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Human Granulocyte Macrophage Colony-Stimulating Factor Enhances Antibiotic Susceptibility of Pseudomonas aeruginosa Persister Cells.

Choudhary GS, Yao X, Wang J, Peng B, Bader RA, Ren D - Sci Rep (2015)

Bottom Line: The DNA microarray and qPCR results indicated that GM-CSF induced the genes for flagellar motility and pyocin production in the persister cells, but not the normal cells of P. aeruginosa PAO1.Consistently, the supernatants from GM-CSF treated P. aeruginosa PAO1 persister cell suspensions were found cidal to the pyocin sensitive strain P. aeruginosa PAK.Collectively, these findings suggest that host immune factors and bacterial persisters may directly interact, leading to enhanced susceptibility of persister cells to antibiotics.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical and Chemical Engineering, Syracuse University, Syracuse, NY 13244, USA.

ABSTRACT
Bacterial persister cells are highly tolerant to antibiotics and cause chronic infections. However, little is known about the interaction between host immune systems with this subpopulation of metabolically inactive cells, and direct effects of host immune factors (in the absence of immune cells) on persister cells have not been studied. Here we report that human granulocyte macrophage-colony stimulating factor (GM-CSF) can sensitize the persister cells of Pseudomonas aeruginosa PAO1 and PDO300 to multiple antibiotics including ciprofloxacin, tobramycin, tetracycline, and gentamicin. GM-CSF also sensitized the biofilm cells of P. aeruginosa PAO1 and PDO300 to tobramycin in the presence of biofilm matrix degrading enzymes. The DNA microarray and qPCR results indicated that GM-CSF induced the genes for flagellar motility and pyocin production in the persister cells, but not the normal cells of P. aeruginosa PAO1. Consistently, the supernatants from GM-CSF treated P. aeruginosa PAO1 persister cell suspensions were found cidal to the pyocin sensitive strain P. aeruginosa PAK. Collectively, these findings suggest that host immune factors and bacterial persisters may directly interact, leading to enhanced susceptibility of persister cells to antibiotics.

No MeSH data available.


Related in: MedlinePlus

Alginate lyase is required for the activity of GM-CSF against persister cells of the mucoid strain P. aeruginosa PDO300.The persister cells were isolated from exponential phase cultures and GM-CSF was tested at 0.17 pM. The viability of persister cells treated with tobramycin (200 μg/mL) alone, tobramycin along with alginate lyase (50 μg/mL), tobramycin along with GM-CSF, or tobramycin along with alginate lyase and GM-CSF is shown. The amount of BSA (0.1%) was adjusted to be the same for all samples. Following the treatment, the viability of persister cells was determined by counting CFU. Tob: tobramycin. AL: alginate lyase. The samples were tested in triplicate (n = 3). Error bars represent SD; *p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA followed by Tukey test.
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f3: Alginate lyase is required for the activity of GM-CSF against persister cells of the mucoid strain P. aeruginosa PDO300.The persister cells were isolated from exponential phase cultures and GM-CSF was tested at 0.17 pM. The viability of persister cells treated with tobramycin (200 μg/mL) alone, tobramycin along with alginate lyase (50 μg/mL), tobramycin along with GM-CSF, or tobramycin along with alginate lyase and GM-CSF is shown. The amount of BSA (0.1%) was adjusted to be the same for all samples. Following the treatment, the viability of persister cells was determined by counting CFU. Tob: tobramycin. AL: alginate lyase. The samples were tested in triplicate (n = 3). Error bars represent SD; *p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA followed by Tukey test.

Mentions: To understand if GM-CSF also affects the mucoid strains of P. aeruginosa, the persister cells of P. aerugionsa PDO300 (henceforth PDO300) isolated from exponential phase cultures were tested following the same protocol used for PAO1 cells. Similar to the results of the wild-type PAO1, treatment with 0.17 pM GM-CSF did not change the viability of PDO300 persister cells (p = 0.77), but sensitized 40.5 ± 18.6% (p = 0.04) of persister cells to 200 μg/mL tetracycline, compared to treatment with tetracycline alone. The decrease in activities of GM-CSF against PDO300 persister cells compared to PAO1 is probably due to the presence of its alginate layer since when 50 μg/mL alginate lyase was added, the reduction in the viability by 200 μg/mL tobramycin and 0.17 pM GM-CSF increased significantly (Fig. 3). Specifically, co-treatment with tobramycin, GM-CSF, and alginate lyase led to killing of persister cells that was 66.9 ± 12.4% (p = 0.0002), 55.7 ± 16.5% (p = 00031), and 64.6 ± 13.2% (p = 0.0003) more than killing by tobramycin alone, tobramycin + GM-CSF, and tobramycin + alginate lyase, respectively (Fig. 3). Prior to the experiment, we had verified that 10, 50, 100, and 200 μg/mL of alginate lyase itself had insignificant (p > 0.05) effect on persister cells of P. aeruginosa PDO300. Overall, these results indicate that GM-CSF can also sensitize persister cells of the mucoid strain P. aeruginosa PDO300 to tobramycin if alginate lyase is present. We believe this is because alginate lyase can help GM-CSF penetrate alginate, as shown in our recent Western results34.


Human Granulocyte Macrophage Colony-Stimulating Factor Enhances Antibiotic Susceptibility of Pseudomonas aeruginosa Persister Cells.

Choudhary GS, Yao X, Wang J, Peng B, Bader RA, Ren D - Sci Rep (2015)

Alginate lyase is required for the activity of GM-CSF against persister cells of the mucoid strain P. aeruginosa PDO300.The persister cells were isolated from exponential phase cultures and GM-CSF was tested at 0.17 pM. The viability of persister cells treated with tobramycin (200 μg/mL) alone, tobramycin along with alginate lyase (50 μg/mL), tobramycin along with GM-CSF, or tobramycin along with alginate lyase and GM-CSF is shown. The amount of BSA (0.1%) was adjusted to be the same for all samples. Following the treatment, the viability of persister cells was determined by counting CFU. Tob: tobramycin. AL: alginate lyase. The samples were tested in triplicate (n = 3). Error bars represent SD; *p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA followed by Tukey test.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4663479&req=5

f3: Alginate lyase is required for the activity of GM-CSF against persister cells of the mucoid strain P. aeruginosa PDO300.The persister cells were isolated from exponential phase cultures and GM-CSF was tested at 0.17 pM. The viability of persister cells treated with tobramycin (200 μg/mL) alone, tobramycin along with alginate lyase (50 μg/mL), tobramycin along with GM-CSF, or tobramycin along with alginate lyase and GM-CSF is shown. The amount of BSA (0.1%) was adjusted to be the same for all samples. Following the treatment, the viability of persister cells was determined by counting CFU. Tob: tobramycin. AL: alginate lyase. The samples were tested in triplicate (n = 3). Error bars represent SD; *p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA followed by Tukey test.
Mentions: To understand if GM-CSF also affects the mucoid strains of P. aeruginosa, the persister cells of P. aerugionsa PDO300 (henceforth PDO300) isolated from exponential phase cultures were tested following the same protocol used for PAO1 cells. Similar to the results of the wild-type PAO1, treatment with 0.17 pM GM-CSF did not change the viability of PDO300 persister cells (p = 0.77), but sensitized 40.5 ± 18.6% (p = 0.04) of persister cells to 200 μg/mL tetracycline, compared to treatment with tetracycline alone. The decrease in activities of GM-CSF against PDO300 persister cells compared to PAO1 is probably due to the presence of its alginate layer since when 50 μg/mL alginate lyase was added, the reduction in the viability by 200 μg/mL tobramycin and 0.17 pM GM-CSF increased significantly (Fig. 3). Specifically, co-treatment with tobramycin, GM-CSF, and alginate lyase led to killing of persister cells that was 66.9 ± 12.4% (p = 0.0002), 55.7 ± 16.5% (p = 00031), and 64.6 ± 13.2% (p = 0.0003) more than killing by tobramycin alone, tobramycin + GM-CSF, and tobramycin + alginate lyase, respectively (Fig. 3). Prior to the experiment, we had verified that 10, 50, 100, and 200 μg/mL of alginate lyase itself had insignificant (p > 0.05) effect on persister cells of P. aeruginosa PDO300. Overall, these results indicate that GM-CSF can also sensitize persister cells of the mucoid strain P. aeruginosa PDO300 to tobramycin if alginate lyase is present. We believe this is because alginate lyase can help GM-CSF penetrate alginate, as shown in our recent Western results34.

Bottom Line: The DNA microarray and qPCR results indicated that GM-CSF induced the genes for flagellar motility and pyocin production in the persister cells, but not the normal cells of P. aeruginosa PAO1.Consistently, the supernatants from GM-CSF treated P. aeruginosa PAO1 persister cell suspensions were found cidal to the pyocin sensitive strain P. aeruginosa PAK.Collectively, these findings suggest that host immune factors and bacterial persisters may directly interact, leading to enhanced susceptibility of persister cells to antibiotics.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical and Chemical Engineering, Syracuse University, Syracuse, NY 13244, USA.

ABSTRACT
Bacterial persister cells are highly tolerant to antibiotics and cause chronic infections. However, little is known about the interaction between host immune systems with this subpopulation of metabolically inactive cells, and direct effects of host immune factors (in the absence of immune cells) on persister cells have not been studied. Here we report that human granulocyte macrophage-colony stimulating factor (GM-CSF) can sensitize the persister cells of Pseudomonas aeruginosa PAO1 and PDO300 to multiple antibiotics including ciprofloxacin, tobramycin, tetracycline, and gentamicin. GM-CSF also sensitized the biofilm cells of P. aeruginosa PAO1 and PDO300 to tobramycin in the presence of biofilm matrix degrading enzymes. The DNA microarray and qPCR results indicated that GM-CSF induced the genes for flagellar motility and pyocin production in the persister cells, but not the normal cells of P. aeruginosa PAO1. Consistently, the supernatants from GM-CSF treated P. aeruginosa PAO1 persister cell suspensions were found cidal to the pyocin sensitive strain P. aeruginosa PAK. Collectively, these findings suggest that host immune factors and bacterial persisters may directly interact, leading to enhanced susceptibility of persister cells to antibiotics.

No MeSH data available.


Related in: MedlinePlus