Chemotherapy/Antimicrobial Section/Original Paper

 Detection of NDM-1 and VIM Genes in Carbapenem-Resistant Klebsiella pneumoniae Isolates from a Tertiary Health-Care Center in Kathmandu, Nepal

 

Sabita Thapa^a Nabaraj Adhikari^b Anil Kumar Shah^c Ishworiya Lamichhane^a Binod Dhungel^b Upendra Thapa Shrestha^b Bipin Adhikari^d Megha Raj Banjara^b Prakash Ghimire^b Komal Raj Rijal^b

 

^aKantipur College of Medical Sciences, Kathmandu, Nepal;

^bCentral Department of Microbiology, Tribhuvan University, Kathmandu, Nepal;

^cAnnapurna Neurological Institute and Allied Sciences, Kathmandu, Nepal; 

^dNuffield Department of Medicine, Centre for Tropical Medicine and Global Health, University of Oxford, Oxford, UK

ABSTRACT

Background: Klebsiella pneumoniae is one of the leading causes of nosocomial infections. Carbapenems are used as the last resort for the treatment of multidrug resistant Gram-negative bacterial infections. In recent years, resistance to these lifesaving drugs has been increasingly reported due to the production of carbapenemase. The main objective of this study was to detect the carbapenem-resistant genes bla_NDM-1 and bla_VIM in K. pneumoniae isolated from different clinical specimens. Methods: A total of 585 clinical specimens (urine, pus, sputum, blood, catheter tips, and others) from human subjects attended at Annapurna Neurological Institute and Allied Sciences, Kathmandu were obtained in the period between July 2018 and January 2019. The specimens were isolated and identified for K. pneumoniae. All K. pneumoniae isolates were processed for antimicrobial susceptibility testing (AST) using the disk diffusion method. The isolates were further phenotypically confirmed for carbapenemase production by the modified Hodge test (MHT) using imipenem (10 μg) and meropenem (10 μg) discs. Thus, confirmed carbapenemase-producing isolates were further screened for the production of bla_NDM-1 and bla_VIM using conventional polymerase chain reaction (PCR). Results: Among the clinical isolates tested, culture positivity was 38.29% (224/585), and the prevalence of K. pneumoniae was 25.89% (58/224). On AST, K. pneumoniae exhibited resistance toward carbapenems including ertapenem, meropenem, and imipenem, while it showed the highest susceptibility rate against to tigecycline (93.1%; 54/58). Overall, AST detected 60.34% (35/58) carbapenem-resistant isolates, while the MHT phenotypically confirmed 51.72% (30/58) isolates as carbapenemase-producers and 48.28% (28/58) as carbapenemase nonproducers. On subsequent screening for resistant genes among carbapenemase-producers by PCR assay, 80% (24/30) and 3.33% (1/30) isolates were found to be positive for bla_NDM-1 and bla_VIM, respectively. In the same assay among 28 carbapenem nonproducing isolates, 9 (32.14%) isolates were positive for bla_NDM-1 gene while none of them were tested positive for bla_VIM gene. Conclusions: Molecular detection of resistant genes provides greater specificity and sensitivity than those with conventional techniques, thus aiding in accurate identification of antimicrobial resistance and clinical management of the disease

 

Keywords: Klebsiella pneumoniae, New Delhi metallo-betalactamase-1, Verona integron-encoded metallo-betalactamase, Carbapenem, Modified Hodge test, Multidrug resistance, Antibiotic susceptibility test

Citation: Thapa S, Adhikari N, Shah A, K, Lamichhane I, Dhungel B, Shrestha U, T, Adhikari B, Banjara M, R, Ghimire P, Rijal K, R: Detection of NDM-1 and VIM Genes in Carbapenem-Resistant <b><i>Klebsiella pneumoniae</i></b> Isolates from a Tertiary Health-Care Center in Kathmandu, Nepal. Chemotherapy 2021. doi: 10.1159/000518256

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Know about the Delta Variant from WHO’s Dr Maria Van Kerkhove

 DELTA VARIANT

Tribhuvan University Journal of Microbiology (TUJM)

Characterization of β-Galactosidase from Lactose Utilizing Yeast Isolated from the Dairy Sample

Bivek Dahal^1#, Sujan Karki^1#, Nabaraj Adhikari², Upendra Thapa Shrestha^{2, 3}*

¹ Universal Science College, Pokhara University, Baneshwor, Kathmandu

² Central Department of Microbiology, Tribhuvan University, Kirtipur, Kathmandu

³ Research Laboratory for Biotechnology and Biochemistry (RLABB), Kathmandu

 

^# Both the authors have equal contributions.

 

*Corresponding author: Upendra Thapa Shrestha, Central Department of Microbiology, Tribhuvan University, Kirtipur, Kathmandu. Email: upendra.thapashrestha@cdmi.tu.edu.np

 

ABSTRACT

 

Objectives: The objective of the study was to isolate lactose positive yeasts from dairy samples collected from local markets of Kathmandu, to extract crude β-galactosidase from the lactose positive yeast and to characterize the enzyme for optimum time duration, pH, temperature, Michaelis-Menten constant (Km) and maximum activity (Vmax).

Methods: Four lactose positive yeast strains were isolated from dairy samples collected from local market of Kathmandu by pour plate method. Single strain having maximum lactose positive activity was selected for the study. The mass culture of the lactose positive yeast strain was lysed by 2% Chloroform and the yeast cell lysate containing β-galactosidase (i.e. crude enzyme extract) was characterized by using ONPG (Ortho-Nitrophenyl-β-D-galactopyranoside) as substrate. ONPG is a colorless substrate for the enzyme assay which is hydrolyzed by the enzyme into yellow colored product ONP (Ortho-Nitrophenol). The concentration of product formed was monitored spectrophotometrically at 420 nm to determine the enzyme activity and to characterize the enzyme.

Results: The enzyme had wide range of working temperature from 0-50ºC, with optimal temperature of 37ºC. However, greater than 50% hydrolyzing ability was maintained in the range of 14-40ºC. Optimum time of reaction was 70 min. The enzyme had maximum activity in the near-neutral pH of 6.8. Michaelis-Menten constant of the enzyme was found to be 2.23 mM of ONPG and Vmax was 58.82 nmol/min/ml. Enzyme activity was 27.88 nmol/min/ml, Specific enzyme activity was 59.97 nmol/ min/mg and total enzyme activity was 3346.33 nmol/min.

Conclusion: The activity over a wide range of temperature 0-50ºC with low Km value shows that the enzyme has a commercial application in clearance of lactose pollution in wastewater in different environmental conditions.

 

Keywords: Yeast, β-galactosidase, enzyme activity, ONPG, Km

DOI: https://doi.org/10.3126/tujm.v7i0.33874

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