Studies On Staphylococcus Aureus And Enterococcus Faecium Resistance Development And Mechanisms To The Investigational Oxazolidinone MRX-I

Staphylococcus aureus and Enterococcus faecium can cause various community and hospital-acquired infections. Recently the cost of the treatment and the rate of mortality are increasing for the infection caused by multi-resistant strains, and which leads to lack of effective drugs. There are very limited choices of drugs to treat infection caused by methicillin-resistant S. aureus (MRSA) and vancomycin-resistant E. faecium (VRE).Oxazolidinone antimicrobial agents are a recent class of synthetic antimicrobial agents that inhibit baterial growth by perturbing bacterial protein synthesis via binding to the 50S ribosomal subunit. To date, the sole approved oxazolidinone linezolid was effective against infections caused by multidrug-resistant Gram-positive bacteria, including MRS A and VRE, but showed adverse effects such as bone marrow toxicity. The new family member of the oxazolidinone antimicrobial agents, MRX-I, is under clinical phase II study recently. The safety profile of MRX-Ⅰ was highly improved than linezoild by structure modification. MRX-Ⅰ exhibited good activity against multidrug-resistant including MRSA and VRE.Although the linezolid-resistant strains are rare, resistance was reported in both clinical use and in vitro studies. To date, the frequently reported three mechanisms were in the region of linezolid target, namely the peptidyl transferase centre (PTC) of the bacterial ribosome, including 23 S rRNA genes mutations, mutations in genes (rplC, rplD and rplV) encoding ribosomal proteins and the acquisition of cfr, a gene encoding a ribosomal methyltransferase.The present study was focused on the following four parts, in vitro resistance development for MRX-I as compared to linezolid and the possible resistance mechanisms, the analysis of spontaneous resistance frequency and the mechanisms of spontaneous resistance mutants, in vitro activity against cfr gene carrying Gram-positive strains and the determination of the mutant prevention concentration (MPC) and the mutant selection window (MSW), to better understand the resistance development and mechanisms of resistance of bacteria against MRX-Ⅰ for reducing or minimizing the risk of developing new antimicrobial agents, supporting MRX-Ⅰ in the ongoing clinical study and future clinical use to decrease the resistance.Part OneSelection and characterization of Staphylococcus aureus and Enterococcus faecium mutants with reduced susceptibility to the investigational oxazolidinone MRX-ⅠBackgroud:Staphylococcus aureus and Enterococcus faecium developed resistance very slow in the continuous presence of oxazolidinones. The dominated mutations were mutations in genes at domain V region of 23 S rRNA and genes encoding ribosomal proteins. We investigated the development of the tested strains in the continuous presence of MRX-Ⅰ through serial passages and studied the resistance mechanisms of the mutants in this part.Methods and Results:Seven tested strains were serially passged in the continuous presence of MRX-I at 1/2xMIC until MIC values increased to≥4 MIC by broth dilution assay. Strains were collected at each point of MIC increased by 2-fold or more and tested MIC stability and susceptibility against other drugs. The stable mutants were amplified and sequenced for 23 S rRNA genes mutations, mutations in genes (rplC, rplD and rplV) encoding ribosomal proteins. Linezolid was as control. The resistant mutants in this study refer to mutants with reduced susceptibility, MICs could reach to the breakpoints recommended by CLSI or not. Seven strains were tested in this part. The four S. aureus strains include methicillin-susceptible S. aureus (MSSA) ATCC 29213, MRSA strain ATCC 33591 and two clinical isolated MRSA strains SA 0016 and SA 0017. The three E. faecium strains include vancomycin-susceptible E. faecium ATCC 35667 and two clinical isolated VRE strains, BM4147, and Efm-HS0661.After 30 passages in the presence of MRX-Ⅰ, MICs of MRX-Ⅰ against MSSA and MRSA strains increased by 8-fold or 16 fold. However, MICs of linezolid against MSSA increased by 128-fold, against MRSA increased by 16-32 fold, the MIC increased earlier than with MRX-Ⅰ. Six mutations were found in MRX-Ⅰ mutants, which were U2504A, C2443U/A2513G/U2571C/U2622C/A2629U (new combined mutation, C2443U, A2513G, U2622C and A2629U were new mutations); Five mutation occurred in ribosomal protein L3, which were G152D, G155R, G155R/M169I (M169I was new mutation), F147L/G155R and F147L/G155R/D159Y (new combined mutation). Seven mutations were found in linezolid mutants, which were C2443U/U2474C/A2513G/U2571C/U2622C/A2629U (new combined mutation, U2474C was new mutation).No mutations were indentified in the genes encoding the ribosomal proteins L4 and L22 in any S. aureus strain.After 35 passages (for VSE,16 passages), MICs of MRX-I and linzoild both increased by 4-fold against VSE; 32-fold, and 8 or 16-fold against VRE. No mutation was detected in 23S rRNA except for mutation U2500A,1% appeared at 8 mg/L of linzolid and 2% at 16 mg/L of linezolid. New mutation F24I was identified in the ribosomal protein L4 for VSE in the presence of MRX-I, and new combined mutation D36N/D70N was detected in the ribosomal protein L22 for VRE in the presense of MRX-Ⅰ. No mutation was found in ribosomal protein L3 with MRX-I. Mutation A73 V was detected in ribosomal protein L4 for both VSE and VRE. No mutation was identified in ribosomal protein L3 and L22 with linezolid. Futher studies are needed to conclusively link these mutations to the observed MIC increase in tested strains.Conclusion:S. aureus resistance to MRX-I developed very slow, slower than did resistance to linezolid. Dominated mutations occurred in 23 S rRNA and ribosomal protein L3. E. faecium resistance to MRX-I developed slow and showed major mutations in ribosomal proteins.Part TwoDetermination of spontaneous resistance frequency of Staphylococcus aureus and Enterococcus faecium and studies on resistance mechanisms with spontaneous mutantsBackgroud:Spontaneous resistance frequency shows intrinsic resistance of the strain to a drug. This part of study focused on spontaneous reistance frequency of Staphylococcus aureus and Enterococcus faecium to MRX-I and the mechanisms of the spontaneous mutants.Methods and Results:The spontaneous resistance frequencies of the seven tested strains (including 4 S. aureus and 3 E. faecium, see Part One) on 4×MIC agar plates containing MRX-I were determined. The spontaneous mutants were amplified and sequenced for 23 S rRNA genes mutations, mutations in genes (rplC, rplD and rplV) encoding ribosomal proteins. Other tested drugs included linezolid, vancomycin, fusidic acid, clindamycin and rifampin.Spontaneous resistance frequencies of MSSA ATCC 29213, MRSA ATCC 33591, MRSA SA 0016 and MRSA SA 0017 against MRX-I were<8.25×10-12, <6.33×10-12,<2.96×10-13 and<4.52×10-13, respectively. The frequencies were the same as linzolid against S. aureus,10000-fold lower than other tested drugs. The spontaneous mutants of ATCC 33591 against MRX-I were detected and an insertion mutation ins2468C was found, mutation G2405U was found against linezolid. No mutation was detected in ribosomal proteins L3, L4 and L22 encoding genes.Spontaneous resistance frequencies of ATCC 35667 (VSE), VRE BM4147 and Efm-HS0661 against MRX-I were<1.54x10-11,<1.43×10-11 and<1.18×10-11, respectively. The frequencies were the same lower as linzolid against E faecium, 10-fold lower than other tested drugs at least. No mutation was detected in spontaneous mutants of ATCC 35667 against MRX-I in allele 1 and 2 of 23S rRNA and the three ribosomal proteins. Allele 3-6 of 23S rRNA genes were amplified unsuccessfully. Futher studies are needed to conclusively link these mutations to the observed MIC increase in tested strains.Conclusion:S. aureus (MSSA and MRSA) and E.faecium (VSE and VRE) exhibited low spontaneous resistance frequency to MRX-I, which indicated that the insintric resistant strains were rare. The dominated mutation might be 23 S rRNA changes.Part ThreeIn vitro activity of MRX-I against cfr carrying Gram positive strainsBackgroud:Cfr (Chloramphenicol-florfenicol resistance) methylation confers resistance to strains against five classes of drugs with different chemical structures including oxazolidinones. We investigated the minimal inhibitory concentration (MIC) of MRX-I against cfr carrying Gram-positive strains in this part.Methods and results:Enterococcus faecalis EF01, Bacillus spp.3-151 and Staphylococcus cohnii 2-141 were tested in MIC experiments determined by microdilution method. Tested drugs included MRX-I, linezolid, vancomycin, methicillin, clindamycin, erythromycin, choloramphenicol, tiamulin, florfenicol rifampin, and norfloxacin.MIC of MRX-I (2 mg/L) against Enterococcus faecalis EF01 was close to that of linezolid (4 mg/L, still sensitive according to CLSI standard, but the MIC increased obviously). The strain was susceptible to vancomycin (2 mg/L) and rifampin (1 mg/L), and resistant to other tested drugs.MIC of MRX-I (2-4 mg/L) against Bacillus spp.3-151 was same to that of linezolid (4 mg/L), The strain was intermediate resistant to chloramphenicol (16 mg/L), resistant to clindamycin (>64 mg/L), and sensitive to other tested drugs, vancomycin, methicillin and erythromycin (all 0.5 mg/L), rifampin (<0.031 mg/L), and norfloxacin (4 mg/L).MIC of MRX-I against Staphylococcus cohnii 2-141 was>64 mg/L, same as those of linezolid and other 8 drugs (64 or>64 mg/L) tested. Norfloxacin had antimicrobial activity against this strain.Conclusion:MICs of MRX-I against cfr genes carrying Gram-positive strains were similar to those of linzolid, and showed resistance or MIC increases, indicating that cross resistance existed between MRX-I and linezolid.Part FourStudies on mutant prevention concentration and mutant selection window of MRX-I against Staphylococcus aureus and Enterococcus faeciumBackgroud:When the concentration of the drug is higher than mutant prevention concentration (MPC), it is difficult for bacteria to mutate. Within the mutant selection window (MSW), the resistant mutants are highly possible to be selected, and subject to cause the increase of the resistance. We investigated the MPC and MSW of MRX-I in this part.Methods and results:ca.1010CFU/mL of the seven tested strains (including 4 S. aureus and 3 E. faecium, see Part One) were plated on agar plates with different MRX-I concentrations to detect the MPC (the concentration without no growth of colony), and determine MSW. Other tested drugs included linezolid, vancomycin, fusidic acid, and clindamycin.The MPC and MSW of MRX-I against MSSA were 4 and 2-4 mg/L, MPCs of MRX-I against MRSA were 2 and 4 mg/L, the MSWs were 1-2 or 1-4 mg/L. MPCs of MRX-I against MSSA and MRSA were very close, which all were 2-fold or 4-fold MIC, indicating that MRX-I had similar MSWs for both MSSA and MRSA, and the MSWs were narrow. MPCs of vancomycin against MSSA and MRSA were 32 mg/L, MSWs were 1-32 mg/L. MPCs of fusidic acid against MSSA and MRSA were>2 or>4 mg/L, MSWs were 0.125->4 or 0.063->2 mg/L. MPC of clindamycin against MSSA was 8 mg/L, MSW was 0.125-8 mg/LMPCs of MRX-I against the three tested E. faecium were 4,2 and 4 mg/L, respectively. MSWs were 2-4,1-2 and 1-4 mg/L. The MSWs of MRX-I against VSE and VRE were similar, and all were narrow. MSW of vancomycin against VSE was 0.5-8 mg/L. MSW of clindamycin against VSE was 0.063-1 mg/L, against BM4147(VRE) was 2-64 mg/L.The MSWs of MRX-I against the seven tested strains were similar compared with linezolid,8-fold or narrower than vancomycin and other control drugs.Conclusion:MSWs of MRX-I against the four S. aureus and three E.faecium strains were all ranged from MIC to 2-fold or to 4-fold MIC, which showed 8-fold or narrower than vancomycin, fusidic acid and clindamycin, indicating that MRX-I had lower selection pressure to the sub-group of mutants.