Study On The Toxicities Of Organic Auxiliary Solvents And A Novel Colorimetric Antibiotic Susceptibility Test In Vitro

ObjectiveMany antimicrobial agents are difficult to dissolve in water,thereby wide application of organic solvents as the auxiliary solvents in the conventional antibiotic susceptibility tests at present.The organic solvents can damage the cell membrane of microorganisms,and reduce the activity of cells or even die,the accuracy of the drug sensitivity test was affected.Therefore,it is necessary to strictly control the type and the used dosage of organic auxiliary solvents in the drug susceptibility tests.This paper attempted to use the conventional drug susceptibility tests,such as broth dilution method and disk diffusion method,making a detailed study on the toxicities of organic auxiliary solvents.The relationship between the solvents of different kinds and the used dosage of the solvent was established by using the effects of different solvents on the tested microorganisms as an indicator.Then all experimental results provided references for the selection of the type and the limited dosage of auxiliary solvent in the antibacterial tests in vitro.A variety of standard drug susceptibility tests often spend a long time,even taking dozens of hours.In addition,the complex process of operation and the requirement of specialized persons are also gating factors in further development of the drug susceptibility tests.So it is very important to establish a rapid,sensitive and simple detecting method of drug efficacy in the scientific research and real application.In our study,a new optical method was constructed to detect drug efficacy in vitro by using a common dye of prussian blue(PB)as a optical probe and Escherichia coli(E.coli)as a model microorganism.Then this new optical method was used to detect the drug efficacy and screen new antibacterial compounds in quick way,which could make up the defects of the existing drug susceptibility tests.MethodsAccording to growth curve method,the conventional drug susceptibility tests(disk diffusion method and broth dilution method)were used to detect inhibitions of organic solvents,such as dimethyl sulfoxide(DMSO),acetone and ethanol,on the activities of E.coli and Staphylococcus aureus(S.aureus).Scanning electron microscopy(SEM)was introduced to observe the changes of bacterial morphology after organic solvents treatment.K3[Fe(CN)6] was a medium in the optical method because it was able to quantitatively transfer the electrons produced by microbial respiration when its ions contacted with the bacterial cells.The reductive production of K4[Fe(CN)6] could react with Fe Cl3,and then PB was generated.The concentration of PB should decrease because the generation of K4[Fe(CN)6] was hindered through the inhibitions of antibacterial drugs on the respirations of microorganisms.Based on the relationship of PB generations and bacterial respirations,the drug susceptibility on the microorganisms could be fixed by detecting the changes of PB concentration at 700 nm optical absorbance.The optimum experimental conditions were screened by studying the bacterial concentrations,incubation time and addition of nutrients.Then the developed optical method with PB as a probe was used to determine the effectiveness of antibiotics.ResultsWhen the solvent inhibitory effects on E.coli and S.aureus were 20%,the concentration(V/V)of auxiliary solvents,such as DMSO,acetone and ethanol,were 1.00%,0.25%,2.00% and 1.0%,1.0%,0.5% using the broth dilution method,respectively.For the growth curve method,the concentrations(V/V)of DMSO,acetone,ethanol were 0.5%,1.0%,0.5% and 1.0%,0.5%,0.5% when 20% inhibitory effects on E.coli and S.aureus were reached.DMSO and ethanol showed obvious inhibitory rings for E.coli at the some concentration(V/V)in 32%,32% acetone could not induced inhibitory ring for E.coli,but no inhibitory rings appeared for S.aureus in the same concentrations of 3 studied solvents with disk diffusion method.To compare three methods mentioned above,the inhibitions of DMSO,acetone and ethanol were lower than 20% when their concentrations(V/V)all lower than 0.5%.Under this condition,effects on the viability of bacteria were slight.Comparing with the broth dilution method,auxiliary solvents showed lower effects on the bacterial viabilities.At the same time,SEM showed that limited solvent concentrations could effectively decrease their effects on the morphology of E.coli.The inhibition of antibiotics on the respirations of E.coli were investigated when 5% efficacy(EC5%)of drugs were defined as the concentration of antibiotics with 5% inhibitions on the objective microorganisms.The applied concentrations of eight antibiotics,such as cefepime,ceftriaxone sodium,cefoperazone sodium,piperacillin sodium,amoxicillin,gentamicin,amikacin and levoloxacin were 2,2,4,4,10,4,8 and 8 mg·L-1 using PB method to detect their EC5% on 20 clinical isolates of E.coli.To compare with the minimum inhibitory concentration results of broth macrodilution method recommended by Clinical and Laboratory Standards Institute(CLSI),the results of PB optical method showed good agreements except with gentamicin.Paired t-test result(P<0.05)also showed that difference between two methods was statistically significant.ConclusionsCompared with DMSO and acetone,ethanol showed a stronger effect on growth and reproduction of microorganisms.After incubation with the same concentrations of organic auxiliary solvents,liquid testing condition,such as broth dilution method and growth curve method,induced the stronger effect on the cells.Therefore,the used type of organic solvents and their suitable usage should be synthetically considered in the drug susceptibility tests.It was different from conventional drug susceptibility tests,the optical method based on PB probe with faster and simpler properties.Moreover,optical method showed a good sensitivity in the detection of antibiotics.So our method is a potential application in the clinical diagnoses and in vitro measurement of antibiotic efficacy against bacteria.