Dithiazole thione derivative as competitive NorA efflux pump inhibitor to curtail multi drug resistant clinical isolate of MRSA in a zebrafish infection model
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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.
KeywordsDithiazole thiones Efflux pump inhibitor MRSA MIC reversal Zebrafish infection Ciprofloxacin
The authors sincerely thank TRR funding provided by management, SASTRA University to NS to carry out this work. TR would like to thank the Science and Engineering Research Board (SERB), Government of India, (SERB/F/1266/2012-13, dated 31st May 2012) for financial support. DST-FIST funding (Grant No SR/FST/ETI-331/2013) provided by DST, Govt of India to SCBT, SASTRA University, is gratefully acknowledged. Infrastructure facility (CRF) established through R&M funds (Grant No R&M/0021/SCBT-007/2012-13) of SASTRA to carry out this work is gratefully acknowledged. R&M grant provided to Microbiology Department by Management SASTRA University is gratefully acknowledged. Research fellowship provided by UGC to L.R.C.Maulana Azad National Fellowship, UGC, New Delhi, India (MANF-2012-13-CHR-WES-13462), is gratefully acknowledged.
CNS synthesized the compounds, SSG helped in purification and characterization of the compounds; NS designed study, LRC carried out the biological part of study, TR and UV designed in vivo and in silico part of study, AM carried out fish toxicity assays; AA helped in design and interpretation of gene expression data, NS, TR and UV wrote the paper.
Compliance with ethical standards
This work is supported by TRR research grant to NS by SASTRA University; DST SERB Funding to TR (SERB/F/1266/2012-13, dated 31st May 2012) and DST-FIST funding (Grant No SR/FST/ETI-331/2013).
Conflict of interest
Author 1 declares no conflict of interest, author 2 declares no conflict of interest, author 3 declares no conflict of interest, author 4 declares no conflict of interest, author 5 declares no conflict of interest, author 6 declares no conflict of interest, author 7 declares no conflict of interest, author 8 declares no conflict of interest and author 9 declares no conflict of interest.
All applicable international, national and/or institutional guidelines for the care and use of animals were followed.
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