Study On The Mutant Selection Window Of Linezolid Combined With Fosfomycin Against Enterococci And The Resistance Mechanism Of The Mutants

Objective: Although the synergistic or additive antibacterial activity of linezolid and fosfomycin has been confirmed in a number of studies,while the ability of the combination to prevent resistance remains unknown.To provide data support for clinical application the combination for enterococcal anti-infective treatment in future,the ability of linezolid combined with fosfomycin to limit the enrichment of enterococci and whether it is consistent with the in vitro antibacterial activity of the combination were studied in this experiment.Meanwhile,the mechanism of for linezolid or fosfomycin resistant mutants obtained from the MPC studies was also explored.Methods:(1)The minimum inhibitory concentration(MIC)of linezolid or fosfomycin against 43 non-repeated clinical isolates of enterococcus were determined by agar dilution method according to CLSI guidelines.The checkerboard assay was performed for the synergy testing and synergy was evaluated by the fractional inhibitory concentration index(FICI).(2)Based on the results of checkerboard assay,five isolates with different value of FICI were selected for the MSW studies.The mutant prevention concentration(MPC)and mutant selection window(MSW)of linezolid,fosfomycin alone and the two agents in combination including different proportions against the five clinical isolates of Enterococcus was tested by the agar dilution method.And the the selection index(SI,MPC/MIC99%)was also calculated.The correlation between SI and FICI was used to analyze whether the ability of the combination to restrict the enrichment of bacterial resistance is in line with the in vitro antibacterial activity of the combination.(3)The resistance mechanism of linezolid-resistant mutants or fosfomycin-resistant mutants was determined by PCR amplification of relevant drug resistance genes and nucleic acid sequencing analysis.Results:(1)The FICI values illustrated that linezolid showed synergy or additivity in combination with fosfomycin against most of the tested strains(69.8%).No antagonistic effect was detected.(2)The results indicated that different proportions of linezolid combined with fosfomycin had presented different MPCs and MSWs.Compared with linezolid or fosfomycin alone,the combination can restrict the enrichment of resistant mutants at a lower concentration.A rough positive correlation between the selection index(SI)of the two agents in combination and the fractional inhibitory concentration index(FICI)of the combination displayed that the smaller FICI of linezolid and fosfomycin,the more probable their MSWs were to close each other.(3)Mutations in ribosomal proteins(L3 and L4)were the mechanisms for linezolid resistant mutants.Among the fosfomycin-resistant mutants,only two fofomycin resistant mutants have detected the Mur A gene mutation related to fosfomycin resistance.Conclusion:(1)In conclusion,the synergistic combination of linezolid and fosfomycin closing each other’s MSW at a certain ratio and could effectively suppress the selection of enterococcus resistant mutants,suggesting that the combination may be an alternative for preventing enterococcal resistance.(2)Research on the resistance mechanism of linezolid or fosfomycin mutants at the gene level shows that the MSW theory has certain adaptability to these two antibacterial drugs.In addition,the resistance mechanism of fosfomycin remains to be further studied.