Diversity and composition of ocular microbiota in contact lens wearers: Efficacy of liposomal ozonated oil

Diversity and composition of ocular microbiota in contact lens wearers: Efficacy of liposomal ozonated oil María Carmen S´ anchez-Gonz´ alez a,* , Inmaculada Gallardo-Real a , Estanislao Guti´ errez-S´ anchez b , Concepci´ on De-Hita-Cantalejo a , Raúl Capote-Puente a , Jos´ e-María S´ anchez-Gonz´ alez a a Department of Physics of Condensed Matter, Optics Area, University of Seville, Reina Mercedes S/N, 41012 Seville, Spain b Department of Surgery, Ophthalmology Area, University of Seville, Doctor Fedriani S/N, 41009, Seville, Spain ARTICLE INFO Keywords: Ozonated Sunflower Seed Oil Ozone Dry Eye Microbiome ocular ABSTRACT Purpose: To characterize the ocular surface microbiota in regular contact lens wearers with dry eyes and assess the effectiveness of reducing bacterial load using a liposomal ozonated oil solution. Methods: This prospective, longitudinal, controlled study randomized subjects into two groups. Group A (45 subjects) received hydroxypropylmethylcellulose (HPMC, Artific®), while Group B (41 subjects) received ozonated sunflower seed oil with soybean phospholipids (OSSO, Ozonest®). Microbial communities were analyzed via DNA metabarcoding of the 16S rRNA gene, and statistical analyses (alpha and beta diversity) were performed in R. Results: Both groups predominantly harbored Staphylococcus caprae, Streptococcus oralis, and Corynebacterium spp., with OSSO and HPMC users showing distinct bacterial profiles. Alpha diversity showed no significant differences, but beta diversity revealed differences in bacterial composition between the groups. Conclusions: The results seem to indicate that the use of ozonized oil reduces the bacterial load compared to the solution used as a control. 1. Introduction The ocular microbiota primarily resides on the eyelids, conjunctiva, and corneal surface. Recent advances in 16S rRNA-based genetic analyses have enabled the detection of over 50 genera and more than 200 species of bacteria in the ocular environment, although this diversity remains significantly lower compared to other body surfaces. The ocular microbiota is relatively stable, as the eye possesses mechanisms that allow it to interact with bacteria without being adversely affected. Tears play a critical role in this defense, containing antibacterial proteins such as lysozyme, lactoferrin, and lipocalin, as well as immunoglobulin A (IgA), which is produced by plasma cells in the lacrimal gland. These components prevent bacterial adhesion to host cells, thus protecting the ocular surface. [1]. Exploring the ocular microbiota is crucial to better understand ocular health and prevent disease [2,3]. A recent study shows that the ocular microbiota of the healthy population is a low diversity microbiome.[4]. Disruptions in the natural microbiota have been associated with various health issues and the onset of diseases [5,6]. Microorganisms normally present on the conjunctiva or in periocular regions can become pathogenic, especially when the eye’s defense mechanisms are compromised due to factors such as contact lens use, surgery, intraocular injections, or other invasive procedures [7,8]. Additionally, exogenous microorganisms can infect the eye through contact with contaminated hands, fomites, or traumatic wounds [9–11]. Ocular infections pose a significant risk to visual health, as some infections can progress rapidly, leading to irreversible damage to ocular tissues if not treated promptly [12]. Although antibiotics are commonly used to manage eye infections, the growing issue of antimicrobial resistance has made treatment more challenging. Antimicrobial resistance, driven by the widespread and improper use of antibiotics, is a major global public health concern [13,14]. To address this issue, the use of molecules with antiseptic and antimicrobial properties, distinct from conventional antibiotics, is being recommended [15]. One such molecule is ozone, formulated as ozonated oil, which has shown particular promise [16–19]. Ozonated oils, * Corresponding author at: University of Seville, Reina Mercedes St. Physic Faculty, University of Seville, Seville, Spain. E-mail address: [email protected] (M.C. S´ anchez-Gonz´ alez). Contents lists available at ScienceDirect Contact Lens and Anterior Eye journal homepage: www.elsevier.com/locate/clae https://doi.org/10.1016/j.clae.2025.102368 Received 1 October 2024; Received in revised form 23 December 2024; Accepted 31 December 2024 Contact Lens and Anterior Eye 48 (2025) 102368 Available online 8 January 2025 1367-0484/© 2025 The Author(s). Published by Elsevier Ltd on behalf of British Contact Lens Association. This is an open access article under the CC BY-NC-ND license ( http://creativecommons.org/licenses/by-nc-nd/4.0/ ). which are more stable than ozone gas, retain the reactivity of the original ozone molecule and exhibit strong oxidant activity against microorganisms. The antiseptic mechanisms of ozonated oil have been extensively studied. Upon contact with biological exudates, ozonides release reactive species such as ozone (O 3 ), trioxolanes, hydroperoxides, and lipoperoxides, which act through oxidation to eliminate various pathogens, including bacteria, viruses, and fungi [20]. These reactive molecules interact with membrane lipids, inducing peroxidation and microbial lysis. Additionally, ozonated oil promotes corneal repair by enhancing collagen synthesis and activating growth factors (PDGF and TGF-β) [21]. The effects of contact lens use and ocular diseases on the ocular surface microbiota have recently been explored using molecular techniques such as 16S rRNA gene sequencing [22–24]. These studies require specific laboratory and bioinformatic protocols to address challenges such as low DNA yield from ocular samples, selecting the right primer pairs for bacterial characterization, and identifying contaminant sequences, often resulting from the co-amplification of human DNA and bacteria from laboratory reagents and the environment [11]. The use of contact lenses is related to changes in the diversity of the conjunctival microbiota that generates a greater risk of ocular infections [10,25]. The purpose of this study was to characterize the diversity and composition of the ocular surface microbiota in contact lens wearers with dry eyes. Additionally, the study sought to assess the effectiveness to reduce the bacterial load of a liposomal ozonated oil solution for ophthalmic use in the eyes of regular contact lens wearers. 2. Materials and methods 2.1. Study design A prospective, longitudinal, and controlled study was conducted to evaluate the efficacy of a solution containing ozonated sunflower seed oil, soybean phospholipids, and hydroxypropylmethylcellulose. The research followed the tenets of the Declaration of Helsinki and was approved by the Andalusian Biomedical Research Ethics Committee (SICEIA-2024–000925) and conducted at the Optics and Optometry facilities of the Pharmacy School, University of Seville (Seville, Spain). 2.2. Subjects Informed consent was obtained from all participants following an explanation of the study’s nature and potential outcomes. An information sheet detailing the study procedures was provided to each subject. The inclusion criteria were: (1) age between 18 and 25 years; (2) contact lens wearers; (3) a Contact Lens Dry Eye Questionnaire-8 (CLDEQ-8) score of 12 or higher (This cut-off point has been validated through clinical studies, showing high sensitivity and specificity in detecting the presence of dry eye symptoms in contact lens wearers) [26–28]; and (4) full understanding of the study’s purpose, along with signed informed consent prior to measurements. Exclusion criteria were: (1) any history of ocular surgery; (2) presence of systemic diseases; (3) use of ophthalmic or systemic medications affecting the tear film or ocular surface; and (4) pregnancy or lactation. 2.3. Examination procedure Participants were randomized into two groups (A and B) using a simple computer-generated randomization process. Group A (n =45) received 1 drop of hydroxypropylmethylcellulose (HPMC) (Artific®, distributed by Bausch & Lomb S.A., Berlin, Germany) three times daily and served as the control group. Group B (n =41) received 1 drop of a solution containing ozonated sunflower seed oil (OSSO), soybean phospholipids, and hydroxypropylmethylcellulose (Ozonest®, distributed by ESTEVE Pharmaceuticals®, Barcelona, Spain) three times daily. For sample collection, a sterile, individualized nylon-flocked swab (Copan Diagnostics, Murrieta, CA) was used. Strict precautions were followed to prevent sample contamination with foreign DNA, including the use of gloves during swab handling and maintaining sterility until collection. Samples were collected from the lower fornix, avoiding contact with the eyelids or eyelashes. Three conjunctival swabs were obtained from each subject: (1) before treatment (Control); (2) after one week of treatment (Eyedrops T1); and (3) one week after treatment discontinuation (Eyedrops T2). The swabs were then placed into Eppendorf tubes, preserved with DNA extraction buffer, and stored at room temperature until processed at the laboratory. 2.4. Molecular and bioinformatic procedures Genomic DNA was extracted from each sample using the QiAamp DNA Mini Kit (Qiagen, Hilden, Germany) strictly following the manufacturer’s instructions. The DNA concentration was quantified in each extract using the Qubit High Sensitivity dsDNA Assay (Thermo Fisher Scientific). The V3-V4 region of the prokaryotic 16S rRNA gene (~460 bp) was amplified using the primers Bakt-341F: 5 ′ CCTACGGGNGGCWGCAG 3 ′ and Bakt-805R: 5 ′ GACTACHVGGGTATCTAATCC 3 ′ [29], both containing Illumina sequencing adapters at their 5 ′ ends. Initial PCR reactions were carried out in a total volume of 12.5 μ L, comprising 4.5 μ L of template DNA, 0.5 μ M of each primer, 7.8 μ L of Supreme NZYTaq 2 × Green Master Mix (NZYTech), and ultrapure water to reach the final volume. The PCR cycling conditions were as follows: an initial denaturation step at 95 ◦C for 5 min, followed by 30 cycles of 95 ◦C for 30 s, 47 ◦C for 45 s, 72 ◦C for 45 s, and a final extension step at 72 ◦C for 7 min. Nine samples from different patients using both ophthalmic solutions failed to amplify during this process and were subsequently excluded from further analysis. A second PCR was performed to add the necessary oligonucleotide indices for multiplexing, following the protocol described by Vierna et al.[30]. The finished libraries were purified using the Mag-Bind RXNPure Plus magnetic beads (Omega Bio-tek) and pooled in equimolar amounts according to the results of a Qubit dsDNA HS Assay (Thermo Fisher Scientific) quantification. The pool was sequenced on a NovaSeq PE250 flow cell (Illumina). The bioinformatics analysis was performed with the QIIME2 v2023.2 [31] pipeline, beginning with Cutadapt v3.5 to trim non-biological DNA sequences such as primers and adapters. The trimmed sequences were then processed with the DADA2 plugin [32], which removes PCR primers, filters reads for quality, denoises, infers Amplicon Sequence Variants (ASVs), merges forward and reverse reads, and eliminates chimeric sequences. To classify the resulting ASVs taxonomically, was used the SILVA reference database [33]; release 138.1 August 2020) in combination with the QIIME2 plugin q2-clawback [34]. Taxonomic weights were calculated by incorporating habitat-specific species abundance data from the Qiita database (release 2022.11) [35], focusing on human “eye” and “ocular” categories. Afterwards, were applied two types of filters to the results. Sequence abundance filters removed singletons and mistagging [36]. Taxonomic filters excluded eukaryotic sequences of plastid and mitochondrial origin, sequences with low taxonomic assignment, sequences from human coamplification, and ASVs present in negative controls. The final filtered ASV table (see Supplementary Table 1) was imported into R v4.2.2 [37] to perform the data analysis. To determine if the sequencing depth was sufficient to capture the existing diversity in the dataset, alpha rarefaction curves were generated. Samples were rarefied to a depth equal to the lowest sequence count recorded among samples (1007 sequences) by random sequence sampling before performing diversity analyses. Alpha diversity for each sample was estimated using the QIIME 2 diversity alpha plugin, calculating the Observed Features and Shannon index metrics. The non-parametric Kruskal-Wallis test was used to identify significant differences among the groups. Box plots to visualize the diversity metrics for different sample types were created with the M.C. S´ anchez-Gonz´ alez et al. Contact Lens and Anterior Eye 48 (2025) 102368 2 ggpubr_0.6.0 package in R [38]. For beta diversity analysis, a BrayCurtis distance dissimilarity matrix was generated, and a Non-Metric Multidimensional Scaling (NMDS) plot was created using the Microbiome v1.24.0 R package [39]. The preparation and sequencing of DNA metabarcoding libraries and the bioinformatic analyses were carried out by AllGenetics & Biology SL (https://www.allgenetics.eu). 3. Results 3.1. Taxonomy A total of 11,851,376 raw reads were obtained by high-throughput sequencing of the V3–V4 region of the 16S rRNA gene from 201 samples. After excluding 115 samples due to insufficient sequencing depth following abundance and taxonomic filtering, a total of 317,409 reads from 86 samples (26 from the control group, 29 from EyedropsT1, and 31 from EyedropsT2) belonging to 48 patients (45 patients receiving eyedrops from HPMC and 41 receiving eyedrops from OSSO corresponded to 2899 unique ASVs and were used for further analyses. Rarefaction curves based on the observed ASVs reached a saturation plateau at a sequencing depth of 1,000 reads per sample as shown in Supplementary Figure 1. The bacterial composition in the ocular samples was similar across all the patients included in this study. At the phylum level, Firmicutes, Actinobacteriota, Proteobacteria, Bacteroidota, Patescibacteria and Fusobacteria were the dominant taxa, at levels greater than 1 % of reads. The majority was accounted for by the top three phyla: Firmicutes (average: 45.15 %), Actinobacteriota (24 %), and Proteobacteria (22.37 %) (see Supplementary Figure 2). At the species level, the taxa with more than 1 % average relative abundance differed in the two groups of patients receiving both opthtalmic solutions. Staphylococcus caprae (18.68 %), Streptococcus oralis (8.68 %), Corynebacterium tuberculostearicum (5.49 %), Haemophilus parainfluenzae (3.68 %), and Corynebacterium accolens (2.01 %) were dominant in both groups. Among the patients receiving OSSO, the most abundant bacteria included also Finegoldia magna, Rothia mucilaginosa, Paracoccus sp., Lactobacillus acidophilus, and Corynebacterium kroppenstedtii. In contrast, Staphylococcus hominis and Neisseria mucosa were also among the most abundant bacteria in patients receiving HPMC. The bacterial community composition of each sample with the dominant species is shown in Fig. 1. 3.2. Alpha diversity The richness and diversity were evaluated based on the number of observed features (ASVs) and the Shannon index. There was no significant difference in alpha diversity observed in any of the comparisons, whether considering the ophthalmic solution received by the patients (see Fig. 2) or the treatment group (Fig. 3). The alpha diversity parameters calculated are shown in Supplementary Table 2. 3.3. Beta diversity In the context of the Bray–Curtis distance, a value of 1 indicates a unique bacterial community composition for an individual, while a value of 0 signifies identical composition. Detailed results can be found in Supplementary Table 3. The analysis revealed a scattered pattern with overlaps between the samples in both comparisons, considering the ophthalmic solution received by the patients (see Fig. 4) or the treatment group (Fig. 5). Comparison between the samples taken at the three treatment times found no difference in bacterial community composition for patients receiving HPMC (PERMANOVA, pseudo-F: 0.836, pvalue: 0.806) and OSSO (PERMANOVA, pseudo-F: 1.047, p-value: 0.365, Fig. 5). However, there was a significant difference in bacterial community composition between the patients receiving the two different ophthalmic solutions (PERMANOVA, pseudo-F: 2.10, p-value: 0.002, Fig. 4). Fig. 1. Bacterial community composition of each sample for patients receiving both ophthalmic solution and the three treatment groups (Control, EyedropsT1 and EyedropsT2) for the dominant species. M.C. S´ anchez-Gonz´ alez et al. Contact Lens and Anterior Eye 48 (2025) 102368 3 4. Discussion Antiseptics provide a safe and effective alternative for the treatment and prevention of ocular infections. Their broad-spectrum activity against various pathogens, lower potential for inducing resistance, and favorable safety profile make them an invaluable tool in ophthalmology. Incorporating antiseptics into eye care protocols not only enhances clinical outcomes but also contributes to the global fight against antimicrobial resistance [15,40,41]. In this investigation, were used two commercially available ophthalmic preparations: an ozonized vegetable oil solution, soybean phospholipids and hydroxypropylmethylcellulose (OSSO) and a hydroxypropylmethylcellulose solution (HPMC). The study describes changes in the ocular surface bacterial composition of contact lens wearers who were administered OSSO. At the ocular level, ozonized oil acts by releasing ozone and peroxides, which create an oxidative environment that disrupts pathogenic bacteria by damaging their cell membranes and impairing their metabolic functions [19,42]. Previous studies have demonstrated its efficacy against both Gram-negative and Gram-positive bacterial strains [17]. While the taxonomic composition of the bacterial communities in both treatment groups remained similar throughout the study, a trend toward a reduction in bacterial load was observed in the OSSO group following administration, with subsequent recovery after discontinuation (Fig. 1). The Richness Index, representing the total number of distinct species, and the Shannon Index [43], which accounts for both species richness and evenness, remained relatively unchanged across all three time points (control, T1, and T2) in the HPMC group. In contrast, the OSSO group demonstrated a reduction in both indices between the control and T1, with a subsequent recovery following treatment cessation (Shannon Fig. 2. Boxplots showing ocular microbiota alpha diversity when treated with different ophthalmic solutions (HPMC and OSSO) using two different indices. Kruskal–Wallis p-values are provided for each comparison. M.C. S´ anchez-Gonz´ alez et al. Contact Lens and Anterior Eye 48 (2025) 102368 4 index, p-value =0.094), Fig. 3). Recent studies corroborate the antimicrobial efficacy of ozonized oil against common pathogens, including Mycobacteria, Staphylococci, Streptococci, Enterococci, and Pseudomonas [18,44,45]. Furthermore, ozonized oil has been shown to reduce bacterial biofilm formation, a natural defense mechanism that bacteria activate, particularly on the ocular surface in cases of abrasions, trauma, or contact lens use. Biofilm removal disrupts the bacteria’s protective environment, making them more vulnerable to the eye’s natural defenses and antibiotics, which may reduce the inflammatory symptoms associated with bacterial infections [46–48]. It is well established that antibiotics alone are often ineffective against biofilms [49]. The results of the study did not show a significant reduction in overall bacterial load, nor did the beta diversity analysis reveal distinct clustering between the samples. However, there was a significant difference in the bacterial community composition between the two treatment groups (p =0.002, Fig. 5). This difference could be attributed to suboptimal concentrations of OSSO that failed to reach the Minimum Inhibitory Concentration (MIC). A rarefaction curve illustrates the relationship between the number of samples and the observed taxa, providing insight into whether the sampling effort adequately captured the dataset’s diversity. A plateauing curve indicates sufficient sequencing depth [50]. In the study, only 86 of the 201 samples reached a saturation plateau, suggesting that the sampling effort may have been insufficient to fully capture the microbial diversity. A larger sample size could have provided a more accurate estimate of species richness. Importantly, although none of the participants showed signs of infection, all were regular contact lens wearers. There is evidence that contact lens use disrupts the conjunctival microbiome, leading to an increased presence of skin-associated bacterial species [10]. This more complex ocular habitat likely required a greater sampling effort to capture its full diversity due to the presence of microhabitats and specialized bacterial species. Currently, chlorhexidine and povidone iodine are the most Fig. 3. Boxplots showing ocular microbiota alpha diversity for patients treated with different ophthalmic solutions (HPMC and OSSO) at three different treatment times, using two different indices. Kruskal–Wallis p-values are provided for global and pairwise comparisons. M.C. S´ anchez-Gonz´ alez et al. Contact Lens and Anterior Eye 48 (2025) 102368 5 commonly used antiseptics for disinfecting the skin and periocular areas in ocular surgery, owing to their efficacy against a broad range of microorganisms [15]. However, their use is limited by corneal toxicity, particularly epithelial toxicity [51]. Animal studies have also reported endothelial toxicity with povidone iodine [52]. In contrast, liposomal ozonized oil has demonstrated antiseptic, anti-inflammatory, and tissuerepair properties without exhibiting corneal toxicity [44]. The results of the study seem to indicate that the use of ozonized oil reduces the bacterial load compared to another solution that was used as a control. In all patients it was well tolerated. Negative controls were not employed for the swab, extraction kit, buffers, or ocular solutions. Nevertheless, the microbial composition of the samples predominantly comprised bacteria commonly associated with the ocular microbiome [53], and this composition was consistent across all patients included in the study. These findings suggest the absence of contamination from external DNA. Furthermore, sterile techniques were rigorously applied during sample collection, and stringent precautions were taken to prevent contamination by foreign DNA. While further research is necessary, ozonized oil could be considered as an antiseptic agent for infection prophylaxis in all types of ophthalmologic interventions. 4.1. Clinical Practice Implications The use of ozonated oil offers a non-antibiotic option to reduce bacterial load, addressing the challenge of antimicrobial resistance. Ozonated oil shows promise as a safe adjunct treatment for managing dry eye in contact lens users, potentially reducing biofilm-related complications. Its safety profile supports its potential inclusion in ocular antiseptic protocols, especially for patients at risk of microbiota imbalances. 4.2. Study Limitations The study compared microbial profiles between eyes treated with different solutions rather than using a reference healthy microbiome. This limits generalizability but allows evaluation of treatment-specific effects in a homogeneous sample. Both groups consisted of contact lens users with dry eye symptoms, preventing extrapolation to non-lens users but ensuring comparability within the study population. Limited sequencing depth and sample size may have restricted the detection of microbial diversity, warranting further studies with larger datasets. The lack of negative controls for the swab, extraction kit, tampons and eye solutions is another limitation of the study. However, the observed microbial composition of the samples predominantly comprised bacteria commonly associated with the ocular microbiome, with consistency across all patients included in the study. While these Fig. 4. NMDS analysis based on the Bray-Curtis distances between samples from patients receiving the two ophthalmic solutions. A significant difference (p <0.05) was observed between the both groups of samples (PERMANOVA, pseudo-F: 2.10, p-value: 0.002). The percentage of variance explained by each component is shown in parentheses. M.C. S´ anchez-Gonz´ alez et al. Contact Lens and Anterior Eye 48 (2025) 102368 6 findings suggest minimal contamination from external DNA, the absence of such controls could limit the certainty of this conclusion. In future research, control and sample collections will be conducted simultaneously under identical conditions and processed together. CRediT authorship contribution statement María Carmen S´ anchez-Gonz´ alez: Conceptualization, Methodology, Investigation, Writing – original draft, Writing – review & editing. Inmaculada Gallardo-Real: Conceptualization, Methodology, Investigation, Writing – review & editing. Estanislao Guti´ errez-S´ anchez: Conceptualization, Writing – review & editing. Concepci´ on De-HitaCantalejo: Conceptualization, Writing – review & editing. Raúl Capote-Puente: Conceptualization, Writing – review & editing. Jos´ eMaría S´ anchez-Gonz´ alez: Conceptualization, Methodology, Investigation, Writing – original draft, Writing – review & editing. Funding The preparation and sequencing of DNA metabarcoding libraries and the bioinformatic analyses were carried out by AllGenetics & Biology SL (https://www.allgenetics.eu) and the research was funded was funded by ESTEVE Pharmaceuticals, S.A. 109, Zona Franca Street, Barcelona, 08038, Catalonia, Spain. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgments All eyedrops were donated by ESTEVE Pharmaceuticals. The authors appreciate the support offered by the members of the Department of Physics of Condensed Matter, Faculty of Physics, University of Seville. In addition, the authors also appreciate the technical support offered by the members and facilities of the Faculty of Pharmacy, University of Seville, with special thanks to María ´ Alvarez-de-Sotomayor. The research was funded by ESTEVE Pharmaceuticals, S.A. 109, Zona Franca Street, Barcelona, 08038, Spain. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.clae.2025.102368. References [1] Dor M, Eperon S, Lalive PH, Guex-Crosier Y, Hamedani M, Salvisberg C, et al. Investigation of the global protein content from healthy human tears. Exp Eye Res 2019;179:64–74. https://doi.org/10.1016/J.EXER.2018.10.006. [2] Borroni D, Romano V, Kaye SB, Somerville T, Napoli L, Fasolo A, et al. Metagenomics in ophthalmology: Current findings and future prospectives. BMJ Open Ophthalmol 2019;4. https://doi.org/10.1136/BMJOPHTH-2018-000248. [3] Borroni D, Paytuví-Gallart A, Sanseverino W, G´ omez-Huertas C, Bonci P, Romano V, et al. Exploring the healthy eye microbiota niche in a multicenter study. Int J Mol Sci 2022;23. https://doi.org/10.3390/IJMS231810229. Fig. 5. 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