Effect Of Subinhibitory Antibiotic Exposure On Resistance And SOS Gene Expression In Staphylococus Aureus

Objectives:Detect the capacity of subinhibitory concentrations of antimicrobials of different families with different molecular targets to facilitate persistence and evolution of resistance and induce SOS response as a new resistant mechanism during antibiotic therapy in Staphylococcus aureus, to investigate corresponding control strategies.Methods:1. detect susceptibility of 50 Staphylococcus aureus isolates(one per patient),30 of methicillin-resistant Staphylococcus aureus (MRSA) and 20 of methicillin- sensitive Staphylococcus aureus (MSSA),to antimicrobial agents:ciprofloxacin,amikacin,vancomycin by determination of the minimum inhibitory concentrations by agar dilution method, to show whether vancomycin "MIC creep" or resistance exists.2. Select one standard strain,one strain of ciprofloxacin-resistant MRSA and one strain of ciprofloxacin-sensitive MSSA respectively. Real-Time quantitative PCR detection of SOS gene recA expression in isolates after action of single exposure or combined therapy of Sub-MIC antibiotics:1/2MIC ciprofloxacin,1/2MIC amikacin,1/2MIC vancomycin,1/4MIC(ciprofloxacin+ amikacin) or 1/4MIC(ciprofloxacin+ vancomycin) for 0h,6h,12h.3. At Oh and 12h, we tested their MICs of the following 12 antimicrobials by agar dilution method:oxacillin,ceftriaxone vancomycin,linezolid,teicoplanin,minocycline ciprofloxacin,levofloxacin,amikacin,tigecycline methoprim/sulfamethorxazole,rifampin.Then made rank correlation analysis between recA transcription and MICs changes by Spearman method.4. To test for differences between MRSA group and MSSA group in the proportion of vancomycin MICs, Fisher's exact test was applied. Statistical significance was defined as P<0.05.Results:1. A total of 50 strains of collected S. aureus was included in this analysis and 30 (60%) strains were found to be MRSA.65.40% and 21.90% were resistant to ciprofloxacin and amikacin while 100% sensitive to vancomycin. The vancomycin MIC ranged from 0.38 to 2.0 mg/L.we grouped the isolates into those with MIC≤0.5 mg/L(13 MRSA and 11 MSSA), those with MIC=1 mg/L(15 MRSA and 9 MSSA) and those with MIC=2 mg/L(2 MRSA). No statistically significant change in the percentage of isolates with a vancomycin MIC of≥1 mg/L was observed between MRSA and MSSA(P>0.05). For MRSA, the geometric mean vancomycin MIC,MIC50,MIC90 were 0.78,1,1. It was assumed that vancomycin MICs did creep in Staphylococcus aureus isolates here comparing with 0.5mg/L,0.5,0.75 in 2006 respectively.2. Sub-MIC ciprofloxacin,amikacin or vancomycin could upregulate SOS gene recA in S. aureus isolates,with 59.3-fold,8.3-fold,4.3-fold in standard strain respectively and 44.5-fold,1.6-fold,12.0-fold in resistant strain respectively. Conclusion combined therapy of ciprofloxacin and other antibiotics down-regulated increased recA expression by single effect of ciprofloxacin.recA expression in 1/4MIC(ciprofloxacin+amikacin) increased by 3.4-fold in standard strain and 26.3-fold in resistant strain,which were 17.4-fold and 1.7-fold respectively lower than that in 1/2MIC ciprofloxacin. recA expression in 1/4MIC(ciprofloxacin+ vancomycin) increased by 3.3-fold in standard strain and 16.9-fold in resistant strain,which were 18.0-fold and 2.6-fold respectively lower than that in 1/2MIC ciprofloxacin. Nevertheless,recA expressions in sensitive strain were very low with and without sub-MIC antibiotics exposure. 3. At 12h of sub-MIC ciprofloxacin,amikacin or vancomycin exposure,in standard strain ciprofloxacin and levofloxacin MICs increased by 2-4-fold, oxacillin and ceftriaxone MICs 1～2-fold, amikacin MIC about 2-fold,the rest drugs MICs 0～2-fold but in the sensitive range;in resistant strain ciprofloxacin,amikacin,ceftriaxone,levofloxacin,oxacillin,rifampin MICs increased by more than 1 fold, still resistant but the rest had no obvious changes;in sensitive strain 12 antibiotics MICs had no obvious changes.Each antibiotic MIC reduced by 0～1 fold in combined therapy.4. In standard strain, relative quantification of recA had no linear dependence relation with change of corresponding ciprofloxacin MIC(r=0.772,p=0.072>0.05).But in the view of single strain,we became conscious of that inducible antibiotics could cause mild MICs increase of not only themselves but also other drugs such asβ-lactams with upregulated recA expression.Conclusions:1. Of Staphylococcus aureus isolates in Jinan, most were resistant to ciprofloxacin but all sensitive to vancomycin. Vancomycin "MIC creep" have existed.2. In addition to fluoroquinolones, sub-MIC aminoglycosides and vancomycin exposure also induced SOS gene recA expression in S. aureus. 3. Inducible sub-MIC antibiotics could cause mild MICs increase of not only themselves but also other drugs such asβ-lactams with upregulated recA expression,that is,SOS response involves in resistance in S. aureus.4. Combined therapy may reduce the capacity of bacterial SOS response as inhibitors of RecA activity,but has no obvious effect on increasing drugs sensitivity.Here with the consideration of the existence of other resistant mechanism, further study and disscusion is necessary.