Research Article | Ecological and Evolutionary Science

Genetic Determinants Enabling Medium-Dependent Adaptation to Nafcillin in Methicillin-Resistant Staphylococcus aureus

Michael J. Salazar, Henrique Machado, Nicholas A. Dillon, Hannah Tsunemoto, Richard Szubin, Samira Dahesh, Joseph Pogliano, George Sakoulas, Bernhard O. Palsson, Victor Nizet, Adam M. Feist
Christopher W. Marshall, Editor
DOI: 10.1128/mSystems.00828-19

Article Figures & Data

Figures

  • FIG 1
    FIG 1

    Medium adaptation of S. aureus TCH1516. (A) Fitness trajectories depicting growth rate increase throughout the course of the medium adaptation ALE in RPMI+. Strains STR 1, 4, and 5 served as progenitors for the medium-adapted starting points in the tolerance evolution. (B) Clonal growth rates for single clones isolated by streaking endpoint populations. Measurements were determined from biological duplicates and an average of two consecutive flasks. STR strains are S. aureus RPMI+-adapted strains. STM strains are S. aureus CA-MHB-adapted strains. Values that are significantly different (P < 0.0001) from the value for the wild type (WT) by two-way ANOVA are indicated by a bar and asterisk. (C) ALE-derived strains maintain parental lineage virulence in a murine pneumonia model of infection. Values that are significantly different (P < 0.05) by t test with Welch’s correction are indicated by a bar and asterisk.

  • FIG 2
    FIG 2

    Nafcillin adaptation of medium-adapted strains derived from S. aureus TCH1516. (A) Fitness trajectory for a typical TALE experiment, showing population growth rate and continuously increasing antibiotic concentration. The selected trajectory depicts SNFR9 exposed to nafcillin in RPMI+. (B) A plot of the MICs for selected clones from endpoint populations after nafcillin tolerization. The MICs for the wild-type TCH1516 (black squares) and TALE strains (green circles) on the respective medium are shown.

  • FIG 3
    FIG 3

    Phenotypic characterization of TALE strains. (A) Growth rates of wild-type, medium-adapted, and nafcillin-adapted strains. The graph shows the measured growth rates of several selected endpoint clones for strains derived from either RPMI+ (STR and SNFR) or CA-MHB (STM and SNFM) evolutionary conditions. White bars represent clonal growth rates in CA-MHB, and gray bars represent the growth rate for the same clones in RPMI+. The graph shows the growth rates of three tolerization endpoint clones from both medium conditions along a lineage. Data presented are averages from triplicates. A comprehensive ANOVA statistical analysis is provided in Table S5 in the supplemental material. (B) Heat map of the nafcillin MIC fold change of TALE strains compared to the wild-type MIC in both medium types. STM, S. aureus CA-MHB-adapted strain; STR, S. aureus RPMI+-adapted strain; SNFM, S. aureus nafcillin-adapted strain in CA-MHB; SNFR, S. aureus nafcillin-adapted strain in RPMI+.

Tables

  • TABLE 1

    Key reproducibly occurring mutations detected in the final populations and clones of S. aureus TCH1516 after adaptive laboratory evolution in RPMI+

    TABLE 1

    • ↵a The gene locus tag corresponds to USA300HOU_RSXXXXX. The gene nomenclature provided by prokka annotation, reflected in the mutation analysis, is shown in the parentheses.

    • ↵b SNP, single nucleotide polymorphism; INS, insertion; DEL, deletion; SUB, substitution.

    • ↵c nt, nucleotide; †, mutation led to formation of a start codon.

    • ↵d p denotes population.

  • TABLE 2

    Tolerance phenotypes for S. aureus USA300_TCH1516 and medium-adapted evolved populations on CA-MHB and RPMI+a

    TABLE 2

    • ↵a Population growth rates for independent replicates were calculated by averaging the initial and final three flasks of the medium adaptation ALEs. An asterisk indicates premature end to experiment due to technical errors.

  • TABLE 3

    Key mutations for final endpoint clones of S. aureus TCH1516 after tolerance adaptive laboratory evolution in CA-MHB to nafcillin (SNFM)

    TABLE 3

    • ↵a The gene nomenclature provided by prokka annotation, reflected in the mutation analysis, is shown in the parentheses.

    • ↵b An asterisk indicates that a mutation led to a stop codon being formed.

  • TABLE 4

    Key mutations for final endpoint clones of S. aureus TCH1516 after tolerance adaptive laboratory evolution in RPMI+ to nafcillin (SNFR)

    TABLE 4

    • ↵a The gene nomenclature provided by prokka annotation, reflected in the mutation analysis, is shown in the parentheses. The gene locus tag corresponds to USA300HOU_RSXXXXX.

    • ↵b An asterisk indicates that a mutation led to a stop codon being formed.

Supplemental Material

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KEYWORDS

Staphylococcus aureus
antibiotic resistance
nafcillin
USA300
adaptive laboratory evolution
drug resistance mechanisms

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