Cooperation and virulence of clinical Pseudomonas aeruginosa populations

Abstract

Bacteria communicate and cooperate to perform a wide range of social behaviors including production of extracellular products (public goods) that are crucial for growth and virulence. Their expression may be switched on by the detection of threshold densities of diffusible signals [Quorum-Sensing (QS)]. Studies using the opportunistic pathogen Pseudomonas aeruginosa suggest that QS "cheats"-individuals that don't respond to the QS signal, but are still able to use public goods produced by others-have a selective advantage in the presence of QS cooperators. It is, however, unclear whether this type of social exploitation is relevant in clinical contexts. Here, we report the evolutionary dynamics and virulence of P. aeruginosa populations during lung colonization of mechanically ventilated patients in the absence of antimicrobial treatments. We observed a large diversity of QS phenotypes among initial colonizing isolates. This diversity decreased over a matter of days, concomitant with a gradual increase in the proportion of QS cheating mutants (lasR mutants), which were found in 80% of the patients after 9 days of colonization. These mutants often evolved from initial wild-type genotypes. The fitness advantage of the lasR mutants is almost certainly due to social exploitation, because this advantage was only apparent in the presence of QS wild-type cells. Crucially, ventilator-associated pneumonia occurred significantly earlier in patients predominantly colonized by QS wild-type populations, highlighting the importance of QS in this clinical situation. These results demonstrate that social interactions can shape the short-term evolution and virulence of bacterial pathogens in humans, providing novel opportunities for therapy.

Fig. 1.

QS phenotype diversity of isolates from intubated patients. (A) Elastase and rhamnolipid production of all 364 isolates was measured by ECR assay and halo diameter on modified SW-blue plates, respectively, and scored with respect to PAO1 activity (set to 100%), as shown in B. Genotypes of isolates from each individual patient were compared by RAPD. (B) The lasR and rhlR genes were sequenced in at least one isolate of each QS class from every patient. The presence of mutated lasR and rhlR alleles in the remaining isolates was assessed by high resolution melting (HRM) analysis of the corresponding PCR amplicons. Isolates of this class may carry either a wild-type (wt) or a mutated (mut) allele of the gene. (C) Diversity (H) of colonizing isolates across the 31 patients as calculated by the Shannon–Weaver index (19).

Fig. 2.

Emergence of QS mutants and population densities. (A) The proportion of patients harboring isolates with mutations in lasR (●) and rhlR (○) genes through time. Day −1 denotes the first day of detectable P. aeruginosa colonization. Note that sample sizes decreased from 31 patients to 5 patients by day 20. (B) The relationship between proportion of patients harboring isolates with lasR mutations and mean elastase production of the given isolates. Each data point represents a single day. (C) Comparison of bacterial loads between patients. Data shows mean (SEM) genomic copy number in patients harboring isolates with only wild-type (●) or mutant (○) lasR alleles. P. aeruginosa genomic copy numbers were determined on total DNA extractions from daily tracheal aspirates and quantified by qRT-PCR. Data only shows the initial periods of colonization because of small sample sizes in the wild-type group after day 5.

Fig. 3.

P. aeruginosa population dynamics in intubated patients. Total P. aeruginosa genomic copy numbers were determined by qRT-PCR on total genomic DNA preparations from tracheal aspirates by using the rpsL primer pair and are expressed as genomic copies/g aspirate (A–D, Upper). (A) QS population dynamics in patient 16101 colonized by a single genotype. To discriminate in the genomic DNA preparations between lasR wild-type and mutant populations, a lasR primer pair was designed which amplifies a 200 bp DNA fragment that is absent in the lasR deletion mutants (isolates from days 3 to 11). The amount of total P. aeruginosa copies was determined in the same DNA samples by using the rpsL primer pair. (Lower) The graph shows the percentage of lasR wild-type copies among the total P. aeruginosa population. (B, C, D) QS population dynamics in patients colonized by 2 genotypes. In patient 21107, populations of clone F469 (lasR wild-type, exoS+, exoU−) and clone 6D92 (lasR mutant, exoS−, exoU+) were quantified by using primer pairs specific for exoS and exoU, respectively. The relative proportions of the 2 populations are expressed as a percentage of the total number of P. aeruginosa genomic copies determined in the same DNA preparations by using the rpsL primer pair (B Lower). The proportions of clones E429 (PA0636+, PA0722−) and 239A (PA0636−, PA0722+) in patient 15108 were quantified by using primer pairs specific for the variable genes PA0636 and PA0722, respectively, and expressed as percentage of total P. aeruginosa genomic copies (C Lower). The proportions of clone OC2E (PA0636+, PA0728−) and clone 239A (PA0636−, PA0728+) in patient 15101 were quantified similarly by using primer pairs specific for variable genes PA0636 and PA0728, respectively (D Lower) (22). Because only 1 isolate was available per day, which usually corresponded to the most abundant clone, the lasR allele of the isolate from the minor population could not be assessed.

Fig. 4.

QS and virulence of clinical P. aeruginosa populations. Intubated patients were colonized by either only QS wild-type isolates (QS+, 6 patients), only QS mutant isolates (QS, 5 patients) or mixed QS wild-type and mutant populations (20 patients). VAP (vertical arrows) occurred earlier (days 4–5) in patients harboring only QS wild-type populations and later (days 9–11) in patients cocolonized by QS mutant isolates.