Abstract: Introduction. The production of β-lactamases by Gram-negative bacteria is among the most important factors of resistance to antibiotics, which has contributed to therapeutic failures that currently threaten human and veterinary medicine worldwide. Antimicrobial photodynamic therapy and antimicrobial blue light have a broad-spectrum antibacterial activity against multidrug-resistant and hypervirulent pathogens. Objective. To investigate the ability of antimicrobial blue light to inhibit the hydrolytic activity of clinically relevant β-lactamase enzymes (i.e., KPC, IMP, OXA, CTX-M, and SHV), with further comparison of the inhibitory effects of antimicrobial blue light with methylene blue-mediated antimicrobial photodynamic therapy. Methods. Blue LED light (λ = 410 ± 10 nm) alone or red LED light (λ = 660 ± 10 nm) in combination with methylene blue were used to inactivate, in vitro, suspensions of Klebsiella pneumoniae strains producing clinically important β-lactamase enzymes assigned to the A, B and D Ambler molecular classes. Furthermore, β-lactamase activity inhibition mediated by antimicrobial blue light and methylene blue-mediated antimicrobial photodynamic therapy was measured by using the chromogenic β-lactam substrate nitrocefin. Results. β-lactamase activities were effectively inactivated by both visible light-based approaches. In this regard, antimicrobial blue light and methylene blue-antimicrobial photodynamic therapy led to a significant reduction in the hydrolysis of nitrocefin (81–98 %). Conclusion. Sublethal doses of antimicrobial blue light and methylene blue-mediated antimicrobial photodynamic therapy are equally effective to inhibit clinically significant β-lactamases, including extended-spectrum β-lactamases and carbapenemases.

Palavras-Chave: antimicrobial agents; methylene blue; antibiotics; lactams; inactivation; enzymes

ANJOS, CAROLINA dos; SELLERA, FABIO P.; RIBEIRO, MARTHA S.; BAPTISTA, MAURICIO S.; POGLIANI, FABIO C.; LINCOPAN, NILTON; SABINO, CAETANO P. Antimicrobial blue light and photodynamic therapy inhibit clinically relevant β-lactamases with extended-spectrum (ESBL) and carbapenemase activity. Photodiagnosis and Photodynamic Therapy, v. 32, p. 1-4, 2020. DOI: 10.1016/j.pdpdt.2020.102086. Disponível em: http://repositorio.ipen.br/handle/123456789/31811. Acesso em: $DATA.

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Abstract: Background Rumenostomy is a useful procedure commonly performed in cattle for medical treatment of domestic ruminants with forestomach diseases. Methylene blue (MB)-mediated antimicrobial photodynamic therapy (APDT) has been broadly investigated to treat infected wounds. Aim The aim of the study was to evaluate the effectiveness of MB-mediated APDT (MB-APDT) combined with chlorhexidine and zinc oxide ointment on wound healing process after rumenostomy. Methods Fourteen Nelore bulls were subjected to rumenostomy procedure. Animals were randomly divided into MB-APDT (MB associated with a red diode laser performed immediately after surgery and repeated on days 3, 5, 7 and 10) and control groups. Daily care included topical cleaning with chlorhexidine 2% followed by topical zinc oxide ointment. Animals were followed-up until the 28th day. Results Wounds presented a better post-surgical profile in MB-APDT group when it was compared with the control group. In MB-APDT group, it was also possible to observe less pain on palpation of wounds borders, less edema and inflammatory exudate. Additionally, animals from MB-APDT group were faster discharged from the cattle care facility. Conclusion Our results support the use of MB-APDT for the post-surgical management of rumenostomy. This pilot study ratifies the use of APDT in cattle and also suggests that it could be performed for other surgical procedures as a complementary approach or an alternative for topical administration of antibiotics.

Palavras-Chave: methylene blue; therapy; cattle; inactivation; veterinary medicine; surgery; wounds; healing

VALANDRO, PATRICIA; MASSUDA, MAYARA B.; RUSCH, ELIDIANE; BIRGEL, DANIELA B.; PEREIRA, PHILIPE P.L.; SELLERA, FABIO P.; RIBEIRO, MARTHA S.; POGLIANI, FABIO C.; BIRGEL JUNIOR, EDUARDO H. Antimicrobial photodynamic therapy can be an effective adjuvant for surgical wound healing in cattle. Photodiagnosis and Photodynamic Therapy, v. 33, p. 1-4, 2021. DOI: 10.1016/j.pdpdt.2020.102168. Disponível em: http://repositorio.ipen.br/handle/123456789/32052. Acesso em: $DATA.

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Abstract: Background: Dental caries are a multifactorial disease that progressively produces tooth destruction as a result of bacterial colonization of enamel surface, especially Streptococcus mutans. The objective of this work was to investigate the role of glucose in antimicrobial photodynamic therapy (aPDT) on S. mutans. Methods: S. mutans ATCC 25175 were cultured on microaerophilia at 37 °C for 48 h, and we tested aPDT in the presence of 50 mM glucose. Bacterial suspension was used to investigate aPDT with 100 μM methylene blue (MB) under LED emitting radiation at ʎ =660 nm and parameters as following (P =473 mW; I =166.8 mW/ cm2, and doses of 5, 10 and 20 J/cm2). A seventy-two hours biofilm was grown on 96 flat buttoned well-plate and irradiation was performed from 10 to 80 J/cm2 at similar conditions. Results: There was no dark toxicity nor bacterial death regarding LED irradiation on suspension and on biofilm. Nevertheless, aPDT presented expressive bacterial inactivation following 1 and 2 min of irradiation on cell suspension. On the other hand, there was no inactivation in the presence of glucose under the same conditions. Biofilm was completely inactivated by MB-mediated aPDT after 6 min of irradiation. However, the presence of glucose delayed the complete inactivation of the biofilm. Conclusion: The presence of glucose in the suspension drastically delayed the effect of aPDT on S. mutans and this effect is more pronounced in bacterial suspension than on biofilm.

Palavras-Chave: antimicrobial agents; therapy; streptococcus; methylene blue; dentistry; caries; teeth; dentin; glucose; photosensitivity

LEAL, CINTIA R.L.; ALVARENGA, LETICIA H.; OLIVEIRA-SILVA, TAMIRES; KATO, ILKA T.; GODOY-MIRANDA, BIANCA; BUSSADORI, SANDRA K.; RIBEIRO, MARTHA S.; PRATES, RENATO A. Antimicrobial photodynamic therapy on Streptococcus mutans is altered by glucose in the presence of methylene blue and red LED. Photodiagnosis and Photodynamic Therapy, v. 19, p. 1-4, 2017. DOI: 10.1016/j.pdpdt.2017.04.004. Disponível em: http://repositorio.ipen.br/handle/123456789/27893. Acesso em: $DATA.

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Abstract: Sir, Antimicrobial photodynamic therapy (aPDT) is emerging as a new promising tool to treat local infections in Veterinary Medicine [1]. In 2016, our group was the pioneer to investigate its use to inactivate pathogens related to bovine mastitis [2]. In this regard, in a recent report published in this journal, Moreira et al. [3] explored the use of toluidine blue (TBO)-mediated aPDT to treat subclinical bovine mastitis. Remarkably, bovine mastitis still remains as the most economically relevant disease for dairy industry worldwide, resulting in substantial economic losses for milk producers due to extensive antibiotic use and product losses. In this sense, antibiotic misuse or overuse to treat bovine mastitis is worrisome because it can simultaneously lead to antibiotic residues in dairy products and increase multidrug-resistant foodborne pathogens. Unfavorably, there is still no consensus on the ideal approach for bovine mastitis treatment; therefore, aPDT represents a viable option to reduce the use of antibiotics in dairy farms. The preliminary results of Moreira et al. [3] are exciting and deserve discussion on some points. In this letter, we intend to highlight other relevant aspects to be addressed in order to consolidate the use of aPDT for management of bovine mastitis. The concentration of the photosensitizer (PS) diluted in the milk plays a pivotal role. In the study by Moreira et al. [3], the authors designed a clinical assay using a 2.5% TBO solution, which promoted a significant reduction of bacterial load. It is noteworthy that clinical mastitis represents a more complex challenge for aPDT than subclinical mastitis since the alterations in the milk composition could influence the PS absorption properties. Additionally, we believe that intermolecular interactions between PS and milk components should be considered to optimize clinical protocols because it could induce aggregation of phenothiazine dyes and leads to lower photodynamic inactivation efficiency [[4], [5]]. Another important point is in respect to the light diffusion within mammary parenchyma. Appropriated light delivery systems need to reach deeper mammary tissue since pathogens normally colonize the entire mammary gland [6]. Therefore, the use of diffuser fibers should be considered to deliver homogeneous light intensities inside the gland parenchyma. Hence, investigations regarding different light delivery systems could optimize clinical protocols and facilitate the establishment of aPDT into the dairy industry. Finally, we encourage the development of innovative strategies to improve selective PS delivery to pathogens using targeted molecular and nanoparticle systems. In summary, we hope that this letter motivates further studies to develop safe and effective protocols for bovine mastitis and promote aPDT applications in Veterinary Medicine.

Palavras-Chave: veterinary medicine; cattle; mammary glands; infectious diseases; bacterial diseases; therapy; photosensitivity

RIBEIRO, MARTHA S.; GARGANO, RONALDO G.; SABINO, CAETANO P.; ANJOS, CAROLINA dos; POGLIANI, FABIO C.; SELLERA, FABIO P. Clinical challenges of antimicrobial photodynamic therapy for bovine mastitis. Photodiagnosis and Photodynamic Therapy, v. 21, p. 327-327, 2018. DOI: 10.1016/j.pdpdt.2018.01.007. Disponível em: http://repositorio.ipen.br/handle/123456789/29010. Acesso em: $DATA.

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Abstract: Background: Candida albicans is an opportunistic commensal microorganism, often associated with severe infections in immunosuppressed individuals. C. albicans has hexose transporters that may favor the intracellular accumulation of photosensitizer (PS). the aims of this study were to investigate the influence of glucose load on photodynamic antimicrobial chemotherapy (PACT); and the role that membrane transport system plays on this therapy in the presence of glucose. Material and methods: Strains of C. albicans were selected: ATCC 10231, YEM 12, YEM 13, YEM 14 and YEM 15. All strains were grown aerobically on Sabouraud agar and incubated at 30 °C for 24 h. The strains were treated with and without glucose, and divided into Control (no treatment), LED light (660 nm, 166 mW/cm2), Photosensitizer (100 μM methylene blue) and PACT at 1, 3 and 6 min of irradiation groups. The colony forming units were counted and data submitted to statistical analysis (ANOVA) and Tukey's test. The concentration of methylene blue (MB) outside the yeast was measured by fluorescence spectroscopy. Results: PACT inactivate C. albicans and the presence of glucose did not affect the killing effect for most strains. Only YEM12 was partially affected by its presence. Regarding efflux systems, ABC overexpressing strain showed a protective effect on the yeast cells. We observed that yeast with overexpression of major facilitator superfamily (MFS) membrane pore tended to accumulate more MB in its cytoplasm, whereas strains that overexpressed ABC pumps (ATP-binding cassette transporters) tended to decrease MB uptake and survive the photodynamic challenge. Conclusion: Presence of glucose showed a small effect on PACT . The accumulation of MB on yeast induces more photodynamic inactivation; however, the photodynamic efficacy depends on the type and characteristics of the microbial strain.

Palavras-Chave: chemotherapy; antimicrobial agents; photosensitivity; yeasts; candida; glucose; methylene blue; x-ray fluorescence analysis

OLIVEIRA-SILVA, TAMIRES de; ALVARENGA, LETICIA H.; LIMA-LEAL, CINTIA; GODOY-MIRANDA, BIANCA; CARRIBEIRO, PAMELA; SUZUKI, LUIS C.; RIBEIRO, MARTHA S.; KATO, ILKA T.; PAVANI, CHRISTIANE; PRATES, RENATO A. Effect of photodynamic antimicrobial chemotherapy on Candida albicans in the presence of glucose. Photodiagnosis and Photodynamic Therapy, v. 27, p. 54-58, 2019. DOI: 10.1016/j.pdpdt.2019.05.032. Disponível em: http://repositorio.ipen.br/handle/123456789/30063. Acesso em: $DATA.

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Abstract: Antimicrobial photodynamic therapy (aPDT) has been used to treat periodontal disease, thus the aim of this study was to investigate red light (ʎ = 660 nm) attenuation in gingival tissue. This clinical trial included 30 patients with chronic periodontitis; three incisors from each patient were selected for the experimental procedures. A laser source with a radiant power output of 100 mW was used. Two digital photographs were taken of each selected incisor (in frontal and occlusal position). The images were analyzed in the ImageJ program. The results demonstrated that at a 3 mm distance from the laser probe, there is an attenuation of light intensity of 50%, along frontal and occlusal views. Light attenuation in gingival tissue should be considered when setting optimal parameters for antimicrobial photodynamic therapy or photobiomodulation.

Palavras-Chave: therapy; dentistry; dosimetry; lasers; light scattering; dentin; bone tissues; antimicrobial agents

ALVARENGA, LETICIA H.; RIBEIRO, MARTHA S.; KATO, ILKA T.; NUNEZ, SILVIA C.; PRATES, RENATO A. Evaluation of red light scattering in gingival tissue – in vivo study. Photodiagnosis and Photodynamic Therapy, v. 23, p. 32-34, 2018. DOI: 10.1016/j.pdpdt.2018.05.016. Disponível em: http://repositorio.ipen.br/handle/123456789/29264. Acesso em: $DATA.

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Abstract: Sporotrichosis is a mycotic infection of humans and animals caused by different fungal species of the genus Sporothrix. Feline sporotrichosis presents a broad spectrum of clinical manifestations and its treatment with classic antifungal drugs is often long and frustrating. Methylene blue-mediated antimicrobial photodynamic therapy (MB-APDT) comes to light as an interesting approach against fungal infections, including sporotrichosis. In this case report, a 1-year-old male cat was diagnosed with sporotrichosis, being confirmed by fungal culture. The cat was treated by MB-APDT combined with oral administration of itraconazole. Following 2 weeks after the end of treatment, the animal was clinically cured, and an additional fungal culture was negative for Sporothrix spp., confirming the total remission of sporotrichosis. No side effects and recurrences were observed after a 3-moth follow-up. MB-APDT is a promising strategy against feline sporotrichosis, however large-scale studies are welcome to confirm its potential.

Palavras-Chave: veterinary medicine; fungal diseases; photodynamic therapy; antimicrobial agents; fungi; cats; methylene blue

CABRAL, FERNANDA V.; SELLERA, FABIO P.; RIBEIRO, MARTHA S. Feline sporotrichosis successfully treated with methylene blue-mediated antimicrobial photodynamic therapy and low doses of itraconazole. Photodiagnosis and Photodynamic Therapy, v. 40, p. 1-3, 2022. DOI: 10.1016/j.pdpdt.2022.103154. Disponível em: http://repositorio.ipen.br/handle/123456789/33457. Acesso em: $DATA.

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