Photodynamic Therapy Combined with Antibiotics or Antifungals against Microorganisms That Cause Skin and Soft Tissue Infections: A Planktonic and Biofilm Approach to Overcome Resistances

pharmaceuticals Review Photodynamic Therapy Combined with Antibiotics or Antifungals against Microorganisms That Cause Skin and Soft Tissue Infections: A Planktonic and Biofilm Approach to Overcome Resistances Vanesa Pérez-Laguna 1,* , Isabel García-Luque 2, Sofía Ballesta 2, Antonio Rezusta 3,4,5,† and Yolanda Gilaberte 3,6,†   Citation: Pérez-Laguna, V.; García-Luque, I.; Ballesta, S.; Rezusta, A.; Gilaberte, Y. Photodynamic Therapy Combined with Antibiotics or Antifungals against Microorganisms That Cause Skin and Soft Tissue Infections: A Planktonic and Biofilm Approach to Overcome Resistances. Pharmaceuticals 2021,14, 603. https://doi.org/10.3390/ ph14070603 Academic Editor: Serge Mordon Received: 15 May 2021 Accepted: 16 June 2021 Published: 23 June 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). 1Max Planck Institute for Evolutionary Biology, 24306 Plön, Germany 2Department of Microbiology, School of Medicine, University of Sevilla and Instituto de Biomedicina de Sevilla (IBIS), 41009 Sevilla, Spain; [email protected] (I.G.-L.); [email protected] (S.B.) 3Aragon Health Research Institute (IIS Aragón), 50009 Zaragoza, Spain; [email protected] (A.R.); [email protected] (Y.G.) 4Department of Microbiology, Miguel Servet University Hospital, 50009 Zaragoza, Spain 5Department of Microbiology, Preventive Medicine and Public Health, University of Zaragoza, 50001 Zaragoza, Spain 6Department of Dermatology, Miguel Servet University Hospital, 50009 Zaragoza, Spain *Correspondence: per[email protected] † These authors contributed equally to this work. Abstract: The present review covers combination approaches of antimicrobial photodynamic therapy (aPDT) plus antibiotics or antifungals to attack bacteria and fungi in vitro (both planktonic and biofilm forms) focused on those microorganisms that cause infections in skin and soft tissues. The combination can prevent failure in the fight against these microorganisms: antimicrobial drugs can increase the susceptibility of microorganisms to aPDT and prevent the possibility of regrowth of those that were not inactivated during the irradiation; meanwhile, aPDT is effective regardless of the resistance pattern of the strain and their use does not contribute to the selection of antimicrobial resistance. Additive or synergistic antimicrobial effects in vitro are evaluated and the best combinations are presented. The use of combined treatment of aPDT with antimicrobials could help overcome the difficulty of fighting high level of resistance microorganisms and, as it is a multi-target approach, it could make the selection of resistant microorganisms more difficult. Keywords: photoinactivation; resistance; antimicrobial agents; SSTI; MDR 1. Introduction 1.1. The Problem of Skin and Soft Tissue Infections Skin and soft tissue infections (SSTIs) are defined as clinical entities of variable presentation, etiology, and severity that involve microbial invasion of the layers of the skin and underlying soft tissues. Related to clinical manifestations of the wide range of pathologies they represent, the minimum diagnostic criteria are erythema, edema, warmth, and pain or tenderness. The affected area may also become dysfunctional depending on the severity of the infection, and, much more relevantly, patient comorbidity can easily transform a normally mild infection into a rapidly advancing threat to life [ 1 – 3 ]. Complicated forms of SSTI (cSSTI) may need, apart from antibiotic or antifungal treatment, surgical procedures, or have significant underlying co-morbidities such as diabetes, systemic immunosuppression, or neurological diseases [4–6]. Their treatment has become more challenging because of the increase in the frequency and severity of infections mainly due to the ageing of the general population, the increased number of critical and immunocompromised patients, and because of the emergence Pharmaceuticals 2021,14, 603. https://doi.org/10.3390/ph14070603 https://www.mdpi.com/journal/pharmaceuticals Pharmaceuticals 2021,14, 603 2 of 21 of resistance to many of the antimicrobial agents commonly used to treat SSTIs in the past [7–9]. 1.2. Antimicrobial Resistance in Skin and Soft Tissue Infections Causal Agents Antimicrobial resistance (AMR) makes treatments more tedious and it has adverse consequences such as prolonged hospitalization, increased medical expenses, overburdened public health system, and even increased mortality rates [10]. The main causative agent of SSTI is Staphylococcus aureus, which is one of the bacteria most frequently involved in the problem of AMR. Other causal agents of a high percentage of SSTI, also involved in AMR problem, are Enterobacteriaceae and Pseudomonas aeruginosa (e.g., 45.9% of hospital-acquired SSTI in North America are caused by S. aureus, 10.8% by P. aeruginosa, and 8.2% by Enterococcus spp.) [ 1 ]. In fact, in 2017 the World Health Organization (WHO) published the first list of bacteria for which new antibiotics were urgently needed [ 11 ] (Table 1). Carbapenem-resistant or third generation cephalosporin-resistant Enterobacteriaceae (e.g., Escherichia coli) and carbapenem-resistant P. aeruginosa are classified in this list within the most dangerous class (Priority 1: critical) and methicillin-resistant or vancomycin intermediate and resistant S. aureus is classified as “Priority 2: high”. Table 1. Global priority list of antibiotic-resistant bacteria (Adapted from: https://www.who.int/ medicines/publications/WHO-PPL-Short_Summary_25Feb-ET_NM_WHO.pdf?ua=1 (accessed on 20 June 2021)). Priority 1: CRITICAL -Acinetobacter baumannii, carbapenem-resistant -Pseudomonas aeruginosa, carbapenem-resistant -Enterobacteriaceae (Klebsiella pneumonia,Escherichia coli,Enterobacter spp., Serratia spp., Proteus spp., and Providencia spp., Morganella spp.), carbapenem-resistant, 3rd generation cephalosporin-resistant Priority 2: HIGH -Enterococcus faecium, vancomycin-resistant -Staphylococcus aureus, methicillin-resistant, vancomycin intermediate and resistant -Helicobacter pylori, clarithromycin-resistant -Campylobacter, fluoroquinolone-resistant -Salmonella spp., fluoroquinolone-resistant -Neisseria gonorrhoeae, 3rd generation cephalosporin-resistant, fluoroquinolone-resistant Priority 3: MEDIUM -Streptococcus pneumoniae, penicillin-non-susceptible -Haemophilus influenzae, ampicillin-resistant -Shigella spp., fluoroquinolone-resistant Carbapenem-resistant Acinetobacter baumannii completes the list of bacteria classified as “Priority 1”. A. baumannii-associated SSTI is an emerging infection in patients who experience trauma; although it causes few cases, usually they are severe [12]. Regarding atypical mycobacteria, which also cause SSTI on certain occasions, they are highly resistant to antibacterial treatments due to the special characteristics of their wall (the mycobacterial cell has four main layers: (i) the plasma membrane, (ii) the peptidoglycan– arabinogalactan complex, (iii) an asymmetrical outer membrane covalently linked with mycolic acids, and (iv) an outermost capsule). They are a globally established priority for which innovative new treatments are urgently needed according to the WHO [11]. On the other hand, the recent emergence of fungi that are resistant to more than one class of antifungal drug is a serious concern, especially because currently only three primary classes of agents are used to treat invasive fungal infections: (1) Azoles (Fluconazole, voriconazole, posaconazole), (2) Echinocandins (Caspofungin, micafungin, anidulafungin) and (3) Polyne (Amphotericin B). Candida glabrata,Candida krusei and Candida auris are species with intrinsic or high rates of resistance against the first, Cryptococcus spp. and Pharmaceuticals 2021,14, 603 3 of 21 Fusarium spp. against the second, and Candida auris and Aspergillus terreus against the last [13]. With regard to yeasts that cause SSTI, Candida spp. stand out as causative agents. They are implicated in the AMR problem due to the increasingly frequent existence of fluconazole resistant strains [13,14]. Finally, dermatophytes and non-dermatophytes molds cause cutaneous and subcutaneous infections than are often chronic and recalcitrant. Invasive infections are rare but occur especially in immunocompromised and debilitated individuals. Its treatment is a great challenge especially in these patients. [15,16]. 1.3. Antimicrobial Photodynamic Therapy Combined with Antibiotics or Antifungals to Treat SSTIs Antimicrobial photodynamic therapy (aPDT) is based on the use of photosensitizer molecules (PS) that are activated by light in the presence of oxygen. Reactive oxygen species (ROS) are generated in the photodynamic reaction resulting in a toxic effect for microorganisms that is capable of destroying them (Figure 1). Hence, aPDT has been proposed as an alternative treatment for SSTIs [17,18]. Pharmaceuticals2021,14,xFORPEERREVIEW3of20   anidulafungin)and(3)Polyne(AmphotericinB).Candidaglabrata,CandidakruseiandCandidaaurisarespecieswithintrinsicorhighratesofresistanceagainstthefirst,Cryptococcus spp.andFusariumspp.againstthesecond,andCandidaaurisandAspergillusterreusagainst thelast[13]. WithregardtoyeaststhatcauseSSTI,Candidaspp.standoutascausativeagents. TheyareimplicatedintheAMRproblemduetotheincreasinglyfrequentexistenceof fluconazoleresistantstrains[13,14]. Finally,dermatophytesandnon‐dermatophytesmoldscausecutaneousandsubcutaneousinfectionsthanareoftenchronicandrecalcitrant.Invasiveinfectionsarerarebut occurespeciallyinimmunocompromisedanddebilitatedindividuals.Itstreatmentisa greatchallengeespeciallyinthesepatients.[15,16]. 1.3.AntimicrobialPhotodynamicTherapyCombinedwithAntibioticsorAntifungalstoTreat SSTIs Antimicrobialphotodynamictherapy(aPDT)isbasedontheuseofphotosensitizer molecules(PS)thatareactivatedbylightinthepresenceofoxygen.Reactiveoxygenspecies(ROS)aregeneratedinthephotodynamicreactionresultinginatoxiceffectformicroorganismsthatiscapableofdestroyingthem(Figure1).Hence,aPDThasbeenproposedasanalternativetreatmentforSSTIs[17,18].  Figure1.Mechanismofactionofantimicrobialphotodynamictherapy:photosensitizermolecules (PS)ingroundstate( 1 PS)areactivatedbylight(hv)ofawavelengththatmatchestheabsorption wavelengthofthemolecule.PSreachesanexcitedstate,firstsinglet( 1 PS*),veryunstable,andrapidlytriplet( 3 PS*).Thisreactswiththeoxygenbytransferringelectrons(TypeI)orenergy(TypeII) initsreversiontothegroundstate( 1 PS*).TypeIreactionischaracterizedbytheformationofreactiveoxygenspecies(ROS)andTypeIIbytheformationofsingletoxygen( 1 O 2− ).Thesemolecules arehighlyreactiveandarecapableofdamagingcellsresultinginatoxiceffectformicroorganisms [18]. AmongtheadvantagesoftheaPDTisthephotoinactivationofthemicroorganisms independentlyoftheirpatternofAMR,withoutitsusecontributingtotheselectionof drug‐resistantstrains,anditsbroadspectrumofactivity;thereforeitdoesnotrequirea precisemicrobialdiagnosisanditisveryusefulforinfectionscausedbyseveralmicroorganisms[19,20].Bycontrast,themostimportlimitationisthepossibilityofmicrobialregrowthbythosewhohavenotbeeninactivatedduringirradiation[17,18,21].ThecombinationofaPDTwithantibioticorantifungaltreatmentisapromisingapproachtothefight againstinfectiousdiseasesduetoaPDT’sabilitytoincreasethesusceptibilityofmicroorganismstoantimicrobialdrugsandminimizethepossibilityoftheregrowthafteraPDT [22–24]. Figure 1. Mechanism of action of antimicrobial photodynamic therapy: photosensitizer molecules (PS) in ground state ( 1 PS) are activated by light (hv) of a wavelength that matches the absorption wavelength of the molecule. PS reaches an excited state, first singlet ( 1 PS*), very unstable, and rapidly triplet ( 3 PS*). This reacts with the oxygen by transferring electrons (Type I) or energy (Type II) in its reversion to the ground state ( 1 PS*). Type I reaction is characterized by the formation of reactive oxygen species (ROS) and Type II by the formation of singlet oxygen ( 1 O 2− ). These molecules are highly reactive and are capable of damaging cells resulting in a toxic effect for microorganisms (Adapted with permission from ref. [18], 2018, Pérez-Laguna et al.). Among the advantages of the aPDT is the photoinactivation of the microorganisms independently of their pattern of AMR, without its use contributing to the selection of drug-resistant strains, and its broad spectrum of activity; therefore it does not require a precise microbial diagnosis and it is very useful for infections caused by several microorganisms [19,20] . By contrast, the most import limitation is the possibility of microbial regrowth by those who have not been inactivated during irradiation [ 17 , 18 , 21 ]. The combination of aPDT with antibiotic or antifungal treatment is a promising approach to the fight against infectious diseases due to aPDT’s ability to increase the susceptibility of microorganisms to antimicrobial drugs and minimize the possibility of the regrowth after aPDT [22–24]. Pharmaceuticals 2021,14, 603 4 of 21 1.4. Objective We present a review of articles related to in vitro activities of aPDT in combination with antibiotics or antifungals, focusing on microorganisms that cause SSTIs. The aim is to combine the information that exists about each microorganism (gather the bases of knowledge). This review serves as a starting point for new combined treatment research (expand and apply knowledge). The questions that are intended to be answered are: (1) Which microorganisms that cause SSTIs have been exposed in vitro to combinations of aPDT and antimicrobials? (2) Which methodologies have been used? (3) Are there an additive or synergistic antimicrobial effect? (4) Which are the best combinations clinically and microbiologically? 2. Methodology 2.1. Eligibility Criteria We have considered in vitro studies that used antimicrobial agents (antibiotics or antifungals) plus aPDT against microorganisms that cause SSTIs. The specific requirements for inclusion of the studies were (1) in vitro studies in planktonic state or forming biofilm and ex vivo studies on the skin or mucous membrane; (2) aimed to inactivate identified bacteria, or fungi that cause SSTIs; (3) used antibiotics or antifungals as a fundamental part of the treatment; (4) used aPDT as a fundamental part of the treatment; and (5) published in indexed journals and written in English or Spanish. 2.2. Study Selection, Data Collection Process, and Characteristics The keywords used for the search in Pubmed and Embase library databases were: photodynamic therapy, PDT, antimicrobial photodynamic therapy, aPDT, photodynamic antimicrobial chemotherapy, PACT, photoinactivation, photodynamic inactivation, PDI, combination, combined treatment, antimicrobial agents, antibiotics, and antifungals. The last search was carried out on 1 June 2021 and no time limits in the past were applied. Research papers that meet the eligibility criteria (Section 2.1) were included. A huge number of studies contained the keywords; nevertheless, after applying the eligibility criteria, the number was drastically reduced to a total of 33. The data recapitulated were: (1) etiological agent of SSTIs; (2) type of study: in vitro (planktonic or forming biofilm) and ex vivo; (3) antimicrobial methodology: antibiotics or antifungals used and their application and concentration; (4) aPDT methodology: PS used and parameters of irradiation (source type, wavelength, intensity, and fluence); (5) effect of combined treatment against microorganism. The 33 included studies were grouped depending on the etiological agent of SSTIs. The structure used is as follows: -Gram-positive bacteria: Staphylococcus spp. (Section 3.1: Section 3.1.1. S. aureus and Section 3.1.2.Staphylococcus epidermidis and Staphylococcus hemolytic) -Mycobacteria: Mycobacterium fortuitum (Section 3.2.) -Gram-negative bacteria: E. coli, P. aeruginosa, and A. baumannii (Section 3.3, Section 3.4 , Section 3.5 respectively) -Fungal infectious agents: Yeast (Section 3.6.Candida spp.), dermatophytes, and molds (Section 3.7). 3. Results of Studies on In Vitro aPDT Combined with Antimicrobial Agents against Infectious Microorganism of Skin and Soft Tissues 3.1. Staphylococcus spp. 3.1.1. Staphylococcus aureus S. aureus, frequently involved in the problem of AMR, is the main causative agent of SSTI and represents one of the most important pathogens involved in cSSTI [ 2 , 25 , 26 ]. For this reason, this is the bacteria with more studies trying the combination of different antibiotics and PS published so far. Table 5 summarizes the studies concerning in vitro aPDT plus other antimicrobial treatments against S. aureus. Pharmaceuticals 2021,14, 603 5 of 21 Porphycene and Porphyrin Studies To our knowledge, one of the most recent articles about aPDT and antimicrobials’ combination was published by Nieves et al. They report the synthesis of a new porphycene namely 2-aminothiazolo[4,5-c]-2,7,12,17-tetrakis(methoxyethyl)porphycene (ATAZTMPo)- gentamicin conjugate. It outperforms the antimicrobial effect against S. aureus and E. coli of the two components delivered separately. This novel photoantimicrobial agent may be used to enhance the therapeutic index of gentamicin, broaden the spectrum of pathogens against which it is effective, and reduce its side effects [27]. The combination of gentamicin with porphyrins also seems to be effective against S. aureus [26,28]. 5-aminolevulinic acid (5-ALA), a pro-drug that, once metabolized by proliferating bacteria, is converted into the natural PS protoporphyrin IX (PpIX), combined with a low concentration of gentamycin (2 µ g/mL) improved qualitative and quantitative antibacterial effects against S. aureus biofilms. The proposed explanation was that photoactivation generated ROS which damages or kills the cells, while gentamicin, even at low doses, completes the eradication. The mechanism of action of gentamicin is based on its capacity to bind tightly to the 30S subunit of the bacterial ribosome impairing protein synthesis and the bacterial cells die. The fundamental requirement for gentamicin to act as an antibiotic is the need to penetrate the membrane of the bacterial cell, and aPDT can possibly damage the membrane thus increasing antibiotic penetration [28]. Deuteroporphyrin-aPDT combined with different antibiotics included gentamicin, vancomycin, rifampicin, fusidic acid, and oxacillin did not detect synergistic effect except for oxacillin. They concluded that only the combination of deuteroporphyrin-aPDT plus oxacillin had potential for aPDT to improve traditional antibiotic treatment with cell wall synthesis inhibitors [29]. Regarding the combination of porphyrins with other antibiotics different from gentamycin, when ALA-aPDT was combined with netilmicin, vancomycin or cefaclor against S. aureus biofilms, synergistic bactericidal effect was observed. Destruction occurred predominantly in the upper layer of the biofilm, and in a strain-dependent manner. That is why they suggested that a drug sensitivity test should be performed in advance [30]. Meso-tetrakis(N-methyl-4-pyridyl) porphine tetra-tosylate (TMP), a cationic porphyrin combined with vancomycin, was highly effective against S. aureus biofilms. It seems that the combination blocks cell wall synthesis, and the damaged biofilms may be more susceptible to host defenses which could be useful for biofilms adhering to medical implant surfaces [31]. In contrast, the combination of other cationic porphyrin, 5,10,15,20-tetrakis[4-(3-N,N- dimethylaminopropoxy) phenyl] porphyrin (TAPP)-aPDT with the cell wall inhibiting antibiotics ceftriaxone or vancomycin did not significantly increase the aPDT effect on planktonic S. aureus. However, the bactericidal effect of TAPP-aPDT was additive with the protein synthesis inhibitors chloramphenicol and tobramycin against S. aureus and E. coli, and was synergistic against methicillin resistant S. aureus (MRSA) and S. epidermidis. It seems that vancomycin and ceftriaxone presented limited effects when combined with TAPP-aPDT; nevertheless, tobramycin and chloramphenicol reached additive effects for S. aureus and E. coli and synergy against MRSA and S. epidermidis, even when sub-minimum inhibitory concentration (MIC) levels of TAPP and antibiotics were used. The results suggest that even sub-MIC levels of photo-activated TAPP could be used to boost the activity of waning antibiotics [32]. Tetracationic porphyrin meso-tetrakis(1-methylpyridinium)porphyrin (Tetra-Py + -Me)- PDT combined with ampicillin significantly reduced S aureus survival in both states (planktonic and biofilm) [33]. Finally, the endogenous porphyrins accumulated by some microorganism, such as E. coli or S. aureus, combined with different wavelengths of light (blue, 470 nm, or red, 625 nm) in the presence of ciprofloxacin were more effective than the antibiotic alone [ 34 ]. Pharmaceuticals 2021,14, 603 6 of 21 Overall, there are multiple promising combinations of antibiotics and porphyrins- PDT which could increase the efficacy in vitro of conventional antibiotics against S. aureus (See Table 5). Phenothiazine Studies Another chemical group of PS for PDT are phenothiazinium dyes, such as methylene blue (MB) and toluidine blue O (TBO). MB-aPDT combined with chloramphenicol showed an additive effect against planktonic S. aureus [ 32 ], while ciprofloxacin plus MB-aPDT was synergistic either in planktonic and in biofilm forms [ 35 ]. As usual, bacteria forming biofilms were less affected than bacteria in planktonic phase and a bigger effect was seen when ciprofloxacin was applied after aPDT rather than before or simultaneously. Kashef et al. using MB or TBO-aPDT with linezolid against biofilm resulted in a greater reduction in the viable count of bacteria than either of them separately. TBO-aPDT plus linezolid gave the greatest reduction (although the killing was only <3 log10 reduction in viable count for any of the strains) [36]. TBO-aPDT combined with gentamicin and incubated 45-min prior to irradiation showed positive effects against S. aureus strains in planktonic state, both in a reference strain and in a multidrug resistant (MDR) clinical isolate; they showed that TBO-aPDT treatment permeabilized the bacterial membranes, promoted gentamicin cellular accumulation and the intracellular ROS generation by the combination was much higher than that of single treatment groups [37]. Our group explored the effect of MB-aPDT, alone or in combination with gentamicin, against planktonic and biofilm S. aureus. The addition of gentamicin (concentrations that by themselves do not achieve any effect) caused the complete photoelimination in the case of planktonic S. aureus, with a lower MB-PDT dose, whereas it did not produce any change in biofilm [ 38 ]. In a previous study, it was proven that concentrations of linezolid or mupirocin which did not harm the bacteria also exert a synergistic effect when they were combined with MB, also reducing the concentration of the PS needed to photoinactivate planktonic S. aureus [ 39 ]. However, recently we have verified in a superficial abrasion mouse model of S. aureus skin infection that the addition of mupirocin to MB-aPDT treatment improved antimicrobial activity but it did not enhance wound healing [40]. Xanthene Studies Rose Bengal (RB, a xanthene dye) has been rehearsed combined with mupirocin, linezolid, or gentamicin with a synergistic bactericidal effect in planktonic S. aureus [ 39 ]. Additionally, the combination of RB-aPDT plus gentamicin was evaluated against S. aureus biofilms. However, only high concentration of RB (64 µ g/mL) and gentamicin (40 µ g/mL) showed a synergistic effect against biofilms [ 41 ]. It is interesting that the combination of RB-aPDT with methicillin significantly reduced the MIC of methicillin either of MRSA or methicillin sensitive S. aureus [26]. 3.1.2. Staphylococcus epidermidis and Staphylococcus haemolyticus S. epidermidis and S. haemolyticus are part of the skin flora of humans but in specific circumstances, such as with immunocompromised or hospitalized patients with catheters or other surgical implants, they are well-known opportunistic pathogens which cause local or systemic infections. The highly antibiotic-resistant phenotype and their ability to form biofilms makes infections difficult to treat [42–45]. The study of Barra et al. has explored 5-ALA-aPDT plus gentamicin at a low concentration against clinical isolates of S. haemolyticus,S. epidermidis, and S. aureus biofilms; they reported a synergistic effect being S. haemolyticus the most sensitive to photoinactivation [28]. Other porphyrins, such as TMP-aPDT combined with vancomycin in biofilms [ 46 ] or TAPP-aPDT combined with chloramphenicol or tobramycin in planktonic cells, showed a higher efficacy against S. epidermidis than any of them separately. The authors hypothesized Pharmaceuticals 2021,14, 603 7 of 21 that TAPP could act in combination with lower concentrations of antibiotics providing a controlled release of the antibiotic, and in consequence improving their efficacy to maintain an antimicrobial effect [32]. In conclusion, multiple antibiotics in combination with different types of PDT based on protoporphyrins showed improved bactericidal effects against planktonic S. epidermidis or forming biofilms [28,32,46]. Table 5 includes a summary of all the studies of aPDT combined with antibiotics. Table 2 summarizes in detail the methodology and the results of different studies that reported the in vitro activity of aPDT plus antibiotics against S. haemolyticus and S. epidermidis (Table 2: Biofilm Studies and biofilm state, planktonic—Table 2: Planktonic Cell Studies). Table 2. Studies combining in vitro aPDT plus other treatment against S. haemolyticus and S. epidermidis comparing the methodology and the results biofilm studies, planktonic cell studies. Biofilm Ttudies Target PS PS Concentration (µM) Antibiotic Antibiotic Concentration (µg/mL) Source Type Wavelength (nm) Intensity (mw/cm2) Fluence (J/cm2) Inactivation Fraction (%) Synergistic Observed Effect (*) Reference S. haemolyticus clinical isolate 5-ALA 40 gentamicin 2 50-LED 630 ±15 83 250 ~70 >inactivation [28] S. epidermidis clinical isolate 5-ALA 40 gentamicin 2 50-LED 630 ±15 83 250 ~75 >inactivation [28] S. epidermidis RP62A & 5179R TMP 10 vancomycin 200 tungsten lamp 400–800 166 300 ~99.9999 >inactivation [46] Planktonic Cell Studies Target PS PS Concentration (µM) Antibiotic Antibiotic Concentration (mg/L) Preincuba tion Time (h) Irradiation Time (h) Source Type Wave length Media CFU/200 µL Well Log10 Reduction S. epidermidis ATCC 35984 TAPP 5 chloram phenicol 2 19 5 100 W, 120 V Sylvania white light Broad spectrum TSB ~106~2 S. epidermidis ATCC 35984 TAPP 5 tobramycin 4.5 19 5 100 W, 120 V Sylvania white light Broad spectrum TSB ~106~3 (*): the combination causes an increase in percentage of microbial inactivation greater than the sum of the antibacterial activity of the aPDT plus the antibiotic treatment when they are applied alone; 5-ALA: 5-aminolevulinic acid; aPDT: antimicrobial photodynamic therapy; CFU: colony forming unit; LED: light-emitting diode; PS: photosensitizer; TAPP: meso-tetra (4-aminophenyl) porphine; TMP: tetra-substituted N-methyl-pyridyl-porphine; TSB: trypticase soy broth. 3.2. Mycobacterium fortuitum M. fortuitum is an atypical mycobacteria involved in skin infections very difficult to treat, thus usually causes refractory infections [47,48]. The combination of MB-aPDT with ciprofloxacin, moxifloxacin, or amikacin against a clinical isolate had in vitro synergistic antimicrobial effect (the latter also was tested in vivo to treat a model of keratitis in rabbits resulting in significantly less bacterial burden). Sublethal concentrations of antibiotic plus sublethal dosage of MB reached a mycobacterial survival reduction in the colony forming units (CFU) of at least 2 log 10 lesser compared to the reduction achieved with antibiotics alone, although this effect became insignificant when higher doses of antibiotics were used. The maximum degree of inactivation was achieved by the combination of MB-aPDT with ciprofloxacin or moxifloxacin ( ≥ 4 log 10 reduction, with amikacin (~3 log 10 reduction) [ 49 ]. Table 5 and Table 3summarize the methodology and the results of this study. Pharmaceuticals 2021,14, 603 8 of 21 Table 3. Comparison of methodology and results of an in vitro study of aPDT followed by culture with antibiotics on Mycobacterium fortuitum. Strain PS PS Concentration (mg/mL) Antibiotic Antibiotic Concentration (mg/mL) Preincu bation Time (h) Source Type Wave length (nm) Intensity (mw/cm2) Fluence (J/cm2) Media/ Culture CFU/ 200µL Well Synergistic Observed Effect (*) Reference M. fortuitum clinical isolate MB 50 amikacin 0–0.5 0 + 72 with antib metal halogen lamp 560–780 100 100 PBS with 0.02% Tween 80 / Muller Hilton ~108 >inactivation (≥2 Log10 reduction) [49] ciprofloxacin hydrochloride 0–0.06 moxifloxacin hydrochloride 0–0.06 (*): the combination causes an increase in percentage of microbial inactivation greater than the sum of the antibacterial activity of the aPDT plus the antibiotic treatment when they are applied alone; aPDT: antimicrobial photodynamic therapy; MB: methylene blue; PBS: phosphate-buffered solution. 3.3. Escherichia coli E. coli is the best-known bacteria of the Enterobacteriaceae family [ 50 ]. Even though some serotypes of E. coli can cause serious disease, most strains are harmless and can only act as opportunistic pathogens [51]. Table 5 includes a summary of the in vitro aPDT plus antibiotics against E. coli studies and Table 4summarizes the methodology and the results of Gram-negatives studies. Table 4. Studies on combination of in vitro aPDT plus other treatment against Gram-negative bacteria that cause SSTIs comparing the methodology and the results. Strain PS Antibiotic Phase Source Type Wavelength (nm) Intensity (mw/cm2) Fluence (J/cm2)Synergy Observed Effect (*) Reference E. coli ATCC 25922 ATAZTMPo gentamicin planktonic LED (Sorisa Photocare) 638 ±9 nm 17 45 yes >inactivation [27] E. coli ATCC 25922 TAPP tobramycin or chloramphenicol planktonic 100 W, 120 V Sylvania white light Broad spectrum ND ND no additivity [32] E. coli ATCC 25922 MB ciprofloxacin planktonic IrradLED® biopdi, São Carlos, SP, Brazil ~660 ND 2.8 and 5.6 yes >inactivation [35] E. coli ATCC 25922 MB ciprofloxacin biofilm IrradLED® biopdi, São Carlos, SP, Brazil 660 ND 11.2 and 22 yes >inactivation [35] E. coli ATCC 9027 and MDR clinical isolates endogenous porphyrins ciprofloxacin or norfloxacin n planktonic LED Dermaled®~470 and ~625 ND ND yes incrase in halo [34] E. coli Chlorin e6 colistin, ciprofloxacin or amikacin planktonic diode laser, Laser Coupler 635 (Wroclaw, Poland) 635 290 120 yes >inactivation [51] E. coli Tetra-Py+- Me ampicillin or chloramphenicol planktonic white light lamps (13 lamps OSRAM 21 of 18 W each) Broad spectrum 380 to 700 40 - yes >inactivation [52] E. coli, E. aerogenes, and K. pneumoniae resistant to 3rd- cephalosporins, clinical isolates MB ceftriaxone planktonic LED (Biopdi/Irrad- Led 660) 660n ±5 25 25 no indifference [53] P. aeruginosa PAO1 TMP tobramycin biofilm mercury vapor lamp Broad spectrum - 220–240 yes >inactivation & tobramycin MIC decreased [54] Pharmaceuticals 2021,14, 603 9 of 21 Table 4. Cont. Strain PS Antibiotic Phase Source Type Wavelength (nm) Intensity (mw/cm2) Fluence (J/cm2)Synergy Observed Effect (*) Reference P. aeruginosa PAO1 endogenous porphyrins gentamicin planktonic Nd:YAG laser continuous / Pulsed-Q switched 532 106 yes >inactivation [55] P. aeruginosa PAO1and others MDR and XDR endogenous porphyrins gentamicin, meropenem or ceftazidime planktonic Singleemitter diode lamp 410 15.7 50 yes >inactivation & antibiotic MIC decreased [56] ATCC 27853 P. aeruginosa ATCC 27853 MB ofloxacin planktonic LED ~637 44 yes >inactivation [57] P. aeruginosa ATCC 27853 MB gentamicin planktonic LED lamp (Showtec LED Par 64 Short 18 x RGB 3-in-1 LED, Highlite International B.V. Spain) 625 ±10 7 18 yes bactericidal effect with lower MB-PDT dose [38] P. aeruginosa ATCC 27853 MB gentamicin biofilms LED lamp (Showtec LED Par 64 Short 18 x RGB 3-in-1 LED, Highlite International B.V. Spain) 625 ±10 7 18 yes bactericidal effect with lower MB-PDT dose [38] P. aeruginosa ATCC 10145 and 35 clinical isolates including MDR and XDR RB camel or pexiganan planktonic LED lamps (SecureMedia, Poland) ~514 23 60 yes >inactivation [58] P. aeruginosa ATCC 25668 and sensitive and resistant clinical isolates RB sulfanilamide planktonic 18 W white luminescent lamp Broad spectrum 400–700 1.25 - no indifference [26] A. baumannii 2 XDR clinical isolates RB gentamycin, doxycicline, trimethoprimsulfamethoxa zole, ciprofloxacin, imipenem, piperacillintazobactam, ceftazidime, ampicillinsulbactam, colistin planktonic LED 515 70 300 yes >inactivation & antibiotic MIC decreased [59] A. baumannii 2 XDR clinical isolates endogenous porphyrins gentamycin, doxycicline, trimethoprimsulfamethoxa zole, ciprofloxacin, imipenem, piperacillintazobactam, ceftazidime, ampicillinsulbactam, colistin planktonic LED 411 130 109.1 yes >inactivation & antibiotic MIC decreased [59] (*) > inactivation: the combination causes an increase in the percentage of microbial inactivation greater than the sum of the antibacterial activity of the aPDT plus the antibiotic treatment when they are applied alone; additivity: the combination causes an increase in percentage of microbial inactivation equal to the sum of the antibacterial activity of the aPDT plus the antibiotic treatment when they are applied alone: Indifference was defined as no change from the most active antibiotic treatment; aPDT: antimicrobial photodynamic therapy; ATAZTMPo; 2-aminothiazolo[4,5-c]-2,7,12,17-tetrakis(methoxyethyl)porphycene; RB: rose bengal; LED: light-emitting diode; MB: methylene blue; MDR: multidrug resistant; MIC: minimum inhibitory concentration; ND: no data; PS: photosensitizer; TAPP: meso-tetra (4-aminophenyl) porphine; TBO: toluidine blue O; Tetra-Py + -Me: 5,10,15,20-tetrakis(1-methylpyridinium-4-yl)porphyrin tetra-iodide; TMP: meso-tetra (N-methyl-4-pyridyl) porphine tetra tosylate; XDR: extensively-drug resistant. Pharmaceuticals 2021,14, 603 16 of 21 Table 5. Cont. Dermatophytes and moulds PS Group PS Antibiotic Phase Synergy Observed Effect (*) Reference Xanthenes RB clotrimazole Planktonic ( in vitro : spores) yes ≥inactivation [63] Porphycene and Porphyrin 5-ALA itraconazole planktonic yes ≥inactivation [64] Phenothiazines MB itraconazole, voriconazole, posaconazole, amphotericin planktonic and biofilms yes MIC decreased [15] MB itraconazole planktonic yes >inactivation [69] (*) > inactivation: the combination causes an increase in the percentage of microbial inactivation, greater than the sum of the antibacterial activity of the aPDT plus the antibiotic treatment when they are applied alone. Additivity: the combination causes an increase in the percentage of microbial inactivation equal to the sum of the antibacterial activity of the aPDT plus the antibiotic treatment when they are applied alone. Indifference was defined as no change from the most active treatment. 5-ALA = 5-aminolevulinic acid; ATAZTMPo = 2-aminothiazolo[4,5- c]-2,7,12,17-tetrakis(methoxyethyl)porphycene; DP = deuteroporphyrin; MB = methylene blue; MIC = minimum inhibitory concentration; MRSA = methicillin resistant S. aureus; MSSA = methicillin sensitive S. aureus; RB = rose bengal; PS = photosensitizer; TAPP = 5 ,10,15,20-tetrakis [4-(3-N,N-dimethylaminopropoxy) phenyl] porphyrin; TBO = toluidine blue O; TAPP = meso-tetra (4-aminophenyl) porphine; TMP = tetra - substituted N-methyl-pyridyl-porphine; Tetra-Py+-Me = 5,10,15,20-tetrakis (1-methylpyridinium-4-yl) porphyrin tetra-iodide. Table 6. Studies on combination of in vitro aPDT plus other treatment against fungus that cause SSTIs comparing the methodology and the results. Strain PS Antifungal Phase Source Type Wavelength (nm) Intensity (mw/cm2) Fluence (J/cm2)Synergy Observed Effect (*) Reference fluconazoleresistant C. albicans and C. glabratai MB fluconazole planktonic InGaAlP LED nd 200 - yes >inactivation [61] fluconazoleresistant C. krusei MB fluconazole planktonic InGaAlP LED nd 200 - no indifference [61] C. albicans SC5314 TMP miconazole planktonic broadband visible light (Sylvania GRO-LUX, 15 W, part no. F15T8/GRO) 575–700 4 1 yes >inactivation [62] C. albicans SC5314 TMP fluconazole planktonic broadband visible light (Sylvania GRO-LUX, 15 W, part no. F15T8/GRO) 575–700 4 1 no indifference [62] C. albicans SC5314 MB miconazole planktonic broadband visible light (Sylvania GRO-LUX, 15 W, part no. F15T8/GRO) 575–700 4 7.2 yes >inactivation [62] T. rubrum clinical isolate RB clotrimazole Planktonic (in vitro: spores) LED 530 13.4 12 yes ≥inactivation [63] F. monophora clinical isolates 5-ALA itraconazole planktonic Zeiss KL 2500 LED 635 36.8 10 yes ≥inactivation [64] E. dermatitidis, F. solani,F. oxysporum clinical isolates MB itraconazole, voriconazole, posaconazole, amphotericin planktonic and biofilms LED 635 ±10 100 12-24 yes MIC decreased [15] S. globosa 5 clinical isolates MB itraconazole planktonic LED 640 ±10 22.2 40 yes >inactivation [69] (*) > inactivation: the combination causes an increase in percentage of microbial inactivation greater than the sum of the antibacterial activity of the aPDT plus the antibiotic treatment when they are applied alone. = inactivation: the combination has no effect on the percentage of inactivation; 5-ALA: 5-aminolevulinic acid; aPDT: antimicrobial photodynamic therapy; nd: no data; RB: rose bengal; LED: light-emitting diode; MB: methylene blue; PS: photosensitizer; TMP: meso-tetra (N-methyl-4-pyridyl) porphine tetra tosylate. Pharmaceuticals 2021,14, 603 17 of 21 Sporothrix globosa is an etiological agent of sporotrichosis whose most common clinical manifestation is cutaneous and subcutaneous nodular lesions with lymphangitis involvement. Currently, AMR and complications are the major concerns, especially in patients who have liver disorders, children, and pregnant women. Li et al. compared the efficacy in the inactivation of S. globosa of MB-aPDT alone or combined with itraconazole in planktonic culture and in a murine model. The combined treatment offered better results in terms of inactivation percentage and improvement of the lesion size. Therefore, they conclude that MB-aPDT could be an effective adjuvant therapy for resistant infections caused by Sporothrix spp. [ 69 ]. In fact, our group treated a patient with cutaneous sporotrichosis using intralesional 1% MB-aPDT in combination with intermittent low doses of itraconazole obtained complete microbiological and clinical response [70]. 4. Summary of Evidence and Limitations In the main, the combination of antibiotics or antifungals with aPDT against in vitro SSTI-etiological agents seems to be beneficial. Combined therapy is more effective than individual treatments alone and often the effects are greater than additivity, i.e., there is synergy. Among the effects reported, the following stand out: (i) the increase in percentage of microbial inactivation; or (ii) the same inactivation percentage is achieved using lower doses of antimicrobials. It is remarkable that in some cases, drug sensitivity of resistant strains can be restored by combining antibiotics/antifungals with aPDT [15,26,59]. The highlight combinations and the best treatment protocols supported by the existing evidence of in vitro studies on combined aPDT therapies against SSTI-causing agents are included in Table 7. Nevertheless, there is not enough evidence to establish the best combination against each causal agent of SSIT according to this review. The number of studies is limited, and the methodologies used are varied, making direct comparison difficult. In addition, they mostly report the effect on inactivation, but the mechanism of action remains unknown. This review provides additional and updated information to the one published by Wozniak and Grinholc in 2018, and it is complementary to the review focused on in vivo studies published by our group in 2019 [ 23 , 24 ]. All three types of review agree on the promising approach of combining both therapies and the need to expand knowledge in this line. The coating of surfaces such as catheters with antimicrobial drugs and aPDT are extremely effective and virtually overcome any resistance build-up. This is more complex on the skin and soft tissues because more variables become part of the process, especially with fungi. However, there is sufficient evidence to support this combined treatment strategy and to lay the foundation for this SSIT treatment approach [18]. Among the obstacles to the incorporation of aPDT as part of the SSIT-treatment, the need to require materials such as lamps for exciting the PS and the need to dedicate more time, because of the irradiation time and because often more than one session of aPDT is required are highlighted. However, it does not require much more clinician specialization, various studies have proven that lamps do not have to be especially expensive and specific, and sources of light with a wide irradiation spectrum or even daylight of radiation can be used effectively [ 18 , 23 ]. The methodology and evolution of these treatments need to be reported to the scientific community to continue expanding knowledge and increasingly implement this combination therapeutic option. Pharmaceuticals 2021,14, 603 18 of 21 Table 7. The highlight combinations and the best treatment protocols supported by the existing evidence of in vitro studies on combined aPDT therapies against SSTI-causing agents. XThe combination of different antibiotics with aPDT in general improves the efficacy against in vitro Gram-positive bacteria. X Different antibiotics combined with aPDT using porphyrins, phenothiaziniums or RB have synergistic effects in vitro against Gram-positive bacteria being the combination with protoporphyrin the most studied. X The combination of aPDT with gentamicin has been extensively tested in both Gram-positive and negative bacteria, reporting positive effects in all cases. XThe combination of MB-aPDT with sublethal concentrations of antibiotics seems to be a good option against mycobacteria. XMB-aPDT combined with ciprofloxacin is the best option in vitro against E. coli. XEndogenous porphyrins or MB based-aPDT used in combination with antibiotics is a promising option against in vitro P. aeruginosa regardless of its antibiotic resistance pattern. XThe administration of aPDT before antifungals seems to enhance their in vitro antimicrobial effect, especially against yeast and dermatophytes. aPDT: antimicrobial photodynamic therapy; RB: rose bengal; MB: methylene blue. 5. Conclusions aPTD combined with antimicrobial agents is promising for the management of microorganisms that cause SSTI. It can help to fight them and to overcome AMR. Author Contributions: Conceptualization and methodology, V.P.-L., Y.G. and A.R.; writing—original draft preparation, V.P.-L.; writing—review and editing V.P.-L., I.G.-L., S.B., Y.G. and A.R.; supervision, Y.G. and A.R.; funding acquisition, I.G.-L., S.B., Y.G. and A.R. All authors have read and agreed to the published version of the manuscript. Funding: Y.G. And A.R. were founded by Aragon Health Research Institute (IIS Aragón), A.R. was funded by Research Groups of Government of Aragón (Spain) B10_17R Infectious Diseases of Difficult Diagnosis and Treatment. I.G.-L. and S.B. were funded by Plan Nacional de I+D+i and Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación, Spanish Network for Research in Infectious Diseases (REIPI; RD16/0016/0001)-co-financed by European Development Regional Fund “A way to achieve Europe”, ERDF. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: No new data were created or analyzed in this study. Data sharing is not applicable to this article. Acknowledgments: The authors thank the research groups of IIS Aragon GIIS-023 and GIS096 for their support. Conflicts of Interest: The authors declare no conflict of interest. Pharmaceuticals 2021,14, 603 19 of 21 References 1. Ki, V.; Rotstein, C. Bacterial skin and soft tissue infections in adults: A review of their epidemiology, pathogenesis, diagnosis, treatment and site of care. Can. J. Infect. Dis. Med. Microbiol. 2008,19, 173–184. [CrossRef] 2. 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