The Combined Antibacterial Effects And Mechanisms Of Fosfomycin And Rifampin Against Carbapenem-resistant Pseudomonas Aeruginosas

BackgroundPseudomonas aeruginosa is a gram-negative pathogenic bacterium that is prone to cause acute and chronic infections.Pseudomonas aeruginosa can survive in human-related environme-nts with minimal nutritional requirements.According to the CHINET surveillance(China Antim-irobial Surveillance Network)in 2020,Pseudomonas aeruginosa was one of the top 4 Gram-ne gative bacteria species isolated from all clinical samples.Importantly,over the last few decad-es,there has been a steady increase in the number of drug-resistant Pseudomonas aeruginos a and carbapenem-resistant Pseudomonas aeruginosa.In 2020,23.2%of Pseudomonas aerugin-osa reported to CHINET were not sus-ceptible to imipenem and 19.3%were not susceptible to meropenem.Drug resistance is usuall-y multifactorial entailing decreased permeability of bacterial outer membrane,increased efflux,and enzymatic activity.Additionally,biofilms of antibiotic-resistant Pseudomonas aeruginosa occur with high frequency,both in vitro and in the lungs of CF patients.The development of brand-new antibacterial drugs is time consuming,costly and requires excellent research team.Therefore,drug combination becomes one of the great strategies against drug resistance.Re-cent years,many researchers found that some ant-imicrobial combinations exhibit great activities against multidrug-resistant Pseudomonas aerugi-nosa.Fosfomycin is an antibiotic that has been used clinically for a long time.It has broad-spectrum antibacterial activity against Gram-positive and Gram-negative bacteria,now mainly used in combination with other classes of antibiotics.Rifampicin shows good activity against staphylococcal biofilms,and has become one of the drugs in the standard treatment method for the treatment of joint infections around prostheses.While rifampin and fosfomycin combination treatment is established for staphylococcal implant-associated infections,the optimal treatment for Pseudomonas aeruginosa infections has not been determined.Objectives:To investigate the antibacterial effects of FOS and RIF against CRPA invitro and preliminary explore the potential antibacterial mechanisms.Method:A broth microdilution method was used to determine antibacterial activity of FOS combined with RIF against Pseudomonas aeruginosa.The FICI model is used to explain the interactions in vitro of drug combinations.Crystal violet assay was used to evaluate the anti-biofilm activity of the drug combination against biofilms.Finally,the swarming motility assay was used to evaluate the swarming motility inhibition effect against CRPA.Results:1.Strains collection,MICs determination and drug resistance backgroundThe MICs of antibiotics were determined by the checkerboard method.According to the outcomes of this assay,10 PA strains(PA11-PA20)resistant to MEM and(or)IPM were defined as CRPA,another 10 strains(PA1-PA10)susceptible to all tested drugs were chosen as control.All CRPA were resistant to CAZ,PIP-T and AZT.Two CRPA strains were intermediate to COL,and one strains was resistant to AMK.2.The synergistic antibacterial effects of rifampin and fosfomycin against Pseudomonas aeruginosaMicro broth dilution method was used to determine bacterial susceptibility in accordance with CLSI M100-S28.This study revealed that rifampin and fos-fomyc in combinations could act synergistically in vitro against all CRPA,the synergistic rat-es was 100%.For strains PA11,the MIC values of FOS alone was 512μg/mL,when RIF wasadded in subinhibitory,the MIC were reduced to 8μg/mL.The final results demonstrated that the combination of rifampin a nd fosfomycin showed synergistic effects on both CRPA and antibiotic-susceptible strains.Time-killing curves verified the antibacterial activity in dynamic.3.FOS and RIF exhibited synergistic anti-biofilm activity against PA pre-formed biofilmsFOS exhibited synergistic effects combined with RIF against PA pre-formed biofilms with FICIs<0.5,resulting in a decrease in the MBIC80 of RIF from 32μg/mL-128μg/mL to 8μg/mL-16μg/mL and the MBIC80 of FOS from>1024μg/mL to 64 μg/mL.4.Inhibition of biofilm formationThe anti-biofilm forming activity of rifampin and fosfomycin was determined by the method of crystal violet staining.Inhibition of biofilm development was assayed in 96-well flat bottom polystyrene plates.Both FOS and RIF exhibited anti-biofilm effects when be used alone at sub-MIC concentrations.At 1/8 MIC of RIF and FOS,data showed that REF alone inhibited biofilm formation(approximately 86%),when combined with FOS,96%biofilm reduction was observed.When rifampicin is used in combination with fosfomycin at 1/32MIC,the inhibition of biofilm is increased by 36%compared with rifampicin alone(p<0.0001).The final results demonstrated that the combination of rifampin and fosfomycin showed significant anti-biofilm effects on CRPA5.Eradication of pre-formed biofilmPA11 biofilms were developed in 96-well flat-bottom polystyreneplates with CAMHB for 24 h.After 24h of biofilm formation.FOS and RIF were added and incubated for another 24h.The eradication of pre-formed biofilms activity of rifampin and fosfomycin was determined by the method of crystal violet staining.We finally could observe that the combination of rifampin and fosfomycin could reduce pre-formed biofilmmass with 47%reduction at 1/8MIC of both antibiotics compared with RIF alone(approximately 11%)(p<0.01).6.Inhibition of swarming motilitySwarming motility is the ability of Pseudomonas aeruginosa to move using flagella on semi-solid surfaces is the ability of bacteria.A 2.5 μL aliquot from an overnight culture was spotted at the center of the agar and plates were incubated at 37℃for 24h and the diameters were measured.we found that FOS alone had no effect on swarming motility in comparison with untreated group.RIF could inhibited swarming movement in comparison with untreated group(p<0.05),when combined with FOS showed high inhibition activity on swarming motility of PA 11 compared with RIF alone(p<0.01).Conclusions:In summary,this study reports for the first time that RIF combined FOS exerted synergistic antibacterial activity against Pseudomonas aeruginosa in vitro.Mechanism study have shown that the synergistic effect was related to inhibition of biofilm formation and swarming motility.More mechanisms of synergism would be investigated in the future.This study will provide ideas for develop new antibacterial strategies.