Genomics of adaptation during experimental evolution of the opportunistic pathogen Pseudomonas aeruginosa - PubMed (original) (raw)

Genomics of adaptation during experimental evolution of the opportunistic pathogen Pseudomonas aeruginosa

Alex Wong et al. PLoS Genet. 2012 Sep.

Abstract

Adaptation is likely to be an important determinant of the success of many pathogens, for example when colonizing a new host species, when challenged by antibiotic treatment, or in governing the establishment and progress of long-term chronic infection. Yet, the genomic basis of adaptation is poorly understood in general, and for pathogens in particular. We investigated the genetics of adaptation to cystic fibrosis-like culture conditions in the presence and absence of fluoroquinolone antibiotics using the opportunistic pathogen Pseudomonas aeruginosa. Whole-genome sequencing of experimentally evolved isolates revealed parallel evolution at a handful of known antibiotic resistance genes. While the level of antibiotic resistance was largely determined by these known resistance genes, the costs of resistance were instead attributable to a number of mutations that were specific to individual experimental isolates. Notably, stereotypical quinolone resistance mutations in DNA gyrase often co-occurred with other mutations that, together, conferred high levels of resistance but no consistent cost of resistance. This result may explain why these mutations are so prevalent in clinical quinolone-resistant isolates. In addition, genes involved in cyclic-di-GMP signalling were repeatedly mutated in populations evolved in viscous culture media, suggesting a shared mechanism of adaptation to this CF-like growth environment. Experimental evolutionary approaches to understanding pathogen adaptation should provide an important complement to studies of the evolution of clinical isolates.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1

Figure 1. Minimum inhibitory concentration (MIC) to ciprofloxacin for each of 48 experimentally evolved populations (“pop”) or single gentoypes (“single”).

Populations were evolved in the presence (black) or absence (grey) of 1 µg/ml ciprofloxacin for 8 days (∼50 generations). Two different media were used, as indicated on the X-axis.

Figure 2

Figure 2. Competitive fitness of experimentally evolved populations (dark bars) or single genotypes (light bars).

Fitness was measured in the absence of antibiotic via direct competitions with a lacZ marked ancestral strain (Pa14). The height of each bar indicates mean fitness for 12 evolved populations (genotypes), with the error bar giving +/−1 SE. Competitions were carried out in scfm for populations (genotypes) evolved in scfm or in scfm+ciprofloxacin, and in scfm+mucin for populations (genotypes) evolved in scfm+mucin or in scfm+mucin+ciprofloxacin. Fitness below one indicates low fitness relative to the ancestor, while fitness above one indicates an overall benefit in the absence of antibiotic.

Figure 3

Figure 3. Numbers of mutations identified in evolved genotypes.

Mean number of mutations by treatment, with error bars giving +/−1 SE. In the “scfm+mucin+Cip” treatment, the filled circle represents all evolved genotypes, and the filled diamond represents the ten non-mutator genotypes.

Figure 4

Figure 4. Locations of mutations in the ciprofloxacin resistance proteins nfxB, orfN, gyrB, and in the putatively mucin-adaptive protein morA.

“S”: location of a single nucleotide polymorphism; “F”: frameshift; “I”: in-frame insertion; “D”: in-frame deletion.

Figure 5

Figure 5. Mutations in known resistance genes are strong predictors of ciprofloxacin resistance, but not fitness.

Fold-increase in MIC (A and B), or relative fitness (C and D), for genotypes bearing the given mutations, with genotypes evolved in scfm (A and C) or scfm+mucin (B and D).

Figure 6

Figure 6. Variation in colony morphology.

Isolates were grown on 1% tryptone plates containing Congo red and Coomassie blue. (A) Ancestral Pa14; (B) strain smA1 carrying a single mutation in morA (H975Y); (C) strain smA2 carrying a different mutation in morA (L1155Q); (D) strain smD6 bearing three mutations, including one in wspF (an out-of-frame deletion); (E) strain smC3 bearing a single mutation in Pa14_56280 (M204I); (F) strain scfmB5 carrying a single mutation in nfxB (G180S).

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