Lysis of methicillin-resistant Staphylococcus aureus by 2,4-diacetylphloroglucinol produced by Pseudomonas sp. AMSN isolated from a marine alga

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Abstract

Previously 2,4-diacetylphloroglucinol (DAPG) produced by Pseudomonas sp. AMSN isolated from a marine alga, had demonstrated a high level of anti-methicillin-resistant Staphylococcus aureus (MRSA) comparable with that of vancomycin. In this study, this substance had bacteriolytic activity against MRSA at 1 μg/ml as well as similar activity against Vibrio parahaemolyticus at 24 μg/ml that suggests a novel antibacterial mode of action by this substance. Heat and pH stability tests showed it to be stable at temperatures ranging from 35 to 70 °C and at pHs ranging from 2–7. It was not acutely toxic to mice at levels up to 100 mg/kg.

Introduction

Methicillin-resistant Staphylococcus aureus (MRSA) causes many problems in public health not only to its common occurrence (<1–80%) [1], but also because it has become resistant to almost all the currently available antibiotics except teicoplanin and vancomycin. Its susceptibility to vancomycin has recently decreased and vancomycin-intermediate and -resistant S. aureus (VISA and VRSA) have been found [1], [2], [3], [4] in an increasing number of countries. This makes the discovery of alternative antibiotics and chemotherapeutics particularly important.

In a previous report, we briefly described the in vitro anti-MRSA activity of 2,4-diacetylphloroglucinol (DAPG) (Fig. 1) produced by Pseudomonas sp. strain AMSN isolated from the surface of a red alga (Ceratodiction spongiosum) [5]. Phloroglucinol is inactive. The anti-MRSA activity of DAPG against ten clinical isolates of MRSA and one isolate of methicillin-sensitive S. aureus were compared with 16 commercial antibiotics, and demonstrated activity comparable with that of vancomycin and higher than those of 15 commercial antibiotics. DAPG also demonstrates bactericidal activity against MRSA. Although DAPG is known to be produced by Pseudomonas spp. [6], [7], [8] and to be an antifungal substance, there has been no report on its anti-MRSA activity and antibacterial mechanism. Therefore, we evaluated the temperature and pH stabilities of DAPG, its mode of action based on bacteriolytic activity, and its acute toxicity.

Section snippets

Bacterial strains

The bacteriolytic activity of DAPG against Vibrio parahaemolyticus (IFO 12711) was compared with that against a clinical isolate of MRSA strain (E 31224). The antibacterial activity of DAPG was also evaluated against Pseudomonas aeruginosa (IFO 13736) and Pseudomonas sp. AMSN, a DAPG-producing bacterium.

2,4-Diacetylphloroglucinol

DAPG was purified from the culture supernatant of Pseudomonas sp. AMSN by silica gel column chromatography and silica gel TLC and HPLC on an Inertsil column (∅ 20×250 mm; GL Science Inc, Tokyo,

MIC of DAPG against MRSA and Gram-negative bacteria

DAPG showed high antibacterial activity against MRSA having a MIC of 1 mg/l. It was less active against V. parahaemolyticus with a MIC of 6 mg/l, and inactive against P.aeruginosa and Pseudomonas sp. AMSN, at 128 mg/l.

Bacteriolytic activity

The reduction of absorbance (at 660 nm) of MRSA and V. parahaemolyticus cell suspensions exposed to DAPG at various concentrations is shown in Fig. 2. The absorbance of MRSA cell suspension decreased drastically at 1–8 μg/ml of DAPG, but the cell suspension of V. parahaemolyticus

Discussion

The phenotypic and phylogenetic analyses based on the 16S rDNA show that the strain AMSN is closely related to P. fluorescens (data not shown), which is known to produce anti-fungal substances such as DAPG, pyoluteorin and penazin [6], [7], [8], [10].

DAPG inhibits the growth of causative fungi of plant root diseases [10], and also the plant pathogenic fungi Pythium ultimatum and Rhizotocnia solani, and a plant pathogenic bacterium Erwinia carotovora subsp. atraceptica [6]. Recently we have also

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