Microbiology
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Item An evaluation of 5% Naoci microscopy method for the laboratory diagnosis of pulmonary tuberculosis(2008) Lama, Suman; Anjana SinghDirect sputum smear microscopy forms the mainstay for pulmonary tuberculosis case-finding in the resource-poor countries like Nepal. However, this method is hampered by the lack of sensitivity. Bleaching of sputum with sodium hypochlorite and concentration of mycobacteria prior to Ziehl-Neelsen staining method is a possible means of improving the sensitivity of direct microscopy. Therefore, this study was performed in National Tuberculosis Centre, Thimi, Bhakatapur, Nepal with an objective to evaluate 5% NaOCl microscopy method for the primary diagnosis of pulmonary tuberculosis. A total 475 sputum were collected from 159 suspected pulmonary tuberculosis patients. Direct smears were prepared from all sputum. ‘On-spot’ sputum was divided equally into three tubes (A, B and C). Tube A and B were treated by NaOCl-centrifugation method and NaOCl-sedimentation method respectively, and ZN smears were prepared. Tube C was treated by Modified Petroff’s method for culture. NaOCl-centrifuged and NaOCl-sedimented ‘on-spot’ sputum showing positive direct smears were also used for culture to ascertain the sterilizing activity of NaOCl. Culture method was employed as a gold standard for pulmonary tuberculosis diagnosis. Direct ‘on-spot’ sputum Ziehl-Neelsen smears, NaOCl-centrifuged Ziehl-Neelsen smears and NaOCl-sedimented Ziehl-Neelsen smears had 17.7%, 16.3% and 10.1% positivity respectively compared to 14.5% of the standard three smears strategy. There was a statistically significant increase in the number of acid-fast bacilli positive sputum samples by NaOCl methods (P<0.05). In addition, there was no statistical significant between two NaOCl methods (P>0.05). With reference to culture, sensitivity of direct ‘on-spot’ sputum ZiehlNeelsen smears, NaOCl-centrifuged Ziehl-Neelsen smears and NaOCl-sedimented ZiehlNeelsen smears were found to be 35.1%, 67.5% and 62.1% compared to 54% of standard three smears strategy. The specificity was found to be same i.e. 97.4% for all methods. The positive and negative predictive values were found to be 81.2%/82.3% for direct ‘on-spot’ sputum Ziehl-Neelsen smears, 89.2%/90.4% for NaOCl-centrifuged Ziehl-Neelsen smears, 88.4%/89% for NaOCl-sedimented Ziehl-Neelsen smears and 87%/87% for standard three smears strategy. No growth of Mycobacteria tuberculosis was seen in culture media (Lowenstein-Jensen) inoculated with NaOCl-centrifuged and NaOCl-sedimented ‘on-spot’ sputum showing the biocidal activity of NaOCl. This study indicates that 5% NaOCl microscopy method i.e. NaOCl-centrifuged ZiehlNeelsen smears andNaOCl-sedimented Ziehl-Neelsen smears could be alternative to direct microscopy and the application of these methods would make a positive impact on the effectiveness of TB control programs. Keywords: Tuberculosis, M. tuberculosis, Sputum smear microscopy, Bleach digestion, Ziehl-Neelsen.Item Determination of ethanol tolerance in yeast cultures isolated from indigenous starter murcha and the effects of uv mutation on ethanol tolerance(2024) Nepal, Ashish; Tika Bahadur KarkiMicroorganisms improved through various methods of strain improvement have been used in industrial production of various organic compounds efficiently for consumption or use in food production. The use of yeast in food grade alcohol production from sugar may be the most known of these processes. In recent years, the research has shifted from the optimization of the reaction parameters to the improvement of the microorganism used. Strain improvement, usually done by gene manipulation are more precise but also tend to be cost-prohibitive. A simpler method to bring about improvement is by mutation of the microorganism. The primary objective of the investigation was the isolation and identification of fermentative yeast from an indigenous starter culture followed by improvement of the alcohol tolerance through UV mutation. The study took place in the laboratory of National College, Khusibu from March 2022 to July 2022. In the present study, 21 isolates were obtained from murcha samples of which 8 were found to be fermentative yeasts. These isolates were subjected to identification, and tolerance tests to sugar and alcohol followed by mutation of best-performing isolates. Isolates Y1 and Y11 were identified as having the best tolerance to sugar and alcohol and were subsequently exposed to UV radiation for mutation. The growth as observed through UV spectrophotometry at 600nm increased for isolate Y10 from 0.89 to 1.55, 0.54 to 0.97, 0.23 to 0.43 at 0%, 5%, 10% respectively, and an increase from 0.73 to 1.1.59, 0.57 to 0.89, 0.23 to 0.6 at 0%, 5%, 10% respectively for isolate Y11. Isolates Y10 and Y11 also grew at 12.5% of ethanol after mutation but they were not able to before UV treatment. The isolates Y10 and Y11 were identified as Saccharomyces spp. The mutated isolates were found to have increased tolerance to ethanol in terms of ethanol percentage while all mutated isolates were found to have more growth in the media than the unmutated isolates after UV irradiation. Keywords: Yeast, Murcha, Indigenous Starter, Sugar Tolerance, Alcohol Tolerance, MutationItem Detection of metallo beta-lactamases producing pseudomonas aeruginosa from different clinical samples(2024) Shrestha, Bonny; Era TuladharPseudomonas aeruginosais an opportunistic bacteria that have an ability to cause nosocomial infection in patient with diverse range of infection such as urine infection, respiratory infection, bacteremia, wound and other central nervous system. Increase in antibiotic resistant has been a nuisance in the hospital settings. More ever, increase in prevalence of metallo beta lactamase producing P. aeruginosa leads to increase in morbidity and mortality due to the limited number of choice of drugs for treatment. This study aims in isolating the P. aeruginosa from different clinical samples and detection of multidrug resistant as well as screening of MBL producing P. aeruginosa. The hospital based cross sectional study was done in Grande International Hospital, Kathmandu from January – June 2024. A total of 1500 different clinical samples such as urine, blood, sputum, pus, tissue, wound etc. were processed during the study time, where 126(8.2%) of Pseudomonas aeruginosa were isolated from different samples of different ages and gender, of either inpatient or outpatient. 34.6% isolates were isolated form urine,31.7% from sputum, 19% from blood, 1.6% from pus, 4.8% from wound swab and 0.8% form tissue were obtained from 66.1% of male and 38.9% of female infected patients. MBL prevalence was found to be 16.6% which were more from ICU (47.6%/) followed by 33.3% from general ward. Levofloxacin had higher sensitive pattern(82.4%) and Nitrofurantoin was more resistant (90.2%).In MBL isolates, Aztrenam was found to be least resistant (33.3%). Infection by P. aeruginosa can cause a medical threat. A multidrug resistant isolates can even be more difficult to treat. Increased MBL isolates has been a nuisance in hospital settings. Therefore, routine evaluation should be done to ensure the control of these isolates. Key words: Pseudomonas aeruginosa, Multidrug resistant,Metallo betalactamases (MBLs),Item Comparative analysis of genexpert and line probeassay for detecting mycobacterium tuberculosis at the national tuberculosis control center inNepal(2024) Baral, Soyuz; Era TuladharTuberculosis is a disease caused by Mycobacterium tuberculosis; a pathogenic bacterial species primarily known for causing chronic respiratory infections. In Nepal, tuberculosis (TB) remains a critical public health challenge, highlighting the urgent need for effective and rapid diagnostic tools. This study is important as it evaluates the effectiveness of GeneXpert and Line Probe Assay (LPA) compared to the culture method, potentially enhancing rapid TB detection and improving patient management. This hospital-based cross-sectional study was conducted from September 2022 to April 2023 at the National Tuberculosis Control Centre. The objective was to evaluate the diagnostic capabilities of GeneXpert and Line Probe Assay (LPA) for detecting Mycobacterium tuberculosis compared to the culture method, which is considered the gold standard. The research method involved collecting and analyzing sputum samples (n=398) using solid culture, GeneXpert and LPA. The culture method identified Mycobacterium tuberculosis in 128 cases. GeneXpert detected it in 129 cases, while LPA detected it in 123 cases. Against culture, GeneXpert demonstrated a sensitivity of 95.49% and a specificity of 99.22%, with results available in approximately 2 hours. The positive predictive value for GeneXpert was 98.45%, and the negative predictive value was 97.70%. In comparison, LPA showed a sensitivity of 90.98% and a specificity of 99.18%, with a turnaround time of up to 6 hours. The positive predictive value for LPA was 98.37%, and the negative predictive value was 95.29%. Additionally, Cohen's Kappa value for GeneXpert was 0.95, and for LPA, it was 0.92, indicating almost perfect agreement with the culture method. The findings suggest that GeneXpert offers higher sensitivity and faster results, making it valuable for rapid TB detection. Both assays exhibited high specificity, supporting their integration into routine TB diagnostics. This enhances rapid and accurate detection, thereby improving patient management and treatment outcomes. Keywords: Mycobacterium tuberculosis, Tuberculosis, GeneXpert, Line Probe Assay, CultureItem SCREENING OF ANTIBIOTIC PRODUCING ACTINOMYCETES FROM THE SOIL SAMPLES AND ANTIBACTERIAL ACTIVITY IN VARYING SODIUM NITRATE CONCENTRATION(Amrit Campus, 2022-08-08) DANGOL, SWORNIMAActinomycetes are gram positive filamentous, slow growing bacteria, best known to produce antibiotic. The aim of this study was to screen antibiotic producing actinomycetes and determine its antibiotic activity against ATCC cultures at different gradient of NaNO3. This study was carried out from March 29 to April 28, 2022. Thirty collected samples were collected and transported and processed in Amrit campus. Primary and secondary screening were performed by perpendicular streaking and agar well diffusion method respectively. Characterization and identification of the isolated actinomycetes were performed. From thirty collected samples, twenty-eight samples were actinomycetes. Only two samples were antibiotic producing actinomycetes MI410 -3 and NP110 -2 showed antibacterial activity against ATCC cultures viz: S. aureus 43300, E. coli 35218, E. coli 25922 and Klebsiella 700603 in primary screening. Antibiotic was produced by sub-merged state fermentation with varying concentration of NaNO3 and secondary screening was done by agar well diffusion against ATCC cultures in comparison to standard streptomycin (100 µg/mL). In comparison to standard streptomycin (100 µg/mL) extract MI410-3 with 1% NaNO3 was effective only against E. coli 25922 while NP110-2 with 0.5% NaNO3 against E.coli 35218 (14.67mm), E.coli 25922 (19.67mm) and S. aureus 43300 (17mm) whereas NP110-2 has also shown antibacterial effect with 1.5% NaNO3 against E. coli 35218 (13mm), Klebsiella spp. ATCC 700603 (11.5mm) and S. aureus 43300 (12.5mm). Statistically, there is significant difference at 5% level of significance between the sample concentration at 0.5% of NaNO3 (.001, P<0.05) and 1.5% of NaNO3 (0.024, P<0.05) in antimicrobial activity. However, there is no significant difference at 5% level of significance between the sample concentration at 1% (0.356, P>0.05). The isolated actinomycetes was presumed as Streptomyces spp. Both MI410-3 and NP110-2 (7.14%) showed antibacterial activity against cultures in primary screening. The indigenous species of actinomycetes, isolated from various places of Kathmandu valley can be used in industrial production of antibiotics which can help in economic growth of Nepal.Item ENUMERATION AND DETECTION OF ANTIBIOTIC SUSCEPTIBILITY PATTERN OF COLIFORM BACTERIA FROM MILK SAMPLES IN KATHMANDU.(Amrit Campus, 2022-08-08) SURAJ, CHAULAGAINThe presence coliforms and their resistance in milk is the big issue in present time. Milk is an excellent source of nutrients and also serves as a good medium for the growth of milk-borne pathogens. Cross-sectional study was conducted to assess and compare microbial quality of raw milk and pasteurized milk and also determine antimicrobial susceptibility patterns of coliforms from the milk samples. For this, 30 milk samples (15 raw and 15 pasteurized milk) were collected from different locations of Kathmandu district. Starch adulteration test and MBRT were done. TCC, FCC for each sample were determined by pour plate technique and interpretated with BIS guidelines (1992), DFTQC guidelines and identification was done. Antibiotic susceptibility testing of isolates was carried out by Kirby Bauer disk diffusion method using 12 different antibiotics. TCC of the 12 raw samples were higher than the guideline and its FCC was also found to be higher in 9 samples. In case of pasteurized samples, TCC was higher in 6 samples and FCC in 4 samples. A total of 31 isolates, 21 from raw samples and rest from pasteurized samples were identified. Out of 31 isolates, 17 (54.84%) were identified as Klebsiella spp., 13 (41.94%) were E. coli and 1 (3.22%) was Citrobacter spp. AST of coliform isolates were 100% sensitive against TE. 96.77% of the isolates were sensitive towards NIT, PIT, COT, C and AK. Out of total, 11 (35.48%) were MDR (Multi-Drug Resistant). Among them, 7 (63.64%) were from raw samples and rest 4 (36.36%) from pasteurized samples. Although, quality of most of the samples were good as per MBRT but the presence of antimicrobial resistant bacteria and adulterants questions the overall quality ofmilk. Thus, it is concluded that the milk produced by small-scale farm from the studied area are not of good quality, caused by coliforms especially the antibiotic resistant. Therefore, such type of study for monitoring the microbial quality of milk should be done in order to safeguard the consumers. Otherwise, it will be hazardous for the consumers and can be a potential source of milk-borne infections.Item BACTERIOLOGICAL ANALYSIS OF MILK SOLD IN KATHMANDU AND ANTIBIOTIC SUSCEPTIBILITY PATTERN OF Staphylococcus species ISOLATED FROM MILK(Amrit Campus, 2022-08-08) Soniya, BohoraMilk is a rich source of nutrients. Milk –borne pathogenic bacteria pose a serious threat to human health. Staphylococcus aureus, Salmonella spp., Listeria monocytogenes with Escherichia coli and Campylobacter are the main microbial hazards associated with contaminated milk. Therefore, it can cause milk borne diseases like scarlet fever, Brucella, diphtheria typhoid etc. This study was conducted to assess and compare microbial quality of raw milk and pasteurized milk and also determine the antimicrobial susceptibility of Staphylococcus species isolated from milk sample consumed in Kathmandu. For this, 30 milk samples (15 raw milk and 15 pasteurized) were collected from different location of Kathmandu district. Total Plate Count and Total Staphylococcus Count for each sample were determined by pour plate technique. While for isolation of Staphylococcus species, samples were isolated by using selective media (MSA) and characterized by biochemical test. Antibiotic susceptibility testing of isolates was carried out by Kirby Bauer disk diffusion method. Total bacterial count of all raw milk samples were within the range while for TBC of pasteurized milk 93% were within the range. 17 Staphylococcus species were isolate from TSC. Among them 15 were identified as Staphylococcus aureus. 17 Staphylococcus species were 100% sensitive to Cotrimoxazole, Amikacin and Levofloxacin but resistant to Penicillin G (100%),Ceftriaxone (52.92%), Tetracycline (17.68%), Cefoxitin (23.58%), Ampicillin (76.82%) Ciprofloxacin (17.68%) and Chloramphenicol (11.79%). 3(17.68%) of Staphylococcus aureus isolated from raw milk samples showed multi-drug resistance and 4(23.58%) MRSA were detected. It is concluded that the milk produced by small scale farm from different places of Kathmandu district are not of quality and can be potential source of milk-borne infection. It is recommended that routine assessment of microbial quality of milk should be done for the safeguard of consumer health.Item EFFICACY OF HAND SANITIZERS AGAINST STANDARD BACTERIAL CULTURES(Amrit Campus, 2022-08-08) Sherpa, Shyronह्याण्ड स्यानिटाइजरहरु हातको स्वच्छता कायम राख्न प्रयोग गरिन्छ । कोरोना महामारीले यसको प्रयोग बारे ध्यान खिचेको थियो । धेरै कम्पनीहरुले उत्पादन गरिरहेका छन् तर प्रभावकारीता राम्रोसँग स्थापित हुन सकेको छैन । यस अध्ययनको मुख्य उद्देश्य काठमाण्डौमा बिक्री हुने ह्याण्ड स्यानिटाइजरहरुको व्याक्टेरियालाई मार्ने क्षमताको प्रभावकारीता मूल्याङ्कन गर्नु थियो । यस अध्ययन मार्च देखि अप्रिल २०२२ सम्म गरिएको थियो । यस अध्ययनमा काठमाण्डौका बजारबाट ३१ वटा अल्कोहल आधारित ह्याण्ड स्यानिटाइजर किनिएको थियो । Hand sanitizers are used to maintain hand hygiene. Corona pandemic had gain the attention; many companies are producing it and are marketed but their efficacy have not been well established. Thus, the main aim of this study was to evaluate the antibacterial efficacy of the hand sanitizers sold in Kathmandu. This study was carried out from March to April, 2022. In this study, 31 alcohol based hand sanitizers were purchased from the markets of Kathmandu. Among them, 15 liquid based hand sanitizers (9 contain alcohol and 6 contain alcohol with additional ayurvedic ingredients) and among 16 gel based hand sanitizers, (10 contain alcohol and 6 contain alcohol and additional ayurvedic ingredients). Efficacy of hand sanitizers were evaluated using standard ATCC cultures: Escherichia coli 35218, Escherichia coli 25922, Staphyloccoccus aureus 43300 and Klebsiella pneumoniae 700603 by agar well diffusion method. In the volume of 50 μl hand sanitizers, (46.67%, 60%, 26.67% and 33.33%) liquid based, (20%, 40%, 33.33% and 26.67%) liquid with ayurvedic ingredients based, (12.5%, 18.75%, 18.75% and 12.5%) gel based and (12.5%, 12.5%, 12.5% and 6.25%) gel with ayurvedic ingredients based hand sanitizers showed the antibacterial effect against E. coli 35218, E. coli 25922, S. aureus 43300 and K. pneumoniae 700603 respectively. In the volume of 100 µl hand sanitizers, (53.33%, 60%, 46.67% and 46.67%) liquid based, (40%, 40%, 40% and 40%) liquid with ayurvedic ingredients based, (50%, 62.5%, 62.5% and 62.5%) gel based and (25%, 31.25%, 25% and 25%) gel with ayurvedic ingredients based hand sanitizers showed the antibacterial effect against E. coli 35218, E. coli 25922, S. aureus 43300 and K. pneumoniae 700603 respectively. In the volume of 150 µl hand sanitizers, (60%, 60%, 60% and 60%) liquid based, (40%, 40%, 40% and 40%) liquid with ayurvedic ingredients based, (62.5%, 62.5%, 62.5% and 62.5%) gel based and (31.25%, 31.25%, 31.25% and 31.25%) gel with ayurvedic ingredients based hand sanitizers showed the antibacterial effect against E. coli 35218, E. coli 25922, S. aureus 43300 and K. pneumoniae 700603 respectively. Comparatively, liquid with ayurvedic ingredients based sanitizers were more effective than gel with ayurvedic ingredients based sanitizers.Item SCREENING OF POTENTIAL ANTIBIOTIC PRODUCING ACTINOMYCETES EXTRACTED FROM SOIL AND WATER SAMPLES FROM DIFFERENT REGIONS OF KATHMANDU VALLEY(Amrit Campus, 2022-08-08) Sardar, Chandra KishoreActinomycetes are Gram-positive, aerobic spore forming bacteria that are characterized by aerial and mycelial growth and are chief antibiotic producers. The aim of this study was to screen antibiotic producing actinomycetes and determine its antibiotic activity against ATCC cultures. A total of 60 samples (30 water and 30 soil) were collected from different regions of Kathmandu Valley viz. Kathmandu, Lalitpur and Bhaktapur spanning from the month of March 2022 to May 2022. Spread plate technique was employed to isolate the actinomycetes on Starch M-Protein Agar, and primary and secondary screening techniques were performed via Perpendicular Streak method and Agar-well diffusion respectively for screening their ability to produce antibiotics. The actinomycetes were confirmed by the macro and microscopic examination, biochemical and physiological tests. The crude extract obtained from the submerged state fermentation was filtered and centrifuged; tested against the Standard cultures viz; Staphylococcus aureus ATCC 43300, Escherichia coli ATCC 35218, Escherichia coli ATCC 25922 and Klebsiella spp. ATCC 700603 via agar well diffusion method. Out of the 28 (93.3%) isolates obtained from 30 soil samples, only two isolates (7.3%) i.e., NP1 and MI4 showed antimicrobial activity against the ATCC cultures which were presumed to be Streptomyces. No actinomycetes were obtained from water samples. ANOVA revealed no significant difference at 5% level of significance (0.535; P>0.05) between the standard streptomycin (100 µg/ml) and NP1.The soil of Kathmandu Valley harbors microbial diversity that encompasses potential antimicrobial producing actinomycetes which in turn can help in booming the economy by enabling the production of indigenous antibiotics.