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 g/ml; vancomycin MICs, <0.25 to 2 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 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.

Four strains of aerobic, Gram-negative rods, motile by means of a single polar flagellum, that produced phenolic anti-methicillin-resistant Staphylococcus aureus (MRSA) substances and brown-pigmented colonies, were isolated from sea water. The G+C content of the DNA ranged from 39?9 to 40?6 mol%. The isolates grew at 18-37˚C and pH 6?5-9?5 (optimal pH 7?5-9) and in medium containing 1-5 % (w/v) NaCl (optimal NaCl concentration 2-3?5 %). The isolates grew optimally in medium dissolved in 40-100 % artificial sea water. Based on 16S rDNA similarities, the novel strains were closely related to Pseudoalteromonas luteoviolacea and Pseudoalteromonas piscicida, with 96?3 and 95?7 % sequence similarity, respectively. However, the strains could be differentiated from P. lutioviolacea by seven traits and from P. piscicida by 10 traits. Analysis of DNA-DNA relatedness to these related species revealed low levels of DNA hybridization (19?6 % to P. luteoviolacea and 22?4 % to P. piscicida). However, the type strain, O-BC30 T , and the other three bacterial isolates showed high DNA relatedness to each other, ranging from 84?8 to 93?7 %. Based on the results of phenotypic characterization, phylogenetic analysis based on 16S rDNA sequences and DNA-DNA hybridization, it is concluded that these isolates represent a novel species in the genus Pseudoalteromonas. Because the type strain, O-BC30 T (=IAM 14989 T =KCTC 12086 T ), produces phenolic anti-MRSA substances, the name proposed for this novel species is Pseudoalteromonas phenolica sp. nov.

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Pseudomonas is a genus of non-fermentative gram-negative Gammaproteobacteria found both on land and in the water. Many terrestrial isolates of this genus have been studied extensively. While many produce bioactive substances, enzymes, and biosurfactants, other Pseudomonas isolates are used for biological control of plant diseases and bioremediation. In contrast, only a few marine isolates of this genus have been described that produce novel bioactive substances. The chemical structures of the bioactive substances from marine Pseudomonas are diverse, including pyroles, pseudopeptide pyrrolidinedione, phloroglucinol, phenazine, benzaldehyde, quinoline, quinolone, phenanthren, phthalate, andrimid, moiramides, zafrin and bushrin. Some of these bioactive compounds are antimicrobial agents, and dibutyl phthalate and di-(2-ethylhexyl) phthalate have been reported to be cathepsin B inhibitors. In addition to being heterogeneous in terms of their structures, the antibacterial substances produced by Pseudomonas also have diverse mechanisms of action: some affect the bacterial cell membrane, causing bacterial cell lysis, whereas others act as acetyl-CoA carboxylase and nitrous oxide synthesis inhibitors. Marine Pseudomonas spp. have been isolated from a wide range of marine environments and are a potential untapped source for medically relevant bioactive substances.

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