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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

GM-CSF enhanced the killing of P. aeruginosa PDO300 and PAO1 biofilm cells by tobramycin in the presence of alginate lyase and DNase I respectively.(a) P. aeruginosa PDO300 cells in 24 h biofilms were treated with (i) 0.17 pM GM-CSF alone, (ii) 200 μg/mL tobramycin alone, (iii) 0.17 pM GM-CSF and 50 μg/mL alginate lyase, (iv) 200 μg/mL tobramycin and 50 μg/mL alginate lyase, (v) 0.17 pM GM-CSF and 200 μg/mL tobramycin, and (vi) 0.17 pM GM-CSF, 200 μg/mL tobramycin, and 50 μg/mL alginate lyase, for 3.5 h. Tob: tobramycin. AL: alginate lyase. (b) P. aeruginosa PAO1 cells in early biofilms (4 h after inoculation) were treated with (i) 0.17 pM GM-CSF alone, (ii) 20 μg/mL tobramycin alone, (iii) 0.17 pM GM-CSF and 5 units/mL DNase I, (iv) 20 μg/mL tobramycin and 5 units/mL DNase I, (v) 0.17 pM GM-CSF and 20 μg/mL tobramycin, and (vi) 0.17 pM GM-CSF, 20 μg/mL tobramycin, and 5 units/mL DNase I for 3.5 h. For both PAO1 and PDO300 biofilm cells, the amount of BSA (0.1%) was adjusted to be the same for all samples. Following the treatment, the viability of biofilm cells was determined by counting CFU. 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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f4: GM-CSF enhanced the killing of P. aeruginosa PDO300 and PAO1 biofilm cells by tobramycin in the presence of alginate lyase and DNase I respectively.(a) P. aeruginosa PDO300 cells in 24 h biofilms were treated with (i) 0.17 pM GM-CSF alone, (ii) 200 μg/mL tobramycin alone, (iii) 0.17 pM GM-CSF and 50 μg/mL alginate lyase, (iv) 200 μg/mL tobramycin and 50 μg/mL alginate lyase, (v) 0.17 pM GM-CSF and 200 μg/mL tobramycin, and (vi) 0.17 pM GM-CSF, 200 μg/mL tobramycin, and 50 μg/mL alginate lyase, for 3.5 h. Tob: tobramycin. AL: alginate lyase. (b) P. aeruginosa PAO1 cells in early biofilms (4 h after inoculation) were treated with (i) 0.17 pM GM-CSF alone, (ii) 20 μg/mL tobramycin alone, (iii) 0.17 pM GM-CSF and 5 units/mL DNase I, (iv) 20 μg/mL tobramycin and 5 units/mL DNase I, (v) 0.17 pM GM-CSF and 20 μg/mL tobramycin, and (vi) 0.17 pM GM-CSF, 20 μg/mL tobramycin, and 5 units/mL DNase I for 3.5 h. For both PAO1 and PDO300 biofilm cells, the amount of BSA (0.1%) was adjusted to be the same for all samples. Following the treatment, the viability of biofilm cells was determined by counting CFU. 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 is also effective against P. aeruginosa biofilm cells, the 24 h biofilm cells of PAO1 and PDO300 were treated with GM-CSF in the presence and absence of antibiotics. Treatment with 0.17 pM GM-CSF alone did not change the viability of biofilm cells of either strain. Since alginate can hinder the penetration of GM-CSF34. Alginate lyase was added at 50 μg/mL in addition to 200 μg/mL tobramycin and 0.17 pM GM-CSF to test the effects of GM-CSF on biofilm cells. Neither GM-CSF nor alginate lyase alone killed biofilm cells of P. aeruginosa PAO1 and PDO300 significantly in the absence of antibiotic. However, co-treatment with 50 μg/mL alginate lyase, 0.17 pM GM-CSF, and 200 μg/mL tobramycin reduced the viability of PDO300 biofilm cells by 97.2 ± 0.4% (35.4-fold reduction of viability; p = 0.0002) compared to the control biofilms without any treatment (Fig. 4a). This is 61.3 ± 6.0% (p = 0.03) more killing than that achieved by treatment with tobramycin alone, and 57.1 ± 6.6% more than treatment with tobramycin and alginate lyase together (Fig. 4a). Thus, GM-CSF is effective against PDO300 biofilm cells if the biofilm matrix is degraded.


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)

GM-CSF enhanced the killing of P. aeruginosa PDO300 and PAO1 biofilm cells by tobramycin in the presence of alginate lyase and DNase I respectively.(a) P. aeruginosa PDO300 cells in 24 h biofilms were treated with (i) 0.17 pM GM-CSF alone, (ii) 200 μg/mL tobramycin alone, (iii) 0.17 pM GM-CSF and 50 μg/mL alginate lyase, (iv) 200 μg/mL tobramycin and 50 μg/mL alginate lyase, (v) 0.17 pM GM-CSF and 200 μg/mL tobramycin, and (vi) 0.17 pM GM-CSF, 200 μg/mL tobramycin, and 50 μg/mL alginate lyase, for 3.5 h. Tob: tobramycin. AL: alginate lyase. (b) P. aeruginosa PAO1 cells in early biofilms (4 h after inoculation) were treated with (i) 0.17 pM GM-CSF alone, (ii) 20 μg/mL tobramycin alone, (iii) 0.17 pM GM-CSF and 5 units/mL DNase I, (iv) 20 μg/mL tobramycin and 5 units/mL DNase I, (v) 0.17 pM GM-CSF and 20 μg/mL tobramycin, and (vi) 0.17 pM GM-CSF, 20 μg/mL tobramycin, and 5 units/mL DNase I for 3.5 h. For both PAO1 and PDO300 biofilm cells, the amount of BSA (0.1%) was adjusted to be the same for all samples. Following the treatment, the viability of biofilm cells was determined by counting CFU. 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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f4: GM-CSF enhanced the killing of P. aeruginosa PDO300 and PAO1 biofilm cells by tobramycin in the presence of alginate lyase and DNase I respectively.(a) P. aeruginosa PDO300 cells in 24 h biofilms were treated with (i) 0.17 pM GM-CSF alone, (ii) 200 μg/mL tobramycin alone, (iii) 0.17 pM GM-CSF and 50 μg/mL alginate lyase, (iv) 200 μg/mL tobramycin and 50 μg/mL alginate lyase, (v) 0.17 pM GM-CSF and 200 μg/mL tobramycin, and (vi) 0.17 pM GM-CSF, 200 μg/mL tobramycin, and 50 μg/mL alginate lyase, for 3.5 h. Tob: tobramycin. AL: alginate lyase. (b) P. aeruginosa PAO1 cells in early biofilms (4 h after inoculation) were treated with (i) 0.17 pM GM-CSF alone, (ii) 20 μg/mL tobramycin alone, (iii) 0.17 pM GM-CSF and 5 units/mL DNase I, (iv) 20 μg/mL tobramycin and 5 units/mL DNase I, (v) 0.17 pM GM-CSF and 20 μg/mL tobramycin, and (vi) 0.17 pM GM-CSF, 20 μg/mL tobramycin, and 5 units/mL DNase I for 3.5 h. For both PAO1 and PDO300 biofilm cells, the amount of BSA (0.1%) was adjusted to be the same for all samples. Following the treatment, the viability of biofilm cells was determined by counting CFU. 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 is also effective against P. aeruginosa biofilm cells, the 24 h biofilm cells of PAO1 and PDO300 were treated with GM-CSF in the presence and absence of antibiotics. Treatment with 0.17 pM GM-CSF alone did not change the viability of biofilm cells of either strain. Since alginate can hinder the penetration of GM-CSF34. Alginate lyase was added at 50 μg/mL in addition to 200 μg/mL tobramycin and 0.17 pM GM-CSF to test the effects of GM-CSF on biofilm cells. Neither GM-CSF nor alginate lyase alone killed biofilm cells of P. aeruginosa PAO1 and PDO300 significantly in the absence of antibiotic. However, co-treatment with 50 μg/mL alginate lyase, 0.17 pM GM-CSF, and 200 μg/mL tobramycin reduced the viability of PDO300 biofilm cells by 97.2 ± 0.4% (35.4-fold reduction of viability; p = 0.0002) compared to the control biofilms without any treatment (Fig. 4a). This is 61.3 ± 6.0% (p = 0.03) more killing than that achieved by treatment with tobramycin alone, and 57.1 ± 6.6% more than treatment with tobramycin and alginate lyase together (Fig. 4a). Thus, GM-CSF is effective against PDO300 biofilm cells if the biofilm matrix is degraded.

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