PREVALENCE AND MOLECULAR CHARACTERIZATION OF BLACTX-M-1, BLATEM, AND BLASHV-1 ESBL-PRODUCING KLEBSIELLA PNEUMONIAE IN BOVINE MASTITIS

Abstract

Background: Bovine mastitis is one of the most significant diseases affecting dairy animals worldwide, leading to considerable economic losses due to reduced milk production and treatment costs. This study aimed to determine the prevalence of ESBL-producing Klebsiella pneumoniae isolated from mastitic milk samples and to detect the presence of β-lactamase genes (blaTEM, blaSHV-1, and blaCTX-M-1) in isolates from District Mardan, Khyber Pakhtunkhwa, Pakistan.

Methods: Klebsiella pneumoniae was isolated through culture and identified using standard biochemical tests (lactose fermentation test, urease, Indole ring and Simmon Citrate), followed by confirmation via PCR. A total of 46 multidrug-resistant (MDR) K. pneumoniae isolates were subjected to antimicrobial susceptibility testing. From these, 6 randomly selected isolates were further analyzed for molecular detection of β-lactamase genes. PCR analysis revealed the presence of blaCTX-M-1, blaTEM, and blaSHV-1 genes, and subsequent sequencing confirmed these resistance determinants.

Results: No significant difference in prevalence was observed between species (p=0.47). Among these isolates, 29.9% (46/154) were confirmed as Extended-Spectrum beta-Lactamase (ESBL) producers. Molecular characterization of representative isolates via PCR detected blaTEM, blaSHV-1, and blaCTX-M-1 in equal proportions (33.3% each). Antibiogram analysis revealed high resistance to cefotaxime (100%), amoxicillin (86.9%), and ceftriaxone (82.6%), though resistance to chloramphenicol and amoxicillin–clavulanic acid remained low. Logistic regression identified previous antibiotic usage as a significant risk factor (AOR = 2.27, p = 0.037$), indicating that a history of treatment significantly increases the likelihood of harboring ESBL-producing K. pneumoniae.

Conclusion:  The present study demonstrates the presence of ESBL-producing Klebsiella pneumoniae harboring blaCTX-M-1, blaTEM, and blaSHV-1genes in mastitic milk samples from dairy animals in Mardan.  Molecular analysis revealed mutation in bla TEM gene  at D10 E at sample 9; E3K at 20 and 34; F131and F22Y at 41; and D10E at samples 47 respectively, while a single nucleotide mutation was observed at bla SHV-1 in sample 9 at M42L. The high prevalence of antimicrobial resistance highlights the urgent need for rational antibiotic use, improved mastitis management practices, and continuous molecular surveillance of antimicrobial resistance in dairy farms to minimize the spread of ESBL-producing pathogens.