Intraclonal competitive fitness of longitudinal cystic fibrosis Pseudomonas aeruginosa airway isolates in liquid cultures

Abstract

The metabolically versatile Pseudomonas aeruginosa inhabits biotic and abiotic environments including the niche of cystic fibrosis (CF) airways. This study investigated how the adaptation to CF lungs affects the within-clone fitness of P. aeruginosa to grow and persist in liquid cultures in the presence of the clonal ancestors. Longitudinal clonal P. aeruginosa isolates that had been collected from 12 CF donors since the onset of colonization for up to 30 years was subjected to within-clone competition experiments. The relative quantities of individual strains were determined by marker-free amplicon sequencing of multiplex PCR products of strain-specific nucleotide sequence variants, a novel method that is generally applicable to studies in evolutionary genetics and microbial ecology with real-world strain collections. For 10 of the 12 examined patient courses, P. aeruginosa isolates of the first years of colonization grew faster in the presence of their clonal progeny than alone. Single growth of individual strains showed no temporal trend with colonization time, but in co-culture, the early isolates out-competed their clonal progeny. Irrespective of the genetic make-up of the clone and its genomic microevolution in CF lungs, the early isolates expressed fitness traits to win the within-clone competition that were absent in their progeny.

From genotype to phenotype: adaptations of Pseudomonas aeruginosa to the cystic fibrosis environment

“…This adjusted metabolism increases P. aeruginosa fitness in the CF environment, but it often results in a slowed growth in laboratory conditions in comparison to non-adapted isolates [7, 8, 12, 13, 18, 107, 110, 114]. This modification of metabolic activities can limit effective detection and treatment of infecting P.…”

“…Development of new metabolic capacities can nonetheless arise through enrichment of the accessory genome in metabolic functions [17,28,60]. This adjusted metabolism increases P. aeruginosa fitness in the CF environment, but it often results in a slowed growth in laboratory conditions in comparison to nonadapted isolates [7,8,12,13,18,107,110,114]. This modification of metabolic activities can limit effective detection and treatment of infecting P. aeruginosa, as illustrated by the emergence of highly resistant small colony variants (SCVs) and viable but non-culturable (VBNC) isolates [115][116][117].…”

Genome-Based Analyses of Fitness Effects and Compensatory Changes Associated with Acquisition of blaCMY-, blaCTX-M-, and blaOXA-48/VIM-1-Containing Plasmids in Escherichia coli

“…Performing competitive growth experiments with progenitor and descendent strain variants in the same experimental setting, such as a combination of recipient and transconjugant cells, allow determining an immediate and interconnected fitness effect and measuring much lower fitness effects [ 28 , 29 , 30 ]. However, in a setting as described here, where conjugative plasmid transfer appears in liquid broth to a substantial amount, measuring growth rates in competitive experiments will always assess effects derived from de novo plasmid transfer and transconjugant propagation as well.…”

Competitive fitness of Pseudomonas aeruginosa isolates in human and murine precision-cut lung slices

“…Based on measurements of the optical density, the serial isolates from early exponential phase were mixed equally (total OD 0.6), and subsequently mPCLS or hPCLS were infected with 50 µl of this bacterial mixture in triplicate (final bacterial concentration 1x10 5 CFU determined to be optimal for harvesting sufficient amounts of bacterial DNA for multiplex PCR in a large background of mammalian DNA without compromising PCLS vitality until the endpoint). The linear correlation between CFU and OD measurements during early exponential phase had been validated in preliminary experiments (Cramer et al, 2020). Prior to inoculation, CFUs of the mixture were assessed by plating on LB agar plates, whereby one aliquot was used for the CFU determination and another aliquot for DNA preparation and subsequent sequencing.…”

“…Next, the sum of all isolates was normalised to 100% within the longitudinal course. The relative changes from 0 h -4 h and from 0 h -8 h and the mean percentage of the three technical replicates (± 2 s) were calculated (if at least two of the three technical replicates showed results) as previously described in Cramer et al, 2020. The relative quantity of a strain at 4 h and 8 h compared to 0 h was visualized by the mean and range of the three replicates in a two-dimensional plot. Outliers were eliminated if individual values of strain-specific SNPs exceeded the range of mean ± 2 s. If the mean number of reads assigned to a strain was below 0.5% of the amplicon reads in at least two of three technical replicates, the bacterial strain was classified as 'detectable, but not quantifiable' (marked without a black line in Figure 2).…”