Novel Glycopolymer Eradicates Antibiotic- and CCCP-Induced Persister Cells in Pseudomonas aeruginosa

Abstract

Antibiotic treatments often fail to completely eradicate a bacterial infection, leaving behind an antibiotic-tolerant subpopulation of intact bacterial cells called persisters. Persisters are considered a major cause for treatment failure and are thought to greatly contribute to the recalcitrance of chronic infections. Pseudomonas aeruginosa infections are commonly associated with elevated levels of drug-tolerant persister cells, posing a serious threat to human health. This study represents the first time a novel large molecule polycationic glycopolymer, poly (acetyl, arginyl) glucosamine (PAAG), has been evaluated against antibiotic and carbonyl cyanide m-chlorophenylhydrazone induced P. aeruginosa persisters. PAAG eliminated eliminated persisters at concentrations that show no significant cytotoxicity on human lung epithelial cells. PAAG demonstrated rapid bactericidal activity against both forms of induced P. aeruginosa persister cells resulting in complete eradication of the in vitro persister cells within 24 h of treatment. PAAG demonstrated greater efficacy against persisters in vitro than antibiotics currently being used to treat persistent chronic infections such as tobramycin, colistin, azithromycin, aztreonam, and clarithromycin. PAAG caused rapid permeabilization of the cell membrane and caused significant membrane depolarization in persister cells. PAAG efficacy against these bacterial subpopulations suggests it may have substantial therapeutic potential for eliminating recurrent P. aeruginosa infections.

Keywords: PAAG; Pseudomonas aeruginosa; chronic infection; glycopolymer; persister cells.

Figures

FIGURE 1

PAAG kills stationary phase Pseudomonas aeruginosa cultures without forming persisters. Stationary phase P. aeruginosa cultures were challenged with tobramycin (16 μg/mL), clarithromycin (256 μg/mL), colistin (256 μg/mL), aztreonam (256 μg/mL), azithromycin (256 μg/mL), or PAAG (100–200 μg/mL) for 24 h. Samples were aliquoted and plated for viable plate count (CFU/mL) at multiple timepoints over 24 h. Unchallenged control samples were aliquoted directly prior to antibiotic treatment. Data represents average of three independent experiments, and the error bars indicate the standard error of the mean.

FIGURE 2

(A,B). Effect of antimicrobials against antibiotic- induced P. aeruginosa persister cells. Activity of PAAG and colistin at a concentration of 200 and 256 μg/mL, respectively, was assessed against stationary phase cultures of P. aeruginosa PA01 pretreated with tobramycin (16 μg/mL), aztreonam (256 μg/mL), or clarithromycin (256 μg/mL) for 3 h. After the addition of PAAG or colistin samples were aliquoted and plated for colony count (CFU/mL) at multiple time points over 24 h. Non-challenged control samples were plated immediately prior to PAAG treatment. T = 0 is 3 h post-antibiotic treatment. Data represents average ± standard error of the mean of three independent experiments.

FIGURE 3

Effect of CCCP to enhance persistence in stationary phase cultures of P. aeruginosa. CCCP at a concentration of 200 μg/mL was used to induce persistence was assessed by evaluating the log reduction of cells surviving the treatment with PAAG (100–200 μg/mL) tobramycin (16 μg/mL), clarithromycin (256 μg/mL), colistin (256 μg/mL), aztreonam (256 μg/mL), azithromycin (256 μg/mL) following 3 h exposure to CCCP. Data are reported as mean ± standard error of the mean of the mean of at least three independent experiments.

FIGURE 4

Effect of PAAG compared to a membrane active antibiotic against CCCP induced persisters of P. aeruginosa PA01. Activity of PAAG at concentrations of 200 and 100 μg/mL and colistin (256 μg/mL) against CCCP-induced P. aeruginosa PA01 cells pretreated with tobramycin (16 μg/mL). Anti-persister activity of PAAG was assessed by CFU counting at 10, 30 min and 1, 2, 4, 8, 24 h. Data represented as average values from three independent experiments ± standard error of the mean.

FIGURE 5

Membrane permeabilization of CCCP- induced P. aeruginosa persister cells using propidium iodide (PI) through spectrophotometry. Permeabilization of persister cells formed in stationary phase cultures of P. aeruginosa cultures were treated with ciprofloxacin (100 μg/mL), tobramycin (16 μg/mL), colistin (256 μg/mL), azithromycin (256 μg/mL), aztreonam (256 μg/mL), and clarithromycin (256 μg/mL) or PAAG (100–200 μg/mL) and measured by spectrophotometry over 4 h. PI fluorescence was measured pre- and post-treatment with antibiotics or PAAG. CCCP-induced cells with PI was used as a baseline control. The experiment was repeated with there independent cultures and the data is represented as the average ±standard error of the mean.

FIGURE 6

PAAG upon permeabilization causes depolarization of the cytoplasmic membrane. Effect of PAAG or colistin on the cytoplasmic membrane of CCCP-induced P. aeruginosa persister cells incubated with DiSC3(5). Results expressed in relative fluorescence units (RFU) observed at 670 nm as compared to negative control. 0.1% Triton X-100 was used as positive control. Cells with DiSC3(5) was used as the baseline control. Data represented as mean ± SEM of three determinations. ∗∗∗∗P < 0.001, ∗∗P < 0.01, ∗P < 0.05. The lines were used to compare the statistical significance between the different concentration of PAAG treatments.

FIGURE 7

Cytotoxicity assay showing the percent cell viability after incubating A549 cells with PAAG for 24 h. A549 cells were plated onto 96-well plate at an initial seeding density of 2.0 × 104 cells/well. PAAG was dissolved in serum-free F12K media at concentrations ranging from 65 to 1,000 μg/mL. The plates were incubated for 24 h and compound mediated cytotoxicity was determined through pierce LDH cytotoxicity assay. No significant difference compared to the control (Untreated A549 cells). 10× lysis buffer (provided by the manufacturer) was used as the positive control. Data represented as mean ± standard error of the mean. The experiment was performed in triplicate (n = 5).