Abstract The worldwide increase in bacterial antibiotic resistance has led to a search for alternative antibacterial therapies. A promising approach to killing antibiotic-resistant bacteria is photodynamic antimicrobial chemotherapy, which uses light in combination with a photosensitizer to induce a phototoxic reaction. We evaluated the photodynamic inactivation (PDI) efficiency of hematoporphyrin monomethyl ether (HMME) on antibiotic-resistant bacteria and biofilms. HMME exhibited no significant dark toxicity and provided dose-dependent inactivation of antibiotic-resistant bacteria and biofilms. After incubation with 100-μM HMME and irradiation with 72- J cm−2 white light, 4.19–7.59 log10 reductions in survival were achieved in planktonic suspension. Antibiotic-resistant strains were as susceptible to PDI in biofilms as in planktonic suspensions, but the inactivation of bacterial cells in biofilms was attenuated. In addition, gram-positive bacterial strains and biofilms were more susceptible than gram-negative strains and biofilms to the PDI effect of HMME. Thus, HMME is a promising photosensitizer for the treatment of infectious diseases caused by antibiotic-resistant bacteria, especially grampositive bacteria.

Introduction 

The extensive and inappropriate use of antibiotics has put tremendous selective pressure on bacteria to devise mechanisms to escape the lethal action of the drugs. As a consequence, infections caused by antibiotic-resistant bacteria are now a serious concern for public health, and 17 million people die each year worldwide from bacterial infections [1]. Several classes of antibiotic-resistant bacteria are emerging as major threats: methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus faecalis (VRE), multidrug-resistant (MDR) mycobacteria and Pseudomonas aeruginosa, and extended-spectrum β-lactamase (ESBL)- producing Klebsiella pneumoniae and Escherichia coli, all of which are responsible for significant morbidity and mortality [2, 3].

Bacterial biofilms are aggregates of microbial cells embedded in a matrix composed of water and extracellular polymeric substances (EPS) [4]. Once a biofilm is established, it constitutes a niche that promotes bacterial growth and survival. Within the biofilm, bacteria become inherently resistant to antibiotics and host immune defenses, thus hampering treatment and clearance of the infection [5]. Biofilm-associated infections can only be resolved by removing the infected device, which exacts a major cost burden on healthcare services. Therefore, to eradicate these recalcitrant bacterial strains and biofilms, we must develop more effective antimicrobial agents or alternative approaches.

Photodynamic antimicrobial chemotherapy (PACT) has been regarded as a promising alternative treatment for various antibiotic-resistant bacteria since its mode of action is markedly different from that of most antibiotics. PACT involves the use of visible or near-infrared light combined with a photosensitizer (PS) and oxygen present in and around the targeted cells. After illumination with light of the appropriate wavelength, the PS is energized to an excited state that can undergo molecular collisions with oxygen, resulting in the formation of reactive oxygen species (ROS) such as singlet oxygen and hydroxyl radicals, which mediate killing of neighboring microbial cells [6]. PACT has the following advantages over traditional antibiotic therapies. First, PACT is more phototoxic to bacteria than to mammalian cells. Human cells (keratinocytes and fibroblasts) survive PACT under conditions that are lethal to microorganisms [7, 8]. Second, PACT possesses a broad spectrum of action; many antibiotic-resistant bacteria are efficiently inactivated by PACT because ROS is toxic to almost all bacteria [9, 10]. Third, the non-specific action of liberated ROS is unlikely to induce the development of protective bacterial mechanisms [11].

Hematoporphyrin monomethyl ether (HMME) is a secondgeneration porphyrin derivative PS, consisting of a mixture of two positional isomers of 7(12)-(1-methoxyethyl)-12(7)-(1- hydroxyethyl)-3, 8, 13, 17-tetramethyl-21H, 23H-porphyrin- 2, 18-dipropionic acid (Fig. 1) [12]. Compared to the firstgeneration PSs, such as Photofrin and HpD, HMME has a known structure, higher photoactivity, stronger photodynamic efficiency (1O2 quantum yield of HMME, ∼0.6 [13]), lower toxicity, and a faster clearance rate. HMME has been used to effectively treat cancerous cells of several types [14–16], although little is known about its photodynamic inactivation (PDI) effect on bacteria, particularly antibiotic-resistant bacterial strains. In this study, we assessed the PDI efficacy of HMME for several important antibiotic-resistant bacterial strains in planktonic and biofilm culture.

Photosensitizer and light source

Drug-grade HMME was purchased from Shanghai Xianhui Pharmaceutical Co., China. A 1-mM stock solution was freshly prepared by dissolving the PS in sterilized PBS (0.1 N, pH 7.2) and stored in the dark. The stock solution was filtered through a 0.22-μm filter disk and properly diluted with sterilized PBS before use. All illuminations were performed with white light from a 150-W xenon lamp (Ceaulight CEL-HXF300, China). A wavelength range between 400 and 780 nm was selected by an optical filter (Ceaulight CEL-UVIRCUT PD-145, China). To avoid heating of the samples, the light was passed through a 1-cm ice-cold water filter. The fluence rate at the level of the samples was 40 mW cm−2 , as measured with a power meter (Ceaulight CEL-NP2000, China).

To avoid heating of the samples, the light was passed through a 1-cm ice-cold water filter. The temperature of samples were 35.1 °C and 32.3 °C before and after irradiation, respectively, as measured by a thermocouple (IKA EST-D5, Germany) at room temperature (25 °C). As shown in Fig. 2, HMME did not exhibit dark toxicity for the six antibiotic-resistant bacterial strains at the tested concentrations. Furthermore, direct exposure of these bacterial strains to light in the absence of HMME produced no significant cytotoxic effect (data not shown). In contrast, the irradiated groups showed reduced bacterial survival with increasing concentrations of HMME. For the gramnegative bacterial strains, 100-μM HMME yielded 5.59 and 4.92 log10 reductions in the survival fraction of ESBLproducing K. pneumoniae and E. coli, respectively. MDR P. aeruginosa was less sensitive to the PDI effect of HMME, and a 4.19 log10 reduction in survival was observed under the same conditions. HMME showed stronger PDI efficacy against the gram-positive strains: 100-μM HMME produced 6.68 and 6.49 log10 reductions in the survival fraction of VRE from clinic and VRE (ATCC 51299). Among the tested strains, MRSA was the most susceptible: 0.1-μM HMME with irradiation yielded a 2.44 log10 reduction in viable MRSA cells. A 7.59 log10 reduction in the survival fraction was achieved at an HMME concentration of 100 μM. All tested strains were treated with 1-mM HMME and irradiated for 30 min, after which no viable cells were detected; thus, the antibiotic-resistant bacteria were completely inactivated.