Increased expression of efflux transport proteins confers the Multi Drug Resistant (MDR) phenotype to drug resistant bacteria, which can be mitigated by efflux pump inhibitors (EPI). EPI's have the dual advantage of restoring efficacy of antibiotics and retarding the evolution of drug resistant mutants. In this study, 17 heterocyclic derivatives synthesized by a polyethylenimine (PEI) catalyzed one-pot protocol were evaluated for their EPI potential. Based on in silico studies and in vitro studies, 5 benzochromene (BC) based compounds (among 17 heterocyclic derivatives), were observed to significantly potentiate the effect of ciprofloxacin (CPX) and displayed a best modulation factor of 32, against the NorA overexpressed mutant SA-1199B. Toxicity analyses using the zebrafish model showed that, from all the benzochromene compounds evaluated, BC9 exhibited low toxicity. BC9 inhibited the NorA efflux pump at a minimum effective concentration (MEC) of 2 μg ml⁻¹ (4.03 μM) and even at subinhibitory concentrations, and it was effective in reversing the CPX MIC of both the ATCC strain and a clinical isolate of MRSA by 128 and 4 fold respectively. Our results show that benzochromene derivative BC9 is a highly specific NorA inhibitor that can be employed to mitigate MDR strains of S. aureus that overexpress the NorA efflux pump.

Buffalo calves have a high mortality rate (~80%) in commercial dairies and testis cryopreservation can provide a feasible option for the preservation of germplasm from immature males that die before attaining sexual maturity. The aim of the present study was to evaluate combinations of 10 or 20% dimethylsulfoxide (DMSO) with 0, 20 or 80% fetal bovine serum (FBS) for cryopreservation of immature buffalo testicular tissues, subjected to uncontrolled slow freezing. Tissues cryopreserved in 20% DMSO with 20% FBS (D20S20) showed total, tubular and interstitial cell viability, number of early apoptotic and DNA-damaged cells, surviving germ and proliferating cells and expression of testicular cell-specific proteins (POU class 5 homeobox (POU5F1), vimentin (VIM) and actin a2 (ACTA2)) similar to that of fresh cultured control (FCC; P > 0.05). Expression of cytochrome P450, family 11, subfamily A (CYP11A1) protein and testosterone assay showed that only tissues cryopreserved in D20S20 had Leydig cells and secretory functions identical to that of FCC (P > 0.05). High expression of superoxide dismutase2 (SOD2), cold-inducible RNA-binding protein (CIRBP) and RNA-binding motif protein3 (RBM3) proteins in cryopreserved tissues indicated involvement of cell signalling pathways regulating cellular protective mechanisms. Similarity in expression of pro-apoptosis proteins transcription factor tumour protein P53 (TP53) and BCL2-associated X protein (BAX) in D20S20 cryopreserved tissues to that of FCC (P > 0.05) suggested lower apoptosis and DNA damage as key reasons for superior cryopreservation.