In Vivo And In Vitro Study On The Inhibitory Effect Of Fosfomycin Combined With Linezolid On Enterococcus Faecalis Biofilm And Its Synergistic Effect

Objectives: The synergistic effect of the combination of fosfomycin and linezolid has been supported in several previous experimental studies,but the combination’s ability to prevent biomembrane formation needs to be further validated.In this experiment,the ability of the combination of fosfomycin and linezolid to limit the synergistic antimicrobial activity of static biofilm in vitro was consistent with the synergistic antibacterial activity of combined medication,and whether the synergistic antibacterial effect of the drug combination in vivo model was consistent with the in vitro additive antibacterial effect,and at the same time,the virulence gene expression of the strain mutant obtained by inducing fosfomycin resistance in vitro was preliminarily studied,which provided an effective theoretical solution for the prevention and treatment of drug-resistant bacteria that may be caused by the use of a single drug to treat biofilm infection.Methods: In this study,（1）we use 19 strains of Enterococcus faecalis and these were clinically isolated from 5 hospitals in Anhui Province in the whole year of 2020,and bacterial identification was carried out by flight mass spectrometry instruments,susceptibility tests were carried out by agar ratio dilution method and FICI determination by chessboard method.（2）To establish the BF infection model of Enterococcus faecalis in vitro,screening strong positive biofilm strains by crystal violet staining,and determining the BF matrix growth curve of strongly positive BF strains by crystal violet staining.We use single drug and two drugs combined with three treatment regimens,also use crystal violet staining,MTT experiments to detect bacteriostatic concentrations and sub-steptost concentrations,the formation of BF matrix and the change of the number of live bacteria in the membrane under conditions of crystal violet staining,and study the effect of the combination of fosfomycin and linezolid on the BF phenotype of Enterococcal BF;Heterogeneous bacterial profiling to analyze the presence of heterogeneous resistant sub-popules of Enterococcus faecalis susceptible to linezolid and fosfomycin.（3）To establish and compare the fosfomycin,linezolid monotherapy,and fosfomycin/linezolid combination of fosfomycin,linezolid,and fosfomycin/linezolid was constructed and compared with the results of the checkerboard test in vitro.Based on the half-lives of fosfomycin and linezolid,single and multiple dosing regimens were used,and the distinction between synergy and additive action was based on a comprehensive assessment of larval survival,hemolymphatic bacterial load,and case scoring index（for internal criteria in the experimental group）.（4）To perform the induced fosfomycin resistance in vitro for 3 clinical isolates in vitro,determining the MIC by micro-broth dilution method,BF production capacity of sensitive bacteria was assessed by crystal violet staining method,the effect of susceptible bacteria and induced drug-resistant bacteria on larval survival was determined by the large wax borer infection model,the up-regulation and down-regulation of virulence genes was determined by q RT-PCR,the in vitro competition experiment and the growth rate determination method of enterococcus were used,and the number of colonies of sensitive bacteria and mutant resistant bacteria within 48 hours was calculated by point plate method.Calculate the in vitro competition index and draw a polyline chart of in vitro competitive growth,and compare the difference between the growth ability of sensitive bacteria and corresponding mutant resistant bacteria in separate culture and mixed culture.Results:（1）In 19 strains of Enterococcus,the results of the MIC assay of linezolid showed that 89.5% of Enterococcus showed sensitivity,5.26% showed mediatation,and 5.26% showed drug resistance.In the assay of fosfomycin,57.9% of enterococcus showed sensitivity,about 36.8% showed median,and 5.26% showed resistance.Fici values for all the strains tested indicate that linezolid combined with fosfomycin has synergistic or additive antimicrobial activity in vitro for most of the strains under test,and no antagonism was seen in the test results of all strains.（2）By constructing an in vitro BF infection model,it was determined that the BF of Enterococcus faecalis that exhibited strong positive BF reached maturity at 24 h,that is,the BF matrix of Enterococcus faecalis reached its peak at this time,and among the 19 clinical isolates,the strong positive BF strains accounted for 57.9%.Under the conditions of bacteriostatic concentration and sub-steptic concentration,the formation of BF and the number of live bacteria in the membrane can be effectively inhibited,and the difference is statistically significant（P<0.05）,under the condition of single treatment,the inhibition rate of linezolid monodrug is better than that of fostomycin,and the difference is statistically significant（P<0.05）Under the condition of combined medication,the two low-dose fostomycin combined with linezolid have a strong synergistic effect on the bacteriostatic effect of the number of live bacteria in the BF membrane.（3）The lethality of Enterococcus faecalis on the larvae of the great wax borer depends on the concentration of bacterial fluid,the lethal dose of different clinical isolates of enterococcus is around 108,reflecting that there is no significant difference in the virulence between different enterococcal bacteria,and the FICI standards for strains measured by in vitro checkerboard method are different,linezolid and fosfomycin show strain-specific synergistic effects in both dosing regimens,and all the synergistic effects observed in vitro can basically be reproduced in the larvae.Even strains that exhibit additive effects in vitro still show good synergy in in vitro experiments.（4）Three strains of fosfomycin-susceptible strains induced drug-resistant mutants,6 strains of Enterococcus can produce biofilm,and the biofilm formation capacity of the mutant strains is much higher than that of the sensitive strains,and in the large wax borer infection model,the virulence of the 3 resistant mutant strains is lower than that of the sensitive strains;q RT-PCR results showed that the expression of two virulence genes was up-regulated;In the experiments of in vivo competition between sensitive strains and drug-resistant strains,the growth competitiveness of sensitive strain 227 was higher than that of drug-resistant mutant strain 227 R,and the above experimental results showed that after the occurrence of fosfomycin-sensitive bacteria 227 in vitro to induce high levels of fosfomycin resistance,there was a biological cost,and the biological costs also occurred after strains 220 and 225 were resistant to fosfomycin.Conclusion:（1）Fosfomycin combined with linezolid in the treatment of enterococcal infection has in vitro synergistic and additive effects.（2）The presence of heterogeneous drug-resistant subpopulations of enterococci susceptible to fosfomycin and linezolid may be one of the important reasons for strain growth during monosotherapy.（3）In the vitro,the fosfomycin and linezolid showed additive effect strains,and in vitro in large wax borer infection model as a synergistic effect.（4）High levels of fosfomycin resistance may have biological costs,which are manifested in weakened virulence of resistant mutant bacteria,as well as upregulation of some virulence genes and growth retardation in vivo and in vivo.High-level fosfomycin-resistant strains are less competitive in growth than susceptible strains.