Background: ESBL problem is increasing worldwide and only limited studies on genes of ESBL are performed in Nepal. Objectives: We aimed to focus on the molecular detection of plasmid-mediated blaTEM, blaSHV and blaCTX-M genes among the ESBL producing Enterobacteriaceae from different clinical samples. Methods: A total of 550 clinical samples were processed and organisms of Enterobacteriaceae were identified using standard microbiological process. ESBL producers were screened and confirmed using modified Kirby Bauer disc diffusion method by CLSI guidelines. Plasmids extracted from the confirmed ESBL positives were the template for PCR. blaSHV, blaTEM and blaCTX-M genes were amplified using specific primers of respective genes by uniplex PCR. The presence of these genes was confirmed by gel electrophoresis. Results: Among 550 different clinical samples 343 (62.36%) were culture positive. Of which, 157 (45.57%) belonged to Enterobacteriaceae. Escherichia coli (45.9%) was predominant. Of these 33.2% (52/157) isolates ESBL positive. ESBL- E. coli (52.8%) were prevalent. All ESBL positive organisms were sensitive to imipenem. Of confirmed ESBL positives, 34.6% harboring blaTEM gene, 30.8% harboring blaSHV gene and blaCTX-M genes were present in all ESBL producers. Twenty-eightout of 52 (53.9%) isolates harbored multiple bla genes, the most common combination being blaCTX-M + blaTEM (21.2%). Conclusion: We report 100% plasmid mediated CTX-M genotype among ESBL producers which might indicate rapid dissemination of blaCTX-Mgenes from the community to the patients. Besides, there is a need for regular monitoring of antibiotic resistance in the country and de-escalate the use of antibiotics so as to preserve the antibiotics for future generation.

Background and Aim: In Nepal not much of study of Thermophilic area and Thermophiles have been done. Thermophilic bacteria are less studied but are important group of microorganisms due to their ability to produce industrially important enzymes. Methods: In this study, thermophilic bacteria were isolated from hot spring of Bhurung, Nepal. Wide range of bacteria that could grow at high temperatures and tolerate extreme temperature were characterized by morphology, biochemistry and sequencing of its 16S rRNA gene sequence. The isolates were screened for production of extracellular enzymes like protease, amylase, lipase, cellulase, caseinase, pectinase and xylanase activity. Phylogenetic tree construction and G+C content evaluation of the isolate was also studied.Results: 15 isolates with ability to tolerate high temperatures were identified as Bacillus sp. by morphology, biochemistry and sequencing of its 16S rRNA gene sequence. BLAST search analysis of the sequence was performed and result showed maximum identity (99% similarity) with Bacillus licheniformis, Bacillus subtilis and Bacillus pumilus. Isolated strains exhibited considerable amount of extracellular exozymes activity. Phylogenetic analysis of the isolates revealed the relatedness among the species. The G+C content of each species was also evaluated and was found to be in range of 54.87 to 55.54%.Conclusion: The study of isolates confirmed that the isolated Bacillus sp. to be a true thermophile and could be a source of various thermostable exozymes which can be exploited for pharmaceutical and industrials applications. Much detailed study of the isolates can Int J Appl Sci Biotechnol, Vol 3(3): 392-397

Background and Aim: Nepal’s geothermal areas are considered as uncharted place for microbiological and biotechnological researchers since not many researches has been conducted on these areas. The main aim of this study was to isolate thermophilic bacteria from hostspring of Nepal. Methods: From one of the hottest natural thermal spring of Nepal, Bhurung Tatopani, 20 different bacteria were isolated and Characterized by both culture dependent and independent approaches. Results: Morphological and biochemical tests along with polyphasic analysis of these thermal isolates confirmed that two of these species belongs to Geobacillus sp. The isolates THG1 and THG2 were found to be gram variable and rod shaped with smooth colony. They were found to be osmotolerant up to 2% NaCl and thermotolerant with optimum temperature of 72°C. They showed significant production of various industrially important enzymes like amylase, lipase, protease and cellulase. 16S rRNA amplification was also performed and sequenced which revealed that the isolate THG1 has 99% similarity with Geobacillus kaustophilus and THG2 has 99% similarity with Geobacillus thermoleovorans. THG1 and THG2 16S rRNA sequences are deposited in genebank with accession id KP764939 and KP764940. For the further confirmation and deeper analysis, fatty acid analysis (FAME) was also performed and their fatty acid composition profile was also created. Conclusion: From the Bhurung hotspring two unique Geobacillus species were isolated.