Emergence and spread of low-level mupirocin resistance in staphylococci have been increasingly reported in recent years. The aim of this study was to characterize missense mutations within the chromosomal isoleucyl-tRNA synthetase gene (ileS) among clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) with low-level mupirocin resistance. A total of 20 isolates of MRSA with low-level mupirocin resistance (minimal inhibitory concentration, 16-64 microgram/mL) were collected from 79 patients in intensive care units for six months. The isolates were analyzed for isoleucyl-tRNA synthetase (IleS) mutations that might affect the binding of mupirocin to the three-dimensional structure of the S. aureus IleS enzyme. All isolates with low-level mupirocin resistance contained the known V588F mutation affecting the Rossman fold, and some of them additionally had previously unidentified mutations such as P187F, K226T, F227L, Q612H, or V767D. Interestingly, Q612H was a novel mutation that was involved in stabilizing the conformation of the catalytic loop containing the KMSKS motif. In conclusion, this study confirms that molecular heterogeneity in ileS gene is common among clinical MRSA isolates with low-level mupirocin resistance, and further study on clinical mutants is needed to understand the structural basis of low-level mupirocin resistance.
Staphylococcus aureus/drug effects/*genetics ; *Mutation, Missense ; Mupirocin/*pharmacology ; Methicillin Resistance ; Isoleucine-tRNA Ligase/*genetics ; Intensive Care Units ; Humans ; Electrophoresis, Gel, Pulsed-Field ; Drug Resistance, Bacterial

OBJECTIVETo investigate the antibacterial activity of silver sulfadiazine (SD-Ag), mupirocin, and clotrimazole used alone or in combination against methicillin-resistant Staphylococcus aureus (MRSA) isolated from burn wounds.

METHODSEighteen MRSA isolates from wound excretion of 18 burn patients hospitalized in our unit from July to December 2011 were collected continuously and non-repetitively. (1) Minimum inhibitory concentration (MIC), 50% MIC (MIC50), and 90% MIC (MIC90) of SD-Ag, mupirocin, and clotrimazole used alone, those of SD-Ag and mupirocin used in combination, and those of SD-Ag, mupirocin, and clotrimazole used in combination to MRSA were determined by checkerboard agar dilution method. (2) Fractional inhibitory concentration (FIC) index was calculated to determine the combined effect of SD-Ag plus mupirocin, and SD-Ag plus mupirocin and clotrimazole. Synergy with FIC index less than or equal to 0.5 or additivity with FIC index more than 0.5 and less than or equal to 1.0 was regarded as effective, and indifference with FIC index more than 1.0 and less than or equal to 4.0 or antagonism with FIC index more than 4.0 was regarded as ineffective. The effective ratio was compared with overall ratio (assumed as 0) by unilateral binomial distribution test.

RESULTSThe MIC, MIC50, and MIC90 of SD-Ag, mupirocin, and clotrimazole used alone against 18 MRSA isolates were respectively 8, 8, 16 µg/mL; 2, 16, 64 µg/mL; 2, 2, 2 µg/mL. MIC of antimicrobial agents used in combination decreased from 3.1% to 50.0% as compared with that of individual agent used alone. Compared with those of single application of SD-Ag and mupirocin, MIC50 of SD-Ag and that of mupirocin both decreased 75.0%, and MIC90 of them decreased 87.5% when SD-Ag and mupirocin were used in combination. Compared with those of single application of SD-Ag, mupirocin, and clotrimazole, MIC50 of SD-Ag, mupirocin, and clotrimazole respectively decreased 75.0%, 87.5%, and 50.0%; MIC90 of them respectively decreased 87.5%, 96.9%, and 50.0% when SD-Ag, mupirocin, and clotrimazole were used in combination. Among the 18 MRSA isolates, the combined effect of SD-Ag and mupirocin was synergic in 9 isolates, additive in 7 isolates, indifferent in 2 isolates, and antagonistic in 0 isolate; the combined effect of SD-Ag, mupirocin, and clotrimazole was additive in 16 isolates, indifferent in 2 isolates, and antagonistic in 0 isolate. There were statistically significant differences between effective ratio and overall ratio of 18 MRSA isolates treated with combined antimicrobial agents (P values all above 0.01).

CONCLUSIONSFor burn wounds at middle and late stages infected with Staphylococcus aureus or Staphylococcus aureus and Fungus, low dose of SD-Ag or combination of above-mentioned antimicrobial agents can effectively control infection and decrease the adverse effect of antimicrobial agents on wound healing.

Adolescent ; Adult ; Aged ; Anti-Bacterial Agents ; administration & dosage ; adverse effects ; pharmacology ; Burns ; microbiology ; Child ; Child, Preschool ; Clotrimazole ; administration & dosage ; adverse effects ; pharmacology ; Drug Therapy, Combination ; Female ; Humans ; Infant ; Male ; Methicillin-Resistant Staphylococcus aureus ; drug effects ; isolation & purification ; Middle Aged ; Mupirocin ; administration & dosage ; adverse effects ; pharmacology ; Silver Sulfadiazine ; administration & dosage ; adverse effects ; pharmacology ; Young Adult