Microbiology

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    Characterization of Antimicrobial Resistance in Escherichia coli Isolated from River Basin of Kathmandu Valley
    (Institute of Science & Technology, TU, 2024-05) Ghimire, Bindu; Prof. Dr. Prakash Ghimire, Dr. Komal Raj Rijal
    Antimicrobial resistance, a global burden, leads to treatment failures and increased healthcare challenges. Antimicrobial resistance in bacterial isolates from water, poultry feces and human pathological specimens has been published in some scattered small studies in Nepal, without any specific correlation. To understand the commonality between the isolates, their antimicrobial resistance pattern and possible correlation between the isolates from human, animal, and environment, the current study has been conducted during Jan 2020 till Sept 2023. The study aimed to characterize Escherichia coli and ESBL E. coli within the Katmandu valley river basin (Bagmati river water, stone spouts, shallow well), poultry feces, and human urine samples and determine their correlation. A total of 264 water samples from 88 sampling sites: upstream (7), midstream (37), downstream (16) and tributaries (6) of Bagmati river, stone spouts (14) and shallow wells (8); 390 poultry feces from 13 poultry farms spread within 5 clusters all along the Bagmati river basin were analyzed following standard methodology, at Central Department of Microbiology Laboratory; while 1,220 human urine samples from patients visiting National Public Health Laboratory, were analyzed at NPHL following CLSI guideline. Out of the 1,295 bacterial isolates detected from the samples, 48 % (617/1,295) were E. coli; of which 288 were from water, 63 were from human urine and 266 were from poultry feces. Analysis revealed that 6 % (80/1295) were ESBL E. coli. A total of 797 bacteria were isolated from water samples: E. coli (36%), K. oxytoca (16%), C. freundii (21%), C. koseri (14%), K. pneumoniae (9%), and E. aerogenes (4%). A total of 402 bacteria were isolated from Poultry feces: E. coli (66%), C. freundii (18%), C. koseri (8%), P. mirabilis (3%), P. vulgaris (1%), S. Typhi (1%), S. Paratyphi (0.7%), S. dysenteriae (2%), and Acinetobacter spp. (0.3%). A total of 96 bacteria were isolated from urine samples: including E. coli (66%), C. freundii (17%), C. koseri (5%), K. pneumoniae (4%), E. aerogenes (3%), K. oxytoca (2%), P. mirabilis (2%), and P. vulgaris (1%).ESBL E. coli was not detected in upstream, stone spout, and shallow well water samples. The MPN count revealed increasing trend from upstream to tributaries: upstream (31), stone spout (69), shallow well (160), midstream (2,400), downstream (17,000), and tributary (70,000), indicating human activity effects across the human inhabitation. In river water 36 % (288/797) of the isolates were E. coli and 5 % (40/797) were ESBL E. coli. E. coli was not detected from about 3 % (3/88) and ESBL E. coli was not detected from 70 % (62/88) of water sampling sites. Among non ESBL E. coli, 99 % (246/248) were resistant to erythromycin; 98 % (244/248) to amoxicillin clavulanic acid and about 98 % (242/248) to ampicillin and 96 % to piperacillin and tetracycline. All ESBL E. coli (40) were resistant to ampicillin, amoxicillin/clavulanic acid, cefotaxime, ceftazidime and piperacillin antibiotics, whereas only about 18 % (7/40) ESBL E. coli were resistant to imipenem. From urine samples, significant growth of Enterobacteriaceae was detected in 8 % (96/1,220) of the samples, with 96 bacterial isolates detected; among which 66 % (63/96) were E. coli and 19 % (18/96) were ESBL E. coli. All ESBL E. coli (18) isolates were resistant to cefotaxime, ceftazidime and ciprofloxacin whereas about 17 % (3/18) ESBL E. coli were resistant to imipenem. Among the poultry feces isolates, 66 % (266/402) were E. coli and 5 % (22/402) were ESBL E. coli. ESBL E. coli was not detected in fecal samples from 15 % (2/13) of the poultry farms. All ESBL E. coli (22) were resistant to amoxicillin/clavulanic acid, cefepime, ceftazidime and piperacillin tazobactam antibiotics whereas about 23 % (5/22) of ESBL E. coli were resistant to imipenem. The ESBL gene bla CTX M-1 was detected in 90 % (72/80), bla TEM was detected in 40 % (32/80) and bla SHV was detected in 15 % (12/80) of the ESBL E. coli isolates. A bla CTX M-9 gene was detected only in ESBL E. coli from one river water sample, while bla SHV gene was not detected in E. coli from human urine samples. The correlation coefficient showed low to moderate correlation between ESBL E. coli in urine and water (r = 0.3), water and feces (r = 0.17) and urine and feces (r = 0.13). The similarity map based upon antibiotic resistance pattern of ESBL E. coli revealed less distance in ESBL E. coli isolates from water and human urine in comparison to water and poultry fecal isolates. The study unveils low but significant correlation in the antibiotic resistance profiles of ESBL E. coli isolates from river water, human urine, and poultry feces, indicating the possible cross-linkage between the bacteria from all 3 sources: human, animal, and environment. The findings of the study clearly indicated the need for strengthening surveillance, monitoring, and containment programs, enhancing sanitation, hygiene, and infection prevention & control practices, and promoting rational use of antibiotics, for public health protection. Addressing the key components of One Health (a healthy environment, healthy humans, and healthy animals) is crucial to reducing the burden of antimicrobial-resistant organisms. जीवाणुले गर्ने सङ्क्रमणको उपचारमा प्रयोग गरिने प्रतिजैविकहरु प्रति जीवाणुले देखाउने प्रतिजैविक प्रतिरोधको अवस्था विश्वव्यापी चुनौतीको रुपमा देखा परेको छ । नेपाल भित्र वातावरण, पशुपंक्षी र सङ्क्रमित मानिसहरुको नमुनाबाट भेटिएका जीवाणुहरुले प्रतिजैविक प्रतिरोध गर्ने पुष्टि गरेका छन् । यो अध्ययन जनवरी, २०२० देखि सेप्टेम्बर, २०२३ सम्म काठमाडौं उपत्यका भित्र वागमती जलधार क्षेत्रमा गरिएको थियो । वागमती नदी अन्तर्गत उपल्लो नदी खण्ड (७)‚ मध्य नदीखण्ड (३७)‚ र तल्लो नदीखण्ड (१६)‚ सहायक नदीहरु (६)‚ तथा ढुंगेधाराहरु (१४), र इनार (८) बाट पानीका तेहोरो नमुनाहरु ;ª\sng गरिएका थिए । राष्ट्रिय जन स्वास्थ्य प्रयोगशालाबाट मूत्रनलीमा शंकास्पद सङ्क्रमण देखिएका विरामीहरुको मूत्रको नमुनाहरु (१‚२२०) र १३ वटा कुखुराको फार्मबाट ३९० कुखुराको सुलीको नमुना पनि ;ª\sng गरिएको थियो । पानीका नमुनाहरुबाट सर्वाधिक संभावित संख्या गणना विधिको प्रयोग गरी तथा उपयुक्त जीवाणु वृद्धि माध्यमको प्रयोग गरेर मानिसको मूत्रको नमुना र कुखुराको सुलीबाट जीवाणुहरु अलगाव गरिएका थिए । जीवाणुहरुलाई उचित जैवरासायनिक परीक्षण गरी प्रजातिको पहिचान पश्चात् E. coli हरुको प्रतिजैविक संवेदनशीलता परीक्षण गरिएको थियो, जसमा १७ वटा प्रतिजैविकहरुलाई ११ वटा श्रेणी र ५ वटा परिक्षण प्रतिवेदन समुहमा विभाजित गरिएको थियो (CLSI, 2020) । विस्तारित स्पेक्ट्रम बिटा ल्याक्टामेज उत्पन्न गर्ने क्षमता भएका E. coli को लक्षण प्रारुप पुष्टिका लागि संयोजन डिस्क र जीव प्रारुप पुष्टिका लागि वहुसंयुक्त पोलिमरेज शृंखला प्रतिक्रिया विधिको प्रयोग गरिएको थियो । माथि उल्लेखित सबै नमुनाहरुको विश्लेषण कार्य त्रिभुवन विश्वविद्यालय अन्तर्गतको सुक्ष्मजीवविज्ञान केन्द्रीय विभागको प्रयोगशालामा गरिएको थियो । यसरी ;ª\slnt नमुनाहरुको विश्लेषण गरी इन्टेरोब्याक्टेरिएसी परिवार भित्र १२ र मोरग्जेलेसी परिवार भित्र १ प्रकारका गरी १,२९५ जीवाणुहरु पहिचान गरीयो । पानीबाट २८८, कुखुराको सुलीबाट २६६ तथा मानवमूत्रबाट ६३ गरी कुल ६१७ E. coli पहिचान भए । यस अध्ययनमा E. coli बाहेक Acinetobacter spp.‚ Citrobacter freundii‚ Citrobacter koseri‚ Enterobacter aerogenes‚ Klebsiella pneumoniae‚ Klebsiella oxytoca‚ Proteus mirabilis‚ Proteus vulgaris‚ Salmonella Typhi‚ Salmonella Paratyphi‚ Shigella dysenteriae जीवाणुहरु पनि भेटिए । कुल १,२९५ जीवाणुहरु मध्ये करिब ६ % (८०/१,२९५) ESBL E. coli को रुपमा पहिचान भए । विशेषतः वागमती नदीको उपल्लो नदी खण्ड, ढुंगेधारा र इनारबाट ;ª\sng गरिएका नमुनाहरुमा ESBL E. coli भेटिएनन् । सय मिलिलिटर पानीको नमुनामा; उपल्लो नदी खण्डमा सबैभन्दा कम (३१) र सहायक नदीहरुका संगमस्थलहरुमा (७०,०००) सबै भन्दा बढी कोलिफर्म्स पाइयो । पानीका नमुनाहरुमा ७९७ वटा जीवाणुहरु फेला परेका थिए, जसमध्ये करीब ३६ % (२८८/७९७) E. coli र करिब ५ % (४०/७९७) ESBL E. coli थिए । E. coli हरु मध्ये करिब १४ % (४०/२८८) ESBL E. coli भेटिए । पानीका नमुनाको विश्लेषण हेर्दा करिब ९७ % (८५/८८) नमुना संग्रहस्थलहरुमा E. coli भेटिए भने करिब ३० % (२६/८८) नमुना संग्रहस्थलहरुमा ESBL E. coli पाइए । प्राप्त E. coli र ESBL E. coli हरुको प्रतिजैविक प्रतिरोध परीक्षण गर्दा परीक्षणमा प्रयोग गरिएका १७ वटा प्रतिजैविक औषधिहरु मध्ये ९५ % भन्दा बढी non ESBL E. coli हरुले एम्पिसिलिन, इराइथ्रोमाइसिन, पाइपेरासिलिन, एमोक्सिलिन क्ल्याभुलिनिक एसिडए र टेट्रासाइक्लिनका विरुद्ध प्रतिरोध देखाए भने करिब १०० % (४०/४०) ESBL E. coli हरुले एम्पिसिलिन‚ एमोक्सिलिन क्ल्याभुलिनिक एसिड‚ सेफ्टाजिडाइम‚ सेफोट्याक्जिमी र पाइपेरासिलिनका विरुद्ध प्रतिरोध देखाए । तर इमिपेनेमका विरुद्ध भने करिब १८ % (७/४०) ESBL E. coli हरुले मात्र प्रतिरोध देखाए । त्यस्तै मानवमूत्रका जम्मा १,२२० वटा नमुनाहरुमध्ये करिब ८ % (९६/१२२०) नमुनाहरुमा उल्लेखनीय रुपमा जीवाणुहरु भेटिए जस मध्ये करीब ६६ % (६३/९६) E. coli र करीब १९ % (१८/९६) ESBL E. coli थिए । शत प्रतिशत (१८/१८) ESBL E. coli हरुले सेफोट्याक्जिमी‚ सेफ्टाजिडाइम र सिप्रोफ्लोजासिनका विरुद्ध प्रतिरोध देखाए भने इमिपेनेमका विरुद्ध करिब १७ % (३ / १८) प्रतिरोध देखाए । अर्को तर्फ कुखुरा फार्मबाट ;ª\slnt कुखुराका सुलीका ३९० वटा नमुनाबाट ४०२ वटा जीवाणुका पहिचान गरियो जसमा करिब ६६ % (२६६/४०२) E. coli र करिब ५ % (२२/४०२) ESBL E. coli थिए । कुखुराका सुली ;ª\sng गरिएका कुखुरा फार्म मध्ये करिब १५ % (२/१३) कुखुरा फार्ममा र ९४ % (३६८/३९०) नमुनाहरुमा ESBL E. coli पाइएन । कुखुराका सुलीबाट प्राप्त ESBL E. coli हरुको प्रतिजैविक प्रतिरोध परीक्षण गर्दा परीक्षणमा प्रयोग गरिएका १७ वटा प्रतिजैविक औषधिहरु मध्ये ESBL E. coli हरुले १०० % (२२/२२) एमोक्सिलिन क्ल्याभुलिनिक एसिड‚ सेफ्टाजिडाइम र सेफिपिमिका विरुद्ध प्रतिरोध देखाए तर इमिपेनेमका विरुद्ध भने करिब २३ % (५/२२) मात्र प्रतिरोध देखाए । बहुसंयुक्त पोलिमरेज शृंखला प्रतिक्रियाबाट प्राप्त नतिजा अनुसार कुल ८० वटा ESBL E. coli मध्ये करिब ९० % (७२/८०) मा bla CTX M-1‚ करिब ४० % (३२/८०) मा bla TEM र १५ % (१२/८०) मा bla SHV आनुवनंशिक तत्त्व देखियो । नदीको पानीको एउटा नमुनामा bla CTX M-9 आनुवनंशिक तत्त्व भेटियो तर मानवमूत्रमा bla SHV आनुवनंशिक तत्त्व भेटिएन । नमुना ;ª\sng गरिएका स्रोतहरु (पानीका स्रोत, मानवमूत्र र कुखुराको सुली) का आधारमा ESBL E. coli बिचको सहसम्बन्ध गुणांक विश्लेषण गर्दा मानवमूत्र र पानी (r=0.3), पानी र कुखुराको सुली (r=0.3), र मानवमूत्र र कुखुराको सुली (r = 0.13) मा कमदेखि मध्यमस्तरसम्मको सहसम्बन्ध भेटियो । ESBL E. coli को प्रतिजैविक प्रतिरोध प्रतिशतको आधारमा तयार गरिएको समानता मानचित्रमा पानी र मानवमूत्रमा नमुनमा भेटिएका ESBL E. coli मा पानी र कुखुरोको सुलिको नमुनमा भेटिएका ESBL E. coli भन्दा कम दुरी भेटियो । यस अध्ययनको नतिजाले मानिस, पशुपंक्षी र वातावरणका विभिन्न स्रोतहरुमा व्याप्त जीवाणु बिचको सम्भावित अन्तरसम्बन्धलाई उजागर गरेको छ । त्यसर्थ यस अध्ययनले प्रतिजैविक प्रतिरोधी जीवाणु जुनसुकै क्षेत्रबाट मानिसमा सर्न सक्ने सम्भावनालाई औंल्याएको छ । प्रतिजैविक प्रतिरोधी जीवाणुको सङ्क्रमणबाट जोगिनका लागि सर्वप्रथम आमजनमानसमा प्रतिजैविक प्रतिरोध सम्बन्धी जानकारी k'/\ofpg र नतीजा उन्मुख अध्ययन अनुसन्धानलाई प्राथमिकता दिन आवश्यक छ । यसका साथै उचित सर्भिलेन्सको व्यवस्था हुन जरुरी देखिन्छ । यसका लागि एक स्वास्थ्यका प्रमुख घटक अन्तर्गत पर्ने स्वास्थ्य वातावरण‚ स्वास्थ्य मानिस र स्वास्थ्य पशुपंक्षीको अवधारणालाई मूर्त रुप दिन जरुरी देखिन्छ ।
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    Virulence genes in staphylococci isolated from paper currency and nasal cavity of bus conductor
    (2024-06) manandhar, Sagarika; Dr. Sarita Manandhar
    Because of their resistance to a variety of medications, Methicillin-Resistant Staphylococci (MRS) poses a serious threat to public health. These organisms also harbor various virulent factors that make them significant concerns in public health. They survive on various environmental surfaces including paper currencies. The purpose of this research was to assess the possible presence of MRS on bus conductor’s nasal swabs and the paper money they carried. A total of 100 samples (50 nasal swab and 50 paper currency) were collected in peptone water and saline water respectively. The samples were processed in Microbiological laboratory of Tri-Chandra Multiple Campus, Ghantaghar and Institute for Research in Science and Technology, Thamel from July 2023 - December 2023. The samples were exposed to standard bacteriological techniques for the purpose of isolation and identification of S. aureus and coagulase negative Staphylococci (CNS). Cefoxitin disc was used to confirm MRS. Modified Kirby- Bauer disc diffusion technique, an antibiotic susceptibility test was carried out and, tsst, sea and pvl gene were amplified by conventional PCR- Polymerase Chain Reaction. Of 50 nasal swab samples, 25 (50%) were identified as CNS and 8 (16%) as S. aureus. Among them, 14 (56%) and 4 (50%) were identified as MR- CNS and MRSA respectively. Similarly, from 50 paper currency, 11(22%) as CNS and 21 (42%) were identified as S. aureus. All CNS were methicillin resistant while only 7 (33.3%) were MRSA. Higher prevalence of MRSA and MRCNS were identified from age group 15-30. All MRSA and MRCNS were resistant to penicillin but sensitive to amikacin, meropenem and chloramphenicol. All MRSA and 9 (81,1%) MRCNS from nasal swab and all MRCNS and 4 (57.1%) MRSA from paper currency were MDR. None of the isolate harbor pvl gene whereas 52.2% MRS isolate carried tsst gene and 50% MRS isolate carried sea gene. High occurrence of MDR staphylococci & virulence gene containing MRS in community shows the need of regular surveillance & implementation of awareness programmed that help to reduce its hazards.
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    Phylogrouping and Antibiogram of Escherichia coli from river water sample
    (Institute of Sciecne & Technology, 2024-06) Thapa,Suchitra; Dr. Dev Raj Joshi
    Water borne diseases like diarrheal infection caused by E. coli is a serious threat to public health. However, in Nepal there are no clear data on characterization of these E. coli based on their virulence or antimicrobial resistance. And phylogrouping will help to track the microbial source and help us understand the route of transmission. Also, antibiotic resistant profile will help to design a better treatment regime for the patients. Therefore, this study aims to characterize the diarrheagenic E. coli that are prevalent in the water sources and gauge their transmission potential to humans. For this E. coli was isolated and identified from water sample following the conventional culture techniques and enumerated by standard membrane filtration method. Further antibiotic susceptibility was determined by disc diffusion method towards different classes of antibiotics that are used in clinical settings following the procedure recommended by CLSI. Further, gene detection was done by PCR using specific primers for phylogroup. The result was then analyzed using SPSS and WHONET software. Descriptive analysis as well as inferential analysis was done to fulfill the objective of the study. The result revealed a high occurrence of thermotolerant E. coli of phylogroup B1 (58.2%) in Bagmati river which is commensal in nature and originates from either human or animals. Also, isolates conferring resistance to multiple antibiotics (n= 6) were detected along with high priority isolates (n=11). Thus, circulation of such high risk isolates in aquatic settings of Nepal is concerning so a comprehensive study of such isolates should be done at genomic level so that to better understand their transmission potential and intervene their transmission.
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    Antimicrobial assay of lactobacillus species against multigrug resistane bacteria
    (Institute of Science & Technology, 2024) Khadka, Rama; Dr. Shyam Prakash Dumre
    Lactic acid bacteria are the group of bacteria which are commonly found in soil and in the guts of animals and they are favorable for both human and animal health. In this study, Lactobacillus species were isolated and identify from soil of a cow farm and perform its antimicrobial activity against MDR bacteria from October 2024 to March 2024 in the microbiology laboratory of Padma Kanya Multiple Campus, Bagbazar, Nepal. A total of 30 soil samples were collected from 3 locations of farm of Kathmandu valley for enumeration and isolation of Lactobacillus species on selective media (MRS). Pure cultures were obtained through sub culturing on MRS media and further morphological as well as biochemical test was be carried to identify the isolated strains. For the confirmation of Lactobacillus species, Gram staining, motility test and catalase test were done. For the further analysis, fermentation test, growth on different temperature, different salt concentration and different pH were also done. Antibiotic susceptibility test (AST) of Lactobacillus species and test bacteria was done by a modified Kirby- Bauer method. The screening test for bacteriocin was done by dot plate technique method. Then, the extraction of bacteriocin from isolated Lactobacillus spp. was done by precipitation method and antimicrobial assay was done against MDR test bacteria. From this study, 10 Lactobacillus species were identified. In this study, bacteriocin like compound were extracted from Lactobacillus spp. were from 24hrs (2day), 48hrs (3day), 72hrs (4day), 96hrs (5days) and 120hrs (6days). However, antimicrobial activity showed against MDR bacteria only after 72hrs (4day), 96hrs (5days) and 120hrs (6days). From 96hrs (5days) of extraction of antimicrobial substance bacteriocin like compound was able to inhibit all test bacteria which were Staphylococcus aureus, Bacillus cereus, Escherichia coli, Pseudomonas aeruginosa and Klebsiella pneumoniae. The highest zone of inhibition was showed by BL10 isolate against B. cereus (30mm) followed by KL1 isolate against E. coli (21mm) and KL5 isolate against B. cereus (20mm). From this study, antimicrobial activity of bacteriocin like compound showed the antimicrobial activity against all MDR bacteria. So, next generation antibiotics can be prepared from bacteriocin like compound extracted from Lactobacillus spp. for targeting the multi drug resistant bacteria.
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    Larvicidal Activity of Native Bacillus thuringiensis Isolated from Soil Samples Of Nepal
    (2024-01) Gharty Chhetri, Ganga; Dr. Komal Raj Rijal
    Climate change is exacerbating the spread of mosquito-borne diseases globally. Vector control with Bacillus thuringiensis var israelensis (Bti) and Lysinibacillus sphaericus (Ls) the biological control agents for larval source management is an effective technique for preventing mosquito-borne diseases by focusing on mosquito larvae in diverse water sources. While these larvicides are used in many other countries, they are not widely available in Nepal for mosquito vector control. This study aims to isolate mosquito larvicidal Bacillus thuringiensis (Bt) from the soil samples collected in Nepal. It was isolated from the soil sample by the acetate selection method. It was identified by observing crystal protein stained by coomassie brilliant blue and further characterized by larvicidal assay and 16srRNA gene sequencing. The larvae were collected from the fields; and reared in the laboratory for the bioassay. Bioassay was carried out in four replicates with the positive control strain (coded as Bt-IPS-82), negative control, and test Bt. The mortality percentage was calculated after 24 hours. Formulated larvicide (Bt-14P2A) in the form of pellet and liquid suspension were evaluated in simulated and open field trials. The lethal concentration (LC), residual effects, and mortality rates were estimated. Bt isolates were isolated from 454 soil samples, with four strains (Bt-14P2A, Bt-7P1A, Bt-8P1A, and Bt-3P2B) demonstrating 100% larvicidal activity against mosquito larvae during primary screening. Bt-14P2A’s LC50 and LC90 against Ae. aegypti larvae in pellet form were 3.98 ppm and 25.11 ppm, respectively, whereas in liquid form they were 6.45 ppm and 37.15 ppm respectively. Simulated field testing revealed a residual impact of seven weeks for the liquid formulation, with 100% mortality for six weeks. Open-field experiments also showed efficacy, with very modest concentrations delivering 100% mortality for up to 14 days and residual effects lasting up to six weeks in household containers such as buckets. The obtained larvicidal Bt strains were found to be genetically identical to those in GenBank. In addition, 11 Ls isolates were isolated from 32 soil samples, one of which demonstrated mild toxicity against Ae. aegypti larvae. Finally, the efficient isolation procedure produced Bt strains with 100% larvicidal activity against Ae. aegypti larvae. These isolates show promise as future strategies for managing larval sources and combating mosquito-borne illnesses in Nepal. जलवायु परिवर्तनका कारण लामखुट्टेबाट सर्ने रोगहरूको संक्रमण बढ्दै गईरहेको अवस्था छ । Malaria, Dengue, Japanese encephalitis आदि रोगहरू लामखुट्टेको माध्यमबाट सर्ने भएकाले यसलाई रोगवाहक (vector) भनिन्छ । लामखुट्टेको वृद्धि रोक्ने विधिलाई भेक्टर नियन्त्रण भनिन्छ । भेक्टर नियन्त्रण गर्न धेरै विधिहरू अपनाउन सकिन्छ । भेक्टर नियन्त्रणबाट लामखुट्टेको लार्भाका स्रोतहरूलाई कटौती गरेर वा नष्ट गरी वयस्क लामखुट्टेको वृद्धि रोक्न सकिन्छ । जैविक कीटनाशक जस्तै Bacillus thuringiensis var israelensis (Bti), Lysinibacillus sphaericus (Ls) जस्ता ब्याक्टेरिया लाई पनि भेक्टर नियन्त्रणका लागि प्रयोग गरिन्छ‚ जसले पानीमा भएका लार्भा नष्ट गरी वयस्क लामखुट्टेको वृद्धि रोक्ने काम गर्दछ । विभिन्न मुलुकले लामखुट्टे नियन्त्रण गर्न जैविक कीटनाशक Bt र Ls को प्रयोग गर्ने गरेका छन् । जैविक कीटनाशकलाई विभिन्न प्रकार जस्तै तरल र ठोसकारूपमा उत्पादन गर्ने गरेका छन । यी जीवाणु मानव र अन्य जीवजन्तुका निम्ति हानिकारक हुँदैनन्‚ तर यिनले उत्पादन गरेका विषालु कीटनाशक क्रिष्टल प्रोटीनले लामखुट्टेको लार्भा नष्ट गरी तिनको स्रोत व्यवस्थापनमा सहयोग पुर्याउँदछ । यस अनुसन्धानको उद्देश्य नेपाली माटोका नमुनाबाट Bt र Ls को खोजी गर्नु र लामखुट्टे नियन्त्रणमा यसको प्रभावकारिता मुल्याङ्कन गर्नु हो । नेपालका विभिन्न वातावरणीय भूभागबाट माटो सङ्कलन गरी फरक फरक प्रविधिद्वारा माटोलाई प्रशोधन गरी Bt र Ls लाई छुट्याई सुरक्षित गरियो । यी दुई जीवाणुलाई सूक्ष्मदर्शक यन्त्र र coomassie brilliant blue (CBB) का सहायताले तिनीहरूको उत्पादन क्रिष्टल प्रोटिन अवलोकन गरी पहिचान गरिएको थियो । यस बाहेक छुट्याउन वा पहिचान गर्न अन्य प्रविधिहरू पनि अपनाइएको थियो । Bt र Ls को लार्भानाशक (Larvicidal) परीक्षण र Bt को16srRNA gene sequence बाट phylogeny को अध्ययन गरिएको थियो । लार्भानाशक क्षमताको परिक्षणका लागि काठमाडौँका विभिन्न स्थानबाट लार्भा सङ्कलन गरिएको थियो । प्रयोगशालामा पनि Aedes aegypti लामखुट्टेका लार्भा पालनपोषण गरिएको थियो । लार्भानाशक क्षमता परीक्षणका लागि विश्व स्वास्थ्य सङ्गठनको मापदण्ड अपनाइएको थियो । परीक्षण गर्दा लार्भालाई Bt का दुई प्रकारका उत्पादन जस्तै : ठोस र तरल जैविक लार्भानाशक (Bio-larvicide) खुवाइएको थियो । प्रयोगशाला र बाहिरी वातावरणमा तरल Bt-14P2A लार्भानाशकको परीक्षण गरिएको थियो । परीक्षणबाट सकारात्मक र नकारात्मक नियन्त्रणका साथै ५०% र ९०% लार्भालाई मार्न चाहिने मात्रा‚ (Lethal concentration dose LC50, LC90) मृत्यू प्रतिशत र प्रभावकारिताको निर्धारण गरिएको थियो । यस अध्ययनका क्रममा ४५४ माटोका नमुना परीक्षण गर्द । गोलाकार क्रिष्टल उत्पादन गर्ने Bt माटोमा प्रशस्त (५८.३३%‚ ८०८/१३८५) भएतापनि केवल चार Bt (Bt-14P2A, Bt-7P1A, Bt-8P1A, Bt-3P1B) को गोलाकार क्रिष्टल प्रोटिनले मात्र शत प्रतिशत (100%) लार्भालाई मार्ने क्षमता राख्ने देखाएको थियो । प्रारम्भिक जाँचका क्रममा Bt-14P2A ले सङ्कलित लार्भा र प्रयोगशालामा पालनपोषण गरिएका Aedes aegypti लामखुट्टेका लार्भा विरुद्ध LC50 ३.९८ र LC90 २.११ PPM देखाएको थियो । तरल बनाइए जैविक लार्भानाशक Bt-14P2A लाई Simulated field trial परीक्षण र खुला स्थलगत परीक्षण (Open field trial) मा प्रयोग गरिएका सबै मात्राहरूले २४ घण्टा भित्र शतप्रतिसत लार्भा नास गरेको थियो । खुला स्थलगत परीक्षणमा सबैभन्दा कम मात्राले १४ दिनका लागि वर्षाको पानी पुन:पूर्ति हुँदा शतप्रतिशत (100%) मृत्युदर देखायो । त्यसैगरी‚ घरेलु प्रयोजनका निम्ति प्रयोग हुने बाल्टिनहरूमा जैविक लार्भानाशकको छ हप्तासम्म residual effect देखिएको थियो । यसै क्रममा 16srRNA gene sequence को परीक्षणबाट ३ ओटै Bt जैविक लार्भानाशक हो भनी प्रमाणित भएको छ । केवल एउटा Ls ले Ae. aegypti लार्भा विरुद्ध २५% मृत्युदर देखाएको थियो । नेपालको माटोबाट पाँचवटा लार्भानासक व्याक्टेरिया (चार) Bt र (एक) Ls प्राप्त भएको थियो । तसर्थ‚ प्राप्त Bt द्वारा पानी भण्डारणका क्रममा भाँडामा हुर्किएका लामखुट्टेका लार्भा नष्ट गरी यसलाई नेपालमा लामखुट्टेबाट सर्ने रोगहरूको नियन्त्रणका लागि प्रयोग गर्न सकिन्छ।
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    Prevalence of intestinal parasitic infections, related risk factors and possible impact on nutritional status among private school children in Kathmandu
    (Department of Microbiology, 2016) Maharjan, Suman
    The present study was conducted among the primary level school children of four private schools of Kathmandu, Nepal to determine the prevalence of intestinal parasitic infections, possible pre-disposing factors as well as its impact on the nutritional status of the school children. This cross sectional study was conducted from July to November, 2014. A total of 329 stool and blood samples were collected and brought to the laboratory of Shi-Gan International College of Science and Technology (SICOST). Anthropometric data were collected and questionnaire related to their personal hygiene, socioeconomic condition were done. Stool samples were processed for microscopy by using formal-ether concentration technique. Hemoglobin was estimated by cyanmethemoglobin method. Statistical analysis was done by using SPSS 16.0. The overall prevalence of intestinal parasitic infection was 17.9% (59/329). Protozoan parasites were dominant (47.5%) over helminthes (22.0%). Altogether eight species of parasites were detected in the study. Giardia intestinalis (29.9%) was the most common protozoan parasite detected whereas Trichuris trichiura (24.7%) topped among the list of helminthes. The prevalence of intestinal parasitic infection was slightly higher among boys (18.6%) than girls (17.1%) (p=0.732). Similarly, the prevalence was significantly higher among the children belonging to age group 6-10 years (p=0.000). No statistically significant association was observed with respect to parents’ literacy, nail trimming, hand washing with soap before eating and after toilet, use of antihelminthic drug, source of drinking water and treatment of drinking water. A total of 41.0%, 29.0% and 11.0% children were found to be stunted, underweight and wasted respectively. Similarly, about 20.5% of the children were anemic without significant association between the parasitic infections. The results indicate the need of periodic deworming programmes, health education and improvement of sanitation and hygienic practices in private schools. Key words: Intestinal parasitic infection, school children, risk factors, nutritional status, Kathmandu.
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    AcrAB ToLC Efflux pump gene in multi drug resistant Escherichia coli isolated from clinical samples
    (Department of Microbiology, 2023) Koirala, Alina
    Escherichia coli is one of the most commonly isolated multi drug resistant (MDR) pathogen and has been categorized as the priority pathogen by WHO. Carbapenem is a drug of choice for treatment of E. coli but now resistance of these drugs is spreading due to various mechanism such as the presence of intrinsic mechanism of antibiotic resistance efflux pump gene. Efflux pump has been illustrated as one of the key mechanism of antibiotic resistance crucially in the gram-negative pathogens. Forty-two carbapenem resistant E. coli were isolated from sample specimens of patients of Kathmandu Model Hospital. Cross sectional descriptive study was conducted and the specimens were processed with respective culture media following the antibiotic susceptibility testing according to CLSI guidelines. Phenotypic detection of ESBL and CRE was done using combination disc diffusion and by using cccp respectively. Chromosomal DNA extraction was done using alkaline hydrolysis method. Amplification of AcrAB TolC was carried out by conventional PCR. Out of 2384 samples the infection rate was found to be (21.8%) that is 520 samples showed significant growth of bacteria.E coli being the major isolates constituting of 78% of the total isolates. Bacterial isolates 89.5% were found mostsensitive to PolymixinB and Colistin (99.8%), however was the most and resistant to Amoxycillin (66%). Out of 42 Carbapenem resistant Escherichia coli isolates, AcrA gene was prevalent in 23 (54.7%) of the isolates. AcrB gene in 30 (71.4%) and 31 (73.8%) of the isolates were found to be positive for TolC gene. Out of 403 Escherichia coli isolates, 42 (10.4%) were found to be Carbapenem resistant. Moreover, out of 147 ESBL isolates, 16 isolates (10.9%) were found to be Carbapenem resistant. This research evaluated the minimum inhibitory concentration (MIC) activity of carbapenem, in the presence or lack of the CCCP (25 g/mL) in order to identify the function of the efflux pump in the carbapenem resistant E. coli isolates. The findings demonstrated that most samples had lower MICs in the presence of the pump inhibitor than in the absence of the efflux pump inhibitor. Key Words: Efflux pump, Carbapenem, antibiotic susceptibility test
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    Formulation, Characterization, and Shelf Life Study of Gluten-Free Cookies Using Hill Grains of Nepal
    (Department of Microbiology, 2023) Pathak, Durga
    The consumption of bakery items made from refined flour has increased in the past decade, resulting in a rapid rise of gluten-related disorders. The purpose of this study was to develop, characterize, and study the shelf life of various gluten-free cookies (GFC) using indigenous hill grain flours of Nepal, fungal α-amylase, baking powder, and baker's yeast as leaveners. Various composite flours were prepared by millet, maize, buckwheat, and taichin rice flour in 75:25%, baked at 200°C for 20 minutes, proofed at 30°C until 1 hour for food grade fungal α-amylase with improver and baker’s yeast treated sample before mixing with cream. The moisture content of all formulated cookies was below 5% except BWBY sample during the 60 days of storage period. BWBY cookies showed highest moisture content, ranging from 6 % to 8.37% during 60 days of storage. The ash content of cookies samples were found to be significantly increased from 0.81% to 2.59%. Cookies made with 75:25 % millet-taichin rice flour blend treated with food grade fungal α-amylase with improver and baker’s yeast, received the highest scores for taste (8.0), texture (7.6) and crunchiness (8.1) out of 9 point headonic scale. Gluten-free cookies (GFC) were found to have a higher ash and moisture content compared to traditional cookies. The bacterial and fungal growth was not observed on both PCA and PDA plates at 0 days of storage. The microbial load and moisture content increased over time in most of the cookie samples. However, the microbial counts remained within acceptable levels (below 1×10 vi 4 cfu/gm) and the moisture content below 5% throughout the entire storage. Despite a marginal decrease, the overall acceptability (OAC) score of the all cookies was still well accepted (7 out of 9) by panelists after 60 days of storage. Based on their nutritional, sensory, and microbial qualities, the cookies made with a combination of millet-taichin rice flour (MABK), and maize-taichin flour (MzABK) were found to be good in quality until the end of the storage period (60 days). Key words: Gluten free cookies, Millet-rice flour cookies, Maize-rice flour cookies, Buckwheat-rice flour Cookies, Whole wheat grain cookies
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    Evaluation of anti fungal activity of plant extracts on fungal pathogens of potato plant
    (Department of Microbiology, 2009) Maharjan, Bijaya Laxmi
    Solanum tuberosum (Potato) is one of the most important tuber crops with the production of 325 million tons worldwide and about 20 million tons in Nepal per year. It is a cool season crop and is considered as a staple food. It is prone to many diseases including late blight (Phytophthora infestans), early blight (Alternaria solani) and fusarial wilt (Fusarium oxysporum) as major ones with a yield loss of 50-90%, 20-50% and 10-40% respectively. Although a large number of synthetic chemicals have been recommended to control these losses, most of these have proved to be pollutive, carcinogenic and the emergence of resistant pathogens towards these chemicals has been a great concern. Biological control is the only safe substitute to be explored to control these phytopathogens. The microbial ones being difficult to produce, maintain and use, the plant materials can be used as an alternative. In the present study an attempt has been made to screen and evaluate the antifungal activity of plant extracts against the fungal pathogens of potato plant. Crude ethanolic extracts of five different fungicidal plant materials viz. Brassica nigra (cake), Cinnamomum camphora (fruits), Eupatorium adenophorum (twigs), Lantana camara (twigs) and Melia azedarach (fruits) were screened and tested against the three fungal pathogens isolated from the leaf samples of infected potato plant collected from the 7 sites of Kathmandu valley. The identification of the isolated fungal organisms was done by the study of colony characteristics and microscopic observation. The antifungal activity of the crude extracts obtained was evaluated by agar well diffusion method and two fold broth dilution method. The moisture content was highest in the twigs of L. camara and lowest in the cake of B. nigra. The yield of crude extracts was obtained highest from C. camphora (70%) and lowest from M. azedarach (9.75%). B. nigra was found most effective against P. infestans with both MIC and MFC values 6.25mg/ml. Similarly, M. azedarach showed the higher antifungal activity against A. solani with both MIC and MFC values 3.125mg/ml. C. camphora was found least effective against P. infestans and A. solani while it was most effective against F. oxysporum with both MIC and MFC values 3.125 mg/ml. Different types of plant extracts with different concentration significantly (P<0.05) inhibited the growth of all the fungal pathogens. The extracts used in this experiment were found to be suitable for the control of these fungal pathogens and those also help to maintain sustainable agriculture and environment.
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    Screening of staphylococus aureus as NASAL carrier from hospital personnel of shree Birendra hospital, Chhauni
    (Department of Microbiology, 2008) Thulunga, Juni
    Staphylococcus aureus is one of the most common human pathogen and is capable of causing wide range of infections in human. S. aureus is the normal flora of the nasal cavity which is carried in the nose of about 40% of healthy people. S. aureus as a nasal carrier has been identified as a risk factor for community acquired and nosocomial infection. The present study was conducted from June 2007 to December 2007 to screen out S. aureus as nasal carrier from hospital personnel of Shree Birendra Hospital Chhauni. All together 264 nasal swab samples were collected from hospital personnel of different wards. All nasal swab samples were cultured on Mannitol Salt Agar and suspected isolates were identified as S. aureus. Antibiotic susceptibility test of the isolated S. aureus was done by Kirby Bauer disc diffusion method. Among 65 isolates of S. aureus from nasal swabs; 27.02% (47/174) isolates were from males and 20% (18/90) were from females. The distribution of S. aureus as a nasal carrier between male and female was not statistically significant (P = 0.21). In both male and female, the highest prevalence (31.66%) of nasal carrier of S. aureus was found in the age group of 26-30 years. Among hospital personnel, maximum nasal carrier rate was found in nursing assistant 33.87% (21/62) followed by intern doctors 31.82% (7/22) and medical trainee 27.59% (16/58). Regarding the department wise distribution of nasal carrier, highest nasal carrier rate of S. aureus was from Medical III 53.84% (7/13) followed by orthopedic 50% (2/4) and surgical officer cabin 42.85% (6/14). The isolates S. aureus showed highest resistant to amoxicillin (76.9%) followed by penicillin (38.5%), cotrimoxazole (21.5%), gentamycin (13.8%), erythromycin (9.2%), ciprofloxacin (9.2%), chloramphenicol (4.6%) and least towards tetracycline (3.1%). Out of 65, only 20% (n=13) S. aureus isolates were multi-drug resistant (MDR) and highest number of MDR S. aureus was isolated from Intensive Care Unit 75% (n=3). No methicillin resistant S. aureus (MRSA) was found among the isolates of S. aureus. Multidrug Resistant (MDR) S. aureus was found but methicillin resistant S. aureus (MRSA) was not found among the isolates. However, regular monitoring of methicillin sensitivity should be carried out. Keywords: S. aureus, MDR, MRSA, Nasal carrier, Shree Birendra Hospital
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    Regulation of thylakoid protein phosphorylation during state transition and photosystem II supercomplex mobilization in rice
    (Department of Integrated Biological Science, 2016) Poudyal, Roshan Sharma
    Recent research identified the role of STN8 kinase for phosphorylation of PSII core proteins such as D1, D2, CP43, PsbH, TSP9, CaS and STN7 kinase for not only phosphorylation of LHCII protein but also PSII core proteins and state transition in Arabidopsis. However complete inhibition of PSII core protein phosphorylation was only observed in the Arabidopsis stn7/stn8 double mutant. Hence, the elucidation of specific roles of STN8 and STN7 kinases has been identified by using T-DNA insertional knock-out mutant of the rice (Oryza sativa) STN8 gene, osstn8, STN7 gene, osstn7 and PPH phosphatase gene, ospph. Here, osstn8 and osstn7 mutants has been used to clarify the role of PSII-LHCII protein phosphorylation in regulation of PSII repair cycle, mobilization of PSII supercomplex, state transition. In the osstn8 mutant, PSII core protein phosphorylation (PCPP) was significantly suppressed and the grana were thin and elongated. During high light (HL) illumination, PSII was strongly inactivated in the osstn8 mutants, yet the D1 protein was degraded more slowly than in wild type, and the mobilization of the PSII supercomplex from the grana to stroma lamellae for repair was also suppressed. Taken together, these data show that the absence of STN8 gene is sufficient to abolish PCPP in osstn8 mutants to produce all of the phenotypes observed in the double mutant of Arabidopsis, indicating the essential role of STN8-mediated PCPP in PSII repair. Using both histochemical and fluorescence probes, ROS production, including superoxide and hydrogen peroxide production was increased in osstn8 mutant’s leaves during HL illumination. When superoxide dismutase was inhibited, superoxide production was increased, indicating that superoxide production is the initial event prior to hydrogen peroxide production. However, singlet oxygen production was not different between WT and osstn8. In addition, PSII-driven superoxide production was more in the thylakoid membranes as well as in isolated PSII and PSII-LHCII supercomplex of osstn8. PSII and PSII-LHCII supercomplex of osstn8 leaves showed strong oxidation of proteins under HL illumination. These results suggest that superoxide is the major form of ROS produced in the osstn8 mutant, and that the damaged PSII in the supercomplex is the primary source of superoxide production during HL illumination. Apart from this, the specific kinase and phosphatase for reversible phosphorylation of CP29 (CP29-P) was still unknown. Therefore T-DNA insertion knock-out mutant of STN7 and STN8 kinase as well as PPH phosphatase mutants were used to confirm it. CP29-P was observed in WT, osstn8 complement, osstn7 and ospph mutant but not in osstn8 mutant during high light and light chilling treatment. However dephosphorylation of CP29-P was observed in all genotypes during dark incubation. The phosphorylation and dephosphorylation of LHCII protein was blocked in osstn7 mutant and ospph mutant, as a consequence, state transition was strongly impaired. Mobilization of PSII supercomplex was observed in osstn7 mutant during high light illumination but it was suppressed in ospph mutant. This result proposed that dephosphorylation of LHCII protein was also involved for mobilization of PSII supercomplex. In control condition, the relative change in maximal fluorescence during state transition, ∆F was similar in WT and osstn8 mutant and mobilization of PSII supercomplex was not observed but ∆F become negligible in osstn8 during high light illumination and mobilization of PSII supercomplex was only observed in WT. Upon high light illumination, CP29-P was observed in PSI complex in WT but not in osstn8 mutant. Therefore it is concluded that lack of CP29-P also impaired high light induced state transition in osstn8 mutant of rice.
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    ANTIBIOTIC SUSCEPTIBILITY TESTING OF ISOLATED Staphylococcus aureus FROM PANEER SOLD IN KATHMANDU
    (Amrit Campus, 2023-08-17) KHANAL, VAGYASHREE
    Paneer is a fresh acid-set, non-aged, non-melting soft indigenous food made from curdling milk. Safety of dairy products is an issue of public health in Nepal. Staphylococcus aureus is an food borne pathogen of importance in dairy products. The main aim of this study is to detect microbial quality of paneer marketed in different localities of Kathmandu district. In present study, 30 paneer samples were randomly collected from dairy shops from 5 different locations in Kathmandu (Bafal, Kalanki, Naikap, Sitapaila and Thamel) during April to May 2022 and processed at laboratory of of Microbiology, Amrit Campus. Microbial analysis of paneer was done by Total bacteria count and total Staphylococcal count S. aureus were identified by biochemical tests and their antibiotic susceptibilty test was done by Kirby-Bauer disc diffusion method. Results obtained from this investigation revealed that out of 30 samples, 39.9% in Total Bacterial Count (TBC) was above specification. Among 30 samples, 29 samples showed Staphylococcus spp. growth and of those only 12 confirmed as Staphylococcus aureus . Among them 6 (20 %) exceeded specification of 100 cfu/gm. Antimicrobial susceptibility pattern of S. aureus showed 100% sensitivity towards Cotrimoxazole and Gentamycin and Levofloxacin, whereas resistance towards Chloramphenicol (8.3%), Cefoxitin (8.3%), Tetracycline(8.3%), Penicillin G (41.6%), Ampicillin (16.6%), Nalidixic acid (41.6%) and Azithromycin (16.6%). MRSA was detected in 1(3.34%). This study concluded that maximum number of sample exceeding microbial specification and antibiotic resistant isolates were reported from Naikap. Irresponsible and over use of antibiotics has lead to an increased the presence of multidrug Regular microbial quality monitoring of marketed paneer should be carried out by authorized Institution of Government of Nepal. Therefore, it is necessary to monitor and control the quality marketed in Kathmandu.
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    ANTIBIOTIC SUSCEPTIBILITYTESTING OF ISOLATED COLIFORMS FROM PANEER MARKETED IN KATHMANDU
    (Amrit Campus, 2023-08-17) PRADHAN, MANISHA
    Paneer is a valuable dairy product due to its high nutritional value, distinctive texture, and flavor makes it a good meat substitute. Poor hygiene during preparation, storage and handling of foods including dairy products can lead to microbial contamination in developing nations like Nepal. The aim of this study was to screen microbial quality of paneer and to determine the antibiotic susceptibility testing of total coliforms and thermotolerant coliforms. Here, 30 paneer samples were randomly collected from dairy shops and other different locations in Kathmandu and samples were processed at Department of Microbiology, Amrit Campus during April to June 2022. Serial dilution of sample was performed and by using pour plate method, total coliform count and thermotolerant coliform count was determined. All isolated organisms were identified by various biochemical tests and antibiotic susceptibility pattern of coliforms was carried out by Kirby-Bauer disc diffusion method. In this study, 16.6% of paneer samples were within requirements of DFTQC and FSSAI with maximum count of 4.5×105 cfu/gm and thermotolerant bacteria with maximum count of 9.6×104 cfu/gm. Coliforms isolated from paneer were Escherichia coli (46.9%), Klebsiella spp. (37.5%) and Citrobacter spp. (15.6%). Thermotolerant coliforms were Escherichia coli (58.5%), Klebsiella oxytoca (11.8%), Klebsiella spp. (17.6%) and Citrobacter spp. (11.8%). All isolates of total coliforms (n=32) and thermotolerant coliforms (n=17) showed 100% sensitivity towards Gentamycin and Cotrimoxazole. Resistance towards Ampicillin (40%), Cefoxitin (30%) and Azithromycin (30%) were shown by thermotolerant E. coli. Resistance towards Cefoxitin (66.7%), Azithromycin (33.3%) were shown by thermotolerant Klebsiella spp. The highest resistance for E. coli was found in Azithromycin (40%) and Ampicillin (40%) in total coliforms and thermotolerant coliforms. However, no multiple- antibiotic resistant was found in isolates of total and thermotolerant coliforms. The majority of the paneer samples in this investigation did not meet the microbiological requirements. Sitapaila provided the greatest number of microbiologically acceptable samples, whereas Naikap provided the least number. Thus, it is necessary to monitor and control the quality of paneer sold in Kathmandu.
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    BACTERIOLOGICAL QUALITY OF DRINKING WATER IN KATHMANDU NEPAL
    (Amrit Campus, 2023-08-17) Gautam, Manisha
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    Identification of mutations in rpoB gene (ß-sub unit of DNA dependent RNA polymerase) in rifampicin resistant mycobacterium tuberculosis among retreatment tuberculosis patients in nepal
    (Faculty of Microbiology, 2018) Khadka, Dhurba Kumar
    Identification of mutations in rpob gene (ß-sub unit of dna dependent rna polymerase) in rifampicin resistant mycobacterium tuberculosis among retreatment tuberculosis patients in nepal streptomycin, isoniazid, rifampicin, ethambutol). There was no significant difference of age wise (p=0.532) and sex wise (p=0.775) distribution of MDR-TB cases identified by Gene Xpert, conventional culture and FLD-DST. Second line antituberculosis drug susceptibility test (SLD-DST) on 29(26.6%) of 109 MDR confirmed M. tuberculosis isolates was performed by Line Probe Assay (LiPA Genotype MTB/DRsl version 1.0), of which 17(58.6%) for gyrA and 8(27.6%) for emb B and all 29(100%) for rrs genes were identified as wild type. Whereas, 12(41.4%) were resistant to gyr A gene i.e. 3 cases resistant to fluoroquinolone alone and 9 cases along with ethambutol. Similarly, 10(34.5%) were resistant to ethambutol (emb B genes) only and 7(24.1%) were susceptible to all SLDs. Out of 29 MDR confirmed M. tuberculosis isolates, 12(41.4%) were found to be pre-extensively drug resistant (pre-XDR-TB) but none was found XDR TB. The maximum number of age/sex wise pre-XDR TB distribution was found in 15-60 years of age group and among females and males equally. There were no differences of age (p=0.531) and sex wise (p=0.428) findings of pre-XDR TB. Among 159 RR/MDR-TB, 128(80.5%) cases were analyzed for time difference between previous cure, current diagnosis of MDR-TB and treatment outcome. The treatment outcomes were categorized into favorable (cured and treatment completed) and unfavorable (died, lost to follow up, not evaluated and treatment failure) groups. The age wise treatment outcomes for a total of 128(100.0%) cases; 1(1.1%), 80(62.5%), 8(9.0%) were among the favorable group and 1(2.6%), 34(26.6%), 4(10.3%) were among unfavorable groups of <15, 1560 and > 60 years of age respectively. Similarly, 2(1.6%) i.e. 1(0.8%) in 5-11 months and 1(0.8%) in 1-4 years groups were not evaluated, 3(2.3%) i.e. 1(0.8% fell in 1-4 years and 2(1.6% fell in ≥5yrs) groups respectively were treatment failure. There were no significant differences of age (p=0.897), sex (p=0.357), time difference (p=0.474), diagnosis by Gene Xpert alone or along with C/DST (p=0.746) and treatment outcomes. This is the first analyzed report on retreatment TB patients in Nepal.
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    Isolation, Identification and characterisation of Amylase enzyme produced by different Microorganisms isolated from soil
    (Department of Microbiology, 2022) Rai, Pratima
    Amylase is one of the most widely used enzyme in industries from decades. Its main function is to hydrolyze the starch molecule into glucose unit and oligosaccharides. Among different types of amylases, α-amylase is in high demand due its wide spread use in food, textile, baking and detergent industries. It can be acquired from microorganisms, plants and animals. This study was conducted with the aim to isolate, identify and to characterize the amylase enzyme produced by microorganisms from soil. The soil sample were collected from the different places of Dharan. The screening of amylase producing microorganisms were carried out by starch hydrolysis test on starch agar plates. The organisms producing highest zone of clearance were identified and further used for amylase production and characterization. Based on colonial characteristics, cultural characteristics, staining and biochemical test the organisms were identified as Bacillus subtilis whereas the isolated fungi was identified as Aspergillus niger on the basis of colonial characteristics and LPCB staining. The identified organisms were further used for the production of amylase on submerged fermentation. The amylase activity was assayed by DNS method. The produced enzyme was centrifuged and partially purified using ammonium sulphate. The amylase production was found to be maximum at 40ºC and 6.5 pH for Bacillus amylase and 30 ºC and pH 7.5 for Aspergillus niger. Further, Characterization of the enzyme was done for the industrial application of the enzyme. Crude amylase showed its maximal activity at pH 7 and 45 ºC For Bacillus subtilis and 30 ºC and pH 6.5 for Aspergillus niger. Crude amylase showed great potential for its application in detergent industry. Keywords –amylase, enzyme, submerged fermentation, DNS method, ammonium sulphate, characterization.
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    Intestinal parasitosis among human immunodeficency virus and tuberculosis infected patients of Dharan, Nepal
    (Department of Microbiology, 2022) Rai, Kishor
    Intestinal parasites are those which must have an intestinal life- cycle stage and usually attach in small and large intestine and produces traumatic damage in the intestinal villi. Parasitic infections caused by protozoa and helminths are the most common infections worldwide. The prevalence of parasitic infections varies with the level of sanitation and is highly prevalent among the general population in Nepal<. The presence study was done to find out the prevalence of intestinal parasitosis among HIV and Tuberculosis (TB) patients of Dharan total of 53 sample (<15 years) from HIV seropositive and 28 samples (<20years) from TB patients were collected in a clean, dry and capped fitted container and subjected to macroscopic and microscopic examination for ova, cyst, adult parasites and or segments of parasites. Samples were fixed in 10% formalin-ether solution. Sedimentation technique along with modified acid fast (Zeihl-Neelsen) staining method was performed for opportunistic intestinal parasites in both patients. Multiparasitic infection was noted in the study. The overall prevalence of intestinal parasites was found to be 54(66.67%) among 81 patients (53 HIV patients and 28 TB patients). The parasitosis in male was higher 25(30.86%) than female 15(81.51%). The study preval 18.51% (G. lambia), 14.81% (E. histolytica), 14.81% (Cryptosporidium pavum), 7.40% (Isosporo beli), 4.93% (Microsporodium), 2.46% (Hookworm), 2.46% (Taenia spp.), 1.23% (Blastocystctis homini). In order to prevent this infection appropriate health education should be given to the patients concerning disease transmission, antiparasitic therapy, personal hygiene and safe drinking water. Keywords: Parasitosis, HIV, TB, formal saline, formalin-ether, ZeihlNeelsen,
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    Anthropogenic impact on Rhesus Macaque (Macaca Mulatta Zimmermann, 1780) behavior in Pashupatinath temple area, Nepal
    (Department of Zoology, 2022) Karki, Melina
    Rhesus macaques are highly commensal non-human primates and they display a close relationship with humans. There is a controversy regarding the human-macaque interactions in the wild and conflict is created due to these interactions. However, the effect of human disturbance in the behavior of the rhesus macaques are understudied. This study is mainly focused on the effect of human disturbance on feeding and grooming behavior of rhesus macaques in Pashupatinath Temple area, Kathmandu. Two groups of macaques from differential disturbance area were taken. Focal animal sampling was performed to note the behavior of the macaques, whereas instantaneous scan sampling was used for the collection of number of people at that exact time. Six focal animals from each group were observed. There was no difference (t= 0.007, P= 0.497) between the time spent in overall activity by two groups regardless of the human presence. Despite the overall activities of males and females being insignificant in high (t= -0.109, P= 0.457) and low (t= -0.038, P= 0.485) disturbance area, there was a noticeable difference in the individual activities. In both the study groups, males rested more than females; whereas females groomed more than males. Assessing the human disturbance effect in grooming activities suggested negative association in overall males and females. In the low disturbance area, the effect on grooming were insignificant for both the sexes (males and females) unlike the macaques in high disturbance area showing negative association of human presence with grooming. Similarly, overall macaques from both high and low disturbance area did not show any effect of human disturbance in feeding whereas the females from both groups showed positive effect of human disturbance on feeding. However, non-significant difference was observed in the feeding of individual sexes from both high and low disturbance area. In conclusion, food provisioning of the people, teasing, scaring off macaques, and throwing stones towards them might be the cause for the reduced grooming time in macaques. However, in presence of humans they get to feed on provisioned food due to which the time spent on feeding might have increased. The reason for insignificant difference between the groups could be due to habituation of the macaques in the anthropogenic environments.