Anti-microbial efficacy of a scientifically developed and standardized herbal-alcohol sanitizer.

Hands are the primary mode of transmission of microbe-based infections, as they harbor normal microbiota and pathogenic microbes. SARS-CoV-2 has endangered lives worldwide, and WHO has recommended good hygiene practices, especially hand hygiene. In addition, other infectious diseases like diphtheria, measles, tuberculosis, HIV, malaria, etc. are spreading in the shadow of the COVID-19 pandemic. The anti-microbial efficiency of two in-house developed herbal-alcohol based hand sanitizers containing Azadirachta indica, Citrus limon, Zingiber officinale, and Aloe vera (HS1) and Zingiber officinale replaced with Ocimum sanctum (HS2) was evaluated. HS1, with Zingiber officinale, and HS2, with Ocimum sanctum, herbal sanitizers showcased in-vitro anti-viral activity on MDCK cells using the reference strain of influenza A virus, A/PR/8/34 (H1N1), and reduced 99.99% of microbial load within 30 s of contact time, estimated by the Antimicrobial Susceptibility Testing Method. On volunteers, HS1 and HS2 were more effective than alcohol-based WHO sanitizers. Moreover, HS2 sanitizer is more effective against viruses and has better efficiency and hedonic qualities in volunteers than HS1. These sanitizers don't irritate or dry up the skin and have a longer shelf life. Overall, findings reveal that herbal-alcohol-based sanitizers are promising hand hygiene products with the capability of reducing microbial load.

Development of immunodetection system for botulinum neurotoxin serotype E.

Background & objectives: Botulism, a potentially fatal paralytic illness, is caused by the botulinum neurotoxins (BoNTs) secreted by Clostridium botulinum. It is an obligate anaerobic, Gram-positive, spore-forming bacterium. BoNTs are classified into seven serotypes based on the serological properties. Among these seven serotypes, A, B, E and, rarely, F are responsible for human botulism. The present study was undertaken to develop an enzyme-linked immunosorbent assay (ELISA)-based detection system for the detection of BoNT/E. Methods: The synthetic gene coding the light chain of BoNT serotype E (BoNT/E LC) was constructed using the polymerase chain reaction primer overlapping method, cloned into pQE30UA vector and then transformed into Escherichia coli M15 host cells. Recombinant protein expression was optimized using different concentrations of isopropyl-ß-D-1-thiogalactopyranoside (IPTG), different temperature and the rBoNT/E LC protein was purified in native conditions using affinity column chromatography. The purified recombinant protein was checked by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and further confirmed by western blot and matrix-assisted laser desorption ionization-tandem time-of-flight (MALDI-TOF). Polyclonal antibodies were generated against rBoNT/E LC using Freund's adjuvant in BALB/c mice and rabbit. Sandwich ELISA was optimized for the detection of rBoNT/E LC and native crude BoNT/E, and food matrix interference was tested. The developed antibodies were further evaluated for their specificity/cross-reactivity with BoNT serotypes and other bacterial toxins. Results: BoNT/E LC was successfully cloned, and the maximum expression was achieved in 16 h of post-induction using 0.5 mM IPTG concentration at 25°C. Polyclonal antibodies were generated in BALB/c mice and rabbit and the antibody titre was raised up to 128,000 after the 2nd booster dose. The developed polyclonal antibodies were highly specific and sensitive with a detection limit about 50 ng/ml for rBoNT/E LC and 2.5×10[3] MLD50 of native crude BoNT/E at a dilution of 1:3000 of mouse (capturing) and rabbit (revealing) antibodies. Further, different liquid, semisolid and solid food matrices were tested, and rBoNT/E LC was detected in almost all food samples, but different levels of interference were detected in different food matrices. Interpretation & conclusions: There is no immune detection system available commercially in India to detect botulism. The developed system might be useful for the detection of botulinum toxin in food and clinical samples. Further work is in progress.

Identification of Cross Reactive Antigens of C. botulinum Types A, B, E & F by Immunoproteomic Approach.

Diseases triggered by microorganisms can be controlled by vaccines, which need neutralizing antigens. Hence, it is very crucial to identify extremely efficient immunogens for immune prevention. Botulism, a fatal neuroparalytic disease, is caused by botulinum neurotoxins produced by the anaerobic, Gram-positive spore-forming bacteria, Clostridium botulinum. Food-borne botulism and iatrogenic botulism are caused by botulinum toxin. Wound botulism, infant botulism, and adult intestinal botulism are caused by primarily C. botulinum followed by secondary intoxication. To identify protective antigens, whole cell proteome of C. botulinum type B was separated by two-dimensional gel electrophoresis. 2-D gel of whole cell proteins was probed with hyper immune sera of whole cell proteins of C. botulinum types A, E, and F. Six cross immunoreactive proteins were identified. These immunoreactive proteins will be further tested for developing vaccines and serodiagnostic markers against botulism.

Immunoproteomics Approach for Screening of Vaccine Candidates against Intestinal Botulism.

Intestinal botulism is an infectious form of botulism in which disease results from ingesting spores, which is followed by spore germination and intraluminal production of botulinum neurotoxins over an extended period. Botulinum neurotoxin is produced by endospore forming bacteria called C. botulinum. Immunoproteomic study was used to screen the cross reactive immunogenic proteins of Clostridium botulinum type B using C. botulinum type B live spore antiserum. The whole cell proteins were separated by two dimensional gel electrophoresis and transferred to polyvinylidene difluoride membranes. Further, the Western blotting was performed with mouse pups immune serum against C. botulinum type B live spores. Eight predominant cross immunoreactive proteins were identified by mass spectrometry. These immunogenic proteins might be used to develop novel subunit vaccine candidates against the intestinal botulism.

Novel Pyrimidine Tagged Silver Nanoparticle Based Fluorescent Immunoassay for the Detection of Pseudomonas aeruginosa.

A simple pyrimidine-based fluorescent probe (R)-4-(anthracen-9-yl)-6- (naphthalen-1-yl)-1,6-dihydropyrimidine-2-amine (ANDPA) was synthesized through the greener one pot reaction and characterized by IR, NMR, and ESI-Mass. Glucose stabilized silver nanoparticles (Glu-AgNPs) were also synthesized and characterized using UV, IR, XRD, SEM, and TEM. When ANDPA was tagged with Glu-AgNPs, the fluorescent intensity of ANDPA decreased drastically. When the monoclonal antibody (Ab) [immunoglobulin G (IgG)] of Pseudomonas aeruginosa (PA) was attached with ANDPA/Glu-AgNPs, the original intensity of the probe was recovered with minimal enhancement at 446 nm. On further attachment of PA with ANDPA/Glu-AgNPs/PA, the fluorescence intensity of the probe was enhanced obviously at 446 nm with red shift. This phenomenon was further supported by SEM and TEM. The linear range of detection is from 8 to 10-1 CFU/mL, and LOD is 1.5 CFU/mL. The immunosensor was successfully demonstrated to detect Pseudomonas aeruginosa in water, soil, and food products like milk, sugar cane, and orange juices.

A green and facile approach for synthesizing imine to develop optical biosensor for wide range detection of bilirubin in human biofluids.

Bilirubin, a key biomarker for the jaundice and its clinical diagnosis needs a better analytical tool. A novel and simple fluorescent platform based on (2,2'-((1E,1'E)-((6-bromopyridine-2,3-diyl) bis(azanylylidene)) bis(methanylylidene diphenol) (BAMD) was designed. BAMD showed a remarkable fluorescent intensity with a very good quantum yield of 0.85 and lifetime of 870ps. Hence, it was applied for the determination of bilirubin using both colorimetric and fluorimetric techniques in physiological and basic pH. Under optimized experimental conditions, the probe detects bilirubin selectively in the presence of other interfering biomolecules and metal ions. The linear range of detection is 1pM-500µM at pH=7.4 and LOD is 2.8 and 3.3 pM at pH=7.4 and 9.0, respectively, which were reported so far. The probe detects the bilirubin through FRET mechanism. The practical application of the probe was successfully tested in the human blood and urine samples. Based on all above advantages, this simple idea can be applied to design a simple clinical diagnostic tool for jaundice.