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The abundance and diversity of antimicrobial-resistant bacteria (ARB) and antimicrobial resistance genes (ARGs) in agricultural landscapes may be important for the spread of antimicrobial resistance (AMR) in the environment. The aim of this study was to apply screening methods for ARB and ARGs to investigate the impact of farming on the prevalence of AMR in a country with low antibiotic usage. We have analyzed samples (n = 644) from soil and wild terrestrial animals and plants (slugs, snails, mice, shrews, earthworms, and red clover) collected over two years in agricultural fields accompanied by nearby control areas with low human activity. All samples were investigated for the occurrence of 35 different ARGs using high-throughput quantitative PCR (HT-qPCR) on a newly developed DNA array. In addition, samples from the first year (n = 415) were investigated with a culture-based approach combined with whole-genome sequencing (WGS) to identify antimicrobial-resistant E. coli (AREC). ARGs were detected in 59.5% of all samples (2019 + 2020). AREC, which was only investigated in the 2019 samples, was identified in 1.9% of these. Samples collected in the autumn showed more ARGs and AREC than spring samples, and this was more pronounced for organic fields than for conventional fields. Control areas with low human activity showed lower levels of ARGs and a lack of AREC. The use of livestock manure was correlated with a higher level of ARG load than other farming practices. None of the soil samples contained antibiotics, and no association was found between AMR and the levels of metals or pesticides. High qualitative similarity between HT-qPCR and WGS, together with the positive controls to the validation of our 35 ARG assays, show that the microfluid DNA array may be an efficient screening tool on environmental samples. In conclusion, even in a country with a very low consumption of antimicrobials by production animals, our results support the hypothesis of these animals being a source of AREC and ARGs in agricultural environments, primarily through the use of manure.

The aim of this study was to model the growth and survival behaviour of Salmonella Reading and endogenous lactic acid bacteria on fresh pre-cut iceberg lettuce stored under modified atmosphere packaging for 10 days at different temperatures (4, 8 and 15 °C). The Baranyi and Weibull models were satisfactorily fitted to describe microbial growth and survival behaviour, respectively. Results indicated that lactic acid bacteria (LAB) could grow at all storage temperatures, while S. Reading grew only at 15 °C. Specific growth rate values (μmax) for LAB ranged between 0.080 and 0.168 h−1 corresponding to the temperatures 4 and 15 °C while for S. Reading at 15 °C, μmax = 0.056 h−1. This result was compared with published predictive microbiology models for other Salmonella serovars in leafy greens, revealing that predictions from specific models could be valid for such a temperature, provided they were developed specifically in lettuce regardless of the type of serovars inoculated. The parameter delta obtained from the Weibull model for the pathogen was found to be 16.03 and 18.81 for 4 and 8 °C, respectively, indicating that the pathogen underwent larger reduction levels at lower temperatures (2.8 log10 decrease at 4 °C). These data suggest that this Salmonella serovar is especially sensitive to low temperatures, under the assayed conditions, while showcasing that a correct refrigeration could be an effective measure to control microbial risk in commercial packaged lettuce. Finally, the microbiological data and models from this study will be useful to consider more specifically the behaviour of S. Reading during transport and storage of fresh-cut lettuce, elucidating the contribution of this serovar to the risk by Salmonella in leafy green products.