Comprehensive analyses of carbapenem-resistant and ESBL-producing bacteria in fresh vegetables and their resistome in the United States.

Carbapenem-resistant and extended-spectrum beta-lactamase (ESBL)-producing bacteria, once largely confined to healthcare settings, are increasingly detected in community environments. Food and the environment may act as important reservoirs for clinically relevant antibiotic-resistant bacteria. A large-scale surveillance study was conducted from 2022 to 2023 to assess antibiotic resistance in retail fresh vegetables across three U.S. regions: the Midsouth, Midwest, and West Coast. A total of 1218 samples representing five vegetable categories (carrots, lettuce, spinach, sprouts/microgreens, and salads) were analyzed for carbapenem-resistant bacteria and ESBL-producing Enterobacterales. Culture-based methods included selective isolation on CHROMagar, antibiotic susceptibility testing, phenotypic evaluation of ESBL and carbapenem resistance, and carbapenemase detection and typing. Whole-genome sequencing of phenotypically resistant isolates was used to identify beta-lactamase genes. Overall, 62 carbapenem-resistant isolates (5.09%) and 70 ESBL-producing Enterobacterales isolates (5.74%) were recovered. Carbapenemase-producing Enterobacterales included 30 Enterobacter strains and one Kluyvera strain, with carbapenem-resistant Enterobacter most frequently isolated from sprouts and microgreens. ESBL-producing strains included 39 Serratia, 20 Enterobacter, 6 Klebsiella, 3 Raoultella, and 2 Rahnella isolates. Comparative genomic analyses showed close similarity between vegetable isolates and human clinical strains. Notably, the carbapenemase gene blaIMI-6 identified in Enterobacter asburiae from microgreens was transferable to Escherichia coli by conjugation. Shotgun metagenomics of 40 samples further confirmed diverse resistance genes. These findings highlight vegetables as potential reservoirs of clinically important antibiotic resistance and emphasize the need for ongoing surveillance in both vegetable products and their production environments.