RESUMEN
BACKGROUND AND OBJECTIVES: Methylene blue (MB) and toluidine blue (TB) form metachromatic complexes with lipopolysaccharides (LPS). The greater photobactericidal efficacy of TB may be explained by its affinity for LPS. This study aims to elucidate the difference in photobactericidal efficacies between the dyes using Ca(2+) as a competitor for dye-binding sites on the bacterial outer membrane. STUDY DESIGN/MATERIALS AND METHODS: Fixed dye concentration solutions with gram-negative bacteria and increasing concentrations of CaCl(2) were exposed to red laser light. Bacterial survival and spectrophotometry were used to describe the effect of Ca(2+) on dye interaction with bacteria and LPS. RESULTS: MB-mediated photokilling was inhibited more significantly than that of TB. CaCl(2) inhibited dye photobleaching and suppressed the metachromatic reaction between the dyes and LPS, in particular TB. CONCLUSIONS: CaCl(2) inhibits bacterial photokilling by binding with LPS, as well as other anionic polymers including outer membrane proteins. LPS is chiefly involved in TB-mediated photokilling, whereas outer membrane proteins probably are more involved in MB-mediated photokilling.
Asunto(s)
Cloruro de Calcio/metabolismo , Colorantes/farmacología , Bacterias Gramnegativas/efectos de los fármacos , Lipopolisacáridos/metabolismo , Azul de Metileno/farmacología , Cloruro de Tolonio/farmacología , Cloruro de Calcio/farmacología , Colorantes/metabolismo , Rayos Láser , Azul de Metileno/metabolismo , Espectrofotometría , Cloruro de Tolonio/metabolismoRESUMEN
BACKGROUND AND OBJECTIVES: The difference in the photobactericidal efficacy of methylene blue and toluidine blue against gram-negative bacteria may result from their primary reaction with lipopolysaccharides (LPS) of the outer bacterial membrane. The aim of the present study was to compare the reactivity of these dyes with LPS extracted from different gram-negative bacteria. STUDY DESIGN/MATERIALS AND METHODS: The interactions of methylene blue and toluidine blue with LPS from Escherichia coli (E. coli), Pseudomonas aeruginosa (P. aeruginosa), Klebsiella pneumoniae (K. pneumoniae), and Serratia marcescens (S. marcescens) were studied spectrophotometrically in 0.45% saline. The dyes were used at the concentration of 10 microM. The concentrations of LPS ranged from 5-100 microg/ml. RESULTS: Methylene blue and toluidine blue enter into a metachromatic reaction with the LPS resulting the in generation of dimers of methylene blue and higher aggregates of toluidine blue. The more significant hypochromic and hypsochromic effects in the reaction of the latter with LPS indicate a greater metachromatic efficacy of toluidine blue than methylene blue. The equilibrium constants of the metachromatic complex between toluidine blue and different LPS were calculated. The spectrophotometric titration of LPS with the dyes was used to estimate the equivalent weight of LPS. CONCLUSIONS: Toluidine blue interacts with LPS more significantly than methylene blue in vitro. This may be one of the main factors determining its greater photobactericidal efficacy against gram-negative bacteria.
Asunto(s)
Antibacterianos/farmacología , Bacterias Gramnegativas/efectos de los fármacos , Lipopolisacáridos/metabolismo , Azul de Metileno/farmacología , Fotoquimioterapia/métodos , Cloruro de Tolonio/farmacología , Bacterias Gramnegativas/efectos de la radiación , Lipopolisacáridos/efectos de la radiación , Resultado del TratamientoRESUMEN
The interactions between the phenothiazine dyes, methylene blue (MB) and toluidine blue (TB), and bacteria (Staphylococcus aureus, Streptococcus pneumoniae, Enterococcus faecalis, Hemophilus influenzae, Escherichia coli and Pseudomonas aeruginosa) were studied spectrophotometrically. This demonstrated that a metachromatic reaction took place between the dyes and bacteria. Furthermore, bacteria induced additional dimerization of MB and TB. The effective dimerization constants of MB and TB were evaluated in the presence of each bacterial strain at a concentration of 10(8) CFU/ml. The analysis of the effective dimerization constants for MB and TB in the presence of bacteria indicated that the ability to form dimers was greater for TB than for MB. Gram-negative bacteria induced the dye dimerization more intensely than gram-positive bacteria. There was a correlation between the ability of each dye to form dimers in the presence of bacteria and the relative photobactericidal efficacy of each dye against these bacteria. These results provide evidence confirming the essential role of the dye dimers in bacterial photodamage.