Protein biomarker elucidation for the verification of biological agents in the taxonomic group of Gammaproteobacteria using tandem mass spectrometry.

Some pathogenic microbes can be used for nefarious applications and instigate population-based fear. In a bio-threat scenario, rapid and accurate methods to detect biological agents in a wide range of complex environmental and clinical matrices, is of paramount importance for the implementation of mitigation protocols and medical countermeasures. This study describes targeted and shot-gun tandem MS based approaches for the verification of biological agents from the environmental samples. The marker proteins and peptides were elucidated by an exhaustive literature mining, in silico analysis of prioritized proteins, and MS/MS analysis of abundant proteins from selected bacterial species. For the shot-gun methodology, tandem MS analysis of abundant peptides was carried from spiked samples. The validation experiments employing a combination of shot-gun tandem MS analysis and a targeted search reported here is a proof of concept to show the applicability of the methodology for the unambiguous verification of biological agents at sub-species level, even with limited fractionation of crude protein extracts from environmental samples.

Synergy of clavine alkaloid 'chanoclavine' with tetracycline against multi-drug-resistant E. coli.

The emergence of multi drug resistance (MDR) in Gram-negative bacteria (GNB) and lack of novel classes of antibacterial agents have raised an immediate need to identify antibacterial agents, which can reverse the phenomenon of MDR. The purpose of present study was to evaluate synergy potential and understanding the drug resistance reversal mechanism of chanoclavine isolated from Ipomoea muricata against the multi-drug-resistant clinical isolate of Escherichia coli (MDREC). Although chanoclavine did not show antibacterial activity of its own, but in combination, it could reduce the minimum inhibitory concentration (MIC) of tetracycline (TET) up to 16-folds. Chanoclavine was found to inhibit the efflux pumps which seem to be ATPase-dependent. In real-time expression analysis, chanoclavine showed down-regulation of different efflux pump genes and decreased the mutation prevention concentration of tetracycline. Further, in silico docking studies revealed significant binding affinity of chanoclavine with different proteins known to be involved in drug resistance. In in silico ADME/toxicity studies, chanoclavine was found safe with good intestinal absorption, aqueous solubility, medium blood-brain barrier (BBB), no CYP 2D6 inhibition, no hepatotoxicity, no skin irritancy, and non-mutagenic indicating towards drug likeliness of this molecule. Based on these observations, it is hypothesized that chanoclavine might be inhibiting the efflux of tetracycline from MDREC and thus enabling the more availability of tetracycline inside the cell for its action.