Effects of different light wavelengths on Bacillus subtilis and Bacillus velezensis, two biocontrol agents isolated from the maize phyllosphere.

In previous studies, two strains isolated from the maize phyllosphere were identified as Bacillus subtilis (EM-A7) and Bacillus velezensis (EM-A8) and selected as potential biocontrol agents against Exserohilum turcicum. This study aimed to assess the ability of EM-A7 and EM-A8 to form biofilm and have antagonistic activity under varying light conditions. LED sources were custom-designed so that each corresponded to a given spectrum at a specific photosynthetically active photon flux density. Significant differences were observed in growth parameters (generation time and constant growth rate) under different LED sources. Blue light inhibited the growth of both strains. Red increased k rate in EM-A8, while the g values increased in EM-A7. Red and white light generally increased biofilm formation, and blue light inhibited it. EM-A7 and EM-A8 significantly reduced their ability to swim under blue LED, but it was not affected by red, green, or white light. The ability to swarm was negatively affected. Fungal growth decreased significantly compared to the control when the bacterium growing on the same plate had been previously incubated under red and white light or in the dark. These results indicate that different light wavelengths clearly influenced the aspects assessed in B. subtilis and B. velezensis, with the effects of blue light being overall negative and those of red and white overall positive. Given that, all these factors can be important for the establishment and survival of Bacillus strains on leaves, as well as for their effectiveness against pathogens, light could be a significant factor to consider in the design of biocontrol strategies.

In vitro studies of biofilm-forming Bacillus strains, biocontrol agents isolated from the maize phyllosphere.

We aimed to assess how biofilm formation by three Bacillus isolates was affected by changes in temperature, water potential, growth media, time, and the combinations between these factors. The strains had been selected as potential biological control agents (BCAs) in earlier studies, and they were identified as B. subtilis and B. velezensis spp. through 16 rRNA sequencing and MALDI-TOF MS. Maize leaves (ML) were used as one of the growth media, since they made it possible to simulate the nutrient content in the maize phyllosphere, from which the bacteria were originally isolated. The strains were able to form biofilm both in ML and biofilm-inducing MSgg after 24, 48, and 72 h. Biofilm development in the form of pellicles and architecturally complex colonies varied morphologically from one strain to another and depended on the conditions mentioned above. In all cases, colonies and pellicles were less complex when both temperature and water potential were lower. Scanning electron microscopy (SEM) revealed that changing levels of complexity in pellicles were correlated with those in colonies. Statistical analyses found that the quantification of biofilm produced by the isolates was influenced by all the conditions tested. In terms of motility (which may contribute to biofilm formation), swimming and swarming were possible for all strains in 0.3 and 0.7% agar, respectively. A more in-depth understanding of how abiotic factors influence biofilm formation can contribute to a more effective use of these biocontrol strains against pathogens in the maize phyllosphere.

Sweet light o' mine: Photothermal and photodynamic inactivation of tenacious pathogens using conjugated polymers.

Each year a rising number of infections can not be successfully treated owing to the increasing pandemic of antibiotic resistant pathogens. The global shortage of innovative antibiotics fuels the emergence and spread of drug resistant microbes. Basic research, development, and applications of alternative therapies are urgently needed. Since the 90´s, light-mediated therapies have promised to be the next frontier combating multidrug-resistance microbes. These platforms have demonstrated to be a reliable, rapid, and efficient alternative to eliminate tenacious pathogens while avoiding the emergence of resistance mechanisms. Among the materials showing antimicrobial activity triggered by light, conjugated polymers (CPs) have risen as the most promising option to tackle this complex situation. These materials present outstanding characteristics such as high absorption coefficients, great photostability, easy processability, low cytotoxicity, among others, turning them into a powerful class of photosensitizer (PS)/photothermal agent (PTA) materials. Herein, we summarize and discuss the advances in the field of CPs with applications in photodynamic inactivation and photothermal therapy towards bacteria elimination. Additionally, a section of current challenges and needs in terms of well-defined benchmark experiments and conditions to evaluate the efficiency of phototherapies is presented.

Photodynamic Inactivation of ESKAPE Group Bacterial Pathogens in Planktonic and Biofilm Cultures Using Metallated Porphyrin-Doped Conjugated Polymer Nanoparticles.

Photodynamic inactivation (PDI) protocols using photoactive metallated porphyrin-doped conjugated polymer nanoparticles (CPNs) and blue light were developed to eliminate multidrug-resistant pathogens. CPNs-PDI protocols using varying particle concentrations and irradiation doses were tested against nine pathogenic bacterial strains including antibiotic-resistant bacteria of the ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) pathogens group. The bactericidal effect was achieved in methicillin-resistant Staphylococus aureus (S. aureus) strains using low light doses (9.6-14.4 J/cm2), while Gram-negative bacteria required a higher light dose (28.8 J/cm2). The bacteria-CPN interaction was studied through flow cytometry, taking advantage of the intrinsic CPN fluorescence, demonstrating that CPNs efficiently bind to the bacterial envelope. Finally, the performance of CPNs-PDI was explored in biofilms; good antibiofilm ability and almost complete eradication were observed for S. aureus and Escherichia coli biofilms, respectively, using confocal microscopy. Overall, we demonstrated that CPNs-PDI is an efficient tool not only to kill superbugs as sessile cells but also to disrupt and eradicate biofilms of highly relevant pathogenic bacterial species.

Conjugated Polymer Nanoparticles as Unique Coinitiator-Free, Water-Soluble, Visible-Light Photoinitiators of Vinyl Polymerization.

The use of conjugated polymer nanoparticles (CP NPs) of poly(9,9-dioctylfluorene-alt-benzothiadiazole) and poly(9,9-di-n-octylfluorenyl-2,7-diyl) as efficient photoinitiator systems (PIS) of vinyl polymerization in water is reported herein. CP NPs are biocompatible, excitable with blue commercial LEDs and, unlike visible light Type II PIS, do not need co-initiators to trigger a monomer chain reaction. CP NPs photoinitiate polymerization of a variety of acrylic monomers with initiation rates comparable to those observed for well-known Type II PIS. Given the extraordinarily large molar absorption coefficients of CP NPs (≈108 m-1 cm-1 ) very low particle concentration is required for effective polymerization. Additionally, CP NPs behave as conventional macrophotoinitiators significantly reducing contamination risks due to leaching of low molecular weight byproducts. These combined features make CP NPs PIS suitable to synthesize polymeric materials for many healthcare and biomedical applications including drug delivery, tissue engineering, prosthetic implants, and food/medicine packaging. These CP NPs PIS are also used to synthesize nano-hydrogels with a relatively narrow and controlled size distribution in the absence of surfactants. It is proposed that polymerization is initiated at the CP NPs surface by photogenerated free polarons, in close analogy to the mechanism previously described for PIS based on inorganic semiconductor NPs.

Metallated porphyrin-doped conjugated polymer nanoparticles for efficient photodynamic therapy of brain and colorectal tumor cells.

AIM: Assess biocompatibility, uptake and photodynamic therapy (PDT) mechanism of metallated porphyrin doped conjugated polymer nanoparticles (CPNs) in human brain and colorectal tumor cells and macrophages. MATERIALS & METHODS: CPNs were developed employing 9,9-dioctylfluorene-alt-benzothiadiazole, an amphiphilic polymer (PS-PEG-COOH),  and platinum octaethylporphyrin. T98G, SW480 and RAW 264.7 cell lines were exposed to CPNs to assess uptake and intracellular localization. Additionally, a PDT protocol using CPNs was employed for the in vitro killing of cancer and macrophage cell lines. RESULTS & CONCLUSION: CPNs were well incorporated into glioblastoma and macrophage cells with localization in lysosomes. SW480 cells were less efficient incorporating CPNs with localization in the plasma membrane. In all cell lines PDT treatment was efficient inducing oxidative stress that triggered apoptosis.

Crosslinked polymer nanoparticles containing single conjugated polymer chains.

Conjugated polymer nanoparticles are widely used in fluorescent labeling and sensing, as they have mean radii between 5 and 100 nm, narrow size dispersion, high brightness, and are photochemically stable, allowing single particle detection with high spatial and temporal resolution. Highly crosslinked polymers formed by linking individual chains through covalent bonds yield high-strength rigid materials capable of withstanding dissolution by organic solvents. Hence, the combination of crosslinked polymers and conjugated polymers in a nanoparticulated material presents the possibility of interesting applications that require the combined properties of constituent polymers and nanosized dimension. In the present work, F8BT@pEGDMA nanoparticles composed of poly(ethylene glycol dimethacrylate) (pEGDMA; a crosslinked polymer) and containing the commercial conjugated polymer poly(9,9-dioctylfluorene-alt-benzothiadiazole) (F8BT) were synthesized and characterized. Microemulsion polymerization was applied to produce F8BT@pEDGMA particles with nanosized dimensions in a ∼25% yield. Photophysical and size distribution properties of F8BT@pEDGMA nanoparticles were evaluated by various methods, in particular single particle fluorescence microscopy techniques. The results demonstrate that the crosslinking/polymerization process imparts structural rigidity to the F8BT@pEDGMA particles by providing resistance against dissolution/disintegration in organic solvents. The synthesized fluorescent crosslinked nanoparticles contain (for the most part) single F8BT chains and can be detected at the single particle level, using fluorescence microscopy, which bodes well for their potential application as molecularly imprinted polymer fluorescent nanosensors with high spatial and temporal resolution.