Environmental Pseudomonads Inhibit Cystic Fibrosis Patient-Derived Pseudomonas aeruginosa

Abstract

Pseudomonas aeruginosa is an opportunistic pathogen which is evolving resistance to many currently used antibiotics. While much research has been devoted to the roles of pathogenic P. aeruginosa in cystic fibrosis (CF) patients, less is known of its ecological properties. P. aeruginosa dominates the lungs during chronic infection in CF patients, yet its abundance in some environments is less than that of other diverse groups of pseudomonads. Here, we sought to determine if clinical isolates of P. aeruginosa are vulnerable to environmental pseudomonads that dominate soil and water habitats in one-to-one competitions which may provide a source of inhibitory factors. We isolated a total of 330 pseudomonads from diverse habitats of soil and freshwater ecosystems and competed these strains against one another to determine their capacity for antagonistic activity. Over 900 individual inhibitory events were observed. Extending the analysis to P. aeruginosa isolates revealed that clinical isolates, including ones with increased alginate production, were susceptible to competition by multiple environmental strains. We performed transposon mutagenesis on one isolate and identified an ∼14.8-kb locus involved in antagonistic activity. Only two other environmental isolates were observed to carry the locus, suggesting the presence of additional unique compounds or interactions among other isolates involved in outcompeting P. aeruginosa This collection of strains represents a source of compounds that are active against multiple pathogenic strains. With the evolution of resistance of P. aeruginosa to currently used antibiotics, these environmental strains provide opportunities for novel compound discovery against drug-resistant clinical strains.

Importance: We demonstrate that clinical CF-derived isolates of P. aeruginosa are susceptible to competition in the presence of environmental pseudomonads. We observed that many diverse environmental strains exhibited varied antagonistic profiles against a panel of clinical P. aeruginosa isolates, suggesting the presence of distinct mechanisms of inhibition among these ecological strains. Understanding the properties of these antagonistic events offers the potential for discoveries of antimicrobial compounds or metabolic pathways important to the development of novel treatments for P. aeruginosa infections.

Keywords: Pseudomonas aeruginosa; antagonistic; kynurenine; pseudomonads; siderophore; thioquinolobactin.

FIG 1

Phylogenetic analysis and antagonistic activity among environmental pseudomonads. Population structure for 330 environmental pseudomonads by neighbor-joining analysis of the gyrB sequence, overlain with data for habitat (inner columns: cream color, soil; blue, water) and antagonistic activity (outer bars: black). The magnitude of antagonism is indicated by bar height. Populations are shaded and numbered. Population 6 (highlighted in yellow) is enriched with strains that antagonize multiple environmental and CF-derived isolates. Strain 06C 126 was used for transposon mutagenesis (indicated by the green arrow). Red squares indicate inhibition of CF-derived P. aeruginosa. Dotted gray lines delineate population boundaries. The top x axis consists of the pathogenic P. aeruginosa strain phylogeny based on the gyrB gene sequence. Both trees were rooted by P. aeruginosa PAO1. The 0.1 and 0.001 represent bar scale values of diversity among environmental and clinical strains, respectively.

FIG 2

Competition plate assay used to determine antagonistic activity among isolates. One strain was spread plated, and 96 other isolates were replicated to it. Strains were cogrown and observed for competitive interactions indicated by a zone of clearing (indicated by arrows). (A and B) Inhibition by water-derived isolates on an environmental (A) or pathogenic (B) P. aeruginosa strain.

FIG 3

Isolates from water and soil environments were competed against intrahabitat strains in a pairwise fashion, as shown in Fig. 2A. (A) A total of 691 antagonistic events (positive events) were observed among 82 water isolates; strain 06C 126 from population 6 antagonized 102 other strains. From all antagonistic events, 146 strains were susceptible to killing (negative events). The mean number of antagonistic events per strains was 4.0. (B) Among soil-derived strains, 295 antagonistic events were observed among 88 isolates (positive events), and 116 were susceptible to killing (negative events). The mean number of antagonistic events per strains was 2.1.

FIG 4

Pathogenic P. aeruginosa isolates antagonized by environmental strains. The competition plate assay was used to determine antagonistic activity. In all, 263 inhibitory events were observed from 88 and 67 water- and soil-derived strains, respectively. One hundred sixty-nine and 94 events were from water-derived (black bars) and soil-derived (gray bars) strains, respectively. Twenty-six out of 33 P. aeruginosa isolates were inhibited by at least one strain, and 23 P. aeruginosa isolates were inhibited by more than one environmental strain. The mean numbers of inhibitory events from water and soil strains were 5.1 and 2.8, respectively.

FIG 5

Loss of antagonistic phenotype by transposon insertion in the quinolobactin gene region in Pseudomonas strain 06C 126. (A and B) Wild-type (A) and mutant (B) strains showing a loss-of-inhibition phenotype. (C) The position of the transposon insertion in the qbsL gene is indicated by the arrow. The qbs locus nucleotide sequence is 99% similar to the region found in P. fluorescens ATCC 17400. The qbsF and qbsH genes that are involved in the kynurenine pathway are homologous to kynA and kynB in P. aeruginosa PAO1, with 72% and 77% nucleotide similarity, respectively.