Comparative Genomics Analysis and Outer Membrane Vesicle-Mediated Horizontal Antibiotic-Resistance Gene Transfer in Avibacterium paragallinarum

Abstract

Avibacterium paragallinarum is the etiological agent of infectious coryza, an acute respiratory disease of chickens that is globally distributed and causes serious economic losses for chicken production. A. paragallinarum is a Gram-negative bacterium that releases outer membrane vesicles (OMVs). In this study, a comparative genomic analysis of A. paragallinarum isolate P4chr1 and its OMVs was carried out, and the ability to transfer antibiotic resistance genes (ARGs) via the OMVs was studied. Sequencing and data analyses demonstrated that the genomic size of A. paragallinarum P4chr1 was approximately 2.77 Mb with a 25 kb tolerance island that covered six types of antibiotics and 11 ARGs. The genomic size of its OMVs was approximately 2.69 Mb, covering 97% of the genomic length and almost all the gene sequences of P4chr1. Purified and DNase-treated A. paragallinarum P4chr1 OMVs were cocultured with the antibiotic-sensitive A. paragallinarum Modesto strain on an antibiotic (chloramphenicol, erythromycin, tetracycline, or streptomycin)-containing plate, and the corresponding ARGs were detected in the colonies grown on the plates. However, using an antimicrobial susceptibility test, we found that ARGs delivered by OMVs were not persistent but only appeared transiently on the antibiotic-containing plates. Antibiotic resistance and ARGs were lost by the second bacterial passage. IMPORTANCE The functions and roles of OMVs on ARG and virulent gene transfer and dissemination have been reported in numerous Gram-negative bacteria. However, the role of OMVs in mediating antibiotic resistance in A. paragallinarum has not been reported. This study is the first report to compare the genomic characteristics of OMVs with its parent A. paragallinarum strain and to study A. paragallinarum ARG transfer via OMVs. This work has provided useful data for further studies focusing on nonplasmid ARG transfer mediated by A. paragallinarum OMVs.

Keywords: A. paragallinarum; antibiotic resistance gene; horizontal gene transfer; outer membrane vesicles; whole genome.

FIG 1

(a) Representative image of A. paragallinarum P4chr1 OMVs visualized by transmission electron microscopy. (b) Representative image showing release of OMVs by A. paragallinarum strain P4chr1. Vesicles were purified from broth culture by ultracentrifugation and filtered through a 0.45-μm filter. The average diameter of the vesicles was 40 nm. The OMVs were free of bacterial contamination. The red circle and arrow indicate OMVs.

FIG 2

(a) Venn diagram showing the A. paragallinarum P4chr1 genome annotated in each database. (b) Venn diagram showing the OMV genome annotated in each database. Different colors represent different databases. Different colored circles have overlapping areas that can be annotated by different databases.

FIG 3

Comparison of A. paragallinarum P4chr1 with the corresponding chromosomal regions of the Pasteurella multocida strain FCf83 from China. The arrows indicate the extents and directions of transcription of the genes. ORFs with different functions are presented in various colors. Regions with >99% homology between A. paragallinarum P4chr1 and the chromosome of Pasteurella multocida strain FCf83 are indicated by gray shading.

FIG 4

(a) Comparative genomic circle diagram of A. paragallinarum P4chr1 and OMVs. The circles show (from outside to inside) OMV genomic sequences, P4chr1 genomic sequences, GC skew, GC content, and scale in kb. (b) Genomic collinearity analysis of A. paragallinarum P4chr1 and OMVs. The two areas connected by a line have similar sequences.

FIG 5

Orthologous cluster analysis of A. paragallinarum P4chr1 (Avibacterium_species) and OMVs (Bacterial_outer_membrane_vesicle).

FIG 6

PCR verification of antibiotic resistance genes. (a) bl2d_oxa1; (b) aph33ib; (c) cml_e3; (d) tetB. Lane 1, marker 2,000 bp; lane 2, P4chr1; lane 3, Modesto; lane 4, Modesto plus OMVs (single colony from TSA plate with antibiotic); lane 5, Modesto plus OMVs (single colony from TSA plate without antibiotic); lane 6, double-distilled water (ddH₂O).

FIG 7

PCR-RFLP profile of the A. paragallinarum strains used in this study. Lane 1, marker 2,000 bp; lane 2, P4chr1; lane 3, Modesto; lane 4, Modesto plus OMVs (single colony from TSA plate with antibiotic); lane 5, Modesto plus OMVs (single colony from TSA plate without antibiotic).

FIG 8

Schematic presentation of the steps involved in vesicle-mediated transfer in an A. paragallinarum strain (12).