Effect of Modified Dayuansan Combined with Imipenem-cilastatin on Biofilm of Clinically Isolated Multidrug-resistant Escherichia coli / 中国实验方剂学杂志

ABSTRACT

ObjectiveTo screen out the extended spectrum beta-lactamase （ESBL）-producing Escherichia coli with the strongest biofilm-forming ability through experiments， and discuss the effect of modified Dayuansan （MDYS） combined with imipenem-cilastatin and cilastatin sodium on the biofilm of E. coli. MethodThe paper diffusion and crystal violet staining methods were used to identify 19 clinically isolated strains of drug-resistant E. coli-induced enzymes and the biofilm-forming ability. The induced enzymes and the E. coli with the strongest biofilm-forming ability were screened out. The minimum inhibitory concentration （MIC） value of MDYS and imipenem-cilastatin and cilastatin sodium was determined by 2，3-bis（2-methoxy-4-nitro-5-sulfophenyl）-2H-tetrazolium-5-carboxamide （XTT） assay. The 1/2， 1/4， and 1/8 MIC of the water extract of MDYS， imipenem-cilastatin and cilastatin sodium alone， and MDYS combined with imipenem-cilastatin and cilastatin sodium was determined by methyl thiazolyl tetrazolium （MTT） assay to obtain the optimum concentration of drugs. BioFlux dynamically observed the effect of the optimum combined drug concentration on the number of bacteria in the biofilm and the biofilm formation of E. coli， and observed the distribution of live/dead bacteria with a laser confocal scanning microscope. Finally， the morphological changes in bacteria after drug treatment were observed statically by scanning electron microscopy. ResultE5E7 strain was ESBL enzyme and the E. coli with the strongest biofilm-forming ability. The results of MTT assay showed that the MIC values of the water extracts of imipenem-cilastatin and cilastatin sodium and MDYS were 1 mg·L-1 and 250 g·L-1， respectively. The results of XTT assay showed that compared with the blank group， the 1/2， 1/4， and 1/8 MIC MDYS groups and the combined drug groups significantly decreased the number of bacteria in the biofilm （P<0.01）. The inhibitory effect diminished as the concentration of imipenem-cilastatin and cilastatin sodium decreased. Compared with the imipenem-cilastatin and cilastatin sodium group with the same concentration， the combined drug group improved the inhibitory effect on the number of bacteria in the biofilm （P<0.01）. Compared with the MDYS group with the same concentration， 1/2 MIC imipenem-cilastatin and cilastatin sodium combined with 1/2， 1/4， and 1/8 MIC MDYS， 1/4 MIC imipenem-cilastatin and cilastatin sodium combined with 1/2 and 1/4 MIC MDYS， and 1/8 MIC imipenem-cilastatin and cilastatin sodium combined with 1/2 and 1/4 MIC MDYS decreased the number of bacteria （P<0.05， P<0.01）. The results of BioFlux showed that compared with the blank group， the 1/2 and 1/8 MIC imipenem-cilastatin and cilastatin sodium groups had an insignificant effect on the area of biofilm， whereas the 1/2 and 1/4 MIC MDYS groups significantly decreased the area of biofilm. The results under the scanning electron microscopy showed that as compared with the blank group and the imipenem-cilastatin and cilastatin sodium group， the division cycle was significantly longer under the action of MDYS combined with imipenem-cilastatin and cilastatin sodium. The length of the division cycle in the combined drug group was higher than that in drug alone group. ConclusionIn vitro studies reveal that MDYS combined with commonly-used antibiotics can inhibit the biofilm status of multi-drug resistant E. coli， and MDYS has the effect of enhancing sensitization and inhibiting bacteria with synergistic antibiotics.