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.

Antibiotic discovery throughout the Small World Initiative: A molecular strategy to identify biosynthetic gene clusters involved in antagonistic activity

“…For our SWI research strategy, pseudomonads were chosen as a model organism to pursue antibiotic discovery because of their high levels of genomic diversity (Gross & Loper, ; Silby, Winstanley, Godfrey, Levy, & Jackson, ) and persistence in diverse habitats such as soil (Chatterjee et al., ; Morris, Monteil, & Berge, ), in association with plants (Berendsen, Pieterse, & Bakker, ; Bulgarelli et al., ; Loper et al., ), and within freshwater ecosystems (Chatterjee et al., ; D'souza et al., ; Morris et al., ). Given the unique physical state of these distinct habitats, we reason that strains adapted to different environments should maintain unique metabolic pathways capable of producing diverse secondary metabolites that may exhibit antimicrobial effects against other bacteria.…”

“…Traditional SWI workflow utilizes chemical approaches to extract inhibitory compounds from antagonistic strains for their subsequent characterization. Previously, we optimized molecular methods for the identification of BGCs involved in antagonistic activity among pathogenic P. aeruginosa strains from cystic fibrosis patients (Chatterjee et al., ). We adapted this methodology, involving transposon (Tn) mutagenesis using the pBAM1 vector (Martinez‐Garcia et al., ), to SWI in order to expand the process of antibiotic discovery by identifying and characterizing BGCs involved in antagonistic activities.…”

“…Using the adapted molecular workflow, undergraduates performed Tn mutagenesis and screened ~10,000 mutants, ultimately identifying one that lost the ability to inhibit the growth of the SR B. subtilis (Figure d) and human pathogens B. cereus , L. monocytogenes , and MRSA (Figure a). Thus, the scientific rigor of this approach is made evident through the identified antagonistic strains and the generation of a loss of inhibition phenotype mutant for novel BGC discovery (Chatterjee et al., ).…”

“…Thus, the scientific rigor of this approach is made evident through the identified antagonistic strains and the generation of a loss of inhibition phenotype mutant for novel BGC discovery (Chatterjee et al, 2017).…”

Environmental vibrios represent a source of antagonistic compounds that inhibit pathogenic Vibrio cholerae and Vibrio parahaemolyticus strains

“…The BGCs encoding the putative NRPs and PKSs exhibited the most diversity, and the PKS in strain 10N.222.47.A9 was non-homologous to all other BGCs. Bacteriocins (Carraturo, Raieta, Ottaviani, & Russo, 2006;Prasad, Morris, Hansen, Meaden, & Austin, 2005), NRPSs (Cordero, Wildschutte, et al, 2012), PKSs (Liaw et al, 2015), and siderophores (Chatterjee et al, 2017) are compounds that have been shown to exhibit antagonistic activity and these BGCs may contribute to the inhibition observed by the 10 strains; moreover, few genes in these cluster have similarity to other known loci (Table 4) suggesting these BGCs may encode novel products. Although these results identify putative regions encoding antagonistic factors on the chromosome, it is also possible that genes encoding inhibitory products originate on plasmids that would be undetected in our genomic analysis.…”