Tribhuvan University Journal of Microbiology

Prevalence of Pangasius Fish Disease in Nepal

Richa Chaudhary, Sabina Puri, Shashi Bhushan Chaturwedi — Sun, 31 Dec 2023 00:00:00 +0000

Objectives: This study was conducted to identify bacterial pathogens in both symptomatic and asymptomatic enteric septicemic infection of Pangasiaus hypophthalmus and evaluate their susceptibility to antibiotics.

Methods: The totals of 120 kidney and liver swab samples of fish were collected from different places of Nepal. The samples were processed to identify the causative agent using pure culture of bacteria through streak plate techniques and different biochemical tests. To assess the susceptibility of the isolated bacteria to antibiotics Ciprofloxacin, Tetracycline, Doxycycline, Gentamycin, Ampicillin/ sulbactam, and Tobramycin were used.

Results: The estimated prevalence of pathogenic bacteria is 27 per 10000 populations. Three pathogenic bacteria i.e. Edwardsiella ictaluri 57.7% (56/97), Aeromonas hydrophila 24.7% (24/97), and Pseudomonas fluorescence 17.5% (17/97) were identified in the studied asymptomatic and symptomatic Pangas fish disease. E. ictaluri was major isolates which was causative agent for the disease, enteric septicemia of catfish. Among the total bacterial isolates 100% bacterial isolates were sensitive to Ciprofloxacin and 84% to Gentamycin. Whereas 100% were resistance to Ampicillin and 84% to Tetracycline. And Tobramycin (60.8%) and Doxycycline (64.9%) showed intermediate result.

Conclusion: Ciprofloxacin and Gentamycin could be administered to control enteric septicemia of catfish in Pangas. This study provides information to strategies to reduce economic losses to farmer and reduce possible health hazards.

Antimicrobial Activity of Traditional Medicinal Plants Available at Banepa and Bhaktapur against Uropathogens

Sun, 31 Dec 2023 00:00:00 +0000

Objectives: The study was aimed to determine the antimicrobial activity of traditional medicinal plants against the uropathogens.

Methods: Overall, 360 urine samples were collected from both outpatient and inpatient for culture and antimicrobial susceptibility testing. All the isolates were processed and identified following standard microbiological procedure and subjected to antibiotic susceptibility testing at Microbiology laboratory of Shree Birendra Hospital following CLSI guidelines. All the three plant extracts were processed by agar well diffusion method and Tube dilution method for antimicrobial activity against Escherichia coli, Klebsiella. pneumoniae, Pseudomonas aeruginosa and Enterobacter spp. at Microbiology laboratory of Sainik Awasiya Mahavidhyalaya following standard laboratory techniques.

Results: Crude extract of plants viz. Centella asiatica, Cuscuta reflexa and Mentha spicata showed good antimicrobial properties against all clinical isolates. Among all plants, ethanolic extract of C. asiatica was found to be most effective against E. coli with zone of inhibition 16 mm and minimum inhibitory concentration (MIC) value 5 mg/ml. Acetone extract of C. reflexa showed good antimicrobial activity against K. pneumoniae with zone of inhibition 14 mm and MIC value 10 mg/ml.

Conclusion: Our research revealed that the crude plant extracts, particularly the acetone and ethanol extracts, had a considerable amount of efficacy against uropathogens. Based on the study results, these traditionally used medicinal plants can overcome the problems of infections caused by multidrug resistant bacteria.

Anti-Bacterial Activity of Different Honey Samples against Bacteria Isolated from Clinical Sources

Pramisha Luitel, Nishaka Luitel, Neha Thapa, Anup Muni Bajracharya — Sun, 31 Dec 2023 00:00:00 +0000

Objectives: To evaluate the antibacterial activities of different honey samples against clinically isolated bacteria and also to compare the antibacterial activity of honey with standard antibiotics

Methods: A study was conducted to evaluate the potent attributes of eight distinct types of honey against five pathogenic bacteria which were collected from Bharatpur Hospital of Chitwan. The susceptibility of isolates to honey was evaluated using the Agar well diffusion method. In addition, the Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of the effective honey samples were determined using tube macro-dilution method. MIC represents the lowest concentration at which visible inhibition of bacterial growth occurs, offering insights into the honey’s inhibitory potential. Furthermore, MBC, determined by sub-culturing the non-turbid wells from the MIC assay, elucidates the minimal concentration required for complete bactericidal activity.

Results: Among the eight types of honey processed, Rudilo honey exhibited remarkable effectivity against Escherichia coli and Staphylococcus aureus, with zone of inhibition of 39 mm and 36 mm, respectively. Conversely, Manuka and Chiuri displayed heightened efficacy against Klebsiella pneumoniae with zone of inhibition 34 mm) and Proteus vulgaris (38 mm). Among the tested bacteria, Pseudomonas aeruginosa demonstrated notable resistance to all honey samples except Rudilo, Manuka, and Multiflora. Furthermore, Manuka and Rudilo exhibited the lowest MIC (6.25% v/v) against Proteus vulgaris and Staphylococcus aureus, while Rudilo displayed the lowest MBC (25% v/v) against the same pathogens. However, Chiuri presented the highest MIC and MBC against the tested bacteria. It was found that honey samples showed a greater zone of inhibition than antibiotics used against Escherichia coli, Proteus vulgaris, Klebsiella pneumoniae, and Staphylococcus aureus but for Pseudomonas aeruginosa antibiotics were found to be more effective than sampled honey.

Conclusion: The study revealed that honey exhibited antibacterial properties even at its minimum inhibitory concentration (MIC), showcasing effectiveness against infections caused by Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae and Proteus vulgaris. However, its efficacy in treating infections attributed to Pseudomonas aeruginosa might be limited.

Screening and Evaluation of Antimicrobial Activity of Medicinal Plants Collected from Lalitpur District

Rojina Tandukar, Sonu Maharjan, Gita Ghimire, Amrit Acharya — Sun, 31 Dec 2023 00:00:00 +0000

Objectives: To screen and evaluate antimicrobial activity of crude ethanol extracts of medicinal plants against various bacteria.

Methods: The study was conducted from February - August 2018 in Pinnacle college, Lalitpur with plan to collect Seven different herbs species; Acorus calamus (Bojho), Aloe vera (Ghiu Kumari), Artermisia indica (Titepate), Azadirachta indica (Neem), Mentha arvensis (Pudhina), Zanthoxylum armatum (Timur) and Zingiber officinale (Ginger) were subjected to 70% ethanol soxhlet extraction, then extract then heated at 78.37°C to remove ethanol, working solution was prepared in DMSO. Test organisms included mainly enteric isolates i.e. Escherichia coli, Pseudomonas aeruginosa, Klebsiella oxytoca, Salmonella Typhi and Staphylococcus aureus were selected. The Antibiotic Susceptibility tests of isolates were done by of Modified Kirby Bauer disc diffusion method. The antimicrobial activities of the extracts were determined by Agar well diffusion technique. Minimum Bactericidal Concentration (MBC) was determined by dilution technique.

Results: Among 7 plants that were tested, 6 plants were found to have activity against test bacteria. Acorus calamus 3/5(60%) was effective against test bacteria. Azadirachta indica and Menthe arvensis inhibited 2/5 (40%). Aloevera and Artermisia indica were effective against S aureus only 1/5(20%) and Zingiber officinale had no antibacterial effects over any tested bacteria. S. aureus was the most susceptible Gram positive bacteria meanwhile K. oxytoca stood among Gram negative, emerged as the most resistant species. S. aureus showed ZOI with 6 plant extracts excluding ginger P. aeruginosa was inhibited by Acorus calamus, Azadirachta indica and Mentha arvensis. The largest ZOI of 15 mm was obtained with Zanthoxylum armatum acting upon S. aureus. While, the smallest diameter of 8 mm was showed by Acorus calamus against S. Typhi. The lowest MBC was given 15.63 mg/ml by Mentha arvensis and Azadirachta indica against Pseudomonas aeruginosa while, Artermisia indica against S. aureus.

Conclusion: Medicinal plants should be possible medication in the future to combat pathogens.

Incidence of Coagulase Negative Staphylococci in Various Clinical Samples in Tertiary Care Hospital, Nepal

Pradeep Kumar Shah, Niru Bhandari, Rajendra D. Joshi — Sun, 31 Dec 2023 00:00:00 +0000

Objectives: To determine incidence of coagulase negative Staphylococci (CoNS) in various clinical samples along with the study of their multi drug resistance, methicillin resistance and biofilm formation.

Methods: A hospital based cross-sectional study was conducted which included 1875 clinical samples. The isolation and identification of isolates was done following standard microbiological protocol. The CoNS were identified phenotypically on the basis of gram staining, slide and tube coagulase test and through various carbohydrates fermentation tests. Antibiotic susceptibility test was done by Kirby Bauer disk diffusion method (Clinical and Laboratory Standards Institute 2020) whereas biofilm production was determined by Tissue Culture Plate (TCP) technique.

Results: A total of 32 CoNS, comprising of 6 species were identified. S. epidermidis (43.8%) was the most common species isolated followed by S. saprophyticus (28.1%), S. haemolyticus (15.6%), S. hominis (6.3%), S. lugdunensis (3.1%) and S. cohini (3.1%). Further, 27 (84.4%) of CoNS were found to be multidrug resistant, 22 (68.8%) methicillin resistant and 8 (25%) showed positive D- test. Strong biofilm production was detected in 9 (28.1%) isolates of CoNS, 10 (31.3%) were moderate biofilm producers and 13 (40.6%) non/weak biofilm producers. The equal distribution, 9 (33.3%) each of strong, moderate and non/weak biofilm producers were found among 27 isolates of MDR. Among 22 methicillin resistant isolates, 9 (40.9%), 7 (31.8%) and 6 (27.3%) were strong, moderate and non/ weak biofilm producers respectively. All isolates were sensitive against Linezolid followed by Cotrimoxazole.

Conclusion: The increasing multi drug resistance among CoNS should be rationally approached with the use of proper antibiotics while treating the patients.

Biofilm Production and Antibiotic Resistance in Clinical Isolates of Acinetobacter Species

Sun, 31 Dec 2023 00:00:00 +0000

Objective: To study the occurrence of the biofilm producing Acinetobacter spp. from different clinical specimens and to assess the antibiotic susceptibility pattern of Acinetobacter species.

Methods: This study was conducted at B&B Hospital Pvt. Ltd., Lalitpur, Nepal from February to September 2018. Various specimens including pus, sputum, urine, catheter tips, body fluids (bile, peritoneal fluid, CSF), suction tube, and blood were collected from the patients (n=5141) visiting B&B hospital. The bacterial isolates were subjected to antibiotic susceptibility testing and Acinetobacter spp. isolates were subjected to biofilm detection by microtiter plate method.

Results: Out of 5141 specimens, 1179 (23%) were culture positive. Escherichia coli (40.8%) was found to be the predominant organism. A total of 83 isolates of Acinetobacter spp. were isolated among which 76(91.57%) were biofilm producers. Biofilm producing isolates of Acinetobacter spp. were found more resistant to the tested antibiotics than non- biofilm producing Acinetobacter spp.

Conclusion: Most Acinetobacter spp. was capable of producing the biofilm. The biofilm producers were more resistant to the antibiotics under study which help to increase the resistivity nature of the bacteria. All of the isolates susceptible to colistin showed that the appropriate therapeutic option for infection caused by biofilm forming Acinetobacter spp.

Molecular Detection of mecA and aac(6’)-aph(2”) Genes in Staphylococcus aureus Isolates from Clinical Specimens

Sun, 31 Dec 2023 00:00:00 +0000

Objectives: The main aim of this study is to assess the prevalence of Staphylococcus aureus in clinical samples and its antimicrobial susceptibility pattern based on the detection of mecA and aac(6’)-aph(2”) genes conferring resistant ability to S. aureus isolates.

Methods: A prospective hospital based cross-sectional study was conducted among the patients visiting Kirtipur hospital for 6 months from May 2020 to November 2020. The clinical samples were collected and processed for culture and identified following standard microbiological procedures. Antibiotic sensitivity testing was performed by Kirby Bauer disc diffusion method and methicillin resistance was determined by using cefoxitin (30 μg) disc and interpreted as per CLSI guidelines. The chromosomal and plasmid DNA were extracted and aac(6’)-aph(2”) and mecA genes were detected by conventional polymerase chain reaction.

Results: Out of 1969 clinical samples, the overall prevalence of S. aureus was 9.82% and among the staphylococci, 50(75.8%) were MRSA. Male patients from 31-45 age group and those visiting OPD were more prone to MRSA infection, although only patient’s status and age group were statistically significant. Most of the MRSA were recovered from wound swab (78.2%). A majority of the MRSA isolates were resistant to Ciprofloxacin (90%). Similarly, resistance to Gentamicin was observed in only 12(24%) isolates. Resistance to Gentamicin was statistically associated with types of samples but not with gender, age group and patient status. Among the 50 MRSA isolates, 10 were identified as co-harboring both the aac(6’)-aph(2”) and mecA genes. Notably, excluding only 2 MRSA isolates, the remaining 48 isolates demonstrated the presence of the mecA gene.

Conclusion: High prevalence of MRSA in this study underscores the need for more commitment towards infection control measures that meet the standard protocols aimed at reducing the spread of infection by MRSA among susceptible individuals.

Recovery of Methicillin Resistant Staphylococci from Public Vehicles in Kathmandu

Aditi Chalise, Charu Arjyal — Sun, 31 Dec 2023 00:00:00 +0000

Objectives: The purpose of this study was to determine the extent of staphylococcal contamination in public vehicles in Kathmandu and to determine the antibiotic susceptibility pattern of the isolates.

Methods: This cross-sectional study was conducted from December 2022 to May 2023 in the Microbiological laboratory of Padma Kanya Multiple Campus, Kathmandu. A total of 120 swab (sterile cotton swab moistened with sterile normal saline) samples were collected by gently rubbing the surfaces from 19 different micro-buses during the time of heavy passenger flow (office time) and transported to the laboratory maintaining a cold chain. Staphylococcus aureus and Coagulase negative Staphylococci (CoNS) were identified by their characteristic yellow pigmented colonies on Mannitol Salt Agar (MSA) and the ability of S. aureus to produce coagulase enzyme. Kirby-Bauer disc diffusion method was employed to test the antibiotic susceptibility pattern of isolates. MRSA were identified on the basis of the zone of inhibition (<21 mm diameter) against Cefoxitin 30mcg disc.

Results: Out of 41 Staphylococcal isolates, 46.36% were S. aureus, among which 84.21% exhibited methicillin resistance (MRSA). All Coagulase Negative Staphylococci (CoNS) isolates (n=22) were resistant to methicillin (MRCoNS). MRSA and MRCoNS isolates were also resistant to antibiotics like Penicillin and Erythromycin.

Conclusion: This study reports relatively high occurrence of MRSA in environmental samples, predominantly in areas frequently touched by people indicating the possibility of easy spread of these organisms and subsequent opportunistic infections.

Phylogeny of a Mosquitocidal Bacillus thuringiensis var israelensis

Ganga GC, Megha Raj Banjara, Ishan Gautam, Prakash Ghimire, Komal Raj Rijal — Sun, 31 Dec 2023 00:00:00 +0000

Objectives: The study aims to compare the 16srRNA sequence of Bacillus thuringiensis var israelensis available in the National Center for Biotechnology Information, database, the sequence length, the relatedness, identity, and ancestral property by constructing a phylogenetic tree and by basic local alignment tool.

Methods: The 16srRNA sequence of all the Bacillus thuringiensis var israelensis was retrieved from the database. A basic local alignment tool was used to study the description of the sequences, the percent identity, graphical summary, and alignment. Mega software was used to draw the phylogenetic tree.

Results: The total number of mosquitocidal 16srRNA gene sequences was 19. One sequence VCRC-B17 strain was the complete genomic sequence deposited other 18 hits were partial sequences. The Expected value, percent identity, maximum, and total score of 18 hits shows a significant match with the query sequence VCRC-B17. The graphic summary result displays that the 18 hits align with the query sequence as they are coded with the highest score coding color bar, red with a ≥ 200 alignment score. The phylogenetic tree did not support the monophyletic status indicating there is a diversity in the strain.

Conclusion: The 19 sequences deposited in the database are identified as a mosquito larvicidal Bacillus thuringiensis var israelensis based on the alignment score or match even though they do not possess an equal base pair length with the query sequence.

Detection of Mycobacterium tuberculosis by GeneXpert in Smear Negative Presumptive Case of Pulmonary Tuberculosis Patients

Sun, 31 Dec 2023 00:00:00 +0000

Objectives: This study aimed to determine the efficacy of GeneXpert to identify smear-negative pulmonary tuberculosis patients

Methods: A cross-sectional descriptive study was carried out among 166 outpatients who visited the laboratory Unit of Jalal-Abad Tuberculosis Centre located in Kyrgyzstan. Non-probability; convenient sampling type was used to collect the data for the study. Standard Procedure that was described by the operating manual (Cepheid Inc, Sunny Vale USA) of GeneXpert machine was used for the procedure.

Results: Out of 166 smear negative cases, 45 (27.1%) cases were shown to be positive by GeneXpert. Among the 45 cases, the GeneXpert analysis revealed that 60% were sensitive towards rifampicin and only 24.4% were resistance, remaining were intermediate.

Conclusion: This research shows that Xpert MTB/RIF can identify TB in patients with negative smear tests with a good detection rate, and results would be available the same day, avoiding patient loss and treatment delays.

blaCTX-M Mediated Extended Spectrum Beta-lactamase Production in Escherichia coli from Urine Samples

Renisha Oli, Sanjit Shrestha, Bigu Kumar Chaudhary, Megha Raj Banjara — Sun, 31 Dec 2023 00:00:00 +0000

Objective: To assess the antimicrobial pattern of Escherichia coli isolates from urine samples, determine the proportion of multidrug resistant (MDR) and extended spectrum beta-lactamase (ESBL) and bla_CTX-Mgene of E. coli from the urine samples.

Methods: A total of 2452 urine samples were collected and processed from April to October 2023 in clinically suspected urinary tract infection (UTI) patients attending B&B Hospital Pvt. Ltd to identify possible bacteria using standard microbiological procedures. E coli isolates were further subjected to antimicrobial susceptibility testing using Kirby Bauer disk diffusion method. Combination disk method was used to identify the ESBL phenotypes. Molecular detection of bla_CTX-Mgene was performed using conventional polymerase chain reaction.

Results: A total of 338 (13.7%) samples showed significant bacterial growth. Maximum bacterial growth was found in outpatients (70.4%) and in patients above 60 years of age. Gram negative bacteria (87.8%) were predominant and E. coli (54.4%) was the most frequent. Nitrofurantoin, Aminoglycosides, Carbapenems and Polymixins were effective antibiotics. Among 184 E. coli isolates, 54 (29%) were MDR, 30 (16.3%) were ESBL producer. bla_CTX-Mgene was detected in 90% of the phenotypically confirmed ESBL producers.

Conclusion: E. coli is the major cause of UTI and bla_CTX-Mis a prime contributor of ESBL production. High rate of resistance to third-generation Cephalosporins, Flouroquinolones and Cotrimoxazole make these antibiotics unsuitable for the treatment of UTI.

Antimicrobial Resistance in Escherichia coli and other Coliform Bacteria Isolated from Bagmati River

Sun, 31 Dec 2023 00:00:00 +0000

Objectives: To explore the presence of fecal indicator bacteria and assess antibiotic resistance status in Bagmati river water.

Methods: In a cross sectional study during a year 2020; a total of 180 water samples were collected from the Bagmati River’s upstream, midstream, and downstream sources. Maintaining reverse cold chain, the samples were transferred to the laboratory of central department of Microbiology, Tribhuvan University. Organisms were isolated on Eosin Methylene Blue (EMB) and Nutrient Agar. The organisms were further identified based on the biochemical properties and antibiotic susceptibility testing was performed following CLSI (2020) guidelines.

Results: Of the 546 isolates, 209 (or 38%) were Escherichia coli. Other coliforms isolated were Enterobacter spp (2%), Citrobacter spp (37%), and Klebsiella spp (23%). Upstream source revealed least percentage 3% (7/209) of E. coli. All the recovered Citrobacter spp. were resistant and E. coli showed >99% resistance towards Tetracycline, Ampicillin and Amoxiclav antibiotics. Klebsiella spp. was 100% resistant towards Ampicillin and Amoxiclav antibiotics. The coliforms exhibited least resistance (10%) towards Chloramphenicol. Based on antibiotic resistance percentage pattern, E. coli showed 27% similarity to Citrobacter spp.

Conclusion: Coliforms showed maximum resistance towards first line antibiotics prescribed in human infection. Immediate water safety plans should be instituted to improve the water quality.

Microbiota Diversity Associated with Midgut and Salivary Gland of Aedes aegypti and Aedes albopictus

Sun, 31 Dec 2023 00:00:00 +0000

Objective: The gut and salivary gland contain diverse micro biota and play an important role in vector competence and disease transmission. In light of this, this study aimed to screen the salivary gland and midgut microbiota associated with Aedes aegypti and Aedes albopictus from Kathmandu and Lalitpur districts of Nepal.

Methods: An analytical cross-sectional study was conducted from April 2017 to October 2017 in Kathmandu and Lalitpur districts of Nepal. The fi eld collected mosquitoes larvae were reared in the laboratory until the adult emergence and identified morphologically using standard key. The dissected salivary gland and gut samples were homogenized, suspended in phosphate buffered saline and inoculated in the culture media for bacterial growth which were further identified.

Results: Pseudomonas aeruginosa was predominant bacteria in the gut and salivary gland of Ae. aegypti. Similarly, in Ae. albopictus, Serratia marcescens was predominant in gut while, Acinetobacter spp. was predominant in salivary gland. Simpsons diversity index (D), Shannon weaver diversity index (H) and Evenness (E) were found to be the highest viz, 0.81, 1.83 and 0.88 in the gut of Ae. aegypti.

Conclusion: This study had provided a comprehensive overview of the bacterial population in the gut and salivary gland of Aedes aegypti and Aedes albopictus. It was found that the most bacterial genera were common to both vectors, although some variation was found in gut and salivary gland. This distribution suggests that there are no host-specific bacterial genera.

Detection of Antibiotic-Resistant E. coli from Ponds of Janakpurdham, Nepal

Raj Kumar Sah, Kavita Shah, Ranjit Kumar Sah — Sun, 31 Dec 2023 00:00:00 +0000

Objectives: The purpose of the study was to determine the extent of antibiotic-resistant E. coli in various sacred ponds of Janakpurdham.

Methods: A cross-sectional study was conducted and 16 samples were processed from 16 different revered ponds around Janakpurdham. Isolation of E. coli 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 16 E. coli isolates, one from each sampling site, were analysed for their antibiotic-resistant pattern against the eight most commonly used antibiotics. From the total samples, 100% (16) were sensitive to tetracycline, Ciprofloxacin, Amikacin, and Gentamicin while 88% (14), 25% (4), 62% (10), and 44% (7) were resistant to Ampicillin, Amoxycillin/Clavulanate, Cefotaxime and Ceftazidime respectively. Two isolates had the lowest estimated multiple antibiotic resistance index (0.125). In addition, 10 isolates showed indices ranging from 0.25 to 0.375. Two isolates showed the highest indices i.e., 0.5.

Conclusion: The holy ponds of Janakpurdham were found to be highly contaminated with pathogenic bacteria. The results may indicate the origin of E. coli isolates from an area of high antibiotic use.

Advanced Molecular Techniques and Diagnosis of Febrile Illness

Komal Raj Rijal, Upendra Thapa Shrestha — Sun, 31 Dec 2023 00:00:00 +0000

No abstract available.