Prevalence And Mechanism Studies For Cefiderocol Heteroresistance

BackgroundAntimicrobial resistance is more serious and critical in recent years,which has become a concerned problem in medical field.As the last line of defense against carbapenem resistant bacteria,such as polymyxin and tegacycline,have been resistant to drugs successively,Carbapenem-resistant bacteria have been included in the list of priority pathogens for the development of Class 1(serious)new antibiotics by WHO,posing a serious threat to the health of infected patients.The research and development of new drugs are increasingly urgent.Cefiderocol(CFD)is a novel siderophore-cephalosporin marketed in the United States in 2019 but has not yet entered the clinic in China.It can simulate the binding of siderophore with iron and enter pericytoplasm with the help of Ton B-dependent extracellular receptors on bacteria.It has better penetration and efficacy against infection by carbapenem-resistant gram-negative bacteria.However,investigation data of phase Ⅲ in abroad showed that CFD treatment failed against some carbapenemresistant bacteria,accompanied by high prevalence of CFD heteroresistance(HR)bacteria.However,the prevalence of CFD HR bacteria in China is rarely reported.HR is a special type of antimicrobial resistance and a phenotype in which the treated bacteria contain subpopulations with lower susceptibility to the antibiotic than the dominant population.And the resistant level of subpopulations is unstable.As its detecting methods has not been widely employed by clinical laboratories,the relative contribution of HR to antibiotic treatment failure and HR mechanism are still poorly understood.and the relevant mechanism remains to be revealed.ObjectivesThe objectives of this study are to investigate the current prevalence of CFD HR bacteria in China,to determine whether CFD HR bacteria can cause treatment failure,and to explore the special mechanism of CFD HR.Methods(1)407 clinical strains were collected from two “AAA” hospitals in southern China,and the samples mainly came from blood,sputum,urine,ascites,etc.Population analysis profiling(PAP)and microtitration population analysis profiling(MPAP)were used to detect the prevalence of HR strains.(2)Carbapenem resistant strains sensitive to CFD(MIC≤4mg/L)were used,including Acinetobacter baumannii(CRAB),Escherichia coli(CRE)and Pseudomonas aeruginosa(CRPA).Each specie of bacteria contained 1 strain of non-heteroresistant(NHR)bacteria and 2 strains of HR bacteria,respectively.The relationship between HR bacteria and treatment failure was explored by in vitro time-kill assay and in vivo mouse infection model.(3)For HR strains(CRAB,CRE and CRPA,2 strains respectively),the HR subpopulation recovery model was constructed to obtain parent bacteria,subpopulation and recovery bacteria,and the mechanism of unstable drug resistance level was further explored.The change in fitness cost of parent bacteria,subpopulation and recovery bacteria was evaluated using growth curves.The whole genome sequencing of parent bacteria,subpopulation and recovery bacteria was conducted to detect the gene mutation.Results(1)HR bacteria were prevalent in areas where CFD was not applied,and the overall HR positive rate was 14.5%(59/407).The distributions of HR bacteria in drug resistant,intermediate and sensitive were 8.5%(5/59),5.1%(3/59)and 86.4%(51/59),respectively.(2)Time-kill assay indicated that NHR strain(including CRAB,CRE and CRPA,1 strain respectively)could be completely killed by 4mg/L CFD.Among HR strains(including CRAB,CRE and CRPA,2 strains respectively),33.3%(2/6)were found to have the minimal bactericidal concentration(MBC)of CFD up to 16mg/L,33.3%(2/6)were found to have the MBC of 8mg/L,and 33.3%(2/6)were found to be completely killed by 4mg/L CFD.It is suggested that higher concentration of bactericidal is needed to treat HR bacteria.In the mouse infection model,the survival rate of mice in all CFD treatment group was significantly higher than that in the PBS control group after the intraperitoneal injection of NHR bacteria.However,the survival rate of mice in CFD group was 16.7%(1/6)higher than that in PBS group after the intraperitoneal injection of HR bacteria,and the rest showed no significant difference,suggesting that HR bacteria could lead to treatment failure.(3)In the HR subpopulation recovery model,the esistant level of the subpopulation was higher than that of the parent bacteria,but after antibiotic-free cultured repeatedly,the resistance level decreased and returned to the level of the parent bacteria(recovery bacteria).Growth curve analysis showed that only 33.3%(2/6)of the subpopulation had a growth rate lower than that of the parent bacteria,and the growth rate of one strain was able to return to that of the parent bacteria.It is suggested that fitness cost can only partially explain the subpopulation recovery phenomenon.Whole genome sequencing results showed that there were gene SNP differences among the parent bacteria,subpopulation and recovery bacteria,among which some gene mutations in the subpopulation were different from those in the parent bacteria and were recovered in the recovery bacteria.Their functions were mainly related to the bacterial type Ⅵ secretion system,quorum sensing system and siderophore receptor.It is suggested that the change of gene repairable SNP may be one of the reasons for the unstable resistant level of HR subpopulation.Among them,there are repairable mutations in siderophore receptor Piu A and type Ⅵ secretion system Vgr G related gene SNPS in HR subpopulation,which can provide a new idea for CFD HR mechanism research.ConclusionsThere is an epidemic of HR bacteria in hospital where CFD is not applied,and HR bacteria that are sensitive to drug susceptibility tests might be neglected and lead to clinical treatment failure.In vitro and in vivo experiments of this study provide evidence that HR bacteria cause CFD treatment failure.The drug resistance level of heteroresistant bacteria subpopulation was unstable and could recover to the level of parent bacteria.The change of adaptive cost could explain part of the subpopulation recovery phenomenon.Gene repairability SNP changes may be one of the mechanisms of unstable resistant levels in HR subpopulation.The results of this study can provide data support for the monitoring and treatment of Chinese CFD HR bacteria,and the exploration for the mechanism of hetero resistance can provide new ideas for subsequent in-depth research.