Antibiotic Resistance Genes and Microbiota in Brassica oleracea var. acephala Cultivated in South Korea: Potential for Resistance Transmission

Abstract

Antimicrobial resistance (AMR) poses a critical global public health challenge. This study investigates the microbiome of Brassica oleracea var. acephala (kale) to evaluate the role of food production systems, particularly plant-derived foods, in AMR dissemination. Using 16S rRNA gene sequencing and metagenomic shotgun sequencing, we analyzed microbial diversity and antimicrobial resistance genes (ARGs) in kale samples. Results showed significant regional differences in microbiota composition and ARG distribution, with traditional fertilizer use linked to higher ARG prevalence in coliform bacteria compared to farms using other fertilization methods. Additionally, we confirmed ARG transfer potential by Klebsiella pneumoniae within coliform populations. Storage conditions notably affected microbial dynamics, with higher temperatures promoting K. pneumoniae growth in washed samples. These findings revealed the importance of AMR research in plant-derived foods and highlight the need for improved agricultural practices to mitigate the risks associated with high ARG abundance in coliform bacteria.

Keywords: Brassica oleracea var. acephala; antimicrobial resistance; coliform; food microbiome; kale; microbiota.

Figure 1

Diversity of kale microbiota. (A) α diversity indices, including observed OTUs, Chao1, and Simpson, were compared to assess regional differences in kale microbiota from Gwangju A–C and Yeoju D–F farms. (B) β diversity was assessed using unweighted and weighted UniFrac principal coordinate analysis (PCoA). The farms were represented as follows: Gwangju A–C in green, Yeoju D in dark blue, and Yeoju E and F in sky blue. (*, p < 0.05; ***, p < 0.001).

Figure 2

Taxonomic profiling and quantification of potential foodborne pathogens. (A) Pie chart showing the proportion of kale microbiota composition at the phylum, class, and family levels. (B) Each column of the heatmap at the genus level shows normalized relative abundance, allowing for a comparison of regional differences between samples. (C) A LEfSe analysis of the kale microbiota identified the most differentially abundant taxa distinguishing the sampling sites. The bar graphs show LDA scores (>3.0). The color of each bar is identical to Figure 1. (D) Potential foodborne pathogens in the kale microbiota were quantified by measuring the gene copies of each specific pathogen relative to the total 16S rRNA gene copies using qRT-PCR, with three technical replicates. The foodborne pathogens targeted for quantitative experiments were A. lwoffii, B. cereus, Enteroaggregative E. coli (EAEC), Enterohemorrhagic E. coli (EHEC), Enterotoxigenic E. coli (ETEC), Enteropathogenic E. coli (EPEC), K. pneumoniae, P. aeruginosa, Pa. agglomerans, S. aureus, and Se. marcescens. Error bars indicate the standard deviation.

Figure 3

Relative abundances of microbial species and overview of antimicrobial resistance (AMR) in kale microbiome. (A) The heatmap compares the dominant taxa at the species level, annotated from shotgun metagenomic sequencing, between the two regions. (B) Relative abundances of bacterial species and subspecies harboring ARGs were determined. (C) The distribution of coliform and noncoliform bacteria harboring antibiotic resistance genes (ARGs) and the classes of antibiotic resistance were compared in kale samples. Values are formulated from the number of reads that aligned to ARGs and normalized to bacterial abundance characterized by alignments to 16S rRNA gene sequences. (For detailed data, please refer to Table S2).

Figure 4

Shift in indigenous kale microbiota depending on washing condition and storage temperature. (A) Change in microbiota composition of kale according to storage temperatures (4 and 30 °C) in nonwashed and washed groups. (B) Total load of bacteria and K. pneumoniae in the nonwashed (solid line) and washed groups (dotted line) over storage time at 4 (blue square) and 30 °C (red circle). (***, p < 0.001).

Figure 5

Comparison of the antibiotic susceptibilities of E. coli and K. pneumoniae strains. The conjugation experiment confirmed the effective transfer of ARGs. Diameters of growth inhibition zones formed in the donor and transconjugant groups were visualized and measured. (***, p < 0.001).