Heterogenous Susceptibility to R-Pyocins in Populations of Pseudomonas aeruginosa Sourced from Cystic Fibrosis Lungs

Abstract

Bacteriocins are proteinaceous antimicrobials produced by bacteria that are active against other strains of the same species. R-type pyocins are phage tail-like bacteriocins produced by Pseudomonas aeruginosa Due to their antipseudomonal activity, R-pyocins have potential as therapeutics in infection. P. aeruginosa is a Gram-negative opportunistic pathogen and is particularly problematic for individuals with cystic fibrosis (CF). P. aeruginosa organisms from CF lung infections develop increasing resistance to antibiotics, making new treatment approaches essential. P. aeruginosa populations become phenotypically and genotypically diverse during infection; however, little is known of the efficacy of R-pyocins against heterogeneous populations. R-pyocins vary by subtype (R1 to R5), distinguished by binding to different residues on the lipopolysaccharide (LPS). Each type varies in killing spectrum, and each strain produces only one R-type. To evaluate the prevalence of different R-types, we screened P. aeruginosa strains from the International Pseudomonas Consortium Database (IPCD) and from our biobank of CF strains. We found that (i) R1-types were the most prevalent R-type among strains from respiratory sources, (ii) a large number of strains lack R-pyocin genes, and (iii) isolates collected from the same patient have the same R-type. We then assessed the impact of intrastrain diversity on R-pyocin susceptibility and found a heterogenous response to R-pyocins within populations, likely due to differences in the LPS core. Our work reveals that heterogeneous populations of microbes exhibit variable susceptibility to R-pyocins and highlights that there is likely heterogeneity in response to other types of LPS-binding antimicrobials, including phage.IMPORTANCE R-pyocins have potential as alternative therapeutics against Pseudomonas aeruginosa in chronic infection; however, little is known about the efficacy of R-pyocins in heterogeneous bacterial populations. P. aeruginosa is known to become resistant to multiple antibiotics and to evolve phenotypic and genotypic diversity over time; thus, it is particularly difficult to eradicate in chronic cystic fibrosis (CF) lung infections. In this study, we found that P. aeruginosa populations from CF lungs maintain the same R-pyocin genotype but exhibit heterogeneity in susceptibility to R-pyocins from other strains. Our findings suggest there is heterogeneity in response to other types of LPS-binding antimicrobials, such as phage, highlighting the necessity of further studying the potential of LPS-binding antimicrobial particles as alternative therapies in chronic infections.

Keywords: Pseudomonas aeruginosa; R-pyocin; bacteriocin; bacteriocins; cystic fibrosis; heterogeneity; lipopolysaccharide; pyocins.

FIG 1

R-pyocin structure. R-type specificity is determined by the sequence on the foot of the tail fiber, which is believed to recognize glycosylated structures on the outer membrane (such as the LPS). Upon binding, the sheath contracts to push the core into the cell membrane. Puncture by the tail spike depolarizes the membrane potential, leading to cell death (1–3, 12, 14, 15, 22–27, 29–36). R-pyocin structure is depicted as described in previous studies (2, 3, 11, 14–21, 27, 29–31, 35, 36).

FIG 2

R-pyocin typing of clinical P. aeruginosa strains and whole populations isolated from CF patients. (A) Using BLAST homology-based alignment, we R-pyocin typed publicly available P. aeruginosa sequences from respiratory sources of the IPCD to determine the prevalence of each pyocin type. Out of the 448 respiratory isolates in the entire database, 185 strains could not be typed. (B) Using multiplex PCR, we R-pyocin typed our own biobank of whole populations of P. aeruginosa isolated from expectorated sputum of CF patients to compare the prevalence of R-types found among CF patients in our cohort. We used 19 longitudinal samples collected from 8 patients and R-pyocin typed 183 isolates in total. We found that in our biobank, R1 was the most prevalent R-pyocin type found among CF patients in our cohort (UT, untyped R-pyocin genotype).

FIG 3

Diverse P. aeruginosa populations from CF exhibit heterogeneity in growth, morphology, and R2-pyocin susceptibility. (A) Growth rates vary between 20 isolates, each from 3 populations collected from expectorated CF sputum. Growth rate was calculated from the optical density (at 600 nm) after 16 h of growth in LB medium and normalized to PAO1. (B) The same isolates from each population are also morphologically diverse, as shown on Congo red agar medium. Both mucoid and nonmucoid isolates are found within the same populations. The populations of patients 1 and 2 were untypeable (UT) by classical R-pyocin typing methods; however, the populations of patient 3 were typed as R1 producers. (C) The diverse isolates vary in susceptibility to the R2-pyocins of PAO1, when R2-pyocins are added to the medium during growth. Each data point represents the means from three independent biological replicates for an individual isolate. PAO1 is denoted as a red star and was measured with each population as a control for R2-pyocin resistance. A fold change of 1 indicates resistance to R2-pyocin, whereas a fold change of less than 1 indicates R2-pyocin susceptibility. UT, untyped R-pyocin genotype.

FIG 4

Clinical CF isolates of P. aeruginosa from the same population exhibit differential susceptibility to R2-pyocins. (A) Three R1-type pyocin-producing clinical CF isolates from the same population (patient 3) were tested for susceptibility to spotted lysates with a standard spot assay and show differences in susceptibility to R2-pyocin lysates. Isolates 2 and 3 show zones of inhibition, indicating susceptibility. Spotted strains include the previously described CF strain A018 (R2), the laboratory strain PAO1 (R2), and a cell-free medium preparation. wt, wild type; ΔR, mutant. (B) The same three isolates were grown in lysogeny broth (LB) and treated with R2-pyocins extracted from PAO1. Each data point depicts a biological replicate (averaged from technical replicates). Log₂ fold change in number of CFU was measured at t = 0 and t = 4 h for each isolate to depict growth or death in the presence or absence of R2-pyocins. Two-tailed, paired Student’s t-test between treatments confirm that isolates 2 and 3 are susceptible to R2-pyocins (**, P = 0.005), while isolate 1 is resistant (ns, not significant). A one-way ANOVA and Tukey-Kramer post hoc test determined that isolates 2 and 3 significantly decrease in CFU numbers from isolate 1 when treated with R2-pyocins (P < 0.05; not shown).

FIG 5

Alginate production and LPS characterization of three CF isolates of P. aeruginosa from the same population. (A) Alginate was isolated and quantified from clinical isolates 1 to 3 (of patient 3). Susceptibility to R2-pyocins is denoted by R (resistance) or S (susceptible). Alginate concentrations measured from three biological replicates are represented by the mean value, and error bars depict standard errors of the means (SEM). The mean alginate concentrations from each isolate were determined by comparison to a standard curve generated and analyzed with a one-way ANOVA and Tukey-Kramer post hoc test. Isolate 3 exhibited a mucoid morphology and produces more alginate (P < 0.025), although it is still susceptible to R2-pyocins, while isolates 1 and 2 did not produce detectable levels of alginate (<50 μg/ml) and differ in R2-pyocin susceptibility. (B) The three isolates each exhibit different LPS phenotypes; isolate 1 presents neither A band nor B band and has a different core than PAO1. Isolate 2 also does not present A band or B band but has a core similar to those of isolate 3 and PAO1. Isolate 3 presents a normal core and presents A-band antigen. mw, molecular weight.

FIG 6

Whole-genome analysis of three CF isolates of P. aeruginosa from the same population. (A) Three R1-type pyocin-producing clinical CF isolates from the same population were sequenced and aligned to a PAO1 reference genome for comparison. For simplicity, only annotated single-nucleotide variants (SNVs) unique to the R2-pyocin-resistant isolate 1 are depicted. (B) Single-nucleotide variant lists between each of the isolates when mapped to PAO1 were compared and revealed that approximately 30,127 SNVs distinguished this population from the laboratory strain PAO1. Out of the 30,646 SNVs between isolate 1 and PAO1, 232 were unique to isolate 1 compared to isolates 2 and 3 from the same population.