Browsing by Author "Alakh Narayan Singh"

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Cadmium content, metabolite profile, biological properties of Eclipta alba (L.) Hassk plant exposed to elevated cadmium in soil
(Elsevier B.V., 2024) Akanksha Dwivedi; Alakh Narayan Singh; Ajay Kumar; Gopal Nath; Rajesh Kumar Sharma
Cadmium (Cd), a highly mobile and hazardous toxic heavy metal negatively affects plant's yield by regulating synthesis of biomolecules, involved in both physiological and biochemical activities. The present study investigated the Cd content, metabolite profile, antioxidant and antibacterial activities of methanolic leaf extracts (MLEs) of Eclipta alba L., plant exposed to elevated soil Cd (eCdS). The tested plants were grown in earthen pot and were weekly treated with Cd as CdCl2. H20 (20 mg/L) till 70 days after the plant transplantation. The results showed that eCdS significantly elevated Cd content in root and shoot tissue of E. alba plant (91 % and 94 %, respectively) as compared to the control. UHPLC-HRMS profile of MLEs revealed a significant decrease in the amino acids and its derivatives with an increased level of phenolic contents, fatty acids, and lipids. Metabolites in MLEs were also upregulated and downregulated (31 and 98, respectively) by an eCdS. An eCdS further increased both total phenolics and total flavonoids content in MLEs of E. alba by 71 % and 31 %, respectively over the control. In-vitro antioxidant activities in MLEs were also found to be significantly increased due to eCdS as DPPH > ABTS > FRAP (30 %, 26 % and 21 %, respectively) as compared to the control. Cd treated plant's MLEs also revealed maximum reduction in an inhibition zone of Pseudomonas aeruginosa (ATCC 27853), among the tested bacterial strains. The present study concludes that eCdS led to the significant changes in the metabolite profile, phenolics content and biological activities of E. alba's MLEs suggesting its ameliorating role in improving the medicinal value of E. alba plant. However, direct consumption of raw material of E. alba plant is not recommended due to high accumulation of Cd in its different parts. © 2024 Elsevier B.V.
Efficacy of bacteriophage cocktails administered through mucosal and non-mucosal routes for urinary tract infections caused by Enterobacter cloacae: A preclinical study
(Academic Press, 2025) Srishti Singh; Alok Kumar Singh; Alakh Narayan Singh; Sudhir Kumar Singh; Virendra Bahadur Yadav; Mayank Gangwar; Minakshi Sahu; Deepak Kumar; Gopal Nath
This preclinical study assessed the effectiveness of a phage cocktail in completely curing Enterobacter cloacae-associated urinary tract infections (UTIs) in a mouse model, employing various routes and dosages (both in quantity and frequency). Three lytic phages, designated ΦENT1, ΦENT2, and ΦENT3, were identified and characterised phenotypically using transmission electron microscopy (TEM) and genotypically through ERIC and restriction enzyme analysis. To induce a UTI, ten groups of female Swiss albino mice were inoculated with 100 μL containing 1 × 109 CFU/mL via the urethral route with E. cloacae GNENT11213. The mice were subsequently treated with the phage cocktail via subcutaneous, oral, transurethral, and rectal routes. The efficacy of these routes was optimised at two doses of phages, namely 1 × 109 PFU/mL (5 mice) and 1 × 105 PFU/mL (5 mice). Furthermore, the levels of Endotoxins and Interleukin-6 (IL-6) were measured to assess the negative impact of phage therapy. Our findings indicated that E. cloacae GNENT11213 could be effectively eliminated with one dosage of 1 × 109Plaque-Forming Units per mouse (PFU/mouse) and two doses of the phage cocktail containing 1 × 105 PFU/mouse administered through the urethra (local mucosa). Interestingly, higher concentrations of phage particles and multiple doses were necessary for other mucosal routes, such as oral and rectal administration, to effectively eradicate E. cloacae GNENT11213 at any stage of acute illness UTI. Furthermore, phage treatment did not significantly alter the levels of IL-6 and Endotoxins. Non-mucosal routes, such as subcutaneous, were ineffective in curing the infection. © 2025 Elsevier Ltd. All rights are reserved, including those for text and data mining, AI training, and similar technologies.
Evaluation of Antimicrobial activity of AAROGYA-5 against the Pan-Drug Resistant Pathogenic Microorganisms
(Association of Ayurvedic Physicians of India, 2025) Priya Mohan; Aprajita Singh; Alakh Narayan Singh; Gopal Nath; Anand Kumar Chaudhary
Background: Antimicrobial resistance (AMR) is emerging as a severe healthcare crisis worldwide. It is a most significant threat to global health, food security, and development, according to the World Health Organization (WHO). The world is entering the “post-antibiotic era”, and over 10 million people will die fromAMR by 2050. This rising resistance has rekindled interest among researchers in discovering and developing novel, more potent antibacterial drugs to combat AMR. Objectives: In this study, the antimicrobial effectiveness of aqueous/hydro-alcoholic extracts of AAROGYA-5 (Rajata Bhasma, powders of Ocimum tenuiflorum, Tinospora cordifolia, Ficus benghalensis, and Azadirachta indica), a Herbo-mineral formulation will be evaluated against the gram-negative, gram-positive bacteria and fungi. Methods: The formulation was studied for its antimicrobial activity by measuring the minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and minimum fungicidal concentration (MFC) against Candida Albicans (fungal strain) and Staphylococcus aureus, Enterococcus faecalis, Klebsiella pneumoniae, and Escherichia coli (bacterial strains) at different concentrations: 100mg/mL, 50mg/mL, 25mg/mL, 12.5mg/mL, 6.25mg/mL, 3.125mg/mL, 1.5625 mg/ mL, and 0.78125 mg/mL by Kirby Bauer and broth microdilution method. Result: AAROGYA-5 in the form of extracts has shown significant activity at a strength of 12.5mg/mL against the Gram-positive bacteria, i.e., S. aureus and E. faecalis, and the hydro-alcoholic extract of AAROGYA-5 was found to be effective at a strength of 6.25 mg/mL against C. albicans. Discussion: The ayurvedic formulation AAROGYA-5 was effective against the Multidrug-resistant (MDR) gram-positive cocci, i.e., S. aureus and E. faecalis. Moreover, the hydro-alcoholic extract is effective against C. albicans. In addition, the AAROGYA-5 has no activity against Gram-negative bacteria in either extract. Conclusion: Based on these results, the present study demonstrated that AAROGYA-5 has significant antimicrobial activity against MDR-S. aureus, E. faecalis, and C. albicans. © 2025, Association of Ayurvedic Physicians of India. All rights reserved.
Evaluation of Bacteriophage Cocktail on Septicemia Caused by Colistin-Resistant Klebsiella pneumoniae in Mice Model
(Frontiers Media S.A., 2022) Aprajita Singh; Alakh Narayan Singh; Nisha Rathor; Rama Chaudhry; Sudhir Kumar Singh; Gopal Nath
Objective: The emergence of resistance against last-resort antibiotics, carbapenem and colistin, in Klebsiella pneumoniae has been reported across the globe. Bacteriophage therapy seems to be one of the most promising alternatives. This study aimed to optimize the quantity and frequency of bacteriophage cocktail dosage/s required to eradicate the Klebsiella pneumoniae bacteria in immunocompetent septicemic mice. Methods: The three most active phages ɸKpBHU4, ɸKpBHU7, and ɸKpBHU14 characterized by molecular and TEM analyses were in the form of cocktail and was given intraperitoneally to mice after inducing the septicemia mice model with a constant dose of 8 × 107 colony-forming unit/mouse (CFU/mouse) Klebsiella pneumoniae. After that, the efficacy of the phage cocktail was analyzed at different dosages, that is, in increasing, variable, constant, and repeated dosages. Furthermore, interleukin-6 and endotoxin levels were estimated with variable doses of phage cocktail. Results: We have elucidated that phage therapy is effective against the Klebsiella pneumoniae septicemia mice model and is a promising alternative to antibiotic treatments. Our work delineates that a single dose of phage cocktail with 1 × 105 plaque-forming unit/mouse (PFU/mouse) protects the mice from fatal outcomes at any stage of septicemia. However, a higher phage dosage of 1 × 1012 PFU/mice is fatal when given at the early hours of septicemia, while this high dose is not fatal at the later stages of septicemia. Moreover, multiple repeated dosages are required to eradicate the bacteria from peripheral blood. In addition, the IL-6 levels in the 1 × 105 PFU/mouse group remain lower, but in the 1 × 1012 PFU/mouse group remains high at all points, which were associated with fatal outcomes. Conclusion: Our study showed that the optimized relatively lower and multiple dosages of phage cocktails with the strict monitoring of vitals in clinical settings might cure septicemia caused by MDR bacteria with different severity of infection. Copyright © 2022 Singh, Singh, Rathor, Chaudhry, Singh and Nath.
Evaluation of bacteriophage cocktail on urinary tract infection caused by colistin-resistant Klebsiella pneumoniae in mice model
(Elsevier Ltd, 2024) Alakh Narayan Singh; Aprajita Singh; Gopal Nath
Objective: The colistin-resistant Klebsiella pneumoniae causes complicated urinary tract infections (UTIs). Of them, 73% of strains of K. pneumoniae formed moderate to strong biofilm. Multidrug-resistant (MDR)/Pandrug-resistant (PDR) bacteria causing UTIs are very challenging to conventional antibiotic therapy. However, bacteriophages may be a promising alternative as they easily disrupt the biofilm and act on receptors unrelated to antibiotic resistance mechanisms. This preclinical study evaluated the efficacy of a phage cocktail with different routes and dosages (in quantity and frequency) to eradicate the K. pneumoniae-associated UTI in the mice model. Methods: The three lytic phages with the broadest spectrum activity (ΦKpnBHU1, ΦKpnBHU2 and ΦKpnBHU3) were meticulously characterized using SEM and sequencing. The cocktails were administered to mice through urethral, rectal, subcutaneous and oral routes after establishing the UTI with 1 × 108 colony-forming unit/mouse (CFU/mouse) of K. pneumoniae (KpnBHU09) resistant to both the drugs carbapenem and colistin. The efficacy of different routes with varying dosages and frequency of administration was thoroughly optimized. Results: We observed that two doses of a phage cocktail containing 1 × 105 Plaque-Forming Unit (PFU/mouse) and a single dose of 1 × 109 PFU/mouse per urethra could eradicate KpnBHU09. Intriguingly, the non-invasive administration through oral and rectal routes required higher concentration and many dosages of phages to eliminate KpnBHU09 at any stage of acute UTI. The subcutaneous route was found unsatisfactory in curing the infection. Conclusion: Bacteriophage cocktails administered through transurethral, oral and rectal routes may cure UTIs. © 2024 The Author(s)
High alcohol-producing Escherichia coli causes obesity and steatotic liver disease in a non-alcoholic cohort -a preclinical study
(BioMed Central Ltd, 2025) Aprajita Singh; Durgesh Narain Singh; Alakh Narayan Singh; Vikas Kailashiya; Dawesh Prakash Yadav; Gopal Nath
Background: Obesity is a global epidemic and multifactorial disease caused by alcohol consumption, a sedentary lifestyle, and genetic and environmental factors. Analyses of gut microbial diversity in obese individuals have been conducted earlier. Unlike non-alcoholic fatty liver diseases, the mechanistic insights into the role of gut microbiota in the development of obesity remain poorly understood. Deciphering the mechanism underlying bacterial-induced obesity will help in targeted therapeutic approaches. We demonstrated bacteriophage therapy as a new offering to treat obesity and hepatic steatosis. Methods: Stool samples from the non-alcoholic obese cohort (BMI > 27 kgm−2) were cultured to analyze gut microbiota. Bacteria were grouped into clusters based on whole-genome-based genotyping. The most predominant, high alcohol-producing E. coli strain, GNBHU03 (ECGNBHU03), was selected for further studies. Genome sequencing and subsequent analyses were performed to understand the pathogenesis of ECGNBHU03. Further, an obesity model was developed by administering ECGNBHU03 into male mice. The induced obesity and liver steatosis were evaluated using weight measurements, blood biochemical tests, and changes in the liver histology. Cocktails of three ECGNBHU03-specific bacteriophages were used to eradicate the ECGNBHU03; blood biochemical tests and histological studies evaluated the effect of eradicating ECGNBHU03 from the gut. Results: Escherichia coli was predominantly detected in the feces of 95.65% of individuals compared to the control group (non-alcoholic healthy cohort with BMI 18.5–22.9 kgm−2). Whole-genome-based genotyping generated five groups. The high alcohol-producing Escherichia coli strain, GNBHU03, was enriched in the gut microbiota of non-alcoholic obese individuals. Genomic analyses showed that GNBHU03 harbours different virulence genes, specifically lpf and hlyE associated with increased intestinal colonization and permeability. In ECGNBHU03-fed mice, colonization with ECGNBHU03 induced obesity, hepatic steatosis, and systemic inflammation, evident by elevated serum aminotransferases, endotoxins, pro-inflammatory cytokines, triglycerides, cholesterol levels, and liver steatosis. Further, targeted bacteriophage therapy effectively eradicated strain ECGNBHU03, reversing obesity and hepatic steatosis. Conclusions: This study highlights the complexity of obesity’s etiology and, for the first time, provides mechanistic insights into obesity and liver dysfunction caused by the high ethanol-producing E. coli strain GNBHU03. Notably, targeted bacteriophage therapy could be a promising strategy to mitigate obesity, offering a novel approach to obesity treatment. © The Author(s) 2025.
In vitro efficacy of bacteriophage and colistin combinations in eradicating biofilm formed by colistin-resistant Enterobacter cloacae complex
(Elsevier B.V., 2025) Srishti Singh; Alok Kumar Singh; Virendra Bahadur Yadav; Sudhir Kumar Singh; Minakshi Sahu; Alakh Narayan Singh; Gopal Nath
In the case of dry antibiotic pipelines and the emergence of resistance against last-resort antibiotics, such as colistin, alternative medicinal techniques have been necessitated. In this context, the potential of phages as a viable alternative to antibiotics is a beacon of hope. Phage and antibiotic combination therapy is a compelling solution to the problem of phage or bacterial mutant generation. In most studies, phage and antibiotic combinations were used simultaneously. Only a few phages have been recently utilised before antibiotics, significantly reducing the bacterial load in vitro and in vivo. Therefore, we investigated the additive action of phage antibiotic combinations in preventing biofilm formation against three different colistin-resistant clinical isolates of the Enterobacter cloacae complex, specifically Enterobacter cloacae and Enterobacter hormaechei, at various time points with varying phage concentrations. We used different phage concentrations and sub-MICs of colistin combinations to determine the optimal phage concentration that shows synergy against three different clinical isolates of the Enterobacter cloacae complex. We also investigated the optimal time for applying antibiotics after phage treatment to eradicate Enterobacter cloacae complex in both planktonic and biofilm states. The cross-colistin susceptibility of colistin-resistant strain after co-treatment with SIM (Phage antibiotic simultaneous) and AF (Antibiotic added 8 h before phage) was also evaluated using the broth dilution method. In brief, applying phage before 8 h or 6 h of colistin, i.e. PF (Phage first followed by antibiotics) treatment at a dosage of 106 PFU/mL, was an effective sequential therapy for eliminating the biofilm and the planktonic form of Enterobacter cloacae complex. The Institutional Ethics Committee of Banaras Hindu University (Institute of Medical Sciences), Varanasi, with reference number Dean/2024/IAEC/6876, has approved this in vitro study. © 2025 The Authors
Klebsiella pneumoniae infections and phage therapy
(Indian Association of Medical Microbiologists, 2024) Alakh Narayan Singh; Aprajita Singh; Sudhir Kumar Singh; Gopal Nath
Objective: Carbapenem-colistin-resistant Klebsiella pneumoniae has emerged as a serious global problem. Klebsiella pneumoniae is a major culprit in healthcare settings and is responsible for septicemia, urinary tract infections, pneumonia, meningitis, burn wound and surgical site infections, and liver abscesses even in younger and healthier population worldwide. The formation of biofilm prevents antibiotics from reaching the bacteria and exerting their effector mechanism. The non-availability of therapeutic alternatives (antibiotic therapy) further complicates the scenario. However, in the era of antibiotic resistance, bacteriophage therapy emerges as a ray of hope against antibiotic-resistant bacteria. Method: The present review focuses on the therapeutic potential of bacteriophages as an antimicrobial agent with special reference to safety, specificity, efficacy, dosage, and dosage frequency against Pan-Drug Resistant (PDR) K. pneumoniae, both in-vitro and in-vivo (animals and human) studies. Result: This review highlights the perspectives therapeutic potential of bacteriophages, their impact on the host immune system, combination therapy, and bacteriophage-encoded gene product endolysin, artificial lysins (Artilysins), polysaccharide depolymerase, and peptidoglycan hydrolases. Conclusion: This review briefly describes the application of bacteriophage and its encoded gene products in clinical trials. © 2024 Indian Association of Medical Microbiologists
Preparation of hydrogel from the hydroalcoholic root extract of Premna integrifolia L. and its mediated green synthesis of silver nanoparticles for wound healing efficacy
(Elsevier Ltd, 2024) Pooja Verma; Jyoti Dixit; Chandrashekhar Singh; Alakh Narayan Singh; Aprajita Singh; Kavindra Nath Tiwari; Madaswamy S. Muthu; Gopal Nath; Sunil Kumar Mishra
The current investigation concentrated on the fabrication of silver nanoparticles through the root of Premna integrifolia L. The antibacterial and wound healing effects of their silver nanoparticles-hydrogel and root extract-hydrogel were evaluated. The hydroalcoholic root extract of P. integrifolia was enriched with crocetin, ferulic acid, oleanolic acid, oleic acid, syringic acid, and vanillin. The silver nanoparticles were biosynthesized through secondary metabolite-enriched hydroalcoholic root extract (5 %) when mixed with 1 mM AgNO3 and kept under sunlight for 10 minutes. They showed an optimum surface plasmon resonance (SPR) at 447 nm. The aldehyde, phenol, and primary amine groups of the extract reduce silver cations into nanoparticles. The nanoparticles band gap of 2.06 eV showed their semi-conductance behavior. The nanoparticles were spherical, uniformly distributed, and stable. The nanoparticles had a good roughness profile and 57.55 % elemental silver. The silver nanoparticles-hydrogel (10 %) showed an efficient 11±0.11 nm zone of inhibition of S. aureus in comparison to the root extract-hydrogel (10 %), i.e., 9±0.15 nm. The nanoparticles-hydrogel and the root extract-hydrogel did not show noticeable symptoms of acute dermal toxicity. The nanoparticles-hydrogel (10 %) and the root extract-hydrogel (10 %) healed 99.9 % and 97.3 % of the S. aureus- infected wound, respectively. The nanoparticle-hydrogel efficiently induced re-epithelialization in the dermis of S. aureus-infected wound compared to the extract-hydrogel. The nanoparticle-hydrogel enhanced the rate of wound closure. © 2024 Elsevier Ltd