The Study On Antibacterial Activity Of 6 Types Of TCM And Their Combination With Sulbactam On Acinetobacter Baumannii

Objective: Acinetobacter baumannii(Aba)is a Gram-negative,non-fermenting coccobacillus,and its pathogenicity depends on capsular polysaccharide,enzymes,pilus,iron body and lipid A,etc.Aba can cause multi-site infections including infection of respiratory,urinary tracts,central nervous system,blood,etc.The commonly used antibiotics for the treatment of Aba infections are cephalosporin,enzyme inhibitors,quinolones,tetracycline,aminoglycosides and carbapenems.With the wide and large dose application of antibiotics,Aba has developed severe resistance to many kinds of antibiotics(e.g.60% of Aba was resistant to carbapenems).After infected by Aba,patients with compromised immunity showed mortality rates of 10-43%.Sulbactam has been widely used because it is an effective antibiotic against Aba.Experts recommended Sulbactam using in combination with other antibiotics to treat infections caused by Aba in order to increase cure rate and reduce mortality rate.To find a solution to deal with the antimicrobial resistance of Aba,we focus on traditional Chinese medicines(TCM)which are known to possess broad spectrum antimicrobial activity with no induction of resistance and even with the capability of reversing resistance.In this study,we screened several TCM granules with high antibacterial activity and analyzed their antibacterial activity on Aba when combined with Sulbactam,providing effective experimental evidence for clinical treatment of Aba infections with TCM and the combination of TCM and Western Medicine.Methods:1 We adopted the RAPID 32 GN ATB to identify the strains.Seventy samples were collected from three municipal hospitals of Xingtai from October,2014 to May,2015,including sputum,cerebrospinal fluid,blood,urine,etc.2 Antibiotic sensitivity test: The K-B method was performed to determine the susceptibility of Aba to 17 antibiotics.On the basis of the results,the selected strains were divided into Sensitive and Resistant Aba.3 The IPM-EDTA disk synergy method was used to determine the strains mentioned above for metallo-β-lactamase(MBL).4 Grouping based on MBL production and sensitivity: resistant and MBL positive Aba was the first group;resistant and MBL negative Aba was the second group;sensitive and MBL negative Aba was the third group;sensitive and MBL positive Aba was the fourth group.5 K-B method was used to determine the antimicrobial activity of 15 TCM granules on strains in each group,then TCM granules of high antimicrobial activity were selected.6 Through the broth dilution method,the selected TCM granules MIC were measured and then compared to see whether the TCM granules had differences on antibacterial activity of strain in different groups.7 MICs of TCM in combination with Sulbactam were measured by micro-broth dilution method.The differences in MICs of TCM were compared by combining with Sulbactam before and after.8 The cumulative inhibitory percentage of different concentrations of TCM was compared by combining with Sulbactam before and after.9 The FIC was calculated to determine the combined effect of the antimicrobials.10 Statistical analysis: the data were analyzed by Whonet5.6 and SPSS13.0 statistical software package,with P value less than 0.05 was considered statistically significant.Results:1 Sixty three strains of Aba with a coincidence rate greater than 99.5% were selected to test their susceptibility to 17 antibiotics.According to test results,7 strains were sensitive and 56 strains resistant.(Table 2,Table3).2 MBL production test: 19 strains were MBL positive(positive rate: 30.2%;19/63);44 strains were MBL negative.3 Based on test results,63 Aba strains were grouped: Group 1,19 strains;group 2,37 strains;group 3,7 strains;group 4,no strains(removed)(Table 3).4 Bacteriostatic activity of 15 TCM granules: 9 TCM granules(loosestrife,patrinia,phellodendron,polygonum aviculare,houttuynia,fritillaria,puffball,fringed pink,trichosanthes)showed weak bacteriostatic activity against Aba.6 TCM granules(Chinese gall,Fructus Schisandra Chinensis,Smoked Plum,Coptis Chinensis,Forsythia Suspensa and Momordica grosvenorii)showed high antimicrobial activity against 63 strains.This study would have a further test on six types of Chinese medicine granules MIC with high antibacterial activity(Table 4).5 TCM granules MIC: MIC50 for Chinese Gall,Fructus Schisandra Chinensis,Smoked Plum,Coptis Chinensis,Forsythia Suspensa and Momordica Grosvenorii were 3.9 mg/ml,7.8 mg/ml,15.6 mg/ml,15.6 mg/ml,31.2 mg/ml,31.2 mg/ml,respectively.MIC90 were 15.6 mg/ml,15.6 mg/ml,31.2 mg/ml,62.5 mg/ml,62.5 mg/ml,62.5 mg/ml,respectively.No statistical difference was observed for the bacteriostatic activity of 6 TCM granules on three groups of Aba(P>0.05)(Table 5,Table 6).6 TCM granules produced similar inhibitory activities against Aba regardless of MBL production(P>0.05)(Table 6).7 MICs of TCM after combining with Sulbactam: MIC50 for Chinese Gall,Fructus Schisandra Chinensis,Smoked Plum,Coptis Chinensis,Forsythia Suspensa and Momordica Grosvenorii were reduced to 0.98 mg/ml,1.95 mg/ml,3.9 mg/ml,3.9 mg/ml,7.8 mg/ml,and 15.6 mg/ml,respectively.MIC90 were reduced to 1.95 mg/ml,7.8 mg/ml,15.6 mg/ml,15.6 mg/ml,15.6 mg/ml and 31.2 mg/ml,respectively.After combining with Sulbactam,MICs of TCM granules were significantly decreased and the antibacterial activity was significantly increased(P<0.01)(Table5、Table7).8 Cumulative percentage of inhibition at various concentrations of TCM: Chinensis Gall at 7.8 mg/ml could inhibit the growth of 88.9% Aba;Fructus Schisandrae Chinensis and Smoked Plum at 31.2 mg/ml could inhibit the growth of more than 90% Aba;Coptis Chinensis,Forsythia Suspensa and Momordica Grosvenorii at 31.2mg/ml could inhibit the growth of 70% Aba.With the same cumulative percentage of inhibition,the concentrations of TCM were lower than combining before(Table8).9 The Fractional Inhibitory Concentration(FIC): After TCM granules were combined with Sulbactam,synergism accounted for 50.0-66.7%,additivity accounted for 30.1-42.9%,indifference accounted for 3.2-9.6%,with no antagonism observed(Fig.7).Conclusions1 Aba shows relatively low resistance to minocycline and cefoperazone/ sulbactam while it shows seriously resistance to other antibiotics.2 TCM granules(Chinese Gall,Fructus Schisandra Chinensis,Smoked Plum,Coptis Chinensis,Forsythia Suspensa and Momordica Grosvenorii)have better antibacterial activity for Aba.6 TCM granules on MBL positive Aba and MBL negative Aba have the same antimicrobial activity.3 Most of 6 TCM granules,after combining with Sulbactam,show increased antibacterial activity against Aba with synergistic and additive effect accounting for more than 90%,indicating the combination of TCM and antibiotics can play a synergistic effect in the treatment of Aba infection.