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IntroductionThe rise in extended-spectrum beta-lactamase (ESBL)-producingEnterobacteriaceaein dairy cattle farms poses a risk to human health as they can spread to humans through the food chain, including raw milk. This study was designed to determine the status, antimicrobial resistance, and pathogenic potential of ESBL-producing -E. coliand -Klebsiellaspp. isolates from bulk tank milk (BTM). MethodsThirty-three BTM samples were collected from 17 dairy farms and screened for ESBL-E. coliand -Klebsiellaspp. on CHROMagar ESBL plates. All isolates were confirmed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and subjected to antimicrobial susceptibility testing and whole genome sequencing (WGS). ResultsTen presumptive ESBL-producing bacteria, eightE. coli, and twoK. pneumoniaewere isolated. The prevalence of ESBL-E. coliand -K. pneumoniaein BTM was 21.2% and 6.1%, respectively. ESBL-E. coliwere detected in 41.2% of the study farms. Seven of the ESBL-E. coliisolates were multidrug resistant (MDR). The two ESBL-producingK. pneumoniaeisolates were resistant to ceftriaxone. Seven ESBL-E. colistrains carry thebla_CTX-Mgene, and five of them co-harboredbla_TEM-1. ESBL-E. colico-harboredbla_CTX-Mwith other resistance genes, includingqnrB19,tet(A),aadA1,aph(3’’)-Ib,aph(6)-Id),floR,sul2, and chromosomal mutations (gyrA, gyrB, parC, parE, and pmrB). MostE. coliresistance genes were associated with mobile genetic elements, mainly plasmids. Six sequence types (STs) ofE. coliwere detected. All ESBL-E. coliwere predicted to be pathogenic to humans. Four STs (three ST10 and ST69) were high-risk clones ofE. coli. Up to 40 virulence markers were detected in allE. coliisolates. One of theK. pneumoniaewas ST867; the other was novel strain.K. pneumoniaeisolates carried three types of beta-lactamase genes (bla_CTX-M,bla_TEM-1andbla_SHV). The novelK. pneumoniaeST also carried a novel IncFII(K) plasmid ST. ConclusionDetection of high-risk clones of MDR ESBL-E. coliand ESBL-K. pneumoniaein BTM indicates that raw milk could be a reservoir of potentially zoonotic ESBL-E. coliand -K. pneumoniae.