Antimicrobial Activity Against Methicillin-Resistant Staphylococcus Aureus In The Culture Broth Of Enterococcus Faecalis Th 10, An Isolate From Malaysian Fermentation Food-Tempeh

By: Iichiroh Ohhira, Ph.D., October 2000

SUMMARY

Bioassay-directed screening for antimicrobial activity against methicillin-resistant Staphylococcus aureus (MRSA) led us to the culture broth of Enterococcus faecalis TH 10, which was isolated from a Malaysian fermentation food, Tempeh. The anti-MRSA component was readily extracted with ethyl acetate from the culture broth at pH 3, and the extract retained the activity when incubated overnight with various proteolytic enzymes. In addition, the extract showed poor antimicrobial activity against lactic acid bacteria, including Enterococcus faecalis. These properties strongly suggest that the active compound is distinct from bacteriocin, an extracellularly released species closely related to the bacteriocin-producing strain. E. faecalis has been reported to produce bacteriocin that has strong hemolytic activity on mammalian erythrocytes, but the active component of the TH10 strain did not mediate the lysis of rabbit and human erythrocytes.

Key words: Enterococcus faecalis, bacteriocin, methicillin-resistant Staphylococcus aureus

Lactic acid bacteria, a physiologically related group of gram-positive bacteria, produce a variety of compounds with antimicrobial activity, and they are termed bacteriocin. Bacteriocin is generally defined as extracellularly released peptide or protein that shows a bacteriocidal activity against species closely related to the bacteriocin-producing strain¹. It has now become evident, however, that many bacteriocins from lactic acid bacteria show somewhat broader spectrum of activity, affecting also more distantly related species². In fact, bacteriocin-producing lactic acid bacteria appears to interfere with the growth of the food-born pathogen Listeria monocygenes during fermentation process^3-5. Researchers have long discussed the potential application of bacteriocins in prevents, and even in treatment of infectious diseases.

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, and these strains are collectively termed methicillin-resistant S. aureus (MRSA). The incidence of MRSA outbreak is particularly high in Japan where the cases are almost four times that reported in Europe. Although the threat to patient care posed by such organisms has stimulated continuing efforts to search for potent anti-MRSA agents, there has been a growing concern that the pharmaceutical industry may no longer be able to develop novel antibiotics sufficiently quickly.

To exploit the potential of anti-microbial metabolite of lactic acid bacteria for the control and chemotherapy of MRSA, we carried out bioassay directly screening of microorganisms insolated from fermented foods and beverages. Several strains were found to produce anti-microbial activity in their culture broth, which inhibited the growth of MRSA on agar medium. Enterococcus faecalis TH 10, an isolate from a Malaysian fermentation food, Tempeh, was especially effective among the lactic acid bacteria isolated. More interestingly, the active component was readily extracted with ethyl acetate, and it retained the activity when treated with proteolytic enzymes. To see whether the anti-MRSA compound falls on the category of bacteriocin, we investigated various properties of the product of E. faecalis TH 10 strain: proteolytic treatment using various proteases and anti-microbial activity against closely related lactic acid bacteria. Hemolytic activity was also assayed for the comparison with previously reported bacteriocin/hemolysin produced by E. faecalis.

MATERIALS AND METHODS

Materials. Reagents were purchased from the following suppliers: proteinase K, V8 protease, achromopeptidase were from, Wako Pure Chemical Industries, Ltd. and trypsin was from Sigma Chemical Company. Other chemicals used were analytical grade reagents. Methicillin-resistant Staphylococcus aureus type-II strain was used in this study.
Culture of Enterococcus faecalis TH 10. A stock culture of E. faecalis TH 10 was inoculated on 10 ml of the seed medium containing 1.5% polypepton, 0.5% yeast extract, 0.25% NaCl, 1.0% glucose, 0.05% sodium thioglycolate, 0.025 9'0 L-cystine, 0.01% sodium sulfite, 0.01% sodium carbonate (pH 6.6 before sterilization). After incubation at 37 degrees C for 24 hours, the broth was transferred to one liter of the same medium, and cultivation was carried out at 37 degrees C for 8 days.
Extraction of Active Component. The fermentation broth was centrifuged at 4000 xg for 20 min at 4 degrees C to remove cells, and the supernatant solution was extracted with ethyl acetate at pH 3.0. The solvent layer was dried over anhydrous NA₂SO₄, and concentrated under reduced pressure. The residue was dissolved in 2 ml of 2 N NaOH solution, neutralized to pH 7, and the 60_pl portion was loaded on a paper disk. After the paper disk was dried, bioassay was performed by placing the disk on an agar plate and seeded with suspension of test organism. After incubation at 30 degrees C for 24 h, a zone of growth inhibition around the disk was presumed to indicate the presence of active compound.
Proteolysis Treatment. The ethyl acetate extract (10 mg in dry weight) was treated overnight at.37 degrees C with 0.1 mg of proteinase K, V8 protease, trypsin; and achromopeptidase, respectively in 100_vL of 20 mM potassium phosphate buffer at pH 7.5, loaded on paper disks and dried for the bioassay. The amount of the proteolytic enzymes used corresponds to 2.2 units for proteinase K, 1, 100 units for trypsin, 2.2 units for V8 protease, and 100 units for achromopeptidase.
Hemolysis Assay. Hemolysis assay was performed with rabbit and human erythrocytes as described for hemolysin/bacteriocin of E. faecalis⁷.

RESULTS AND DISCUSSION

Properties of anti-MRSA component in the culture broth of E. faecalis TH 10. Enterococcus faecalis TH 10, an isolate from a Malaysian fermentation food Temper was especially effective among the lactic acid bacteria tested. The active component was readily extracted with ethyl acetate from the culture broth of the TH 10 strain at pH3. The extract showed potent growth inhibition against MRSA, and a moderate activity against methicillin-sensitive S aureus. Since the extract contains various compounds in it, the result does not prove that an identical substance inhibited MRSA and methicillin-sensitive S aureus. It is rather more important to show inhibitory activity against the lactic acid bacteria tested except for two species, Streptococcus salivarius and Pediococcus ocidilactici. Enterococcus species were not inhibited by the extract, either. This anfmicrobial spectrum indicates that the anti-MRSA component appears to be distinct from bacteriocin molecules that are usually inhibitory against closely related lactic acid bacteria.
Proteolytic enzymes are frequently used for conventional diagnosis for bacteriocin production; proteolytic enzymes catalyze hydrolysis of protein or peptide-like components, and cause dramatic decrease in the bacteriocidal activity. We found that the ethyl acetate extract retained the anti-MRSA activity after the treatment with V8 protease, proteinase K, trypsin, and achromopeptidase. These proteolytic enzymes alone do not affect the growth of MRSA. The results suggest that the active component is not a peptide or a protein, but it rather appears to be an acidic low molecular weight substance, which is extracted with ethyl acetate at pH3.
Thus, the active component produced in the culture broth of E. faecalis TH 10 strain appears to differ from bacteriocins, a protein or peptide-line anti-microbial metabolite with potent activity against species closely related to the bacteriocin-producing strain.

Hemolysis Assay. In previous papers, E. faecalis has been isolated from natural food samples such as traditional French cheese8, mozzarella cheese whey 9, and foods in a retail supermarket. The species has also been recognized as human pathogens with multiple antibiotic-resistance and high-level aminoglycoside resistant^11-13. These clinical isolates of E. faecalis are reported to produce bacteriocin that mediates the lysis of a broad range of gram-positive bacteria^14-16 and the molecule also acts as a hemolysin that effectively lyses human, rabbit and horse erythrocytes. The anti-MRSA component of the TH 10 strain, however, failed to mediate the lysis of human and rabbit erythrocytes, indicating that the anti-MRSA compound is distinct from ever-reported bacteriocins produced by E. faecalis.
Our present study shows that E. faecalis produces a potent anti-microbial product against MRSA, whose identity appears to be a low molecular weight substance. Raceinic lactate did not show anti-microbial activity against MRSA even when 30 mg of the sodium salt was loaded on the paper disk, so the commonly distributed metabolite of lactic acid bacteria can be ruled out as the candidate for the active substance. 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 novel anti-microbial agents is becoming increasingly difficult, the present study suggests that food-born lactic acid bacteria may offer some potential applicability in chemotherapy and control of MRSA.

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Antimicrobial Activity Against Methicillin-Resistant Staphylococcus Aureus In The Culture Broth Of Enterococcus Faecalis Th 10, An Isolate From Malaysian Fermentation Food-Tempeh

By: Iichiroh Ohhira, Ph.D., October 2000

SUMMARY

MATERIALS AND METHODS

RESULTS AND DISCUSSION