Dairy Cattle and the Iconic Autochthonous Cattle in Northern Portugal Are Reservoirs of Multidrug-Resistant Escherichia coli

<b>Background/Objectives:</b> Animals destined for human consumption play a key role in potentially transmitting bacteria carrying antibiotic resistance genes. However, there is limited knowledge about the carriage of antibiotic-resistant bacteria in native breeds. We aimed to characterize the phenotypic profiles and antibiotic resistance genes in <i>Escherichia coli</i> isolated from bovines, including three native Portuguese bovine breeds. <b>Methods:</b> Forty-nine <i>E. coli</i> isolates were selected from 640 fecal samples pooled by age group (eight adult or eight calf samples) from each farm, representing both dairy cattle raised in intensive systems and meat cattle raised in extensive systems in Northern Portugal. The presumptive <i>E. coli</i> colonies plated onto MacConkey agar were confirmed using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). The antibiotic resistance profiles were screened by antimicrobial susceptibility testing (EUCAST/CLSI guidelines), and the antibiotic resistance genes by PCR. <b>Results:</b> Most isolates showed resistance to ampicillin (69%), tetracycline (57%), gentamicin (55%), and trimethoprim + sulfamethoxazole (53%), with no resistance to imipenem. Resistance to at least one antibiotic was found in 92% of isolates, while 59% exhibited multidrug resistance. Most calf isolates, including those from native breeds, showed a multidrug-resistant phenotype. Among the adults, this was only observed in Holstein-Friesian and Barrosã cattle. None of the Holstein-Friesian isolates were susceptible to all the tested antibiotics. ESBL-producing <i>E. coli</i> was identified in 39% of isolates, including those from Holstein-Friesian calves and adults, Cachena calves and Minhota adults. The <i>sul</i>2 gene was detected in 69% of isolates, followed by <i>bla</i>_CTX-M (45%), <i>aac</i>(3')-IV (41%), and <i>aac</i>(6')-Ib-cr (31%), with a higher prevalence in adults. <b>Conclusions:</b> This pioneering study highlights the concerning presence of multidrug-resistant <i>E. coli</i> in native Portuguese cattle breeds.