Severe infections emerge from commensal bacteria by adaptive evolution

Abstract

Bacteria responsible for the greatest global mortality colonize the human microbiota far more frequently than they cause severe infections. Whether mutation and selection among commensal bacteria are associated with infection is unknown. We investigated de novo mutation in 1163 Staphylococcus aureus genomes from 105 infected patients with nose colonization. We report that 72% of infections emerged from the nose, with infecting and nose-colonizing bacteria showing parallel adaptive differences. We found 2.8-to-3.6-fold adaptive enrichments of protein-altering variants in genes responding to rsp, which regulates surface antigens and toxin production; agr, which regulates quorum-sensing, toxin production and abscess formation; and host-derived antimicrobial peptides. Adaptive mutations in pathogenesis-associated genes were 3.1-fold enriched in infecting but not nose-colonizing bacteria. None of these signatures were observed in healthy carriers nor at the species-level, suggesting infection-associated, short-term, within-host selection pressures. Our results show that signatures of spontaneous adaptive evolution are specifically associated with infection, raising new possibilities for diagnosis and treatment.

Keywords: Staphylococcus aureus; adaptation; human; infection; infectious disease; microbiology; pathogen genomics; virulence; within-host evolution.

Figure 1.. Infection-causing S. aureus form closely related but distinct populations descended from nose-colonizing bacteria in the majority of infections.

Bacteria sampled from the nose and infection site of 105 patients formed one of three population structures, illustrated with example haplotrees: (A) Unrelated populations differentiated by many variants. (B) Highly related populations separated by few variants. (C) Highly related populations with one genotype in common. Reconstructing the ancestral genotype in each patient helped identify the ancestral population: (D) Nose-colonizing bacteria ancestral. (E) Ambiguous ancestral population. (F) Infection site bacteria ancestral. (G) Phylogeny illustrating the working hypothesis that variants differentiating highly related nose-colonizing and infection-causing bacteria would be enriched for variants that promote, or are promoted by, infection. In A–F, haplotree nodes represent observed genotypes sampled from the nose (white) or infection site (grey), with area proportional to genotype frequency, or unobserved intermediate genotypes (black). Edges represent mutations. Patient identifiers and sample sizes (n) are given. In A–G, edge color indicates that mutations occurring on those branches correspond to B-class variants between nose-colonizing and infection-causing bacteria (blue), C-class variants among nose-colonizing bacteria (gold) or D-class variants among infection-causing bacteria (red). Black dashed edges indicate ancestral lineages. A B C.

Figure 1—figure supplement 1.. Distribution of the number of variants identified within 105 severely infected patients, by class.

Three classes of variants were identified: those representing genuine differences between nose-colonizing and infection populations (B-class), variants specific to the nose-colonizing population (C-class) and variants specific to the disease-causing infection population (D-class). The number of variants is shown on a piecewise-linear axis, with horizontal positioning permuted to assist visualization. Where nose-colonizing and infecting bacteria possessed different multilocus sequence types, the number of variants between those populations is colored red. When the number of B-class variants was 66 or less, nose-colonizing and infecting bacteria were considered related, since a similar range of (C-class) diversity was observed within the nose-colonizing populations of bacteria with the same multilocus sequence type. When the number of B-class variants was 1104 or more, nose-colonizing and infecting bacteria were considered unrelated.

Figure 2.. Genes, ontologies and pathways enriched for protein-altering substitutions between nose-colonizing and infection-causing bacteria within infected patients.

(A) Significance of enrichment of 2650 individual genes. (B) Significance of enrichment of 552 gene sets defined by BioCyc gene ontologies. (C) Significance of enrichment of 248 gene sets defined by SAMMD expression pathways. Genes, pathways and ontologies that approach or exceed a Bonferroni-corrected significance threshold of α = 0.05, weighted for the number of tests per category, (red lines) are named.

Figure 2—figure supplement 1.. Genes, ontologies and pathways enriched for protein-altering transient variants within nose-colonizing and infection-causing bacteria.

(A) Significance of enrichment of 2650 individual genes. SAR1461 encodes Pbp2, penicillin-binding protein 2. (B) Significance of enrichment of 552 gene sets defined by BioCyc gene ontologies. (C) Significance of enrichment of 248 gene sets defined by SAMMD expression pathways. C-class variants among nose-colonizing bacteria are colored gold, D-class variants among infection-causing bacteria are colored red. Genes, pathways and ontologies that approach or exceed a Bonferroni-corrected significance threshold of α = 0.05, weighted for the number of tests per category, (red lines) are named.

Figure 2—figure supplement 2.. Gene set enrichment analysis of B-class mutants occurring in the nose or the infection site.

Each point indicates the –log10 p-values of two tests for enrichment of protein-altering variants found among mutants in nose-colonizing bacteria vs infection-causing bacteria. The shape of each point represents the type of enrichment tested (squares: within 2650 genes in MRSA252, triangles: 552 BioCyc gene ontologies, circles: 248 SAMMD expression pathways). A line of 1:1 correspondence is plotted in red. A -log₁₀p-value above 5.2, 4.5 or 4.2 was considered genome-wide significant for loci, gene ontologies or expression pathways, respectively.

Figure 2—figure supplement 3.. Genes, ontologies and pathways enriched for protein-altering variants among longitudinally sampled asymptomatic nasal carriers.

(A) Significance of enrichment of 2650 individual genes. (B) Significance of enrichment of 552 gene sets defined by BioCyc gene ontologies. (C) Significance of enrichment of 248 gene sets defined by SAMMD expression pathways. Genes, pathways and ontologies that approach or exceed a Bonferroni-corrected significance threshold of α = 0.05, weighted for the number of tests per category, (red lines) are named.

Figure 3.. All genes contributing to the pathways and ontologies most significantly enriched for protein-altering substitutions between nose-colonizing and infection-causing bacteria.

The pathogenesis ontology, in which significant enrichments were observed in infection-causing but not nose-colonizing bacteria, is shown for comparison. Every gene with at least one substitution between nose-colonizing and infection-causing bacteria and which was up- (red) or down- regulated (blue) in one of the pathways or a member of one of the ontologies (blue) is shown. To the left, the number of altering (yellow/orange) and truncating (pink/red) B-class variants is shown, broken down by the population in which the mutant allele was found: nose (B_C; yellow/pink) or infection site (B_D; orange/red).

Figure 3—figure supplement 1.. Genes enriched for substitutions between nose-colonizing and infection-causing bacteria within patients are not the most rapidly evolving at the species level.

An estimate of the d_N/d_S ratio between unrelated bacteria is shown for each gene, color-coded by the number of protein-altering substitutions between nose-colonizing and infection-causing bacteria within patients. There was a negative Spearman rank correlation between d_N/d_S ratio and substitutions within patients (ρ = –0.04, p=0.02).

Figure 3—figure supplement 2.. Gene set enrichment analysis is robust to species-level differences in d_N/d_S between genes.

For every locus, expression pathway and gene ontology, we estimated d_N/d_S between unrelated S. aureus. There was no relationship between d_N/d_S and enrichment of protein-altering substitutions between nose-colonizing and infection-causing bacteria in (A) loci, (B) ontologies nor (C) pathways (non-significant correlations, p>0.05). When we incorporated variability in d_N/d_S between genes in the gene set enrichment analyses, the results were robust for (D) loci, (E) ontologies and (F) pathways, showing only small differences in significance (-log₁₀ p-value) between the analyses that correct for locus length only (horizontal axes) and those that correct for locus length and d_N/d_S (vertical axes).