Host and antibiotic jointly select for greater virulence in Staphylococcus aureus
- Emory University School of Medicine, Division of Infectious Diseases, United States
- Department of Biology, Emory University, United States
- Science Department, Penn State Scranton, United States
Figures
Figure 1
Experimental evolution design.
(A) Growth curves and (B) total growth of ancestral methicillin-resistant S. aureus (MRSA) and methicillin-sensitive S. aureus (MSSA) populations in vitro with and without sub-minimum inhibitory concentration (sub-MIC) oxacillin. Different letters indicate significant differences ( = 6.39, p=0.01). (C) MRSA and MSSA were passaged 12 times with or without hosts, in the presence or absence of a sub-MIC of the antibiotic oxacillin. Each treatment consisted of six independently evolving replicate populations. Experimental evolution treatment abbreviations are indicated in the purple and yellow boxes. Error bars indicate standard errors.
Figure 2 with 1 supplement
Evolution of virulence is facilitated by exposure to both host and sub-minimum inhibitory concentration (sub-MIC) antibiotic.
(A) Virulence in terms of C. elegans mortality. Dashed and dotted lines indicate respective ancestral virulence. Shaded areas indicate standard errors of technical replicates of ancestral virulence. Different letters indicate significant differences ( = 460.43, p<0.001; methicillin-resistant S. aureus [MRSA] Levene’s test for homogeneity of variance: F3,20 = 4.06, p=0.021; methicillin-sensitive S. aureus [MSSA]: F3,20 = 0.99, p=0.417). (B) Virulence in terms of the ability to hemolyze sheep’s blood, assayed at the population level (oxacillin: = 5.82, p=0.016). The y-axis indicates the number of evolved populations for each category. (C). Host mortality from (A) grouped by hemolysis status in (B) (Kruskal-Wallis = 3.97, p=0.046). (D) The proportion of colonies sampled from each evolved population that are able to hemolyze sheep’s blood (oxacillin: = 23.30, p<0.001; Levene’s test for homogeneity of variance: F1,22 = 26.06, p<0.001). Error bars indicate standard errors. *p<0.05.
Figure 2—figure supplement 1
Most colonies sampled matched their respective population in terms of the ability to hemolyze sheep’s blood (i.e. over 50% of colonies having the same hemolysis status as the population they were sampled from).
Figure 3 with 1 supplement
Host and sub-minimum inhibitory concentration (sub-MIC) antibiotic selection facilitated pathogen growth in antibiotics.
Pathogen in vitro growth (A) without oxacillin ( = 9.24, p=0.24) and (B) in sub-MIC oxacillin ( = 106.16, p<0.001). Different letters indicate significant differences. (C). Oxacillin MIC of evolved populations (Fisher’s exact test, p<0.001). The y-axis indicates the number of evolved populations for each category. Error bars indicate standard errors. **p<0.01.
Figure 3—figure supplement 1
Zone of inhibition from Kirby-Bauer disk diffusion susceptibility test with oxacillin for colonies sampled from each of the four populations with the most number of mutations (two from methicillin-resistant S. aureus [MRSA] and two from methicillin-sensitive S. aureus [MSSA]), and two additional randomly selected populations.
Figure 4 with 3 supplements
Regulatory genes likely played an important role in pathogen adaptation.
(A) Mutations swept to fixation, excluding intergenic and synonymous mutations, grouped by general function. The size of each point indicates how many populations had acquired at least one mutation in the gene. Colored shapes next to genes indicate whether these genes are regulatory or have been implicated in virulence or antibiotic resistance in the literature (see Supplementary file 1).
Figure 4—figure supplement 1
Count of all mutations (from 10% to 100% frequency) arisen in evolved populations.
Figure 4—figure supplement 2
Mutations at frequencies between 0.1 and <1, excluding synonymous or intergenic mutations, in each evolved population.
Points with darker shades indicate more than one mutation present. All mutations in methicillin-resistant S. aureus (MRSA) +host + ox populations between 0.1 and <1 are all either synonymous or intergenic. A table of all mutations and frequencies is available on figshare (10.6084/m9.figshare.28745558).
Figure 4—figure supplement 3
Mutations in evolved populations.
(A) Count and (B) mean of mutations swept to fixation in evolved populations.
Figure 5
Correlation between mutations and phenotypes.
(A) In vitro growth of populations with SCCmec and ACME deletions with or without sub-MIC oxacillin (one-sample t-test t=2.73, df = 8, p=0.026). (B) Change from ancestral pathogen-induced mortality vs. mutations (codY one-sample t-test=19.56, df = 7, p<0.001; gdpP: 17.02, df = 3, p=0.002; pbpA: 7.54, df = 3, p=0.012). (C) Hemolysis status vs. mutations (Fisher’s exact test p<0.001; agr vs. alr: p=0.036; argR: p=0.006; codY: p=0.005; gdpP: p=0.006; purR: p=0.006). (D) Oxacillin MIC vs. mutations (Fisher’s exact test p=0.0015; codY vs. brnQ1: p=0.043). (E) Biofilm production of evolved populations. Different letters indicate significant differences (methicillin-resistant S. aureus [MRSA]: = 7.24, p=0.06; methicillin-sensitive S. aureus [MSSA]: = 17.91, p<0.001). The column widths in (C) and (D) correspond to the number of mutations. Error bars indicate standard errors. All evolved populations were sequenced except for one population from the -host-ox treatment. *p<0.05, **p<0.01.
Figure 6
Mutations that arose during the experiment are enriched in isolates associated with systemic infection in humans.
(A) Number of genomes in our public S. aureus genome dataset (see Materials and methods) grouped by isolation source. General host-association refers to descriptions not specific enough to assign to other categories. We excluded samples that were ambiguously specified or missing information. (B–H) Number of genomes in the database containing mutations in the respective gene. (I) Number of genomes in the dataset containing the mutations arisen in agr. This gene is separate from the others to facilitate ease of visualization due to the magnitude of the y-axis. Asterisks indicate significant differences in the proportion of blood/systemic-associated genomes compared to the expected distribution in the dataset. *p<0.05, **p<0.01, ***p<0.001.
Figure 7 with 1 supplement
Methicillin-resistant S. aureus (MRSA) and methicillin-sensitive S. aureus (MSSA) underwent distinct evolutionary trajectories.
Principal component analysis of traits evolved from (A) MRSA ancestors (PERMANOVA F(3,23) = 8.38, r2=0.557, p=0.001) and (B) MSSA ancestors (PERMANOVA F(3,23) = 2.20, r2=0.248, p=0.064). Phylogenetic tree constructed from frequencies of mutations in populations evolving from (C) MRSA ancestors (distance to ancestor: F(3,20) = 2.39, p: 0.099; distance between populations: Kruskal-Wallis = 21.09, p<0.001) and (D) MSSA ancestors (distance to ancestor: F(3,19) = 10.44, p<0.001; distance between populations: F(3,51) = 54.20, p<0.001). Scale bar indicates Euclidean distance.
Figure 7—figure supplement 1
Genetic distance between the ancestor and evolved populations for (A) methicillin-resistant S. aureus (MRSA) and (B) methicillin-sensitive S. aureus (MSSA) genotypes.
Genetic distance between replicate populations within each treatment for (C) MRSA and (D) MSSA genotypes. Different letters indicate significant differences. Error bars indicate standard errors.
Author response image 1
Schematic of procedural steps involved in one passage of S. aureus through nematodes (+host -ox) compared to without nematodes (-host -ox).
Additional files
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Supplementary file 1
Nonsynonymous mutations occurred in genes with known roles in virulence and antibiotic resistance.
- https://cdn.elifesciences.org/articles/107936/elife-107936-supp1-v1.docx
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Supplementary file 2
Fixed mutations in genes and intergenic regions appearing in more than two populations.
SYN = synonymous, NONSYN = nonsynonymous.
- https://cdn.elifesciences.org/articles/107936/elife-107936-supp2-v1.docx
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Supplementary file 3
Chi-square goodness-of-fit test results for comparison between the proportion of blood/systemic infection-associated mutations vs. skin/nose/throat-associated mutations against the expected proportion (i.e. all BioSamples in the dataset).
All degrees of freedom equaled 1.
- https://cdn.elifesciences.org/articles/107936/elife-107936-supp3-v1.docx
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Supplementary file 4
Categories for ‘isolation_source’ terms from metadata for BioSamples linked to our dataset of public S. aureus genomes in Figure 6A.
- https://cdn.elifesciences.org/articles/107936/elife-107936-supp4-v1.docx
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MDAR checklist
- https://cdn.elifesciences.org/articles/107936/elife-107936-mdarchecklist1-v1.docx
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Host and antibiotic jointly select for greater virulence in Staphylococcus aureus
eLife 14:RP107936.
https://doi.org/10.7554/eLife.107936.3
