Research ArticleMICROBIOLOGY

A multihost bacterial pathogen overcomes continuous population bottlenecks to adapt to new host species

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Science Advances  27 Nov 2019:
Vol. 5, no. 11, eaax0063
DOI: 10.1126/sciadv.aax0063
  • Fig. 1 Experimental model of bacterial host switching and transmissions.

    (A) From the S. aureus human-associated parental strains, host switches were reconstructed by infecting ewes (represented with an asterisk). (B) Serial passages from sheep to sheep were performed every 3 to 5 weeks (red dashed lines). (C) Animals were housed with other infected animals and their lambs that frequently milk-fed from their mothers. (D) The parental human S. aureus strains were also passaged in vitro in nutrient-rich medium. (E) We selected three clones from some intermediate and the last isolation plates of every lineage for genomic DNA sequencing. (F) Representation of the entire transmission chains performed in the study for each strain.

  • Fig. 2 Mutations acquired during the infections and passages in sheep.

    (A) Distribution of different types of mutations across the genomes and genes affected, colored by type of mutation (bottom legend). SNPs are plotted in the outer circle, and indels are plotted in the inner circle. Protein products involved in pathogenesis and cell wall association (CWA) or that are exported/bacterial surface related are shown in bold. (B) Frequency of various types of mutations (FS: frameshift; Inter: intergenic; Intra: intragenic; Nonsys: nonsynonymous; Syn: synonymous). (C) Bar plots of frequency of mutation effects on the protein level (High: stop codon, frameshift; Moderate: nonsynonymous; Low: synonymous and intragenic SNPs) or in intergenic regions (modifier effect).

  • Fig. 3 Evolutionary dynamics of infection passages.

    (A) Minimum evolution trees of the passages constructed using SNP data only are consistent with the topologies and lineages of the transmission chains. Differences in branch lengths for isolates from specific sheep indicate the coexistence of different alleles within the animals, supporting genetic variability arisen from single clones after a transmission. Clones c422 and c221 also presented some indels not represented in these trees. (B) Lineages from which intermediate isolates were sequenced are marked in red. Substitution rates for in vivo and in vitro passages of the strains N315 and NCTC8325. (C) SNP accumulation over time during transmissions and/or within host only. (D) Distribution of pairwise genetic distance between weeks 8 and 9 (early) and weeks 10 and 11 (late) indicates late accumulation of diversity within hosts.

  • Fig. 4 S. aureus has undergone adaptive evolution during passage in a new host species.

    (A) Competition experiments were performed by coinfecting sheep with equal number of progenitor and passaged bacteria derived from wild-type (wt) and passaged strain, and 40 days later, infected sheep were assessed for strain carriage. (B) Significant differences were obtained when compared to the 50-50% outcome expected for a null hypothesis of no adaptation (P = 0.039, one-tailed Fisher’s exact test; P = 0.027, Barnard’s test). (C) Proportions of the wt strain (green) and the strain with a synonymous SNP (ss) for coinfection experiments of 20 sheep. Dashed lines indicate the last time bacteria were isolated from milk. (D) WGS of 100 isolates after coinfection revealed the within-host genetic variation.

  • Fig. 5 Simulations of genomic populations under transmission and feeding bottlenecks.

    (A) Average pairwise genetic distances between randomly selected isolates from the populations simulated. (B) Accumulation of fixed and variable SNPs over time. (C) Types of variable and fixed SNPs determined from the selection coefficients associated with every nucleotide.

  • Table 1 Mutations with moderate and high effect acquired during the infections and passages.

    StrainMutation*bpTypeGeneEncoded productGroupLineage
    NCTC8325SNP27,460MissensewalKSensor protein kinase WalKGR | TM | ST
    NCTC8325SNP98,945MissenseSAOUHSC_00092Conserved hypothetical proteinHP | TM | SEc222
    NCTC8325SNP120,749Missensecap5BCapsular polysaccharide synthesis enzyme Cap5BSE | HP
    NCTC8325SNP24,5287MissensetarIConserved hypothetical proteinSE | HP
    NCTC8325SNP280,449MissenseessBESAT-6 secretion machinery protein EssBTM | SE | P
    NCTC8325SNP393,634MissenseSAOUHSC_00390Conserved hypothetical proteinHP | P
    NCTC8325SNP420,058MissenseSAOUHSC_00417Conserved hypothetical proteinTM
    NCTC8325SNP464,931MissensernmVRibonuclease M5TM
    NCTC8325SNP546,984Missenseazo1FMN-dependent NADPH-azoreductaseTM
    NCTC8325SNP568,990MissenseSAOUHSC_00560Conserved hypothetical proteinc222
    NCTC8325SNP588,150MissenseSAOUHSC_00586Conserved hypothetical proteinSE
    NCTC8325SNP706,223MissenseSAOUHSC_00723Conserved hypothetical proteinTMc222
    NCTC8325SNP721,884MissenseSAOUHSC_00736Putative lipid kinaseTMc222
    NCTC8325SNP778,092MissenseSAOUHSC_00795Glyceraldehyde-3-phosphate dehydrogenaseTM | HPc222
    NCTC8325SNP813,228MissensemetN2Methionine import ATP-binding
    protein MetN2
    NCTC8325SNP1,030,094MissenseSAOUHSC_01064Pyruvate carboxylaseTM
    NCTC8325SNP1,067,459MissensesdhBIron-sulfur subunit of succinate dehydrogenase%2C putativeTM
    NCTC8325SNP1,121,026MissensecarBCarbamoyl phosphate synthase large subunitTM
    NCTC8325SNP1,138,999MissenseSAOUHSC_01187Conserved hypothetical proteinSE
    NCTC8325SNP1,550,169Stop-gainSAOUHSC_01628Conserved hypothetical proteinSE
    NCTC8325SNP1,727,352MissenseSAOUHSC_01821Conserved hypothetical proteinTMc222
    NCTC8325SNP1,757,024MissenseccpACatabolite control protein AGR | Pc222
    NCTC8325SNP1,773,253MissenseSAOUHSC_01866Conserved hypothetical proteinTM
    NCTC8325SNP2,033,426MissensescniStaphylococcal complement inhibitorHP | P | SE
    NCTC8325SNP2,045,511MissenseyqbOTail length tape measure proteinc222
    NCTC8325SNP2,095,322MissenseagrCAccessory gene regulator protein CGR | ST | P | SE
    NCTC8325SNP2,249,603MissenseSAOUHSC_02420Conserved hypothetical proteinSEc222
    NCTC8325SNP2,286,918MissensehysAHyaluronate lyaseTM | CWA | Pc222
    NCTC8325SNP2,311,214MissenserpsHRibosomal protein S8TMc222
    NCTC8325SNP2,624,646MissenseSAOUHSC_02849Putative pyruvate oxidaseTM
    NCTC8325SNP2,756,289Missenseasp2Accessory Sec system protein Asp2TM | SE
    NCTC8325Ins487,126FrameshiftSAOUHSC_00488Cysteine synthaseTM
    NCTC8325Del513,207FrameshiftcysESerine acetyltransferaseTM
    NCTC8325Del624,930DisruptiveSAOUHSC_00634ABC transporterTM | SE
    NCTC8325Ins658,822DisruptiveSAOUHSC_00670Conserved hypothetical proteinTM | SEc222
    NCTC8325Del764,412DisruptivelgtProlipoprotein diacylglyceryl transferaseTM | SE
    NCTC8325Del1,323,611FrameshiftoppC2Oligopeptide transporter putative
    membrane permease domain
    TM | SE
    NCTC8325Del2,096,058FrameshiftagrAAccessory gene regulator protein AGR | ST | P
    NCTC8325Ins2,188,845FrameshiftSAOUHSC_02366Conserved hypothetical proteinTM
    N315SNP274,258Stop-gainSA_RS01330Acetyl-CoA/acetoacetyl-CoA transferaseTMn222
    N315SNP377,083MissenseSA_RS01835PTS lactose transporter subunit IIBTM
    homocysteine methyltransferase
    N315SNP572,540MissensecysSCysteine—tRNA ligaseTM
    N315SNP580,166MissenserpoBDNA-directed RNA polymerase subunit betaTM
    N315SNP758,669MissenseSA_RS03785Hypothetical proteinSE
    N315SNP863,049MissenseosmCOrganic hydroperoxide resistance proteinHP
    N315SNP949,546MissenseLysR TRLysR family transcriptional regulatorGR
    N315SNP1,343,567MissensesbcCNuclease SbcCD subunit CTM
    N315SNP1,471,383MissensenorBQuinolone resistance protein NorBTM | P | SE
    N315SNP1,486,091MissenserecUHolliday junction resolvase RecUTM
    N315SNP1,735,479MissensepykAPyruvate kinaseTMn222
    N315SNP1,817,943MissenseleuSLeucine—tRNA ligaseTM
    N315SNP1,948,268MissensevraSTwo-component sensor histidine kinaseGR | ST | SE | P
    N315SNP2,035,898Stop-gainSA_RS10285Hypothetical proteinn222
    N315SNP2,193,397MissenseSA_RS11160EVE domain–containing proteinTM
    N315SNP2,320,771MissenseacrAcrB/AcrD/AcrF family proteinTM | SE
    N315SNP2,393,851MissenseSA_RS12225MOSC domain–containing proteinTM
    N315SNP2,575,229MissenseSA_RS13145Hypothetical proteinCWA | HP
    N315SNP2,626,125MissenseSA_RS13405Membrane proteinSE
    N315SNP2,637,600MissenseSA_RS13450N-succinyldiaminopimelate aminotransferaseTMn222
    N315SNP2,743,605Stop-gainSA_RS13980Hypothetical proteinCWA | SE
    N315SNP2,751,067Missenseasp2Accessory Sec system protein Asp2TM | SEn222
    N315Del90,901FrameshiftSA_RS00555Anion membrane transporterTM | SE
    N315Del1,085,977FrameshiftSA_RS05425Hypothetical proteinSE
    N315Del1,473,471FrameshifttdcBl-Threonine dehydratase catabolic TdcBTM
    N315Ins1,475,240Frameshiftald1Alanine dehydrogenaseTM | CWA | SE
    N315Del1,947,711FrameshiftvraSTwo-component sensor histidine kinaseGR | ST | P | SE
    N315Del2,568,566DisruptivefnbAFibronectin-binding protein ACWA | HP | SE | P

    *Only mutations with moderate effect (missense SNPs and disruptive indels) and high effect (stop-gained and frameshifts) are listed because they are more likely to play a role in host adaptation. Mutations include SNPs, deletions (Del), and insertions (Ins). Group: HP (host-pathogen interaction), GR (gene regulation), ST (signal transduction), TM (transport and metabolism), P (pathogenesis), CWA (cell wall associated), and SE (surface associated/exported). Lineage indicates the fittest clone in which mutations were identified.

    Supplementary Materials

    • Supplementary material for this article is available at

      Fig. S1. Distribution of selection coefficients in the computer simulations of evolving populations.

      Fig. S2. Growth curves in ewe milk.

      Table S1. Detailed transmission chains of the infections.

      Table S2. Information on the isolates used in this study.

      Table S3. Remaining mutations acquired during the infections and passages.

      Table S4. Coinfection experiment results.

      Table S5. Coinfection experiment results with isogenic strains.

      Table S6. SNP fixed in the population at different times.

      Table S7. Counts of the SNP found in 3% of the population.

      Table S8. Bacterial strains, plasmids, and oligonucleotides used in this study.

    • Supplementary Materials

      The PDFset includes:

      • Fig. S1. Distribution of selection coefficients in the computer simulations of evolving populations.
      • Fig. S2. Growth curves in ewe milk.
      • Legends for tables S1 and S2.
      • Table S3. Remaining mutations acquired during the infections and passages.
      • Table S4. Coinfection experiment results.
      • Table S5. Coinfection experiment results with isogenic strains.
      • Table S6. SNP fixed in the population at different times.
      • Table S7. Counts of the SNP found in 3% of the population.
      • Table S8. Bacterial strains, plasmids, and oligonucleotides used in this study.

      Download PDF

      Other Supplementary Material for this manuscript includes the following:

      • Table S1 (Microsoft Excel format). Detailed transmission chains of the infections.
      • Table S2 (Microsoft Excel format). Information on the isolates used in this study.

      Files in this Data Supplement:

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