.Susceptibility test:K-B dise diffusion method was used to detect their resistance to 20antimicrobial agents against isolates through antimicrobial agents susceptibility standards of CLSI2010.3.Genotypie characterization:Built up a more appropriate operation for Acinetobacter baumanii PFGE typing, with which we carried out operating on isolates;The un-weighted Pair-group mean arithmetic (UPGMA) of Bionumerics V4.o software was used to cluster analysis.4. Detect the resistance genes of extensively drug-resistant strains:Used PCR amplification assay to detect the common resistant gene of Extended-spectrum-β-lactamas,Metallo-β-lactamas, Cephalosporins enzyme, et al.Results:1. Susceptibility testing results of Acinetobacter baumanii from2009-2011in part of Xiamen area.1.1Susceptibility testing on Acinetobacter baumanii.Totally we collected386Acinetobacter baumanii from hospital infections, each strain showed varying degrees of drug-resistance among20common used antibioties (Penicillins, CePhalosPorins, otherβ-lactamas, aminoglyeosides, quinolones, sulfonamides, colistin, minocyclin). No strain of Pan-resistance or whole drug-sensitive were found in this experiment.The drug resistance situations were as follows:AZM(87.05%)、 PIP(77.72%)、FEP(77.72%)、CTX(72.28%)、ETP(71.50%)、TIM(69.17%)、 SXT(68.91%)、LVX(67.62%)、TOB(67.62%)、CP(67.36%)、CAZ(66.32%)、 GN(65.54%)、AMC(64.51%)、MEM(63.73%)、IMP (63.47%)、AK(57.77%)、SCF (52.85%)、PB (41.19%)、MIN (0.00%).Isolated strains had high resistant rates to common β-lactam antibiotics and enzyme inhibitors, aminoglycosides, quinolones, sulfonamides, most weremore than50%, some were close to90%; drug resisitance tor carbapenem antibiotics was also serious, resistance rate was more than60%; resistant rate to colistin was less than50%, resistance rate to minocyclin was0%. 1.2Time trends of antibiotics-resistance on Acinetobacter baumanii.The drug resistance of B A in Xiamen region was relatively serious, during the last3years, the drug resistance rates to most common antibiotic were increasing, especially drug resistance rate to imipenem, meropenem, ertapenem and polymyxin growth were more obvious.1.3The detection of multi-drug resistance and extensive-drug resistanceThere were118strains of multi-drug resistance and97strains of extensive-drug resistance Acinetobacter baumanii among total386, rate were30.57%and25.13%.2. Genotyping of Acinetobacter baumanii with pulse field gel electrophoresis2.1Appropriate operation for Acinetobacter baumanii PFGE typing exploringWhen OD4.5, cell disruption for20h, Spa Ⅰ20U digest more than3h, initial switch time value of2.16sece, final switceh time of63.8sec at a gradient of6V/cm and an included angle of120°electrophoresed for20hours in0.5×TBE at14℃. Results came out to be perfect (including bandnumber, size, brightness, and, length).2.2Genotyping results of Acinetobacter baumanii.With the same reagents, equipments and Parameters, each strain showed21-33bands ranging from30kb to800kb after20U Spa Ⅰ digestion.Clustered by Bionumeries V4.0, similar values were among45.9%and100%with349types (consider to be a same type when similar value was100%).2.3The most common genotype had40strains of bacteria,12strains separated in2009,15strains in2010,13strains in2011.2.4Genotyping of97strains extensive-drug resistance Acinetobacter baumanii: clustered by Bionumeries V4.0, similar values were among62.5%and100%, with67types.3. Resistant gene of extensively drug-resistant Acinetobacter baumanii.There were23strains carrying TEM gene,17strains carrying CTX-M-13gene in the97strains of extensively drug-resistant Acinetobacter baumanii, the positive rates were23.71%and17.53%.30strains carrying VIM-2gene, the positive rate was30.93%;39strains carrying IMP-1gene, the positive rate was40.20%;10strains carrying OXA-58gene, the positive rate was10.31%;80strains carrying OXA-51gene, the positive rate was82.47%;85strains carrying OXA-23gene, the detection rate was87.63%;11strains carrying OXA-24gene, the detection rate was11.34%. No detectable NDM-1gene.Clusions:1. In part of Xiamen area from2009to2011Acinetobacter baumanii were isolated from respiratory specimens mainly, distributing mainly in the Departmen of ICU and Respiratory medicine, isolated mostly from patients over the age of45.2. Isolated strains had high resistant rates to common β-lactam antibiotics and enzyme inhibitors, aminoglycosides, quinolones, sulfonamides, most weremore than50%, some were close to90%; drug resisitance tor carbapenem antibiotics was also serious, resistance rate was more than60%; resistant rate to colistin was less than50%, resistance rate to minocyclin was0%. During the last3years, the drug resistance rates to most common antibiotic were increasing, especially drug resistance rate to imipenem, meropenem, ertapenem and polymyxin growth were more obvious.3. PFGE experimental technique has a strong awareness of the differences in molecular level.It is really a stable and accurate genotyping method which was suitable for homology analysis of nosocomial infections.4. In Xiamen regions, there was no dominant clone strains a from2009-2011. Small-scale spread of nosoeomial infection took place during the last yeas, especially in wards of ICU, still cross-infection could suffer different wards. On the other hand, completely homologous strains were found in different place which reminded us the importance of epidemiological investigation.PFGE patterns showed that Acinetobacter baumanii,especially those carrying resistance gene strains, had a higher degree genetic homology, which reveal that it was more likely carrying out cross-infection.5. The extensively drug-resistant Acinetobacter baumanii isolated in part of Xiamen area carrying resistance genes including TEM, CTX-M-13, OXA-23, OXA-24, OXA-51, OXA-58and VIM-2, IMP-1, OXA-23and OXA-51were the the predominant genotypes.