RESEARCH PAPER TWO

ANTIMICROBIAL ACTIVITY AGAINST MRSA

BACTERIOCIDAL ACTIVITY AGAINST
METHICILLIN-RESISTANT STAPHYLOCOCCUS AUREUS
IN THE CULTURE BROTH OF ENTEROCOCCUS FAECALIS TH10

Authors: Iichiroh Ohhira, Ph.D., Takashi Tamura, Ph.D., Nozomi Fujii, Ph.D.,
Kenji Inagaki, Ph.D., and Hidehiko Tanaka, Ph.D.
Correspondence Author: Hidehiko Tanaka

Address: Department of Biological Resources Chemistry, Faculty of Agriculture
Okayama University, Okayama 700, Japan

Bioactive Research Institute, 2-1-1, Gakunan-cho, Okayama 700, Japan

(Thesis Published in 1996)

In recent years, there has been a dramatic increase in the incidence of hospital-associated (nosocomial) infections caused by Staphylococcus aureus strains that are resistant to multiple antibiotics (1-3); some strains now demonstrate resistance to as many as 20 antimicrobial compounds, including antiseptics and disinfectants (4). These strains are collectively termed methicillin resistant S.aureus (MRSA). The incidence of MRSA out-break varies throughout the world (5-7), but it is particularly high in Japan where the cases are almost four-times that reported in Europe (8). Although the threat to patient care posed by such organisms has stimulated continuing efforts to search for potent anti-MRSA agents (9,10), there has been a growing concern that the pharmaceutical industry may no longer be able to develop novel antibiotics sufficiently quickly (4). It is also pointed out, however, that most of the antimicrobial compounds, to which MRSA shows intransigent resistance, has been produced by soil bacteria such as Streptomyces and Bacillus genera and fungi, Penicillium and Cephalosporium, thus novel anti-MRSA agents may be discovered from other microorganisms that have not been examined so intensively (11,12).

Lactic acid bacteria, a physiologically related group of Gram positive bacteria, produce a variety of compounds with antimicrobial activity (13,14). Some of these are proteins or peptides, and they are termed bacteriocin. Bacteriocin is generally defined as extracellularly released peptide or protein that shows a bacteriocidal activity against those closely related to the producer species (15,16). However, it has now become evident that many bacteriocins from lactic acid bacteria have somewhat broader spectrum of activity, affecting also more distantly related species (17,18). In fact, bacteriocin-producing lactic acid bacteria appear to interfere with the growth of the food-borne pathogen Listeria monocytogenes during fermentation process (19-21). Researchers have long discussed the potential application of bacteriocins in prevention and even in treatment of various infectious diseases (22).

To exploit the potential of bacteriocins for the control and chemotherapy of MRSA, bioassay directed screening of lactic acid bacteria was carried out. Several strains were found to produce antimicrobial activity in their culture broths, which inhibited the in vitro growth of MRSA on agar medium. Enterococcus faecalis TH10, an isolate from a Malaysian fermentation food, tempeh, was especially among the lactic acid bacteria tested. More interestingly, the active component was readily extracted by ethyl acetate at pH 3. The present communication describes the cultivation of E. faecalis TH10, extraction of anti-MRSA component, and its inhibitory spectrum to various lactic acid bacteria.

A stock culture of E. faecalis TH10 was inoculated in 10 ml of the seed medium containing 1.5% polypeptone, 0.5% yeast extract, 0.25% NaCl, 1.0% glucose, 0.05% sodium thioglycolate, 0.025% L-cystine, 0.01% sodium carbonate (pH 6.6 before sterilization.) After incubation at 37 degrees centigrade for 24 hours, the broth was transferred to one liter of the same medium, and cultivation was carried out at 37 degrees centigrade for eight days. The fermentation broth was centrifuged to remove cells, and the supernatant solution was extracted with ethyl acetate of pH3. The solvent layer was dried over anhydrous Na2SO4, and concentrated under reduced pressure. The residue was dissolved with 2 ml of 2 N NaOH solution, and neutralized to pH 7. Bioassay was performed by placing paper disks saturated with the solution on a nutrient agar plate and seeded with suspension of test organism. After incubation at 30 degrees centigrade for 24 hours, a zone of growth inhibition around the disk was presumed to indicate the presence of active compound. The minimum inhibitory dosage was determined by the dilution method with 2-fold dilution.

Bacteriocidal activity of the ethyl acetate extract was tested using MRSA and a variety of lactic acid bacteria as test organisms (Table). Contradictory to the common properties of bacteriocins, the extract did not show potent activity against closely related lactic acid bacteria. The growth inhibition was potent against MRSA, while most of the lactic acid bacteria tested were not affected. Streptococcus salivarius and Pediococcus acidilactici were affected, but only when the dosage was twice the amount required for the inhibition of MRSA. In addition, we found that the active component retained the bacteriocidal activity against MRSA when it was treated with various proteases such as proteinase K, V8 protease, trypsin and achromopeptidase. The extract of minimum inhibitory dosage was treated at 37 degrees centigrade overnight with 0.1 mg of each proteolytic enzyme, and the activity was assayed as described above. The results suggest that the active component is not peptide or a protein. It rather appears to be an acidic low molecular weight substance, since it was extracted with ethyl acetate of pH 3.

E. faecalis has been isolated from natural food samples such as traditional French cheese (23), mozzarella cheese whey (24) and foods in a retail supermarket (25). However, in most of the cases the species has been recognized as human pathogens with multiple antibiotic-resistance and high-level aminoglycoside resistance (26-28). Many clinical isolates of Enterococcus faecalis have been reported to produce bacteriocin which mediates the lysis of a broad range of Gram-positive bacteria (29-31), and the molecule also acts as a hemolysin which effectively lyses human, rabbit and horse erythrocytes (32). The active component of the TH10 strain, however, failed to show hemolytic activity toward human and rabbit erythrocytes.

Further study is in progress to purify and characterize the active substance using silica gel column, activated charcoal and anion-exchange resin and the results will be reported soon. In the current situation where the discovery of new antimicrobial agents are becoming increasingly difficult, the present study suggests that investigation of lactic acid bacteria may offer some potential applicability to chemotherapy of MRSA.

Acknowledgement from the research team:

This work was supported by a grant from Bioactive Research Institute. The authors are grateful to Dr. Taku Miyamoto, Faculty of Agriculture, Okayama University, for providing us various lactic acid bacteria.

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