Vancomycin-sensitized photooxidation in the presence of the natural pigment vitamin B2: Interaction with excited states and photogenerated ROS

Abstract

Sensitized photooxidation processes in the presence of natural pigments may provide an alternative to antibiotics degradation since these compounds are transparent to natural light irradiation, therefore, they can be degraded by the action of photosensitizers which absorb light and produce highly reactive species, especially those derived from molecular oxygen (ROS). Most antibiotics used currently belong to a group of pharmaceutical substances that have been considered a new type of contaminants due to their persistence and bioaccumulation in the environment.

Objective: In this context, we decided to investigate the kinetic and mechanistic aspects of Vancomycin (Vanco) photosensitized degradation in the presence of the natural pigment Riboflavin (Vitamin B2, Rf) and the artificial dye Rose Bengal (RB) for comparative purposes.

Methods: The study have been done by using Stationary photolysis, Laser flash photolysis, Time-resolved phosphorence detection of O₂(1Δg) experiments and Bactericidal activity evaluation. The experiments were carried out in aqueous solution at different pH values in order to establish relationships between the structure of the compound and its susceptibility to ROS-mediated photooxidation.

Results: Experimental evidence indicates that in the presence of Rf there is considerable contribution of the radical-mediated mechanism, while in the presence of RB the photooxidation process occurs exclusively through O₂(1Δg) and the reactivity to this excited species increases with increasing pH of the environment.

Discussion: The results obtained, have been shown that Rf can raise the photodegradation of Vanco by both the radical pathway and the O₂(¹Δ_g) mediated. Furthermore, the antibiotic is able to interact with the excited electronic states of Rf as well as O₂(¹Δ_g) generated by energy transfer between the excited triplet state of the photosensitizer and the oxygen ground state. The predominant mechanism for photodegradation of Vanco in the presence of the Rf is the radical via because of the considerable interaction with the excited triplet state of the photosensitizer demonstrated by laser flash photolysis experiments. Microbiological test on Staphylococcus aureus ATCC25923 showed that the bactericidal activity of the antibiotic on the strain studied was affected by the sensitized photodegradation process, suggesting that photoproducts generated eventually do not retain the bactericidal properties of the original antibiotic.

Keywords: Antibiotics; Microbiological assay; Photooxidation; Reactive oxygen species; Riboflavin; Rose Bengal; Vancomycin; pH effect.

Scheme 1

Chemical structure of vancomycin (Vanco).

Scheme 2

Possible reactions pathways for the visible-light mediated photosensitized degradation of Vanco (A) and reaction of ROS with specific auxiliary quenchers. S: sensitizer (Rose Bengal or riboflavin).

Figure 1

Spectral changes of 0.1 mM vancomycin + 0.02 mM riboflavin vs. 0.02 mM riboflavin upon visible light photoirradiation in aqueous solution at pH 7.4. The numbers on the spectra represent irradiation time in min. Inset I: Relative rates of oxygen uptake (v_−ΔO2) of: (a) 0.02 mM riboflavin + 0.5 mM vancomycin; (b) 0.02 mM riboflavin + 0.5 mM vancomycin + 1 mM sodium azide; (c) 0.02 mM riboflavin + 0.5 mM vancomycin + 1 mg/100 ml superoxide dismutase; (d) 0.02 mM riboflavin + 0.5 mM vancomycin + 10 mM d-mannitol; (e) 0.02 mM riboflavin + 0.5 mM vancomycin + 1 mg/100 ml catalase. Inset II: Spectral changes in Argon-saturated solution of 0.02 mM riboflavin upon visible light photoirradaition (a) without vancomycin and non-irradiated; (b) without vancomycin, 5 minutes irradiation; (c) in the presence of 0.5 mM vancomycin, 5 minutes irradiation. Solvent: aqueous solution at pH 7.4.

Figure 2

Transient absorption spectra of (a) 0.01 mM riboflavin and (b) 0.01 mM riboflavin + 0.05 mM vancomycin taken at 1 and 30 μs after the laser pulse, respectively, in Argon-saturated aqueous solutions. Inset I: Stern–Volmer plot for the interaction of ³Rf* with vancomycin. Inset II: transient absorption spectra of (a) 0.01 mM riboflavin (1 μs) and (b) 0.01 mM riboflavin + 0.05 mM vancomycin (30 μs) normalized at 670 nm, in Argon-saturated aqueous solutions.

Figure 3

Spectral evolution of 0.1 mM vancomycin upon sensitized photoirradiation with visible light in aqueous solution in the presence of Rose Bengal (A_549  nm= 0.5) of vancomycin/Rose Bengal vs. Rose Bengal at pH 11. Inset: Spectral evolution of 0.1 mM vancomycin upon sensitized photoirradiation with visible light in aqueous solution in the presence of Rose Bengal (A_549  nm= 0.5) of vancomycin/Rose Bengal vs. Rose Bengal at pH 7.4. The numbers on the spectra represent irradiation time in minuite.

Figure 4

First-order plots for 0.5 mM vancomycin consumption plus Rose Bengal (A_549  nm= 0.5) in aqueous solution at (a) pH 7.4 and (b) pH 11. Inset: Stern–Volmer plots for the quenching of O₂ (¹Δ_g)-phosphorescence emission by vancomycin in Rose Bengal (A_532  nm= 0.3)/D₂O at (a) pD 7.4 and (b) pD 11.

Figure 5

Effect of 0.75 mg/l vancomycin: (c) before and (d) after 2.5 h Rose Bengal-sensitized photolysis on S. aureus ATCC 25923; (a) cellular control without vancomycin; (b) cellular control with vancomycin and without Rose Bengal.