Contribution of Microbiologists in Nepal during COVID-19 Pandemic

No abstract available.

Characterization of β-Galactosidase from Lactose Utilizing Yeast Isolated from the Dairy Sample

Objectives: The objective of the study was to isolate lactose positive yeasts from dairy samples collected from local markets of Kathmandu, to extract crude β-galactosidase from the lactose positive yeast and to characterize the enzyme for optimum time duration, pH, temperature, Michaelis-Menten constant (Km) and maximum activity (Vmax). Methods: Four lactose positive yeast strains were isolated from dairy samples collected from local market of Kathmandu by pour plate method. Single strain having maximum lactose positive activity was selected for the study. The mass culture of the lactose positive yeast strain was lysed by 2% Chloroform and the yeast cell lysate containing β-galactosidase (i.e. crude enzyme extract) was characterized by using ONPG (Ortho-Nitrophenyl-β-D-galactopyranoside) as substrate. ONPG is a colorless substrate for the enzyme assay which is hydrolyzed by the enzyme into yellow colored product ONP (Ortho-Nitrophenol). The concentration of product formed was monitored spectrophotometrically at 420 nm to determine the enzyme activity and to characterize the enzyme. Results: The enzyme had wide range of working temperature from 0-50ºC, with optimal temperature of 37ºC. However, greater than 50% hydrolyzing ability was maintained in the range of 14-40ºC. Optimum time of reaction was 70 min. The enzyme had maximum activity in the near neutral pH of 6.8. Michaelis-Menten constant of the enzyme was found to be 2.23 mM of ONPG and Vmax was 58.82 nmol/min/ml. Enzyme activity was 27.88 nmol/min/ml, Specific enzyme activity was 59.97 nmol/ min/mg and total enzyme activity was 3346.33 nmol/min. Conclusion: The activity over a wide range of temperature 0-50ºC with low Km value shows that the enzyme has a commercial application in clearance of lactose pollution in waste water in different environmental conditions.

Field Evaluation of Native B. thuringiensis Isolates Against Aphids (Aphis fabae)

Objectives: The purpose of this study was to evaluate the aphicidal activity of native Bacillus thuringiensis (Bt) strains. Methods: Soil samples of Provinces 2 and 3 of Nepal were collected randomly for isolation of Bt by acetate selection method. Bt were identified by observing insecticidal crystal proteins (ICPs) by Commassie Brilliant Blue (CBB) staining technique. Aphicidal activity of 12 B. thuringiensis isolates was evaluated by two processes. The preliminary screening was done by spraying the suspension containing the spore and ICPs mixture in Phaseolus species heavily infested with black aphids (Aphis fabae) in fi elds. The second process (selective bioassay) was done by counting the number of aphids (nymphs, instar, winged, wingless) before and after spraying 5ml of suspension containing the spore and ICPs mixture on the leaf or on the beans pods surface infested by Aphids. The mortality percentage of Aphids after treatment was calculated on the 4th day, by counting the live aphids and the result was recorded. Results: Preliminary screening for aphicidal activity revealed that 4 isolates ML5(1), CW1(1), SN2(1) and MP2(1) producing spherical crystal protein, showed 100% mortality against nymphs, instar, winged and wingless Aphids. Isolates were effective in controlling the Aphid (Aphis fabae) within 4 days and the part of the plant that was sprayed becomes free of Aphids. Selective bioassay of native isolate MP3(3) was most effective in killing 95.83% of aphids followed by CW2(1), 85.71%, ML5(1), 77.34%, SN3(1), 72.72%, CW1(1), 70.21%. Conclusion: This study revealed that indigenous Bacillus thuringiensis of Terai region of Nepal are effective in controlling Aphids.

Bacteriological Profile and Antibiotic Susceptibility Pattern of Isolates of Wound Infection In Children Visiting Kanti Children Hospital

Objectives: The objectives of this study was to isolate and identify the bacterial etiological agent of wound infection and explore the status of methicillin-resistant Staphylococcus aureus ( MRSA), multidrug Resistant (MDR) and extended spectrum β-lactamase (ESBL) producers’ strains in clinical specimens and to find the antibiotic susceptibility pattern. Methods: A prospective cross sectional study design was conducted from period of February 2014 to October 2014 at Kanti Children Hospital, Kathmandu. The organisms were isolated and identified from pus sample by standard microbiological methods. Antimicrobial susceptibility test was performed by modified the Kirby Bauer disc diffusion method to evaluate the status of MRSA and MDR. ESBL detection was performed by the combined disc diffusion method. Results: Out of 365 specimens collected between the age group below < 2 to 15 years, 210 (57.73%) samples from male patients and 155 (42.47%) from female patients. In the total samples processed, Gram-positive organisms were found to be more prevalent in which Staphylococcus aureus accounts for 135(47.20%), followed by P. aeruginosa 62 (21.67%), E. coli 29 (10.20%), K. pneumoniae 27 (9.44%), Acinetobacter spp. 20 (6.70%), P. vulgaris 7 (2.44%) and CoNS 6 (2.10%). Among the S. aureus isolates, 29 (21.48%) were found MRSA. Of the total Gram-negative organisms isolated, 74 (51.03%) were MDR and 14 (100%) ESBL producer, (P<0.01). S. aureus was found to be the most important and leading cause of wound infection in this study. Conclusion: Thus, routine antibiotic susceptibility testing is recommended for empirical drug therapy and proper management of disease.

Screening of Indigenous Yeast From Different Ecological Region of Kathmandu Valley and Its Application in Wine Production

Objectives: The aim of the study was to isolate and screen the potent yeast from the air for implementing new yeast in wine fermentation. Methods: In this study, 35 air samples collected in sterile grape juice in glass jar and left over for four days exposure for the growth of yeast from different locations around the Kathmandu Valley. Yeasts were screened by culturing on selective Ethanol Sulfite Agar (ESA) media at 30°C for 2-3 days in Microbiology Lab of Pinnacle College. Yeast isolates were characterized based on colony morphology, microscopic characteristics, Fermentative capacity, Hydrogen sulfide production. Selected yeast isolates were subjected to ethanol fermentation and tested for alcohol tolerance capacity. Wine quality was assessed by sensory evaluation. Results: Of 35 samples, only 20 yeast isolates were isolated. Among these isolates, the variation in colony characteristics along with oval and ellipsoidal microscopic appearance was observed. All the isolates were able to ferment major sugars such as glucose, fructose and sucrose, but few could not ferment galactose and maltose, while none-fermented lactose and xylose. Here, isolates showing no H2S (L29, L34) and mild H2S producer (isolate L31) were subjected to ethanol fermentation. Also, Comparative analysis was made by using commercial standard wine yeast (STAN). Rapid fermentation of grape juice with initial 21 0Brix was observed in L31 isolate, which produced 12.99% v/v alcohol with titratable acidity (TA) 5.25 g/L, followed by L29 strain with 11.99%v/v alcohol and 4.5 g/L TA which were higher than STAN (10.99% alcohol). These isolates specified as Ethanol tolerance up to 13%v/v, while none of them were able to grow at 15% v/v ethanol concentration and 45°C temperature. However, significant growth was observed at pH 3 along with sugar tolerance capacity at 30 0Brix. The wine produced by these isolates was found to be remarkably different among each other. While the sensory analysis of wine led to isolate L31 being congenial to tasters. Conclusion: L31 isolate was found to be efficient and advantageous for wine production indicating its industrial application.

Evaluation of Ground Water Quality of Kathmandu Valley and Antibiotic Susceptibility test against Klebsiella pneumoniae

Objectives: The aim of this study was to assess quality status of ground water in Kathmandu valley and describe the antibiotic susceptibility of the isolated Klebsiella pneumoniae. Methods: A total of 100 samples were collected from different places of Kathmandu valley with 50 each from two different groundwater sources namely boring and well. This study was conducted from June to September, 2019 at Environment and Climate Study Laboratory, Nepal Academy of Science and Technology (NAST). The physicochemical analysis of the samples was done according to standard methodology. Membrane filtration technique was performed for the enumeration of total coliform and different biochemical tests were performed for isolation and identification of Klebsiella pneumoniae followed by Kirby-Bauer disc diffusion method for antibiotic susceptibility test. Results: This study reveals the poor microbiological aspects of ground water sources as 98% of total water samples crossed the standard value for total coliform count. The pH, turbidity, ammonia, nitrate and iron content were found to be higher than Nepal Drinking Water Quality Standard (NDWQS 2005) in 15%, 26%, 34%, 7% and 26% of total water samples respectively. The chloride and arsenic content in all the water samples were within the NDWQS, 2005. The 12 out of 18 isolates of Klebsiella pneumoniae from ground water source were highly resistant against first generation Cefazolin however, 15 out of 18 isolates were sensitive to Chloramphenicol. Additionally, four isolates showed zone of inhibition in intermediate range provided by Clinical and Laboratory Standard Institute (CLSI) guideline towards Imipenem and Meropenem. Conclusion: This study concludes that ground water sources were heavily contaminated by coliform bacteria and most of the physicochemical aspects were under standard limit. Although Klebsiella pneumoniae isolated from ground water were not multidrug resistant, one isolate was recorded to be resistant to Meropenem. These results alarm for circulation of antibiotic resistance in environmental bacterial isolates. Therefore, the appropriate water purification methods should be applied before consumption and should be examined periodically.

Characterization of Intestinal Parasitosis in Pregant Women at Ram Janaki Hospital, Janakpurdham

Objectives: The objective of this study was designed to focus the prevalence, detection and identification of intestinal parasites and its associated factors among pregnant women. Methods: Total 264 stool samples were collected in a labeled dry, clean disinfectant free wide mouthed plastic container during antenatal visits at Ram Janaki Hospital, Janakpurdham and were examined by macroscopically and microscopically. The detection and identification of protozoal cysts, oocysts, trophozoites and helminthic eggs or larva was done by wet preparation and formalether sedimentation concentration technique. The data was analysed using SPSS 20 version and Microsoft Excel 2007. A Chi-square test was performed to predict the parasite detection using predictor variables. The p-values <0.05 was considered as significant. Results: The prevalence of intestinal parasitosis among pregnant women was 42%. There was positive association of symptoms of intestinal parasitosis among pregnant women (p < 0.05). The most predominant intestinal parasites among study participants were E. histolytica (20%) slightly dropped by G. lamblia (16%) followed by Hook worm (13%) and A. lumbricoides (11%). The correlation between all the variables with intestinal parasites presence and absence was statistically significant (p<0.05) but statistically insignificant for age and consumption of green leafy vegetables (p>0.05). Conclusion: The overall prevalence of intestinal parasitosis was relatively moderate. Lack of awareness, low hygienic and sanitation habits regarding parasitic infections were the major determinant factors for higher prevalence. Improving sanitation, awareness creation and public health programes should be organized at regular interval in community.

Effectiveness of Commonly Used Antibiotics in Combination with Honey Against Bacterial Infection

Objectives: The study was carried out to compare the inhibitory effects between commonly used antibiotics and bee honey samples, so as to correlate the inhibitory effects between bee honey alone and in combination with antibiotics. Methods: This study was carried out between December 2012 to September 2013. A total of one hundred and twenty-two clinical microbiological specimens and five different floral sourced honey samples were collected between December 2012 to September 2013. Twenty-three multi-drug resistant organisms were selected. Then, AST for commonly used antibiotics, honey alone and combination of honey-antibiotics discs was done. The difference in ZOI of antibiotic contrasting with the antibiotics containing honey were statistically analysed to define the synergism. Results: The inhibition due to honey is variable among bacteria types (F=39.17, p<0.05). From means plot, Staphylococcus and Acinetobacter were recognized as highly susceptible bacteria for honey (Χ = 21.1 ± 6.2 mm and Χ = 18.3 ± 3.3 mm respectively) but Acinetobacter species could not show synergism to honey-antibiotic combination. The tested organisms from Enterobacteriaceae family showed effective susceptibility to Chloramphenicol-honey mixture. Imipenem-honey combination and Gentamicin-honey combination showed significant effects against Pseudomonas aeruginosa. Conclusion: Thus, honey can be used in various bacteria-directed infections and found to be effective in various infections. Incorporation of honey in antibiotics like Chloramphenicol, Imipenem, and Gentamicin work better in healing various infection.

Antibiotic Susceptibility Pattern of Salmonella Enterica serovars Typhi and Paratyphi A Isolated From Patients Suspected of Enteric Fever

Objectives: The study aimed to assess the antibiotic susceptibility profile of Salmonella spp isolated from patients suspected of enteric fever. Methods: This cross-sectional prospective study was carried out from April to June, 2014among 484 patients clinically suspected of enteric fever visiting Bir Hospital, Kathmandu, Nepal. Blood sample collected from each patient was processed for culture in bile broth. Identification of Salmonella spp was done by conventional microbiological techniques including colony characteristics, Gram's staining and biochemical tests. Antibiotic susceptibility testing of identified isolates was done by Kirby-Bauer disk diffusion method following the 2014 CLS I guideline. Results: Out of 484 blood samples, 36 (7.43%) cases showed the growth of Salmonella spp; of which 27 (75%) were Salmonella enterica serovar Typhi (ST) and 9 (25%) were Salmonella enterica Paratyphi A (SPA). Among the Salmonella isolates, 5.55% were multidrug resistant and 41.66% were fluoroquinolone resistant. More than 80% of isolates were sensitive to chloramphenicol, amoxicillin, and cotrimoxazole whereas 58%, 50% and 6% of isolates were sensitive to fluoroquinolone antibiotics i.e. ciprofloxacin, ofloxacin and nalidixic acid respectively. All the isolates were susceptible to ceftazidime. All SPA and 89% of ST were sensitive to azithromycin. Conclusion: Higher percentage of susceptible isolates to chloramphenicol, cotrimoxazole, and amoxicillin suggests the reconsideration of these antibiotics for the treatment of enteric fever. Azithromycin can be considered as drug of choice for the treatment of enteric fever.

Incidence of Intestinal Parasites in Government and Private School Going Children

Objective: Aim to assess the incidence of intestinal parasites in government and private school going children. Methods: The work was conducted from October, 2018 to March, 2019 at Microbiology Laboratory of DAV College, Dhobighat, Lalitpur. A total of 100 stool samples of children aged between 5-12 years were collected from both government and private schools situated in Lalitpur metropolitan city, during school hours. The stool samples were examined for intestinal parasites by Saline wet mount; Iodine wet mount and Formal – ether sedimentation technique. The questionnaires accompanying the queries related to the study were filled. Results: Of the total 100 stool samples examined, intestinal parasites were observed in 7% (7/100) of the total stool samples. Among the positive stool samples, 71% (5/7) of the stool samples were from government school’s children whereas 29% (2/7) were from private school’s children. Fifty seven percentage 57% (4/7) girls and 43% (3/7) boys were found to be infected with intestinal parasite in the tested stool samples. Out of total parasite detected, 57% (4/7) were eggs of Ancylostoma duodenale, 29% (2/7) were eggs of Ascaris lumbricoides and 14% (1/7) were cysts of Giardia lamblia. The study indicates that Ancylostoma is the most commonly infecting parasite followed by Ascaris and Giardia. Conclusion: Personal hygiene and sanitary condition were responsible for the incidence of intestinal parasites in the school going children. Environmental sanitation improvement and health education promotion will be helpful to reduce the parasitic infection rate.