Fitness Characteristics Of Cfr-Positive MRSA And PK/PD-Based Treatment Regimens

Methicillin-resistant Staphylococcus aureus(MRSA)is an important zoonotic germ that causes a variety of diseases,ranging from skin and skin structure infections(SSSIs),hospital-acquired pneumonia to life-threatening septicemia,endocarditis and toxic shock syndrome.In the recent years,with the increasing use of antimicrobial drugs in clinical and livestock industry,the incidence of multidrug resistant MRSA isolates,especially isolates harbouring cfr gene that confers phenicols,lincosamide,oxazolidinone,pleuromutilin and streptogramin A(‘Ph LOPS_A')resistance,posed a great challenge to global public health.Of note,the adaptive changes of cfr-positive MRSA strains may be an important factor in their widespread dissemination.In this study,we systematically compared the in vitro and in vivo phenotype characteristics at different stages of infection between cfr-positive and negative MRSA strains on the basis of drug-resistance phenotypes and population analysis profiles.In addition,we investigated the pharmacokinetic/pharmacodynamic(PK/PD)relationship of linezolid when combined with rifampicin and defined combinational dosing strategies to successfully treat infections due to linezolid-resistant MRSA carrying the cfr plasmid.In this study,total 150 MRSA strains with human and animal origins isolated from 7different provinces of China from 2011 to 2016 were tested for cfr gene,and 27 cfr-positive strains were obtained with a positive rate of 18.0%.Results from drug resistance analysis of cfr-positive strains showed that resistance rates to florfenicol,clindamycin and valnemulin were>80%,while resistance rate to linezolid was only 14.8%.Notably,cfr-positive strains MRSA strains exhibited low-level resistance to linezolid,with the MICs slightly higher even lower(for some animal-origin isolates)than the CLSI linezolid breakpoint.However,the MIC distribution and MIC₉₀ of linezolid against cfr-positive strains(0.5?64?g/m L and8?g/m L,respectively)were significantly higher than those in cfr-negative MRSA strains(0.5?2?g/m L and 2?g/m L;P<0.01).In vitro time-kill assays showed that linezolid alone at clinically achievable steady state concentration(C_ss)of 16?g/m L had less activity against cfr-positive strains compared with cfr-negative strains.In addition,the population analysis profile(PAP)demonstrated that cfr-negative MRSA strains had a higher ability to acquire the drug-resistant subpopulation under the antibiotic pressure of linezolid,with an AUC_PAPof 34.3 vs.15.5 that observed for cfr-negative strains(P<0.05).We supposed that in vitro PD difference between cfr-positive and negative strains may due to the adaptability change.The phenotype characteristics associated with host fitness were therefore evaluated.As a group,cfr-positive MRSA isolates exhibited significantly reduced susceptibilities to the host defense cationic peptides including thrombin-induced platelet microbicidal proteins(t PMP),human neutrophil peptide 1(h NP-1)and cathelicidin LL-37(P<0.01,0.01 and 0.005 vs.cfr-negative strains,respectively).In addition,increased binding to fibronectin and endothelial cells in parallel with robust biofilm formations and hemolysis activity were observed in cfr-positive vs.-negative MRSA group.These specific in vitro phenotypic profiles correlated with poor outcomes of linezolid monotherapy in the murine pneumonia,bacteremia and SSSI models due to cfr-positive MRSA strains.To find out effective therapeutic strategies to overcome infections due to such resistant strains,synergistic activity between linezolid and rifampicin combination was evaluated by chequerboard method,time-kill assays and Mac Synergy program.Increased susceptibility of cfr-positive MRSA strains to linezolid was observed with rifampicin combination(MIC decreased 2?16 fold vs.linezolid alone).The combination had synergistic activity with fractional inhibitory concentration index(FICI)<0.5 against all cfr-positive MRSA strains.In murine pneumonia model,linezolid demonstrated excellent pulmonary penetration with an ELF/fplasma AUC ratio of 2.68±0.17.The addition of rifampicin significantly improved the efficacy of linezolid in the murine pneumonia model due to MRSA strains,especially the cfr-positive ones.Linezolid f AUC/MIC targets in both plasma and ELF were 2.4?6.7times lower in linezolid and rifampicin combination therapy vs.linezolid monotherapy(P<0.005).When combined with rifampicin,the f AUC/MIC targets of linezolid associated with stasis,1.0 log₁₀ and 2.0 log₁₀ kill were 2.07,6.19 and 14.3 in plasma,and 5.92,17.1and 38.7 in ELF,respectively.In summary,widespread distribution,potential cross-host transmission between human and animals and high risk of cfr-positive MRSA strains in clinics are not only related to the selection pressure of antibiotics,but also to the enhanced host fitness.Our results provided new observations for host adaptation studies of multidrug resistant MRSA strains.More importantly,the magnitudes of combinatorial PK/PD targets determined in this study will provide a framework for the development and optimization of linezolid-based treatment strategies in the treatment of infections due to cfr-positive MRSA strains.