Riva Francesco, Dechesne Arnaud, Eckert Ester M, Riva Valentina, Borin Sara, Mapelli Francesca, Smets Barth F, Crotti Elena
Department of Food, Environmental and Nutritional Sciences (DeFENS), University of Milan, Milan, Italy.
Department of Environmental and Resource Engineering, Technical University of Denmark, Kongens Lyngby, Denmark.
Front Microbiol. 2024 Aug 29;15:1457854. doi: 10.3389/fmicb.2024.1457854. eCollection 2024.
Horizontal gene transfer (HGT) of antibiotic resistance genes (ARGs) is one of the primary routes of antimicrobial resistance (AMR) dissemination. In the One Health context, tracking the spread of mobile genetic elements (MGEs) carrying ARGs in agri-food ecosystems is pivotal in understanding AMR diffusion and estimating potential risks for human health. So far, little attention has been devoted to plant niches; hence, this study aimed to evaluate the conjugal transfer of ARGs to the bacterial community associated with the plant rhizosphere, a hotspot for microbial abundance and activity in the soil. We simulated a source of AMR determinants that could enter the food chain via plants through irrigation.
Among the bacterial strains isolated from treated wastewater, the strain EEF15 was selected as an ARG donor because of the relevance of in the AMR context and the One Health framework. The strain ability to recolonize lettuce, chosen as a model for vegetables that were consumed raw, was assessed by a rifampicin resistant mutant. EEF15 was genetically manipulated to track the conjugal transfer of the broad host range plasmid pKJK5 containing a fluorescent marker gene to the natural rhizosphere microbiome obtained from lettuce plants. Transconjugants were sorted by fluorescent protein expression and identified through 16S rRNA gene amplicon sequencing.
EEF15 was able to colonize the lettuce rhizosphere and inhabit its leaf endosphere 7 days past bacterial administration. Fluorescence stereomicroscopy revealed plasmid transfer at a frequency of 10; cell sorting allowed the selection of the transconjugants. The conjugation rates and the strain's ability to colonize the plant rhizosphere and leaf endosphere make strain EEF15:: with pKJK5::Plac:: an interesting candidate to study ARG spread in the agri-food ecosystem. Future studies taking advantage of additional environmental donor strains could provide a comprehensive snapshot of AMR spread in the One Health context.
抗生素抗性基因（ARGs）的水平基因转移（HGT）是抗菌药物耐药性（AMR）传播的主要途径之一。在“同一健康”背景下，追踪携带ARGs的移动遗传元件（MGEs）在农业食品生态系统中的传播对于理解AMR扩散和评估对人类健康的潜在风险至关重要。到目前为止，植物生态位很少受到关注；因此，本研究旨在评估ARGs向与植物根际相关的细菌群落的接合转移，植物根际是土壤中微生物丰度和活性的热点区域。我们模拟了一个AMR决定因素的来源，该来源可能通过灌溉经植物进入食物链。
在从处理后的废水中分离出的细菌菌株中，菌株EEF15被选为ARGs供体，这是因为其在AMR背景和“同一健康”框架中的相关性。通过利福平抗性突变体评估该菌株在生菜（选为生食蔬菜的模型）上重新定殖的能力。对EEF15进行基因操作，以追踪携带荧光标记基因的广宿主范围质粒pKJK5向从生菜植物获得的天然根际微生物群中的接合转移。通过荧光蛋白表达对接合子进行分选，并通过16S rRNA基因扩增子测序进行鉴定。
在施用细菌7天后，EEF15能够定殖在生菜根际并栖息于其叶内圈。荧光立体显微镜显示质粒转移频率为10；细胞分选允许选择接合子。接合率以及该菌株在植物根际和叶内圈定殖的能力使携带pKJK5::Plac::的菌株EEF15成为研究ARGs在农业食品生态系统中传播的一个有趣候选对象。利用其他环境供体菌株的未来研究可以提供“同一健康”背景下AMR传播的全面概况。
