Study On Venenum Bufonis Extracts In Combination Of Beta-lactam Antibiotics Against Multi-drug Resistant Acinetobacter Baumannii In Vitro

Objective To investigate the prevalence of Acinetobacter baumannii infection,resistance to commonly used antimicrobial and phenotypes of beta-lactam resistance in multi-drug resistant isolations.The efficacies of Venenum Bufonis extracts in combination of beta-lactam antimicrobials effected on multi-drug resistant Acinetobacter baumannii was analyzed.The underlying mechanisms of synergy between Venenum Bufonis extracts and beta-lactam antimicrobials for multi-drug resistant isolations was clarified preliminarily.Methods A retrospective study was conducted to know the distributions of Acinetobacter baumannii clinical isolations and the antibiotics resistance were analyzed.The minimum inhibitory concentrations(MIC)of 4 kinds of beta-lactamase antibiotics including SCF,CAZ,TZP and IMP/CS for 100 multi-drug resistant Acinetobacter baumanni strains were determined by agar dilution method.The broth micro-dilution method was used to assay the MICs of water extracts and alcohol extracts from Venenum Bufonis on multi-drug resistant Acinetobacter baumannii,as well as the efficacies of which combined 4 antibacterials effected on multi-drug resistant Acinetobacter baumannii.The resistant genotypes of 40 strains of Acinetobacter baumannii were examined by polymerase chain reaction(PCR).The Real-time PCR was used to detect the drug-resistance genes expression of antibacterials against multi-drug resistant Acinetobacter baumannii in different treatment times.Results A total of 1112 clinical isolations were detected,of those,sputum samples were detected dominantly(82.37%,916/1112),and intensive care unit(61.6%,685/1112),elderly cadres ward(11.6%,129/1112)and respiratory department(9.98%,111/1112)were detected respectively.The drug sensitivity results showed Acinetobacter baumannii were highly resistant to antibiotics with respect to cephalosporin,aminoglycosides and quinolones.The prevalence of cefoxitin-resistant,cefmetazole-resistant,amoxicillin/clavulanate-resistant strains reached 99.7%,100%and 98.8%,respectively.Water extracts and alcohol extracts from Venenum Bufonis had effective on multi-drug resistant Acinetobacter baumannii directly,and the difference in effect between two kinds of liquid had no significance(P>0.05).The results of subinhibitory concentration of Venenum Bufonis combined with four kinds of beta-lactam antibiotics on multi-drug resistant Acinetobacter baumannii showed that the MIC of imipenem/cilastatin,piperacillin/tazobactam and Cefoperazone/sulbactam could be reduced by one or two titer within 0.5,1,2 or 4 hours of administration.According to the different effective time points,the MIC of SCF,CAZ,TZP and IMP/CS on multi-drug resistant Acinetobacter baumannii showed obviously difference by repetitive measure analysis(F=43.179,P=0.000).Beta-lactam antibiotics related resistance genes including TEM,SHV,CTX-M-I,CTX-M-9,OXA-2,VIM-2,SIM,OXA-23,OXA-24 and IMP were detected for 65%,72.5%,7.5%,70%,5%,12.5%,12.5%,72.5%,37.5%and 10%,respectively.The expressions of OXA-23 and OXA-24 were both decreased after 2 h of water extracts or alcohol extracts administration,and OXA-24 expression decreased more obviously after 2 h of water extracts administration and the effect gradually reduced after 2 h.OXA-23 and OXA-24 expressions were suppressed quickly after 0.5 h of alcohol extracts administration,and the inhibition time maintained longer(more than 16 h)and the strength was superior to that of water extracts administration.Conclusion Bauman Acinetobacter clinical isolations were prevalent,and the phenomenon of multi-drug resistance was serious.A variety of beta-lactame genotypes were detected in multi-drug resistant Acinetobacter baumannii.Both of Venenum Bufonis extracts by water and alcohol methods have antibacterial effect on multi-drug resistant Acinetobacter baumannii in virto.Venenum Bufonis extracts in combination of beta-lactam antimicrobials had synergistic antibacterial effect on multi-drug resistant Acinetobacter baumannii.The potential mechanism may be Venenum Bufonis extracts could inhibit OXA-23,OXA-24 gene expression which showed time-dependent effect.