Contagious Antibiotic Resistance: Plasmid Transfer among Bacterial Residents of the Zebrafish Gut

Abstract

By characterizing the trajectories of antibiotic resistance gene transfer in bacterial communities such as the gut microbiome, we will better understand the factors that influence this spread of resistance. Our aim was to investigate the host network of a multidrug resistance broad-host-range plasmid in the culturable gut microbiome of zebrafish. This was done through in vitro and in vivo conjugation experiments with Escherichia coli as the donor of the plasmid pB10::gfp When this donor was mixed with the extracted gut microbiome, only transconjugants of Aeromonas veronii were detected. In separate matings between the same donor and four prominent isolates from the gut microbiome, the plasmid transferred to two of these four isolates, A. veronii and Plesiomonas shigelloides, but not to Shewanella putrefaciens and Vibrio mimicus When these A. veronii and P. shigelloides transconjugants were the donors in matings with the same four isolates, the plasmid now also transferred from A. veronii to S. putrefaciensP. shigelloides was unable to donate the plasmid, and V. mimicus was unable to acquire it. Finally, when the E. coli donor was added in vivo to zebrafish through their food, plasmid transfer was observed in the gut, but only to Achromobacter, a rare member of the gut microbiome. This work shows that the success of plasmid-mediated antibiotic resistance spread in a gut microbiome depends on the donor-recipient species combinations and therefore their spatial arrangement. It also suggests that rare gut microbiome members should not be ignored as potential reservoirs of multidrug resistance plasmids from food.IMPORTANCE To understand how antibiotic resistance plasmids end up in human pathogens, it is crucial to learn how, where, and when they are transferred and maintained in members of bacterial communities such as the gut microbiome. To gain insight into the network of plasmid-mediated antibiotic resistance sharing in the gut microbiome, we investigated the transferability and maintenance of a multidrug resistance plasmid among the culturable bacteria of the zebrafish gut. We show that the success of plasmid-mediated antibiotic resistance spread in a gut microbiome can depend on which species are involved, as some are important nodes in the plasmid-host network and others are dead ends. Our findings also suggest that rare gut microbiome members should not be ignored as potential reservoirs of multidrug resistance plasmids from food.

Keywords: conjugation; gut microbiome; horizontal gene transfer; plasmid; plasmid network; plasmid persistence; zebrafish.

FIG 1

All 29 unique zebrafish gut isolates grouped into four different genera based on their 16S rRNA gene sequences: Aeromonas, Plesiomonas, Vibrio, and Shewanella. ZFG, bacterial strains isolated from the zebrafish gut; DOMINANT, numerically dominant colony morphology types, originally identified on R2A agar, which successfully acquired the plasmid following conjugation with the donor E. coli; BHI, CHOC (chocolate), R2A, and TSA, media used to isolate the strains (see Materials and Methods). Red indicates culture collection strains most similar to isolates, and blue indicates zebrafish core microbiome reference strains (44). The tree was rooted using the archaean Methanococcus jannaschii.

FIG 2

Agarose gel electrophoresis of plasmid DNA extracted from plasmid-free and plasmid-containing strains.

FIG 3

The persistence of plasmid pB10::gfp in zebrafish gut isolates over 100 generations of growth in the absence of antibiotic selection varied greatly from high (P. shigelloides [solid triangles]), to moderate (A. veronii [solid squares]), to poor (S. putrefaciens [solid circles]). Each experiment consisted of three replicate populations, and the error bars represent the standard deviation.

FIG 4

Achromobacter was present at low abundance among the many genera in the gut microbiota of the treated zebrafish populations [(7.9 ± 4.2) × 10⁻³% in samples A to D] and untreated populations [(8.2 ± 5.6) × 10⁻²% in samples E to H], and not detectable in the water control (WC). Only the relevant cultured genera or operational taxonomic units (OTUs) are indicated. The relative abundance corresponds to the number of paired-end reads for each taxa normalized to the total number of paired-end reads per sample. A complete list of all the OTUs can be found in Table S1.

FIG 5

Plasmid pB10::gfp displays good persistence in Achromobacter. The experiment consisted of three replicate populations, and the error bars represent the standard deviation.