Rationale and mechanism for the low photoinactivation rate of bacteria in plasma

Abstract

The rate of bacterial photoinactivation in plasma by methylene blue (MB), especially for Gram-negative bacteria, has been reported to be lower, by about an order of magnitude, than the rate of inactivation in PBS and water solutions. This low inactivation rate we attribute to the bleaching of the 660-nm absorption band of MB in plasma that results in low yields of MB triplet states and consequently low singlet oxygen generation. We have recorded the change of the MB 660-nm-band optical density in plasma, albumin, and cysteine solutions, as a function of time, after 661-nm excitation. The transient triplet spectra were recorded and the singlet oxygen generated in these solutions was determined by the rate of decrease in the intensity of the 399-nm absorption band of 9, 10-anthracene dipropionic acid. We attribute the bleaching of MB, low singlet oxygen yield, and consequently the low inactivation rate of bacteria in plasma to the attachment of a hydrogen atom, from the S-H group of cysteine, to the central nitrogen atom of MB and formation of cysteine dimer.ADPA | photoinactivation deficiency | Leuco methylene blue | electrophilic attack | transient spectroscopy E ven though infections from bacterially contaminated plasma are thought to be very few (1) and the frequency of patient sepsis caused by transfusion of blood contaminated by bacteria is as low or less than the transfusion-associated hepatitis C virus infection (2), the possibility of such infection cannot be entirely discounted in view of the fact that new or even more resistant strains of known bacteria are being encountered. To a large extent, pooled human plasma is sterilized by the solvent-detergent method (3-5), whereas single-donor human plasma is often decontaminated by methylene blue (MB) photoexcitation, which generates reactive oxygen species that safely inactivate many viruses (6, 7). However, several Gram-positive and especially Gram-negative bacteria are found to be very resistant to photoinactivating agents such as porphyrins and thiazine dyes, possibly because the outer walls of Gram-negative bacteria, such as Serratia marcescens (SM), are composed of negatively charged lipopolysaccharides, which are resistant to photoinactivation by these dyes (8).Gram-positive bacteria and several Gram-negative bacteria are relatively easily inactivated, especially in high-pH MB-PBS solutions, after irradiation with 661-nm light for a rather short period, i.e., 10 min (9). Gram-negative bacteria usually require longer exposure times for inactivation. Table 1 lists the time required for the inactivation of two representative bacteria, coagulasenegative Staphilcocci, CoNS (Gram-positive) and SM (Gramnegative). Inactivation of bacteria dispersed in PBS at pHs 7 and 9 occurs within 20 min or less; however, note that when the same concentration of bacteria, 10 7 /mL, was dispersed in a solution of human plasma containing 2 × 10 −5 M MB and irradiated with the same photon flux of 6.8-mW, 661-nm light-emitting diode (LED) light, the inactivation rate was found ...

The low photo-inactivation rate of bacteria in human plasma II. Inhibition of methylene blue bleaching in plasma and effective bacterial destruction by the addition of dilute acetic acid to human plasma

“…These time resolved spectra changes indicate that while the 660 nm MB band OD decreases with light exposure at a rather high rate, the intensity of the 400 nm ADPA absorption band remains constant. These data are in agreement with the previously published data 24 that attribute the bleaching of the MB excited state to the loss of the MB ring conjugation, possibly, by the addition of an H-atom to the MB central ring nitrogen atom, forming the leuco-MB type molecule which does not absorb at 660 nm, Scheme 1. The expected very low photogeneration of singlet oxygen, if any, in the plasma/MB solu-Scheme 2 tions is substantiated by the unaltered OD of the 400 nm absorption band of ADPA with illumination, Fig.…”

“…The relative amount of singlet oxygen generated by each MB solution, after 661 nm LED illumination was determined by the slope of the decay of the 400 nm ADPA band OD as a function of irradiation time. 23 ADPA is known to react with singlet oxygen, to form endoperoxide, ADPA-O 2 , which does not absorb in the 400 nm region where ADPA has its first excited singlet state absorption band, 23,24 Scheme 2. Therefore the decrease of the 400 nm ADPA absorption band is a measure of the amount of singlet oxygen formed.…”

Aluminum plasmonic nanoshielding in ultraviolet inactivation of bacteria

“…Bacterial inactivation has recently received much attention due to the rising concern of antibiotic resistance 1 . Many alternative bacterial inactivation techniques have been developed, including the promising light-induced photo-inactivation methods such as photodynamic 2 – 6 and photothermal 7 – 11 treatments. The former relies on the interaction of light at frequencies typically in the visible and near-infrared range with photosensitizers which produce reactive species.…”

A nano phototheranostic approach of toluidine blue conjugated gold silver core shells mediated photodynamic therapy to treat diabetic foot ulcer

“…10 μM AMDA was added to the solutions containing monomicrobial and polymicrobial S. aureus and P. aeruginosa (10 8 CFU/mL) biofilms in PBS followed by the treatment as explained above. The reduction in the 399-nm absorption peak of AMDA after treatment corresponds to the amount of singlet oxygen produced 68 .O.D in untreated group (control) was taken as 100%. …”