Efficacy of the combination of flashlight (365 nm) and riboflavin in reducing microorganisms isolated from canine corneas: in vitro study Ultraviolet Flashlight for Riboflavin Photoactivation

Efficacy of the combination of flashlight (365 nm) and riboflavin in reducing microorganisms isolated from canine corneas: in vitro study Ultraviolet Flashlight for Riboflavin Photoactivation | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Efficacy of the combination of flashlight (365 nm) and riboflavin in reducing microorganisms isolated from canine corneas: in vitro study Ultraviolet Flashlight for Riboflavin Photoactivation Laís Fernanda Sargi, Romário Alves Rodrigues, Pamella Almeida Freire Casemiro, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8604527/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The photoactivation of riboflavin induces genetic and structural damage to microbial cell membranes, potentially leading to the death of bacteria, viruses, and fungi. However, the conventional approach requires the use of a Photoactivated Chromophore for Corneal Collagen Crosslinking (PACK-CXL), a high-cost device with limited availability in clinical practice. As a low-cost alternative, this study evaluated photoactivation using a portable ultraviolet flashlight (365 nm) against isolates obtained from cases of canine ulcerative keratitis. Riboflavin at different concentrations (0.025, 0.05, 0.1, 0.2, 0.4, and 0.8%) were tested and exposed to UVA radiation (365 nm) at a distance of 3 cm for 15 or 30 minutes, followed by analysis using the time-kill curve method. Results showed that the flashlight was able to activate riboflavin and completely inhibit the growth of Staphylococcus pseudintermedius at a concentration of 0.4% for both exposure periods, and also caused effects at 0.2% after 15 minutes and at 0.05% after 30 minutes of UVA exposure. For Streptococcus canis , significant reductions were observed at 0.4% after 15 minutes of exposure, although these effects were insufficient to kill the bacterium completely. Klebsiella pneumoniae showed reduced growth at 0.2% (after 30 minutes), whereas Pseudomonas aeruginosa did not respond to any tested concentrations. The comparative analysis between exposure times (15 vs. 30 min) revealed no significant differences (p > 0.05). The handheld ultraviolet flashlight (365 nm) was able to photoactivate riboflavin, and this low-cost approach may represent a potential alternative in the absence of conventional PACK-CXL therapy, although further studies are warranted. corneal cross-linking bacterial keratitis corneal ulcer keratomalacia infectious keratitis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. INTRODUCTION Infectious ulcerative keratitis is a common clinical condition in veterinary ophthalmology This injury is associated with several risk factors, including ocular trauma, lagophthalmos, reduced corneal sensitivity, and ocular surface disorders such as keratoconjunctivitis sicca and eyelid abnormalities. Other complications, such as corneal ulcers, also referred to as melting ulcers or keratomalacia, liquefactive necrosis of the cornea, or acute stromal collagenolysismay also develop [ 1 , 2 ]. Several bacterial species have been implicated in infectious keratitis in dogs and cats, with Pseudomonas aeruginosa , Staphylococcus spp., and Streptococcus spp. being the most commonly isolated pathogens [3, 4, 5]. In addition, other microorganisms such as Escherichia coli , Bacillus spp., Klebsiella pneumoniae , and Proteus spp. have also been associated with this condition [ 6 , 7 ]. Gram-positive bacteria, such as Staphylococcus spp. and Streptococcus spp., are commonly isolated in canine keratitis and are often associated with less aggressive infections. In contrast, Gram-negative bacteria, including Pseudomonas aeruginosa and Klebsiella spp., are typically implicated in acude infections, posing greater therapeutic challenges. Clinical management generally involves the use of antibiotics, lubricants, and protease inhibitors; however, antimicrobial resistance has emerged as an increasing concern in veterinary ophthalmology [ 4, 6, 8]. In cases of severe keratomalacia or corneal perforation, surgical techniques such as conjunctival flaps, amniotic membrane transplantation, or synthetic membrane transplantation are employed to provide tectonic support. However, these procedures may result in varying degrees of corneal opacity, depending on the size, location, and depth of the lesion, making the exploration of novel antimicrobial strategies crucial for maintaining the effectiveness of infectious keratitis treatment [ 9 , 10 , 11 ]. A non-surgical photodynamic therapy known as Photoactivated Chromophore for Corneal Collagen Crosslinking (PACK-CXL) represents an innovative approach in the treatment of ophthalmological conditions in both humans and animals. This technique combines ultraviolet-A light and vitamin B2 (riboflavin) to enhance the stability of collagen fibers in the cornea and promote the inactivation of corneal pathogens [ 12 , 13 ]. In crosslinking (CXL), riboflavin, when exposed to ultraviolet-A light (UVA) with a wavelength of approximately 365 nanometers, acts as a photosensitizer. This process also results in the formation of covalent bonds between collagen fibers in the cornea, imparting increased biomechanical strength and biochemical stability [ 14 , 15 ]. Recently, this technique has found application in veterinary medicine to address cases of keratomalacia [ 16 , 17 , 18 , 19 ]. The interaction of riboflavin with ultraviolet light induces damage to the genetic material and cell membranes of microorganisms, potentially leading to the demise of bacteria, viruses, parasites, and fungi [ 20 , 21 ]. Following exposure to UVA (λ = 365nm), riboflavin by-products irreversibly penetrate cell membranes, interacting with the nucleic acids of bacteria, causing harm through the action of reactive oxygen species [ 22 , 23 ]. However, PACK-CXL is a high-cost device with limited accessibility for veterinarians and patients in clinical practice A study investigated the use of an ultraviolet flashlight (365 nm) as a cost-effective alternative for treating keratomalacia in dogs, yielding noteworthy results [ 24 ]. To date, there are no in vitro published data addressing the efficacy of the UVA flashlight (Nitecore Chameleon CU6®, Guangdong, China) as an alternative to the PACK-CXL device in canine ulcerative keratitis. Therefore, the aim of this study was to assess the in vitro antibacterial efficacy of the UVA flashlight (365 nm) on bacterial isolates obtained from ulcerative keratitis in dogs. 2. MATERIALS AND METHODS 2.1 Pathogen Selection Four distinct bacterial strains were selected from canine ocular samples obtained from ulcerative keratitis. These strains included Staphylococcus pseudintermedius, Streptococcus canis, Pseudomonas aeruginosa and Klebsiella pneumoniae . They were previously identified using microbiological and biochemical techniques and confirmed by MALDI-TOF (Matrix-Assisted Laser Desorption/Ionization–Time of Flight). 2.2 Riboflavin Preparation and Dilution Riboflavin (R9504-25G, Sigma-Aldrich) was diluted in Mueller-Hinton Broth (MHB) at concentrations of 0.8%, 0.4%, 0.2%, 0.1%, 0.05%, and 0.025%. Initially, 0.8 grams of riboflavin were weighed and dissolved in 100 mL of MHB, resulting in an initial solution at a concentration of 0.8%. Subsequently, a 2 mL aliquot was withdrawn. Next, 1 mL of the initial solution was transferred to a Falcon tube containing 1 mL of diluent, and thereafter, serial double dilutions were performed up to 0.025%, ensuring that all dilutions maintained a volume of 1 mL. Two tubes with MHB were prepared, one for positive control and one for the negative control. Finally, 10 µL was removed from all samples, that is, from the concentrations and controls. Throughout the procedure, riboflavin was protected from exposure to external light (Fig. 1 A). 2.3 Bacterial Suspension in Riboflavin Solution Each cryopreserved bacterial isolate was inoculated into Brain Heart Infusion Broth (BHI Broth) and incubated under aerobic conditions at 37°C for 18 hours, followed by incubation on BHI Agar under the same conditions. The bacterial suspension was standardized to 0.08–0.12 absorbance, equivalent to 0.5 on the MacFarland scale, by spectrophotometry (Eppendorf BioPhotometer Plus, OD 600) in a 0.9% sodium chloride solution at pH 7.3. After preparation, 10 µL of the bacterial suspension was dispensed into the different riboflavin concentrations and the positive control prior to exposure to the flashlight UVA light (Figura 1A). 2.4 UVA Irradiation and Exposure Time Subsequently, 300 µL of each concentration (%riboflavin + bactéria) was transferred to an Elisa plate (86 x 128mm) for exposure to UVA light. The flashlight used was designed to illuminate only 4 wells at a time. To achieve this precision, we divided the sample into two separate ELISA plates: the first plate contained higher concentrations (0.8%, 0.4%, 0.2%, and 0.1%), while the second plate contained lower concentrations (0.05%, 0.025%, positive control, and negative control. This approach allowed the flashlight to directly target the four wells of interest in each plate, preventing light from overlapping onto adjacent wells. This ensured precise and controlled light exposure. The light was directed from a distance of 3 cm from the surface, mimicking light emission on the cornea of dogs with infectious ulcerative keratitis. The entire process was carried out under aseptic conditions and protected from interference by other light sources (Fig. 1 B). For the UVA irradiation assessment, a CU6 Nitecore® flashlight (Guangdong, China) with a 365 nm wavelength was utilized. The light intensity was measured at 19.1 mW/cm² using a photometer. The flashlight was shone on the ELISA plate from a distance of 3 cm for two durations: 15 and 30 minutes. The administered energy dose was calculated to be 5.763 mW/cm², based on the formula (intensity x exposure time), considering the distance of 3 cm. 2.5 Time-Kill Curve The time-kill curve was conducted following the protocol published by Blondeau et al. 29 with minor modifications. The ELISA plates were sealed, protected from light, and incubated at 36 ± 1°C, being removed only at the designated times to withdraw an aliquot for performing the serial dilution (Fig. 1 B). Microbial growth was assessed through plate microdilution at the following time points (hours): pre-exposure (T0) and post-exposure to UVA light (2 (T2), 8 (T8), 16 (T16), 32 (T32), and 64 (T64) (Fig. 1 B). For each analyzed period, 20 µL from the initial column subjected to light (riboflavin and bacteria concentrations, positive and negative controls) was transferred to another Elisa plate filled with 180 µL of 0.9% saline, conducting decimal serial dilution (Fig. 1 C). Subsequently, a 5 µL aliquot from each well of the ELISA plate was withdrawn and transferred to a Petri dish containing Mueller-Hinton agar (MHA), in triplicate The MHA Petri plates were incubated and subsequently used for quantification (Fig. 1 D). The incubation time for each bacterium for plate microdilution and, consequently, total bacterial counting was determined through a pilot study. P. aeruginosa was incubated for 9 hours, S. pseudointermedius and K. pneumoniae for 16 hours, and Streptococcus canis for 24 hours, the latter under anaerobic conditions. The counting considered microbial growth between 5 and 30 colonies and was used a colony counter with magnifying glass, with the value multiplied by the respective dilution and the correction factor (200, i.e., 1000 µL/5 µL). The counting result was expressed in Colony Forming Units (CFU)/mL. The entire process was carried out for both the 15- and 30-minute exposure periods and for each bacterial species. 2.6 Statistical Analysis Statistical analyses were performed using the R 4.1.2 program [ 25 ]. Means were log-transformed, and normality and homogeneity patterns were assessed using Shapiro-Wilk and Levene tests, respectively. Based on the obtained data, the Kruskall-Wallis test was conducted, followed by the Dunn test corrected by the Bonferroni test. Values of p less than 0.05 were considered significant. 3. RESULTS Following 15 minutes of UVA exposure, Staphylococcus pseudintermedius exhibited reduced growth at riboflavin concentrations between 0.05% and 0.4%, with 0.2% and 0.4% causing complete bacterial death from 32 hours post-exposure. After 30 minutes of exposure, a 0.4% riboflavin concentration produced a similar bactericidal effect with 64 hours, while 0.05% also resulted in complete bacterial killing within 32 hours. The positive control also showed statistically significant differences (p < 0.05) with higher bacterial growth curves, especially at riboflavin concentrations of 0.025%, 0.05%, 0.2%, and 0.4% at the T2/15UVA and T64/30UVA time points, respectively. Therefore, it was observed that, after UVA exposure, there was a reduction in the population of microorganisms, with a pronounced decrease at concentrations of 0.4%, 0.2%, and 0.1%, starting 8 hours after exposure. This resulted in a decrease of up to three logs in the count (Fig. 2 and and Supplementary Material 1). In the analysis of Streptococcus canis , the concentration of 0.4% showed a decrease in bacterial count at 32 hours (for 15UVA) and at 16 hours (for 30UVA), both observations significantly differing from the positive control group (p < 0.05). However, although not pronounced, riboflavin at a concentration of 0.4% appeared to exert a stronger effect against Streptococcus canis , particularly after 15 minutes of flashlight exposure. Nevertheless, this effect was insufficient to achieve complete microbial killing, resulting only in a reduction of viable cell counts. No other noticeable growth patterns were observed during either the 15- or 30-minute exposure periods (Fig. 3 and Supplementary Material 1). Regarding K. pneumoniae , there was no significant variation in its overall growth. However, the growth curve analysis revealed a reduction in bacterial counts at all tested concentrations, particularly at concentrations of 0.025% and 0.2% at the 16-hour time point (30UVA). It is worth noting that only the concentration of 0.2% showed a statistically significant difference compared to the positive control (p < 0.05). On the other hand, at concentrations of 0.8% and 0.4%, there was an increase in bacterial growth after 2 and 16 hours (15UVA), although this increase did not reach statistical significance compared to the positive control (p < 0.05) (Fig. 4 and Supplementary material 1). In the analyses conducted with Pseudomonas aeruginosa , bacterial counts at different concentrations remained high over the 64 hours, with a slight reduction observed at concentrations of 0.025% between 2 to 16 hours (15UVA) and 0.5% at 32 hours (15UVA), which were not statistically significant. In treatments with concentrations of 0.8% and 0.4%, higher counts than the positive control were observed at the early time points of 2 and 8 hours (Fig. 5 and Supplementary material 1). Concerning the exposure periods, the results indicated no statistically significant difference between the 15UVA and 30UVA exposure periods for the four tested bacterial strains (p > 0.05). 4. DISCUSSION The results highlighted distinctions among the assessed microorganisms isolated from ulcerative keratitis in dogs. Gram-positive bacteria ( S. pseudintermedius and S. canis ) exhibited greater sensitivity compared to Gram-negative bacteria ( P. aeruginosa and K. pneumoniae ) to the light emission effect from the UVA lantern (Nitecore Chameleon CU6®, Guangdong, China) in combination with riboflavin. In veterinary ophthalmology, riboflavin at a 0.1% concentration is routinely used in PACK-CXL protocols, which have been shown not only to exert significant antimicrobial effects against various pathogens but also to induce beneficial structural changes in the cornea, such as increased stromal stiffness and enhanced resistance to enzymatic degradation [ 19 , 26 , 27 ]. In the present study, in vivo effects (such as potential benefits to the córnea) were not assessed. The results demonstrated that a 0.4% riboflavin concentration was the most effective alternative against Gram-positive microorganisms. These findings suggest that the handheld flashlight is capable of photoactivating riboflavin; however, its effects in the cornea may differ, considering factors such as tissue characteristics and light penetration capacity. The effect of riboflavin was evaluated at different concentrations (0.025%, 0.05%, 0.1%, 0.2%, 0.4%, and 0.8%). It was observed that the sensitivity of bacterial strains to UVA radiation varied considerably depending on the concentration of the photosensitizer. P. aeruginosa showed a slight reduction in bacterial count at concentrations of 0.025% between 2 and 16 hours and at 0.5% in 32 hours (15UVA). However, at concentrations of 0.8% and 0.4%, higher counts than the positive control were observed in the early phases of 2 and 8 hours. Similarly, high bacterial counts were observed at a 0.8% riboflavin concentration for S. pseudintermedius and S. canis . Thus, that higher concentrations of riboflavin may inhibit the penetration of UVA light into the solution, compromising its activation and, consequently, its action against all microorganisms [ 28 ]. In vitro studies have unveiled the favorable impacts of crosslinking on ulcerative keratitis in dogs. Three crosslinking protocols (UVA 3 mW/cm² for 60 minutes, UVA 3 mW/cm² for 30 minutes, and UVA 30 mW/cm² for 3 minutes) demonstrated growth inhibition for Streptococcus spp ., Staphylococcus spp ., E. coli , and S. canis . However, in the case of P. aeruginosa and S. pseudintermedius , a response was observed only after prolonged exposure to 3 mW/cm² for 60 minutes [ 29 ]. In human studies, this approach exhibited significant efficacy against S. aureus and S. epidermidis but showed reduced effectiveness against P. aeruginosa , suggesting a higher efficiency against Gram-positive microorganisms [ 14 ]. Consequently, these findings align with the outcomes presented in the current study, indicating that the action of the UVA lantern yields similar effects to those observed with PACK-CXL light. However, other studies in dogs and humans have noted that no differences in susceptibility to PACK-CXL were found among bacterial genera such as Staphylococcus spp , Streptococcus spp , P. aeruginosa , and Pasteurella , as well as P. aeruginosa , S. aureus , and S. epidermidis . In these cases, all these bacterial agents exhibited equivalent sensitivity to their reactions to the treatments tested in their respective studies [ 19 , 30 , 31 ]. In this study, strains of Streptococcus canis and Staphylococcus pseudintermedius were selected to represent Gram-positive bacteria. Notably, S. canis showed a marked reduction in bacterial counts after 16 hours following 15 minutes of UVA exposure, whereas S. pseudintermedius exhibited greater susceptibility after 30 minutes of exposure. These species, along with other Gram-positive bacteria (such as Staphylococcus aureus , Staphylococcus epidermidis , and beta-hemolytic Streptococcus spp.), are frequently implicated in cases of ulcerative keratitis and keratomalacia. Therefore, the handheld flashlight may also exert beneficial effects against these additional pathogens [ 32 , 33 ]. On the other hand, Pseudomonas spp . stands out as the primary infectious agent identified in corneal melting samples [ 34 ]. P. aeruginosa secretes a spectrum of proteases, including alkaline protease, elastase A and B, protease IV, and P. aeruginosa small protease, among others, which are recognized as significant virulence factors contributing to the pathogenesis of ulcerative keratitis. These proteases play a crucial role in degrading essential host defense proteins and collagen fibers, leading to epithelial loss, corneal erosion, and keratomalacia [ 35 , 36 ]. In this study, P. aeruginosa demonstrated resistance to the proposed adjuvant therapy. Consequently, the combined action of riboflavin (0.025–0.8%) and lantern exposure (15 and 30 minutes) proved inefficacious. This suggests that the effect of adjuvant therapy on each bacterium may be attributed to their distinct virulence factors. Gram-negative bacteria commonly found in corneal ulcers include K. pneumoniae and Escherichia coli , recognized as highly virulent agents with the potential to induce endophthalmitis [ 37 ]. In our study of K. pneumoniae , minimal reduction in bacterial counts was observed across the tested concentrations compared to the positive control, suggesting that riboflavin may have had an impact on bacterial growth. Importantly, there is a lack of studies on corneal cross-linking evaluating the response of Klebsiella pneumoniae to photodynamic therapy in veterinary ophthalmology. However, human studies have indicated that Klebsiella oxytoca demonstrates significantly lower sensitivity to PACK-CXL compared to other investigated strains, such as S. aureus, P. aeruginosa e Bacillus subtilis [ 38 ]. Additionally, the virulence of K. pneumoniae has been associated with the production of cell wall-associated factors and capsules [ 39 ]. This underscores the need for further research specific to veterinary ophthalmology to better understand the effectiveness of photodynamic therapy in addressing infections caused by K. pneumoniae and similar pathogens. Several factors can play a crucial role in the varied response of different bacterial species to UVA light and riboflavina [ 31 ]. Among these factors, diversity in metabolism, sensitivity to oxidative stress, duration of the cell cycle, cell wall structure, and others stand out [ 30 ]. In Gram-negative bacteria, the membrane structures are more complex compared to the peptidoglycan layer found in Gram-positive bacteria. This complexity may impede the penetration of riboflavin by-products into the bacterial cell [ 40 ]. In a study involving Gram-positive bacteria, S. epidermidis , and S. aureus , it was observed that the survival rate of S. epidermidis was significantly lower than that of S. aureus when subjected to the same UVA light parameters. This difference may be associated with the formation of a bacterial biofilm, acting as a barrier preventing the penetration of the photosensitizer and, consequently, the activation of riboflavina [ 41 ]. Furthermore, microbial sensitivity to oxidative stress induced by riboflavin and UV light is not influenced by acquired antibiotic resistance, making this approach a viable alternative in such cases. Additionally, the flashlight-based methodology has not yet been evaluated in vivo or in combination with other commonly used therapies that contribute positively to clinical outcomes, such as antibiotics, lubricants, and metalloproteinase inhibitors [ 30 ]. In in vivo studies using PACK-CXL for the treatment of ulcerative keratitis in dogs and cats, several protocols have been documented. These include UVA irradiance of 3 mW/cm² for 30 minutes and an accelerated protocol with UVA irradiance of 30 mW/cm² administered for 3 minutes [ 13 , 17 ]. It is important to note that the flashlights used in the study do not allow for power adjustment, which limits their application. Increasing the exposure time to UVA light from 15 to 30 minutes, with the same power setting (light intensity), did not provide a statistically significant difference in the results of this study. A similar finding was reported, where there was no significant difference in the areas of bacterial growth inhibition between the 3-minute and 30-minute UVA/riboflavin protocols [ 29 ]. This result diverges from findings in the literature regarding the use of PACK-CXL, which suggests that longer exposure periods to UVA light are more effective in eradicating bactéria [ 28 , 30 ]. Therefore, understanding the differential responses among bacterial species and the available therapeutic alternatives is crucial for optimizing photodynamic therapy approaches, particularly in veterinary ophthalmology, where treatment efficacy can be influenced by the specific characteristics of both the infectious pathogens and the host. 5. CONCLUSION The results indicate that UVA radiation from the handheld flashlight (Nitecore Chameleon CU6®, Guangdong, China) combined with riboflavin can effectively eliminate S. pseudintermedius counts, particularly at a 0.4% concentration, and can reduce and partially inhibit S. canis (0,4%) e Klebsiella pneumoniae (0,2%). However, the protocol was not effective against Pseudomonas aeruginosa . Declarations 6. FUNDING This work was carried out with the support of the Coordination for the Improvement of Higher Education Personnel - Brazil (CAPES) - Financing Code 001. 7. CONFLICT OF INTEREST The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. References O’Neill DG, Lee MM, Brodbelt DC, Church DB, Sanchez RF. Corneal ulcerative disease in dogs under primary veterinary care in England: epidemiology and clinical management. Canine Genet Epidemiol. 2017;4(1):1–12. Martin de Bustamante MG, Good KL, Leonard BC, Hollingsworth SR, Edwards SG, Knickelbein KE, et al. Medical management of deep ulcerative keratitis in cats: 13 cases. J Feline Med Surg. 2019;21(4):387–93. Ekapopphan D, Srisutthakarn A, Moonarmart W, Buddhirongawatr R, Bangphoomi N. Identification and antimicrobial susceptibility of microorganisms isolated from severe corneal ulcers of dogs in Thailand. J Vet Med Sci. 2018;80(8):1259–65. Hewitt JS, Allbaugh RA, Kenne DE, Sebbag L. Prevalence and Antibiotic Susceptibility of Bacterial Isolates From Dogs With Ulcerative Keratitis in Midwestern United States. Front Vet Sci. 2020;7(November):1–10. Verdenius CY, Broens EM, Slenter IJM, Djajadiningrat-Laanen SC. Corneal stromal ulcerations in a referral population of dogs and cats in the Netherlands (2012–2019): Bacterial isolates and antibiotic resistance. Vet Ophthalmol. 2024;27(1):7–16. Park J, Kim D, Kwon M, Kwon H, Yong J, Yoon H, et al. Bacterial isolates and antibiotic sensitivity in canine bacterial keratitis in Korea. Vet Ophthalmol. 2024;(May):1–12. Kyung SM, Lee JH, Lee ES, Xiang XR, Yoo HS. Emergence and genomic chion of Proteus mirabilis harboring blaNDM-1 in Korean companion dogs. Vet Res [Internet]. 2024;55(1):50. Available from: https://doi.org/10.1186/s13567-024-01306-w McKeever JM, Ward DA, Hendrix DVH. Comparison of antimicrobial resistance patterns in dogs with bacterial keratitis presented to a veterinary teaching hospital over two multi-year time periods (1993–2003 and 2013–2019) in the Southeastern United States. Vet Ophthalmol. 2021;24(6):653–8. Soontornvipart K, Tuntivanich N, Kecová H, Raušer P. Conjunctival pedicle graft in dogs and cats: A retrospective study of 88 cases. Acta Vet Brno. 2003;72(1):63–9. Dorbandt DM, Moore PA, Myrna KE. Outcome of conjunctival flap repair for corneal defects with and without an acellular submucosa implant in 73 canine eyes. Vet Ophthalmol. 2015;18(2):116–22. Costa D, Leiva M, Sanz F, Espejo V, Esteban J, Vergara J, et al. A multicenter retrospective study on cryopreserved amniotic membrane transplantation for the treatment of complicated corneal ulcers in the dog. Vet Ophthalmol. 2019;22(5):695–702. Pot SA, Gallhöfer NS, Matheis FL, Voelter-Ratson K, Hafezi F, Spiess BM. Corneal collagen cross-linking as treatment for infectious and noninfectious corneal melting in cats and dogs: Results of a prospective, nonrandomized, controlled trial. Vet Ophthalmol. 2014;17(4):250–60. Spiess BM, Pot SA, Florin M, Hafezi F. Corneal collagen cross-linking (CXL) for the treatment of melting keratitis in cats and dogs: A pilot study. Vet Ophthalmol. 2014;17(1):1–11. Martins SAR, Combs JC, Noguera G, Camacho W, Wittmann P, Walther R, et al. Antimicrobial efficacy of riboflavin/UVA combination (365 nm) in vitro for bacterial and fungal isolates: A potential new treatment for infectious keratitis. Investig Ophthalmol Vis Sci. 2008;49(8):3402–8. Alio JL, Abbouda A, Valle DD, del Castillo JMB, Fernandez JAG. Corneal cross linking and infectious keratitis: A systematic review with a meta-analysis of reported cases. J Ophthalmic Inflamm Infect. 2013;3(1):1–7. Famose F. Evaluation of accelerated collagen cross-linking for the treatment of melting keratitis in eight dogs. Vet Ophthalmol. 2014;17(5):358–67. Famose F. Evaluation of accelerated collagen cross-linking for the treatment of melting keratitis in ten cats. Vet Ophthalmol. 2015;18(2):95–104. Zibura AE, Cullen MA, Rutledge H, Lassalle L, Salmon JH, Gilger BC, et al. Optimizing corneal riboflavin administration in ex vivo horse, dog, rabbit, and pig samples for use in corneal collagen cross-linking. Vet Ophthalmol. 2020;23(5):840–8. Suter A, Schmitt S, Hübschke E, Kowalska M, Hartnack S, Pot S. The bactericidal effect of two photoactivated chromophore for keratitis-corneal crosslinking protocols (standard vs. accelerated) on bacterial isolates associated with infectious keratitis in companion animals. BMC Vet Res [Internet]. 2022;18(1):1–10. Available from: https://doi.org/10.1186/s12917-022-03397-z Sharma A, Sharma R, Chander J, Nirankari VS. In vitro antimicrobial efficacy of riboflavin, ultraviolet-A radiation, and combined riboflavin/ultraviolet-A radiation on ocular pathogens. Taiwan J Ophthalmol [Internet]. 2023 Jan;13(1):21–7. Available from: https://journals.lww.com/10.4103/tjo.tjo_28_21 Mundt JM, Rouse L, Van Den Bossche J, Goodrich RP. Chemical and Biological Mechanisms of Pathogen Reduction Technologies. Photochem Photobiol. 2014;90(5):957–64. Kumar V, Lockerbie O, Keil SD, Ruane PH, Platz MS, Martin CB, et al. Riboflavin and UV-Light Based Pathogen Reduction: Extent and Consequence of DNA Damage at the Molecular Level. Photochem Photobiol. 2004;80(1):15. Tabibian D, Richoz O, Hafezi F. PACK-CXL: Corneal cross-linking for treatment of infectious keratitis. J Ophthalmic Vis Res. 2015;10(1):77–80. Lembo JA, Sapienza J, Wolfer J, Allgoewer I. A rapid and cost-effective method for corneal cross-linking for treatment of canine keratomalacia. In: Annual Scientific Meeting Of The American College Of Veterinary Ophthalmologists; September 2018; Minneapolis, Minnesota. R Core Team. R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria; 2020. Available from: https://www.R-project.org/. Kowalska ME, Pot SA, Hartnack S. Photoactivated Chromophore for Keratitis-Corneal Cross-linking (PACK-CXL)—A Scoping Review Based on Preclinical Studies. Transl Vis Sci Technol. 2024;13(7):11–6. Shukla AK, Kowalska ME, Arteaga K, Crasta M, Dixon C, Famose F, et al. Evaluation of photoactivated chromophore for keratitis–corneal cross-linking (PACK-CXL) in feline infectious keratitis—patient demographics, treatment protocols, risk factors, and treatment outcome: a retrospective study. Vet Ophthalmol. 2025;28(2):330–40. Bäckman A, Makdoumi K, Mortensen J, Crafoord S. The efficiency of cross-linking methods in eradication of bacteria is influenced by the riboflavin concentration and the irradiation time of ultraviolet light. Acta Ophthalmol. 2014;92(7):656–61. Large TP, Mack S, Villiers E, Oliver J. In vitro susceptibility of canine corneal bacterial pathogens to three cross-linking protocols. Vet Ophthalmol. 2023;26(S1):134–42. Makdoumi K, Bäckman A. Photodynamic UVA-riboflavin bacterial elimination in antibiotic-resistant bacteria. Clin Exp Ophthalmol. 2016;44(7):582–6. Makdoumi K, Mortensen J, Crafoord S. Infectious keratitis treated with corneal crosslinking. Cornea. 2010;29(12):1353–8. Tolar EL, Hendrix DVH, Rohrbach BW, Plummer CE, Brooks DE, Gelatt KN. Evaluation of clinical characteristics and bacterial isolates in dogs with bacterial keratitis: 97 Cases (1993-2003). J Am Vet Med Assoc. 2006;228(1):80–5. Wang Z, Guo L, Li J, Li J, Cui L, Dong J, et al. Antibiotic resistance, biofilm formation, and virulence factors of isolates of staphylococcus pseudintermedius from healthy dogs and dogs with keratitis. Front Vet Sci. 2022;9. Tsvetanova A, Powell RM, Tsvetanov KA, Smith KM, Gould DJ. Melting corneal ulcers (keratomalacia) in dogs: A 5-year clinical and microbiological study (2014–2018). Vet Ophthalmol. 2021;24(3):265–78. Hobden JA. Pseudomonas aeruginosa proteases and corneal virulence. DNA Cell Biol. 2002;21(5):391–6. O’callaghan R, Caballero A, Tang A, Bierdeman M. Pseudomonas aeruginosa keratitis: Protease iv and pasp as corneal virulence mediators. Microorganisms. 2019;7(9). Chee SP, Jap A. Endogenous endophthalmitis. Curr Opin Ophthalmol [Internet]. 2001 Dec;12(6):464–70. Available from: http://journals.lww.com/00055735-200112000-00012 Kling S, Hufschmid FS, Torres-Netto EA, Randleman JB, Willcox M, Zbinden R, et al. High Fluence Increases the Antibacterial Efficacy of PACK Cross-Linking. Cornea. 2020;39(8):1020–6. Pomakova DK, Hsiao CB, Beanan JM, Olson R, MacDonald U, Keynan Y, et al. Clinical and phenotypic differences between classic and hypervirulent Klebsiella pneumonia: An emerging and under-recognized pathogenic variant. Eur J Clin Microbiol Infect Dis. 2012;31(6):981–9. Hamblin MR, Hasan T. Enacting strategies for graduate employability: A look at multiple issues and disciplines. Photochem Photobiol [Internet]. 2004;3(5):436–50. Available from: http://pubs.rsc.org/en/Content/ArticleHTML/2004/PP/B311900A%5Cnhttp://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=3071049&tool=pmcentrez&rendertype=abstract Shen J, Liang Q, Su G, Zhang Y, Wang Z, Liang H, et al. Effect of Ultraviolet Light Irradiation Combined with Riboflavin on Different Bacterial Pathogens from Ocular Surface Infection. J Biophys. 2017;2017. Additional Declarations No competing interests reported. Supplementary Files S1.jpg Supplementary Material 1. Boxplot illustrating the variation in mean antibacterial activity of riboflavin at different concentrations, photoactivated by ultraviolet A light from a flashlight (CU6 Nitecore®, Guangdong, China), after 15 minutes (T15) and 30 minutes (T30) against Staphylococcus pseudintermedius, Streptococcus canis, Klebsiella pneumoniae and Pseudomonas aeruginosa. Light exposure times include T0, T2, T8, T16, T32, and T64. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8604527","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":581710547,"identity":"0934c51d-3636-4042-89d7-c7a4fd985931","order_by":0,"name":"Laís Fernanda Sargi","email":"","orcid":"","institution":"São Paulo State University","correspondingAuthor":false,"prefix":"","firstName":"Laís","middleName":"Fernanda","lastName":"Sargi","suffix":""},{"id":581710548,"identity":"7073d908-7bcc-4203-9b10-74ff6a21af16","order_by":1,"name":"Romário Alves Rodrigues","email":"","orcid":"","institution":"São Paulo State University","correspondingAuthor":false,"prefix":"","firstName":"Romário","middleName":"Alves","lastName":"Rodrigues","suffix":""},{"id":581710549,"identity":"41280c00-16b6-47da-a236-cfbb3c6204f0","order_by":2,"name":"Pamella Almeida Freire Casemiro","email":"","orcid":"","institution":"São Paulo State University","correspondingAuthor":false,"prefix":"","firstName":"Pamella","middleName":"Almeida Freire","lastName":"Casemiro","suffix":""},{"id":581710550,"identity":"b8b3e7c1-bebc-4f2d-9d59-279941da05c4","order_by":3,"name":"Lucas José Luduverio Pizauro","email":"","orcid":"","institution":"State University of Santa Cruz","correspondingAuthor":false,"prefix":"","firstName":"Lucas","middleName":"José Luduverio","lastName":"Pizauro","suffix":""},{"id":581710551,"identity":"2461081d-b5cf-4ac2-a6c2-4ae95cbbefbd","order_by":4,"name":"Ivan Ricardo Martinez Padua","email":"","orcid":"","institution":"State University of Santa Cruz","correspondingAuthor":false,"prefix":"","firstName":"Ivan","middleName":"Ricardo Martinez","lastName":"Padua","suffix":""},{"id":581710552,"identity":"5556f79e-b645-43f2-a411-39c1c94ba343","order_by":5,"name":"Gabriela Moraes Madruga","email":"","orcid":"","institution":"Madruga Veterinary Ophthalmology","correspondingAuthor":false,"prefix":"","firstName":"Gabriela","middleName":"Moraes","lastName":"Madruga","suffix":""},{"id":581710553,"identity":"1dcb3964-ef2d-4cdb-952a-eb0c4e999fb3","order_by":6,"name":"Cinthya de Andrade Gujanwski","email":"","orcid":"","institution":"São Paulo State University","correspondingAuthor":false,"prefix":"","firstName":"Cinthya","middleName":"de Andrade","lastName":"Gujanwski","suffix":""},{"id":581710554,"identity":"0cb0eb18-c118-4a0f-9fda-ef3cf42a667c","order_by":7,"name":"Marita Vedovelli Cardozo","email":"","orcid":"","institution":"São Paulo State University","correspondingAuthor":false,"prefix":"","firstName":"Marita","middleName":"Vedovelli","lastName":"Cardozo","suffix":""},{"id":581710555,"identity":"5001acb8-e55b-44a5-bc75-3bd062d8d50a","order_by":8,"name":"Paola Castro Moraes","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6UlEQVRIiWNgGAWjYHACxgNw5gcIxUZQD1wL4wyStTDzEKPFnP3wgQM/GLbJmbcfPvzZtu2OnPzsBrbHFXi0WPakJRzsYbhtLHMmLU06t+2ZscGdA+yGZ/BoMTiQY3CAh+F24gyGHDPm3LbDiRskEtgkG/BpOf/G4OAfkBb+958/W7Ydrp8/g5CWGzkGh8G2SOQwSDO2HU5guEFAi+WMZwmHZQxuG0tIPDOT7Dl32HDDnYPthvi0mPMnH3z4puK2nAR/8uMPP8oOy8vPbj72EK/DkEgokGDEpwFNMVQLXg2jYBSMglEwAgEAHFVRnEUxleoAAAAASUVORK5CYII=","orcid":"","institution":"São Paulo State University","correspondingAuthor":true,"prefix":"","firstName":"Paola","middleName":"Castro","lastName":"Moraes","suffix":""}],"badges":[],"createdAt":"2026-01-14 18:53:12","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8604527/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8604527/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":101434153,"identity":"561a6d60-dc96-4540-978a-f03a40b316b4","added_by":"auto","created_at":"2026-01-29 16:04:00","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":227820,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic drawing of the methodology of this study. A) \u003c/strong\u003ePreparation of riboflavin concentration and dilution, positive and negative control \u003cstrong\u003eB\u003c/strong\u003e) Time-kill curve: Elisa plates were evaluated at pre-exposure (T0), exposed to UVA flashlight irradiation four wells at a time for designated periods (15 and 30 minutes, in different plates) and incubated for post-exposure analysis in hours at times 2 (T2), 8 (T8), 16 (T16), 32 (T32) and 64 (T64). UVA light with a wavelength of 365 nm in the wells of the Elisa plate containing Mueller-Hinton Broth, bacterial sample and riboflavin at concentrations of 0.8%, 0.4%, 0.2%, 0.1% and 0.5% and 0.025%, negative and positive controls. \u003cstrong\u003eC and D) \u003c/strong\u003ePlate microdilution, decimal serial dilution: at each evaluation time, the tested concentrations were diluted for bacterial counting (CFU)/mL.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-8604527/v1/48040238ff51c01540a3635c.png"},{"id":101434152,"identity":"3212cd83-0d98-479f-8984-319c83d50f1b","added_by":"auto","created_at":"2026-01-29 16:04:00","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":343719,"visible":true,"origin":"","legend":"\u003cp\u003eTime–kill curve – Bactericidal effect of riboflavin at different concentrations, photoactivated by UVA light from a handheld flashlight (CU6 Nitecore®, Guangdong, China), after 15 (T15) and 30 (T30) minutes of exposure against \u003cem\u003eStaphylococcus pseudintermedius\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-8604527/v1/6aa601b58ee71ec79fdcb0f8.png"},{"id":101434157,"identity":"a1b79e34-c5d1-423a-8279-f1d94919ede0","added_by":"auto","created_at":"2026-01-29 16:04:00","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":405295,"visible":true,"origin":"","legend":"\u003cp\u003eTime–kill curve – Bactericidal effect of riboflavin at different concentrations, photoactivated by UVA light from a handheld flashlight (CU6 Nitecore®, Guangdong, China), after 15 (T15) and 30 (T30) minutes of exposure Against \u003cem\u003eStreptococcus canis.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-8604527/v1/e0ed96164d5744d607173112.png"},{"id":101751517,"identity":"682cfc62-c82d-4f36-a2ed-989bdd397890","added_by":"auto","created_at":"2026-02-03 10:20:57","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":458377,"visible":true,"origin":"","legend":"\u003cp\u003eTime–kill curve – Bactericidal effect of riboflavin at different concentrations, photoactivated by UVA light from a handheld flashlight (CU6 Nitecore®, Guangdong, China), after 15 (T15) and 30 (T30) minutes of exposure Against \u003cem\u003eKlebsiella pneumoniae.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-8604527/v1/a92d176edd8e23348527e3e2.png"},{"id":101434154,"identity":"870068da-ebf8-4ce1-aaec-51cb52544013","added_by":"auto","created_at":"2026-01-29 16:04:00","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":415097,"visible":true,"origin":"","legend":"\u003cp\u003eTime–kill curve – Bactericidal effect of riboflavin at different concentrations, photoactivated by UVA light from a handheld flashlight (CU6 Nitecore®, Guangdong, China), after 15 (T15) and 30 (T30) minutes of exposure Against \u003cem\u003ePseudomonas aeruginosa.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-8604527/v1/81aefc6173e30db29b3a67b7.png"},{"id":104186213,"identity":"3a1aaaa2-7677-4558-aae6-9a83a3ced8f2","added_by":"auto","created_at":"2026-03-08 20:24:21","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2294380,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8604527/v1/6f6ebc50-bcad-41e0-92ae-df7cdaf1a315.pdf"},{"id":101752039,"identity":"276f4553-e034-452b-8dcd-8deaeec48e29","added_by":"auto","created_at":"2026-02-03 10:25:01","extension":"jpg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":449248,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Material 1. \u003c/strong\u003eBoxplot illustrating the variation in mean antibacterial activity of riboflavin at different concentrations, photoactivated by ultraviolet A light from a flashlight (CU6 Nitecore®, Guangdong, China), after 15 minutes (T15) and 30 minutes (T30) against \u003cem\u003eStaphylococcus pseudintermedius, Streptococcus canis, Klebsiella pneumoniae \u003c/em\u003eand\u003cem\u003ePseudomonas aeruginosa.\u003c/em\u003e Light exposure times include T0, T2, T8, T16, T32, and T64.\u003c/p\u003e","description":"","filename":"S1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8604527/v1/236da8039c775b7f365e3631.jpg"}],"financialInterests":"No competing interests reported.","formattedTitle":"Efficacy of the combination of flashlight (365 nm) and riboflavin in reducing microorganisms isolated from canine corneas: in vitro study Ultraviolet Flashlight for Riboflavin Photoactivation","fulltext":[{"header":"1. INTRODUCTION","content":"\u003cp\u003eInfectious ulcerative keratitis is a common clinical condition in veterinary ophthalmology This injury is associated with several risk factors, including ocular trauma, lagophthalmos, reduced corneal sensitivity, and ocular surface disorders such as keratoconjunctivitis sicca and eyelid abnormalities. Other complications, such as corneal ulcers, also referred to as melting ulcers or keratomalacia, liquefactive necrosis of the cornea, or acute stromal collagenolysismay also develop [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSeveral bacterial species have been implicated in infectious keratitis in dogs and cats, with \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e, \u003cem\u003eStaphylococcus\u003c/em\u003e spp., and \u003cem\u003eStreptococcus\u003c/em\u003e spp. being the most commonly isolated pathogens [3,\u003c/p\u003e \u003cp\u003e4, 5]. In addition, other microorganisms such as \u003cem\u003eEscherichia coli\u003c/em\u003e, \u003cem\u003eBacillus\u003c/em\u003e spp., \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e, and \u003cem\u003eProteus\u003c/em\u003e spp. have also been associated with this condition [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eGram-positive bacteria, such as \u003cem\u003eStaphylococcus\u003c/em\u003e spp. and \u003cem\u003eStreptococcus\u003c/em\u003e spp., are commonly isolated in canine keratitis and are often associated with less aggressive infections. In contrast, Gram-negative bacteria, including Pseudomonas aeruginosa and Klebsiella spp., are typically implicated in acude infections, posing greater therapeutic challenges. Clinical management generally involves the use of antibiotics, lubricants, and protease inhibitors; however, antimicrobial resistance has emerged as an increasing concern in veterinary ophthalmology [ 4, 6, 8].\u003c/p\u003e \u003cp\u003eIn cases of severe keratomalacia or corneal perforation, surgical techniques such as conjunctival flaps, amniotic membrane transplantation, or synthetic membrane transplantation are employed to provide tectonic support. However, these procedures may result in varying degrees of corneal opacity, depending on the size, location, and depth of the lesion, making the exploration of novel antimicrobial strategies crucial for maintaining the effectiveness of infectious keratitis treatment [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eA non-surgical photodynamic therapy known as Photoactivated Chromophore for Corneal Collagen Crosslinking (PACK-CXL) represents an innovative approach in the treatment of ophthalmological conditions in both humans and animals. This technique combines ultraviolet-A light and vitamin B2 (riboflavin) to enhance the stability of collagen fibers in the cornea and promote the inactivation of corneal pathogens [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. In crosslinking (CXL), riboflavin, when exposed to ultraviolet-A light (UVA) with a wavelength of approximately 365 nanometers, acts as a photosensitizer. This process also results in the formation of covalent bonds between collagen fibers in the cornea, imparting increased biomechanical strength and biochemical stability [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Recently, this technique has found application in veterinary medicine to address cases of keratomalacia [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The interaction of riboflavin with ultraviolet light induces damage to the genetic material and cell membranes of microorganisms, potentially leading to the demise of bacteria, viruses, parasites, and fungi [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Following exposure to UVA (λ\u0026thinsp;=\u0026thinsp;365nm), riboflavin by-products irreversibly penetrate cell membranes, interacting with the nucleic acids of bacteria, causing harm through the action of reactive oxygen species [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHowever, PACK-CXL is a high-cost device with limited accessibility for veterinarians and patients in clinical practice A study investigated the use of an ultraviolet flashlight (365 nm) as a cost-effective alternative for treating keratomalacia in dogs, yielding noteworthy results [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. To date, there are no \u003cem\u003ein vitro\u003c/em\u003e published data addressing the efficacy of the UVA flashlight (Nitecore Chameleon CU6\u0026reg;, Guangdong, China) as an alternative to the PACK-CXL device in canine ulcerative keratitis. Therefore, the aim of this study was to assess the \u003cem\u003ein vitro\u003c/em\u003e antibacterial efficacy of the UVA flashlight (365 nm) on bacterial isolates obtained from ulcerative keratitis in dogs.\u003c/p\u003e"},{"header":"2. MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Pathogen Selection\u003c/h2\u003e \u003cp\u003eFour distinct bacterial strains were selected from canine ocular samples obtained from ulcerative keratitis. These strains included \u003cem\u003eStaphylococcus pseudintermedius, Streptococcus canis, Pseudomonas aeruginosa\u003c/em\u003e and \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e. They were previously identified using microbiological and biochemical techniques and confirmed by MALDI-TOF (Matrix-Assisted Laser Desorption/Ionization\u0026ndash;Time of Flight).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Riboflavin Preparation and Dilution\u003c/h2\u003e \u003cp\u003eRiboflavin (R9504-25G, Sigma-Aldrich) was diluted in Mueller-Hinton Broth (MHB) at concentrations of 0.8%, 0.4%, 0.2%, 0.1%, 0.05%, and 0.025%. Initially, 0.8 grams of riboflavin were weighed and dissolved in 100 mL of MHB, resulting in an initial solution at a concentration of 0.8%. Subsequently, a 2 mL aliquot was withdrawn. Next, 1 mL of the initial solution was transferred to a Falcon tube containing 1 mL of diluent, and thereafter, serial double dilutions were performed up to 0.025%, ensuring that all dilutions maintained a volume of 1 mL. Two tubes with MHB were prepared, one for positive control and one for the negative control. Finally, 10 \u0026micro;L was removed from all samples, that is, from the concentrations and controls. Throughout the procedure, riboflavin was protected from exposure to external light (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Bacterial Suspension in Riboflavin Solution\u003c/h2\u003e \u003cp\u003eEach cryopreserved bacterial isolate was inoculated into Brain Heart Infusion Broth (BHI Broth) and incubated under aerobic conditions at 37\u0026deg;C for 18 hours, followed by incubation on BHI Agar under the same conditions. The bacterial suspension was standardized to 0.08\u0026ndash;0.12 absorbance, equivalent to 0.5 on the MacFarland scale, by spectrophotometry (Eppendorf BioPhotometer Plus, OD 600) in a 0.9% sodium chloride solution at pH 7.3. After preparation, 10 \u0026micro;L of the bacterial suspension was dispensed into the different riboflavin concentrations and the positive control prior to exposure to the flashlight UVA light (Figura 1A).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 UVA Irradiation and Exposure Time\u003c/h2\u003e \u003cp\u003eSubsequently, 300 \u0026micro;L of each concentration (%riboflavin\u0026thinsp;+\u0026thinsp;bact\u0026eacute;ria) was transferred to an Elisa plate (86 x 128mm) for exposure to UVA light. The flashlight used was designed to illuminate only 4 wells at a time. To achieve this precision, we divided the sample into two separate ELISA plates: the first plate contained higher concentrations (0.8%, 0.4%, 0.2%, and 0.1%), while the second plate contained lower concentrations (0.05%, 0.025%, positive control, and negative control. This approach allowed the flashlight to directly target the four wells of interest in each plate, preventing light from overlapping onto adjacent wells. This ensured precise and controlled light exposure. The light was directed from a distance of 3 cm from the surface, mimicking light emission on the cornea of dogs with infectious ulcerative keratitis. The entire process was carried out under aseptic conditions and protected from interference by other light sources (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eFor the UVA irradiation assessment, a CU6 Nitecore\u0026reg; flashlight (Guangdong, China) with a 365 nm wavelength was utilized. The light intensity was measured at 19.1 mW/cm\u0026sup2; using a photometer. The flashlight was shone on the ELISA plate from a distance of 3 cm for two durations: 15 and 30 minutes. The administered energy dose was calculated to be 5.763 mW/cm\u0026sup2;, based on the formula (intensity x exposure time), considering the distance of 3 cm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Time-Kill Curve\u003c/h2\u003e \u003cp\u003eThe time-kill curve was conducted following the protocol published by Blondeau et al.\u003csup\u003e29\u003c/sup\u003e with minor modifications. The ELISA plates were sealed, protected from light, and incubated at 36\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C, being removed only at the designated times to withdraw an aliquot for performing the serial dilution (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Microbial growth was assessed through plate microdilution at the following time points (hours): pre-exposure (T0) and post-exposure to UVA light (2 (T2), 8 (T8), 16 (T16), 32 (T32), and 64 (T64) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). For each analyzed period, 20 \u0026micro;L from the initial column subjected to light (riboflavin and bacteria concentrations, positive and negative controls) was transferred to another Elisa plate filled with 180 \u0026micro;L of 0.9% saline, conducting decimal serial dilution (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eSubsequently, a 5 \u0026micro;L aliquot from each well of the ELISA plate was withdrawn and transferred to a Petri dish containing Mueller-Hinton agar (MHA), in triplicate The MHA Petri plates were incubated and subsequently used for quantification (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). The incubation time for each bacterium for plate microdilution and, consequently, total bacterial counting was determined through a pilot study. \u003cem\u003eP. aeruginosa\u003c/em\u003e was incubated for 9 hours, \u003cem\u003eS. pseudointermedius\u003c/em\u003e and \u003cem\u003eK. pneumoniae\u003c/em\u003e for 16 hours, and \u003cem\u003eStreptococcus canis\u003c/em\u003e for 24 hours, the latter under anaerobic conditions.\u003c/p\u003e \u003cp\u003eThe counting considered microbial growth between 5 and 30 colonies and was used a colony counter with magnifying glass, with the value multiplied by the respective dilution and the correction factor (200, i.e., 1000 \u0026micro;L/5 \u0026micro;L). The counting result was expressed in Colony Forming Units (CFU)/mL. The entire process was carried out for both the 15- and 30-minute exposure periods and for each bacterial species.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Statistical Analysis\u003c/h2\u003e \u003cp\u003eStatistical analyses were performed using the R 4.1.2 program [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Means were log-transformed, and normality and homogeneity patterns were assessed using Shapiro-Wilk and Levene tests, respectively. Based on the obtained data, the Kruskall-Wallis test was conducted, followed by the Dunn test corrected by the Bonferroni test. Values of p less than 0.05 were considered significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. RESULTS","content":"\u003cp\u003eFollowing 15 minutes of UVA exposure, \u003cem\u003eStaphylococcus pseudintermedius\u003c/em\u003e exhibited reduced growth at riboflavin concentrations between 0.05% and 0.4%, with 0.2% and 0.4% causing complete bacterial death from 32 hours post-exposure. After 30 minutes of exposure, a 0.4% riboflavin concentration produced a similar bactericidal effect with 64 hours, while 0.05% also resulted in complete bacterial killing within 32 hours. The positive control also showed statistically significant differences (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) with higher bacterial growth curves, especially at riboflavin concentrations of 0.025%, 0.05%, 0.2%, and 0.4% at the T2/15UVA and T64/30UVA time points, respectively.\u003c/p\u003e \u003cp\u003eTherefore, it was observed that, after UVA exposure, there was a reduction in the population of microorganisms, with a pronounced decrease at concentrations of 0.4%, 0.2%, and 0.1%, starting 8 hours after exposure. This resulted in a decrease of up to three logs in the count (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and and Supplementary Material 1).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn the analysis of \u003cem\u003eStreptococcus canis\u003c/em\u003e, the concentration of 0.4% showed a decrease in bacterial count at 32 hours (for 15UVA) and at 16 hours (for 30UVA), both observations significantly differing from the positive control group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). However, although not pronounced, riboflavin at a concentration of 0.4% appeared to exert a stronger effect against \u003cem\u003eStreptococcus canis\u003c/em\u003e, particularly after 15 minutes of flashlight exposure. Nevertheless, this effect was insufficient to achieve complete microbial killing, resulting only in a reduction of viable cell counts. No other noticeable growth patterns were observed during either the 15- or 30-minute exposure periods (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and Supplementary Material 1).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eRegarding \u003cem\u003eK. pneumoniae\u003c/em\u003e, there was no significant variation in its overall growth. However, the growth curve analysis revealed a reduction in bacterial counts at all tested concentrations, particularly at concentrations of 0.025% and 0.2% at the 16-hour time point (30UVA). It is worth noting that only the concentration of 0.2% showed a statistically significant difference compared to the positive control (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). On the other hand, at concentrations of 0.8% and 0.4%, there was an increase in bacterial growth after 2 and 16 hours (15UVA), although this increase did not reach statistical significance compared to the positive control (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e and Supplementary material 1).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn the analyses conducted with \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e, bacterial counts at different concentrations remained high over the 64 hours, with a slight reduction observed at concentrations of 0.025% between 2 to 16 hours (15UVA) and 0.5% at 32 hours (15UVA), which were not statistically significant. In treatments with concentrations of 0.8% and 0.4%, higher counts than the positive control were observed at the early time points of 2 and 8 hours (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e and Supplementary material 1).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eConcerning the exposure periods, the results indicated no statistically significant difference between the 15UVA and 30UVA exposure periods for the four tested bacterial strains (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e"},{"header":"4. DISCUSSION","content":"\u003cp\u003eThe results highlighted distinctions among the assessed microorganisms isolated from ulcerative keratitis in dogs. Gram-positive bacteria (\u003cem\u003eS. pseudintermedius and S. canis\u003c/em\u003e) exhibited greater sensitivity compared to Gram-negative bacteria (\u003cem\u003eP. aeruginosa and K. pneumoniae\u003c/em\u003e) to the light emission effect from the UVA lantern (Nitecore Chameleon CU6\u0026reg;, Guangdong, China) in combination with riboflavin.\u003c/p\u003e \u003cp\u003eIn veterinary ophthalmology, riboflavin at a 0.1% concentration is routinely used in PACK-CXL protocols, which have been shown not only to exert significant antimicrobial effects against various pathogens but also to induce beneficial structural changes in the cornea, such as increased stromal stiffness and enhanced resistance to enzymatic degradation [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. In the present study, in vivo effects (such as potential benefits to the c\u0026oacute;rnea) were not assessed. The results demonstrated that a 0.4% riboflavin concentration was the most effective alternative against Gram-positive microorganisms. These findings suggest that the handheld flashlight is capable of photoactivating riboflavin; however, its effects in the cornea may differ, considering factors such as tissue characteristics and light penetration capacity.\u003c/p\u003e \u003cp\u003eThe effect of riboflavin was evaluated at different concentrations (0.025%, 0.05%, 0.1%, 0.2%, 0.4%, and 0.8%). It was observed that the sensitivity of bacterial strains to UVA radiation varied considerably depending on the concentration of the photosensitizer. \u003cem\u003eP. aeruginosa\u003c/em\u003e showed a slight reduction in bacterial count at concentrations of 0.025% between 2 and 16 hours and at 0.5% in 32 hours (15UVA). However, at concentrations of 0.8% and 0.4%, higher counts than the positive control were observed in the early phases of 2 and 8 hours. Similarly, high bacterial counts were observed at a 0.8% riboflavin concentration for \u003cem\u003eS. pseudintermedius\u003c/em\u003e and \u003cem\u003eS. canis\u003c/em\u003e. Thus, that higher concentrations of riboflavin may inhibit the penetration of UVA light into the solution, compromising its activation and, consequently, its action against all microorganisms [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cem\u003eIn vitro\u003c/em\u003e studies have unveiled the favorable impacts of crosslinking on ulcerative keratitis in dogs. Three crosslinking protocols (UVA 3 mW/cm\u0026sup2; for 60 minutes, UVA 3 mW/cm\u0026sup2; for 30 minutes, and UVA 30 mW/cm\u0026sup2; for 3 minutes) demonstrated growth inhibition for \u003cem\u003eStreptococcus spp\u003c/em\u003e., \u003cem\u003eStaphylococcus spp\u003c/em\u003e., \u003cem\u003eE. coli\u003c/em\u003e, and \u003cem\u003eS. canis\u003c/em\u003e. However, in the case of \u003cem\u003eP. aeruginosa\u003c/em\u003e and \u003cem\u003eS. pseudintermedius\u003c/em\u003e, a response was observed only after prolonged exposure to 3 mW/cm\u0026sup2; for 60 minutes [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. In human studies, this approach exhibited significant efficacy against S. \u003cem\u003eaureus\u003c/em\u003e and \u003cem\u003eS. epidermidis\u003c/em\u003e but showed reduced effectiveness against \u003cem\u003eP. aeruginosa\u003c/em\u003e, suggesting a higher efficiency against Gram-positive microorganisms [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Consequently, these findings align with the outcomes presented in the current study, indicating that the action of the UVA lantern yields similar effects to those observed with PACK-CXL light.\u003c/p\u003e \u003cp\u003eHowever, other studies in dogs and humans have noted that no differences in susceptibility to PACK-CXL were found among bacterial genera such as \u003cem\u003eStaphylococcus spp\u003c/em\u003e, \u003cem\u003eStreptococcus spp\u003c/em\u003e, \u003cem\u003eP. aeruginosa\u003c/em\u003e, and \u003cem\u003ePasteurella\u003c/em\u003e, as well as \u003cem\u003eP. aeruginosa\u003c/em\u003e, \u003cem\u003eS. aureus\u003c/em\u003e, and \u003cem\u003eS. epidermidis\u003c/em\u003e. In these cases, all these bacterial agents exhibited equivalent sensitivity to their reactions to the treatments tested in their respective studies [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this study, strains of \u003cem\u003eStreptococcus canis\u003c/em\u003e and \u003cem\u003eStaphylococcus pseudintermedius\u003c/em\u003e were selected to represent Gram-positive bacteria. Notably, \u003cem\u003eS. canis\u003c/em\u003e showed a marked reduction in bacterial counts after 16 hours following 15 minutes of UVA exposure, whereas \u003cem\u003eS. pseudintermedius\u003c/em\u003e exhibited greater susceptibility after 30 minutes of exposure. These species, along with other Gram-positive bacteria (such as \u003cem\u003eStaphylococcus aureus\u003c/em\u003e, \u003cem\u003eStaphylococcus epidermidis\u003c/em\u003e, and beta-hemolytic \u003cem\u003eStreptococcus\u003c/em\u003e spp.), are frequently implicated in cases of ulcerative keratitis and keratomalacia. Therefore, the handheld flashlight may also exert beneficial effects against these additional pathogens [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOn the other hand, \u003cem\u003ePseudomonas spp\u003c/em\u003e. stands out as the primary infectious agent identified in corneal melting samples [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. \u003cem\u003eP. aeruginosa\u003c/em\u003e secretes a spectrum of proteases, including alkaline protease, elastase A and B, protease IV, and \u003cem\u003eP. aeruginosa\u003c/em\u003e small protease, among others, which are recognized as significant virulence factors contributing to the pathogenesis of ulcerative keratitis. These proteases play a crucial role in degrading essential host defense proteins and collagen fibers, leading to epithelial loss, corneal erosion, and keratomalacia [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. In this study, \u003cem\u003eP. aeruginosa\u003c/em\u003e demonstrated resistance to the proposed adjuvant therapy. Consequently, the combined action of riboflavin (0.025\u0026ndash;0.8%) and lantern exposure (15 and 30 minutes) proved inefficacious. This suggests that the effect of adjuvant therapy on each bacterium may be attributed to their distinct virulence factors.\u003c/p\u003e \u003cp\u003eGram-negative bacteria commonly found in corneal ulcers include K. \u003cem\u003epneumoniae\u003c/em\u003e and \u003cem\u003eEscherichia coli\u003c/em\u003e, recognized as highly virulent agents with the potential to induce endophthalmitis [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. In our study of \u003cem\u003eK. pneumoniae\u003c/em\u003e, minimal reduction in bacterial counts was observed across the tested concentrations compared to the positive control, suggesting that riboflavin may have had an impact on bacterial growth.\u003c/p\u003e \u003cp\u003eImportantly, there is a lack of studies on corneal cross-linking evaluating the response of \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e to photodynamic therapy in veterinary ophthalmology. However, human studies have indicated that \u003cem\u003eKlebsiella oxytoca\u003c/em\u003e demonstrates significantly lower sensitivity to PACK-CXL compared to other investigated strains, such as \u003cem\u003eS. aureus, P. aeruginosa e Bacillus subtilis\u003c/em\u003e [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Additionally, the virulence of \u003cem\u003eK. pneumoniae\u003c/em\u003e has been associated with the production of cell wall-associated factors and capsules [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. This underscores the need for further research specific to veterinary ophthalmology to better understand the effectiveness of photodynamic therapy in addressing infections caused by \u003cem\u003eK. pneumoniae\u003c/em\u003e and similar pathogens.\u003c/p\u003e \u003cp\u003eSeveral factors can play a crucial role in the varied response of different bacterial species to UVA light and riboflavina [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Among these factors, diversity in metabolism, sensitivity to oxidative stress, duration of the cell cycle, cell wall structure, and others stand out [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. In Gram-negative bacteria, the membrane structures are more complex compared to the peptidoglycan layer found in Gram-positive bacteria. This complexity may impede the penetration of riboflavin by-products into the bacterial cell [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn a study involving Gram-positive bacteria, \u003cem\u003eS. epidermidis\u003c/em\u003e, and \u003cem\u003eS. aureus\u003c/em\u003e, it was observed that the survival rate of \u003cem\u003eS. epidermidis\u003c/em\u003e was significantly lower than that of \u003cem\u003eS. aureus\u003c/em\u003e when subjected to the same UVA light parameters. This difference may be associated with the formation of a bacterial biofilm, acting as a barrier preventing the penetration of the photosensitizer and, consequently, the activation of riboflavina [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFurthermore, microbial sensitivity to oxidative stress induced by riboflavin and UV light is not influenced by acquired antibiotic resistance, making this approach a viable alternative in such cases. Additionally, the flashlight-based methodology has not yet been evaluated in vivo or in combination with other commonly used therapies that contribute positively to clinical outcomes, such as antibiotics, lubricants, and metalloproteinase inhibitors [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. In \u003cem\u003ein vivo\u003c/em\u003e studies using PACK-CXL for the treatment of ulcerative keratitis in dogs and cats, several protocols have been documented. These include UVA irradiance of 3 mW/cm\u0026sup2; for 30 minutes and an accelerated protocol with UVA irradiance of 30 mW/cm\u0026sup2; administered for 3 minutes [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. It is important to note that the flashlights used in the study do not allow for power adjustment, which limits their application.\u003c/p\u003e \u003cp\u003eIncreasing the exposure time to UVA light from 15 to 30 minutes, with the same power setting (light intensity), did not provide a statistically significant difference in the results of this study. A similar finding was reported, where there was no significant difference in the areas of bacterial growth inhibition between the 3-minute and 30-minute UVA/riboflavin protocols [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. This result diverges from findings in the literature regarding the use of PACK-CXL, which suggests that longer exposure periods to UVA light are more effective in eradicating bact\u0026eacute;ria [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Therefore, understanding the differential responses among bacterial species and the available therapeutic alternatives is crucial for optimizing photodynamic therapy approaches, particularly in veterinary ophthalmology, where treatment efficacy can be influenced by the specific characteristics of both the infectious pathogens and the host.\u003c/p\u003e"},{"header":"5. CONCLUSION","content":"\u003cp\u003eThe results indicate that UVA radiation from the handheld flashlight (Nitecore Chameleon CU6\u0026reg;, Guangdong, China) combined with riboflavin can effectively eliminate \u003cem\u003eS. pseudintermedius\u003c/em\u003e counts, particularly at a 0.4% concentration, and can reduce and partially inhibit \u003cem\u003eS. canis\u003c/em\u003e (0,4%) \u003cem\u003ee Klebsiella pneumoniae\u003c/em\u003e (0,2%). However, the protocol was not effective against \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003e6. FUNDING\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was carried out with the support of the Coordination for the Improvement of Higher Education Personnel - Brazil (CAPES) - Financing Code 001.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e7. CONFLICT OF INTEREST\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eO\u0026rsquo;Neill DG, Lee MM, Brodbelt DC, Church DB, Sanchez RF. Corneal ulcerative disease in dogs under primary veterinary care in England: epidemiology and clinical management. Canine Genet Epidemiol. 2017;4(1):1\u0026ndash;12.\u003c/li\u003e\n \u003cli\u003eMartin de Bustamante MG, Good KL, Leonard BC, Hollingsworth SR, Edwards SG, Knickelbein KE, et al. Medical management of deep ulcerative keratitis in cats: 13 cases. J Feline Med Surg. 2019;21(4):387\u0026ndash;93.\u003c/li\u003e\n \u003cli\u003eEkapopphan D, Srisutthakarn A, Moonarmart W, Buddhirongawatr R, Bangphoomi N. Identification and antimicrobial susceptibility of microorganisms isolated from severe corneal ulcers of dogs in Thailand. J Vet Med Sci. 2018;80(8):1259\u0026ndash;65.\u003c/li\u003e\n \u003cli\u003eHewitt JS, Allbaugh RA, Kenne DE, Sebbag L. Prevalence and Antibiotic Susceptibility of Bacterial Isolates From Dogs With Ulcerative Keratitis in Midwestern United States. Front Vet Sci. 2020;7(November):1\u0026ndash;10.\u003c/li\u003e\n \u003cli\u003eVerdenius CY, Broens EM, Slenter IJM, Djajadiningrat-Laanen SC. Corneal stromal ulcerations in a referral population of dogs and cats in the Netherlands (2012\u0026ndash;2019): Bacterial isolates and antibiotic resistance. Vet Ophthalmol. 2024;27(1):7\u0026ndash;16.\u003c/li\u003e\n \u003cli\u003ePark J, Kim D, Kwon M, Kwon H, Yong J, Yoon H, et al. Bacterial isolates and antibiotic sensitivity in canine bacterial keratitis in Korea. Vet Ophthalmol. 2024;(May):1\u0026ndash;12.\u003c/li\u003e\n \u003cli\u003eKyung SM, Lee JH, Lee ES, Xiang XR, Yoo HS. Emergence and genomic chion of Proteus mirabilis harboring blaNDM-1 in Korean companion dogs. Vet Res [Internet]. 2024;55(1):50. Available from: https://doi.org/10.1186/s13567-024-01306-w\u003c/li\u003e\n \u003cli\u003eMcKeever JM, Ward DA, Hendrix DVH. Comparison of antimicrobial resistance patterns in dogs with bacterial keratitis presented to a veterinary teaching hospital over two multi-year time periods (1993\u0026ndash;2003 and 2013\u0026ndash;2019) in the Southeastern United States. Vet Ophthalmol. 2021;24(6):653\u0026ndash;8.\u003c/li\u003e\n \u003cli\u003eSoontornvipart K, Tuntivanich N, Kecov\u0026aacute; H, Rau\u0026scaron;er P. Conjunctival pedicle graft in dogs and cats: A retrospective study of 88 cases. Acta Vet Brno. 2003;72(1):63\u0026ndash;9.\u003c/li\u003e\n \u003cli\u003eDorbandt DM, Moore PA, Myrna KE. Outcome of conjunctival flap repair for corneal defects with and without an acellular submucosa implant in 73 canine eyes. Vet Ophthalmol. 2015;18(2):116\u0026ndash;22.\u003c/li\u003e\n \u003cli\u003eCosta D, Leiva M, Sanz F, Espejo V, Esteban J, Vergara J, et al. A multicenter retrospective study on cryopreserved amniotic membrane transplantation for the treatment of complicated corneal ulcers in the dog. Vet Ophthalmol. 2019;22(5):695\u0026ndash;702.\u003c/li\u003e\n \u003cli\u003ePot SA, Gallh\u0026ouml;fer NS, Matheis FL, Voelter-Ratson K, Hafezi F, Spiess BM. Corneal collagen cross-linking as treatment for infectious and noninfectious corneal melting in cats and dogs: Results of a prospective, nonrandomized, controlled trial. Vet Ophthalmol. 2014;17(4):250\u0026ndash;60.\u003c/li\u003e\n \u003cli\u003eSpiess BM, Pot SA, Florin M, Hafezi F. Corneal collagen cross-linking (CXL) for the treatment of melting keratitis in cats and dogs: A pilot study. Vet Ophthalmol. 2014;17(1):1\u0026ndash;11.\u003c/li\u003e\n \u003cli\u003eMartins SAR, Combs JC, Noguera G, Camacho W, Wittmann P, Walther R, et al. Antimicrobial efficacy of riboflavin/UVA combination (365 nm) in vitro for bacterial and fungal isolates: A potential new treatment for infectious keratitis. Investig Ophthalmol Vis Sci. 2008;49(8):3402\u0026ndash;8.\u003c/li\u003e\n \u003cli\u003eAlio JL, Abbouda A, Valle DD, del Castillo JMB, Fernandez JAG. Corneal cross linking and infectious keratitis: A systematic review with a meta-analysis of reported cases. J Ophthalmic Inflamm Infect. 2013;3(1):1\u0026ndash;7.\u003c/li\u003e\n \u003cli\u003eFamose F. Evaluation of accelerated collagen cross-linking for the treatment of melting keratitis in eight dogs. Vet Ophthalmol. 2014;17(5):358\u0026ndash;67.\u003c/li\u003e\n \u003cli\u003eFamose F. Evaluation of accelerated collagen cross-linking for the treatment of melting keratitis in ten cats. Vet Ophthalmol. 2015;18(2):95\u0026ndash;104.\u003c/li\u003e\n \u003cli\u003eZibura AE, Cullen MA, Rutledge H, Lassalle L, Salmon JH, Gilger BC, et al. Optimizing corneal riboflavin administration in ex vivo horse, dog, rabbit, and pig samples for use in corneal collagen cross-linking. Vet Ophthalmol. 2020;23(5):840\u0026ndash;8.\u003c/li\u003e\n \u003cli\u003eSuter A, Schmitt S, H\u0026uuml;bschke E, Kowalska M, Hartnack S, Pot S. The bactericidal effect of two photoactivated chromophore for keratitis-corneal crosslinking protocols (standard vs. accelerated) on bacterial isolates associated with infectious keratitis in companion animals. BMC Vet Res [Internet]. 2022;18(1):1\u0026ndash;10. Available from: https://doi.org/10.1186/s12917-022-03397-z\u003c/li\u003e\n \u003cli\u003eSharma A, Sharma R, Chander J, Nirankari VS. In vitro antimicrobial efficacy of riboflavin, ultraviolet-A radiation, and combined riboflavin/ultraviolet-A radiation on ocular pathogens. Taiwan J Ophthalmol [Internet]. 2023 Jan;13(1):21\u0026ndash;7. Available from: https://journals.lww.com/10.4103/tjo.tjo_28_21\u003c/li\u003e\n \u003cli\u003eMundt JM, Rouse L, Van Den Bossche J, Goodrich RP. Chemical and Biological Mechanisms of Pathogen Reduction Technologies. Photochem Photobiol. 2014;90(5):957\u0026ndash;64.\u003c/li\u003e\n \u003cli\u003eKumar V, Lockerbie O, Keil SD, Ruane PH, Platz MS, Martin CB, et al. Riboflavin and UV-Light Based Pathogen Reduction: Extent and Consequence of DNA Damage at the Molecular Level. Photochem Photobiol. 2004;80(1):15.\u003c/li\u003e\n \u003cli\u003eTabibian D, Richoz O, Hafezi F. PACK-CXL: Corneal cross-linking for treatment of infectious keratitis. J Ophthalmic Vis Res. 2015;10(1):77\u0026ndash;80.\u003c/li\u003e\n \u003cli\u003eLembo JA, Sapienza J, Wolfer J, Allgoewer I. A rapid and cost-effective method for corneal cross-linking for treatment of canine keratomalacia. In: Annual Scientific Meeting Of The American College Of Veterinary Ophthalmologists; September 2018; Minneapolis, Minnesota.\u003c/li\u003e\n \u003cli\u003eR Core Team. R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria; 2020. Available from: https://www.R-project.org/.\u003c/li\u003e\n \u003cli\u003eKowalska ME, Pot SA, Hartnack S. Photoactivated Chromophore for Keratitis-Corneal Cross-linking (PACK-CXL)\u0026mdash;A Scoping Review Based on Preclinical Studies. Transl Vis Sci Technol. 2024;13(7):11\u0026ndash;6.\u003c/li\u003e\n \u003cli\u003eShukla AK, Kowalska ME, Arteaga K, Crasta M, Dixon C, Famose F, et al. Evaluation of photoactivated chromophore for keratitis\u0026ndash;corneal cross-linking (PACK-CXL) in feline infectious keratitis\u0026mdash;patient demographics, treatment protocols, risk factors, and treatment outcome: a retrospective study. Vet Ophthalmol. 2025;28(2):330\u0026ndash;40.\u003c/li\u003e\n \u003cli\u003eB\u0026auml;ckman A, Makdoumi K, Mortensen J, Crafoord S. The efficiency of cross-linking methods in eradication of bacteria is influenced by the riboflavin concentration and the irradiation time of ultraviolet light. Acta Ophthalmol. 2014;92(7):656\u0026ndash;61.\u003c/li\u003e\n \u003cli\u003eLarge TP, Mack S, Villiers E, Oliver J. In vitro susceptibility of canine corneal bacterial pathogens to three cross-linking protocols. Vet Ophthalmol. 2023;26(S1):134\u0026ndash;42.\u003c/li\u003e\n \u003cli\u003eMakdoumi K, B\u0026auml;ckman A. Photodynamic UVA-riboflavin bacterial elimination in antibiotic-resistant bacteria. Clin Exp Ophthalmol. 2016;44(7):582\u0026ndash;6.\u003c/li\u003e\n \u003cli\u003eMakdoumi K, Mortensen J, Crafoord S. Infectious keratitis treated with corneal crosslinking. Cornea. 2010;29(12):1353\u0026ndash;8.\u003c/li\u003e\n \u003cli\u003eTolar EL, Hendrix DVH, Rohrbach BW, Plummer CE, Brooks DE, Gelatt KN. Evaluation of clinical characteristics and bacterial isolates in dogs with bacterial keratitis: 97 Cases (1993-2003). J Am Vet Med Assoc. 2006;228(1):80\u0026ndash;5.\u003c/li\u003e\n \u003cli\u003eWang Z, Guo L, Li J, Li J, Cui L, Dong J, et al. Antibiotic resistance, biofilm formation, and virulence factors of isolates of staphylococcus pseudintermedius from healthy dogs and dogs with keratitis. Front Vet Sci. 2022;9.\u003c/li\u003e\n \u003cli\u003eTsvetanova A, Powell RM, Tsvetanov KA, Smith KM, Gould DJ. Melting corneal ulcers (keratomalacia) in dogs: A 5-year clinical and microbiological study (2014\u0026ndash;2018). Vet Ophthalmol. 2021;24(3):265\u0026ndash;78.\u003c/li\u003e\n \u003cli\u003eHobden JA. Pseudomonas aeruginosa proteases and corneal virulence. DNA Cell Biol. 2002;21(5):391\u0026ndash;6.\u003c/li\u003e\n \u003cli\u003eO\u0026rsquo;callaghan R, Caballero A, Tang A, Bierdeman M. Pseudomonas aeruginosa keratitis: Protease iv and pasp as corneal virulence mediators. Microorganisms. 2019;7(9).\u003c/li\u003e\n \u003cli\u003eChee SP, Jap A. Endogenous endophthalmitis. Curr Opin Ophthalmol [Internet]. 2001 Dec;12(6):464\u0026ndash;70. Available from: http://journals.lww.com/00055735-200112000-00012\u003c/li\u003e\n \u003cli\u003eKling S, Hufschmid FS, Torres-Netto EA, Randleman JB, Willcox M, Zbinden R, et al. High Fluence Increases the Antibacterial Efficacy of PACK Cross-Linking. Cornea. 2020;39(8):1020\u0026ndash;6.\u003c/li\u003e\n \u003cli\u003ePomakova DK, Hsiao CB, Beanan JM, Olson R, MacDonald U, Keynan Y, et al. Clinical and phenotypic differences between classic and hypervirulent Klebsiella pneumonia: An emerging and under-recognized pathogenic variant. Eur J Clin Microbiol Infect Dis. 2012;31(6):981\u0026ndash;9.\u003c/li\u003e\n \u003cli\u003eHamblin MR, Hasan T. Enacting strategies for graduate employability: A look at multiple issues and disciplines. Photochem Photobiol [Internet]. 2004;3(5):436\u0026ndash;50. Available from: http://pubs.rsc.org/en/Content/ArticleHTML/2004/PP/B311900A%5Cnhttp://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=3071049\u0026amp;tool=pmcentrez\u0026amp;rendertype=abstract\u003c/li\u003e\n \u003cli\u003eShen J, Liang Q, Su G, Zhang Y, Wang Z, Liang H, et al. Effect of Ultraviolet Light Irradiation Combined with Riboflavin on Different Bacterial Pathogens from Ocular Surface Infection. J Biophys. 2017;2017.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"corneal cross-linking, bacterial keratitis, corneal ulcer keratomalacia, infectious keratitis","lastPublishedDoi":"10.21203/rs.3.rs-8604527/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8604527/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe photoactivation of riboflavin induces genetic and structural damage to microbial cell membranes, potentially leading to the death of bacteria, viruses, and fungi. However, the conventional approach requires the use of a Photoactivated Chromophore for Corneal Collagen Crosslinking (PACK-CXL), a high-cost device with limited availability in clinical practice. As a low-cost alternative, this study evaluated photoactivation using a portable ultraviolet flashlight (365 nm) against isolates obtained from cases of canine ulcerative keratitis. Riboflavin at different concentrations (0.025, 0.05, 0.1, 0.2, 0.4, and 0.8%) were tested and exposed to UVA radiation (365 nm) at a distance of 3 cm for 15 or 30 minutes, followed by analysis using the time-kill curve method. Results showed that the flashlight was able to activate riboflavin and completely inhibit the growth of \u003cem\u003eStaphylococcus pseudintermedius\u003c/em\u003e at a concentration of 0.4% for both exposure periods, and also caused effects at 0.2% after 15 minutes and at 0.05% after 30 minutes of UVA exposure. For \u003cem\u003eStreptococcus canis\u003c/em\u003e, significant reductions were observed at 0.4% after 15 minutes of exposure, although these effects were insufficient to kill the bacterium completely. \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e showed reduced growth at 0.2% (after 30 minutes), whereas \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e did not respond to any tested concentrations. The comparative analysis between exposure times (15 vs. 30 min) revealed no significant differences (p \u0026gt; 0.05). The handheld ultraviolet flashlight (365 nm) was able to photoactivate riboflavin, and this low-cost approach may represent a potential alternative in the absence of conventional PACK-CXL therapy, although further studies are warranted.\u003c/p\u003e","manuscriptTitle":"Efficacy of the combination of flashlight (365 nm) and riboflavin in reducing microorganisms isolated from canine corneas: in vitro study Ultraviolet Flashlight for Riboflavin Photoactivation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-29 16:03:55","doi":"10.21203/rs.3.rs-8604527/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ee96759e-53d0-4583-bc9d-3c6d33b9fbfb","owner":[],"postedDate":"January 29th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-03-08T20:23:59+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-29 16:03:55","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8604527","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8604527","identity":"rs-8604527","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}