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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 sensitized the persister cells of P. aeruginosa PAO1 to antibiotics.The wild-type PAO1 obtained from two different sources were tested including one (a) from Prof. Thomas K. Wood at Pennsylvania State University and another (b) from Prof. Matthew Parsek at University of Washington. The persister cells were isolated from exponential phase cultures by killing the normal cells with 200 μg/mL ciprofloxacin for 3.5 h, and then treated with 0.17 pM GM-CSF alone for 1 h, followed by additional treatment with GM-CSF plus an antibiotic as indicated for 3.5 h (all tested at 200 μg/mL). The samples without GM-CSF or antibiotic were used as controls. 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. Cip: ciprofloxacin. Tob: tobramycin. Tet: tetracycline. Gen: gentamicin. The samples were tested in triplicate (n = 3). Error bars represent SD; **p < 0.01, ***p < 0.001, ****p < 0.0001, one-way ANOVA followed by Tukey test.
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f1: GM-CSF sensitized the persister cells of P. aeruginosa PAO1 to antibiotics.The wild-type PAO1 obtained from two different sources were tested including one (a) from Prof. Thomas K. Wood at Pennsylvania State University and another (b) from Prof. Matthew Parsek at University of Washington. The persister cells were isolated from exponential phase cultures by killing the normal cells with 200 μg/mL ciprofloxacin for 3.5 h, and then treated with 0.17 pM GM-CSF alone for 1 h, followed by additional treatment with GM-CSF plus an antibiotic as indicated for 3.5 h (all tested at 200 μg/mL). The samples without GM-CSF or antibiotic were used as controls. 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. Cip: ciprofloxacin. Tob: tobramycin. Tet: tetracycline. Gen: gentamicin. The samples were tested in triplicate (n = 3). Error bars represent SD; **p < 0.01, ***p < 0.001, ****p < 0.0001, one-way ANOVA followed by Tukey test.

Mentions: We started this study using the P. aerugionsa PAO1 strain32 obtained from Prof. Thomas K. Wood at Pennsylvania State University. Treatment of this strain with 0.17 pM GM-CSF alone did not affect the viability of persister cells isolated (by treatment with 200 μg/mL ciprofloxacin for 3.5 h) from the exponential phase cultures [p = 0.36; One-way ANOVA followed by Tukey test used throughout this study] (Fig. 1a). However, the treatment with GM-CSF significantly sensitized the persister cells to antibiotics. For example, treatment with 0.17 pM recombinant human GM-CSF (henceforth GM-CSF) sensitized 96.2 ± 5.9% (26.3-fold reduction of viability; p = 0.0002), 91.3 ± 1.2% (p < 0.0001), 61.4 ± 16.6% (p = 0.0119), and 47.6 ± 14.9% (p = 0.0030) of the persister cells to 200 μg/mL of ciprofloxacin, tobramycin, tetracycline, and gentamicin respectively, compared to treatments with antibiotic alone, which were found ineffective in killing persister cells (p > 0.05 for all antibiotics tested) (Fig. 1a). To test if GM-CSF is also effective against the mucoid strains of P. aeruginosa, we tested another wild-type strain of P. aeruginosa PAO133 obtained from Prof. Matthew Parsek at the University of Washington, so that the isogenic mucoid strain P. aeruginosa PDO30033 can be compared. Similar results were obtained for the PAO1 strain from Parsek lab; e.g., treatment with 0.17 pM GM-CSF alone did not affect the viability of persister cells isolated from exponential phase cultures (p = 0.37) (Fig. 1b), but sensitized the persister cells to the above antibiotics except for gentamycin. For example, treatment with 0.17 pM GM-CSF for 1 h sensitized 79.3 ± 8.3% (p = 0.0002), 72.2 ± 12.7% (p = 0.0012), and 45.7 ± 7.8% (p = 0.001) of persister cells to 200 μg/mL of ciprofloxacin, tobramycin, and tetracycline, respectively, compared to treatments with antibiotic alone (Fig. 1b). Treatment with any of these antibiotics alone did not cause significant killing of persister cells (p > 0.05 for all). Since the results are generally consistent with the other PAO1 strain and an isogenic mucoid strain is available, the PAO1 strain from Parsek lab (henceforth PAO1) was used for the rest of this study. Furthermore, because persister population is larger in stationary phase, we also tested if GM-CSF is effective against persister cells isolated from stationary phase cultures. The concentrations of antibiotics were also reduced to better understand the potential of GM-CSF. As shown in Table 1, GM-CSF itself did not significantly affect the viability of persisters in the absence of an antibiotic (0.17, 1.7, and 17 pM GM-CSF tested; p > 0.3 for all conditions), and synergistic effects were observed between GM-CSF and antibiotics in killing PAO1 persister cells isolated from stationary phase cultures. Specifically, treatment with 0.17 pM GM-CSF sensitized 61.5 ± 14.5% (p = 0.0003) and 77.1 ± 2.0% (p = 0.0048) of persister cells to 5 μg/mL ciprofloxacin and tobramycin respectively, compared to treatments with antibiotic alone (Table 1). At a higher concentration of 17 pM, GM-CSF sensitized 74.0 ± 2.9% (p = 0.0005) and 86.5 ± 1.7% (p = 0.0002) of PAO1 persister cells to 5 μg/mL ciprofloxacin and tobramycin, respectively, compared to antibiotic treatments alone (Table 1). Thus, GM-CSF appeared to be effective against persister cells in both exponential and stationary phase cultures of P. aeruginosa PAO1 and exhibited synergy in persister killing with low concentrations of ciprofloxacin and tobramycin.


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 sensitized the persister cells of P. aeruginosa PAO1 to antibiotics.The wild-type PAO1 obtained from two different sources were tested including one (a) from Prof. Thomas K. Wood at Pennsylvania State University and another (b) from Prof. Matthew Parsek at University of Washington. The persister cells were isolated from exponential phase cultures by killing the normal cells with 200 μg/mL ciprofloxacin for 3.5 h, and then treated with 0.17 pM GM-CSF alone for 1 h, followed by additional treatment with GM-CSF plus an antibiotic as indicated for 3.5 h (all tested at 200 μg/mL). The samples without GM-CSF or antibiotic were used as controls. 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. Cip: ciprofloxacin. Tob: tobramycin. Tet: tetracycline. Gen: gentamicin. The samples were tested in triplicate (n = 3). Error bars represent SD; **p < 0.01, ***p < 0.001, ****p < 0.0001, one-way ANOVA followed by Tukey test.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: GM-CSF sensitized the persister cells of P. aeruginosa PAO1 to antibiotics.The wild-type PAO1 obtained from two different sources were tested including one (a) from Prof. Thomas K. Wood at Pennsylvania State University and another (b) from Prof. Matthew Parsek at University of Washington. The persister cells were isolated from exponential phase cultures by killing the normal cells with 200 μg/mL ciprofloxacin for 3.5 h, and then treated with 0.17 pM GM-CSF alone for 1 h, followed by additional treatment with GM-CSF plus an antibiotic as indicated for 3.5 h (all tested at 200 μg/mL). The samples without GM-CSF or antibiotic were used as controls. 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. Cip: ciprofloxacin. Tob: tobramycin. Tet: tetracycline. Gen: gentamicin. The samples were tested in triplicate (n = 3). Error bars represent SD; **p < 0.01, ***p < 0.001, ****p < 0.0001, one-way ANOVA followed by Tukey test.
Mentions: We started this study using the P. aerugionsa PAO1 strain32 obtained from Prof. Thomas K. Wood at Pennsylvania State University. Treatment of this strain with 0.17 pM GM-CSF alone did not affect the viability of persister cells isolated (by treatment with 200 μg/mL ciprofloxacin for 3.5 h) from the exponential phase cultures [p = 0.36; One-way ANOVA followed by Tukey test used throughout this study] (Fig. 1a). However, the treatment with GM-CSF significantly sensitized the persister cells to antibiotics. For example, treatment with 0.17 pM recombinant human GM-CSF (henceforth GM-CSF) sensitized 96.2 ± 5.9% (26.3-fold reduction of viability; p = 0.0002), 91.3 ± 1.2% (p < 0.0001), 61.4 ± 16.6% (p = 0.0119), and 47.6 ± 14.9% (p = 0.0030) of the persister cells to 200 μg/mL of ciprofloxacin, tobramycin, tetracycline, and gentamicin respectively, compared to treatments with antibiotic alone, which were found ineffective in killing persister cells (p > 0.05 for all antibiotics tested) (Fig. 1a). To test if GM-CSF is also effective against the mucoid strains of P. aeruginosa, we tested another wild-type strain of P. aeruginosa PAO133 obtained from Prof. Matthew Parsek at the University of Washington, so that the isogenic mucoid strain P. aeruginosa PDO30033 can be compared. Similar results were obtained for the PAO1 strain from Parsek lab; e.g., treatment with 0.17 pM GM-CSF alone did not affect the viability of persister cells isolated from exponential phase cultures (p = 0.37) (Fig. 1b), but sensitized the persister cells to the above antibiotics except for gentamycin. For example, treatment with 0.17 pM GM-CSF for 1 h sensitized 79.3 ± 8.3% (p = 0.0002), 72.2 ± 12.7% (p = 0.0012), and 45.7 ± 7.8% (p = 0.001) of persister cells to 200 μg/mL of ciprofloxacin, tobramycin, and tetracycline, respectively, compared to treatments with antibiotic alone (Fig. 1b). Treatment with any of these antibiotics alone did not cause significant killing of persister cells (p > 0.05 for all). Since the results are generally consistent with the other PAO1 strain and an isogenic mucoid strain is available, the PAO1 strain from Parsek lab (henceforth PAO1) was used for the rest of this study. Furthermore, because persister population is larger in stationary phase, we also tested if GM-CSF is effective against persister cells isolated from stationary phase cultures. The concentrations of antibiotics were also reduced to better understand the potential of GM-CSF. As shown in Table 1, GM-CSF itself did not significantly affect the viability of persisters in the absence of an antibiotic (0.17, 1.7, and 17 pM GM-CSF tested; p > 0.3 for all conditions), and synergistic effects were observed between GM-CSF and antibiotics in killing PAO1 persister cells isolated from stationary phase cultures. Specifically, treatment with 0.17 pM GM-CSF sensitized 61.5 ± 14.5% (p = 0.0003) and 77.1 ± 2.0% (p = 0.0048) of persister cells to 5 μg/mL ciprofloxacin and tobramycin respectively, compared to treatments with antibiotic alone (Table 1). At a higher concentration of 17 pM, GM-CSF sensitized 74.0 ± 2.9% (p = 0.0005) and 86.5 ± 1.7% (p = 0.0002) of PAO1 persister cells to 5 μg/mL ciprofloxacin and tobramycin, respectively, compared to antibiotic treatments alone (Table 1). Thus, GM-CSF appeared to be effective against persister cells in both exponential and stationary phase cultures of P. aeruginosa PAO1 and exhibited synergy in persister killing with low concentrations of ciprofloxacin and tobramycin.

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