Virginiamycin acetyltransferase D (VatD) plays a vital rule in streptogramins resistance by chemically inactivating streptogramin A. Therefore, it is desirable to discover novel small molecular weight inhibitors of VatD via state-of-the-art virtual screening techniques. This "cocktail" strategy by combining VatD inhibitor with streptogramins may provide new therapeutic opportunity for resistant bacteria infections. Structure-based virtual screening method (molecular docking) was applied to rank and score a chemical database containing 300 000 commercially available compounds against the VatD substrate binding site. Twenty six out of the 200 top scored compounds from the docking calculation were selected and submitted to the VatD enzymatic inhibition assay. The plasmid pRSET B/vatD was constructed and transformed into E. coli (trxB) host cells for over-expression, and VatD enzyme was purified and validated by showing acetyltransferase activity to Virginiamycin M1. Three out of these 26 tested compounds showed enzymatic inhibition on VatD with IC50 168.6, 91.0 and 55.2 micromol x L(-1), separately. Other compounds could not be dissolved in the system and/or had little effect on the enzyme (IC50 > 200 micromol x L(-1)). To our knowledge, it is first time that small molecular weight organic compounds were identified as VatD inhibitors. It is expected that the VatD inhibitors identified at present study could serve as lead compounds for the further development of the novel therapeutic agents to overcome streptogramins resistance.
Acetyltransferases ; antagonists & inhibitors ; genetics ; metabolism ; Catalysis ; drug effects ; Drug Design ; Drug Resistance, Bacterial ; Enzyme Inhibitors ; chemistry ; metabolism ; pharmacology ; Escherichia coli ; genetics ; Genetic Vectors ; Kinetics ; Molecular Structure ; Plasmids ; Streptogramin Group A ; chemistry ; metabolism ; pharmacology ; Transformation, Genetic

OBJECTIVES: Genetic determinants conferring resistance to macrolide, lincosamide, and streptogramin B (MLSB) via ribosomal modification such as, erm, msrA/B and ereA/B genes are distributed in bacteria. The main goals of this work were to evaluate the dissemination of MLSB resistance phenotypes and genotypes in methicillin-resistant Staphylococcus aureus (MRSA) isolates collected from clinical samples. METHODS: A total of 106 MRSA isolates were studied. Isolates were recovered from 3 hospitals in Tehran between May 2016 to July 2017. The prevalence of MLSB-resistant strains were determined by D-test, and then M-PCR was performed to identify genes encoding resistance to macrolides, lincosamides, and streptogramins in the tested isolates. RESULTS: The frequency of constitutive resistance MLSB, inducible resistance MLSB and MSB resistance were 56.2%, 22.9%, and 16.6%, respectively. Of 11 isolates with the inducible resistance MLSB phenotype, ermC, ermB, ermA and ereA were positive in 81.8%, 63.6%, 54.5% and 18.2% of these isolates, respectively. In isolates with the constitutive resistance MLSB phenotype, the prevalence of ermA, ermB, ermC, msrA, msrB, ereA and ereB were 25.9%, 18.5%, 44.4%, 0.0%, 0.0%, 11.1% and 0.0%, respectively. CONCLUSION: Clindamycin is commonly administered in severe MRSA infections depending upon the antimicrobial susceptibility findings. This study showed that the D-test should be used as an obligatory method in routine disk diffusion assay to detect inducible clindamycin resistance in MRSA so that effective antibiotic treatment can be provided.
Bacteria ; Clindamycin ; Diffusion ; Drug Resistance ; Genotype ; Lincosamides ; Macrolides ; Methicillin-Resistant Staphylococcus aureus ; Methods ; Phenotype ; Prevalence ; Staphylococcus aureus ; Staphylococcus ; Streptogramin B ; Streptogramins