Enhancing Antibiotic Effect by Photodynamic: The Case of Klebsiella pneumoniae

Abstract

Background: The effect of antibiotics can be severely affected by external factors. Combining the oxidative impact of photodynamic therapy with antibiotics is largely unexplored, which may result in positive results with great impact on clinical applications. In particular, that can be relevant in the case of antibiotic resistance. Objectives: In this study, we examined the effects of aPDT using the photosensitizers (PSs), methylene blue (MB) or Photodithazine (PDZ), both alone and in combination with the antibiotics ciprofloxacin (CIP), gentamicin (GEN), and ceftriaxone (CEF), against the Gram-negative bacterium Klebsiella pneumoniae. Methods: A standard suspension of K. pneumoniae was subjected to PDT with varying doses of MB and PDZ solutions, using a 75 mW/cm² LED emitting at 660 nm with an energy of 15 J/cm². The MICs of CIP, GEN, and CEF were determined using the broth dilution method. We also tested the photosensitizers MB or PDZ as potentiating agents for synergistic combinations with antibiotics CIP, GEN, and CEF against K. pneumoniae. Results: The results showed that MB was more effective in inhibiting survival and killing K. pneumoniae compared to PDZ. The tested antibiotics CIP, GEN, and CEF suppressed bacterial growth (as shown by reduced MIC values) and effectively killed K. pneumoniae (reduced Log CFU/mL). While antibiotic treatment or aPDT alone showed a moderate effect (1 Log₁₀ to 2 Log₁₀ CFU reduction) on killing K. pneumoniae, the combination therapy significantly increased bacterial death, resulting in a ≥3 Log₁₀ to 6 Log₁₀ CFU reduction. Conclusions: Our study indicates that pre-treating bacteria with PDT makes them more susceptible to antibiotics and could serve as an alternative for treating local infections caused by resistant bacteria or even reduce the required antibiotic dosage. This work explores numerous possible combinations of PDT and antibiotics, emphasizing their interdependence in controlling infections and the unique properties each PS-antibiotic combination offers. Clinical application for the combination is a promising reality since both are individually already adopted in clinical use.

Keywords: Klebsiella pneumoniae; combination of PDT and antibiotics; photodynamic therapy; photosensitizers; synergetic effect.

Figure 1

Evaluation of the photodynamic activity of photosensitizers (MB and PDZ) on K. pneumoniae irradiated with red LED light (660 nm). Varying the photosensitizer concentration of MB (0, 0.25, 0.5, 0.75, and 1 µg/mL), or PDZ (0, 25, 50, and 100 µg/mL). All assays used a light dose of 15 J/cm². Calculated using mean and standard deviation, statistical significance was compared between each treated group (PS + PDT) to the “only PS” control group for each concentration level.

Figure 2

The combined effect of antibiotics (CEF, GEN, and CEF) and Photodynamic Therapy (PDT) on the viability of K. pneumoniae. The graph shows the count of colony-forming units (CFU/mL) as a function of tested antibiotics concentration (µg/mL) at a constant light dose of 15 J/cm². The profile for GEN, indicates that it reaches full elimination much faster than the last point indicated.

Figure 3

The combined effect of photosensitizers (MB or PDZ) and antibiotics (CEF, GEN, and CEF) on the viability of K. pneumoniae. The graph shows colony-forming units (CFU/mL) as a function of photosensitizer and antibiotic concentrations (µg/mL) at an energy dose of 15 J/cm². Statistical significance was determined by comparing treated groups (PS + antibiotics) to the “only PS” control group for each concentration. Error bars represent standard deviation across replicates. (A) Sequence of different experiments using the three selected antibiotics as the concentration of Methylene Blue is varied. (B) Sequence of different experiments using the three selected antibiotics as the concentration of PDZ is varied.

Figure 4

Equivalence plot between PDT under the described conditions and the concentration of antibiotics. Combination effect of Photosensitizers (MB or PDZ) and antibiotics (CEF, GEN, and CEF) against K. pneumoniae. (A) for 1 log reduction, (B) for 2 log reduction, (C) for 2, 4, 6 log reduction in each antibiotic. As the photosensitizer concentration increases, less gain is obtained, indicating a saturation tendency for high concentrations of photosensitizer.

Figure 5

Diagram of the experimental irradiation setup used for photodynamic therapy (PDT). Irradiation was performed with a PineTek Biotable^®—PINETEK LLC, College Station, TX, USA, a red light-emitting diode (LED)-based device that consists of 24 emitting centers with a wavelength of 660 nm. LED arrays were arranged so that each LED array was placed under a well from the well plate, providing the same average irradiation. The RSD values of irradiance obtained with this device were 75 mW/cm². Variation from well to well remains at about 10%. The configuration allows for consistent and homogeneous light exposure, ensuring reproducibility of the photoinactivation experiments. The light dose applied was 15 J/cm² at an exposure time of 3 min 20 s. In opposite to illuminating a single well each time, the device is more precise and reproducible.

Figure 6

The schematic presentation of the combination effect of photosensitizers and antibiotics. At the beginning, the bacterial samples are treated with different doses of PS and incubated for 20 min in the dark at 37 °C and irradiated with a light dose of 15 J/cm², except the control samples. Then the samples are incubated with antibiotics for 8 h at 37 °C. The samples were performed in triplicate and analyzed.