Breaking down antibiotic resistance in methicillin-resistant Staphylococcus aureus: Combining antimicrobial photodynamic and antibiotic treatments

Abstract

The widespread use of antibiotics drives the evolution of antimicrobial-resistant bacteria (ARB), threatening patients and healthcare professionals. Therefore, the development of novel strategies to combat resistance is recognized as a global healthcare priority. The two methods to combat ARB are development of new antibiotics or reduction in existing resistances. Development of novel antibiotics is a laborious and slow-progressing task that is no longer a safe reserve against looming risks. In this research, we suggest a method for reducing resistance to extend the efficacious lifetime of current antibiotics. Antimicrobial photodynamic therapy (aPDT) is used to generate reactive oxygen species (ROS) via the photoactivation of a photosensitizer. ROS then nonspecifically damage cellular components, leading to general impairment and cell death. Here, we test the hypothesis that concurrent treatment of bacteria with antibiotics and aPDT achieves an additive effect in the elimination of ARB. Performing aPDT with the photosensitizer methylene blue in combination with antibiotics chloramphenicol and tetracycline results in significant reductions in resistance for two methicillin-resistant Staphylococcus aureus (MRSA) strains, USA300 and RN4220. Additional resistant S. aureus strain and antibiotic combinations reveal similar results. Taken together, these results suggest that concurrent aPDT consistently decreases S. aureus resistance by improving susceptibility to antibiotic treatment. In turn, this development exhibits an alternative to overcome some of the growing MRSA challenge.

Keywords: antibiotic resistance; antimicrobial-resistant bacteria; methicillin-resistant Staphylococcus aureus; methylene blue; photodynamic therapy.

Fig. 1.

Schematic representation of single and combined antibiotic and aPDT treatment options for infections. Antibiotics serve an important role in medicine, successfully treating many common bacterial infections. However, their use leads to increased resistance, necessitating more dangerous antibiotic classes and higher doses. Likewise, aPDT can successfully eliminate bacteria, regardless of antibiotic resistance, but is not always applicable at large doses due to drug and light side effects. These two treatments used concurrently have been found to significantly lower average antibiotic resistance and broaden the resistance distribution, permitting the use of smaller doses of each for a more effective treatment.

Fig. 2.

Reduction in tetracycline (Tet) and chloramphenicol (Chl) MIC for S. aureus strains (A) USA300 and (B) RN4220 exposed to $0$ to 10.8 J/cm² photoactivating light at $650 \text{nm}$ and containing $1 \mathrm{\mu }\text{M}$ MB and $0.5 \mathrm{\mu }\text{M}$ MB in the treated groups, respectively. “Control” denotes samples exposed to light but without PS, while “Treated” indicates the presence of PS in addition to light. For each sample series, distributions were compared with the Friedman’s test and post hoc Dunn’s multiple comparisons test relative to the no-exposure point. ns is not significant; *P < 0.05; **P < 0.01; ****P < 0.0001. Individual comparison P values are provided in Table 2. $n=12$ for all samples.

Fig. 3.

(A) Mean antibiotic MIC concentrations from Fig. 2 where each exposure data point is divided by that sample’s baseline MIC prior to exposure. (B) The same data are also given numerically for all mean fold MIC reductions as described in the above paragraphs. As only means are used in calculating fold MIC reduction, no statistical considerations can be made. $n=12$ for all samples.

Fig. 4.

Cultured assessment of viable cells [${\log }_{10}(\text{CFU/mL})$] immediately after aPDT treatment. (A) USA300 incubated with $1.0 \mathrm{\mu }\text{M}$ MB and RN4220 incubated with $0.5 \mathrm{\mu }\text{M}$ MB were exposed to $0$ to 14.4 J/cm² photoactivating light. At 14.4 J/cm² exposure, both cultures were found to produce under the ${10}^5 \text{CFU/mL}$ limit of detec0074ion; thus, these data are not shown graphically. (B) An example of culture dishes is given, where arrows indicate the progressive twofold dilution of bacteria concentration by volume between spots. The left plate is USA300 given no light exposure, while the right plate is given 10.8 J/cm², both containing methylene blue. For each sample series of viable cells, distributions were compared with the Kruskal–Wallis test and post hoc Dunn’s multiple comparisons test relative to the respective no-exposure point. ns is not significant; *P < 0.05; **P < 0.01; ****P < 0.0001. Individual comparison P values are provided in Table 2. $n=9$ for all data points.

Fig. 5.

aPDT treatment was combined with the antibiotics Amp, Kan, Tet, and Chl for the S. aureus strains (A) USA300, (B) RN4220, (C) ΔSaeR, (D) MW2, and (E) JE2. Each of the strain–antibiotic combinations was examined only once ($n=1$) in this early survey trial. This surface-level observation was taken only to determine combinations of interest prior to more in-depth study. Each strain was tested with an MB concentration of $2 \mathrm{\mu }\text{M}$ and was exposed to $0$ to 14.4 J/cm² photoactivating light, but aPDT control cultures were completely eliminated at 14.4 J/cm², and thus, data stop at 10.8 J/cm². Since all data are single points, no statistical tests were completed.

Fig. 6.

The (Left) plate holder and LED array along with (Right) LED driver and power controller.