In 2020, the World Health Organization warned about the appearance of strains increasingly resistant and difficult to control. The indiscriminate use of antimicrobial drugs is facilitated by inadequate medical prescriptions and substandard medications ^[1].

Considering the challenges related to antimicrobial resistance, other strategies for controlling infections have been suggested ^[2][3][4][5]. Antimicrobial photodynamic therapy (aPDT) has been used to inactivate microorganisms and treat infections ^[2][3][4][5]. aPDT involves the application of a photosensitizing agent (PS), an LED source corresponding to the absorption band of the PS, and the presence of oxygen. This therapy has several advantages in the treatment of infections from microorganisms, such as the wide spectrum of action and a low mutagenic potential in exposed cells ^[5].

When comparing aPDT with other therapies, it has the advantage of local PS application, restricting the treatment to the area of interest, thus preventing systemic side effects. There is also an immediate onset of action and elimination of virulence factors secreted by resistant microorganisms ^[6]. Lastly, the literature did not report the development of bacteria and fungi resistance to aPDT ^[3][7].

Studies have shown that microbial biofilms reduce the susceptibility to aPDT compared to planktonic cultures ^[3]. Considering the protection endowed by the extracellular matrix (ECM), it is difficult for the PS to penetrate the deeper layers of the microbial biofilm, impairing aPDT activity ^[8]. To overcome this limitation, aPDT associated with enzymes or antifungal agents was more effective for microbial inactivation than aPDT alone ^[4][8]. Additionally, antimicrobial peptides (AMP) have been used alone ^[9][10], combined with aPDT ^[11][12], or by conjugating a PS to the AMP molecule ^{[13][14][15][16][17][18][19][20][21][22][23][24][25][26][27][28][29][30]}, presenting satisfactory results in pathogenic microorganism inactivation.

AMP are molecules expressed by all living organisms and responsible for the innate defense system against pathogen infection, including viruses, bacteria, fungi, and parasites ^[31]. AMP are oligopeptides with up to 50 amino acids with a broad spectrum of action against microorganisms ^[32][33]. This new class of compounds has boosted science for new methodologies for synthesizing, isolating, purifying, analyzing, and quantifying peptides ^[34]. The presence of cationic residues (Arg and Lys) in AMP promotes a positive liquid charge for this structure, resulting in the interaction with the negative cell membrane of the target organism, such as bacteria ^[34]. Another important aspect of the construction of the AMP amphipathic structure is the high fraction of hydrophobic amino acids (>50%) ^[35], which is vital for membrane penetration. The biological activity of AMP is closely related to their structure, and these could be classified as α-helix, β-sheet, extended peptides, and both α-helix and β-sheet peptides ^[36], with the first two appearing more frequently ^[37]. Although the molecular target of some peptides is inside the cell, as non-membrane disruptive AMP ^[38], most peptides interact with the anionic components of the membranes of microorganisms and damage this structure ^[31].

The literature has described the association of AMP and aPDT to explore the best properties of both treatments, increasing the effectiveness and decreasing the time of application ^[11][12]. AMP can form pores in cell membranes and present biofilm activity ^[10], which leads to the penetration of the PS into the membrane, facilitating the inactivation of structures through LED photoexcitation ^[11]. Other advantages of association treatments are reduced effective dose, minimized toxicity potential, and reduced treatment costs ^[11][39].

2. Synthesis of Results

The results of the systematic review show that all articles had an in vitro experimental design and 3 of them were both in vitro and in vivo experimental studies ^[27][29][30]. Moreover, of the 20 articles analyzed, 18 performed the therapy with a portion of the PS redirected to AMP and only 2 studies performed the therapy combined with AMP ^[11][12]. The shortest and longest irradiation times were 30 s ^[13] and 20 h ^[20][21], respectively. The most commonly used PS were chlorin e6 ^{[11][12][23][27][29][30]} and porphyrin ^{[13][15][16][18][20][21][24][25]}. Additionally, the most frequently used microorganism in the assay was Staphylococcus aureus ^{[11][13][14][16][17][18][19][20][21][23][25][26][28][29][30]}, followed by Escherichia coli ^{[11][13][15][16][17][18][19][20][21][25][26][29][30]}. Most of the studies analyzed evaluated the microorganisms in suspension (planktonic culture) and only 4 evaluated the therapy in a biofilm culture ^{[11][23][27][30]} (Table 1).

Table 1. Summary of the characteristics of the studies included.

Study (Year)	Study Design	Peptide	Irradiation Time	Wavelength	Photosensitizer	Microorganism	Culture Type	Sample Size	Outcomes
Bourré et al. 2010 [13]	In vitro	Tat	30, 43, 60, and 120 s	410 nm	Tetracks (phenol) and porphyrin	Escherichia coli Staphylococcus aureus Pseudomonas aeruginosa Streptococcus pyogenes	Suspension	ND	Reduction in the concentration of 1 uM from 3 to 6 log10 CFU/mL. The greatest effect was in the first 30 s.
Yang et al. 2011 [14]	In vitro	WLBU2	100 s	652 nm	Temoporfin + WLBU2	S. aureus (methicillin resistant) P. aeruginosa	Suspension	3	Reduction by 100% for S. aureus (aPDT only and aPDT + peptide) and reduction by 2 log10 CFU/mL for P. aeruginosa (aPDT + peptide).
Liu et al. 2012 [15]	In vitro	WI13WF (YVLWKRKRKFCFI-amide)	2, 5, and 10 min	400 to 900 nm	Protoporphyrin IX	E. coli Salmonella enteric Klebsiella pneumoniae	Suspension	ND	Peptide and PS conjugate 99% lethal.
Dosseli et al. 2013 [16]	In vitro	Apidaecin	ND	600–750 nm 390–460 nm	Porphyrin	E. coli S. aureus	Suspension	ND	Reduction by 100% for E. coli.
Johnson et al. 2013 [17]	In vitro	(KLAKLAK)2	30 min	525 nm	(KLAKLAK)2 + Eosin Y	Acinetobacter baumannii P. aeruginosa E. coli S. aureus Staphylococcus epidermidis	Suspension	ND	Reduction by 99% for all microorganisms.
Dosseli et al. 2014 [18]	In vitro	Magainin Buforin	ND	390–460 nm	Porphyrin	E. coli S. aureus (methicillin resistant)	Suspension	ND	Reduction by 100% for all microorganisms.
Johnson et al. 2014 [19]	In vitro	(KLAKLAK)2	2 min 5 min 30 min	525 nm	(KLAKLAK)2 + Eosin Y	E. coli S. aureus	Suspension	3	Reduction by 50% for all microorganisms (2 min of irradiation). Reduction by 90% (5 min of irradiation). Reduction by 99.99% (30 min of irradiation).
Le guern et al. 2017 [20]	In vitro	Polymyxin B	20 h	420 nm	Porphyrin	S. aureus E. coli P. aeruginosa	Suspension	ND	Antibactericidal activity of the PS and peptide association on 3 strains.
De Freitas et al. 2018 [11]	In vitro	Aurein 1.2 (AU)	ND	660 nm	Methylene blue Chlorin e6	S. aureus A. baumannii E. coli Enterococcus faecium	Suspension	9	S. aureus reduction - MB ~ 1.0 log10 CFU/mL - MB + AU ~ 6.0 log10 CFU/mL - Ce6 and Ce6 + Au = total reduction A. baumannii reduction - MB ~ 1.0 log10 CFU/mL - MB + AU ~ 6.0 log10 CFU/mL - Ce6 and Ce6 + AU no significant results E. coli reduction - MB ~ 4.0 log10 CFU/mL - MB + AU ~ 4.0 log10 CFU/mL - Ce6 and Ce6 + AU no significant results E. faecium reduction - MB ~ 1.0 log10 CFU/mL - MB + AU ~ 3.0 log10 CFU/mL - Ce6 ~ 1.0 log10 CFU/mL - Ce6 + AU = total reduction
Le guern et al. 2018 [21]	In vitro	Polymyxin B modified by lysine	20 h	420 nm	Porphyrin	S. aureus E. coli P. aeruginosa	Suspension	ND	Reduced antibacterial activity of polymyxin modified by lysine.
Nakonieczana et al. 2018 [22]	In vitro	CAMEL Pexiganan	668 s 1335 s 2668 s	514 nm	Rose-bengal (RB)	P. aeruginosa	Suspension	3	Reduction by 2.06 log10 CFU/mL for RB + CAM. Reduction by 6.00 log10 CFU/mL for RB + PEX.
Gao et al. 2019 [23]	In vitro	Magainin I	2 min 4 min 8 min	660 nm	Magainin I + Chlorin e6	P. aeruginosa S. aureus (methicillin resistant)	Biofilm	ND	P. aeruginosa 2 min (0.385 log10 CFU/mL reduction) 4 min (1.645 log10 CFU/mL reduction) 8 min (6.724 log10 CFU/mL reduction) S. aureus 2 min (0.922 log10 CFU/mL reduction) 4 min (3.796 log10 CFU/mL reduction) 8 min (6.586 log10 CFU/mL reduction)
De Freitas et al. 2019 [12]	In vitro	AU (GLFDIIKKIAESF-NH2) (AU)2K[(GLFDIIKKIAESF)2-k]	ND	664 nm	Methylene blue Chlorin e6	Enterococcus faecalis S. aureus E. faecium	Biofilm	9	Reducing the early biofilm stage - 95.5%—(Ce6-aPDT + (AU)2K) - 78%—Ce6-aPDT - 30%—MB-aPDT + AU - 20%—MB-aPDT - 30%—AU - 70%—(AU)2K)
Feese et al. 2019 [25	In vitro	Alkyne 1-Zn TMPYP	5, 15, and 30 min	400 to 700 nm	Porphyrin	Mycobacterium smegmatis	Suspension		Inactivation of 4 Log10 CFU/mL when associated with porphyrin and 1-Zn.
Zhang et al. 2019 [25]	In vitro	(KLAKLAK)2(KLA)	5 min (in vivo) 10 min (in vitro)	660 nm	PpIX PPK = PpIX + (KLAKLAK)2(KLA)	S. aureus E. coli	Suspension	ND	Inhibition rate S. aureus = 100% for both PS E. coli = 100% (PPK)/50% (PpIX)
Chu et al. 2021 [26]	In vitro	Bacitracin	5 and 30 min	610 nm	Phthalocyanine	E. coli S. aureus	Suspension	9	High phototoxicity of the Peptide with PS. The group without light 99% reduced.
Gao et al. 2021 [27]	In vitro/in vivo	PEGylated polypeptide	5 min	660 nm	PEGylated polypeptide + Chlorin e6	P. aeruginosa	Biofilm	ND	Total eradication of P. aeruginosa biofilms.
Judzewitsch et al. 2021 [28]	In vitro	ZnTTP-AC	30 min	Green-light irradiation	ZnTTP-AC	S. aureus P. aeruginosa	Suspension	3	4.5 log10 CFU/mL reduction for S. aureus. Total reduction for P. aeruginosa.
Qiu et al. 2021 [a] [29]	In vitro/in vivo	GKRWWKWWR-RPLGVRG	5 min	660 nm	GKRWWKWWR-RPLGVRG + Chlorin e6	S. aureus E. coli	Suspension	3	Total reduction for S. aureus 90% reduction for E. coli
Qiu et al. 2021 [b] [30]	In vitro/in vivo	GKRWWKWWRR	10 min 20 min 30 min	660 nm	GKRWWKWWRR + Chlorin e6 + AuNPs	S. aureus E. coli	Biofilm	3	S. aureus 10 min (~50% viability) 20 min (~20% viability) 30 min (~2.5% viability) E. coli 10 min (~60% viability) 20 min (~42.5% viability) 30 min (~10% viability)

ND: not documented; s: seconds; min: minutes: h: hour; PS: photosensitizer; ~: approximately; MB: methylene blue; RB: rose-bengal; Ce6: chlorin e6.

3. Risk of Bias Assessments for In Vitro Studies

The criteria from the OHAT Rob tool were applied to all articles included in the systematic review. The most frequent biases regarded blinding procedures. Moreover, the problem with internal validity was the lack of methodological details in the statical analyses and the performance of treatments only in microorganism suspensions (Table 2).

Table 2. Risk of bias assessment in the articles included, according to the OHAT criteria.

Studies/Questions	Was the Dose or Exposure Level Administered Adequately Randomized?	Was the Allocation to Study Groups Adequately Concealed?	Were the Experimental Conditions Identical Across Study Groups?	Were Research Personnel Blind to the Study Group During the Study?	Were the Outcome Data Complete without Attrition or Exclusion from the Analysis?	Is the Exposure Characterization Reliable?	Is the Outcome Assessment (Including Blinding of Assessors) Reliable?	Were There No Other Potential Threats to Internal Validity?
Bourré et al. 2010 [13]	++	++	++	--	++	++	--	--
Yang et al. 2011 [14]	++	++	++	--	++	++	--	--
Liu et al. 2012 [15]	++	++	++	--	++	++	--	--
Dosseli et al. 2013 [16]	++	++	++	--	--	++	--	--
Johnson et al. 2013 [17]	++	++	++	--	++	++	--	--
Dosseli et al. 2014 [18]	++	++	++	--	++	++	--	--
Johnson et al. 2014 [19]	++	++	++	--	++	++	--	--
Le Guern et al. 2017 [20]	++	++	++	--	++	++	--	--
De Freitas et al. 2018 [11]	++	++	++	--	++	++	--	--
Le Guern et al. 2018 [21]	++	++	++	--	++	++	--	--
Nakonieczana et al. 2018 [22]	++	++	++	--	++	++	--	--
Gao et al. 2019 [23]	++	++	++	--	++	++	--	--
De Freitas et al. 2019 [12]	++	++	++	--	++	++	--	--
Fesse et al. 2019 [24]	++	++	++	--	++	++	--	--
Zhang et al. 2019 [25]	++	++	++	--	++	++	--	--
Chu et al. 2021 [26]	++	++	++	--	++	++	--	--
Gao et al. 2021 [27]	++	++	++	--	++	++	--	--
Judzewitsch et al. 2021 [28]	++	++	++	--	++	++	--	--
Qiu et al. 2021a [29]	++	++	++	--	++	++	--	--
Qiu et al. 2021b [30]	++	++	++	--	++	++	--	--

++: direct evidence of positive finding; --: direct evidence of negative finding.

4. Meta-Analysis

The meta-analysis was performed only in 3 studies ^[12][14][22]. The reduced number of studies included in the quantitative analysis is due to the lack of data (e.g., sample size) and the absence of a study group evaluating only aPDT application. The experimental group included microorganisms treated with aPDT associated with peptides (aPDT + AMP), while the control group included microorganisms treated only with aPDT (aPDT). The microbial load was the outcome evaluated in the meta-analysis.

The Peto method was used to perform the meta-analysis due to the sparse data. The results were transformed into odds, and, therefore, the odds ratio (OR) was used as the effect measure. The result was significant (OR = 0.14/p = 0.0235/I-squared = 0%), showing better outcomes for aPDT associated with peptides than those for aPDT alone for controlling the microbial load (Figure 1A). Moreover, small-study effects in the meta-analysis and consequently publication and meta-analysis biases were verified with the trim-and-fill method. However, there were no biases (Figure 1B).

Figure 1. Ilustration of the results of the quantitative analysis. The experimental group (positive events) included microorganisms that received the association therapy (aPDT + AMP), while the control group included microorganisms that received only aPDT. (A) results of the meta-analysis illustrated in a forest plot. OR: odds ratio; CI: confidence interval; W: weight, ^[12][14][22]. (B) trim-and-fill method results illustrated in a forest plot. TE: estimated mean; seTE: estimated standard deviation; OR: odds ratio; CI: confidence interval; W: weight, ^[12][14][22].