Impact of PVC microplastics in photodynamic inactivation of Staphylococcus aureus and MRSA.

Photodynamic processes have found widespread application in therapies. These processes involve photosensitizers (PSs) that, when excited by specific light wavelengths and in the presence of molecular oxygen, generate reactive oxygen species (ROS), that target cells leading to inactivation. Photodynamic action has gained notable attention in environmental applications, particularly against pathogens and antibiotic-resistant bacteria (ARB) that pose a significant challenge to public health. However, environmental matrices frequently encompass additional contaminants and interferents, including microplastics (MPs), which are pollutants of current concern. Their presence in water and effluents has been extensively documented, highlighting their impact on conventional treatment methods, but this information remains scarce in the context of photodynamic inactivation (PDI) setups. Here, we described the effects of polyvinyl chloride (PVC) microparticles in PDI targeting Staphylococcus aureus and its methicillin-resistant strain (MRSA), using curcumin as a PS under blue light. The presence of PVC microparticles does not hinder ROS formation; however, depending on its concentration, it can impact bacterial inactivation. Our results underscore that PDI remains a potent method for reducing bacterial concentrations in water and wastewater containing ARB, even in highly contaminated scenarios with MPs.

Hydrogen peroxide preoxidation as a strategy for enhanced antimicrobial photodynamic action against methicillin-resistant Staphylococcus aureus.

Antimicrobial photodynamic treatment (aPDT) is a photooxidative process based on the excitation of a photosensitizer (PS) in the presence of molecular oxygen, under specific wavelengths of light. It is a promising method for advanced treatment of water and wastewater, particularly targeting disinfection challenges, such as antibiotic-resistant bacteria (ARB). Research in improved aPDT has been exploring new PS materials, and additives in general. Hydrogen peroxide (H2O2) a widely applied disinfectant, mostly in the food industry and clinical settings, present environmentally negligible residuals at the usually applied concentrations, making it friendly for the water and wastewater sectors. Here, we explored the effects of preoxidation with H2O2 followed by blue light-mediated (450 nm) aPDT using curcumin (a natural-based PS) against methicillin-resistant Staphylococcus aureus (MRSA). Results of the sequential treatment pointed to a slight hampering in aPDT efficiency at very low H2O2 concentrations, followed by an increasing cooperative effect up to a deleterious point (≥7 log10 inactivation in CFU mL-1), suggesting a synergistic interaction of preoxidation and aPDT. The increased performance in H2O2-pretreated aPDT encourages studies of optimal operational conditions for the assisted technology and describes potentials for using the described strategy to tackle the issue of ARB spread.

Photon spectra effects tested on the vegetal model Allium cepa.

The use of artificial light sources in plants is considered a type of photobiomodulation (PBM), a trend in agriculture and food industries, aiming at decontamination, pest control, and increased production yield. However, literature lacks a broader assessment to address the effects of photon light spectra on plant characteristics. Here, we aimed to describe the effects of visible light, infrared, and ultraviolet light upon Allium cepa, a known bioindicator, under various light doses. Samples irradiated under visible and infrared light did not show cytotoxicity, genotoxicity, or mutagenicity in any of the evaluated doses. Light induction at 460 and 635 nm significantly stimulated root development of the test organism. In contrast, 254 nm irradiation proved to be cytotoxic, genotoxic, and mutagenic. This work reveals and quantifies the spectral response of A. cepa seeds, suggesting that it can be proposed as a model for future research on mechanisms of PBM in plants.

Analytical challenges and perspectives of assessing viability of Giardia muris cysts and Cryptosporidium parvum oocysts by live/dead simultaneous staining.

Giardia and Cryptosporidium are pathogenic protozoa often present in the environment in their infective form(cysts and oocysts). These parasites are very resistant to disinfection, which makes them important target organisms in environmental quality monitoring and sanitation. Viability assessment provides an interpretation of cell inactivation, and it can be evaluated by membrane integrity as well as enzyme activity, using different staining methods. These are straightforward and adequate to laboratories that lack infrastructure for molecular-based technologies or animal infectivity tests. This study investigated simultaneous staining by a commercial live/dead kit, in order to assess viability of Cryptosporidium parvum oocysts and Giardia muris cysts, comparing it to propidium iodide (PI) incorporation, a common stain applied in viability estimation. Results suggested that, although the central hypothesis of one-panel visualization (α = 0.05) was met, simultaneous staining impaired (oo)cyst detection by immunofluorescence assay (IFA), which was found to be essential to enumeration, as the live/dead test led to poor (oo)cyst labelling or a 10-fold lower recovery when carried out concomitantly to IFA. As for the viability assessment itself, although red dye uptake occurred as expected by dead or weakened organisms, neither live G. muris cysts or C. parvum oocysts present any green fluorescence by esterase metabolism. This may have been caused by low enzyme activity in the infective form and/or wall thickness of these parasites. The results do not exclude the possibility of simultaneous fluorescence staining for protozoa, but it is a starting point for a broader analysis, that may consider, for instance, different incubation conditions.

Wastewater treatment performance in microbiological removal and (oo)cyst viability assessed comparatively to fluorescence decay.

Municipal wastewater is a source of pathogenic protozoan (oo)cysts and may play a significant role in spreading waterborne diseases. This scenario becomes more critical as treated sewage from municipal wastewater treatment plants (WWTP) is discharged into springs, which are often used for water supply, irrigation, recreation and, further downstream, indirect potable reuse, quite common in Brazil. This study aimed to elucidate, regarding microbiological quality, the performance of a full-scale WWTP, consisting of preliminary treatment, upflow anaerobic sludge blanket (UASB) reactor, activated sludge system and ultraviolet (UV) radiation disinfection. Pathogenic protozoa (Giardia spp. cysts and Cryptosporidium spp. oocysts), as well as microbiological indicators (Escherichia coli and Clostridium perfringens), were evaluated in terms of their removal. In addition, (oo)cyst viability and fluorescence reduction were assessed. By using the data obtained from this research, the prevalence of infection estimated for the population served by the WWTP was between 7.4% and 14.8% for giardiasis, and between 0.055% and 0.11% for cryptosporidiosis.

Giardia spp. cysts and Cryptosporidium spp. oocysts in drinking water treatment residues: comparison of recovery methods for quantity assessment.

Water treatment plant (WTP) residues, e.g. sludge and filter backwash water (FBW), may contain pathogenic microorganisms, as Giardia spp. and Cryptosporidium spp. However, recovering protozoa from such matrices lacks a formal and precise protocol, which is imperative to improve research in their detection, removal and inactivation. The latter includes a deeper challenge as some recovery methods may compromise viability. This study applied different recovery methods for G. muris cysts and C. parvum oocysts spiked into settled sludge and FBW obtained from a bench treatment. Procedures in sludge involved direct centrifugation, alkaline and acid flocculation, including purification by immunomagnetic separation (IMS). FBW samples were tested for membrane filtration (MF) and heated Tween® scrapings followed or not by IMS. Propidium iodide (PI) inclusion was used for oocyst viability evaluation prior and after recovery. Results with purified suspensions lead to higher recovery efficiencies (RE) for C. parvum, which was assumed to relate to poor G. muris fluorescence. Analytical quality assessments were carried out with ColorSeed® for the methods that stood out for each matrix and the results indicated lower RE than when organisms from purified suspensions were recovered. Ferric sulphate flocculation and MF, both followed by IMS reached 32.25% and 11.00% RE for Giardia spp. and 19.61% and 2.00% for Cryptosporidium spp., respectively. All of the tested methods affected oocyst viability. These results encourage further research to overcome the matrices complexity explained in this paper and increase RE, taking effects in protozoa viability into consideration.

Cryptosporidium spp. and Giardia spp. (oo)cysts as target-organisms in sanitation and environmental monitoring: A review in microscopy-based viability assays.

Cysts and (oo)cysts are the infective forms of parasitic protozoa, as Giardia and Cryptosporidium, which are widespread and associated to worldwide waterborne diseases outbreaks. These microorganisms pose a challenge to public health, as they are resistant to conventional disinfection methods, which make them important parameters when evaluating inactivation efficiency. However, when (oo)cysts are targets, it is challenging to infer inactivation efficacy, as it may require infectivity tests that are not often an option for laboratory routine analysis. In this scene, (oo)cyst viability based on induced excystation, membrane integrity and enzyme activity evaluated by dye inclusion and/or exclusion, as well as fluorescence reduction consist on microscopy-based techniques that may be options to estimate inactivation in the environmental context. This scoping review presents applications, advantages and limitations of these methodologies for viability assessment, in order to shed light on the (oo)cyst viability topic and provide insight strategies for choosing protocols in the environmental and sanitation field, in laboratory applications and novel research.