Screening of Potential Plant Growth Promoting Properties of Bacillus Species Isolated from Different Regions of Nepal

The deleterious effects of intensive use of chemical fertilizers and pesticides in agriculture has led to the substantial research efforts on finding the alternatives to these agrochemicals. This study was aimed to isolate Bacillus species from soil of different regions of Nepal and screen for their ability to promote plant growth directly or indirectly by testing their ability to produce plant growth hormone indole acetic acid, hydrogen cyanide, ammonia and protease as well as phosphate solubilization. Thirty nine Bacillus strains were isolated from 25 soil samples of different regions of Kathmandu and Chitwan districts of Nepal. These isolates were tested for plant growth promoting traits in vitro. Among the total isolates, about 48.7% were indole acetic acid producers, 38.4% of the isolates showed the ability to solubilize the phosphate, 71.8% were able to produce ammonia and all the isolates had the ability to produce hydrogen cyanide and protease. The isolated strains showed positive results to maximum PGPR traits and exhibited a potential to be used as alternatives to chemical fertilizers and pesticides and could be used as low-cost bio-based technology to promote plant growth in the agricultural sector

Assessment of phytochemicals, antimicrobial, antioxidant and cytotoxicity activity of methanolic extract of Tinospora cordifolia (Gurjo)

Traditionally used medicinal plants are the major resources of biologically active metabolites which are widely used for the cure of numerous diseases especially in developing countries where health facilities are rare. Many plants are in use for centuries but there is not enough scientific evidence and exploration. This research is focused on phytochemicals, antibacterial, antioxidant and cytotoxicity activity analysis of one of the most commonly used ethnomedicine Tinospora cordifolia collected from the Kavrepalanchok district of Nepal. Phytochemicals analysis of methanol extract of T. cordifolia showed the presence of alkaloids, coumarin saponins, glycosides, reducing sugar, and triterpenes. Antibacterial activity performed by disc diffusion method exhibited the highest activity against Streptococcus with a zone of inhibition are 10.3mm, 8.5mm, 6.5mm, and 6mm at 200mg/ml, 100mg/ml, 50mg/ml, and 25mg/ml of concentration respectively. DPPH radical scavenging activity increased with increasing concentration of extract. When compared with ascorbic acid at equivalent concentration, the extract shows a lower scavenging profile (56.07% for the extract and 98.01% for ascorbic acid at 320 ppm). Cytotoxicity was evaluated in terms of LC50 (lethality concentration). The result showed that the extract of T. cordifolia was found to be toxic with an LC50 value of 232.64μg/ml.  The bioactive component present in the plants could be the result of its pharmacological effects that support the traditional use of plants

Antibiogram and Phytochemical Analysis of Cinnamon, Clove, and Sichuan Pepper Extracts

A wide range of medicinal plant extracts has phytochemicals that possess antimicrobial properties and these plants are used to treat several infections. The study aimed to assess the antimicrobial activities of some spices extracts and to evaluate the phytochemicals present in them. The extracts of spices were prepared using Soxhlet apparatus refluxing with methanol and ethanol. The well diffusion technique was implemented for the evaluation of antimicrobial activities of the extracts and the zone of inhibitions was recorded in millimeters. The antimicrobial test was done against five bacterial isolates: Escherichia coli, Proteus mirabilis, Pseudomonas aeruginosa, Salmonella enterica serotype Typhi, and Staphylococcus aureus and a fungal isolate: Candida albicans. The extracts were concentrated by Rotary Vacuum Evaporator and a stock solution of 200 mg/mL was prepared by dissolving in 10 % DMSO. Concentrations of 40, 60, 80 and 100 mg/mL extracts were used for antimicrobial activity. The result of this study showed that clove extracts had the highest antimicrobial property against all the test microorganisms. Methanolic extract of clove had the highest inhibitory effect against Proteus mirabilis (24.21±0.15 mm), Pseudomonas aeruginosa (19.78±0.23 mm), and Candida albicans (20.07±0.08 mm) whereas ethanolic extract was effective against Escherichia coli (20.44±0.16 mm), Salmonella Typhi (21.66±0.31 mm) and Candida albicans (21.11±0.09 mm). Cinnamon and pepper extracts, leaving some exceptions, also had antimicrobial properties. The presence of phytochemicals: polyphenols, flavonoids, and tannins are the major components responsible for antimicrobial activity. Thereby, this study successfully demonstrated the possibilities of using spices extracts in the treatment of microbial infections

Phytochemical Analysis and α-Amylase Inhibitory Activity of Young and Mature Leaves of Cinnamomum tamala

The bioactive chemical components of the plant\u27s origin have been used as primary remedies for a wide array of human diseases including diabetes. The present research deal to evaluate and compare anti-diabetic potential of ethanolic and methanolic, young and mature leaves of medicinally valuable Cinnamomum tamala. Total phenolic and flavonoid contents of young and mature leaves were determined. In vitro α-amylase inhibition was carried out using 2-chloro-4-nitrophenyl-α-D-maltotrioside (CNPG3) as substrate. Phytochemical screening revealed the presence of polyphenols, flavonoids, terpenoids, quinones, carbohydrates, glycosides, diterpenes, tannins, and reducing sugars. The highest total phenolic content and flavonoid content were observed in methanolic extract of mature leaves (13.725 ± 0.54 mg GAE/g) and young leaves (12.591 ± 0.71 mg QE/g) respectively. Methanolic young leaves extract showed α-amylase inhibition with IC50 value 224.6 ± 2.76 μg/mL as compared to acarbose with IC50 value 5.93 ± 0.14 μg/mL. The result suggests that young leaves of C. tamala had anti-diabetic activity so further work should be carried out

Isolation, Identification and Screening of Bacillus species with Antimicrobial Activity from Different Soil Samples of Kathmandu Valley

Bacillus species are one of the predominant soil bacteria that are able to produce essential secondary metabolites that have antagonistic effects on other microorganisms. They are Gram-positive, endospore-forming, chemoheterotrophic, aerobic or facultative anaerobic rods usually consisting of peritrichous flagella for motility. The major aim of this study was to isolate the antimicrobials producing Bacillus spp. from soil samples of different parts of the Kathmandu Valley, identify them and to assess their antimicrobial activity against different pathogenic bacteria. The test organisms used were Staphylococcus aureus (ATCC 25923), E. coli (ATCC 25922), Pseudomonas spp., Salmonella spp., methicillin-resistant Staphylococcus aureus (MRSA) and extended spectrum beta-lactamase (ESBL) producing E. coli. Twenty four isolates from 9 soil samples identified as Bacillus spp. showed the zone of inhibition around their growth on Nutrient agar during isolation. These 24 isolates were chosen for primary screening of production of antimicrobial by perpendicular streaking method using four test organisms. . Of these 24 isolates, six isolates showing a significant zone of inhibition (≥1mm) against two or more test organisms from the primary screening were chosen for secondary screening which was further tested with six test organisms including ESBL E.coli and MRSA. They were further characterized through different physiological and biochemical tests. All 6 isolates showed inhibitory action against MRSA and the largest zone of inhibition (30mm) was shown by isolate U6. Isolate U3 was found to have broad spectrum antimicrobial activity with inhibitory effect against gram negative organisms- Pseudomonas and Salmonella and gram positive organism S. aureus (ATCC 25923). Isolate U5 showed a zone of inhibition of about 25mm against S. aureus which was comparable to that of erythromycin. Hence, this study determines the soil in Kathmandu Valley as a potential source of antimicrobial producing Bacillus spp. and recommends isolation and further characterization of Bacillus isolates as a possible source of novel drug to combat with the emergence of multidrug resistant strains

Sero-prevalance of Cryptococcal Antigenemia in HIV Positive Individual having CD4 Counts

Cryptococcus neoformans is one of the foremost common opportunistic infectious agents in people living with Acquired Immuno Deficiency Syndrome (AIDS). It has been reported to cause about 1 million cases of cryptococcal meningitis per year among HIV/AIDS and 600,000 deaths annually. This study was done to find the prevalence of Cryptococcal antigenemia among HIV positive individuals having CD4counts <100 cells/mm3. A cross-sectional study was conducted in the HIV Reference unit, National public health laboratory from July to December 2015. The study comprised of 99 HIV positive individuals having CD4counts <100 cells/mm3. CD4 T cell count was performed by flow cytometry (BD Biosciences, San Jose, CA, USA) and Cryptococcal antigen test by Latex agglutination assay. The overall prevalence of cryptococcal antigenemia was found to be 18.2%. Of the total ninety-nine subjects enrolled in the study, 72 (72.8%) were males and 27 (27.2%) were females. The mean age of the patients was 38 years ranging from 13 to 69 years. Higher percentage of female (22.2%) showed Cryptococcal infection in our study as compared to male (16.7%). The study concludes higher prevalence of Cryptococcal antigenemia among HIV infected individuals and recommends Cryptococcal antigen screening to be made mandatory in HIV positive patients having CD4 T cells count below 100/μl

Potential surface active agent production using very low grade and cheap substrate by Bacillus subtilis as microbial cell factory

Bio-surfactants are surface-active molecules which are produced by the wide range of microbes including bacteria, fungi, moulds, and yeast. This study was conducted to identify bio-surfactants by Bacillus subtilis combined with use of cheap substrates and industrial wastes (Mustard cake, Whey and Soya cake) which are found locally in Nepal. Bacillus subtilis, one of the most potential bio-surfactants producer; was isolated from soil sample of hydrocarbon contaminated site. Isolates were grown in a Minimal Salt Media (MSM) with 10% (v/v) mustard oil cake, whey and soya cake separately. The presence and potential of surfactant was determined by the oil spreading technique, emulsification index (%E24) and surface tension measurement. It was revealed that the surface tensions of cell free extract were 54.41, 60.02 and 56.64 mN/m for from mustard cake, whey and soya cake respectively as compared to distilled water (72.09) at 25oC. The emulsification index values was found to be highest in engine oil from the bio-surfactant extracted from mustard cake, soya cake and whey respectively. Similarly, mustard oil showed the lowest value of emulsification index. The highest emulsification activity was shown in mustard oil i.e. 1.13 from the cell free extract from mustard oil and lowest in engine oil i.e., 0.07, by the extract from soya cake medium, when measured in spectrophotometer at 540 nm. In conclusion, strain of Bacillus subtilis was found to be the potential surface active agent producers on the mustard oil cake, which can be useful medium for various environmental, food, medicinal and industrial processes

Antibiotic Susceptibility Pattern of Staphylococcus aureus Isolated from Pus/Wound Swab from Children Attending International Friendship Children's Hospital

A wound gets infected when the organism gets invaded through the breached skin, proliferated and production of various enzymes, toxins, etc. In order to treat the wound infection, antibiotic susceptibility pattern of organism should be determined before the prescription of the medicine. The present study was conducted from September 2017 to March 2018 with an aim to determine antibiotic susceptibility pattern of Staphylococcus aureus identified from the pus/wound swab among the patients visiting the International Friendship Children's Hospital, Kathmandu, Nepal. Total 270 sample were processed, isolated and identified using standard microbiological procedure and biochemical test. Antibiotic susceptibility test was carried out by using Modified Kirby Bauer's Disc Diffusion Method. Out of total sample, 51.48% (139) showed growth. The growth distribution was found to be high in out-patient department 84.9% (118) than in-patient department 15.1% (21). Among 139 positive growth, 83.5% were gram positive and 16.5% were gram negative. All together 12 different organisms were identified, among which S. aureus was found to be predominant organism 105 (75.5%). S. aureus was found to be sensitive towards Linezolid followed by Doxycycline whereas it was found resistant towards Ciprofloxacin. Among S. aureus identified, 50% were Multidrug resistant (MDR) S. aureus and 55% were Methicillin resistance S. aureus (MRSA). MRSA was found to be sensitive towards Linezolid followed by Doxycycline and resistant towards Ciprofloxacin. The association between MDR and MRSA was found positively significant (i.e. p-value = 0.000). All strains of S. aureus were found to be sensitive towards Vancomycin.  22.86% were double disk diffusion test (D-test) positive. The prevalence of D-test was found to be high in MRSA (75%). The relationship between D-test and MRSA was found to be significantly correlated with each other (r = 0.39). Linezolid, Chloramphenicol, Vancomycin and Doxycycline is a drug of a choice for both S. aureus and MRSA infection

Potential surface active agent production using very low grade and cheap substrate by Bacillus subtilis as microbial cell factory

Bio-surfactants are surface-active molecules which are produced by the wide range of microbes including bacteria, fungi, moulds, and yeast. This study was conducted to identify bio-surfactants by Bacillus subtilis combined with use of cheap substrates and industrial wastes (Mustard cake, Whey and Soya cake) which are found locally in Nepal. Bacillus subtilis, one of the most potential bio-surfactants producer; was isolated from soil sample of hydrocarbon contaminated site. Isolates were grown in a Minimal Salt Media (MSM) with 10% (v/v) mustard oil cake, whey and soya cake separately. The presence and potential of surfactant was determined by the oil spreading technique, emulsification index (%E24) and surface tension measurement. It was revealed that the surface tensions of cell free extract were 54.41, 60.02 and 56.64 mN/m for from mustard cake, whey and soya cake respectively as compared to distilled water (72.09) at 25oC. The emulsification index values was found to be highest in engine oil from the bio-surfactant extracted from mustard cake, soya cake and whey respectively. Similarly, mustard oil showed the lowest value of emulsification index. The highest emulsification activity was shown in mustard oil i.e. 1.13 from the cell free extract from mustard oil and lowest in engine oil i.e., 0.07, by the extract from soya cake medium, when measured in spectrophotometer at 540 nm. In conclusion, strain of Bacillus subtilis was found to be the potential surface active agent producers on the mustard oil cake, which can be useful medium for various environmental, food, medicinal and industrial processes

Isolation, Identification and Screening of Bacillus species with Antimicrobial Activity from Different Soil Samples of Kathmandu Valley

Bacillus species are one of the predominant soil bacteria that are able to produce essential secondary metabolites that have antagonistic effects on other microorganisms. They are Gram-positive, endospore-forming, chemoheterotrophic, aerobic or facultative anaerobic rods usually consisting of peritrichous flagella for motility. The major aim of this study was to isolate the antimicrobials producing Bacillus spp. from soil samples of different parts of the Kathmandu Valley, identify them and to assess their antimicrobial activity against different pathogenic bacteria. The test organisms used were Staphylococcus aureus (ATCC 25923), E. coli (ATCC 25922), Pseudomonas spp., Salmonella spp., methicillin-resistant Staphylococcus aureus (MRSA) and extended spectrum beta-lactamase (ESBL) producing E. coli. Twenty four isolates from 9 soil samples identified as Bacillus spp. showed the zone of inhibition around their growth on Nutrient agar during isolation. These 24 isolates were chosen for primary screening of production of antimicrobial by perpendicular streaking method using four test organisms. . Of these 24 isolates, six isolates showing a significant zone of inhibition (≥1mm) against two or more test organisms from the primary screening were chosen for secondary screening which was further tested with six test organisms including ESBL E.coli and MRSA. They were further characterized through different physiological and biochemical tests. All 6 isolates showed inhibitory action against MRSA and the largest zone of inhibition (30mm) was shown by isolate U6. Isolate U3 was found to have broad spectrum antimicrobial activity with inhibitory effect against gram negative organisms- Pseudomonas and Salmonella and gram positive organism S. aureus (ATCC 25923). Isolate U5 showed a zone of inhibition of about 25mm against S. aureus which was comparable to that of erythromycin. Hence, this study determines the soil in Kathmandu Valley as a potential source of antimicrobial producing Bacillus spp. and recommends isolation and further characterization of Bacillus isolates as a possible source of novel drug to combat with the emergence of multidrug resistant strains