Tribhuvan University Journal of Microbiology

COVID-19 Pandemic: Lesson Learned and Next Plan of Actions

Komal Raj Rijal — 2022-07-25

No Abstract.

Prevalence of Staphylococci in Environmental Surfaces and Characterization of Isolates by Antibiotic Susceptibility

Charu Arjyal — 2021-12-31

Objectives: The purpose of the study was to determine the extent of staphylococcal contamination in various environmental sites and to characterize the isolates by antibiotic susceptibility.

Methods: A cross-sectional study was conducted and 123 samples were collected from 9 different sites around Kathmandu valley. Isolation of S. aureus was done through cultural and biochemical analysis. Kirby-Bauer disc diffusion test was employed to test the susceptibility of isolates to antibiotics.

Results: A total of 25 S. aureus (20.33%) were isolated; among which 12 isolates exhibited methicillin resistance i.e. 48% (MRSA) and 13 isolates were methicillin susceptible, 52% (MSSA). Similarly, 53 Coagulase Negative Staphylococci (CoNS) were isolated; among which 17(32.07%) were resistant to methicillin. The antibiotic resistance patterns of MRSA were reported as: erythromycin(n=2;16.6%.), clindamycin (n=2;16.6%), cotrimoxazole (n=2;16.6%), ciprofloxacin (n=2;16.6%) and gentamicin (n = 1;8.3%). MRCoNS showed high resistance to erythromycin (n=6; 35.2%), followed by co-trimoxazole (n=4; 23.5%), novobiocin (n=4; 23.5%) and ciprofloxacin (n=3; 17.6%). All MRSA and MRCoNS isolates were susceptible to linezolid and clindamycin.

Conclusion: This study reports relatively high prevalence of MRSA on environmental surfaces, pre-dominating in areas having heavy crowds. There may be a likely connection between humans and the environment to share MRSA and MSSA.

Knowledge and Perception of COVID-19 Pandemic during the First Wave: a Cross-Sectional Study among Nepalese Healthcare Workers

Biplav Aryal — 2021-12-31

Objectives: This study aimed to assess knowledge and perception of COVID 19 among frontline health care workers in Nepal.

Methods: A web-based cross-sectional study was conducted among frontline healthcare workers involved in managing and treating COVID-19 in Nepal by adapting the questionnaire from the World Health Organization. Data were analyzed using the Statistical Package for Social Science version 26.0. A chi-square test was used to investigate the association level among variables at 95% level of significance.

Results: A total of 285 health professionals participated in this study, among them male (50.5%), and female (49.5%). The mean (± SD) age of participants was 27.21 ± 5.42 years old. Most of the participants got information on COVID-19 from social media (50.5%) and online news/newspapers (40.3%). On the other hand, a significant proportion of Health care workers (HCWs) had poor knowledge about its transmission (n=170, 59.6%) and an incubation period (n=129, 45.3%).

Conclusion: There was a significant gap in knowledge and perception, particularly about the transmission and incubation period. A concerned national initiative to respond the pandemic could help better compliance with behavioral guidelines to respond to this public health emergency.

Microbiological Study of Food Packaging Paper of Kathmandu Valley

Anupa Kumari Budhathoki — 2021-12-31

Objectives: The purpose of this study was to isolate and identify microorganisms of food packaging papers of Kathmandu valley and determine antibiotic susceptibility of the isolates.

Methods: A total of 34 food packaging paper samples were collected aseptically from hotels, bakeries and sweet shops (considered as closed shop) and open street vendors and were transported to microbiology laboratory of Golden Gate International College for processing. The isolates were identified by standard microbiological procedures and subjected to antimicrobial susceptibility testing by modified Kirby-Bauer disk diffusion method following CLSI guidelines. The rate of Extended Spectrum Beta- lactamase (ESBL) producing and multiple drug resistant (MDR) isolates were also determined.

Results: All 34 samples yielded microbial growth with average microbial count of 4.145×105 CFU/g. Among 103 microbial isolates, 78 were bacteria, 15 molds and 10 yeasts. The predominant bacterial and mold isolates were Bacillus spp (43.59%) and Cladosporium spp (46.67%) respectively. Ciprofloxacin (42/43) and Amikacin (42/43) were the most effective and ampicillin (39/43) was most resistant antibiotics for Gram negative bacteria. A total of 9.30% Gram negative isolates were identified as ESBL producing and MDR strains.

Conclusion: This result indicates that potential pathogens are found in food packaging papers which can be threat to health of consumers as they may act as a source of food borne infection.

Antimicrobial Activity of Some Common Spices

Shashi Bhusan Chaturwedi — 2021-12-31

Objective: Antibiotic toxicity and multi drug resistant pathogens are the two greatest challenges that today's medical world has been facing. As a consequence of the haphazard use of antimicrobials, the spread of antimicrobial resistance is now a global issue. This study aimed to investigate antimicrobial activity of some common spices.

Methods: During the study period Five commonly used spices were collected from local market of Lagankhel, Lalitpur. The antimicrobial activity of selected naturally grown spices was done against two gram positive and three gram-negative pathogenic bacteria. The extracts of the spices were obtained by using absolute ethanol (99.9%) to carry out the antibacterial susceptibility assay using agar well diffusion method.

Results: The result of agar well diffusion method showed Clove and Cinnamon were found to possess relatively higher antimicrobial activities by preventing the growth of all 5 tested bacteria. Gram positive bacteria were found to be more sensitive to spices than Gram negative bacteria.

Conclusion: The finding of this study showed that extract of spices can be alternative to synthetic drugs to control infectious diseases.

Biofilm Producing Pseudomonas aeruginosa in Patients with Lower Respiratory Tract Infections

Sabina Chhunju — 2021-12-31

Objectives: To determine the prevalence of Gram-negative bacteria in lower respiratory tract infections and study the biofilm producing Pseudomonas aeruginosa.

Methods: This study was conducted at B & B Hospital Pvt. Ltd., Lalitpur, Nepal from February to September 2018. The samples were collected from the patients (n=420) with signs and symptoms suggestive of LRTIs. The isolated organisms were identified and antimicrobial sensitivity was performed. Among all the isolates, P. aeruginosa isolates were subjected for biofilm detection by microtitre plate method.

Results: Out of 420 specimens, 90 (21.6%) were culture positive. Klebsiella pneumoniae (42.9%) was found to be the predominant organism with higher rate of resistance to antibiotics. A total of 25 isolates of P. aeruginosa were isolated among which 15 (60%) were biofilm producers. Biofilm-producing isolates of P. aeruginosa were found more resistant to the tested antibiotics.

Conclusion: Gram-negative bacteria were found to be the predominant etiological agents in causing the LRTIs; K. pneumoniae, being the most commonly isolated bacteria. Most P. aeruginosa were capable of producing the biofilm. The biofilm producers were more resistant to the antibiotics. The biofilm may help increase the resistivity nature of the bacteria.

Microbial Quality Analysis of Panipuri Samples Collected from Different Parts of Bhaktapur

Punam Ghimire — 2021-12-31

Objectives: The study was aimed to identify potential bacterial pathogens from the panipuri samples collected from different vendors of Bhaktapur district and determine their antibiogram patterns.

Methods: Altogether 120 (40 puri, 40 stuffing and 40 pani) samples of panipuri were collected from different vendors of Bhaktapur district in a cold chain and were transported to the microbiology laboratory. Puri and stuffing were then pre-enriched, enriched and cultured while pani samples were processed using the MPN method. All the isolates were identified following standard microbiological procedure and subjected to antibiotic susceptibility testing following CLSI guidelines.

Results: The result revealed contamination of 77.5% stuffing, 67.5% puri and 52.5% pani samples with pathogenic bacteria. Among bacterial pathogens, the highest number was Staphylococcus aureus followed by Escherichia coli and Salmonella spp. 100% of S. aureus were found to be resistant to ampicillin and novobiocin. Similarly, 94.1% of E. coli were resistant to ampicillin followed by ciprofloxacin (64.7%). A very few isolates of Salmonella spp, Shigella spp and Vibrio spp were resistant to tetracycline. The highest number of multidrug-resistant bacteria were S. aureus, followed by Klebsiella spp and E. coli.

Conclusion: The study showed that the panipuri samples from street vendors were found to be highly contaminated with pathogenic bacteria which might affect consumers’ health. Thus, to prevent any food-borne illness in the future, frequent evaluation and regulation of the quality of such foods should be carried out.

Antimicrobial Resistance in Escherichia coli: a Cross Sectional Study in Chicken Poultry of Kirtipur, Nepal

Manisha Kharel — 2021-12-31

Objectives: The study was conducted to determine antimicrobial susceptibility pattern and prevalence of Extended Spectrum Beta Lactamase (ESBL) producing E. coli from fecal sample of different chicken poultry farm located at Kirtipur.

Methods: The cross sectional study was conducted from November 1st 2019 to February 29th 2020. The samples were collected from 27 different poultry farms and processed at Microbiology laboratory at Trichandra Multiple Campus. Identified E. coli were subjected to antimicrobial susceptibility test by using Kirby and Bauer Disc Diffusion technique and Combined disk method was used to determine ESBL E. coli.

Results: From all 27 poultry farms, E. coli was isolated from broiler (n=13), layer (n=10), and local (n=4) breeds, of which 23 (85.18%) were not registered. The chickens were fed with vitamin and calcium as growth promoter along with antibiotics; Piperacillin, Colistin and Doxycycline. Altogether 85.18% (n=23) isolates showed sensitivity towards Nitrofurantoin, Cefotaxime and Ceftazidime followed by Tigecycline 77.7% (n=21). Among these isolates 66.6% (n=18) were resistance towards Piperacillin followed by Ampicillin 37% (n=10). A statistically significant correlation was seen in resistance rate between broiler and layers. Among total isolates 37.03% (n=10) were Multi Drug Resistance (MDR) and 14.81% (n=4) were ESBL producer. Unregistered poultry farms were associated with MDR and ESBL Ec isolates.

Conclusion: Unregistered poultry farms and irrational use of antibiotics has influenced development of MDR and ESBL isolates. Timely monitoring and surveillance is suggested to decrease the trend of antimicrobial resistance (AMR) in poultry system.

Antifungal Susceptibility and Biofilm Formation of Candida albicans Isolated from Different Clinical Specimens

Shirshak Lamsal — 2021-12-31

Objective: Increasing antifungal resistance and biofilm formation among Candida species is an intimidating public health concern, especially at the hospital settings. In light of this, the current study was designed to assess the biofilm-forming ability of clinically isolated Candida albicans and determine their antifungal susceptibility against both the planktonic and sessile forms.

Methods: A total of 58 Candida isolates from different clinical samples received at the Microbiology laboratory of KIST Medical College and Teaching Hospital, Lalitpur, Nepal in between April to October 2018 were included in the study. Isolation and identification of C. albicans was done following standard microbiological procedures that comprised of microscopic observations along with germ tube formation and biochemical tests. Besides qualitative investigation of biofilm by tube method, it was also investigated quantitatively by crystal violet staining method and metabolic activity of the biofilm was assayed by tetrazolium (XTT) salt reduction method. Antifungal susceptibility pattern against common antifungal drugs was determined as planktonic and sessile Minimum Inhibitory Concentrations (MICs) by broth micro-dilution method.

Results: Out of 58 Candida recovered from the total samples, 21(36.2%) were identified as C. albicans. The vaginal swabs showed a higher prevalence (57.14%, 4/7) of C. albicans whereas none were recovered from the wound swabs. Qualitative study of biofilm formation showed that 4 (19.1%) Candida albicans were strong biofilm producers, 11 (52.3%) isolates were moderate and 6 (28.6%) produced weak or none biofilms, whereas a majority (85.7%) of the isolates gave biofilm positive test in microtiter plate assay. The metabolic activity of the biofilm revealed that the average absorbance following the metabolic reduction of tetrazolium salt was 0.577. Interestingly, both the methods used for assessing biofilm productions correlated well (r=0.569, p=0.007). Most of the isolates were susceptible to Fluconazole (80.9%) at MIC 0.12 μg/mL, Amphotericin B (76.19 %) at MIC 0.25 μg/mL and Clotrimazole (80.9%) at MIC 0.25 μg/mL. In addition, sessile forms of C. albicans was found to have 2 to 8 fold increases in MIC compared to the planktonic cells.

Conclusion: High prevalence of C. albicans in vaginal swabs may implicate that the women are more prone to vaginosis. The sessile forms are more resistant to antifungal agents and proper administration of antifungal targeting the biofilms should be prioritized only with susceptibility result interpretations.

Susceptibility to Fluoroquinolones among Salmonella enterica Serovars in Blood Culture

Monika Maharjan — 2021-12-31

Objectives: The study was designed to analyze the antibiotic susceptibility pattern of fluoroquinolones among Salmonella enterica.

Methods: A cross-sectional study was carried out at National Public Health Laboratory, Kathmandu. Blood samples were collected from suspected enteric fever patients and cultured in BACTEC standard/10 Aerobic/F culture vials. Isolates obtained from the vials with bacterial growth were identified by standard procedure. Serotyping of the identified isolates Salmonella enterica was done. An antibiotic susceptibility test was done by Kirby-Bauer disc diffusion method and results were interpreted according to Clinical Laboratory Standards Institute (CLSI 2014) guidelines.

Results: Among 404 samples, 17 (4.2%) were positive for Salmonella enterica in which 9 (52.9%) were Salmonella Typhi and 8 (47.1%) were Salmonella Paratyphi A. All the Salmonella isolates showed resistance to nalidixic acid and ampicillin and showed sensitivity to ceftriaxone and chloramphenicol. No multi-drug resistant isolates were identified in this study. All isolates of Salmonella Typhi and Salmonella Paratyphi A showed the reduced susceptibility to ciprofloxacin and ofloxacin.

Conclusion: It is concluded that fluoroquinolones cannot be considered as the drug of choice for the treatment of Salmonella infections due to their high level of reduced susceptibility and resistance to fluoroquinolones and third generation cephalosporin antibiotics like ceftriaxone remains better choice of drugs against fluoroquinolone-resistant Salmonella Typhi and Paratyphi.

Prevalence of Methicillin Resistant Staphylococcus aureus among Dumpsite Workers in Kathmandu Valley

Dinju Manandhar — 2021-12-31

Objectives: The main objective of this study was to determine prevalence of methicillin resistance Staphylococcus aureus among dumpsite workers.

Methods: Total 60 nasal swab samples were collected. Conventional microbiological methods were used to isolate and identify S. aureus. Antibiotic susceptibility test was performed by Kriby Bauer disc diffusion method. MRSA was confirmed by using Cefoxitin disc. The organism showing resistance against three or more class were considered as MDRSA.

Results: The prevalence rate of S. aureus and MRSA was found to be 46.67% (28/60) and 6.67% (4/60) in total population. The nasal carriage rate of S. aureus was found to be higher in age group 28-37 (70.06%, 12/17), gender male (47.5%, 19/40), district Kathmandu (70%, 14/20) and dumpsite workers (50%, 15/30). The isolated S. aureus were resistant to Penicillin (35.7%), Erythromycin (35.7%), Cefoxitin (14.3%) and Oxacillin (10.7%). S. aureus was susceptible (100%) to Tetracycline, Gentamycin, Ciprofloxacin, Co-trimoxazole, Chloramphenicol, and Vancomycin. Multidrug resistant S. aureus was not found in community of Kathmandu Valley.

Conclusion: The study shows prevalence of MRSA strains of S. aureus in Kathmandu Valley. The occurrence of MRSA indicates development of Community acquired-antibiotic resistant bacteria.

Seroprevalence of Brucellosis among Pigs of Commercial Farms in Chitwan District of Nepal

Kiran Pokhrel — 2021-12-31

Objectives: This study aimed to determine the seroprevalence of brucellosis among pigs of commercial farms in Chitwan district of Nepal.

Methods: This cross sectional prospective study was conducted among 100 pigs of commercial farms located in western region of Chitwan district of Nepal. Blood sample was collected from each pig by the trained veterinarians and serum was extracted. Each serum sample was processed for Rose Bengal plate test (RBPT) and ELISA for the detection of Brucella spp. Data was analyzed using SPSS software version 21.0 and a p-value of less than 0.05 was considered as significant.

Results: Prevalence of brucellosis in pigs was found to be 15% (15/100) by RBPT and 10% (10/100) by ELISA. There was statistically insignificant difference (p=0.98) in gender wise prevalence of brucellosis among pigs. Younger pigs below one year of age were more susceptible to brucellosis than the older pigs. Landrace breed showed more positive test results compared to other breeds.

Conclusion: Pigs can be the potential source of transmission of brucellosis to humans. Considering the high economic loss on livestock sector and possible transmission to humans, awareness program and appropriate control strategies is warranted. Breed and age factors should be considered while adopting the control measures of brucellosis among pigs.

Antibiogram and Biofilm Development among Klebsiella pneumoniae from Clinical Isolates

Subash Paudel — 2021-12-31

Objectives: This study was aimed to evaluate antibiotic resistance pattern and biofilm formation in K. pneumoniae strains isolated from different clinical specimens and to study on association of drug resistance pattern with biofilm formation.

Methods: A total of 944 clinical samples from patients attending Sahid Gangalal National Heart Center were processed from September 2019 to March 2020 to identify possible bacterial pathogens following standard microbiological procedures. K. pneumonaie isolates were further subjected to antibiotic susceptibility testing using modified Kirby Bauer disc diffusion technique. Biofilm formation was evaluated by tissue culture plate technique.

Results: Of the total 944 samples, 15.47% (146) samples showed bacterial growth, among which 23.97% (35) were K. pneumoniae. Out of 35 K. pneumoniae isolates, 45.71% (16) were multidrug-resistant and 42.86% (15) were extensively drug-resistant. Sixty percent (21) of K. pneumoniae feebly produced biofilm. Significant association was observed between biofilm production and exhibition of multidrug resistance (p value<0.05).

Conclusion: Prevalence of antibiotics resistant K. pneumoniae in hospital setting is high and alarming. Significant association between drug resistance pattern and biofilm production implicates need of an immediate response to limit growth and spread of drug resistant microbes in clinical settings.

Determination of Inhibitory Effects of Allium sativum Extract on Biofilm Production by Clinical Staphylococcus aureus Isolates

Ashim Rai — 2021-12-31

Objectives: To determine anti-biofilm effect of fresh garlic extract (FGE) on Staphylococcus aureus biofilm production and the relationship between methicillin resistance and biofilm production intensity.

Methods: Clinical S. aureus isolates were identified methicillin resistant S. aureus (MRSA) by cefoxitin disc diffusion method. The anti-biofilm effects of FGE on S. aureus biofilm biomass determination was done using crystal violet assay.

Results: Among 71 S. aureus isolates, MRSA were 37 (52.1%). Among biofilm producers, comparison of biofilm biomass (absorbance at 492 nm) showed no significant differences in biofilm formation ability between MRSA and MSSA (p=0.325). Use of 10% FGE decreased biofilm production in MRSA and MSSA by 40.4% (p<0.001) and 48.1% (p<0.001) respectively. Detachment assay using sodium dodecyl sulfate showed that control group biofilm biomass was decreased by 41.2%, while test group was decreased by 61.7% (p<0.001).

Conclusion: Garlic extracts has potency as an anti-biofilm agent and could be developed and used to manage different S. aureus biofilm related infections.

Antifungal Susceptibility Testing of Pathogenic Aeromycoflora Isolated from Kathmandu

Manju Shree Shakya — 2021-12-31

Objective: To identify the predominant pathogenic aeromycoflora present in dense areas of Kathmandu, and perform their antifungal susceptibility test.

Methods: Aeromycoflora were isolated by the Gravity Plate method and identified by observing colony morphology and microscopic methods. For pathogenic mycoflora, MIC test was carried out following “Reference Method for Broth Dilution Antifungal Susceptibility Testing of Filamentous Fungi; Approved Standard. NCCLS document M38-A”. The antifungal agents used were Cotrimazole, Fluconazole, Ketoconazole, Itraconazole and Terbinafine available at pharmaceutical companies of Nepal during study period.

Results: Penicillium spp (43.8%) was found to be the most predominant aeromycoflora followed by Cladosporium spp (35.5%), and pathogenic Aspergillus species (21.4%). Among Aspergillus species, Aspergillus niger (71.7%) was predominant, followed by A. flavus (17.1%) and A. fumigatus (11.1%). A. fumigatus and A. flavus were found to be most sensitive towards Itraconazole (MIC range – <0.0625 - 1μg/ml) while A. niger to Ketoconazole (MIC- 0.0625 - >32μg/ml).

Conclusion: The finding of this study helped to identify the potent antifungal drugs available against the pathogenic aeromycoflora.

Antimicrobial Activity of Lactic Acid Bacteria Isolated from Traditional Fermented Food

Pratibha Sharma — 2021-12-31

Objective: The main objective is to isolate Lactic acid bacteria from traditional fermented food of Kathmandu valley and to study their antimicrobial properties by agar well diffusion method.

Methods: A total of 30 samples of 4 different types of traditional fermented food (Gundruk and sinki, Pickles and Dahi) were obtained from Kathmandu valley and processed in Microbiology Laboratory of Padma Kanya Multiple Campus. For identification of Lactic acid bacteria (LAB), Gram staining, catalase and motile tests were done. In the carbohydrate fermentation test, all isolates were processed for fermentation of glucose, lactose, sucrose and fructose. Bacteriocin was extracted by precipitation method. Antibiotic susceptibility of the isolates was determined by using modified Kirby-Bauer disc diffusion method. The antimicrobial activity of Lactic acid bacterial (LAB) was done by agar well diffusion method.

Results: A total of 21 LAB isolates were identified which were 10 Lactobacillus spp (47.6%), 8 Pediococcus spp (38.0%) and 3 Streptococcus spp (14.3%). The antimicrobial activity of bacteriocin showed inhibitory activity against Shigella spp, Escherichia coli and Bacillus spp but did not show inhibition to Staphylococcus aureus, Salmonella spp. and Klebsiella pneumoniae. For the quality control, the antimicrobial activity of bacteriocin was done on ATCC (25923) Staphylococcus aureus. All isolates were susceptible to ampicillin while resistant to nalidixic acid and Co-trimoxazole.

Conclusion: The present study showed the bacteriocin produced by LAB from traditional fermented food (Gundruk and Sinki, yogurt and Pickle) showed antimicrobial activity against different bacteria which underline its important role in improving food quality and increasing safety.

Prevalence of Candida Carriage and in Vitro Evaluation of Phospholipase and Haemolysin Activity of Oral Candida albicans among Tobacco Users and Smokers in Dharan, Nepal

Bijay Kumar Shrestha — 2021-12-31

Objectives: The main objective of this study was to determine the prevalence of Candida carriage among tobacco users and smokers along with in vitro evaluation of phospholipase and hemolysin activity of Candida albicans.

Methods: A laboratory based cross-sectional study was carried out in Dharan Sub-Metropolitan city, Eastern Nepal from June 2018 to November 2018. During the study 150 oral rinse samples were obtained from smokers (50), smokeless tobacco consumers (50) and non-tobacco users (50) as control group. The participants were provided 10 ml of normal saline and were asked to oral rinse for 1 minute. Oral rinse was collected in a sterile screw capped container and was transported to microbiology laboratory by maintaining the cold chain. The oral rinse sample was inoculated onto the Sabouraud dextrose agar with chloramphenicol and was incubated at 37°C for 3-4 days. The number of colonies of Candida was counted and C. albicans were identified by cultural characteristics, staining, germ tube test and chlamydospore formation test.

Results: The prevalence of Candida carriage was reported to be 22 (44%) in smoker group, 26 (52%) among smokeless tobacco users and 13 (26%) among control groups. The prevalence of Candida carriage was found to be significantly higher in the study group associated with tobacco chewers (P=0.008). However, the Candida carriage among smoker’s group was not found to be statistically significant (P=0.059). Isolation of Candida albicans was higher among smokeless tobacco users 15 (30%), smokers 5 (10%) and non-users 6 (12%).

Conclusion: Colonization and carriage of Candida in the oral cavity of smokers and tobacco chewers were found to be higher than in controls. In addition, individuals with poor oral hygiene increase the risk of Candida colonization and infection under host debilitated condition.