Resistome variations between endophytic and epiphytic bacteria in apple fruits

Introduction: In recent years, there has been increasing concerns about antibiotic resistance. Despite the widespread public awareness about antibiotic resistance, little is known about resistome associated with endophytic and epiphytic microbial communities of fresh produce. Objectives: To this end, we tested the antibiotic resistance profiles of bacterial isolates recovered from Golden Reinders and Mandy. We aimed to investigate the effects of orchards, apple cultivars and microbe lifestyle on the antibiotic resistance profiles. Methods: 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 resistome. Results: A total of 516 bacterial isolates were screened for susceptibility against seven different classes of antibiotics. Results showed that 298 were resistant to at least one antibiotic. From those, 203 were epiphytes and 95 classified as endophytes, 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. Ten multi-drug resistant isolates were whole genome sequenced to identify the presence and localization of antibiotic resistance genes (ARGs). We found ARGs encoding resistance to 14 main antibiotic classes, with the majority of the confirmed resistances attributed to multi-drug resistance. 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. Conclusion: Overall, the study highlights the prevalence of antibiotic resistance in apple microbiomes. The presence of multi-resistance genes further underscores the persistent threat of ‘antibiotic resistance’, underlining the necessity for deeper insight into antibiotic resistance within food chain.