Molecular Detection of CTX-M Type ESBL Genes in Clinical Isolates of Klebsiella Species

 Molecular Detection of CTX-M Type ESBL Genes in Clinical Isolates of Klebsiella Species

Neesha Shrestha¹, Kuntala Shrestha¹, Upendra Thapa Shrestha², Komal Raj Rijal², Gayatri Karki³, Ishworiya Lamichhane¹, Kiran Sapkota⁴, Sanjib Adhikari² ,Shyam Prakash Dumre², Nabaraj Adhikari^2*

¹Kantipur College of Medical Science, Tribhuvan University, Sitapaila, Kathmandu, Nepal

²Central Department of Microbiology, Tribhuvan University

³Himal Hospital Pvt. Ltd., Thirbum Marg, Kathmandu

⁴Sam Houston State University, Huntsville, Texas, USA

^*Corresponding author: Nabaraj Adhikari, Assistant Professor, Central Department of Microbiology, TU, Email: nabaraj.adhikari@cdmi.tu.edu.np

 

ABSTRACT

Objective: The objective of this study is to determine the prevalence of Extended-Spectrum β-Lactamase (ESBL) production and CTX-M genes among Klebsiella species isolated from clinical specimens.

Methods: A total of 1,815 clinical samples—including urine, blood, sputum, pus, and body fluids were collected at Himal Hospital Kathmandu, during 2019–2020. Standard microbiological techniques were used for isolation and identification of bacterial pathogens. Antimicrobial susceptibility testing was performed using the modified Kirby–Bauer disk diffusion method following CLSI (2019) guidelines. ESBL screening was conducted using third-generation cephalosporins, and confirmation was done via the Double Disk Synergy Test (DDST). Molecular detection of the CTX-M gene was performed using PCR with specific primers targeting a 544 bp amplicon.

Results: Among 1,815 clinical samples, urine constituted the majority (65.8%), followed by blood (25.1%). Escherichia coli was the predominant isolate (89.1%), while Klebsiella pneumoniae (6.2%) and Klebsiella oxytoca (0.74%) comprised a smaller proportion. Of the 28 Klebsiella spp. isolates, the highest antibiotic sensitivity was observed toward Amikacin (60.7%) and Meropenem (57.1%), whereas complete resistance to Amoxicillin (100%) and high resistance to Cefixime (89.3%) and Cefotaxime (75.0%) were recorded. ESBL screening identified 22 (78.6%) potential ESBL producers, of which 18 (64.3%) were confirmed phenotypically. PCR analysis revealed the CTX-M gene in 7 of the 18 ESBL-positive isolates, demonstrating a notable presence of CTX-M–mediated resistance among Klebsiella spp.

Conclusion: The findings highlight a concerning prevalence of ESBL production and CTX-M genes in Klebsiella species in the study population, underscoring the need for continuous surveillance, rational antibiotic use, and strengthened antimicrobial stewardship programs to limit the spread of multidrug-resistant strains.

Keywords: Klebsiella spp., ESBL producer and CTX-M

 

Date of Submission: November 12, 2025     Date of Acceptance: December 22, 2025

Published Online: December, 2025               DOI: https://doi.org/10.3126/tujm.v12i1.88388

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Investigation of the blaNDM-1 and armA Genes in Carbapenem-Resistant Klebsiella pneumoniae Isolated from the Clinical Samples in Kathmandu, Nepal

Rohit Ghimire,¹ Subash Kumar Thakur,² Upendra Thapa Shrestha,¹* Komal Raj Rijal,¹ Prakash Ghimire,¹ and Megha Raj Banjara¹*

¹Central Department of Microbiology, Tribhuvan University, Kirtipur, Kathmandu, Nepal; 

²Paropakar Maternity and Women’s Hospital, Thapathali, Kathmandu, Nepal

Abstract

Klebsiella pneumoniae carbapenemase (KPC) and metallo-β-lactamase production are major causes of carbapenem resistance, whereas aminoglycoside resistance is caused by extrinsically acquired 16S-rRNA methyltransferase. K. pneumoniae coharboring resistance genes are serious health care issues that can cause multidrug resistance (MDR). This study aimed to describe the resistance genes (New Delhi metallo-beta-lactamase 1 [blaNDM-1] and armA) in the plasmids of K. pneumoniae from patients visiting the Paropakar Maternity and Women’s Hospital, Kathmandu, Nepal. Altogether, 8,017 clinical specimens were processed following standard microbiological procedures to identify K. pneumoniae. Antibiotic susceptibility testing and detection of phenotypic carbapenemase production in K. pneumoniae isolates were performed using the modified Kirby-Bauer disc diffusion method. The resistance genes were detected by conventional polymerase chain reaction. Of 8,017 clinical specimens, 6.8% (n = 545) had bacterial growth, and 70 were K. pneumoniae. Colistin (100%, n = 70) and imipenem (80%, n = 56) were the most effective antibiotics. Thirty percent (n = 21) of the isolates were MDR, whereas 66.7% (n = 14) were carbapenemase producers, among which 38.1% (n = 8) had a minimum inhibitory concentration of 64 mg/mL to imipenem. Among carbapenemase producers, 23.8% (n = 5) were KPC and 66.7% (n 5 14) were metallo-β-lactamase producers. Out of 21 MDR K. pneumoniae, 19.5% (n = 4) harbored the blaNDM-1 and armA genes, and 14.3% (n = 2) had both genes. Detection of the coexistence of the resistance genes from K. pneumoniae reveals that there might be increased antibiotic resistance, leading to multidrug resistance and an increased resistance to imipenem. In conclusion, advancing antimicrobial-resistance surveillance in maternity wards and minimizing the use of last-line antibiotics are crucial for safeguarding maternal and neonatal health.

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Evaluation of Antibiotic Resistance Patterns and Biofilm Formation among the Clinical Isolates of Pseudomonas aeruginosa

 Asuma Gurung^{1 ߙ}, Samjhana Gurung^{1 ߙ}, Umesh Kaji Manandhar¹, Raina Chaudhary², Anand Kumar Mandal³ Avinash Chaudhary¹, Dinesh Dhakal¹, Anup Muni Bajracharya⁴, Upendra Thapa Shrestha⁵ *

¹ Department of Microbiology, Sainik Awasiya Mahavidhyalaya, Bhaktpur, Nepal

² Shree Birendra Hospital Chhauni, Kathmandu, Nepal

³ Department of Pathology, Bhaktapur Hospital, Bhaktapur, Nepal

⁴ Balkumari College, Chitwan, Nepal

⁵ Central Department of Microbiology, Tribhuvan University, Kathmandu, Nepal

 

† These authors contributed equally.

 

*Corresponding author: Upendra Thapa Shrestha, Assistant Professor, Central Department of Microbiology, Tribhuvan University, Kathmandu, Nepal, E-mail: upendra.thapashrestha@cdmi.tu.edu.np

ABSTRACT

Objectives: To evaluate the antibiotic resistance pattern of Pseudomonas aeruginosa isolated from clinical specimen and to detect Metallo beta lactamase producers as well as to accesses their biofilm forming capacity by both qualitative and quantitative analysis.  

Methods: The study was conducted in Shree Birendra Hospital, Chhauni, from June to August 2025. The total of 6444 specimens was cultured and isolates of P. aeruginosa were subjected to antibiotic susceptibility tests. Metallo beta lactamase producers were identified by modified Hodge and EDTA synergy tests. Biofilm was detected by the Congo Red Agar and Microtiter Plate Assay method.

Results: Out of 671 positive isolates (15.05%) from pus, urine and wound, 101 isolates of P. aeruginosa were obtained. The highest rate of distribution was observed in in-patients as well as in the age group of 61-70 years. Among the isolates, high resistance was observed against Aztreonam (65.59%) whereas isolates were most sensitive against Tobramycin (76%). 37 were found to produce Metallo beta lactamase enzyme and almost 46% were MDR. The biofilm isolates accounted for 34 by CRA but MPA detected 100 biofilm producers. The biofilm producers showed high resistance against Aztreonam (59.41%) and Levofloxacin (56.44%). Furthermore, the MBLS were the most resistant against Levofloxacin (28.7%) followed by Aztreonam (27.7%), Cefepime (27.7%), Ceftazidime (25.7%), Imipenem (25.7%) and Meropenem (25.7%). Out of all the isolates, 36 biofilm isolates were highlighted to produce MBL enzyme as well.

 Conclusion: Pseudomonas aeruginosa was most frequent in sputum and pus samples from inpatients and older patients, with rising resistance to monobactams, fourth-generation cephalosporins, and fluoroquinolones. High rates of MBL production and biofilm formation contributed to marked β-lactam resistance, emphasizing the need for alternative therapeutic strategies.

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Keywords: Pseudomonas aeruginosa, Metallo beta lactamase, Microtitre plate, Biofilm

Published in TUJM Vol12, 2025

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