MC21-A, a bactericidal antibiotic produced by a new marine bacterium, Pseudoalteromonas phenolica sp. nov. O-BC30(T), against methicillin-resistant Staphylococcus aureus

Abstract

We previously reported a new marine bacterium, Pseudoalteromonas phenolica sp. nov. O-BC30(T), which produced a bactericidal antibiotic against methicillin-resistant Staphylococcus aureus (MRSA). In the present study, we purified an anti-MRSA substance (MC21-A) from the methanol extract of the cells of P. phenolica O-BC30(T) and analyzed its chemical structure. MC21-A was determined to be 3,3',5,5'-tetrabromo-2,2'-biphenyldiol by spectrometric analyses. Its anti-MRSA activity against 10 clinical isolates of MRSA was comparable to that of vancomycin (MC21-A MICs, 1 to 2 micro g/ml; vancomycin MICs, <0.25 to 2 micro g/ml). This substance was also high active against Enterococcus serolicida, Enterococcus faecium, and Enterococcus faecalis but was less active against Streptococcus spp. A time-kill study also demonstrated that MC21-A was bactericidal and that its killing rate was much higher than that of vancomycin. The postantibiotic effect (PAE) of MC21-A against a clinical MRSA isolate, strain E 31243, was also comparable to that of vancomycin (MC21-A PAEs, 1.46 to 1.65 h; vancomycin PAEs, 0.84 to 1.43 h). However, a lysis experiment demonstrated that this substance failed to lyse MRSA cells. This substance also did not lyse human erythrocytes. A SYTOX Green staining experiment implied that this substance permeabilized the cell membrane of MRSA as its mode of action. When its activities against a hypersensitive Escherichia coli mutant (KO 1489) and wild-type strains were tested, MC21-A exhibited higher levels of activity against the former. Furthermore, MC21-A was not cytotoxic to human normal fibroblast, rat pheochromocytoma, and Vero cells at concentrations up to 50 micro g/ml. These results suggest that MC21-A might be useful as a lead compound in the development of new types of anti-MRSA substances with modes of action different from those of vancomycin and teicoplanin.

FIG. 1.

Chemical structure of MC21-A.

FIG. 2.

Comparative bactericidal activities of MC21-A and vancomycin against a reference strain of MRSA ATCC 33591 (A) and a clinical isolate of MRSA, E 31243 (B). •, growth control; □, MIC; ▵, two times the MIC; ×, four times the MIC; ○, eight times the MIC. The values with standard error bars are mean values from duplicate experiments.

FIG. 3.

Bacteriolytic activities of MC21-A against a reference strain of MRSA (ATCC 33591) and a clinical isolate of MRSA (E 31243). •, positive control (1 μg of lysostaphin per ml); ○, the MIC; ▵, two times the MIC; □, four times the MIC. Relative absorbance was calculated by dividing the absorbance for the treated tube by that for the negative control tube. The values with standard error bars are mean values from triplicate experiments.

FIG. 4.

Cells membrane permeabilization of MRSA induced by MC21-A (A), chloramphenicol (B), amoxicillin (C), and vancomycin (D). ▵, untreated control; □, 1/16 the MIC; ○, 1/4 the MIC; ▪, the MIC; •, 4 times the MIC. The MICs of chloramphenicol and amoxicillin were each 64 μg/ml. The fluorescence values with standard error bars are the corrected mean values from quadruplet measurements. The corrected fluorescence values are each fluorescence value subtracted from the background value.

FIG. 5.

Hemolytic activity of MC21-A (•) compared to those of amphotericin B (▪) and gramicidin D (♦) against human red blood cells. The values are represented as the percentage of total lysis compared to the lysis caused by 0.1% Triton X-100. Each value with a standard error bar is the mean value from triplicate experiments.

FIG. 6.

Cytotoxicity of MC21-A to HDFs (♦), human leukemic cells (MOLT-4 cells) (○), MDCK cells (□), African green monkey kidney cells (Vero cells) (•), and rat pheochromocytoma cells (PC12D cells) (▪). The relative growth rates with standard error bars are mean values from triplicate experiments.