Evolution of P. Aeruginosa in CF Infections: Phenotypes and Mutations

Phenotypes of P. Aeruginosa in CF Infections

1. In Pseudomonas aeruginosa infections of cystic fibrosis (CF) patients, what is the acute phenotype for P. aeruginosa? What is the chronic phenotype for P. aeruginosa? What are the selective pressures for this phenotype change? The ‘acute’ phenotype is characterized by motility, protease production, and type III secretion and slowly transitions to a ‘chronic’ phenotype which is characterized by biofilm formation, decreased antibiotic susceptibility, loss of virulence factor production, altered pro-inflammatory effect, and hypermutability. One of the key features of P. aeruginosa evolution in the lung is caused by the emergence of mutations in the las quorum sensing system, which are associated with a loss in production of virulence factors, such as proteases and exotoxins. If selective pressures present in the CF lungs, i.e. viscous mucus, hyperinflammation, prolonged antibiotic treatment, the presence of bacteriophages and a complex microbial community.

Biofilm Setup for P. Aeruginosa Evolution

2. Describe how the biofilms were set up in these experiments to generate an evolved population of P. aeruginosa. A large portion of the frozen biofilm (stored as glycerol stock at −80 °C) was plated on LB agar and incubated overnight at 37 °C. The total growth on the agar plate was then divided into two microbank cryovials with beads and frozen at −80 °C. A single bead was then used to inoculate 5ml LB broth (incubated at 37 °C, 250 rpm, 16 hours) to serve as inoculum for phenotypic tests and DNA extraction. These cultures are designated ‘evolved populations’ throughout the text for the sake of clarity.

Mutations in P. Aeruginosa Genes

3. Which genes were mutated in P. aeruginosa during the evolution experiment? What types of mutations were identified? (Table 1) Genes lasR, pqsR, gntR, mexL, sahH, and pilA were mutated. Mutations in lasR were already present with a frequency of 33% in the starting culture, these mutations in lasR were the only mutations present in T0 or T1. Mutations in pqsR (PA1003), another QS regulator gene, were found in all samples at frequencies of up to 93% at T10 and T18, these were not found at T1 or T0 (Tables 1, S3). The types of mutations in lasR and pqsR include insertions of A/G or a transposase, several deletions, and different SNPs (single nucleotide polymorphisms). Mutations were also found in two other transcriptional regulators. The tetR gene encoding MexL, a specific transcriptional repressor of the multidrug efflux operon mexJK, was mutated at T18 in all samples that evolved in the absence of the microbiome (at frequencies up to 61%), and only in one sample that evolved in the presence of the microbiome. A deletion was also found in pilA (PA4525), the major pilin of the type IV pili, in all lineages regardless of the presence of the microbiome at T10 and T18, at frequencies of up to 97%. In all but one sample, a mutation (SNP of G to A, or deletion of G) upstream of sahH (PA0432), encoding an adenosyl homocysteinase, was found. This mutation consistently occurred at lower frequency in the presence of the microbiome.

Comparisons in Evolved P. Aeruginosa

4. Compare 3-O-C12-HSL production in evolved and unevolved strains. (Figure 2) Production of 3-O-C12-HSL was significantly reduced in the evolved populations compared to the evolved populations at T1 in all lineages. This showed that the observed mutations led to a null phenotype for the population only at T18, although lasR is already mutated at T1.
5. Compare virulence factor production in evolved and unevolved strains. (Figure 3) In all lineages, no differences in growth were observed between all populations in the lag and exponential phases. In the stationary growth phase significant differences in absorbance values were observed between T0 and populations from T1 compared to the evolved populations at T18.

Mutations and Phenotypes

6. When individual colonies were tested for mutations in lasR and pqsR, how many colonies had mutations in both genes? What effect did these mutations have on pyocyanin and protease production? (Figure 4) All isolates had a mutation in the lasR gene (10/10, 100%), six out of ten (60%) isolates also had a pqsR mutation. Both pyocyanin production and protease activity were significantly lower in the isolates that contained both mutations.
7. To which antibiotics did evolved populations show an increase in resistance? What class of antibiotics are these? Why do you think P. aeruginosa showed increased resistance to these antibiotics? (Figure 5) AmpR, a β-lactamase expression regulator, and LasR could explain the observed increase in resistance. P. aeruginosa is known to be resistant to a variety of antibiotics, it causes infections to express various virulence factors.
8. Describe the competition of P. aeruginosa with Staphylococcus aureus in evolved and unevolved cultures. Why do you think you see these results based on the data presented? (Figure 6) S. aureus was significantly more present than with unevolved populations. Unevolved P. aeruginosa populations could outcompete S. aureus completely, showing no S. aureus growth in the biofilm, while S. aureus growth up to 7 log CFU/mL was observed in biofilms with the evolved populations. S. Aureus is notorious for its ability to resist antibiotics which makes sense in this scenario.

Conclusions and Learnings

9. What are the main conclusions of this paper? Does the data presented support these conclusions? Why or why not? In vitro evolution of P. aeruginosa AA2 biofilms can lead to pathoadaptive mutations and phenotypes commonly found in in vivo chronic infections, such as CF. The genotypic and phenotypic changes are related to the las and pqs QS machineries and occurred regardless of the presence of other members of the CF microbiome. These conclusions are proven within in the tables, figures, and supplementary data provided. CF is a common infection and the evolution of biofilms within P. Aeruginosa makes sense as a label for causation.
10. Describe two things you learned from this paper. I learned that In vivo factors that drive and/or modulate the evolutionary behavior of P. Aeruginosa can lead to therapeutic avenues that direct evolution towards a genotype/phenotype that is susceptible to treatment. It was also interesting to learn that CF is caused by the evolution of microbiomes in the absence or presence of CF lung microbiomes. I did not know the causation of CF before reading this article, this is an interesting POV and overall an interesting experiment.