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Antimicrobial Agents and Chemotherapy, May 2009, p. 2218-2220, Vol. 53, No. 5
0066-4804/09/$08.00+0     doi:10.1128/AAC.00829-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Triple Combinations of Lower and Longer Alkyl Gallates and Oxacillin Improve Antibiotic Synergy against Methicillin-Resistant Staphylococcus aureus{triangledown}

Hirofumi Shibata,1 Tatsuro Nakano,1 M. Anowar Khasru Parvez,1 Yoshihiro Furukawa,1 Akio Tomoishi,1 Shingo Niimi,2 Naokatu Arakaki,1 and Tomihiko Higuti1*

Department of Molecular Cell Biology and Medicine, Institute of Health Biosciences, The University of Tokushima Graduate School, 1-78 Sho-machi, Tokushima 770-8505, Japan,1 Division of Biological Chemistry and Biologicals, National Institute of Health Sciences, Kamiyoga, Tokyo 158-8501, Japan2

Received 24 June 2008/ Returned for modification 22 October 2008/ Accepted 2 February 2009


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ABSTRACT
 
Using liposome systems, we found that gallates with short alkyl chains were located in the external medium and those with longer alkyl chains were located in the surface region of lipid bilayer. Combinations of these gallates remarkably reduced oxacillin MICs against methicillin-resistant Staphylococcus aureus to below the antibiotic breakpoint (≤2 µg/ml).


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INTRODUCTION
 
We have shown that some alkyl gallates at sub-MIC concentrations, in combination with β-lactams, modulated the activities of antibiotics by reducing the concentration of antibiotics needed to inhibit the growth of methicillin-resistant Staphylococcus aureus (MRSA) (6, 9). The effect, as well as the intrinsic antimicrobial activity, of alkyl gallates was associated with the length of the alkyl chain (9). An alkyl gallate is characterized molecularly by the amphiphilic property. We considered that such a property is presumably important for the establishment of a molecular interaction with the phospholipid acyl chains of the cell membrane of S. aureus (1).

In this study, we aimed to obtain some clue for demonstration of the location of alkyl gallates in the staphylococcal cell and to confirm whether or not combinations of alkyl gallates have a capability to improve the synergistic effect with oxacillin on MRSA strains.

Commercially unavailable alkyl gallates were synthesized as described previously (7, 9). 2-Anthroyloxystearic acid (2-AS), 12-AS, and phosphatidylcholine from egg yolk (egg PC) were obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Other reagents were of the highest grade commercially available.

All strains of MRSA (19 strains) and methicillin-sensitive S. aureus (MSSA; seven strains) used throughout this work were recovered from our laboratory stock culture collection (8), including clinical isolates of MRSA (17 strains) and MSSA (6 strains), MRSA COL and Mu 3, and MSSA RN4220.

The MICs of the chemicals and antibiotics were determined as described previously (9). Fractional inhibitory concentration (FIC) indices for triple combinations were calculated as follows: FIC = FICOXA + FICB + FICC = (CcombOXA/MICOXA) + (CcombB/MICB) + (CcombC/MICC), where CcombB and CcombC are the concentrations of alkyl gallates tested; CcombOXA is the lowest concentration of oxacillin in the combination that inhibited growth; and MICOXA, MICB, and MICC are the MICs of the compounds when used alone. For combinations of two compounds, the term CcombC/MICC was omitted. An FIC index of ≤0.5 indicates synergy.

Liposome was prepared using egg PC in the presence of either 2-AS or 12-AS, as described previously (3-5) with modifications. The resulting small unilamellar vesicles were suspended in phosphate-buffered saline (PBS; pH 7.4). The final concentration of egg PC in the liposome solution was adjusted to 1 mg/ml. After incubation with an alkyl gallate for 10 min, the liposomes were purified by centrifugation and suspended with PBS. The fluorescence of 2-AS (excitation, 362 nm; emission, 446 nm) or 12-AS (excitation, 381 nm; emission, 446 nm) in the lipid bilayer was measured with a Hitachi fluorescence spectrophotometer F4500 (Hitachi, Ltd., Tokyo, Japan). The fluorescence intensity was calculated as a percentage of the fluorescence intensity of 2-AS or 12-AS in the absence of alkyl gallates.

The anthroyl moieties include in this study are linked to carbons 2 and 12 of the base fatty acid and report the location of alkyl gallates at a defined depth within the membrane bilayer. Hence, if an alkyl gallate is located on the surface of the lipid bilayers, it quenches the fluorescence of 2-AS. On the other hand, if it is located in the hydrophobic core of the lipid bilayers, it quenches that of 12-AS (3-5). In addition, the degree of fluorescence quenching is closely correlated with the amount of compound incorporated into the lipid bilayers (3, 4).

As shown in Fig. 1, gallic acid and gallates with the short alkyl chain, such as methyl, ethyl, propyl, and butyl gallates, had no quenching effect on the fluorescence of 2-AS or that of 12-AS, indicating that gallates with the short alkyl chain are located near the lipid-water interface. The results obtained using a model membrane system are quite suggestive of the behavior of alkyl gallates upon the staphylococcal cell membrane, even though there are some differences in membrane compositions between them: it would be probable that gallates with short alkyl chains would act from the exterior part, which is near the lipid-water interface, of the staphylococcal cell membrane, while gallates with longer alkyl chains would act from the polar part of the phospholipid palisade in the staphylococcal cell membrane.


Figure 1
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  		FIG. 1. Effects of alkyl gallates on the fluorescence intensity of 2-AS or 12-AS. An alkyl gallate in 0.3 ml of PBS (2 mM) was added to 2.7 ml of liposomal solution containing 2-AS (open circles) or 12-AS (closed circles). After incubation of the mixture for 10 min at 20°C, the fluorescence intensity of 2-AS or 12-AS in the lipid bilayers was measured. The results are expressed as mean values of two independent experiments.

We predicted that such a difference in location of gallates with distinct lengths of alkyl chains would be reflected in their mechanisms of action, although the mechanisms are unknown. Consequently, the combination of the gallates with the distinct lengths of alkyl chains would cause a dramatic reduction in the MICs of β-lactams against MRSA strains. For this, we used propyl gallate (PG) and octyl gallate (OG) as representatives of a gallate with a short alkyl chain and one with a longer alkyl chain, respectively.

Table 1 summarizes MIC ranges, MIC50s, and MIC90s of oxacillin from testing the antimicrobial activities of mixtures of oxacillin, PG, and OG against 19 strains of MRSA and 7 strains of MSSA, as well as the resulting FIC indices. The oxacillin MICs against MRSA and MSSA strains examined in this study were 64 to 512 and 0.125 to 1 µg/ml, respectively. The combination of PG, OG, and oxacillin showed a remarkable improvement of antimicrobial activity of the antibiotic against MRSA strains, and the FIC indices indicated strong synergy between oxacillin, PG, and OG against all MRSA strains. In particular, the combination of 25 µg/ml PG and 6.25 µg/ml OG with oxacillin is able to reduce the antibiotic MICs of MRSA strains from high levels of resistance (64 to 512 µg/ml) to below the antibiotic breakpoint (≤2 µg/ml).


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  		TABLE 1. MICs of oxacillin in combination with PG and OG

The mechanism of alkyl gallate-mediated β-lactam resistance modulation is at present unclear; however, this study highlights the cooperative nature of the interactions between gallates with a longer alkyl chain and gallates with a short alkyl chain with regard to their membrane-associated biological effects. According to the report of the 28th Session of the Codex Committee on Food Additives and Contaminants (2), the temporary acceptable daily intake of PG in mg/kg of body weight is 0 to 1.4, whereas that of OG was not allocated. Therefore, further studies are needed to establish the pharmacokinetics and toxicity of octyl gallate.


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ACKNOWLEDGMENTS
 
We thank Katsuko Kajiya, Tatsuhiro Ishida, and Satoru Ueno for help with the preparation of liposome.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Molecular Cell Biology and Medicine, Institute of Health Biosciences, The University of Tokushima Graduate School, 1-78 Sho-machi, Tokushima 770-8505, Japan. Phone: 81-88-633-7254. Fax: 81-88-633-9550. E-mail: higuchi{at}mibct.com Back

{triangledown} Published ahead of print on 17 February 2009. Back


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Antimicrobial Agents and Chemotherapy, May 2009, p. 2218-2220, Vol. 53, No. 5
0066-4804/09/$08.00+0     doi:10.1128/AAC.00829-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.




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