ABSTRACT
Objective To investigate the mechanism of drug resistance of sulbactam mediated by TEM-1 beta-lactamases and porin CarO in clinical strains of acinetobacter baumannii. Methods Twenty-four unrepeated clinical acinetobacter baumannii strains were divided into sensitive strain group (n=6) and insensitive strain group (n=18) by susceptibility testing to sulbactam.Antibiotics susceptibility test was carried out using the Kirby-Bauer method.BlaTEM-1and carO genes were amplified by PCR.Four blaTEM-1positive strains(A3,A5,1327 and C1)were selected,and their amplified products were sequenced.The quantitative real-time RT-PCR was used to analyze mRNA transcriptional levels of blaTEM-1 and carO genes. Results Resistant rates of the sensitive strain group for meropenem, imipenem, cefoperazone, ciprofloxacin,gentamicin and ampicillin were 1/6,2/6,3/6,1/6,5/6 and 5/6,and resistant rates of the insensitive strain group were 6/18,10/18,18/18,18/18 and 18/18.BlaTEM-1genes were amplified in 16 insensitive strains,and blaTEM-1was negative in sensitive strains. The carO genes were amplified in all 24 strains.There was no significative mutation in the 4 strains of blaTEM-1genes. The promoters of the strains A3, A5 and 1327 were P4, and C1 was P3. There was a positive correlation between the mRNA expression of blaTEM-1and the MIC value of sulbactam (rs=0.551, P=0.027). There was no difference in the mRNA expression of carO between the two groups.Conclusion The clinical strains are seriously resistant to antibiotics.The main resistance mechanism of clinical strains to sulbactam is the high mRNA expression of blaTEM-1,and the promoter may be one of the reasons of high expression of TEM-1.
ABSTRACT
Objective To Screen the carbapenem-resistant acinetobacter baumannii so as to understand the drug resistance,and to analyze the outer membrane protein expression between the carbapenem-resistant Acinetobacter baumannii and the sensitive strains.Methods The twofold agar dilution method was used to screen the carbapenem-resistant acinetobacter baumannii.PCR technique was used to detect the outer membrane protein-encoding gene carO.SDS-PAGE was used to analyze the expression of outer membrane protein.Results 32 strains were carbapenem-resistant acinetobacter baumannii separated from 1 10 strains,which were 100% sensitive to polymyxin B and were resistant to other drugs with different degrees O.They all carried carO gene.There were different protein bands between the carbapenem-resistant strains and the sensitive strains,in which two protein bands of 50× 10 3 and 22×10 3 were recognized as outer membrane proteins through the analysis of SDS-PAGE.Conclusion Drug resistance of acinetobacter baumannii is serious,and the outer membrane protein expression between the carbapenem-resistant strains and the sensitive strains presents discrepancy.
ABSTRACT
ObjectiveTo study the characteristics of the distribution and drug resistance ofAcinetobacter baumannii, and the epidemiology of the main strains among wards and hospitals, and to investigate the role of outer membrane protein in producing resistance against carbapenems.Methods 145Acinetobacter baumannii strains were collected from July 2013 to July 2014 from Huangdao Hospital Affiliated to Qingdao University and 401st Army Hospital. Antimicrobial susceptibility test was carried out with K-B disk diffusion method. Enterobacterial repetitive intergenic consensus polymerase chain reaction (ERIC-PCR) was used for DNA typing and test of homology. The carO gene of outer membrane protein was amplified by PCR, and the outer membrane proteins were extracted by ultrasonication and ultracentrifuge method from 30 randomly selected carbapenem-resistantAcinetobacter baumannii and 17 carbapenem-sensitive strains. Sodium dodecyl sulfate-polyacrylamide gel electropheresis (SDS-PAGE) was used to analyze the expressions of outer membrane proteins.Results 145Acinetobacter baumannii strains were generally resistant to 16 common antimicrobial agents, with the highest susceptibility rate of 79.3% for minocycline, followed by susceptibility rate of 40.7% for amikacin. There were 128 carbapenem-resistant strains (resistance rate of 88.3%), 137 multidrug-resistant strains and 126 extensively drug-resistant strains. The detection rates of carO gene were 97.7%(125/128)and 17.6%(3/17) in carbapenem-resistant and sensitive strains respectively. Around position of relative molecular mass 47 000, 16 of 17 sensitive isolates were expressed this protein, while 20 of 30 resistant ones were detected nothing or fade; 13 of 17 sensitive isolates were expressed around position of relative molecular mass 37 000 and 29 000 while 25 were detected nothing or fade around position of relative molecular mass 37 000 and 23 were detected nothing or fade around position of relative molecular mass 29 000 in 30 resistant ones. 145Acinetobacter baumannii were classified into 8 types based on ERIC-PCR electrophoresis patterns, and the major prevalence types were type A (71 strains) and type E (37 strains).Conclusions Drug resistance of clinically isolatedAcinetobacter baumannii is a serious problem in two hospitals; drug-resistant strains are spread and epidemic among wards and hospitals. The carO gene of outer membrane protein is widespread in carbapenem-resistantAcinetobacter baumannii. The loss or fading of outer membrane protein may play an important role inAcinetobacter baumannii resistance to carbapenems drugs.