Antibiotic resistance determinants among carbapenemase producing bacteria isolated from wastewaters of Kathmandu, Nepal.

The emergence of carbapenem resistant bacteria (CRB) possesses a remarkable threat to the health of humans. CRB and carbapenem resistance genes (CRGs) have frequently been reported in clinical isolates from hospitals, however, their occurrence and distribution in wastewaters from various sources and river water have not been emphasized in Nepal. So, this study aimed to detect carbapenem resistant bacterial isolates and their resistance determinants in river water and different types of wastewaters. River water and both untreated and treated wastewater samples from hospitals, pharmaceutical industries, and municipal sewage were collected in summer and winter seasons. From 68 grab wastewater samples, CRB were detected only in 16 samples, which included eight hospital wastewater, and four each from untreated municipal sewage and river water. A total of 25 CRB isolates were detected with dominance of E. coli (44.0%) and K. pneumoniae (24.0%). The majority of the isolates harbored blaNDM-1 (76.0%), followed by blaOXA (36.0%) and blaKPC (20.0%) genes. Hospital wastewater majorly contributed to the presence of blaNDM-1, blaKPC, and blaOXA along with intI1 genes compared to river water and untreated municipal sewage, especially during the winter season. However, CRB were not detected in treated effluents of hospitals and municipal sewage, and both influents and effluents from pharmaceutical industries. The combined presence of each blaNDM-1 & blaOXA and blaKPC & blaOXA occurred in 16.0% of the bacterial isolates. The increased minimum inhibitory concentration (MIC) of meropenem was significantly associated with the presence of CRGs. The results of this study highlight the significance of carbapenem resistance in bacteria isolated from wastewater and river water, and underscore the necessity for efficient monitoring and control strategies to prevent the dispersion of carbapenem resistance in the environment and its potential consequences on human health.

Characterization of Thermostable Cellulase from Bacillus licheniformis PANG L Isolated from the Himalayan Soil.

This study aimed to isolate, purify, and characterize a potential thermophilic cellulase-producing bacterium from the Himalayan soil. Eleven thermophilic bacteria were isolated, and the strain PANG L was found to be the most potent cellulolytic producer. Morphological, physiological, biochemical, and molecular characterization identified PANG L as Bacillus licheniformis. This is the first study on the isolation of thermostable cellulase-producing Bacillus licheniformis from the Himalayan soil. This bacterium was processed for the production of cellulase enzyme. The optimum conditions for cellulase production were achieved at 45°C after 48 h of incubation at pH 6.5 in media-containing carboxymethyl cellulose (CMC) and yeast extract as carbon and nitrogen sources, respectively, in a thermo-shaker at 100 rpm. The enzyme was partially purified by 80% ammonium sulphate precipitation followed by dialysis, resulting in a 1.52-fold purification. The optimal activity of partially purified cellulase was observed at a temperature of 60°C and pH 5. The cellulase enzyme was stable within the pH ranges of 3-5 and retained 67% of activity even at 55°C. Cellulase activity was found to be enhanced in the presence of metal ions such as Cd2+, Pb2+, and Ba2+. The enzyme showed the highest activity when CMC was used as a substrate, followed by cellobiose. The Km and Vmax values of the enzyme were 1.8 mg/ml and 10.92 µg/ml/min, respectively. The cellulase enzyme obtained from Bacillus licheniformis PANG L had suitable catalytic properties for use in industrial applications.

Production Optimization and Biochemical Characterization of Cellulase from Geobacillus sp. KP43 Isolated from Hot Spring Water of Nepal.

This study is aimed at isolating and identifying a thermophilic cellulolytic bacterium from hot spring water and characterizing thermostable cellulase produced by the isolate. Enrichment and culture of water sample was used for isolation of bacterial strains and an isolate with highest cellulase activity was chosen for the production, partial purification, and biochemical characterization of the enzyme. Different staining techniques, enzymatic tests, and 16s ribosomal DNA (16s rDNA) gene sequencing were used for the identification of the isolate. The cellulase producing isolate was Gram positive, motile, and sporulated rod-shaped bacterium growing optimally between 55°C and 65°C. Based on partial 16s rDNA sequence analysis, the isolate was identified as Geobacillus sp. and was designated as Geobacillus sp. KP43. The cellulase enzyme production condition was optimized, and the product was partially purified and biochemically characterized. Optimum cellulase production was observed in 1% carboxymethyl cellulose (CMC) at 55°C. The molecular weight of the enzyme was found to be approximately 66 kDa on 12% SDS-PAGE analysis. Biochemical characterization of partially purified enzyme revealed the temperature optimum of 70°C and activity over a wide pH range. Further, cellulase activity was markedly stimulated by metal ion Fe2+. Apart from cellulases, the isolate also depicted good xylanase, cellobiase, and amylase activities. Thermophilic growth with a variety of extracellular enzyme activities at elevated temperature as well as in a wide pH range showed that the isolated bacteria, Geobacillus sp. KP43, can withstand the harsh environmental condition, which makes this organism suitable for enzyme production for various biotechnological and industrial applications.

First report on the molecular detection of Entamoeba bovis from the endangered wild water buffalo (Bubalus arnee) in Nepal.

BACKGROUND: The Asiatic wild water buffalo (Bubalus arnee) is an endangered species that is conserved in the Koshi Tappu Wildlife Reserve (KTWR), Nepal, and was recently translocated to the Chitwan National Park (CNP). Gastrointestinal (GI) parasites are the cause of significant negative health and production impacts on animals worldwide. METHODS: A coprological survey of GI parasites of wild water buffalo was carried out in the CNP in 2020. Fresh dung samples (n = 25) were collected from wild water buffaloes and analysed using sedimentation and flotation techniques for morphological identification of parasite cysts, oocysts and eggs. RESULTS: Nine different GI parasites were recorded of which Entamoeba spp. (20 samples, 80%) were the most common. The presence of Entamoeba spp. was further validated using polymerase chain reaction (PCR) analysis and DNA sequencing. The PCR results were positive for all of the microscopically positive samples, and the species was identified as Entamoeba bovis. Three samples were sequenced and formed a cluster of E. bovis, which was separated from other Entamoeba spp. in phylogenetic analysis. CONCLUSION: This is the first report for molecular detection of E. bovis from wild water buffaloes in Nepal. Future work should focus on the prevalence of such infections in water buffaloes in forest environments.

Molecular study of 2019 dengue fever outbreaks in Nepal.

BACKGROUND: Dengue cases have been continuously reported in Nepal, including some large outbreaks, since its first introduction in 2004. The disease is now expanding towards newer locations above 1400 m high, especially the country's capital city, Kathmandu. In 2019, >14,000 dengue cases including six deaths were reported. This study was aimed at the detection and molecular characterization of dengue virus (DENV) in dengue patients. METHODS: A total of 451 patients were enrolled in this study. Demographic, clinical and laboratory information was collected from dengue patients. Dengue infection was confirmed by antibody/antigen detection assays followed by RT-PCR analysis. RESULTS: The DENV patients showed fever, body ache, headache, myalgia, retro-orbital pain and arthralgia. The platelets were decreased, serum liver enzymes were increased and leucopenia was seen. Out of 195 patients, 111 (57.0%) were positive for DENV RNA by consensus PCR. We found DENV-2, 70 (63.1%) as the predominant serotype responsible for the 2019 outbreak, while DENV-3 was detected in two patients. CONCLUSION: Our findings suggest that DENV-2 was the major serotype causing the 2019 massive outbreak in Nepal. This information will help in disease control programs to understand the molecular epidemiology and its changing trend.

Thiazolidinedione derivatives as PTP1B inhibitors with antihyperglycemic and antiobesity effects.

Benzylidene-2,4-thiazolidinedione derivatives with substitutions on the phenyl ring at the ortho or para positions of the thiazolidinedione (TZD) group were synthesized as PTP1B inhibitors with IC50 values in a low micromolar range. Compound 3e, the lowest, bore an IC50 of 5.0 microM. In vivo efficacy of 3e as an antiobesity and hypoglycemic agent was evaluated in a mouse model system. Significant improvement of glucose tolerance was observed. This compound also significantly suppressed weight gain and significantly improved blood parameters such as TG, total cholesterol and NEFA. Compound 3e was also found to activate peroxisome proliferator-activated receptors (PPARs) indicating multiple mechanisms of action.

Isoxazol-5(4H)one derivatives as PTP1B inhibitors showing an anti-obesity effect.

In developing inhibitors of therapeutic target enzymes, significant time and effort are committed to the preparation of large numbers of compounds. In an effort to develop a potent inhibitor of protein tyrosine phosphatase (PTP) 1B as an anti-obesity and/or anti-diabetic agent, we constructed an isoxazolone chemical library by using a simplified procedure that circumvents tedious workup and purification steps. The 10×7 isoxazolone derivatives were synthesized by coupling the two halves of the target compounds. When mixed and heated in test tubes, the precursors produced the reaction products as precipitates. After brief washing, the products were pure enough to be used for enzymatic experiments. With the precursors for the coupling reactions prepared, the 10×7 library compounds could be prepared in a day by using the present protocol. The library compounds thus obtained were examined for their inhibitory activities against PTP1B. Among them, compound C3 was the most potent inhibitor of PTP1B with an IC(50) of 2.3 µM. The in vivo effect of C3 was also examined in an obesity-prone mouse strain. Diet-induced obese (DIO)/diabetic mice were divided into two groups and each group was fed a high-fat diet (HFD) or HFD+C3 for four weeks. The group of C3-fed mice gained significantly less weight relative to the HFD-fed control group during the four weeks of the drug feeding period. In contrast to the anti-obesity effect of C3, no difference was observed in the glycemic control of the HFD and HFD+C3 mice groups.