RANDOM AMPLIFIED POLYMORPHIC DNA-POLYMERASE CHAIN REACTION ANALYSIS OF BACILLUS THURINGIENSIS ISOLATES FROM THE KHUMBU REGION OF EVEREST BASE CAMP

(Published in Journal of Institute of Science and Technology, Vol. 15, 2007-08, pp. 90-102.)

Random Amplified Polymorphic DNA-polymerase chain reaction analysis of Bacillus thuringiensis isolates from the Khumbu region of Everest Base Camp

G. S. Sahukhall, U. T. Shrestha1, B. Lekhak2, A. Singh2, V. P. Agrawal1

1. Research Laboratory for Biotechnology and Biochemistry (RLABB);
Maitidevi Plaza, Maitidevi, Kathmandu, Nepal
2. Central Department of Microbiology, Tribhuvan University;
Kirtipur, Kathmandu, Nepal.

ABSTRACT
Genetic similarity and diversity among the Bacillus thuringiensis isolates were examined using Random Amplified Polymorphic DNA-polymerase chain reaction (RAPD-PCR). Relationship between the species was determined by comparing their unique RAPD fingerprint information. A total of 86 endotoxin producing B. thuringiensis strains isolated from soil samples of Khumbu region were tested against a series of 100 decamer RAPD primers (codes 201-300). Nine primers: 208, 254, 256, 268, 275, 276, 284, 292 and 299 with GC%: 50-80, were found to amplify genomic DNA fragments with reproducible polymorphisms. Primer 284 with GC% 70 that produced more polymorphic bands was used to obtain RAPD profiles. The reaction mixture was optimized in 25ul system with PCR buffer containing 10mM Tris HCl, 50 mM KCl, 3 mM MgCl2 with pH 8.3.; 200μm dNTPs each, 1U Taq polymerase, 40 pmol decamer primers, 20 ng template DNA and 1% DMSO. The thermal programme was set as: initial denaturation temperature at 94oC for 5 mins followed by 35 cycles with denaturation at 94oC for 1 min, annealing at 36oC for 1 min and extension at 72oC for 2 mins with final extension temperature at 72oC for 10 mins. Higher polymorphic fragments were produced in the range between 700-900 base pairs. The range of 400-700 and 1200-1600 bp were also highly polymorphic. The Discriminatory capacity (D) of the RAPD-PCR was 0.9901 and hence the cold tolerant B. thuringiensis isolates from high altitude regions were very rich in genomic polymorphism.

Antimicrobial Efficacies of Nepalese Toothpastes against Dental Pathogens and Prevalence of Dental caries

(Abstract published in the Fifth National Conference on Science and Technology, Nov.10-12, 2008, Kathmandu pp. 353-354)

Antimicrobial Efficacies of Nepalese Toothpastes against Dental Pathogens and Prevalence of Dental caries

A. Acharya1, B. Subedi1, B.Paudyal1, M. Jnawali1, P. Shakya1, U. KC 1,
U. T. Shrestha2, K.B. Tiwari1, 2, A. Singh1 and V. P. Agrawal2
1Central Department of Microbiology, Tribhuvan University, Kirtipur, Kathmandu
2Research Laboratory for Biotechnology and Biochemistry (RLABB), Maitidevi, Kathmandu
E-mail: md.jnawali@gmail.com

ABSTRACT
Dental caries is a multifactorial oral disease developed by the localized dissolution of the tooth hard tissues, caused by acids produced by bacteria in the biofilms (dental plaque) which eventually leads to cavities. Frequently encountered bacteria in caries are Streptococci (S. mutans, S. salivarious, S. sanguis, S. sobrinus and S. mitis) and others including some species of Actinomyces, Lactobacillus, Candida albicans and some anaerobes together with Bacteroids, Fusobacterium, and Prevotella etc. Maintainace of oral health is highly interrelated with use of toothpaste. Toothpaste need to contain various antimicrobial agents in order to reduce, control and prevent different kind of dental caries. Different brands have their own composition and concentration of ingredients for their efficacy. The consumers should aware about the facts associated with their health. Present study was carried out to determine the antimicrobial status of locally used toothpastes against common dental pathogens and to know dental caries condition of school children of Kathmandu valley. The dental pathogens were isolated and identified from various dental samples. Antibacterial activities of 25 different toothpastes available locally in markets were assessed against the isolates by standard agar well diffusion method. Altogether 638 students (325 of age group 12-13 years and 313 of age group 5-6 years) from 30 different schools of Kathmandu valley were included in the study and their dental status was examined and recorded by using WHO caries recording form with DMFT and DMFS as standard tools. Both monomicrobial and polymicrobial infections were observed in all cases. The dental pathogens isolates and identified were Streptococcus mutans, S. salivarious, S. sanguis, S. sobrinus, S. mitis, Lactobacillus species and Candida albicans. Out of 25 assayed toothpastes only 7 were found to be highly effective against all the tested pathogens. DMFS and caries percentage of age group 5-6 years and 12-13 years were found to be 3.79, 69% and 1.60, 53% respectively. But no significance difference at p less than 0.001 was observed among male and female of both age group. Strong correlation was found in dental caries status and type of schools, diet and toothpaste using habit. The result showed that the toothpastes containing Triclosan followed by fluoride as a major chemical ingredient possess significant antibacterial activities. In both age group caries percentage was found to be above the recommended level of WHO where as DMFS and DMST varies within the level.

Mutagenic effects of sodium azide in a cold tolerant Streptomyces spp.

(Published in The fifth National conference on Science and Technology, Nov. 10-12, 2008 Kathmandu pp.185)

Mutagenic effects of sodium azide in a cold tolerant Streptomyces spp.

K. B. Tiwari1, K Bhattarai2, U. T. Shrestha1 and V. P. Agrawal1
1Research Laboratory for Biotechnology and Biochemistry (RLABB)
Maitidevi, Kathmandu, Nepal
2Universal Science College
Maitidevi, Kathmandu, Nepal

ABSTRACT
Streptomyces spp. produces most of the known antibiotics that has therapeutic importance. The work explores the sodium azide induced beneficial mutations in a Streptomyces strain isolated from Khumbu, Everest region. A Streptomyces strain was isolated from a soil sample and purified at the Research Laboratory for Agricultural Biotechnology and Biochemistry (RLABB), Kathmandu, Nepal. The bacterium was then grown separately on plates containing 12.5, 25, 37.5 or 50ppm sodium azide. Subcultured colonies from plates containing sublethal concentrations of sodium azide were more brittle, irregular, smaller in size, yellowish in color, and asporogenic in comparison to the wild strain. Treatment of Streptomyces with 12.5ppm of sodium azide enabled bacteria to utilize nitrate, whereas alternate carbon sources (sucrose, mannitol and salicin) could be metabolized by those bacteria treated with 37.5ppm of the chemical. To explore possible mutations a RAPD primer (sequence CTGGCGTGAC; GC 70%; mp 34ºC) was used to investigate the DNA polymorphisms. Compared to that of the wild strain with five bands on a 1% agarose gel, two bands from a mutant were missing. Although point mutations on the primer binding site most likely resulted in the loss of bands, the RAPD-PCR technique can not reveal mutations in sequence between flanking ends. While this study explored loss-of-function (LOF) and gain-of-function (GOF) mutations, the GOF changes are more likely to result in strain improvement.