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

Abstract

Background/Objectives: 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 Escherichia coli isolated from bovines, including three native Portuguese bovine breeds. Methods: Forty-nine E. coli 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 E. coli 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. Results: 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 E. coli was identified in 39% of isolates, including those from Holstein-Friesian calves and adults, Cachena calves and Minhota adults. The sul2 gene was detected in 69% of isolates, followed by bla_CTX-M (45%), aac(3')-IV (41%), and aac(6')-Ib-cr (31%), with a higher prevalence in adults. Conclusions: This pioneering study highlights the concerning presence of multidrug-resistant E. coli in native Portuguese cattle breeds.

Keywords: Escherichia coli; One Health; antimicrobial resistance; livestock; native breeds.

Figure 1

Phenotypic characterization of antimicrobial resistance in E. coli from cattle breeds (n = 49): (a) antimicrobial susceptibility profiles; and (b) antimicrobial resistance of E. coli isolates. Pan-sus—pan-susceptibility; Res ≥1—resistance to ≥1 antibiotic; MDR—multidrug-resistant phenotype; ESBL—extended-spectrum β-lactamase production phenotype; AMP: ampicillin; AMC: amoxicillin + clavulanic acid; CTX: cefotaxime; ATM: aztreonam; IPM: imipenem; CIP: ciprofloxacin; CN: gentamicin; AK: amikacin; TE: tetracycline; SXT: trimethoprim + sulfamethoxazole.

Figure 2

Antimicrobial susceptibility profiles of E. coli isolates from cattle breeds: (a) Holstein-Friesian breed (n = 29, with 16 from adults and 13 from calves); (b) Barrosã breed (n = 12, with 6 from adults and 6 from calves); (c) Cachena breed (n = 5, with 2 from adults and 3 from calves); and (d) Minhota breed (n = 3, with 1 from an adult and 2 from calves). Pan-sus: pan-susceptibility; Res ≥ 1: resistance to ≥1 antibiotics; MDR: multidrug-resistant phenotype; ESBL: extended-spectrum β-lactamase production phenotype.

Figure 3

Antibiotic susceptibility of E. coli isolates from cattle breeds: (a) Holstein-Friesian breed (n = 29, with 16 from adults and 13 from calves); (b) Barrosã breed (n = 12, with 6 from adults and 6 from calves); (c) Cachena breed (n = 5, with 2 from adults and 3 from calves); and (d) Minhota breed (n = 3, with 1 from an adult and 2 from calves). AMP: ampicillin; AMC: amoxicillin + clavulanic acid; CTX: cefotaxime; ATM: aztreonam; IPM: imipenem; CIP: ciprofloxacin; CN: gentamicin; AK: amikacin; TE: tetracycline; SXT: trimethoprim + sulfamethoxazole.

Figure 4

Genotypic characterization of the antimicrobial resistance profile of the E. coli (n = 49) isolated from all the cattle breeds: (a) Holstein-Friesian (n = 29, with 16 from adults and 13 from calves); (b) Barrosã (n = 12, with 6 from adults and 6 from calves); (c) Cachena (n = 5, with 2 from adults and 3 from calves); and (d) Minhota (n = 3, with 1 from an adult and 2 from calves).

Figure 5

Spatial distribution of the bovine farms based on the four breeds included in this study. Each circle represents a sample collection region.