Use of Antimicrobial Photodynamic Therapy to Inactivate Multidrug-Resistant Enterobacter spp.: Scoping Review

Abstract

Background/Objectives: Multidrug-resistant (MDR) Enterobacter spp. are critical pathogens within the ESKAPE group, frequently exhibiting resistance to carbapenems. Antimicrobial photodynamic therapy (aPDT) represents a promising non-antibiotic strategy to circumvent these resistance mechanisms. This scoping review aims to map the current evidence regarding the efficacy of aPDT in inactivating Enterobacter spp., identifying the most effective photosensitizers (PS), light parameters, and existing research gaps. Methods: A systematic search was performed across PubMed, Scopus, and Google Scholar (2013–2025) following PRISMA-ScR guidelines and registered on OSF. Studies were included if they evaluated aPDT against Enterobacter spp. (in vitro or in vivo) and provided quantitative data on microbial reduction. Data was extracted using a standardized charting form covering bacterial strains, PS type, light source, and viability reduction. The results from the eligible sources of evidence were synthesized narratively to address the review objectives. Results: Despite the clinical priority of Enterobacter, only seven studies met the eligibility criteria. Methylene Blue remains the most frequently studied PS, achieving reductions of 3–8 log₁₀. Emerging evidence highlights the synergistic efficacy of monocationic chlorins and graphene-based nanomaterials in enhancing the bactericidal effect of light-based treatments. Notably, aPDT demonstrated the ability to inactivate carbapenemases, the bacterial enzymes responsible for carbapenem resistance. However, only two studies evaluated in vivo applications, primarily within dental settings. Conclusions: aPDT is a promising method against MDR Enterobacter spp. and bypasses traditional resistance mechanisms. However, the limited number of studies indicates a significant knowledge gap. Future research should focus on standardized in vivo protocols and the synergy between aPDT and conventional antibiotics to support clinical translation.

1. Introduction

Multidrug-resistant (MDR) bacteria are the primary cause of healthcare-associated infections (HAIs) [1,2]. Among them, the group termed ESKAPE—an acronym for six bacterial pathogens notorious for “escaping” the effect of antimicrobials and responsible for most nosocomial infections—includes Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species (spp.) [3,4]. Enterobacteriaceae account for half of all clinical isolates in HAIs, with 80% of them Gram-negative. Enterobacter is a genus in the family Enterobacteriaceae that causes nosocomial infections and, less commonly, community-acquired infections, including urinary tract infections (UTIs), respiratory infections, soft tissue infections, osteomyelitis, and endocarditis, among others [5]. Enterobacter aerogenes (E. aerogenes), E. cloacae, and E. hormaechei are the most frequently isolated species in clinical infections, particularly among immunocompromised patients and those hospitalized in intensive care units (ICUs) [6]. Nevertheless, species of these bacteria can be part of the normal microflora of the mammalian gastrointestinal tract and are also associated with a variety of environmental habitats [5,7].

The World Health Organization (WHO) estimates that by 2050, infections caused by antimicrobial resistance (AMR) will result in 10 million deaths annually. Meanwhile, the Centers for Disease Control and Prevention (CDC) predicts that in the United States, infections caused by antibiotic-resistant microorganisms are responsible for at least 23,000 deaths each year [8]. Additionally, the WHO highlighted carbapenem-resistant Enterobacteriaceae (CRE) on its 2017 list of priority antibiotic-resistant bacteria. In this group, Enterobacter has developed significant resistance to many previously effective antibiotics, such as ampicillin, amoxicillin, first-generation cephalosporins, and cefoxitin [6].

The clinical significance of Enterobacter spp. has escalated dramatically due to their remarkable ability to acquire and disseminate mobile genetic elements encoding carbapenemases, such as KPC (Klebsiella pneumoniae carbapenemase) [9], NDM (New Delhi metallo-beta-lactamase) [10], and OXA-48 [11]. These enzymes not only confer resistance to almost all beta-lactam antibiotics but are also frequently associated with co-resistance to aminoglycosides and fluoroquinolones, leaving clinicians with extremely limited therapeutic options, often restricted to polymyxins, which carry significant nephrotoxic risks [12]. Unlike other Gram-negative bacteria, Enterobacter species possess an inducible chromosomal AmpC beta-lactamase, which, when combined with porin loss or efflux pump overexpression, creates a formidable barrier against conventional pharmacology [9]. As a result, current antibiotics are often ineffective against infections caused by MDR Enterobacter spp. Therefore, developing non-antibiotic antimicrobial therapies is essential. Several innovative strategies have been devised to combat MDR bacteria, including photothermal therapy and photodynamic therapy (PDT) [13,14]. Specifically, PDT uses non-toxic photosensitizers (PSs), such as metallic nanoparticles, cationic polymers, and nanocarriers, which are locally activated using phototherapy devices [15]. Cationic compounds are particularly popular as PSs because they can weaken the bacterial outer membrane’s permeability barrier, enhancing PS penetration [16,17].

Antimicrobial Photodynamic Therapy (aPDT) (Figure 1) represents a paradigm shift, as its mechanism of action is primarily physicochemical and multi-targeted, theoretically bypassing genetically encoded resistance mechanisms in Enterobacter [13]. PDT requires both a specific photosensitizer (PS) and a light source matching the PS absorption spectrum to induce local oxidative stress. Upon light irradiation, the PS becomes excited and transfers energy to molecular oxygen [15,18]. This process generates reactive oxygen species (ROS), including superoxide (O₂^•−), hydrogen peroxide (H₂O₂), hydroxyl radicals (^•OH), and singlet oxygen (¹O₂^•). The accumulation of ROS damages bacterial cells by oxidizing essential macromolecules, such as membrane lipids, proteins, and nucleic acids, thereby contributing to the broad-spectrum, non-specific antimicrobial effect of aPDT [19].

This scoping review aims to summarize the progress of aPDT in treating this bacterium and to identify gaps and opportunities for future research. The main research question guiding this review was: What information is available in the literature about aPDT treatment of MDR Enterobacter spp., in both in vitro and in vivo studies, and how many different PSs have been employed for this purpose?

2. Results

2.1. Choosing Sources of Evidence

As shown in Figure 2, 375 articles were identified through searches across three databases: 335 from Google Scholar, 7 from PubMed, and 34 from Scopus. Of these, 20 were duplicates, leaving 355 articles. After applying the inclusion and exclusion criteria, an additional 184 articles were removed, leaving 171 for review. Additional criteria, such as the availability of the full article, incomplete information, non-research content, or lack of clinical significance, eliminated 168 articles, leaving 10. Among these, 6 were in conflict, and C.E.P. resolved them, resulting in a final count of 8 articles for revision. Data were extracted from the selected articles, processed, and summarized in

Table 1. List of articles and development of aPDT against Enterobacter spp.

Bacteria	PS/Dose	Dose/aPDT	Effect/Reduction	Applications	Ref.
Isolates from infected wounds (Enterobacter spp.)	MB 0.1 mg/mL	50 J/cm2 LED red light 660 nm, 1 h	In vitro 100% Reduction of CFU	aPDT for treating infected wounds	[21]
E. aerogenes E. coli K. pneumoniae (MDR)	MB 100 μg/mL	25 J/cm2 LED red light 660 nm, 15 min	In vitro >3 log10 Reduction in CFU	aPDT to treat MDR bacteria from clinical isolates	[22]
E. aerogenes S. aureus S. marcescens K. pneumoniae (MDR)	MB 100 μM	10 J/cm2 Laser red light 660 nm, 20 min	In vitro 3 log10 Reduction in CFU	aPTD for MDR bacteria	[23]
Isolates from diabetic foot ulcers (MDR E. cloacae)	MB 16 μM	54 J/cm2 Red Light OLED 669–737 nm	In vitro 8 log10 Reduction of CFU	OLED light source implementation for aPDT	[24]
Clinical isolates from dental samples (MDR E. cloacae)	TB 1 mg/mL	200 mW/cm2 Laser red light 630 nm, 1 min	In vivo 3 log10 Reduction in CFU	In vivo aPDT for dental infections (FotoSan 630®)	[25]
E. cloacae P. aeruginosa E. coli A. baumanii (HAI, MDR)	McChl 25 and 50 μM	40–80 J/cm2 LED red light 660 nm 7–14 min	In vitro 99.99% inactivation with both concentrations	For in vivo studies of antitumor activity	[26]
Various Gram + and – (E. aerogenes)	GQD 10 mg/mL CQD and N-CQD 5 mg/mL	10 W Blue LED light 465–470 nm	MBC for GQD and for CQD 15.6 μg/mL, and N-CQD 7.8 μg/mL	Graphene NPs with bactericidal activity	[27]
Isolates from Wastewater Treatment Plants (E. tabaci E2)	5BZN 20 μg/mL	12,5 mW/cm2 Blue LED light ~500 nm	In vitro 40 min–2 log10 80 min–3.9 log10	aPDT for in situ MDR bacteria from WWTPs	[28]

Abbreviations: min: minute(s); h: hour(s); MB: Methylene Blue; MDR: Multidrug-resistance; aPDT: antimicrobial Photodynamic Therapy; CFU: Colony Forming Units; TB: Toluidine Blue; McChl: Mono-cationic Chlorin; HAI: Health-associated infections; OLED: Organic Light Emitting Diodes; 5BZN: 5-Bromoindole-capped Zinc Oxide; WWTPs: Wastewater Treatment Plants; GQD: Graphene Quantum Dot; CQD: Carbon Quantum Dot; N-CQD: Nitrogen-doped Carbon Quantum Dot; NPs: Nanoparticles.

to facilitate reading. The fact that we found only 8 articles shows that research in this area remains limited, especially compared with other ESKAPE pathogens, such as Klebsiella pneumoniae, Staphylococcus aureus, or Pseudomonas aeruginosa, which have significantly more published work [17,20].

2.2. PSs Used for Enterobacter spp. aPDT

2.2.1. Methylene Blue as the Most Common PS

As shown in Table 1, methylene blue (MB) is one of the most commonly used photosensitizers in antimicrobial photodynamic therapy (aPDT). When exposed to an appropriate light source, MB becomes a powerful oxidizing agent that can cause cellular damage, disrupt membrane integrity, and deactivate key proteins, ultimately killing microbes. Its widespread use in clinical settings is supported by its low cost, hospital availability, and Food and Drug Administration (FDA) approval for intravenous use in humans, making MB the primary PS used in aPDT [29,30]. However, it is contraindicated for pregnant individuals and those taking serotonergic medications. MB has low toxicity and high quantum efficiency in producing singlet oxygen. It absorbs light in the red spectrum, typically between 590 and 690 nm, with a peak near 668 nm, allowing effective action against both Gram-positive and Gram-negative bacteria [18]. For example, Pereira de Lima and colleagues evaluated the effects of two photosensitizers, methylene blue (MB) and photodithazine (PDZ), on the in vitro photoinactivation of microorganisms isolated from infected wounds [21]. The study included both Gram-positive and Gram-negative bacteria, including Enterobacter spp. PDT mediated by PDZ effectively inhibited the growth of Gram-positive bacteria but showed limited activity against Gram-negative strains, which exhibited bacterial counts exceeding 5 log₁₀ CFU. In contrast, MB-mediated PDT was effective against both Gram-positive and Gram-negative bacteria, achieving significant growth inhibition in all tested groups. The authors suggested that these differences may be attributed to the physicochemical properties of the photosensitizers, as well as to structural and physiological differences between Gram-positive and Gram-negative bacteria [21]. Another example is the study by Costa Magacho and collaborators which evaluated the in vitro effect of aPDT combined with ceftriaxone against third-generation cephalosporin-resistant Gram-positive and Gram-negative bacteria [22]. Using a fixed methylene blue (MB) concentration (100 μg/mL), the authors assessed the impact of different light doses (10 and 25 J/cm²). In E. aerogenes, aPDT at 10 J/cm² resulted in a 0.82 log₁₀ reduction in bacterial counts, whereas 25 J/cm² achieved a 2.85 log₁₀ reduction compared to the dark control. Notably, a reduction of ≥3 log₁₀ CFU is generally considered indicative of significant bactericidal activity (1000 times decrease in CFU). When combined with ceftriaxone, reductions of 1 log₁₀ and 3.14 log₁₀ were observed at 10 and 25 J/cm², respectively, suggesting that the addition of the antibiotic did not substantially enhance the photodynamic effect under the conditions tested. The authors concluded that MB-mediated aPDT may represent a promising alternative for inactivating Gram-negative strains [22]. In the same year, Feng and colleagues investigated the effect of aPDT on carbapenemase activity in multidrug-resistant (MDR) bacteria [23]. They emphasized the importance of understanding the relationship between bacterial killing and enzyme inactivation, as resistance genes released from lysed bacteria may be horizontally transferred to other pathogens, promoting the dissemination of carbapenem resistance. The authors evaluated different MB concentrations under both irradiated (aPDT) and dark conditions. In E. aerogenes, MB-mediated aPDT at 50 μM resulted in an approximately 3 log₁₀ reduction in bacterial survival compared to the no-MB control, with similar results at 100 μM. Under the same conditions, carbapenemase activity decreased from 0.15 (dark control) to 0.1 (MB 50–100 μM with aPDT), confirming that the observed effects were dependent on photodynamic activation. Similar results were obtained for Serratia marcescens. In Klebsiella pneumoniae, aPDT with 100 μM MB achieved an approximately 7 log₁₀ reduction in bacterial survival, along with a threefold decrease in carbapenemase activity, measured by nitrocefin hydrolysis (absorbance at 486 nm) [23]. Overall, this study demonstrated that aPDT can inhibit bacterial carbapenemase activity, highlighting a potential mechanism that may contribute to restoring antibiotic susceptibility, although this effect was not directly validated in functional assays.

Another study used MB (16 μM) with clinical bacterial isolates from diabetic foot ulcers. Piksa and collaborators implemented a new light source, Organic Light-Emitting Diodes (OLEDs), that emitted red light in the 669–737 nm range at a fluence of 54 J/cm² for PDT treatment of MDR bacteria [24]. For pathogenic bacteria, reductions of 1.1 to 8 log₁₀ were observed, and for the E. cloacae strain, a decrease of >8 log₁₀ was observed with this new light source. They concluded that aPDT using MT under OLED-activation is effective against pathogens and opportunistic bacteria, especially for treating difficult-to-heal ulcers resistant to antibiotic therapies [24].

In general, MB-based aPDT consistently resulted in reductions of approximately 3 log₁₀, similar to results observed in earlier studies of MB-PDT against Gram-negative bacteria, specifically when treating Enterobacter species [31].

2.2.2. Other PSs Used Against Enterobacter spp.

FotoSan^® 630 is a dental product that combines a powerful red light with the photosensitizer Toluidine Blue (TB) at a concentration of 1 mg/mL (CMS Dental APS, Copenhagen, Denmark) [32]. In 2021, Gueorgieva and Gergova used in vivo treatment with FotoSan^® to disinfect root canals (endodontic treatment) and promote healing of apical periodontitis [25]. They applied 5 mL of PS in the root canals and irradiated with an intense laser (200 mW/cm²) at 665 nm for 1 min, which reduced the survival of all microbial isolates by 77%, with a slightly lower antimicrobial effect against MDR Enterobacter spp. and Klebsiella spp. Specifically, a 3 log₁₀ reduction was observed for E. cloacae. They found that PDT with FotoSan^® exhibited some of the strongest antimicrobial activities, comparable to 2.5% sodium hypochlorite (NaOCl), a widely used antimicrobial agent in dentistry [33], concluding that PDT with FotoSan^® could serve as a complementary method to routine endodontic treatment [25].

Kustov and colleagues used a monocationic chlorin PS (McChl), derived from methylpheophorbide α (MPh), which in turn is a commonly used PS in cancer PDT [34], as a new PS agent for both antitumor and antimicrobial PDT [26]. In vitro studies showed that at 25 and 50 μM McChl, E. cloacae survival was 0 CFU (a 5–6 log₁₀ reduction compared to control) after irradiation with both 40 and 80 J/cm². They also examined McChl in K-562 myeloid leukemia cells and in M1 sarcoma-bearing rats, demonstrating that McChl could be a promising candidate for further preclinical studies [26].

2.3. Nanoparticles with aPDT Activity Against Enterobacter spp.

The development of PSs has resulted in three generations of PS types: the first includes porphyrins; the second features chlorins, porphyrinoids, and some transition-metal complexes [35]; and the third consists of biomolecule conjugates and covalently attached peptides [36].

Over the past decade, new PSs have been developed to enhance PDT. Among these, graphene-based materials, particularly graphene oxide (GO), graphene quantum dots (GQDs), and carbon quantum dots (CQDs), have attracted attention due to their biomedical applications, including cancer therapy and antibacterial activity [37,38,39]. GO consists of a single-atom-thick carbon layer (~0.8 nm) arranged in a honeycomb lattice with sp² hybridization. Its antibacterial activity is primarily attributed to membrane disruption and the generation of reactive oxygen species (ROS). GQDs are typically produced via top-down approaches, with lateral dimensions up to ~100 nm, offering a high surface area and tunable photoluminescence properties depending on size, edge structure, and surface functionalization [27]. In contrast, CQDs are small carbon nanoparticles synthesized through bottom-up methods, generally with diameters below 10 nm, and exhibit strong photoluminescence and favorable optical properties, with scalable and cost-effective production.

In 2018, Markovic and colleagues evaluated the antibacterial activity of four graphene-based nanomaterials: GO, GQDs, CQDs, and nitrogen-doped CQDs (N-CQDs), against a broad panel of Gram-positive and Gram-negative bacteria (19 strains) [27]. They also assessed ROS generation under dark conditions, ambient light, and blue light irradiation (~470 nm). CQDs and N-CQDs exhibited the highest antibacterial activity. Notably, N-CQDs showed strong photodynamic effects against Enterobacter aerogenes and other pathogens, including Proteus mirabilis, Staphylococcus saprophyticus, Listeria monocytogenes, Salmonella typhimurium, and Klebsiella pneumoniae. E. aerogenes and P. mirabilis were the most sensitive species, with minimum inhibitory concentrations (MICs) of 3.9 μg/mL for aPDT with N-CQDs, compared to 500 μg/mL for the antibiotic amracin (a tetracycline derivative). Under blue light irradiation, all nanoparticles except GO generated singlet oxygen, leading to bacterial inactivation via ROS-mediated oxidative stress. These findings highlight GQDs and CQDs as promising candidates for antibacterial applications [27].

Another nanoparticle, published in 2022 by Manoharan and colleagues, is 5-bromoindole (5B)-capped zinc oxide (ZnO) (5BZN), synthesized to improve antibacterial, antibiofilm, and disinfection properties for controlling microorganisms in wastewater treatment [28]. They tested 5BZN against multidrug-resistant (MDR) bacteria and observed that, even at low doses (20 μg/mL), after 12.5 mW/cm² blue LED irradiation, the nanoparticle demonstrated superior photodynamic inactivation of two wastewater MDR bacteria, Enterobacter tabaci E2 and Klebsiella quasipneumoniae SC3, with cell densities reduced by 3.9 and 4.7 log₁₀ in CFU/mL, respectively. Additionally, 5BZN showed greater photodynamic inactivation of total coliform bacteria in wastewater effluents under a blue light intensity of 12.5 mW/cm², achieving almost complete inactivation of coliform cells within 40 min. Moreover, 5BZN was found to be safe in an animal model. They concluded that this study indicates significant potential for 5BZN as a self-healing agent in large-scale photodynamic disinfection [28].

Beyond MB, newer PSs and nanomaterials demonstrated high bactericidal potential under specific illumination conditions. In fact, nanoparticles, such as titanium dioxide (TiO₂), fullerenes, or chitosan, often produce more significant antibacterial effects than conventional PSs, which aligns with recent nano-photodynamic research indicating enhanced ROS production, better membrane penetration, and synergistic photothermal effects [40,41,42]. However, most of these findings remain limited to in vitro settings and lack standardized comparisons with MB or among each other.

2.4. Uses of PSs Against Enterobacter spp.

2.4.1. In Vivo Studies

Few studies have used animal models to evaluate in vivo PDT, and only one has focused on Enterobacter spp. infections. This study was conducted on patients diagnosed with pulp necrosis or chronic periapical periodontitis who needed endodontic treatment. Gueorgieva and Gergova in 2021,reported the use of FotoSan^® in 36 patients [25]. They collected microbiological samples by inserting a sterile paper point into the root canal before and after treatment (Group 1: used FotoSan^®, and Group 2: used the standard protocol with 2,5% NaOCl and 17% EDTA). The results showed that NaOCl was the most effective in vivo, with only 10% of the initially isolated microorganisms remaining after treatment, indicating 90% efficacy. In comparison, with FotoSan^®, 23% of the initially isolated microorganisms persisted after PDT, indicating a 70% effectiveness. They concluded that their findings demonstrate an excellent antibacterial effect of PDT with FotoSan^®, which is essential for considering PDT as an additional step in endodontic treatment for root canal disinfection, especially when lower concentrations of rinsing solutions are required [25].

Unlike other ESKAPE pathogens, for which several animal and ex vivo infection models are available [17,43], our review found no ex vivo studies evaluating MB-based PDT specifically against Enterobacter spp. This gap highlights a significant limitation in translating MB-mediated aPDT into real-world clinical settings.

2.4.2. PSs for Environmental Improvement

Photodynamics can also be used to decrease bacterial levels in wastewater. Manoharan and colleagues utilized the novel 5BZN PS to enhance not only antibacterial but also antibiofilm and disinfection processes for controlling microorganisms in wastewater treatment. They demonstrated that exposure to low doses of 5BZN (20 μg/mL) significantly reduced biofilm density and cell attachment in MDR bacteria. Additionally, they conducted toxicity studies using nematodes as the animal model and seed germination assays, concluding that 5BZN is harmless and emphasizing its great potential as a self-healing agent in large-scale photosynthetic disinfection systems [28].

2.5. Limitations and Future Perspectives

The limited number of eligible studies (n = 8) highlights not only the fragmented nature of the field but also the importance of critically interpreting current evidence to guide future research. Key methodological parameters, such as PS concentration, fluence rate, and exposure time, varied substantially across studies, hindering direct comparisons (Table 1). Additionally, only one study addressed wastewater isolates, suggesting an underexplored opportunity for environmental applications, while the few in vivo studies were restricted to dental settings and did not exclusively target Enterobacter infections.

Despite these limitations, the available evidence consistently supports the ability of aPDT to induce broad-spectrum, ROS-mediated killing of MDR Enterobacter spp., including strains resistant to last-resort antibiotics. Notably, no study reported the emergence of resistant mutants, reinforcing the potential of aPDT as a complementary or alternative antimicrobial strategy.

From a translational perspective, advancing this field will require the establishment of standardized in vitro protocols to enable cross-study comparisons, as well as the development of robust in vivo infection models, which remain largely absent for Enterobacter. Furthermore, mechanistic studies focused on ROS-mediated damage in this genus, and the evaluation of synergistic effects between aPDT and conventional antibiotics are essential to determine whether photodynamic approaches can effectively contribute to restoring antibiotic susceptibility [44,45].

3. Discussion

3.1. Summary of Evidence

This scoping review synthesizes evidence from studies published between 2013 and 2025, mapping the current landscape of aPDT against MDR Enterobacter spp. Our findings highlight emerging trends supporting aPDT as an effective, “orthogonal” therapeutic strategy. The available evidence describes two main mechanistic pathways: Type I reactions (electron transfer that generates superoxide and hydroxyl radicals) and Type II reactions (energy transfer that produces singlet oxygen). aPDT relies on light-mediated activation of PSs; therefore, tissue accessibility is a key determinant of its clinical applicability. Superficial infections caused by Enterobacteriaceae, such as surgical wounds, chronic ulcers, or burns, are well suited for aPDT due to direct light exposure. In contrast, infections in hollow organs (e.g., the urinary tract) require specialized light-delivery systems, such as fiber optics, to achieve effective PS activation, which may limit broader clinical implementation [46,47].

A key theme that emerged is the structural barrier imposed by the outer membrane of Enterobacter spp. In contrast to Gram-positive organisms such as Enterococcus spp., which lack an outer membrane and are generally more susceptible to photosensitizer uptake, Enterobacter spp. possesses an additional permeability barrier that can reduce treatment efficacy [48]. However, the use of cationic photosensitizers and nanomaterials, such as graphene-based quantum dots and ZnO nanoparticles, can enhance membrane interactions and promote localized ROS generation. This synergistic effect results in amplified multi-target damage, including lipid peroxidation, protein oxidation, and DNA damage. Because these oxidative events occur simultaneously across multiple cellular targets, the likelihood of developing resistance is considered low. This evidence is particularly relevant for clinical microbiology and pharmacology, as it underscores the potential of aPDT to inactivate carbapenemases and overcome genetically encoded resistance mechanisms in one of the most challenging ESKAPE pathogens.

3.2. Limitations

Despite following PRISMA-ScR guidelines (Supplementary Material), several limitations inherent to the review process should be acknowledged. First, the search was restricted to studies published in English and Spanish, potentially excluding relevant evidence in other languages. Second, the small number of eligible studies specifically targeting Enterobacter spp. (n = 8) limits the robustness of evidence synthesis and reduces the generalizability of the findings. Third, the inclusion criteria required complete reporting of photosensitizer and irradiation parameters, which may have excluded studies with incomplete methodological descriptions. In addition, agents in very early stages of development or lacking consistent clinical data were excluded. Other promising PSs (curcumin, riboflavin, nanomaterials, etc.) remain to be investigated.

Moreover, heterogeneity in study design and reporting, particularly regarding light dosimetry and bacterial growth conditions, precluded direct quantitative comparisons across studies. Finally, the limited number of in vivo studies restricts the ability of this review to draw conclusions about the clinical applicability of aPDT in complex infection settings.

4. Materials and Methods

4.1. Protocol and Registration

The methodology adheres to the PRISMA-ScR (PRISMA Extension for Scoping Reviews) guideline (Supplementary Material) [49]. This type of review was selected because it allows systematic reviews to synthesize the literature, provide an overview of existing research, and identify potential gaps in current knowledge. This study has been registered on the Open Science Framework (OSF) platform (Registration DOI: 10.17605/OSF.IO/US6Z4).

4.2. Search Strategy and Eligibility Criteria

The search focused on works published no earlier than 2013; considering the PICO criterion [50], we agreed on a 10-year range with a 20% buffer (12 years old). Original articles were searched in PubMed (MeSH), Google Scholar, and Scopus databases from January 2013 to June 2025. The search used the terms: “Enterobacter spp.” AND “photodynamic therapy”.

Studies were included if they met the following criteria: (1) original articles published after 2013 that examined photodynamic therapy related to antimicrobial activity against Enterobacter species, including in vitro or in vivo clinical applications and synergism with antibiotics, other antimicrobial agents, or PS linked to other particles; (2) articles written in English and Spanish; and (3) peer-reviewed articles. The exclusion criteria were: (1) studies published before 2013 or research not published in peer-reviewed journals and not original articles; (2) articles that were not accessible; (3) studies that did not specify the photosensitizer compound/concentration or light source/doses used; (4) studies involving bacteria from the ESKAPE group but not including Enterobacter species; and (5) studies without clinical relevance, such as those showing no significant reduction in Enterobacter species growth (≥3 log₁₀ CFU reduction).

4.3. Information Sources and Search

All data were extracted exclusively from the databases listed above, and the most recent research was conducted in June 2025.

In the Scopus database, in the Documents section, the Search within was set to “All fields,” and the terms mentioned above were entered in order in the Search document. Then, the date range was set from “2013” to “Present,” and the search was executed. The “Article” button was selected for Document type, along with “English” Language and “Final” Publication status. The same criteria were applied across all three databases.

4.4. Selection of Sources of Evidence and Data Charting Process

All documents retrieved from the search were exported as *.csv files and uploaded to Rayyan [51] for filtering articles. The PRISMA criteria were as follows: two researchers (ARB and MFC) independently used Rayyan to remove duplicates and select final documents based on inclusion and exclusion criteria.

As shown in Figure 2, of the 375 references collected from the three databases, 20 duplicates were identified and removed. The remaining 355 references were read individually in Rayyan to determine inclusion or exclusion based on the criteria outlined above, with decisions guided by PRISMA-ScR, resulting in 8 articles. Two reviewers independently extracted the following relevant characteristics: 1. Authors’ names; 2. Year of publication; 3. Study objective; 4. Methods used to analyze the aPDT treatment; 5. Photosensitizer used, with doses; 6. Light source and doses; 7. Range of bacterial growth reduction reported; 8. Current and potential future applications; and 9. Clinical significance of the findings.

4.5. Data Items and Synthesis of Results

After the screening was finished, the senior researcher (CEP) resolved the conflicts and reviewed all the articles included by the two researchers (ABR and MFC). The data extracted were summarized in a table (Table 1), and a narrative summary was created.

5. Conclusions

In conclusion, this review confirms that aPDT is a promising and practical method for controlling MDR Enterobacter spp., achieving bacterial reductions of 3–8 log₁₀ across various PSs and illumination conditions. The results answer our review questions by identifying Methylene Blue and newer monocationic chlorins as the most effective and promising agents, while determining the inactivation of carbapenemases as a breakthrough implication for drug discovery.

The next steps for this field must prioritize bridging the gap between innovative materials science and clinical microbiology. Future research should focus on:

• Engineering: Development of next-generation light delivery systems, such as flexible Organic Light-Emitting Diodes (OLEDs) and biocompatible optical fibers for internal infections or colonized medical devices.
• Synergy: Investigating the potential of sub-lethal aPDT to “resensitize” MDR strains to previously ineffective carbapenems or aminoglycosides through membrane permeabilization. In addition, it would be of great value to explore other promising and/or novel photosensitizers, as well as the synergism with inorganic salts or antibiotics to potentiate aPDT.
• Translational Research: Implementing standardized in vivo models to establish definitive dosimetry protocols. Harnessing the full potential of light-based therapies is essential to mitigate the escalating threat posed by carbapenem-resistant Enterobacteriaceae in the 21st century.