Phenotypic resistance profiles and resistome variations between endophytic and epiphytic bacteria in apple fruits

In recent years, there has been increasing concerns about antibiotic resistance. Although studies have investigated resistance in food-associated bacteria, fresh produce microbes remain underexplored as potential hub of resistance genes capable for horizontal transfer to human via consumption. To this end, we tested the antibiotic resistance profiles of bacterial isolates recovered from Golden Reinders and Mandy apple cultivars. We aimed to investigate the effects of orchard-cultivar combinations and microbial lifestyle on the antibiotic resistance profiles. The apples (Golden Reinders and Mandy) were sampled from four separate orchards (EEL-Lleida, Esterri, Fruits-de-Ponent and Gotarta) in Spain. We used combination of culture-dependent and whole genome sequencing approaches to analyse the antibiotic resistance profiles. A total of 516 bacterial isolates were screened for susceptibility against seven different classes of antibiotics. Results showed that 272 isolates were resistant to at least one antibiotic. From those, 203 were epiphytes and 95 classified as endophytes (isolated from surface-sterilized apple peels), whereas 26 isolates were shared between the groups. The resistance profiles varied across the antibiotics, with over 50% of the isolates exhibiting resistance to tetracycline, quinolones and cephalosporins. In contrast, none of the isolates showed resistance to imipenem. Whole genome sequencing (WGS) was performed on 18 isolates, however, only 10 genomes passed quality-control thresholds and were included in subsequent resistome analyses. We found ARGs encoding resistance to 14 main antibiotic classes, with the majority of the confirmed resistances attributed to multidrug resistance (MDR). Only few target-specific ARGs were annotated, including (Rif)iri (rifampicin), lnu(A) (lincomycin) and FosD (Fosfomycin). Pantoea agglomerans possessed higher number of ARGs, while Staphylococcus arlettae exhibited notable prevalence of plasmid-encoded ARGs. Overall, the study highlights the prevalence of antibiotic resistance in apple microbiomes. The presence of multidrug-resistance (MDR) genes further underscores the persistent threat of ‘antibiotic resistance’, underlining the necessity for deeper insight into antibiotic resistance within food chain.