Study on Phytochemical, Antibacterial, Antioxidant and Toxicity Profile of Viscum album Linn Associated with Acacia catechu

This study focuses on antibacterial, antioxidant and toxic potentials of Viscum album Linn, commonly known as European mistletoe associated with Acacia catechu (Khayer in Nepali). Methanol extract of the aerial parts of the Mistletoe was prepared by cold percolation method. The resulting extract was simultaneously subjected to phytochemical screening; anti-microbial activity; anti-oxidant potential and Brine shrimp toxicity test. The major biologically active phyto-constituents observed were alkaloids, glycosides, saponins, polyphenols, flavonoids, tannins, terpenoids and cardiac glycosides. Upon antibacterial activity screening, the plant extract was found to be highly effective against Pseudomonas aeruginosa with the zone of inhibition 16±1mm compared to 17±1mm of chloramphenicol (50 mcg). The antioxidant activity as EC50 value by DPPH (1,1-diphenyl-2-picrylhydrazyl) free radical scavenging activity was found to be 1.58 mg/ml while the ferric reducing capacity was measured to be 282.83±19.55 mg FeSO4.7H2O eqvt/g dry wt. of the extract during Ferric Ion Reducing Antioxidant Power (FRAP) Assay. The LC50 value for Brine Shrimp Toxicity Assay was found to be 31.62 ppm. This study shows the medicinal value of the mistletoe associated with Acacia catechu. Further meticulous analysis of this plant might lead to identification of active biomolecules effective as drugs for various ailmentsNepal Journal of Biotechnology. Dec. 2015 Vol. 3, No. 1: 60-6

Pharmacokinetics and Pharmacodynamics of a Novel Virulent Klebsiella Phage Kp_Pokalde_002 in a Mouse Model

Published: 16 August 2021Phage therapy is one of the most promising alternatives to antibiotics as we face global antibiotic resistance crisis. However, the pharmacokinetics (PK) and pharmacodynamics (PD) of phage therapy are largely unknown. In the present study, we aimed to evaluate the PK/PD of a locally isolated virulent novel øKp_Pokalde_002 (Podoviridae, C1 morphotype) that infects carbapenem-resistant Klebsiella pneumoniae (Kp56) using oral and intraperitoneal (IP) route in a mouse model. The result showed that the øKp_Pokalde_002 rapidly distributed into the systemic circulation within an hour via both oral and IP routes. A higher concentration of phage in plasma was found after 4 h (2.3 x 105 PFU/ml) and 8 h (7.3 x 104 PFU/ml) of administration through IP and oral route, respectively. The phage titer significantly decreased in the blood and other tissues, liver, kidneys, and spleen after 24 h and completely cleared after 72 h of administration. In the Kp56 infection model, the bacterial count significantly decreased in the blood and other organs by 4–7 log10 CFU/ml after 24 h of øKp_Pokalde_002 administration. Elimination half-life of øKp_Pokalde_002 was relatively shorter in the presence of host-bacteria Kp56 compared to phage only, suggesting rapid clearance of phage in the presence of susceptible host. Further, administration of the øKp_Pokalde_002 alone in healthy mice (via IP or oral) did not stimulate pro-inflammatory cytokines (TNF-a and IL-6). Also, treatment with øKp_Pokalde_002 resulted in a significant reduction of pro-inflammatory cytokines (TNF-a and IL-6) caused by bacterial infection, thereby reducing the tissue inflammation. In conclusion, the øKp_Pokalde_002 possess good PK/PD properties and can be considered as a potent therapeutic candidate for future phage therapy in carbapenem-resistant K. pneumoniae infections.Gunaraj Dhungana, Roshan Nepal, Madhav Regmi and Rajani Mall

Maculosin, a non-toxic antioxidant compound isolated from Streptomyces sp. KTM18

Context Streptomyces species are prolific sources of bioactive secondary metabolites known especially for their antimicrobial and anticancer activities. Objective This study sought to isolate and characterize antioxidant molecules biosynthesized by Streptomyces sp. KTM18. The antioxidant potential of an isolated compound and its toxicity were accessed. Materials and methods The compound was purified using bioassay-guided chromatography techniques. Nuclear magnetic resonance (NMR) experiments were carried out for structure elucidation. The antioxidant potential of the isolated compound was determined using DPPH free radical scavenging assay. The toxicity of the isolated compound was measured using a brine shrimp lethality (BSL) assay. Results Ethyl acetate extract of Streptomyces sp. KTM18 showed more than 90% inhibition of DPPH free radical at 50 mu g/mL of the test concentration. These data were the strongest among 13 Streptomyces isolates (KTM12-KTM24). The active molecule was isolated and characterized as maculosin (molecular formula, C14H16N2O3 as determined by the [M + H](+) peak at 261.1259). The DPPH free radical scavenging activity of pure maculosin was higher (IC50, 2.16 +/- 0.05 mu g/mL) than that of commercial butylated hydroxyanisole (BHA) (IC50, 4.8 +/- 0.05 mu g/mL). No toxicity was observed for maculosin (LD50, <128 mu g/mL) in brine shrimp lethality assay (BSLA) up to the compound's antioxidant activity (IC50) concentration range. The commercial standard, berberine chloride, showed toxicity in BSLA with an LD50 value of 8.63 +/- 0.15 mu g/mL. Conclusions Maculosin may be a leading drug candidate in various cosmetic and therapeutic applications owing to its strong antioxidant and non-toxic properties

Factors associated with neonatal deaths in Chitwan district of Nepal

Background: Neonatal mortality has remained unchanged since 2006 in Nepal. Reducing neonatal mortality is indispensable to reduce child mortality. The objective of this study was to investigate the factors associated with neonatal mortality. This study assesses socio-demographic factors, maternal health care and newborn care practices contributing to neonatal deaths in Chitwan district of Central Nepal. Methods: A case–control study was conducted during April–July 2012. The study used a mixed-method approach, in which records of neonatal deaths were obtained from the District Public Health Office and a comparison group, survivors, was obtained from the same community. A total of 198 mothers (of 99 neonatal deaths and 99 survivor neonates) were included in the survey. Focus group discussions, in-depth interviews and case studies were also conducted. Maternal characteristics were analyzed using descriptive statistics, Mc Nemar’s Chi square test and multivariable backward conditional logistic regression analysis. Qualitative data were analyzed by narrative analysis method.Results: More than four-fifth of mothers (86 %) had antenatal check-up (ANC) and the proportion of four or more ANC was 64 %. Similarly, the percentage of mothers having institutional delivery was 62 %, and postnatal check-up was received by 65 % of mothers. In multivariable analysis, low birth weight [adjusted odds ratio: 8.49, 95 % CI (3.21–22.47)], applying nothing on cord [adjusted odds ratio: 5.72, 95 % CI (1.01-32.30)], not wrapping of newborn [adjusted odds ratio: 9.54, 95 % CI (2.03–44.73)], and no schooling of mother [adjusted odds ratio: 2.09, 95 % CI (1.07–4.11)] were significantly associated with an increased likelihood of neonatal mortality after adjusting for other confounding variables. Qualitative findings suggested that bathing newborns after 24 h and wrapping in clean clothes were common newborn care practices. The mothers only attended postnatal care services if health problems appeared either in the mother or in the child. Conclusion:L Results of this study suggest that the current community based newborn survival intervention should provide an even greater focus to essential newborn care practices, low birth weight newborns, and female education

Antifungal Factor Produced by Pseudomonas fluorescens against an Endophytic Fungus

Growth characteristics of Piriformospora indica an endophytic fungus with plant growth promoting rhizobacteria in an aspergillus medium were studied. P. indica showed promotional effects with species of Azotobacter chroococcum, Azospirillum brasilensis, Bradyrhizobium spp., and inhibitory effect with Pseudomonas fluorescens. The biochemical and molecular level of inhibitory factor produced by P. fluorescence suggests that the antifungal substance was thermolabile and the protease test showed it to be protease resistant in nature. Dialysis test indicated that this substance had a molecular weight of more than 12,000 kDa. The inhibitory substance turns out to be ‘fungistatic ’ as well as ‘fungicidal ’ in nature. Scanning electron microscopy (SEM) and Transmission electron microscopy (TEM) studies showed the cell wall in damaged form suggesting the high potency of this antifungal substance. The inhibitory compound produced by P. fluorescence was suspected to be pyoverdine, a kind of siderophore, an iron containing substance

Isolation and Physicochemical Characterization of Laccase from Ganoderma lucidum-CDBT1 Isolated from Its Native Habitat in Nepal

At present, few organisms are known to and capable of naturally producing laccases and white rot fungi are one such group. In the present study, three fungal species, namely, Ganoderma lucidum-CDBT1, Ganoderma japonicum, and Lentinula edodes, isolated from their native habitat in Nepal were screened for laccase production, and G. lucidum-CDBT1 was found to express highest levels of enzyme (day 10 culture media showed 0.92 IU/mg total protein or 92 IU/mL laccase activity with ABTS as substrate). Lignin extracted from rice straw was used in Olga medium for laccase production and isolation from G. lucidum-CDBT1. Presence of lignin (5 g/L) and copper sulfate (30 μM) in the media increased the extracellular laccase content by 111% and 114%, respectively. The laccase enzyme produced by G. lucidum-CDBT1 was fractionated by ammonium sulfate and purified by DEAE Sepharose anion exchange chromatography. The purified enzyme was found to have a molecular mass of 43 kDa and exhibits optimal activity at pH 5.0 and 30°C. The isolated laccase was thermally stable for up to 70°C for 1 h and exhibited broad pH stability. The kinetic constants, Km, Vmax, and Kcat, determined using 2,2′-azinobis-(-3-ethylbenzothiazoline-6-sulfonic acid) as substrate were found to be 110 μM, 36 μmol/min/mg, and 246 min−1, respectively. The isolated thermostable laccase will be used in future experiments for delignification process

Re-emergence of dengue virus serotype 2 strains in the 2013 outbreak in Nepal

Background & objectives: Epidemiological interventions and mosquito control are the available measures for dengue control. The former approach uses serotype and genetic information on the circulating virus strains. Dengue has been frequently reported from Nepal, but this information is mostly lacking. The present study was done to generate a comprehensive clinical and virological picture of a dengue outbreak in Nepal during 2013. Methods: A hospital-based study involving patients from five districts of Nepal was carried out. Demographic information, clinical details and dengue serological status were obtained. Viral RNA was characterized at the molecular level by reverse-transcription polymerase chain reaction (RT-PCR), nucleotide sequencing and phylogenetic analysis. Results: From among the 2340 laboratory-confirmed dengue cases during the study period, 198 patients consented for the study. Clinically they had fever (100%), headache (59.1%), rashes (18.2%), retro-orbital pain (30.3%), vomiting (15.1%), joint pain (28.8%) and thrombocytopenia (74.3%). Fifteen (7.5%) of them had mucosal bleeding manifestations, and the rest were uncomplicated dengue fever. The patients were mostly adults with a mean age of 45.75 ± 38.61 yr. Of the 52 acute serum samples tested, 15 were positive in RT-PCR. The causative virus was identified as DENV serotype 2 belonging to the Cosmopolitan genotype. Interpretations & conclusions: We report here the involvement of DENV serotype 2 in an outbreak in Nepal in 2013. Earlier outbreaks in the region in 2010 were attributed to serotype 1 virus. As serotype shifts are frequently associated with secondary infections and severe disease, there is a need for enhancing surveillance especially in the monsoon and post-monsoon periods to prevent large-scale, severe dengue outbreaks in the region

Detection of Pathogenic Viruses, Pathogen Indicators, and Fecal-Source Markers within Tanker Water and Their Sources in the Kathmandu Valley, Nepal

Tanker water is used extensively for drinking as well as domestic purposes in the Kathmandu Valley of Nepal. This study aimed to investigate water quality in terms of microbial contamination and determine sources of fecal pollution within these waters. Thirty-one samples from 17 tanker filling stations (TFSs) and 30 water tanker (WT) samples were collected during the dry and wet seasons of 2016. Escherichia coli was detected in 52% of the 31 TFS samples and even more frequently in WT samples. Of the six pathogenic viruses tested, enteroviruses, noroviruses of genogroup II (NoVs-GII), human adenoviruses (HAdVs), and group A rotaviruses were detected using quantitative PCR (qPCR) at 10, five, four, and two TFSs, respectively, whereas Aichi virus 1 and NoVs-GI were not detected at any sites. Index viruses, such as pepper mild mottle virus and tobacco mosaic virus, were detected using qPCR in 77% and 95% out of 22 samples, respectively, all of which were positive for at least one of the tested pathogenic viruses. At least one of the four human-associated markers tested (i.e., BacHum, HAdVs, and JC and BK polyomaviruses) was detected using qPCR in 39% of TFS samples. Ruminant-associated markers were detected at three stations, and pig- and chicken-associated markers were found at one station each of the suburbs. These findings indicate that water supplied by TFSs is generally of poor quality and should be improved, and proper management of WTs should be implemented

Presence of Human Enteric Viruses, Protozoa, and Indicators of Pathogens in the Bagmati River, Nepal

Quantification of waterborne pathogens in water sources is essential for alerting the community about health hazards. This study determined the presence of human enteric viruses and protozoa in the Bagmati River, Nepal, and detected fecal indicator bacteria (total coliforms, Escherichia coli, and Enterococcus spp.), human-fecal markers (human Bacteroidales and JC and BK polyomaviruses), and index viruses (tobacco mosaic virus and pepper mild mottle virus). During a one-year period between October 2015 and September 2016, a total of 18 surface water samples were collected periodically from three sites along the river. Using quantitative polymerase chain reaction, all eight types of human enteric viruses tested—including adenoviruses, noroviruses, and enteroviruses, were detected frequently at the midstream and downstream sites, with concentrations of 4.4–8.3 log copies/L. Enteroviruses and saliviruses were the most frequently detected enteric viruses, which were present in 72% (13/18) of the tested samples. Giardia spp. were detected by fluorescence microscopy in 78% (14/18) of the samples, with a lower detection ratio at the upstream site. Cryptosporidium spp. were detected only at the midstream and downstream sites, with a positive ratio of 39% (7/18). The high concentrations of enteric viruses suggest that the midstream and downstream regions are heavily contaminated with human feces and that there are alarming possibilities of waterborne diseases. The concentrations of enteric viruses were significantly higher in the dry season than the wet season (p &lt; 0.05). There was a significant positive correlation between the concentrations of human enteric viruses and the tested indicators for the presence of pathogens (IPP) (p &lt; 0.05), suggesting that these IPP can be used to estimate the presence of enteric viruses in the Bagmati River water

Occurrence and Reduction of Shiga Toxin-Producing Escherichia coli in Wastewaters in the Kathmandu Valley, Nepal

Inadequately treated effluents discharged from wastewater treatment plants (WWTPs) severely affect the environment and the surrounding population. This study analyzed the presence of the Shiga toxin-producing Escherichia coli (STEC) genes, stx1, and stx2, and the E. coli gene, sfmD, in municipal WWTP A (n = 11) and B (n = 11) where the reductions were also evaluated; hospitals (n = 17), sewage treatment plants (STPs) (n = 4) and non-functional WWTPs (not-working WWTPs) (n = 5) in the Kathmandu Valley, Nepal. The sfmD gene was detected in 100% of the samples in WWTPs, hospitals, and not-working WWTPs and 50% of STP samples. The highest detection of stx1 and stx2 was shown in the WWTP influents, followed by WWTP effluents, not-working WWTP wastewater, hospital wastewater, and STP wastewater. Log10 reduction values of sfmD, stx1, and stx2 in WWTP A were 1.7 log10, 1.7 log10, 1.4 log10, whereas those in WWTP B were 0.5 log10, 0.6 log10, 0.5 log10, respectively, suggesting the ineffective treatment of STEC in the wastewater in the Kathmandu Valley. The high concentrations of the stx genes in the wastewaters suggest the increasing presence of aggressive STEC in the Kathmandu Valley, which should be a major public health concern