Tribhuvan University Journal of Microbiology
The Tribhuvan University Journal of Microbiology is published by the Central Department of Microbiology, Tribhuvan University, Kirtipur, Kathmandu, Nepal. Full text content available here and on the journal's own website www.microbiotu.edu.np
Central Department of Microbiology, Tribhuvan University
© Copyright Central Department of Microbiology, Tribhuvan University
Integrated Disease Surveillance — Current Need in Vector-Borne Viral Diseases in Nepal
There has been no systematic study comprised of hospital cases, community (population immunity) and mosquito surveillance to look at the actual dynamics of dengue in Nepal. In fact, there is no data on ZIKA, no molecular data on CHIKV and only single report available on WNV from Nepal. This indicates a huge research gap in mosquito-borne viral diseases in Nepal. Therefore, to understand the full-extent of virus transmission dynamics and potential roles of climate change in virus migration, an integrated virus surveillance system that comprises both human and mosquito component of multiple viruses at a time is essential apart from the classical case-based disease surveillance at hospitals.
Copyright (c) 2024 Central Department of Microbiology, Tribhuvan University
Trends of Hepatitis Virus Infections in a Tertiary Care Hospital in Nepal, 2012-2023
Objectives: To monitor the trends of incidence of different hepatitis virus infections during 2012–2023 in a tertiary care hospital in Nepal. Methods: A descriptive study was conducted using demographic and serological data from 2012 to 2023 at Tribhuvan University Teaching Hospital. The serological tests were performed by enzyme-linked immunosorbent assays from the patient’s serum. Results: The incidence of HAV infections showed a downward trend from 2012–2016 (37.6%- 17.7%), increased in 2017–2019 (33.9%-27.8%), and then gradually decreased to 10.2% in 2023. In case of HEV infection, the incidence did not show any trend over the ten years period (from 2012-2018: 5.4%-13.3%; 2020: 7.1%; 2021:14.7%; 2023: 4.2%). The incidence of HBV infection was nearly unchanged between 2.5-3.1% from 2012-2023, the incidence of HCV infection was between 0.7-1.8% during the same period. The proportion of male cases was higher for all hepatitis infections and adult cases (15-47 years old) were in higher proportions except hepatitis A. Conclusion: While the incidence trends of hepatitis B infections have been nearly the same over the past ten years, the trends of hepatitis A, E, and C infections are declining. Extensive preventive efforts are essential for reducing the infection rates to a low level.
Comparison of Beta-lactamase Resistance Gene Detection in MDR Escherichia coli Isolates in Nepal
Objectives: To compare ESBL and carbapenemase gene detection using phenotypic (double disc diffusion and VITEK-2AES), and genotypic (PCR) methods in the E. coli isolates in Nepal. Methods: 132 multidrug resistant E. coli isolates with phenotypic extended-spectrum-β-lactamase (ESBL) and carbapenemase archived during 2018 to 2023 at National Public Health Laboratory were analyzed to detect three extended-spectrum-β-lactamase genes (CTX-M, SHV and OXA 10/11) and eight carbapenemase genes (NDM, KPC, IMP, VIM, OXA‐51, OXA‐23, OXA‐48 and OXA‐58). Vitek 2 generated Advanced Expert SystemTM reports were compared with PCR findings, to evaluate their predictive diagnostic values. Results: Out of the 132 isolates tested, CTX-M gene was detected in 60/80 (75%), OXA 10/11 and SHV gene in none (0/80); while no genes were detected in 20 ESBL-non producers. NDM gene was detected in 12/22(55%) and OXA-48 in 2/22(9%) metallo-beta-lactamase (MBL) producers, while other carbapenemase genes were not detected in 10 MBL non-producers. Five (5%; 5/102) of the isolates had both CTX-M and NDM genes. The Vitek 2 detection of CTX-M-like ESBLs showed a sensitivity of 75% (60/80) and specificity of 100% (20/20) PPV of 100% and NPV of 50% for the E. coli isolates. Conclusion: blaCTX-M and blaNDM were the most common genes responsible for third-generation cephalosporins and carbapenem resistance respectively with low co-existence. Vitek2 AES detects ESBL with adequate sensitivity, however more evidences are required for use in routine screening.
Can Cycle Threshold Value of RT-PCR for SARS-CoV-2 Predict Secondary Bacterial Infections in Patients with Coronavirus Disease?
Objectives: This study aims to investigate the relationship between cycle threshold (Ct) values of RT-PCR for SARS-CoV-2, as a marker of viral load, and the occurrence of secondary bacterial infections in hospitalized COVID-19 patients. Methods: A cross-sectional study was conducted among 141 COVID-19 patients at Sukraraj Tropical and Infectious Disease Hospital, Nepal, between July 2022 and June 2023. Bacterial cultures were performed for hospitalized patients. Ct values for SARS-CoV-2 N and Orf 1ab genes were compared between patients with and without secondary bacterial infections. Results: Out of 32 hospitalized patients, 11 (34.37%) had secondary bacterial infections. The median Ct values of the N gene were slightly lower in patients with secondary bacterial infections (19.5) compared to those without (19.0), though this difference was not statistically significant (p = 0.2113). Similar trends were observed for the Orf1ab gene (20.5 vs. 19.5; p = 0.3125). The most frequently isolated bacterial pathogen was Klebsiella pneumoniae, while Acinetobacter baumanii and E. coli were associated with the lowest Ct values. Conclusion: While patients with secondary bacterial infections tended to have lower Ct values, no statistically significant relationship was observed between Ct values and the occurrence of secondary bacterial infections. However, Ct values might still provide supplementary insights into disease severity and the risk of secondary infections.
Antibacterial Action of Citrus Fruits Against Selected Pathogenic Bacteria
Objectives: This study's primary goal was to assess citrus fruits' antibacterial effectiveness against particular microorganisms. Methods: The juice and peel of the seven chosen citrus fruits were extracted using a conventional qualitative method in an ethanol solvent. The resulting extracts were then diluted in an aliquot of 1 mL of 10% v/v DMSO. The antimicrobial test was conducted using the Agar well diffusion method. This study was conducted from February and April, 2022. Results: The peel and juice extracts shown varying degrees of inhibitory zones on particular bacteria. Citrus aurantium juice extract was the most efficient citrus fruit extract against Staphylococcus aureus, with a 27mm zone of inhibition, out of the ones that were chosen. On the other hand, a 19mm zone of inhibition was demonstrated by the Citrus reticulata peel extract, which was more efficient against Escherichia coli. Conclusion: Based on the findings, we conclude that citrus fruits may be helpful in preventing bacterial growth because of their strong antibacterial properties.
Microbial Assessment of the Ice Cream: A focus on Bacillus cereus Contamination in Pokhara, Nepal
Objectives: The study aims to assess microbial contamination in ice cream sold in Pokhara, with a specific focus on Bacillus cereus and to determine its antibiotic susceptibility pattern. Methods: The cross- sectional study was conducted on 108 ice cream samples of different brands collected from different shops in Pokhara. The samples were cultured on plate count agar for heterotrophic count and were also cultured on Mannitol Egg Yolk Polymyxin Agar for B. cereus identification. B. cereus was identified through cultural characteristics, gram staining and various biochemical tests. Antibiotic susceptibility test was performed by Kirby Bauer disc diffusion technique. Results: Heterotrophic plate count revealed 91.67% (n=99) ice-cream samples were contaminated with average count of 5.25±0.78 log CFU/gm. Open cup ice-cream samples of all nine brands showed the maximum bacterial count with average of 5.49±0.73 log CFU/gm whereas that of minimum was found in sealed cone samples (4.99±0.51 log CFU/gm). B. cereus contamination was observed in 16.67% (n=18) ice-cream samples with average count of 3.52±0.25 log CFU/gm. Out of nine brands, 5 brands were contaminated with B. cereus. Sealed cone ice cream did not show any growth of B. cereus and heterotrophic plate count was also minimum. All the isolates were susceptible toward used antibiotic except Piperacillin/Tazobactum. Conclusion: The present study reveals the occurrence of microorganism in the ice cream which is the frozen dairy product. Sealed cone ice cream is generally considered safer compared to other types.
Enzyme Screening and Antimicrobial Potential of Thermophiles Isolated from Different Environmental Samples
Objectives: This study aimed to examine the growth, enzyme activities, and antimicrobial properties of thermophilic bacteria from compost, soil, and hot springs. Methods: Six samples each from compost, soil, and hot springs were collected and thermophiles were isolates. These isolates were tested for growth in extreme conditions (high pH, high salt concentrations, temperature up to 65ºC) and identified by morphological and biochemical characteristics. Enzyme production such as amylase, gelatinase, cellulase, pectinase, and lipase were evaluated, and the antimicrobial properties against test organisms were assessed. Results: From 43 isolates, 17 grew well in high pH, 16 in high salt, and 5 in 65ºC. Bacillus licheniformis had the highest amylase and lipase activity. Bacillus stearothermophilus and Thermoactinomyces had the highest gelatinase activity, Bacillus polymyxa had the highest cellulase activity, and Bacillus macquariensis and Bacillus stearothermophilus had the highest pectinase activity. Antimicrobial activity was observed in 54% of isolates, with 95.7% inhibiting Staphylococcus aureus ATCC 25923, 34.8% inhibiting Escherichia coli, and 1 isolate inhibiting Salmonella spp. Conclusion: The study showed that compost-derived thermophilic isolates had high enzymatic activity. Furthermore, many isolates were able to inhibit the growth of Staphylococcus aureus ATCC 25923 by producing an antimicrobial substance.
Comparison of Phenotypic Methods for the Detection of Carbapenem-Resistant Enterobacteriaceae and Molecular Detection of OXA-181 Gene
Objective: To compare different phenotypic methods used for detecting carbapenem-resistant Enterobacteriaceae and to detect the presence of the OXA-181 gene among them. Methods: A hospital-based cross-sectional prospective study was conducted at Shahid Gangalal National Heart Center, Kathmandu, from September 2021 to March 2022. Altogether, 143 Enterobacteriaceae were isolated from 1042 clinical specimens and subjected to antibiotic susceptibility tests following CLSI guidelines. Several phenotypic methods (Modified Hodge Test (MHT), modified carbapenem inactivation method (mCIM), EDTA-CIM (eCIM) and CarbaNP test were performed for the detection of CRE and the results were compared. In addition, MIC of imipenem was carried out against MDR Enterobacteriaceae using agar dilution method and resistant isolates were also tested by PCR for presence of OXA-181 gene. Results: Among all the Enterobacteriaceae isolates, Klebsiella pneumoniae was the predominant isolate. Gentamycin, nitrofurantoin and carbapenem were found to be most effective, whereas the majority of the isolates were highly resistant to ß-lactam antibiotics along with ciprofloxacin. In total, 43.36% (62/143) isolates were carbapenem-resistant, showing 48.39% (30/62) positivity by MHT, 72.58% (45/62) by mCIM and 61.29% (38/62) by CarbaNP method. The OXA-181 gene was detected in 12 carbapenem-resistant isolates (MIC ≥4 µg/ml), predominantly in K. pneumoniae and E. coli isolates. Conclusion: mCIM is the most effective phenotypic method for detecting carbapenemase production. Presence of OXA-181 gene among clinical isolates is a serious concern, especially in the hospital setting.
Prevalence and Factors Associated with Intestinal Parasitic Infections among School Children in Sauka and Non-Sauka Communities of Darchula, Nepal
Objective: To determine the prevalence of intestinal parasitic infections among the school children of Sauka and non Sauka communities of Khalanga, Darchula, Sudurpaschim Province, Nepal. Methods: This was a cross-sectional study conducted among school children from Sauka and non Sauka communities in Darchula district of Nepal. Stool samples were processed by using formal-ether sedimentation technique followed by normal saline and Iodine wet mount methods. Identification of parasites and/or ova in the stool samples was performed by microscopy. Results: The overall prevalence of parasitic infection was 21.3% (74/348), with significantly higher in males (28.3%) than females (16.3%) (p = 0.01). Sauka children exhibited a significantly higher prevalence (26%) of parasitic infection compared to non Sauka (14.9%) (p = 0.02). Lack of toilet at home, occupation, hand washing habits, and anti-helminthic drug use were significantly associated with infection. Monoparasitism (17.8%) dominated over multi parasitism (3.4%), with protozoan (21.6%) more prevalent than helminthes (3.2%). Giardia lamblia (9.2%) and Trichuris trichiura (1.4%) were the most prevalent protozoan and helminthic parasites, respectively. Conclusions: High prevalence of parasitic infection in the Sauka community indicates poor health hygiene and socio-economic status. It is high time to implement effective programs including awareness, anti-parasitic drugs distribution, and sanitation and hygiene improvement in this remote community.
Bacteriological Analysis of Vegetables found in Kathmandu Valley
Objectives: The study was conducted to determine the bacteriological analysis of vegetables by comparing the number of bacterial count between farm and market along with antibiotic susceptibility pattern of the isolates and evaluate the status of MDR and MRSA. Methods: The cross-sectional study was done from February to July of 2023. A total of 60 samples were gathered from three distinct districts: Kathmandu, Bhaktapur, and Lalitpur, consisting of 10 samples from each farm and market and the research was carried out at the Nepalese Farming Institute, Kathmandu. The bacterial count was compared and identified by standard microbiological approaches such as serial dilution, spread plate method, and other biochemical tests. The antimicrobial profile was carried out by the Kirby-Bauer disc diffusion method to analyze the status of MDR and MRSA. Results: From all 60 samples the highest total plate count and total coliform count (mean) was found in spinach (9.3×106) and cabbage (4.9×106) from market. A total of 35 bacteria were identified i.e. 16 (46%) from farms including E. coli, Klebsiella spp., Pseudomonas spp. and Staphylococcus aureus and 19(54%) from markets such as E. coli, Klebsiella spp., Pseudomonas spp., Serratia spp. and Staphylococcus aureus. Antibiotic susceptibility pattern shown that Tetracycline was the most effective antibiotic as 80% Enterobacteriaceace isolates were sensitive but contradicted to Serratia marcescens and S. aureus was 100% susceptible to Linezolid, Cotrimoxazole followed by Gentamicin and Tetracycline. Out of 35 isolates, 14 showed MDR Strains i.e., Enterobacteriaceace (n=7; 35%) and S. aureus (n=7; 46.67%) and 3 isolates were screened as suspected ESBL producers but none were confirmed. A total of 15 S. aureus were isolated; among which 12 exhibited methicillin resistance (MRSA). Conclusion: The present study shows that the vegetables from the studied area contain a wide variety of bacteria raising significant public health concerns. It highlights the need for improved protocols and practices throughout the entire process of vegetable production, distribution and consumption.
Evaluation of Antioxidant and Antibacterial Properties of Rhododendron arboreum, Acmella calva and Trifolium repens of Nepal
Objectives: To evaluate the antioxidant and antibacterial properties of Rhododendron arboreum, Acmella calva and Trifolium repens. Methods: Different extracts of the R. arboreum, A. calva and T. repens were used for detection of total phenolic content and total flavonoid content. 2, 2-diphenyl-1-picryl hydrazyl (DPPH) assay was performed to evaluate the antioxidant activity. Antimicrobial activity was carried out by agar well diffusion method. Minimum Bactericidal Concentration (MBC) was determined by broth micro dilution assay. Results: Ethyl acetate extract of R. arboreum had high total phenolic content (128.36±1.65) and total flavonoid content (107.3±0.92). The high antioxidant activity with IC50 value of 34.97µg/mL was shown by ethyl acetate extract of R. arboreum. The ethyl acetate extract of R. arboreum showed maximum zone of inhibition against E. coli, K. pneumoniae, P. aeruginosa, A. baumanii, S. aureus and E. fecalis with the lowest MBC values. Conclusion: R. arboreum, A. calva and T. repens have antimicrobial activity and compounds with antioxidant activity and should be investigated further for potential medicinal use.
Antibiotic Resistance Bacteria and Revealing Resistant Genes from Surface Water
Objectives: To detect antibiotic resistance genes (ARG) genes and bacteria from surface water in Kathmandu. Methods: Bacteria were isolated on MacConkey agar and Eosin Methylene Blue (EMB) agar, further identified by biochemical test. Antibiotic susceptibility tests, and ESBL production was detected following CLSI guidelines. Plasmid was extracted by phenol and chloroform method and blaTEM, blaSHV, blaCTX-M, blaNDM-1 and blaOXA-48 genes were amplified by PCR. Results: Out of 100 isolates of Escherichia coli (60) and Klebsiella spp (40), 15 strains were ESBL producers. Of isolated ESBL positive isolates, total isolated strains were sensitive to imipenem and ofloxacin, whereas, from 85 non ESBL producers, all strains were sensitive to imipenem, ofloxacin, cefotaxime, ceftazidime, and ceftriaxone. The most prevalent gene distributed in surface water includes blaTEM, followed by blaNDM-1 and blaCTX-M genes. The combination of blaCTX-M and blaTEM (97.7%) was the most prevalent, followed by blaSHV and blaTEM (5.4%) genes. Conclusion: The detection of ESBL positive genes and bacteria in surface water indicates the growing challenge of antibiotic resistance. It requires a comprehensive response involving environmental management, public health strategies, policy changes, and global cooperation.
Isolation and Identification of Microorganisms from High Vaginal Swab of Pregnant Women Visiting Paropakar Maternity and Women’s Hospital, Kathmandu, Nepal
Objectives: The primary objective was to isolate and identify the bacteria present in high vaginal swabs (HVS) of pregnant women and assess their antibiotic susceptibility patterns. Methods: A cross-sectional study was conducted between January and June 2024, involving 300 HVS samples collected from pregnant women attending Paropakar Maternity and Women’s Hospital (PMWH). The samples were cultured on Mac Conkey and Blood agar plates. Antibiotic susceptibility test of the identified bacterial colonies was carried out by Kirby Bauer disc diffusion method. Results: Of the 300 samples, 79 tested positive for bacterial growth, revealing a diverse range of organisms, with Escherichia coli (29.11%), Citrobacter koseri (22.79%), Proteus mirabilis (20.25%), and Klebsiella pneumoniae (17.72%) as the predominant species. The highest incidence of bacterial presence was observed in the 21-25 age group (35.44%). Conclusion: This study highlights the concerning prevalence of bacteria and their antibiotic resistance in HVS from pregnant women, underscoring the need for improved infection control measures and targeted antibiotic strategies.
Etiological Characterization of Infectious Vaginitis among Reproductive-Aged Women Visiting a Tertiary Care Center in Nepal
Objectives: To characterize the etiology of infectious vaginitis among reproductive-aged women visiting a tertiary care hospital in Nepal. Methods: The vaginal samples were collected from woman with abnormal vaginal discharge. Nugent's score system and Amsel’s criteria were used in diagnosing bacterial vaginosis (BV), wet mount preparation was done to diagnose trichomonal vaginitis (TV), and Aerobic vaginitis (AV), and vulvovaginal candidiasis (VVC) were diagnosed by culture method. Clinical and Laboratory Standards Institute (CLSI) guidelines were used to assess the antimicrobial susceptibility profile of aerobic bacteria and Candida isolates. Results: Out of 141 reproductive-aged women included, 60.3% presented with thin and scanty vaginal discharge, 52.5% had clear to white discharge, and 39.0% had vulvovaginal itching. The overall rate of any type of infectious vaginitis was 44.0% with the predominance of BV (19.8%) followed by AV (12.8%), VVC (11.3%), and TV (7.1%). Infectious vaginitis was frequent among women between the age of 25 and 34 years. BV was significantly associated with malodorous discharge (p = 0.002) and VVC was significantly higher in women with vulvovaginal itching (p = 0.009). The most common aerobic bacteria that caused AV was Escherichia coli, while Candida albicans represented the most common cause of VVC. Conclusion: Incorporating simple laboratory test in management of vaginal discharge cases helps identify correct etiology minimizing the disease burden with appropriate use of antibiotics.
Antibiogram Profile of Respiratory Pathogens and Identifying Predisposing Factors of Respiratory Tract Infections among Patients Visiting Bhaktapur Hospital
Objectives: To address the current problem of MDR infections and respiratory bacterial pathogens and identify the risk factors associated with respiratory tract infections (RTIs). Methods: A total of 327 RTI specimens were cultured using a conventional microbiological method to identify potential bacterial pathogens. The isolates were then subjected to antibiotic susceptibility testing using modified Kirby Bauer’s disc diffusion method. In addition, the risk factors associated with RTIs were obtained by direct interviews with patients using a structured questionnaire. Results: The overall prevalence of RTIs among the study population was found to be 11.9%. The highest rate of infections was observed among patients of age group 61-70 years in both sexes and from ICU wards. Among the bacterial pathogens, Acinetobacter baumannii was isolated in the highest number followed by Pseudomonas aeruginosa. 64.2% A. baumannii isolates were resistant to Azithromycin but 100% sensitive to meropenem. P. aeruginosa resistance rate was 50% to Piperacillin-tazobactam, ciprofloxacin, Polymyxin B and Ceftriaxone, and had the highest MDR. Half of the Staphylococcus aureus isolates were MRSA. Risk factors such as previous infections, family history with RTIs, chronic use of antibiotics, and smoking were found to be significantly associated with RTIs. Conclusion: We observed high MDR among the respiratory pathogens with growing resistance to β-lactam, macrolides, and Polymyxins whch necessitates alternative drugs in management. There is also need for targeted preventive strategies, prohibition of irrational use of antibiotics, and avoidance of exposure to risk factors such as smoking, outdoor air pollution, and chronic use of antibiotics.
Assessment of Microbial Quality, Physiochemical Properties and Adulteration of Raw Milk in Pokhara
Objectives: This study was conducted to assess the microbial quality, physiochemical properties and adulteration status of raw milk collected from different farms of Lekhanath, Pokhara, Nepal. Methods: Total of 30 milk samples (15 Cow and 15 Buffalo) were collected from five different farms of Lekhanath, Pokhara. Total coliform count, isolation and identification of Escherichia coli and Staphylococcus aureus were determined by pour plated and spread plate technique and different biochemical and enzymatic tests, respectively. Additionally, physiochemical properties and adulteration parameters were also tested. Result: The laboratory analysis revealed that, the total coliform count of raw cow and buffalo milk samples were found as 0.55´104 to 2.13´106 CFU/ml and 0.34´104 to 1.76´106 CFU/ml respectively which is higher than the DFTQC and BIS guidelines. E. coli and S. aureus were found as 40%, 13.33% and 46.67%, 33.33% for cow and buffalo milk respectively. 20% of the raw buffalo milk sample were adulterated with Neutralizer. However, no adulteration was detected for table sugar, formalin, and starch. The average fat content of cow and buffalo milk were found to be 4.13% and 5.47% respectively meeting the minimum requirement set by guidelines. The solid-not-fat and Total solid of the samples were within the acceptable limit and acidity level were within the expected range indicating proper handling and storage of the milk sample. Conclusion: Thus, the findings suggest that the raw milk samples analysed in this study may not meet the microbiological safety standards for consumption. Proper hygiene practice and quality control measures should be implemented to ensure the safety and quality of milk.
Association of mecA Gene and pvl Gene in Methicillin Resistant Staphylococcus aureus Isolated from Clinical Specimens
Objectives: The aim of this study was to explore the association of mecA and pvl genes in clinical isolates of MRSA. Materials and Methods: A total of 748 clinical samples were processed by standard microbiological techniques. Colony morphology, Gram’s staining and biochemical tests including catalase, oxidase and biochemical tests were used for the identification. Antimicrobial susceptibility test was performed by Kirby-Bauer disc diffusion technique and their resistance to Methicillin was evaluated using the Cefoxitin disc diffusion method. DNA was extracted by using a modified phenol-based DNA extraction method and finally, the presence of pvl and mecA genes were investigated by the polymerase chain reaction method. Results: Out of 748 clinical samples, (29.4%; 220/748) samples had bacterial growth. Among 220 culture positive isolates, 19.1% (42/220) were positive for S. aureus. Among 42 S. aureus (66.7%; 28/42) were multiple drug resistant (MDR) and 38.1% (16/42) were MRSA. Among 16 MRSA; mecA and pvl genes were positive for 16 (100%) and 7 (43.7%) of the isolates respectively. Seven of mecA positive isolates was positive for pvl gene. Conclusions: It can be concluded from these results that the prevalence of pvl gene is high in MRSA isolates in this study and there is association between the presence of pvl and mecA genes in these isolates.
Extended Spectrum Beta-Lactamase Producing Gram Negative Pathogens in Urine Samples of Patients Visiting B.P. Koirala Memorial Cancer Hospital, Bharatpur, Nepal
Objectives: To isolate and identify extended spectrum beta-lactamases producing Gram negative bacteria in urine samples of patient visiting B.P. Koirala Memorial Cancer Hospital, Chitwan, Nepal. Methods: Urine samples were processed to isolate and identify Gram negative bacteria by different biochemical tests (Gram stain, catalase test, oxidase test, indole test, MR test, VP test, citrate utilization test, TSIA test), antimicrobial susceptibility test was done as recommended by Clinical Laboratory Standard Institute (CLSI) and production of ESBL was determined by combination disk diffusion method. Results: Out of total 142 samples, prevalance of UTI in cancer and cancer suspected patients was found to be 24.64%. Of the total Gam negative isolates, the most predominant organism was found to be E. coli 63% followed by Klebsiella 26% and others 11.11%. Among 27 Gram negative isolates, MDR was found to be 81.48%. The prevalence of ESBL producers among the total Gram negative isolates was 11.11%. The higher rate of growth was seen in age group of 60- 70 (29.62%). Higher prevalence of bacterial growth was observed in male. Polymyxin B, Gentamycin and Nitrofurantoin were the most effective antibiotics towards Gram negative bacteria. Conclusion: The prevalence of MDR and ESBL among Gram negative uropathogens isolated from cancer and cancer related patients are quite high. Therefore, it is essential to have a regular monitoring of ESBL producing clinical isolates in laboratory practice.
Antibiogram and Minimum Inhibitory Concentration of Selected Antibiotics against Vibrio cholerae Isolated from Sewage Samples
Objectives: The purpose of this study was to perform antibiogram profiling and determine minimum inhibitory concentration (MIC) of selected antibiotics against Vibrio cholerae isolated from sewage samples. Methods: A cross-sectional study was carried out at the microbiology and molecular biology laboratory of Central Campus of Technology. A total of 45 sewage samples were collected. The samples for this study was aseptically collected from different places of Sunsari district of Eastern Nepal and subjected to isolation and identification of V. cholerae., followed by antibiotic susceptibility testing by Kirby Bauer disc diffusion method. MIC was performed by selecting three antibiotics ampicillin, tetracycline and streptomycin and was done by microtiter plate method. Results: A total of 24 V. cholerae (53.33%) were isolated. From the total samples, 53.33% (n=13) were found to be resistant against Erythromycin and Tetracycline. 66.67% (n=16) samples showed that the bacterium was resistant to cefotaxime. 93.33% (n=22;) samples were sensitive to ciprofloxacin where as 66.67% (n=16) samples were sensitive to ampicillin and cotrimoxazole. 14 isolates were inhibited by Ampicillin at concentrations below 8 μg/ml, while 8 and 10 isolates were inhibited by Tetracycline and Streptomycin, respectively at concentrations below 4 μg/ml. Conclusions: This sewage-based V. cholerae antibiogram study underscores sewage as a reservoir and urges sewage control. ampicillin, erythromycin, and tetracycline show limited efficacy, in contrast to ciprofloxacin, ampicillin, and cotrimoxazole, which remain viable for treating much of cholera.
Antibacterial Efficacy of Selected Herbal Plants against Common Pathogenic Bacteria
Objectives: The purpose of this study was to ascertain the effectiveness of particular herbal plants as antibacterial agents. Methods: Different herbal plants were collected from Asan Bazar, Kathmandu, then the samples were dried, reduced to a powder, and combined with the appropriate amounts of hexane, ethyl acetate, and methanol to produce the extract accordingly. To obtain the desired extract, filtration, incubation, and vaporization were used. Escherichia coli, Staphylococcus aureus, and Bacillus spp. were the three test organisms. The test organism was uniformly swabbed onto a Muller Hinton Agar (MHA) plate. With the use of a borer, agar wells were created on MHA plates. The extracts of about 0.1 ml were applied to those wells using sterile micro pipette, which were then incubated at 37°C for 24 hours. Each well’s zone of inhibition (ZOI) was evaluated in relation to test organisms. Using the dilution approach, the minimum inhibitory concentration (MIC) was determined. Results: Bacillus spp. were found to be significantly inhibited by ethyl acetate extract of Coriandrum sativum (Coriander) with a ZOI of 25 mm. The ZOI 14mm test revealed that ethyl acetate extract of C. sativum had minimum efficacy against E. coli. The methanolic extract of C. sativum was reported to have MICs of 1:16, 1:32, and 1:64 against S. aureus, Bacillus spp., and E. coli, respectively. Conclusion: C. sativum is the most effective herbal plant against common pathogenic bacteria. Ethyl acetate extract was found to be more effective when compared to methanolic and hexane extracts. To update its medicinal value, additional research on its antibacterial characteristics may be required.
Antimicrobial Susceptibility Profile of Salmonella enterica Serovars and Determination of Minimum Inhibitory Concentration of Ciprofloxacin and Cefotaxime
Objectives: To study the antibiotic susceptibility profile of Salmonella enterica serovar Typhi and Paratyphi A and determine the minimum inhibitory concentration of Cefotaxime and Ciprofloxacin. Methods: Blood samples from patients suspected of enteric fever were cultured following standard microbiological techniques. Antibiotic susceptibility tests for commonly used drugs were performed by modified Kirby Bauer disc diffusion technique following the guidelines of Clinical and Laboratory Standard Institute (CLSI). Determination of minimum inhibitory concentration (MIC) was done by agar dilution method. Results: A total of 1,328 blood samples were processed out of which 64 were culture positive. Among the 33 isolates of Salmonella enterica, 22 were Salmonella Typhi while the rest 11 were Salmonella Paratyphi A. This study showed that 96.96% of isolates were resistant to Nalidixic acid and 87.87% were resistant to Ciprofloxacin. In contrast, 93.93% isolates were sensitive to Ampicillin and 90.90% were sensitive to Cefixime. 65.62% isolates were resistant with MIC ≥1 µg/ml against Ciprofloxacin while against Cefotaxime, 53.12% isolates were sensitive with MIC ≤1 µg/ml. Conclusion: Fluoroquinolones have shown the reduced susceptibility towards Salmonella Typhi and Salmonella Paratyphi A making them inappropriate to use for treating enteric fever. Ampicillin and Cefixime were found to be highly susceptible suggesting them for possible treatment. However, susceptibility testing must be done before the administration of any antibiotics.
Viability of Herbal Treatments for Bacterial Wound Infections
Objectives: This study was conducted to investigate whether the commonly used kitchen spices can be used as a potential source of antimicrobial agents or not. Methods: The three spice extracts (aqueous, methanolic and ethanolic) of cinnamon, black pepper, turmeric, ginger and clove were prepared for their antimicrobial properties against common bacteria. Antimicrobial susceptibility testing was done by Well Diffusion Method on Muller Hinton Agar (MHA). Then the plates were incubated at 37°C for 24 hours. The zone of inhibition for each spice was measured. For Minimum Inhibitory Concentration (MIC), the crude extracts of ethanolic extract of clove were considered as having 100% concentration of antimicrobials. The extract was then diluted in Mueller Hinton broth serially. Standard Suspension of test organism was kept in each dilutions and incubated at 37°C for 24 hours. The highest dilution of extract showing inhibition of growth was considered as MIC. Results: In comparison to all extract, the methanolic extract of ginger showed the largest zone of inhibition (3.6 cm) against Escherichia coli. Similarly, ethanolic extract of clove was the most effective against E. coli with the zone of inhibition (3 cm). In aqueous extraction, clove showed higher antimicrobial activity against Staphylococcus aureus (1.9 cm) and cinnamon was effective against Klebsiella spp. (1.9 cm). Among all the extracts, aqueous extract generally exhibited lower antimicrobial activity. The MIC of ethanolic extract of clove against S. aureus, Bacillus spp., Klebsiella spp. and E. coli found to be the dilutions 1: 0.78, 1: 0.048, 1:0.39 and 1:1.56, respectively. Conclusion: This study concludes that clove and ginger extracts could be potential source of antimicrobials that could be used for treatment of bacterial wound infections.
Biofilm Formation, Inhibition and Multi Drug Resistance in Escherichia coli Isolated from Clinical Specimens
Objectives: This study aimed to determine the prevalence of biofilm formation, assess the inhibitiory action of EDTA on biofilm production and to asses the antimicrobial resistance profiles of Escherichia coli from clinical samples. Methods: A descriptive study was conducted at Microbiology Department of Modern Technical College, Sanepa, Lalitpur. A total of 300 E. coli isolated from different clinical specimens were included in the study for biofilm formation by Tissue Culture Plate (TCP) method and inhibition by EDTA solution. Antimicrobial susceptibility testing was performed using the Kirby-Bauer disc diffusion method on Mueller Hinton agar according to CLSI guidelines. Results: Among the 300 E. coli isolates, 130 (43.3%) were found to be biofilm producers. Biofilm-producing E. coli showed significantly (p < 0.05) higher resistance to several antibiotics, including Amoxicillin (98.5%), Cefalexin (91.5%), Co-trimoxazole (87.7%), Cefixime (86.2%), Ceftriaxone (83.1%), Ciprofloxacin (73.8%), and Ofloxacin (73.8%). However, lesser drug resistance was observed among non-biofilm-producing E. coli. EDTA at concentrations of 5 mM and 10 mM greatly inhibited and reduced biofilm formation. Conclusion: Among the isolated E. coli, 43.3 % were biofilm producer and those strains exhibited higher antimicrobial resistance compared to non-biofilm producers. EDTA was found to be effective in inhibiting biofilm formation. Hence, EDTA can act as antibiofilm agent to control and minimize biofilm asssociated bacterial infections.
Antibiogram of ESBL-Producing Klebsiella Species and Pseudomonas aeruginosa from Clinical Specimens
Objectives: The study aims to determine the antibiotic susceptibility pattern of extended spectrum beta-lactamase (ESBL)-producing isolates of Klebsiella and Pseudomonas from clinical specimens. Methods: A total of 230 samples from different bodily fluids (urine, sputum, drain, pus, BAL, CSF, blood, and ascitic fluid) of patients were collected from various wards of Alka Hospital Private Limited, Lalitpur, Nepal. The samples were analyzed in the microbiological laboratory as per the CLSI guidelines. The Kirby-Bauer disc diffusion technique.was used to perform antibiotic susceptibility testing. Results: Thirty-four (14.78%) samples showed culture positivity. Urine had the highest culture positivity; the patients of the age group 90-100 years were 66.67% culture positive. 100% of the samples from the SH/C ward were culture-positive. Of 34 isolates, 88.24% were ESBL producers, including 61.76% K. pneumoniae, 11.76% K. oxytoca, and 26.47% P. aeruginosa. MDR was observed in 96.3% of ESBL producers. Amikacin, Gentamicin, and Piperacillin-tazobactam were the most effective against ESBL-producing P. aeruginosa. Colistin and Polymyxin-B were the most effective antibiotics against Klebsiella species. Conclusion: The study reports ESBL production significantly affected MDR development in selected bacterial specimens.
Antibacterial Activity of Locally Available Spices against Common Pathogenic Bacteria
Objectives: The objective of this study was to determine the antibacterial activity of spices against common pathogenic bacteria and Minimum Inhibitory Concentration (MIC) of spices extract. Methods: Dried buds of Syzygium aromaticum, bulb of Allium sativum, root of Allium sativum and rhizome of Curcuma longa were collected from different locality in Kathmand, Nepal. They were dried, crushed to powder form and mixed with adequate ethanol and methanol to get the extract respectively. After processing, the extracts were tested against Bacillus spp, Klebsiella pneumoniae, S. aureus and Escherichia coli by disc diffusion on Muellar Hinton Agar plate. Minimum Inhibitory Concentration (MIC) was determined by dilution technique. Results: All the extract of spices were found effective against Bacillus spp, K. pneumoniae, S. aureus and E. coli. However, the ethanolic extract of clove was found to be highly effective against E. coli with Zone of Inhibition (ZOI) of 28 mm. Similarly, the methanolic extract of clove was found to be highly effective against K. pneumoniae (ZOI= 22 mm). The MIC against S. aureus, Bacillus spp, K. pneumoniae and E. coli, of the ethanolic extract of clove was found to be 1:32, 1:8, 1:4 and 1:16 and methanolic extract was found to be 1:64, 1:16, 1:8 and 1:4, respectively. Conclusion: Clove was found to be the most effective spice against common pathogenic bacteria. Ehanolic extract was found to be more effective compared to methanolic suggesting ethanol as a suitable solvent. Further study on its antibacterial characteristics may be required to update its medicinal value.