The characterization of outer membrane vesicles (OMVs) and their role in mediating antibiotic-resistance gene transfer through natural transformation in Riemerella anatipestifer

Abstract

Riemerella anatipestifer (R. anatipestifer, RA) is the etiological agent of duck serositis, an acute multisystemic disease in ducks that is globally distributed and causes serious economic losses in the duck industry. Despite exhibiting multidrug resistance, the transmission mechanism of its antibiotic resistance genes (ARGs) remains incompletely identified. To contribute to addressing this gap, in this study, outer membrane vesicles (OMVs) from the RA strain CH-1 were isolated and characterized to investigate their involvement in ARG transfer in RA. Sequencing and data analysis revealed that RA CH-1 OMVs had ∼2.04 Mb genomic size, representing 88.3 % of the RA CH-1 genomic length. Proteomic analysis showed that OMVs contained 577 proteins, representing 27.2 % of the bacterial proteins. Subsequent investigations demonstrated that OMVs from antibiotic-resistant strains transferred ARG fragments and plasmids to the sensitive strain RA ATCC11845, relying on the natural transformation system, and the transformants exhibited corresponding resistance. Overall, OMV-mediated horizontal transfer of ARGs serving as a significant mechanism for acquiring multiple resistance genes in R. anatipestifer.

Keywords: Horizontal gene transfer; Outer membrane vesicles; Plasmid transfer; Riemerella anatipestifer.

Fig. 1

Transmission electron microscopy of R. anatipestifer CH-1 and purified OMVs. (A) The arrows indicate OMV budding from the RA CH-1 surface. Scale bars = 200 nm. (B) The arrows point to several OMVs of varying sizes. Scale bars = 100 nm. (C) Diameter analysis of OMVs ranging from 80 to 150 nm. The x-axis shows the number of OMVs, while the y-axis shows their diameters.

Fig. 2

KEGG pathway classification and GO category analysis of the OMV genome. (A) 43 % of OMV genes are involved in the KEGG metabolic pathway classification, divided into 47 pathways. The y-axis represents the names of the pathways, and the x-axis represents the number of genes in each pathway. (B) 63 % of OMV genes are involved in GO classification and are divided into 76 functional annotations. The y-axis represents the names of the functional classifications, and the x-axis represents the number of genes in each functional classification.

Fig. 3

Venn diagram showing the OMV genome annotated in each database, genomic collinearity analysis, and subcellular localization of OMV proteins. (A) Different colors represent different databases, including KEGG, eggNOG, Nr, Swiss-prot, VFDB, CARD, and GO. The overlapping areas of the differently colored circles are annotated with the respective databases. (B) Genomic collinearity analysis of RA CH-1 and its OMVs, and the two areas connected by a line have similar sequences. (C) Predicted subcellular localizations of the proteins in the OMVs, as determined by PSORTb software. Various localization categories, including cytoplasmic, cytoplasmic membrane, outer membrane, periplasmic, extracellular, and unknown, are represented. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 4

Genomic distribution of resistance genes and colony PCR from recipient strain RA ATCC11845 incubated with OMVs of RA CH-1. (A) Distribution of resistance genes tet(X), ermF, and bla_RASA on the genome of RA CH-1. (B–H) DNA gel showing PCR products of ARGs with the expected lengths. (B) Colonies grown on erythromycin resistance plates were identified using primers ErmF F and ErmF R. Lane M: MD104 DNA marker (BioMed); Lane 1-8: the ermF product (801 bp). (C) Colonies grown on tetracycline resistance plates were identified using primers TetXF and TetXR. Lane M: MD104 DNA marker (BioMed); Lane 1-8: the tet(X) product (1,137 bp). (D) Colonies grown on an aztreonam resistance plate were identified using primers bla_RASA F and bla_RASA R. Lane M: MD104 DNA marker (BioMed); Lane 1-10: the bla_RASA product (882 bp). (E) Colonies grown on erythromycin resistance plates were identified using primers TetXF and TetXR. Lane M: MD104 DNA marker (BioMed); Lane 1-8: the tet(X) product (1,137 bp). (F) Colonies grown on erythromycin resistance plates were identified using primers bla_RASA F and bla_RASA R. Lane M: MD104 DNA marker (BioMed); Lane 1-8: the bla_RASA product (882 bp). (G) Colonies grown on tetracycline resistance plates were identified using primers bla_RASA F/bla_RASA R and ErmF F/ErmF R. Lane M: MD104 DNA marker (BioMed); Lane 1-4: the bla_RASA product (882 bp). Lane 5: the bla_RASA positive control (882 bp); Lane 6-9: the ermF product (801 bp). Lane 10: the ermF positive control (801 bp). (H) Colonies grown on an aztreonam resistance plate were identified using primers TetXF/TetXR and ErmF F/ErmF R. Lane M: MD104 DNA marker (BioMed); Lane 1-4: the tet(X) sequence amplicon; Lane 5: the tet(X) positive control (1,137 bp); Lane 6-9: the ermF sequence amplicon. Lane 10: the ermF positive control (801 bp).

Fig. 5

PCR identification of plasmid transformation mediated by OMVs and quantitative analysis of plasmid copy numbers in OMVs. (A) Colony PCR from RA ATCC11845 treated with OMVs-pLM03, DNA gel showing PCR products with expected lengths. Lane M: MD104 DNA marker (BioMed); Lane 1-7: the cfx product (638 bp). (B) Colony PCR from RA ATCC11845 treated with OMV-pRCAD0416RA-1, DNA gel showing PCR products with expected lengths. Lane M: MD104 DNA marker (BioMed); Lane 1: the ermF product (801 bp). (C) Standard curves were generated using qPCR for plasmid pLMF03, with a linear regression equation of y = −2.837x + 37.69, R² = 0.9908. (D) The plasmid copy number (PCN) for pLMF03 was 4.29 × 10¹⁰ copies/μL, while the PCN for OMV-pLMF03 was 1.44 × 10⁶ copies/μL.