Stein Maria, Brinks Erik, Habermann Diana, Cho Gyu-Sung, Franz Charles M A P
Department of Microbiology and Biotechnology, Max Rubner-Institut, Federal Research Institute of Nutrition and Food, Hermann-Weigmann, Kiel, Germany.
Front Microbiol. 2025 Feb 5;16:1538973. doi: 10.3389/fmicb.2025.1538973. eCollection 2025.
This study aimed to characterize antibiotic-resistance plasmids present in microorganisms from sprout samples using exogenous plasmid capture. Fresh mung bean sprouts were predominantly colonized by bacteria from the phyla and . To capture plasmids, a plasmid-free () CV601 strain, containing a green fluorescent protein gene for selection, was used as the recipient strain in exogenous plasmid capture experiments. Transconjugants were selected on media containing cefotaxime or tetracycline antibiotics. While no cefotaxime-resistant transconjugants were obtained, 40 tetracycline-resistant isolates were obtained and sequenced by Illumina NextSeq short read and Nanopore MinION long read sequencing. Sequences were assembled using Unicycler hybrid assembly. Most of the captured long plasmids carried either the (A) or (D) resistance gene, belonged to the IncFI or IncFII replicon types, and were predicted as conjugative. While the smaller plasmids contained the (A) tetracycline resistance gene as well as additional quinolone (S1), sulfonamide (1) and trimethoprim (A1) resistance genes, the larger plasmids only contained the (D) resistance gene. An exception was the largest 192 kbp plasmid isolated, which contained the (D), as well as sulfonamide () and streptomycin (A1) resistance genes. The smaller plasmid was isolated from different sprout samples more often and showed a 100% identity in size (71,155 bp), while the 180 kbp plasmids showed some smaller or larger differences (in size between 157,683 to 192,360 bp). This suggested that the plasmids obtained from the similar sprout production batches could be clonally related. Nanopore MinION based 16S metagenomics showed the presence of () , , , () , , () and , which have previously been isolated from fresh produce in Germany. These bacteria may harbor antibiotic resistance genes on plasmids that could potentially be transferred to similar genera. This study demonstrated that bacteria present in sprouts may act as the donors of antibiotic resistance plasmids which can transfer resistance to other bacteria on this product via conjugation.
本研究旨在利用外源质粒捕获技术对豆芽样品中微生物所携带的抗生素抗性质粒进行特征分析。新鲜绿豆芽主要被来自 门和 门的细菌定殖。为了捕获质粒，在质粒捕获实验中，使用不含质粒的 （ ）CV601菌株作为受体菌株，该菌株含有用于筛选的绿色荧光蛋白基因。在含有头孢噻肟或四环素类抗生素的培养基上筛选接合子。虽然未获得对头孢噻肟耐药的接合子，但获得了40株对四环素耐药的分离株，并通过Illumina NextSeq短读长测序和Nanopore MinION长读长测序进行测序。使用Unicycler混合组装法对序列进行组装。大多数捕获的长质粒携带（A）或（D）抗性基因，属于IncFI或IncFII复制子类型，并被预测为可接合的。较小的质粒除了含有（A）四环素抗性基因外，还含有额外的喹诺酮（S1）、磺胺（1）和甲氧苄啶（A1）抗性基因，而较大的质粒仅含有（D）抗性基因。一个例外是分离出的最大的192 kbp质粒，它含有（D）抗性基因以及磺胺（ ）和链霉素（A1）抗性基因。较小的质粒更常从不同的豆芽样品中分离出来，并且在大小上显示出100%的一致性（71,155 bp），而180 kbp的质粒在大小上显示出一些较小或较大的差异（大小在157,683至192,360 bp之间）。这表明从相似的豆芽生产批次中获得的质粒可能具有克隆相关性。基于Nanopore MinION的16S宏基因组学显示存在 （ ）、 、 、 （ ）、 、 （ ）和 ，这些菌株先前已在德国从新鲜农产品中分离出来。这些细菌可能在质粒上携带抗生素抗性基因，这些基因有可能转移到相似的属中。本研究表明，豆芽中存在的细菌可能作为抗生素抗性质粒的供体，通过接合作用将抗性转移到该产品上的其他细菌。
