A Possible Phenomenon of Persistence in Pseudomonas aeruginosa Treated with Methylene Blue and Red Light

Abstract

Planktonic Pseudomonas aeruginosa cells harvested in stationary phase were exposed to red light in the presence of methylene blue to study the potential occurrence of persistence in bacterial populations submitted to photodynamic antimicrobial therapy. Survival curves revealed the existence of small subpopulations of cells exhibiting increased ability to tolerate the treatment. These subpopulations were detected even using high concentrations of photosensitizer, whether added in a single step or following a fractionated scheme, and when the irradiation medium was modified to delay the photodecomposition of methylene blue. When cells grown from survivors to the treatment were cultured and exposed to red light and dye, their responses were similar to that of the original strain. These results exclude exhaustion of the photosensitizer and selection of resistant mutants as explanations for the features of the survival curves. Cells able to tolerate the treatment were found even when radiation was imparted at a high-dose rate. They exhibit a response typical of persisters, which tolerate antimicrobial agents due to transient and reversible changes in their phenotype, suggesting that persistence is a factor to consider upon evaluating the efficacy of photodynamic antimicrobial therapy.

The ineffectiveness of antimicrobial photodynamic therapy in the absence of preincubation of the microorganisms in the photosensitizer

“…In general, this study agrees with what is usually found in the literature regarding APDT with MB. The fact that Gram-negative bacteria are less susceptible to APDT compared to Gram-positive is already established in the literature and probably occurs due to structural differences in their cell membranes [12,19,25,27]; this pattern was observed in our study, since only the Gram-positive bacterium was affected by the experimental procedure adopted.…”

“…In our experiment, the procedure adopted was not able to generate P. aeruginosa inhibition. This opportunistic pathogen is also capable of generating several types of infection and can generate resistance to antibiotics with ease, factors that make it the target of APDT protocols [25]. In general, it is possible to observe that this bacterium has a certain susceptibility to MBmediated APDT [12,[24][25][26][27]29], but, as in other Gram-negative bacteria, the action of the ADPT for P. aeruginosa is not as intense as for bacteria such as S. aureus [12,19,21,24,25,28,30].…”

The ineffectiveness of antimicrobial photodynamic therapy in the absence of preincubation of the microorganisms in the photosensitizer

“…In general, this study agrees with what is usually found in the literature regarding APDT with MB. The fact that Gram-negative bacteria are less susceptible to APDT compared to Gram-positive is already established in the literature and probably occurs due to structural differences in their cell membranes [12,19,25,27]; this pattern was observed in our study, since only the Gram-positive bacterium was affected by the experimental procedure adopted.…”

“…In our experiment, the procedure adopted was not able to generate P. aeruginosa inhibition. This opportunistic pathogen is also capable of generating several types of infection and can generate resistance to antibiotics with ease, factors that make it the target of APDT protocols [25]. In general, it is possible to observe that this bacterium has a certain susceptibility to MBmediated APDT [12,[24][25][26][27]29], but, as in other Gram-negative bacteria, the action of the ADPT for P. aeruginosa is not as intense as for bacteria such as S. aureus [12,19,21,24,25,28,30].…”

Lethal Effect of Photodynamic Treatment on Persister Bacteria

“…Survival curves reported for P. aeruginosa treated with OFX (22), as well as those obtained when it was exposed to light and MB (13), exhibit decreased rates of cell death at low survival fractions. From such observations, the presence of minority subpopulations of cells able to tolerate these treatments can be inferred.…”

“…The presence of a tolerant subpopulation, whose ability to survive depends on a noninheritable condition , strongly resembles the response of persister bacteria to antibiotics . If there were a common mechanism responsible for the phenotypic tolerance to antibiotics and photodynamic inactivation, survivors of one of these treatments should have increased tolerance to the other.…”

Combined Effects of Amikacin and Methylene Blue-Mediated Photodynamic Therapy on Pseudomonas aeruginosa Biofilms Mimicking Mono- and Polymicrobial Diabetic Foot Ulcer Infections

“…Several studies have demonstrated that antimicrobial photodynamic therapy (aPDT) using methylene blue (MB) effectively reduces the viability of both Gram-negative and Gram-positive bacteria in planktonic cultures, including P. aeruginosa [19,23] and E. faecalis [24,25]. In contrast, our results indicate that, in biofilm-associated cells, aPDT with MB induced a significant reduction in viability only when a high photosensitizer concentration (6000 μM) was applied.…”

The ineffectiveness of antimicrobial photodynamic therapy in the absence of preincubation of the microorganisms in the photosensitizer

“…In general, this study agrees with what is usually found in the literature regarding APDT with MB. The fact that Gram-negative bacteria are less susceptible to APDT compared to Gram-positive is already established in the literature and probably occurs due to structural differences in their cell membranes [12,19,25,27]; this pattern was observed in our study, since only the Gram-positive bacterium was affected by the experimental procedure adopted.…”

“…In our experiment, the procedure adopted was not able to generate P. aeruginosa inhibition. This opportunistic pathogen is also capable of generating several types of infection and can generate resistance to antibiotics with ease, factors that make it the target of APDT protocols [25]. In general, it is possible to observe that this bacterium has a certain susceptibility to MBmediated APDT [12,[24][25][26][27]29], but, as in other Gram-negative bacteria, the action of the ADPT for P. aeruginosa is not as intense as for bacteria such as S. aureus [12,19,21,24,25,28,30].…”

Lethal Effect of Photodynamic Treatment on Persister Bacteria

“…Survival curves reported for P. aeruginosa treated with OFX (22), as well as those obtained when it was exposed to light and MB (13), exhibit decreased rates of cell death at low survival fractions. From such observations, the presence of minority subpopulations of cells able to tolerate these treatments can be inferred.…”

“…The presence of a tolerant subpopulation, whose ability to survive depends on a noninheritable condition , strongly resembles the response of persister bacteria to antibiotics . If there were a common mechanism responsible for the phenotypic tolerance to antibiotics and photodynamic inactivation, survivors of one of these treatments should have increased tolerance to the other.…”

Combined Effects of Amikacin and Methylene Blue-Mediated Photodynamic Therapy on Pseudomonas aeruginosa Biofilms Mimicking Mono- and Polymicrobial Diabetic Foot Ulcer Infections

“…Several studies have demonstrated that antimicrobial photodynamic therapy (aPDT) using methylene blue (MB) effectively reduces the viability of both Gram-negative and Gram-positive bacteria in planktonic cultures, including P. aeruginosa [19,23] and E. faecalis [24,25]. In contrast, our results indicate that, in biofilm-associated cells, aPDT with MB induced a significant reduction in viability only when a high photosensitizer concentration (6000 μM) was applied.…”

The ineffectiveness of antimicrobial photodynamic therapy in the absence of preincubation of the microorganisms in the photosensitizer

“…In general, this study agrees with what is usually found in the literature regarding APDT with MB. The fact that Gram-negative bacteria are less susceptible to APDT compared to Gram-positive is already established in the literature and probably occurs due to structural differences in their cell membranes [12,19,25,27]; this pattern was observed in our study, since only the Gram-positive bacterium was affected by the experimental procedure adopted.…”

“…In our experiment, the procedure adopted was not able to generate P. aeruginosa inhibition. This opportunistic pathogen is also capable of generating several types of infection and can generate resistance to antibiotics with ease, factors that make it the target of APDT protocols [25]. In general, it is possible to observe that this bacterium has a certain susceptibility to MBmediated APDT [12,[24][25][26][27]29], but, as in other Gram-negative bacteria, the action of the ADPT for P. aeruginosa is not as intense as for bacteria such as S. aureus [12,19,21,24,25,28,30].…”

Lethal Effect of Photodynamic Treatment on Persister Bacteria

“…Survival curves reported for P. aeruginosa treated with OFX (22), as well as those obtained when it was exposed to light and MB (13), exhibit decreased rates of cell death at low survival fractions. From such observations, the presence of minority subpopulations of cells able to tolerate these treatments can be inferred.…”

“…The presence of a tolerant subpopulation, whose ability to survive depends on a noninheritable condition , strongly resembles the response of persister bacteria to antibiotics . If there were a common mechanism responsible for the phenotypic tolerance to antibiotics and photodynamic inactivation, survivors of one of these treatments should have increased tolerance to the other.…”

Combined Effects of Amikacin and Methylene Blue-Mediated Photodynamic Therapy on Pseudomonas aeruginosa Biofilms Mimicking Mono- and Polymicrobial Diabetic Foot Ulcer Infections

“…Several studies have demonstrated that antimicrobial photodynamic therapy (aPDT) using methylene blue (MB) effectively reduces the viability of both Gram-negative and Gram-positive bacteria in planktonic cultures, including P. aeruginosa [19,23] and E. faecalis [24,25]. In contrast, our results indicate that, in biofilm-associated cells, aPDT with MB induced a significant reduction in viability only when a high photosensitizer concentration (6000 μM) was applied.…”