In vitro resistance selection studies of RLP068/Cl, a new Zn(II) phthalocyanine suitable for antimicrobial photodynamic therapy

Abstract

Resistance to antimicrobial agents is emerging in a wide variety of nosocomial and community-acquired pathogens. The development of alternative therapies against nosocomial infections caused by clinically relevant pathogens represents a major public health concern. RLP068/Cl is a novel Zn(II) phthalocyanine proposed as a photosensitizer suitable for antimicrobial photodynamic therapy (APDT) for localized infections. Its ability, following activation by light, to induce resistance in three major human pathogens after 20 daily passages was studied. Simultaneously for the same strains, the ability of daily sequential subcultures in subinhibitory concentrations of RLP068/Cl to develop resistant mutants without illumination was evaluated. We demonstrate that 20 consecutive APDT treatments with RLP068/Cl did not result in any resistant mutants and that, in dark conditions, only Staphylococcus aureus strains had increased MICs of RLP068/Cl. However, even in this case, the susceptibility of the mutated bacteria to APDT was not affected by their MIC increase.

FIG. 1.

Efficacy of killing of Staphylococcus aureus 26A7 (a) and Staphylococcus aureus 28C65 (b) after 5 min of incubation in the dark with concentrations of RLP068/Cl ranging from 0 ng/ml to 64 ng/ml, with illumination (gray bars) and without illumination (white bars). Values below the horizontal line represent ≥99.9% (MBCs) efficacy of bacterial killing relative to the untreated controls (bacteria, but no light and no photosensitizer).

FIG. 2.

Efficacy of killing of Pseudomonas aeruginosa GiaS (a) and Pseudomonas aeruginosa GW-1 (b) after 5 min of incubation in the dark with concentrations of RLP068/Cl ranging from 0 μg/ml to 26.4 μg/ml, with illumination (gray bars) and without illumination (white bars). Values below the horizontal line represent ≥99.9% (MBCs) efficacy of bacterial killing relative to the untreated controls (bacteria, but no light and no photosensitizer).

FIG. 3.

Efficacy of killing of Candida albicans 3518 (a) and Candida albicans 17 (b) after 1 h of incubation in the dark with concentrations of RLP068/Cl ranging from 0 μg/ml to 3.3 μg/ml, with illumination (gray bars) and without illumination (open bars). Values below the horizontal line represent ≥99.9% (MFCs) efficacy of yeast killing relative to the untreated controls (yeast, but no light and no photosensitizer).