Extensive horizontal gene transfer during Staphylococcus aureus co-colonization in vivo

Abstract

Staphylococcus aureus is a commensal and major pathogen of humans and animals. Comparative genomics of S. aureus populations suggests that colonization of different host species is associated with carriage of mobile genetic elements (MGE), particularly bacteriophages and plasmids capable of encoding virulence, resistance, and immune evasion pathways. Antimicrobial-resistant S. aureus of livestock are a potential zoonotic threat to human health if they adapt to colonize humans efficiently. We utilized the technique of experimental evolution and co-colonized gnotobiotic piglets with both human- and pig-associated variants of the lineage clonal complex 398, and investigated growth and genetic changes over 16 days using whole genome sequencing. The human isolate survived co-colonization on piglets more efficiently than in vitro. During co-colonization, transfer of MGE from the pig to the human isolate was detected within 4 h. Extensive and repeated transfer of two bacteriophages and three plasmids resulted in colonization with isolates carrying a wide variety of mobilomes. Whole genome sequencing of progeny bacteria revealed no acquisition of core genome polymorphisms, highlighting the importance of MGE. Staphylococcus aureus bacteriophage recombination and integration into novel sites was detected experimentally for the first time. During colonization, clones coexisted and diversified rather than a single variant dominating. Unexpectedly, each piglet carried unique populations of bacterial variants, suggesting limited transmission of bacteria between piglets once colonized. Our data show that horizontal gene transfer occurs at very high frequency in vivo and significantly higher than that detectable in vitro.

Keywords: bacteriophage; experimental evolution; host adaptation; plasmid; population dynamics.

Fig. 1.—

Coculture with S0385 and H398 isolates. (a) Origin and genetic features of parental S. aureus strains. (b) Growth of S. aureus strains during co-colonization of gnotobiotic piglets. (c) Growth of S. aureus strains during in vitro coculture in BHIB. Error bars represent one standard error of the mean. Comparison of bacterial growth was assessed using Student’s two-tailed t-test, *P < 0.05, **P < 0.01, ns, nonsignificant.

Fig. 2.—

Highly diverse mobilomes of S. aureus progeny generated during co-colonization of gnotobiotic piglets. The unique mobilomes of 20 S0385 progeny (pig clade, black circle) and 20 H398 progeny (human clade, gray circle) for each piglet (shown as four different panels) at each sample point are shown. The frequency of each mobilome at a specific sample point is represented by a percentage above the mobilome. Individual MGEs have a colored border indicative of the parental strain they originate from, red border = originates from S0385, blue border = originates from H398. Parental mobilomes are represented by a gray shadow.

Fig. 3.—

Minimum MGE transfer events (acquisition or loss) accounting for mobilome profiles detected in piglets and BHI. Significantly more HGT events were detected in the H398 background (red) in piglets than in the S0398 background (blue, t-test, P < 0.05). Significantly more HGT events were detected in the H398 background in piglets than in BHI (t-test, P < 0.01).

Fig. 4.—

Stable mobilomes of S. aureus CC398 progeny generated during in vitro coculture in BHIB. The unique mobilomes of 20 S0385 progeny (pig clade, black circle) and 20 H398 progeny (human clade, gray circle) for each replication (shown as four different panels) at each sample point (4 h, 2, 4, 12, and 16 days postinoculation) are shown. The frequency of each mobilome at a specific sample point is represented by a percentage above the mobilome. Individual MGEs have a colored border indicative of the parental strain they originate from, red border = originates from S0385, blue border = originates from H398. Parental mobilomes are represented by a gray shadow.

Fig. 5.—

Incidence of bacteriophages (a) and plasmids (b) in H398 progeny during colonization of gnotobiotic piglets over time. A red border indicates an MGE that originated from S0385, and a blue border indicates an MGE that originated from H398. There was no evidence that SCCmec, SaPI5, or Tn916 elements moved into the H398 progeny.

Fig. 6.—

Bacteriophage acquisition and genome remodeling in H398 progeny. Parental genomes are represented by a single line (red = S0385 genome, blue = H398 genome) with the integration site of φ2, φ6, and φ3 bacteriophages shown. The majority of H398 progeny have the entire φ2 and φ6 bacteriophage integrated at known integration sites. AP1510 and AP1660 strains have unique φ2/φ6 recombinant bacteriophages integrated in their genomes. AP1503 has a φ2 bacteriophage integrated at a novel integration site.