An insight into the photodynamic approach versus copper formulations in the control of Pseudomonas syringae pv. actinidiae in kiwi plants.

In the last decade, the worldwide production of kiwi fruit has been highly affected by Pseudomonas syringae pv. actinidiae (Psa), a phytopathogenic bacterium; this has led to severe economic losses that are seriously affecting the kiwi fruit trade. The available treatments for this disease are still scarce, with the most common involving frequently spraying the orchards with copper derivatives, in particular cuprous oxide (Cu2O). However, these copper formulations should be avoided due to their high toxicity; therefore, it is essential to search for new approaches for controlling Psa. Antimicrobial photodynamic therapy (aPDT) may be an alternative approach to inactivate Psa. aPDT consists in the use of a photosensitizer molecule (PS) that absorbs light and by transference of the excess of energy or electrons to molecular oxygen forms highly reactive oxygen species (ROS) that can affect different molecular targets, thus being very unlikely to lead to the development of microbe resistance. The aim of the present study was to evaluate the effectiveness of aPDT to photoinactivate Psa, using the porphyrin Tetra-Py+-Me and different light intensities. The degree of inactivation of Psa was assessed using the PS at 5.0 µM under low irradiance (4.0 mW cm-2). Afterward, ex vivo experiments, using artificially contaminated kiwi leaves, were conducted with a PS at 50 µM under 150 mW cm-2 and sunlight irradiation. A reduction of 6 log in the in vitro assays after 90 min of irradiation was observed. In the ex vivo tests, the decrease was lower, approximately 1.8 log reduction at an irradiance of 150 mW cm-2, 1.2 log at 4.0 mW cm-2, and 1.5 log under solar radiation. However, after three successive cycles of treatment under 150 mW cm-2, a 4 log inactivation was achieved. No negative effects were observed on leaves after treatment. Assays using Cu2O were also performed at the recommended concentration by law (50 g h L-1) and at concentrations 10 times lower, in which at both concentrations, Psa was efficiently inactivated (5 log inactivation) after a few minutes of treatment, but negative effects were observed on the leaves after treatment.

Single and combined effects of photodynamic therapy and antibiotics to inactivate Staphylococcus aureus on skin.

In this study, the effect of antimicrobial photodynamic therapy (aPDT) and aPDT combined with antibiotics to inactivate Staphylococcus aureus in vitro and ex vivo was compared. The tetracationic porphyrin 5,10,15,20-tetrakis(1-methylpyridinium-4-yl)porphyrin tetra-iodide (Tetra-Py+-Me) was used to inactivate S. aureus in phosphate buffer solution (PBS) (in vitro) and in pork skin artificially contaminated with S. aureus (ex vivo). The results showed an efficient reduction of 8 log in PBS, after 180 min of irradiation under white light at 4.0 mW cm-2 with Tetra-Py+-Me at 5.0 µM. When aPDT was repeated in the presence of antibiotics, an increased effect was observed with ampicillin at 0.5 and 1.0 µg mL-1 (MIC 0.25 µg mL-1) - the full inactivation (8 log) occurred faster, respectively, after 60 and 30 min of irradiation. In ex vivo, a reduction of ∼4 log of S. aureus was observed after 180 min with 50 µM of Tetra-Py+-Me at 150 mW cm-2, but this efficiency increased significantly with the application of three successive light cycles (reduction to the detection limit ∼6 log). The photoinactivation efficiency observed in ex vivo was also significantly improved when the experiments with 50 µM of Tetra-Py+-Me were repeated in the presence of 5.0 µg mL-1 of ampicillin (inactivation of ∼5.6 log). The results showed that aPDT is an effective approach to control S. aureus infection in skin, inactivating the bacterium to the detection limit after three successive cycles of treatment or after one cycle by using the combination aPDT and ampicillin.

Effects of single and combined use of bacteriophages and antibiotics to inactivate Escherichia coli.

A major concern of phage therapy is the emergency of phage-resistant mutants. This limitation can be overcome by the combined use of phages and antibiotics. It has been shown that the combination of antibiotics and phages is an alternative that cannot only be effective at reducing bacterial numbers, but also to contribute to the management of resistance levels. However, this view has only been discussed with regard to antibiotic resistance and not to control phage-mutant emergence. In our study we compared not only the resistance of the bacteria to the four antibiotics tested with and without phages addition, but also the resistance to the phages in the presence and absence of antibiotics. The aim of this study was to evaluate the potential synergistic effect of phages and antibiotics in the inactivation of Escherichia coli in order to control infections, namely urinary tract infection (UTI), and to reduce the development of bacterial resistance to phages. Phage therapy combined with antibiotics (ampicillin, piperacillin, kamanycin, tetracycline, chloramphenicol and ciprofloxacin) was evaluated in the inactivation of E. coli, both in saline solution and urine samples. Phage and antibiotic combinations could result in high synergistic effects in the inactivation of bacteria. The combination of phage and ciprofloxacin at sublethal concentration decreased the bacterial counts in urine samples by 7.8±0.1 log CFU/ml after 8h, but when phages or the antibiotic were tested alone, the decrease was of 3.9±0.3 log CFU/mL and 1.2±0.1 log CFU/mL, respectively, after the same time. The efficacy of the combination of the two therapies depends on the antibiotic resistance status of the targeted bacteria to the employed antibiotic and of the antibiotic type (bactericide or bacteriostatic), causing the same or less bacterial resistance than phages and antibiotics applied alone (1.2±1.0×10-5 to 2.4±1.5×10-7 CFU/mL for the combined treatment, 2.7±0.2×10-4 CFU/mL for the antibiotics and 5.0±1.5×10-6 CFU/mL for the phages). The addition of antibiotics, at subinhibitory concentration, during phage treatment can control the phage-mutant. The high bacterial inactivation efficiency of these combined techniques and the long periods of phage survival in urine, pave the way for depth studies to control UTI and to overcome the development of resistances by bacteria.