Detection of Methicillin Resistant Staphylococcus aureus and Determination of Minimum Inhibitory Concentration of Vancomycin for Staphylococcus aureus Isolated from Pus/Wound Swab Samples of the Patients Attending a Tertiary Care Hospital in Kathmandu, Ne

The present study was conducted to evaluate the performance of cefoxitin disc diffusion method and oxacillin broth microdilution method for detection of methicillin resistant S. aureus (MRSA), taking presence of mecA gene as reference. In addition, inducible clindamycin resistance and beta-lactamase production were studied and minimum inhibitory concentration (MIC) of vancomycin for S. aureus isolates was determined. A total of 711 nonrepeated pus/wound swab samples from different anatomic locations were included in the study. The Staphylococcus aureus was identified on the basis of colony morphology, Gram's stain, and biochemical tests. A total of 110 (15.47%) S. aureus isolates were recovered, of which 39 (35.50%) isolates were identified as MRSA by cefoxitin disc diffusion method. By oxacillin broth microdilution method, 31.82% of the Staphylococcus aureus isolates were found to be MRSA. However, mecA gene was present in only 29.1% of the isolates. Further, beta-lactamase production was observed in 71.82% of the isolates, while inducible clindamycin resistance was found in 10% of S. aureus isolates. The MIC value of vancomycin for S. aureus ranged from 0.016 g/mL to 1 g/mL. On the basis of the absolute sensitivity (100%), both phenotypic methods could be employed for routine diagnosis of MRSA in clinical microbiology laboratory; however cefoxitin disc diffusion could be preferred over MIC method considering time and labour factor

Isolation and characterization of antibacterial actinomycetes from soil samples

Seventy-nine Actinomycetes were isolated from soils of Kalapatthar (5545m), Mount Everest region. Twenty seven (34.18%) of the isolates showed an antibacterial activity against at least one test-bacteria among two Gram positive and nine Gram negative bacteria in primary screening by perpendicular streak method. Thirteen (48.15%) showed antibacterial activity in secondary screening. The result showed that three of the isolates, K.6.3, K.14.2, and K.58.5 were highly active with an inhibition zone e”20mm and broad spectrum antibacterial activity including two methicillin resistant Staphylococcus aureus (MRSA) strains. Minimum inhibitory concentration (MIC) of antibacterial metabolite

Drug susceptibility patterns of the Mycobacterium tuberculosis isolated from previously treated and new cases of pulmonary tuberculosis at German-Nepal tuberculosis project laboratory, Kathmandu, Nepal

Abstract Background Multidrug resistant tuberculosis (MDR-TB) is a serious public health problem in Nepal. It is a major obstacle for the control of the tuberculosis. The main objectives of this study were to determine the prevalence of the multidrug resistant pulmonary tuberculosis and to evaluate the drug susceptibility patterns of Mycobacterium tuberculosis isolated from previously treated and newly diagnosed cases of pulmonary tuberculosis. Methods A cross-sectional study was conducted from March 2013 to August 2013 at German-Nepal tuberculosis project (GENETUP) laboratory, Kathmandu, Nepal. For this the sputum samples from total of 153 (49 new and 104 previously treated) suspected pulmonary tuberculosis patients were used. The diagnosis of the tuberculosis was performed by using fluorescent microscopy and culture, while the drug susceptibility testing of Mycobacterium tuberculosis was performed by proportion method. Lowenstein-Jensen (L-J) medium was used for the culture of Mycobacterium tuberculosis and the colonies grown were identified on the basis of the colony morphology, pigment production and biochemical characteristics. Results The prevalence of MDR-TB among all the cases of culture positive pulmonary tuberculosis was 15.6 %. The rate of MDR-TB among previously treated culture positive tuberculosis patients was 19.4 % and that among newly diagnosed culture positive pulmonary tuberculosis cases was 7.1 %. The highest rate of resistance of Mycobacterium tuberculosis, was toward streptomycin (24.4 %) followed by isoniazid (23 %), rifampicin (17.8 %) and ethambutol (15.6 %). Among the total of MDR-TB cases among previously treated patients, highest percentage of the cases were relapse (61.1 %) followed by chronic (16.7 %). Conclusions The high prevalence of DR/MDR-TB in our study reflects poor implementation of tuberculosis control program. On the basis of the drug susceptibility patterns of M. tuberculosis we found in our study, we recommend to include ethambutol instead of streptomycin in the multidrug therapy for the treatment of tuberculosis patients in Nepal. Further, due to high rate of MDR-TB among previously treated patients, we do not recommend to use first line drugs for the treatment of pulmonary tuberculosis among previously treated patients

Detection of TEM and CTX-M Genes in Escherichia coli Isolated from Clinical Specimens at Tertiary Care Heart Hospital, Kathmandu, Nepal

Background: Antimicrobial resistance (AMR) among Gram-negative pathogens, predominantly ESBL-producing clinical isolates, are increasing worldwide. The main aim of this study was to determine the prevalence of ESBL-producing clinical isolates, their antibiogram, and the frequency of ESBL genes (blaTEM and blaCTX-M) in the clinical samples from patients. Methods: A total of 1065 clinical specimens from patients suspected of heart infections were collected between February and August 2019. Bacterial isolates were identified on colony morphology and biochemical properties. Thus, obtained clinical isolates were screened for antimicrobial susceptibility testing (AST) using modified Kirby–Bauer disk diffusion method, while ESBL producers were identified by using a combination disk diffusion method. ESBL positive isolates were further assessed using conventional polymerase chain reaction (PCR) to detect the ESBL genes blaTEM and blaCTX-M. Results: Out of 1065 clinical specimens, 17.8% (190/1065) showed bacterial growth. Among 190 bacterial isolates, 57.4% (109/190) were Gram-negative bacteria. Among 109 Gram-negative bacteria, 40.3% (44/109) were E. coli, and 30.2% (33/109) were K. pneumoniae. In AST, 57.7% (n = 63) Gram-negative bacterial isolates were resistant to ampicillin and 47.7% (n = 52) were resistant to nalidixic acid. Over half of the isolates (51.3%; 56/109) were multidrug resistant (MDR). Of 44 E. coli, 27.3% (12/44) were ESBL producers. Among ESBL producer E. coli isolates, 58.4% (7/12) tested positive for the blaCTX-M gene and 41.6% (5/12) tested positive for the blaTEM gene. Conclusion: Half of the Gram-negative bacteria in our study were MDR. Routine identification of an infectious agent followed by AST is critical to optimize the treatment and prevent antimicrobial resistance

Re-emergence of the susceptibility of the Salmonella spp. isolated from blood samples to conventional first line antibiotics

Abstract Background Enteric fever is an important public health problem in Nepal. Due to emergence of multidrug resistant strains of Salmonella spp. the conventional first-line drugs, ampicillin, chloramphenicol, and cotrimoxazole have not been used as empiric therapy for treatment of enteric fever for last two decades and there have been increased uses of fluoroquinolones as the drugs of choice. The aim of this study was to evaluate and analyze the antimicrobial susceptibility patterns of Salmonella spp. Methods A total of 620 blood samples collected from the patients suspected of suffering from enteric fever were cultured using standard microbiological techniques. Antibiotic susceptibility testing of the Salmonella spp., was performed by Kirby Bauer disc diffusion technique following Clinical and Laboratory Standard Institute (CLSI) guidelines. Minimum inhibitory concentrations of ciprofloxacin, ofloxacin and nalidixic acid were determined by agar dilution method. Results Of the total 83 Salmonella spp., 48 (57.83 %) were S. Typhi and 35 (42.26 %) were S. Paratyphi A. Among 83 Salmonella isolates, 98.8 % of the Salmonella spp. were susceptible to chloramphenicol and co-trimoxazole and about 97.6 % of the isolates were susceptible to ampicillin. Similarly, 69 (83.13 %) isolates were resistant to nalidixic acid. Only 16.9 % of the isolates were susceptible to ciprofloxacin. One S. Typhi isolate was multidrug resistant. Conclusion The present study revealed the decreased susceptibility of the S. Typhi and S. Paratyphi A to fluoroquinolones, proving them to be inappropriate for empirical therapy for the treatment of enteric fever in our setting. Further the higher susceptibility of the isolates to first line drugs, ampicillin, chloramphenicol, and cotrimoxazole suggests the possibility of using these drugs for empirical therapy

Antimicrobial Susceptibility Pattern of Salmonella spp. Isolated from Enteric Fever Patients in Nepal

Introduction: Enteric fever, a systemic infection caused by Salmonella enterica Typhi and S. enterica Paratyphi is one of the most common infections in developing countries such as Nepal. Aside from irrational practices of antibiotic use, mutations in chromosomal genes encoding DNA gyrase and Topoisomerase IV and by plasmid mediated quinolone resistant (PMQR) genes are suggested mechanisms for the development of resistance to nalidixic acid and reduced susceptibility to ciprofloxacin. Regardless of high endemicity of enteric fever in Nepal, there is paucity of studies on prevalence and drug-resistance of the pathogen. Therefore, this study aimed to assess the antibiotic susceptibility pattern of Salmonella isolates and determine the minimum inhibitory concentration of ciprofloxacin. Methods: A total of 1298 blood samples were obtained from patients with suspected enteric fever, attending Sukraraj Tropical and Infectious Disease Hospital (STIDH) during March–August, 2019. Blood samples were inoculated immediately into BACTEC culture bottles and further processed for isolation and identification of Salmonella Typhi and S. Paratyphi. Axenic cultures of the isolates were further subjected to antimicrobial susceptibility testing (AST) by using the modified Kirby–Bauer disc diffusion method based on the guidelines by CLSI. The minimum inhibitory concentration (MIC) of ciprofloxacin was determined by agar-dilution method. Results: Out of 1298 blood cultures, 40 (3.1%) were positive for Salmonella spp. among which 29 (72.5%) isolates were S. Typhi and 11 (27.5%) isolates were S. Paratyphi A. In AST, 12.5% (5/40), 15% (6/40) and 20% (8/40) of the Salmonella isolates were susceptible to nalidixic acid, ofloxacin and levofloxacin, respectively, whereas none of the isolates were susceptible to ciprofloxacin. The MIC value for ciprofloxacin ranged from 0.06-16 µg/mL in which, respectively, 5% (2/40) and 52.5% (21/40) of the isolates were susceptible and resistant to ciprofloxacin. None of the isolates showed multidrug-resistance (MDR) in this study. Conclusion: This study showed high prevalence of quinolone-resistant Salmonella spp., while there was marked re-emergence of susceptibilities to traditional first option drugs. Hence, conventional first-line-drugs and third-generation cephalosporins may find potential usage as the empirical drugs for enteric fever. Although our reporting was free of MDR strains, extensive surveillance, augmentation of diagnostic facilities and treatment protocol aided by AST report are recommended for addressing the escalating drug-resistance in the country

Genetic Fingerprinting of Bacillus thuringiensis Isolates by Randomly Amplified Polymorphic DNA Polymerase Chain

Random Amplified Polymorphic DNA (RAPD) is a method of producing a genetic fingerprint of a particular species without its prior genetic information. Relationship between species may be determined by comparing their unique fingerprint information. B. thuringiensis was isolated from soil samples of Khumbu base camp of Everest region, Nepal. Crystal protein (delta endotoxin) producing strains (46 from Phereche and 40 from Sagarmatha national park) were tested against a series of 100 decamer RAPD primers (codes 201-300, obtained from University of British Columbia) by RAPD PCR. Primer 284 was found the best among the tested primers and the reaction condition for PCR was optimized with a PCR buffer containing 10mM Tris HCl, 50 mM KCl, 3 mM MgCl 2 with pH 8.3.; 200ìm dNTPs each, 1U Taq polymerase, 40 pmol decamer primers, 20 ng template DNA and 1 % DMSO as a final concentrations in 25ìl reaction mixture. The thermal programme was programmed as initial denaturation temperature at 94 o C for 5 min followe

Detection of Methicillin Resistant Staphylococcus aureus and Determination of Minimum Inhibitory Concentration of Vancomycin for Staphylococcus aureus Isolated from Pus/Wound Swab Samples of the Patients Attending a Tertiary Care Hospital in Kathmandu, Nepal

The present study was conducted to evaluate the performance of cefoxitin disc diffusion method and oxacillin broth microdilution method for detection of methicillin resistant S. aureus (MRSA), taking presence of mecA gene as reference. In addition, inducible clindamycin resistance and beta-lactamase production were studied and minimum inhibitory concentration (MIC) of vancomycin for S. aureus isolates was determined. A total of 711 nonrepeated pus/wound swab samples from different anatomic locations were included in the study. The Staphylococcus aureus was identified on the basis of colony morphology, Gram’s stain, and biochemical tests. A total of 110 (15.47%) S. aureus isolates were recovered, of which 39 (35.50%) isolates were identified as MRSA by cefoxitin disc diffusion method. By oxacillin broth microdilution method, 31.82% of the Staphylococcus aureus isolates were found to be MRSA. However, mecA gene was present in only 29.1% of the isolates. Further, beta-lactamase production was observed in 71.82% of the isolates, while inducible clindamycin resistance was found in 10% of S. aureus isolates. The MIC value of vancomycin for S. aureus ranged from 0.016 μg/mL to 1 μg/mL. On the basis of the absolute sensitivity (100%), both phenotypic methods could be employed for routine diagnosis of MRSA in clinical microbiology laboratory; however cefoxitin disc diffusion could be preferred over MIC method considering time and labour factor