Photodynamic inactivation of multidrug-resistant strains of Klebsiella pneumoniae and Pseudomonas aeruginosa in municipal wastewater by tetracationic porphyrin and violet-blue light: The impact of wastewater constituents

PLOS ONE RESEARCH ARTICLE Photodynamic inactivation of multidrugresistant strains of Klebsiella pneumoniae and Pseudomonas aeruginosa in municipal wastewater by tetracationic porphyrin and violet-blue light: The impact of wastewater constituents a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 Martina Mušković1☯, Matej Planinić ID2☯, Antonela Crepulja2, Marko Lušić1, Marin Glad3, Martin Lončarić ID4, Nela Malatesti ID1*, Ivana Gobin ID2 1 Department of Biotechnology, University of Rijeka, Rijeka, Croatia, 2 Department of Microbiology and Parasitology, Faculty of Medicine, University of Rijeka, Rijeka, Croatia, 3 Department for Environmental Protection and Health Ecology, Teaching Institute of Public Health, Rijeka, Croatia, 4 Photonics and Quantum Optics Unit, Center of Excellence for Advanced Materials and Sensing Devices, Ruđer Bošković Institute, Zagreb, Croatia ☯ These authors contributed equally to this work. * OPEN ACCESS Citation: Mušković M, Planinić M, Crepulja A, Lušić M, Glad M, Lončarić M, et al. (2023) Photodynamic inactivation of multidrug-resistant strains of Klebsiella pneumoniae and Pseudomonas aeruginosa in municipal wastewater by tetracationic porphyrin and violet-blue light: The impact of wastewater constituents. PLoS ONE 18(8): e0290080. https://doi.org/10.1371/journal. pone.0290080 Editor: Adelaide Almeida, Universidade de Aveiro, PORTUGAL Received: May 5, 2023 Accepted: July 31, 2023 Published: August 15, 2023 Peer Review History: PLOS recognizes the benefits of transparency in the peer review process; therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. The editorial history of this article is available here: https://doi.org/10.1371/journal.pone.0290080 Copyright: © 2023 Mušković et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Abstract There is an increasing need to discover effective methods for treating municipal wastewater and addressing the threat of multidrug-resistant (MDR) strains of bacteria spreading into the environment and drinking water. Photodynamic inactivation (PDI) that combines a photosensitiser and light in the presence of oxygen to generate singlet oxygen and other reactive species, which in turn react with a range of biomolecules, including the oxidation of bacterial genetic material, may be a way to stop the spread of antibiotic-resistant genes. The effect of 5,10,15,20-(pyridinium-3-yl)porphyrin tetrachloride (TMPyP3) without light, and after activation with violet-blue light (VBL) (394 nm; 20 mW/cm2), on MDR strains of Pseudomonas aeruginosa, Klebsiella pneumoniae and K. pneumoniae OXA-48 in tap water and municipal wastewater was investigated. High toxicity (~2 μM) of TMPyP3 was shown in the dark on both strains of K. pneumoniae in tap water, while on P. aeruginosa toxicity in the dark was low (50 μM) and the PDI effect was significant (1.562 μM). However, in wastewater, the toxicity of TMPyP3 without photoactivation was much lower (12.5–100 μM), and the PDI effect was significant for all three bacterial strains, already after 10 min of irradiation with VBL (1.562–6.25 μM). In the same concentrations, or even lower, an anti-adhesion effect was shown, suggesting the possibility of application in biofilm control. By studying the kinetics of photoinactivation, it was found that with 1,562 μM of TMPyP3 it is possible to achieve the complete destruction of all three bacteria after 60 min of irradiation with VBL. This study confirmed the importance of studying the impact of water constituents on the properties and PDI effect of the applied photosensitiser, as well as checking the sensitivity of targeted PLOS ONE | https://doi.org/10.1371/journal.pone.0290080 August 15, 2023 1 / 23 PLOS ONE Photoinactivation of Klebsiella pneumoniae and Pseudomonas aeruginosa in wastewater Data Availability Statement: All relevant data are within the paper and its Supporting Information files. bacteria to light of a certain wavelength, in conditions as close as possible to those in the intended application, to adjust all parameters and perfect the method. Funding: Our study was financed by the University of Rijeka grants (UNIRI-INOVA to NM, and uniribiomed-18-171 to IG) and the Ministry of Science and Education of Croatia (ERDF) grant for CEMS No. KK.01.1.1.01.0001. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Introduction Competing interests: The authors have declared that no competing interests exist. Photodynamic therapy (PDT) uses photoactive dye (= photosensitiser, PS), light and oxygen to create reactive oxygen species (ROS), most importantly singlet oxygen (1O2), to induce a cytotoxic effect. Although the PDT effect was discovered on the microorganism (Paramecium), PDT has been developed primarily for applications in antitumor therapies [1]. In recent years, antimicrobial PDT (aPDT), known also as photodynamic inactivation (PDI) and photodynamic antimicrobial chemotherapy (PACT), has been increasingly developed to treat various (usually local) infections, and for disinfection purposes, such as the disinfection of water, blood, (hospital) surfaces and medical devices, food, and crops [2]. New antimicrobial approaches, such as aPDT, are needed because the excessive and inappropriate use of antibiotics has led to the emergence of antibiotic resistance (ABR), which has been declared by the World Health Organization (WHO) as one of the greatest dangers to global health and development [3]. Shortly thereafter, WHO declared this kind of danger for antimicrobial resistance (AMR) in general, which includes multidrug resistance (MDR) [4]. Among the most notorious pathogens that pose a particularly significant health threat due to their MDR and an elevated risk to cause nosocomial or healthcare-associated infections (HAI), are ESKAPE bacteria and biofilms they produce. ESKAPE is an acronym that stands for the names of Gram-positive bacteria, Enterococcus faecium and Staphylococcus aureus, and Gram-negative, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter species [5], and at the same time connects their high tendency to “escape” from the bactericidal activity of many antibiotics used in clinics [6]. Municipal wastewater from public sewage contains stormwater and wastewater from households, industries, and hospitals, and can be burdened by MDR bacterial strains. A recent study of facultative pathogenic bacteria at twenty-three wastewater treatment plants in Germany found a link between clinically relevant antibiotic resistance genes (ARGs) and hospital wastewaters [7]. Not only are ARGs present in municipal and hospital waters, but (...truncated)