Effectsof sub-inhibitory concentrations of nafcillin, vancomycin, ciprofloxacin,and rifampin on biofilmformation of clinical methicillin-resistant Staphylococcus aureus

Methicillin-resistant Staphylococcus aureus (MRSA) infections are often difficultto treat because of their biofilm-formingability and antimicrobial resistance. We investigated the effectsof sub-minimal inhibitory concentrations (MICs) of antibiotics on MRSA biofilmformation. Clinical MRSA isolates were grown with sub-MICs (1/256-1/2 × MICs) of nafcillin, vancomycin, ciprofloxacin,and rifampin. The biofilmbiomass was measured using crystal violet staining. Of the 107 MRSA isolates tested, 63 (58.9%) belonged to sequence type 5 (ST5), and 44 (41.1%) belonged to ST72. The MIC50/MIC90 values of nafcillin, vancomycin, ciprofloxacin,and rifampin were 256/512, 1/2, 64/512, and 0.008/0.03 mg/L, respectively. The sub-MICs of nafcillin, vancomycin, ciprofloxacin,and rifampin promoted biofilmformation in 75 (70.1%), 49 (45.8%), 89 (83.2%), and 89 (83.2%) isolates, respectively. At sub-MICs of nafcillin, the factors associated with strong biofilminduction were the ST5 strain (P = 0.001) and agr dysfunction (P = 0.005). For the sub-MICs of ciprofloxacin,the associated factors were the ST5 strain (P = 0.002), staphylococcal protein A type t002 strain (P < 0.001), and ciprofloxacinresistance (P < 0.001). Among the sub-MICs of rifampin, only ST5 was associated with strong biofilminduction (P = 0.006). Because the sub-MICs of rifampin were much lower than clinically relevant concentrations, we further tested the capability of biofilminduction in 0.03-32 mg/L of rifampin. At these concentrations, rifampin-induced biofilmformation was rare in rifampin-susceptible MRSA [1.0% (1 of 100)] but common in rifampin-resistant MRSA [71.4% (5 of 7), P < 0.001]. Induction of biofilmbiomass at sub-MICs of antibiotics is common in clinical MRSA isolates and is differentiallyaffectedby the MRSA strain and antibiotic class.