In order to estimate herd-level prevalence of extended-spectrum β-lactamase/AmpC β-lactamase (ESBL/AmpC)- and carbapenemase-producing commensal Escherichia coli in ruminants in the Basque Country (northern Spain), a cross-sectional survey was conducted in 2014 to 2016 in 300 herds using selective isolation. ESBL-/AmpC-producing E. coli was isolated in 32.9% of dairy cattle herds, 9.6% of beef cattle herds, and 7.0% of sheep flocks. No carbapenemase-producing E. coli was isolated. Phenotypic antimicrobial susceptibility determined by broth microdilution using EUCAST epidemiological cutoff values identified widespread coresistance to extended-spectrum cephalosporins and other antimicrobials (110/135 isolates), particularly tetracycline, sulfamethoxazole, trimethoprim, and ciprofloxacin. All isolates were susceptible to tigecycline, imipenem, meropenem, and colistin. The genomes of 66 isolates were sequenced using an Illumina NovaSeq 6000 and screened for antimicrobial resistance determinants against ResFinder and PointFinder. The plasmid/chromosomal locations of resistance genes were predicted with PlasFlow, and plasmid replicons were identified using PlasmidFinder. Fifty-two acquired resistance genes and point mutations in another four genes that coded for resistance to 11 antimicrobial classes were identified. Fifty-five genomes carried ESBL-encoding genes, blaCTX-M-14 being the most common, and 11 carried determinants of the AmpC phenotype, mostly the blaCMY-2 gene. Additionally, genes coding for β-lactamases of the CTX-M group 9 were detected as well as the sporadic presence of blaSHV-12, blaCMY-4, and a point mutation in the ampC promoter. Only a bovine isolate coharbored more than one ESBL/AmpC genetic determinant (blaCTX-M-14 and a mutation in the ampC promoter), confirming its ESBL- and AmpC β-lactamase-producing phenotype. Most ESBL/AmpC genes were located in IncI1 plasmids, which also carried a great variety of other antimicrobial resistance genes. IMPORTANCE Extended-spectrum β-lactamase (ESBL)- and AmpC β-lactamase (AmpC)-producing E. coli isolates have emerged in recent years as some of the fastest spreading antimicrobial resistance determinants in humans and food-producing animals, becoming a concern for animal and public health. This study provided insight into the prevalence of cefotaxime-resistant E. coli in cattle and sheep in the Basque Country and the associated genetic determinants of antimicrobial resistance. These constituted an important contribution to the limited repository of such data for cattle in the region and for sheep worldwide. Antimicrobial susceptibility testing by phenotypic and molecular methods is key in surveillance programs to enhance early detection of resistance development, monitor resistance trends, and provide guidance to clinicians in selecting the adequate therapy.

Mycobacteria include obligate and opportunistic pathogens that cause significant human and animal disease. The burden of tuberculosis has been largely reduced in developed territories but remains a huge problem worldwide. The significance of nontuberculous mycobacteria is growing considerably, especially in developed regions with higher life expectancy and more therapy-related immunosuppressed individuals. Due to their robustness mycobacteria can contaminate animal products by direct transmission from infected individuals or by environmental contamination during processing. The situation at market level is poorly known. Most studies analyzing commercially available foods are limited to a small or local scale and mainly focused on a particular mycobacterial species. There is a need to investigate if animal products that have passed the established controls to be for sale at main supermarkets could represent a route of contact with any mycobacteria. Thus, our goal was to study the prevalence of mycobacteria in these foods to assess if this could represent a source of human exposure. Five stores from the main supermarket chains in Spain were selected. 138 dairy and 119 meat products were purchased. All were processed using culture and multiplex real-time PCR methods. Additional molecular methods were used to specifically identify any positive result. Mycobacterium avium subsp. hominissuis (2), M. avium subsp. avium (1), and M. fortuitum (1) were isolated from powdered infant formula and ground beef, chicken sausage, and mortadella cold cut, respectively. Mycobacterial DNA (M. avium, M. tuberculosis complex and other nontuberculous mycobacteria) was detected in 15% of dairy products and 2% of meat products. These results show that the prevalence of viable mycobacteria in foods of animal origin obtained at the supermarket was not substantial although a considerable proportion of them contained mycobacterial DNA. Contact with mycobacteria through this route could be ensured over time. Further investigation is necessary to determine the real impact of foodborne mycobacterial exposure on human health and identify critical points in the food production system to enable setting up more stringent control measures.

Paratuberculosis is a chronic enteric infection, caused by Mycobacterium avium subsp. paratuberculosis (MAP), affecting virtually all ruminants as well as other animals. MAP is also suspected to be involved in the etiology of some human diseases, like Crohn’s disease and others. In surveillance studies, different analytical methodologies were employed to detect MAP, showing different results and incidence in dairy products. The aim of this study was to evaluate the performance characteristics of three analytical methods [culture, quantitative PCR (qPCR) and peptide-mediated magnetic separation (PMS) phage-based assay] for MAP detection in raw, heat-treated and powdered milk. The methods were evaluated according to performance characteristics defined for qualitative methods in ISO 16140-2:2016. To estimate sensitivity (including trueness) and LOD, 720, and 900 test portions, respectively, were blind tested by two laboratories. Considering all matrices, different sensitivities, expressed as the percentage of positives from the total of true positive test portions, were obtained for IS900 qPCR (94%), f57 qPCR (76%), culture (83%), and PMS-phage (40%). Trueness, expressed as results correctly assigned (including positive and negative) to the reference value, was 93% for the IS900 qPCR method, 89% for culture and 49% for the PMS-phage. The LODs obtained in this study were similar to the LODs previously published for cultural and qPCR methods. However, for the PMS-phage method, the obtained results showed higher LOD values compared to the limited data available in the scientific literature. Our results highlight that while the PMS-phage assay is workable in pure liquid culture for estimation of MAP counts, its usage for surveillance of dairy matrices should be treated with a lot of caution as performance characteristics obtained were lower than for the two other methods tested. qPCR and culture are the most appropriate methods to detect MAP in milk-based matrices according to ISO 16140 methodology. Cultural techniques are considered the gold standard for detection of viable MAP, but qPCR, which is widely used in analytical and surveillance studies, can be considered a suitable and recommendable alternative to cultural methods for screening, if confirmation of MAP’s viability is not requested.