Loci Encoding Compounds Potentially Active against Drug-Resistant Pathogens amidst a Decreasing Pool of Novel Antibiotics

Abstract

Since the discovery of penicillin, microbes have been a source of antibiotics that inhibit the growth of pathogens. However, with the evolution of multidrug-resistant (MDR) strains, it remains unclear if there is an abundant or limited supply of natural products to be discovered that are effective against MDR isolates. To identify strains that are antagonistic to pathogens, we examined a set of 471 globally derived environmental Pseudomonas strains (env-Ps) for activity against a panel of 65 pathogens including Achromobacter spp., Burkholderia spp., Pseudomonas aeruginosa, and Stenotrophomonas spp. isolated from the lungs of cystic fibrosis (CF) patients. From more than 30,000 competitive interactions, 1,530 individual inhibitory events were observed. While strains from water habitats were not proportionate in antagonistic activity, MDR CF-derived pathogens (CF-Ps) were less susceptible to inhibition by env-Ps, suggesting that fewer natural products are effective against MDR strains. These results advocate for a directed strategy to identify unique drugs. To facilitate discovery of antibiotics against the most resistant pathogens, we developed a workflow in which phylogenetic and antagonistic data were merged to identify strains that inhibit MDR CF-Ps and subjected those env-Ps to transposon mutagenesis. Six different biosynthetic gene clusters (BGCs) were identified from four strains whose products inhibited pathogens including carbapenem-resistant P. aeruginosa BGCs were rare in databases, suggesting the production of novel antibiotics. This strategy can be utilized to facilitate the discovery of needed antibiotics that are potentially active against the most drug-resistant pathogens.IMPORTANCE Carbapenem-resistant P. aeruginosa is difficult to treat and has been deemed by the World Health Organization as a priority one pathogen for which antibiotics are most urgently needed. Although metagenomics and bioinformatic studies suggest that natural bacteria remain a source of novel compounds, the identification of genes and their products specific to activity against MDR pathogens remains problematic. Here, we examine water-derived pseudomonads and identify gene clusters whose compounds inhibit CF-derived MDR pathogens, including carbapenem-resistant P. aeruginosa.

Keywords: Pseudomonas; antagonistic; antibiotic; biosynthetic gene cluster; multidrug resistance; transposon mutagenesis.

FIG 1

Phylogenetic analysis and antagonistic activity among env-Ps. Population structure for 471 environmental pseudomonads by neighbor-joining analysis of the gyrB sequence, merged with data for habitat (inner bars: purple, United States; dark blue, Germany; light blue, Hungary) and antagonistic activity (outer bars; black and red) against 65 CF-Ps. The magnitude of antagonism is indicated by bar height. Strains that inhibit more than nine pathogens are indicated by black bars. Populations are shaded and numbered 1 to 11. Tn mutagenesis was used to identify BGCs involved in antagonistic activity in strains 06C126, 09C129, LG1D9, and LH1G9 (indicated by the green arrows).

FIG 2

Antagonistic events of env-Ps against CF-Ps. A competition plate assay was used to determine antagonistic activity (inset). A total of 471 env-Ps were competed against 65 CF-Ps that resulted in 30,615 individual competitions. Of the 1,530 antagonistic events observed, activity originated from 579 U.S. (purple bars), 359 German (dark-blue bars), and 592 Hungarian (light-blue bars) env-Ps that inhibited the growth of nine Achromobacter, 19 Burkholderia, 31 P. aeruginosa, and three Stenotrophomonas strains. Black arrows indicate activity by three antagonistic strains. Only one Burkholderia strain and one P. aeruginosa strain were not inhibited by any env-P. P. aeruginosa strains were tested for susceptibility against eight antibiotics using the Kirby-Bauer disk assay. Numbers above the antagonistic bar data represent the number of drugs a particular pathogen resists, indicated in Table 1. Linear regression analysis showed that the number of antagonistic events to a pathogen was inversely related to antibiotic resistance.

FIG 3

Tn insertions were identified in strains 06C126, LH1G9, 09C129, and LG1D9. (A to C) Tn-mutated BGCs with JGI ID numbers 161819466 (A) and 161819467 (B) were identified in strain 02C26, and BGC 161848994 (C) was identified in LH1G9. All three loci were predicted to encode a nonribosomal peptide. (D and E) In 09C129, BGCs 161816930 (D) and 161816936 (E) were identified and predicted to encode a phenazine and nonribosomal peptide, respectively. (F) With strain LG1D9, four Tn insertions were identified in genes that were not predicted to be a BGC. ORFs are represented by solid-color filled arrows; different colors represent dissimilar proteins (listed in Tables S4 to S9). Right and left pointed arrows signify loci on forward and reverse DNA strands, respectively. Red lines indicate the position of the Tn insertion. Predicted compound structures follow each BGC and were determined using PRISM.

FIG 4

Predicted domains of NRPSs in env-Ps. (A and B) Strain 02C26 has two BGCs which were each predicted to encode NRPSs, as follows: ORF 23 was identified in BGC with JGI ID 161819466 (A) and ORFs 20, 22, and 23 were identified in BGC 161819467 (B). (C) Strain LH1G9 has two loci that encode an NRPS which were identified in ORFs 14 and 15 of BGC 161848994. (D) In strain 09C129, ORF 16 was found in BGC 161816936 and was predicted to encode an NRPS with associated ORFs 11 to 13 and 18 to 20. NRPS domains are shown as individual circles with predicted functions involved in condensation (C; in pink), adenylation (OHBu, 3-hydroxybutanoic acid; Dab, 2,4-diamino-butyric acid; and OHOm, N⁵-hydroxy-l-ornithine; in red), thiolation (T; in gray), epimerization (E; in dark red), and thioesterase (TE; in black). Other domains identified were antibiotic resistance (AMR235, MacB subunit of efflux pump; AMR262, MatE: efflux protein; and AMR116, puromycin major facilitator superfamily [MFS] transporter; in maroon), and dehydratase (DH; blue). Numbered ORFs correspond to Tables S4, S5, S6, and S8.

FIG 5

Heat map of similar Pfam protein families in the JGI ABC to 02C26, LH1G9, 09C129, and LG1D9 BGCs. The 95 most similar BGCs were identified and used to show similarities and differences within each cluster. Five clades were generated in the analysis, C1 to C5. The JGI ABC Pfam designations are listed on the top x axis and correspond to Table S11. The heat map color shading represents the number of each Pfam protein family in a BGC. Unique, single, and multiple protein families in a BGC range from 0 to 5, with yellow representing 0 protein families and lightest green to darkest green representing 1 to 5 protein families. The left y axis consists of BGC phylogeny determined by number and content of similar protein families. Tree branch lengths were determined using the Jaccard index scores of all protein families. The right y axis lists strains, with 02C26, LH1G9, and 09C129 in red; their BGC is boxed with a black dashed line.