Strategy for the Enrichment of Protein Biomarkers from Diverse Bacterial Select Agents.

BACKGROUND: Some pathogenic bacteria can be potentially used for nefarious applications in the event of bioterrorism or biowarfare. Accurate identification of biological agent from clinical and diverse environmental matrices is of paramount importance for implementation of medical countermeasures and biothreat mitigation. OBJECTIVE: A novel methodology is reported here for the development of a novel enrichment strategy for the generally conserved abundant bacterial proteins for an accurate downstream species identification using tandem MS analysis in biothreat scenario. METHODS: Conserved regions in the common bacterial protein markers were analyzed using bioinformatic tools and stitched for a possible generic immuno-capture for an intended downstream MS/MS analysis. Phylogenetic analysis of selected proteins was carried out and synthetic constructs were generated for the expression of conserved stitched regions of 60 kDa chaperonin GroEL. Hyper-immune serum was raised against recombinant synthetic GroEL protein. RESULTS: The conserved regions of common bacterial proteins were stitched for a possible generic immuno-capture and subsequent specific identification by tandem MS using variable regions of the molecule. Phylogenetic analysis of selected proteins was carried out and synthetic constructs were generated for the expression of conserved stitched regions of GroEL. In a proof-of-concept study, hyper-immune serum raised against recombinant synthetic GroEL protein exhibited reactivity with ~60 KDa proteins from the cell lysates of three bacterial species tested. CONCLUSION: The envisaged methodology can lead to the development of a novel enrichment strategy for the abundant bacterial proteins from complex environmental matrices for the downstream species identification with increased sensitivity and substantially reduce the time-to-result.

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.

Paradoxical Reaction to Midazolam in Preterm Neonates: A Case Series.

Midazolam is a drug belonging to the benzodiazepine group and is used commonly for seizure control as well as preoperative and procedure-related sedation in neonates. Many adverse effects of midazolam have been reported in the past. Paradoxical stimulation of the central nervous system such as restlessness, nightmare, and hallucinations as well as hypomanic behavior has been reported in adults and children. Seizure is a rare adverse effect of midazolam. Cases of myoclonic movements associated with midazolam have been published worldwide; however, none so far have been reported from India. We report two newborns in our Neonatal Unit, who developed myoclonic seizure after the administration of midazolam. Both of these neonates were preterm, require multiple invasive and noninvasive investigations also leads to parent and clinician stress.

Endotracheal Aspirate Microscopy, Cultures and Endotracheal Tube Tip Cultures for Early Prediction of Ventilator Associated Pneumonia in Neonates.

OBJECTIVE: To evaluate the utility of endotracheal aspirate microscopy, culture and endotracheal tube tip culture for early diagnosis of ventilator-associated pneumonia in neonates. METHODS: Inborn ventilated neonates were followed-up for ventilator-associated pneumonia using Center for Disease Control and Prevention (CDC) criteria. Endotracheal aspirate microscopy, culture and endotracheal tube tip cultures were performed. RESULTS: Ventilator-associated pneumonia occurred in 28/68 (41%) neonates as per CDC criteria. Endotracheal aspirate microscopy (≥5 polymorphonuclear cells per high power field) and endotracheal aspirate culture had 78.6% and 75% sensitivity, 87.5% and 90% specificity, positive predictive value of 81.5% and 84%, and negative predictive value of 85.4% and 83.72%, respectively. Mean (SD) time of result of microscopy and endotracheal aspirate culture was 55.7 (4.3) h and 108.3 (19.7) h, respectively in comparison to diagnosis made at 143.5 (23.3) h, as per CDC criteria. CONCLUSION: Endotracheal aspirate microscopic examination and culture can be supportive in objective diagnosis of ventilator-associated pneumonia with an added advantage of earlier prediction.