title: Dithiazole thione derivative as competitive NorA efflux pump inhibitor to curtail multi drug resistant clinical isolate of MRSA in a zebrafish infection model.
abstract: Multi drug resistant (MDR) pathogens pose a serious threat to public health since they can easily render most potent drugs ineffective. Efflux pump inhibitors (EPI) can be used to counter the MDR phenotypes arising due to increased efflux. In the present study, a series of dithiazole thione derivatives were synthesized and checked for its antibacterial and efflux pump inhibitory (EPI) activity. Among 10 dithiazole thione derivatives, real-time efflux studies revealed that seven compounds were potent EPIs relative to CCCP. Zebrafish toxicity studies identified four non-toxic putative EPIs. Both DTT3 and DTT9 perturbed membrane potential and DTT6 was haemolytic. Among DTT6 and DTT10, the latter was less toxic as evidenced by histopathology studies. Since DTT10 was non-haemolytic, did not affect the membrane potential, and was least toxic, it was chosen further for in vivo study, wherein DTT10 potentiated effect of ciprofloxacin against clinical strain of MRSA and reduced bacterial burden in muscle and skin tissue of infected zebrafish by ~ 1.7 and 2.5 log fold respectively. Gene expression profiling of major efflux transport proteins by qPCR revealed that clinical isolate of MRSA, in the absence of antibiotic, upregulated NorA, NorB and MepA pump, whereas it downregulates NorC and MgrA relative to wild-type strain of Staphylococcus aureus. In vitro studies with NorA mutant strains and substrate profiling revealed that at higher concentrations DTT10 is likely to function as a competitive inhibitor of NorA efflux protein in S. aureus, whereas at lower concentrations it might inhibit ciprofloxacin efflux through NorB and MepA as implied by docking studies. A novel non-toxic, non-haemolytic dithiazole thione derivative (DTT10) was identified as a potent competitive inhibitor of NorA efflux pump in S. aureus using in silico, in vitro and in vivo studies. This study also underscores the importance of using zebrafish infection model to screen and evaluate putative EPI for mitigating MDR strains of S. aureus.
authors.affiliation: School of Chemical and Biotechnology, SASTRA University, Thirumalaisamudram, Thanjavur, Tamil Nadu, 613 401, India.|Centre for Research in Infectious Diseases (CRID), School of Chemical & Biotechnology, SASTRA University, Thanjavur, Tamil Nadu, 613401, India.
authors.forename: Rene Christena
authors.initials: RC
authors.lastname: Lowrence
authors.affiliation: School of Chemical and Biotechnology, SASTRA University, Thirumalaisamudram, Thanjavur, Tamil Nadu, 613 401, India.|Centre for Research in Infectious Diseases (CRID), School of Chemical & Biotechnology, SASTRA University, Thanjavur, Tamil Nadu, 613401, India.
authors.forename: Thiagarajan
authors.initials: T
authors.lastname: Raman
authors.affiliation: School of Chemical and Biotechnology, SASTRA University, Thirumalaisamudram, Thanjavur, Tamil Nadu, 613 401, India.
authors.forename: Himesh V
authors.initials: HV
authors.lastname: Makala
authors.affiliation: School of Chemical and Biotechnology, SASTRA University, Thirumalaisamudram, Thanjavur, Tamil Nadu, 613 401, India.
authors.forename: Venkatasubramanian
authors.initials: V
authors.lastname: Ulaganathan
authors.affiliation: School of Chemical and Biotechnology, SASTRA University, Thirumalaisamudram, Thanjavur, Tamil Nadu, 613 401, India.
authors.forename: Selva Ganesan
authors.initials: SG
authors.lastname: Subramaniapillai
authors.affiliation: School of Chemical and Biotechnology, SASTRA University, Thirumalaisamudram, Thanjavur, Tamil Nadu, 613 401, India.
authors.forename: Ashok Ayyappa
authors.initials: AA
authors.lastname: Kuppuswamy
authors.affiliation: School of Chemical and Biotechnology, SASTRA University, Thirumalaisamudram, Thanjavur, Tamil Nadu, 613 401, India.
authors.forename: Anisha
authors.initials: A
authors.lastname: Mani
authors.affiliation: Department of Chemistry, Sri Sathya Sai Institute of Higher Learning, Brindavan Campus, Kadugodi, Bengaluru, 560 067, India.
authors.forename: Sundaresan
authors.initials: S
authors.lastname: Chittoor Neelakantan
authors.affiliation: School of Chemical and Biotechnology, SASTRA University, Thirumalaisamudram, Thanjavur, Tamil Nadu, 613 401, India. sai@scbt.sastra.edu.|Centre for Research in Infectious Diseases (CRID), School of Chemical & Biotechnology, SASTRA University, Thanjavur, Tamil Nadu, 613401, India. sai@scbt.sastra.edu.
authors.forename: Saisubramanian
authors.initials: S
authors.lastname: Nagarajan
mesh_terms: D000818:Animals; D000900:Anti-Bacterial Agents / Q000008:administration & dosage*; D058491:Bacterial Load; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors*; D002939:Ciprofloxacin / Q000008:administration & dosage; D004195:Disease Models, Animal; D024901:Drug Resistance, Multiple, Bacterial / Q000187:drug effects*; D004791:Enzyme Inhibitors / Q000008:administration & dosage / Q000138:chemical synthesis / Q000494:pharmacology*; D020869:Gene Expression Profiling; D055624:Methicillin-Resistant Staphylococcus aureus / Q000187:drug effects*; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000037:antagonists & inhibitors*; D009132:Muscles / Q000382:microbiology; D060888:Real-Time Polymerase Chain Reaction; D012867:Skin / Q000382:microbiology; D013203:Staphylococcal Infections / Q000188:drug therapy* / Q000382:microbiology; D013844:Thiazoles / Q000008:administration & dosage / Q000494:pharmacology*; D013871:Thiones / Q000008:administration & dosage / Q000494:pharmacology*; D016896:Treatment Outcome; D015027:Zebrafish
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D002939:Ciprofloxacin; D004791:Enzyme Inhibitors; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D013844:Thiazoles; D013871:Thiones; C075040:NorA protein, Staphylococcus
keywords: Ciprofloxacin; Dithiazole thiones; Efflux pump inhibitor; MIC reversal; MRSA; Zebrafish infection