Genome mining to identify valuable secondary metabolites and their regulation in Actinobacteria from different niches.

Actinobacteria are the largest bacteria group with 18 significant lineages, which are ubiquitously distributed in all the possible terrains. They are known to produce more than 10,000 medically relevant compounds. Despite their ability to make critical secondary metabolites and genome sequences' availability, these two have not been linked with certainty. With this intent, our study aims at understanding the biosynthetic capacity in terms of secondary metabolite production in 528 Actinobacteria species from five different habitats, viz., soil, water, plants, animals, and humans. In our analysis of 9,646 clusters of 59 different classes, we have documented 64,000 SMs, of which more than 74% were of unique type, while 19% were partially conserved and 7% were conserved compounds. In the case of conserved compounds, we found the highest distribution in soil, 79.12%. We found alternate sources of antibiotics, such as viomycin, vancomycin, teicoplanin, fosfomycin, ficellomycin and patulin, and antitumour compounds, such as doxorubicin and tacrolimus in the soil. Also our study reported alternate sources for the toxin cyanobactin in water and plant isolates. We further analysed the clusters to determine their regulatory pathways and reported the prominent presence of the two component system of TetR/AcrR family, as well as other partial domains like CitB superfamily and HTH superfamily, and discussed their role in secondary metabolite production. This information will be helpful in exploring Actinobacteria from other environments and in discovering new chemical moieties of clinical significance.

Identification of Arg32Ser, His92Tyr and Leu147Phe novel mutations in chromosomally mediated ß-lactamase SHV and in silico characterization to understand their substrate activity imparting resistance.

The emergence of ß-lactam resistance is yearning for clinical significance in Enterobacteriaceae, which are categorized under global priority pathogen lists by the World Health Organization. Likewise, the prevalence of numerous ß-lactamase enzymes, mutational propensity in such bacteria, and their role in accelerating resistance is still a major concern. Thus, the present work intends to characterize the ß-lactamase producing bacteria isolated from acute diarrheal patients to understand their chromosomally acquired resistance pattern through molecular characterization and in silico approaches. The current study highlights the first identified Escherichia fergusonii and Escherichia marmotae species and their ß-lactamase encoding genes, blaOKP-A, blaNDM and blaOXA from the unexplored Enterobacteriaceae family from North East India. First-ever reported point mutations such as Arg32Ser, His92Tyr, and Leu147Phe were observed in BlaSHV protein of two Klebsiella pneumoniae isolates S-35 and S-46. In molecular docking, non-catalytic site H-bond interactions of Arg 218, Ala 223, Asn 128, Ser 126, Gln 95, Asp 100, Tyr 101, Ser 102, Ala 274 with a low binding affinity towards BlaSHV was found. This correlates with the high imipenem, ceftazidime, cefuroxime, ceftriaxone, and cefpodoxime resistance in Klebsiella pneumoniae S-35 with the complementary effect of mutations Arg32Ser and Leu147Phe. Besides, the role of His92Tyr mutation in controlling the resistance in Klebsiella pneumoniae S-46 is also illustrated. Thus, our study highlights the novel mutations of ß-lactamase and its clinical importance with altered resistance profiles. This could be useful to design better therapeutics and to readjust antibiotic treatment regimes against them and control to grow more resistance under selective pressure.Communicated by Ramaswamy H. Sarma.

Potentiating the intracellular killing of Staphylococcus aureus by dihydroquinazoline analogues as NorA efflux pump inhibitor.

Staphylococcus aureus is an emerging human pathogen that has become difficult to treat due to its high resistance against wide range of drugs. Emergence of drug resistant isolates has further convoluted the treatment process. Among different resistance mechanisms, efflux pump proteins play a central role and has made itself a direct approach for therapeutic exploration. To demarcate the role of dihydroquinazoline analogues as NorA efflux pump inhibitor in S. aureus1199B (NorA over producing) strain total seventeen analogues were synthesized and tested for their modulatory effects on norfloxacin and Etbr resistance. Further accumulation assays, bacterial time kill kinetics, cytotoxicity assay were also carried out. The intracellular killing ability of analogues, as EPI was determined using THP-1 monocytes. The binding interaction of analogues with NorA was also predicted. Dihydroquinazoline analogues notably reduced the MIC of norfloxacin and Etbr in S. aureus1199B. In addition to their very low toxicity, they showed high Etbr and norfloxacin accumulation respectively. Further effective over time log reduction in bacterial kill kinetics in presence of these analogues confirmed their role as NorA efflux pump inhibitor. FESEM analysis clearly depicted their effect on the cell surface morphology owing to its lyses. The most significant finding of this study was the ability of analogues to significantly reduce the intracellular S. aureus1199B in human THP-1 monocytes in presence of norfloxacin. Our study has shown for the first time the possibility of developing the dihydroquinazoline analogues as NorA efflux pump inhibitors for S. aureus and control its infection.