In Vitro Antibacterial Activities of Phloxine B and Other Halogenated Fluoresceins against Methicillin-Resistant Staphylococcus aureus

Abstract

Fluorescein dyes in which the benzoic acid moiety has been tetrachlorinated (50 to 100 μg/ml) inhibit in vitro Staphylococcus aureus growth (MIC, 25 μg/ml). Specifically, under standard room illumination, phloxine B at a concentration of 100 μg/ml killed 99% of the cultures (mid-log phase). It also reduced S. aureus CFU by 10⁴. Structure-activity analysis revealed that the activity against S. aureus increases with the increase in the number of the substituting halogens in the hydroxyxanthene moiety.

Staphylococcus aureus is one of the most important bacterial pathogens. It is found in 30 to 50% of the general population (14), and in a healthy carrier, it is commonly located in mucous membranes, such as the upper respiratory tract (10) and on skin and on clothing (11). The only antibiotics to which methicillin-resistant S. aureus (MRSA) did not develop resistance by 1996 were the glycopeptides vancomycin and teicoplanin (13). In 1996, the first case of MRSA with intermediate resistance to vancomycin was reported in Japan, followed by cases in Slovakia, France, the United States, Spain, and Turkey (8).

Because of the emergence of increased antibiotic resistance of S. aureus and other bacteria and since there are few therapeutic alternatives (9) for infections produced by vancomycin-resistant S. aureus, development of new antibacterial drugs has become the top priority for many pharmaceutical companies (6). In the context of these research goals, the present study investigates the effect of a series of halogenated fluorescein dyes, especially phloxine B, against S. aureus. Phloxine B (color index no. 45410) is a hydroxyxanthene color additive used for coloring foods in Japan (food red 104) (7), and its Food and Drug Administration (FDA)-certified counterpart, D&C red no. 28, is used for coloring drugs and cosmetics in the United States (1). It is identified principally as the disodium salt of 2′,4′,5′,7′-tetrabromo-4,5,6,7-tetrachlorofluorescein (compound 14 [see Table 1]). Under the name phloxine B, this dye is used as a biological stain (3) (Sigma-Aldrich handbook of stains, dyes, and indicators; Aldrich, Milwaukee, Wis.). Due to its photoactive properties (12), phloxine B was found to be a useful pesticide (5) and a bactericidal agent against the plant pathogen Agrobacterium tumefaciens (21). The aim of the present study was to determine the in vitro anti-S. aureus activity of phloxine B and other halogenated fluorescein dyes against seven strains of MRSA and a strain of enterotoxigenic S. aureus under conditions of standard room illumination, with no special light activation, to explore its potential as a new antibiotic.

TABLE 1.

Chemical structure of the main component of the halogenated fluorescein dyes used in the present study

^aFluorescein disodium salt.

The seven clinical strains of MRSA used in this study were obtained from the FDA (Washington, D.C.) collection. The enterotoxigenic strain used was ATCC 13565 (American Type Culture Collection, Manassas, Va.).

The chemical structures of the fluorescein dyes tested are shown in Table 1. Their source varied: some were purchased directly from the manufacturer, others were selected from batches submitted to FDA for certification during the past 6 years, and still others were obtained through separation using pH zone-refining countercurrent chromatography. Specifically, the compounds used were as follows. Compound 1 is uranine (fluorescein disodium salt) (Fluka, Buchs, Switzerland). Compound 2 is 4′-bromofluorescein (4′-BrF) (15). Compound 3 is the purified 4′,5′-dibromofluorescein (4′,5′-Br₂F). Compound 4 is 2′,4′,5′-tribromofluorescein (2′,4′,5′-Br₃F). Compound 5 is the main component of eosin, 2′,4′,5′,7′-tetrabromofluorescein (2′,4′,5′,7′-Br₄F) (20); eosin was also purchased from two companies (Fluka and Aldrich [Milwaukee, Wis.]). Compound 6 is 4′,5′-dichlorofluorescein (4′,5′-Cl₂F) and was a stock sample of D&C orange no. 8, which is no longer certified by the FDA. Compound 7 is 2′,7′-dichlorofluorescein (2′,7′-Cl₂F) (Fluka). Compound 8 is 4′,5′-diiodofluorescein (4′,5′-I₂F) and a portion of D&C orange no. 10. Compound 9 is erythrosine (2′,4′,5′,7′-tetraiodofluorescein) (Kodak, Rochester, N.Y.). Compound 10 is 4,5,6,7-tetrachlorofluorescein (4,5,6,7-Cl₄F) (TCF) (17). Compound 11 is 4′-bromo-4,5,6,7-tetrachlorofluorescein (4′-BrTCF) (19). Compound 12 is 4′,5′-dibromo-4,5,6,7-tetrachlorofluorescein (4′,5′-Br₂TCF) (18). Compound 13 is 2′,4′,5′-tribromo-4,5,6,7-tetrachlorofluorescein (2′,4′,5′-Br₃TCF) (16). Compound 14 is phloxine B (from the following three manufacturers: Fluka, Sigma [St. Louis, Mo.], and Pfaltz & Bauer [Waterbury, Conn.]). The purified main component of phloxine B (2′,4′,5′,7′-Br₄TCF) was obtained as described previously (16). Compound 15 is Rose Bengal (from Kodak and Aldrich).

For the preparation of each solution of dye, a test portion (approximately 10 mg) was placed in a 10-ml volumetric flask, which was wrapped in aluminum foil, and dissolved by adding aqueous sodium hydroxide (≈0.4% NaOH) (1 ml) and water (9 ml) (high-pressure liquid chromatography grade; Burdick & Jackson, Muskegon, Mich.). The solutions obtained by this procedure were found to be stable for at least 6 months in a refrigerator at 4°C.

For the growth of bacteria, brain heart infusion broth (BHI) (Difco Laboratories, Detroit, Mich.) was used. Cultures were incubated shaking (200 rpm) at 37°C. All the experiments were conducted under conditions of standard room illumination (fluorescent ceiling light, Sylvania Octron 4100K) in tubes containing 3 ml of medium. Growth was analyzed by measuring turbidity (absorbance at a wavelength of 660 to 680 nm) with a spectrophotometer (Spectronic 21D; Milton Roy, Riviera Beach, Fla.). Experiments were initiated with ∼0.2 ml of inoculum from a fresh culture grown aerobically at an optical density (OD) of ∼1. Colony counts were measured on BHI plates. For the susceptibility tests, an S. aureus culture grown on a BHI plate (1 to 14 days old) was used to inoculate 20 ml of BHI in a 200-ml flask (OD, ∼0.05). Starter culture was grown for 2 to 3 h to late log phase (OD, ∼1.8) with vigorous aeration (210 rpm). One milliliter of this culture was used to inoculate 25 ml of BHI, and the resulting OD was measured. Two-milliliter aliquots of this diluted culture were placed in sterile tubes. For testing, a 300-μl aliquot of dye solution (1 mg/ml) was mixed with 0.7 ml of BHI and then added to a tube of diluted culture. The tubes were aerated vigorously, and the OD was measured every 45 min to check culture growth. The survival rate was measured by plating serial dilutions of a sample taken during the experiment on BHI plates.

The effect of each dye (100 μg/ml) on S. aureus (ATCC 13565) is shown in Fig. 1. All the dyes used are closely related structurally, but they differ in the following halogenation characteristics: type of halogen, number of the substituting halogen molecules, and position of the substitution. They were chosen in such a way as to facilitate the in vitro assessment of the relationship between chemical structure and activity against enterotoxigenic S. aureus and MRSA. All experiments were performed in triplicate on separate days to ascertain reproducibility. The nonhalogenated fluorescein (compound 1) and most of the mono-, di-, tri- and tetrahalogenated fluoresceins in the hydroxyxanthene moiety (halogens in some or all of the 2′, 4′, 5′, and 7′ positions [Table 1]) (compounds 2 to 9) have no activity against S. aureus, that is, they are no different than the control (buffered culture to which no dye was added), as shown in Fig. 1a. The only exceptions are 2′,7′-dichlorofluorescein (compound 7) and 2′,4′,5′,7′-tetraiodofluorescein (compound 9), which show some anti-S. aureus activity. In contrast, with the exception of compound 10, which has no substitutions in the hydroxyxanthene moiety, all the tetrachlorinated compounds in the benzoic acid moiety (chlorine atoms in positions 4, 5, 6, and 7 [Table 1], compounds 11 to 15) showed anti-S. aureus activity (Fig. 1b). In addition, the activity seemed to increase, in the latter group, with the increase in the number of substituting halogens in the hydroxyxanthene moiety.

FIG. 1.

Comparison of the effects of variously halogenated fluoresceins (100 μg/ml) on the growth of S. aureus. (a) Fluorescein disodium salt (compound 1) and the fluoresceins in which the hydroxyxanthene moiety has been brominated, chlorinated, and iodinated (compounds 2 to 9). Symbols: ▪, compound 1; ▿, compound 2; ▾, compound 3; ○, compound 4; •, compound 5; ⧫, compound 6; □, compound 7; , compound 8; , compound 9; ◊, control culture (no dye added). (b) TCF (compound 10) and the brominated and iodinated TCFs (compounds 11 to 15). Symbols: ▿, compound 10; ▾, compound 11; ▪, compound 12; •, compound 13; ○, compound 14; ◊, compound 15; □, control culture (no dye added).

Rose Bengal, compound 15, is the only dye in this series that was shown previously to be cytotoxic to S. aureus and to inactivate it in the dark and in illuminated conditions (2, 22). Rose Bengal is used as a biological stain and as a photosensitizing dye (Sigma-Aldrich handbook of stains, dyes, and indicators; Aldrich), but is not permitted for human consumption in the United States.

This study concentrates on the anti-S. aureus activity of phloxine B, compound 14. FDA-certified batches of phloxine B (D&C red no. 28) are permitted in the United States for use in coloring cosmetics and ingested drugs with an acceptable daily intake for humans set by the FDA at 1.25 mg/kg of body weight (4). To determine the dose response to phloxine B, the growth curves of mid-log-phase S. aureus cultures treated with various concentrations of the dye were determined. The results are shown in Fig. 2. The responses of the mid-log cultures are dose dependent. The lowest concentration tested, 10 μg/ml, had little effect on growth (Fig. 2a) or on cell count (Fig. 2b). At 25 μg/ml, the dye slowed the growth of the culture and decreased the cell count by approximately 10-fold during 160 min. The concentration of phloxine B that inhibited 90% of S. aureus culture growth, based on this data, is 25 μg/ml. When dye was added at concentrations of 50 or 100 μg/ml, bacterial growth stopped completely and the cell count decreased by 10⁴ and 10⁵, respectively, within 80 min. These values point to a MIC of 25 μg/ml.

FIG. 2.

Bactericidal activity of phloxine B against S. aureus in mid-log-phase cultures. Cultures were incubated with various concentrations of phloxine B. The OD (a) and the CFU count (b) of the cultures were determined. Symbols: •, control culture (no phloxine B added); ○,10 μg/ml; ▾, 25 μg/ml; ▿, 50 μg/ml; ▪, 100 μg/ml.

To assess the effect of phloxine B on MRSA bacteria, seven MRSA strains were incubated with phloxine B. The response of these strains was similar to the response obtained from the control strain, ATCC 13565, which was used throughout the study. Similarly, colony counts show reductions from 6 × 10⁹ CFU to 2 × 10³ CFU within 3 h (data not shown). These results demonstrate that phloxine B has inhibitory activity against MRSA strains as well.

This study demonstrated that at a concentration of 100 μg/ml, phloxine B and other fluoresceins tetrachlorinated in the benzoic acid moiety, significantly inhibit S. aureus growth. It was also shown that there is a structure-activity relationship in this system, since the activity against S. aureus increases with the increase in the number of the substituting halogens in the hydroxyxanthene moiety. Furthermore, the chemical structure of phloxine B could be modified to potentially result in compounds with more potent antimicrobial activity. As for the safety of phloxine B, its current use as the main component of a color additive in food (7) and in drugs and cosmetics (1, 4) indicates that it has already met safety specifications. On the other hand, the common usage of phloxine B as a color additive in food, drugs, and cosmetics does bring the risk that bacteria might have the opportunity for exposure to small amounts of it over long periods of time, which might result in their developing resistance to its antimicrobial activity. The antibacterial properties of phloxine B could also be applied in veterinary medicine. Currently, various antibiotics are used in large quantities to control animal bacterial infections, which may contribute to the spread of multidrug-resistant bacteria. The use of alternative antimicrobial compounds, such as phloxine B, might be an efficient treatment that does not provide an opportunity for bacteria to acquire resistance to well-established antibiotics.