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Abstract
Infections caused by multidrug-resistant (MDR) Klebsiella pneumoniae are difficult to treat with conventional antibiotics. Thus, alternative strategies to control the growth of MDR Klebsiella are warranted. We hypothesized that activation of innate effector systems could sensitize MDR K. pneumoniae to conventional antibiotics. Thus, human primary macrophages were stimulated with compounds known to activate innate immunity (vitamin D3, phenylbutyrate [PBA], and the aroylated phenylenediamine HO53) and then infected with MDR Klebsiella in the presence or absence of antibiotics. Antibiotics alone were ineffective against MDR Klebsiella in the cellular model, whereas vitamin D3, PBA, and HO53 reduced intracellular growth by up to 70%. The effect was further improved when the innate activators were combined with antibiotics. Vitamin D3- and PBA-induced bacterial killing was dependent on CAMP gene expression, whereas HO53 needed the production of reactive oxygen species (ROS), as shown in cells where the CYBB gene was silenced and in cells from a patient with reduced ROS production due to a deletion in the CYBB gene and skewed lyonization. The combination of innate effector activation by vitamin D3, PBA, and HO53 was effective in sensitizing MDR Klebsiella to conventional antibiotics in a primary human macrophage model. This study provides new evidence for future treatment options for K. pneumoniae.
Conventional antibiotics lack activity against MDR Klebsiella inside primary human macrophages but can…
FIG 1 Conventional antibiotics lack activity against MDR Klebsiella inside primary human macrophages but can rapidly kill susceptible Klebsiella. MDMs were infected with MDR (AO15200) and wild-type (ATCC 25955) Klebsiella strains for 30 min with an MOI of 1:5. The remaining extracellular bacteria were eliminated with gentamicin (25 μg/ml) treatment for 30 min. The MICs of the antibiotics (azithromycin, 1 mg/liter; cefotaxime, 16 mg/liter; ciprofloxacin, 16 mg/liter; and fosfomycin, 0.0625 g/liter) were administered after bacterial uptake. CFU were measured after 5 h of infection. (A and B) Bacterial survival is presented as CFU/ml of (A) MDR Klebsiella and (B) wild-type Klebsiella. Each bar represents a median value of 9 independent experiments ± interquartile range (IRQ). Statistical significance was calculated using the Mann-Whitney U test. **, P < 0.01. The untreated control (black) was used for all statistical tests in relation to treatment with the different antibiotics.
FIG 2
Activators of innate effector systems…
FIG 2
Activators of innate effector systems enhance antibacterial activity of infected human macrophages. Vitamin…
FIG 2 Activators of innate effector systems enhance antibacterial activity of infected human macrophages. Vitamin D3, PBA, and HO53 enhance the antimicrobial response in MDMs. MDMs were stimulated with 10 nM vitamin D3, 2 mM PBA, and 12.5 μM HO53 for 24 h and then infected with the two strains of Klebsiella for 30 min. The remaining extracellular bacteria were eliminated with gentamicin (25 μg/ml) treatment for 30 min. CFU were measured after 5 h of infection. Bacterial survival is presented as CFU/ml (A and B). Each bar represents a median value of 9 independent experiments ± interquartile range (IQR). Statistical significance was calculated using the Mann-Whitney U test. *, P < 0.05; **, P < 0.01; ***, P < 0.001. The untreated control (black) was used for all statistical tests in relation to the various treatments.
FIG 3
Activation of innate effector systems…
FIG 3
Activation of innate effector systems increases the effects of conventional antibiotics against MDR …
FIG 3 Activation of innate effector systems increases the effects of conventional antibiotics against MDR Klebsiella. MDMs were pretreated with 10 nM vitamin D3, 2 mM PBA, and 12.5 μM HO53 for 24 h separately or in combination before infection with the MDR Klebsiella strain for 30 min. The MICs of the antibiotics (azithromycin, 1 mg/liter; cefotaxime, 16 mg/liter; ciprofloxacin, 16 mg/liter; and fosfomycin, 0.0625 g/liter) were administered after bacterial uptake. The additive effect of antibiotics with the innate inducers was analyzed by comparison with the control group (without antibiotics). Survival of MDR Klebsiella is shown in the presence (white) and absence (black) of antibiotics. (A) azithromycin; (B) cefotaxime; (C) ciprofloxacin; (D) fosfomycin. Bacterial survival is presented as CFU/ml. Each bar represents a median value of 9 independent experiments ± interquartile range (IQR). Statistical significance was calculated using the Mann-Whitney U test. *, P < 0.05; **, P < 0.01; #, significant compared to untreated control (black bar); ###, P < 0.001.
FIG 4
Vitamin D 3 , PBA,…
FIG 4
Vitamin D 3 , PBA, and HO53 induce the expression of antimicrobial peptides,…
FIG 4 Vitamin D3, PBA, and HO53 induce the expression of antimicrobial peptides, oxidative stress, and autophagy-related genes in human macrophages infected with Klebsiella. MDMs were stimulated with 10 nM vitamin D3, 2 mM PBA, and 12.5 μM HO53 for 24 h and then were infected with the MDR Klebsiella strain for 30 min. The remaining extracellular bacteria were eliminated with gentamicin (25 μg/ml) treatment for 30 min. The mRNA expression of the different genes was measured before infection and at 1 h and 5 h after bacterial uptake. (A to D) Expression of antimicrobial peptide gene (A) CAMP/LL-37 mRNA expression, (B) hBD1 mRNA expression, (C) hBD2 mRNA expression, and (D) hBD4 mRNA expression. (E to H) Expression of oxidative stress-related gene (E) NOS2 mRNA expression, (F) duox2 mRNA expression, (G) catalase mRNA expression, and (H) GPX3 mRNA expression. (I and J) Expression of autophagy-related gene (I) Atg5 mRNA expression and (J) Atg12 mRNA expression (normalized to 18S rRNA expression). Each bar represents a median value of 6 independent experiments ± interquartile range (IQR). Statistical significance was calculated using the Mann-Whitney U test compared with the untreated control. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
FIG 5
Vitamin D 3 alone and…
FIG 5
Vitamin D 3 alone and in combination with PBA depends on LL-37 for…
FIG 5 Vitamin D3 alone and in combination with PBA depends on LL-37 for killing of MDR Klebsiella in human macrophages. (A) LL-37 mRNA expression and (B) intracellular killing of the MDR-Klebsiella in CAMP-siRNA-transfected (siRNA-CAMP versus siRNA-mock) macrophages. MDMs were transfected with siRNA (30 pmol) for 24 h before treatment with 10 nM vitamin D3, 2 mM PBA, and 12.5 μM HO53 for 24 h before infection. The mRNA expression was determined using qRT-PCR and presented as CAMP mRNA fold-induction in siRNA-CAMP-transfected macrophages compared to the siRNA-mock-transfected controls. Intracellular killing was determined using CFU/ml counts. Results were obtained from n = 4 donors. Data (median ± IQR) are presented in bar graphs. Statistical significance was calculated using the Mann-Whitney U test. *, P < 0.05; #, significant compared to untreated control (black bar); ##, P < 0.01.
FIG 6
Reactive oxygen species (ROS) are…
FIG 6
Reactive oxygen species (ROS) are essential for HO53-, vitamin D 3 -, and…
FIG 6 Reactive oxygen species (ROS) are essential for HO53-, vitamin D3-, and PBA-mediated killing of Klebsiella in human macrophages. (A) Intracellular killing of the MDR Klebsiella in ROS-deficient versus healthy donor macrophages. Data are presented as the median ± IQR. (B) NOX2 mRNA expression. (C) Intracellular killing of the MDR Klebsiella in CYBB-siRNA-transfected (siRNA-mock versus siRNA-CYBB) macrophages. MDMs were transfected with siRNA (30 pmol) for 24 h before treatment with 10 nM vitamin D3, 2 mM PBA, and 12.5 μM HO53 for 24 h, followed by the infection. The mRNA expression was determined using qRT-PCR and presented as NOX2 (CYBB) mRNA fold-induction in siRNA-CYBB-transfected macrophages compared to the siRNA-mock-transfected controls. Intracellular killing was determined using CFU/ml. Data were obtained from n = 1 donor for panel A and n = 4 donors for panels B and C. Statistical significance was calculated using the Mann-Whitney U test. *, P < 0.05; **, P < 0.01; #, significant compared to untreated control (black bar); ##, P < 0.01; ###, P < 0.001.
FIG 7
Vitamin D 3 , PBA,…
FIG 7
Vitamin D 3 , PBA, and HO53 depend on autophagy to mediate killing…
FIG 7 Vitamin D3, PBA, and HO53 depend on autophagy to mediate killing of MDR Klebsiella in human macrophages. MDMs were treated with 10 nM vitamin D3, 2 mM PBA, and 12.5 μM HO53 prior to infection with MDR Klebsiella. After infection, MDMs were treated with 0.5 μM bafilomycin A1. Intracellular killing was determined using CFU/ml. Data were obtained from n = 5 donors. Data represent the median ± IQR. Statistical significance was calculated using the Mann-Whitney U test. **, P < 0.01; ***, P < 0.001; #, significant compared to untreated control (black bar); ###, P < 0.001.
FIG 8
Expression of antimicrobial peptides and…
FIG 8
Expression of antimicrobial peptides and autophagy-related genes in ROS-deficient human macrophages. (A) Expression…
FIG 8 Expression of antimicrobial peptides and autophagy-related genes in ROS-deficient human macrophages. (A) Expression of antimicrobial peptide genes (LL-37, hBD1, hBD2, and hBD4). (B) Expression of autophagy-related genes (Atg5 and Atg12) (normalized to 18S rRNA expression). (C) A representative Western blot of autophagy protein LC3-II expression in healthy and ROS-deficient macrophages and the housekeeping protein β-actin. Data were obtained from 6 healthy donors and 1 ROS-deficient patient (samples tested in triplicate). Statistical significance was calculated using the Mann-Whitney U test. *, P < 0.05. All figures (8) See this image and copyright information in PMC
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