Bacteriocins are secreted oligopeptides, proteins or protein complexes with antimicrobial activity against strains taxonomically related to the producer organism (2, 10). In recent years, numerous reports have been published on antimicrobial peptides or proteins produced by gram-positive bacteria, including lactic acid bacteria (LAB) (4, 7), and bacteriocins for lactobacilli from different environments have been described (3). However, only two reports mention bacteriocin production in Lactobacillus salivarius isolates, which were obtained from Japanese grass leaves (1) and from a human vaginal sample (8). The objective of this study was to detect bacteriocin production in LAB isolated from animal fecal samples in order to determine their spectrum of action, including human pathogens, and their significance in the ecology of the gastrointestinal environment.
Twenty-nine LAB isolates recovered from fecal samples of 20 pigs and 9 pets (cats and dogs) were tested for antimicrobial activity (one LAB isolate per fecal sample). These isolates were the following: 18 Lactobacillus salivarius, 1 Lactobacillus fructivorans, 3 Lactobacillus fermentum, 1 Lactobacillus viridescens, 1 Lactobacillus brevis, 2 Lactobacillus spp., 2 Pediococcus pentosaceus, and 1 Pediococcus acidilactici. Fifty-two bacterial isolates belonging to eight different genera and 26 different species were used as indicators for bacteriocin production. Human pathogens and LAB of animal fecal origin were included, among others, in the group of indicator strains (Table 1). Bacteriocin production was evaluated by a method previously described (5).
TABLE 1.
Spectrum of growth inhibition by bacteriocin-producing L. salivarius isolates
| Indicator isolatea | No. of isolates | No. of isolates inhibited by bacteriocin-producing L. salivarius
|
|||||
|---|---|---|---|---|---|---|---|
| X13 | X61 | X19 | X24 | X25 | X8, X16, X19, X20, X21, X63 | ||
| Staphylococcus aureus METr | 2 | 2 | 2 | 2 | 2 | 2 | 2 |
| Staphylococcus aureus METs | 1 | 1 | 1 | 1 | 1 | 1 | 1 |
| Staphylococcus epidermidis METr | 2 | 2 | 2 | 0 | 2 | 1 | 0 |
| Staphylococcus epidermidis METs | 5 | 3 | 1 | 1 | 1 | 0 | 0 |
| Micrococcus luteus | 1 | 1 | 0 | 0 | 0 | 0 | 0 |
| Listeria innocua | 1 | 0 | 0 | 0 | 0 | 0 | 0 |
| Listeria monocytogenes | 1 | 0 | 0 | 1 | 0 | 0 | 0 |
| Listeria grayi | 1 | 0 | 0 | 0 | 0 | 0 | 0 |
| Listeria murrayi | 1 | 1 | 0 | 0 | 0 | 0 | 0 |
| Lactobacillus salivarius | 5 | 2 | 4 | 0 | 0 | 0 | 0 |
| Lactobacillus fermentum | 2 | 1 | 1 | 0 | 0 | 0 | 0 |
| Lactobacillus paracasei | 2 | 1 | 1 | 0 | 0 | 0 | 0 |
| Lactobacillus acidophylus | 1 | 0 | 0 | 0 | 0 | 0 | 0 |
| Lactobacillus brevis | 1 | 0 | 0 | 0 | 0 | 0 | 0 |
| Lactobacillus viridescens | 1 | 0 | 0 | 0 | 0 | 0 | 0 |
| Lactobacillus plantarum | 1 | 0 | 0 | 0 | 0 | 0 | 0 |
| Lactobacillus fructivorans | 1 | 0 | 0 | 0 | 0 | 0 | 0 |
| Lactobacillus spp. | 3 | 0 | 0 | 0 | 0 | 0 | 0 |
| Pediococcus acidilactici | 1 | 1 | 1 | 0 | 0 | 0 | 0 |
| Pediococcus pentosaceus | 2 | 1 | 1 | 0 | 0 | 0 | 0 |
| Enterococcus faecium | 3 | 0 | 0 | 0 | 0 | 0 | 0 |
| Enterococcus faecalis | 5 | 0 | 0 | 0 | 0 | 0 | 0 |
| Enterococcus durans | 2 | 0 | 0 | 0 | 0 | 0 | 0 |
| Enterococcus gallinarum | 1 | 0 | 0 | 0 | 0 | 0 | 0 |
| Enterococcus hirae | 1 | 0 | 0 | 0 | 0 | 0 | 0 |
| Bacillus sphaericus | 1 | 0 | 0 | 0 | 0 | 0 | 0 |
| Bacillus subtillis | 1 | 0 | 0 | 0 | 0 | 0 | 0 |
| Bacillus thuringiensis | 1 | 0 | 0 | 0 | 0 | 0 | 0 |
| Escherichia coli | 2 | 0 | 0 | 0 | 0 | 0 | 0 |
METr, methicillin resistant; METs, methicillin susceptible.
Bacteriocin activity was detected in 11 of 18 (61%) L. salivarius isolates tested (all of them recovered from pigs) but not among the other species of LAB studied. Table 1 shows the spectrum of antimicrobial activity of these 11 isolates against the 52 indicator isolates used. All bacteriocin-producing L. salivarius isolates strongly inhibited the growth of the three clinical Staphylococcus aureus isolates tested as indicators (two methicillin resistant and one methicillin susceptible), and for five of them, antimicrobial activity was also detected against clinical Staphylococcus epidermidis isolates (methicillin resistant and susceptible). None of these 11 bacteriocin-producing L. salivarius isolates showed growth inhibition activity against Enterococcus spp., Bacillus spp., or E. coli. Two of these isolates (X13 and X61) showed the widest range of action, inhibiting the growth of nine and seven different species of indicators tested, respectively. Curiously, both strains inhibited the growth of four different LAB species of animal intestinal origin, and X13 also inhibited the growth of Listeria murrayi and Micrococcus luteus.
A negative PCR result was obtained with these 11 bacteriocin-producing L. salivarius isolates when the specific plnA primers for plantaricine A were used (9). Bacteriocin activity of X13 and X61 could not be demonstrated after filtration (Millipore filter; 45-μm pore size), and this fact could be explained by its adsorption to the cell membrane.
Therefore, antimicrobial peptides constitute a potent adaptation advantage for those strains that dominate in a medium and could play an important role in the ecology of the gastrointestinal tract. In fact, L. salivarius is a species frequently found in the intestinal tract of pigs (6), and this could be explained by the production of bacteriocins in fecal L. salivarius isolates active against other animal fecal LAB. On the other hand, the high inhibitory activity of these bacteriocins against methicillin-resistant and -susceptible staphylococci could be of clinical interest.
Acknowledgments
We thank Carmen Tenorio for providing us with lactic acid bacteria for this study.
This work was financed in part by a grant of the Fondo de Investigaciones Sanitarias (00/0545), and Beatriz Robredo has a fellowship from the University of La Rioja, Logroño, Spain.
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