JOURNAL OF CLINICAL MICROBIOLOGY, Oct. 2000, p. 39083909 0095-1137/00/$04.00 0 Copyright 2000, American Society for Microbiology.

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Vol. 38, No. 10

Bacteriocin Production by Lactobacillus salivarius of Animal Origin

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 signicance 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). 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 ve 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 specic plnA primers for plantaricine A were used (9). Bacteriocin activity

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TABLE 1. Spectrum of growth inhibition by bacteriocin-producing L. salivarius isolates

Indicator isolatea No. of isolates
r

No. of isolates inhibited by bacteriocin-producing L. salivarius X13 X61 X19 X24 X25 X8, X16, X19, X20, X21, X63

Staphylococcus aureus MET Staphylococcus aureus METs Staphylococcus epidermidis METr Staphylococcus epidermidis METs Micrococcus luteus Listeria innocua Listeria monocytogenes Listeria grayi Listeria murrayi Lactobacillus salivarius Lactobacillus fermentum Lactobacillus paracasei Lactobacillus acidophylus Lactobacillus brevis Lactobacillus viridescens Lactobacillus plantarum Lactobacillus fructivorans Lactobacillus spp. Pediococcus acidilactici Pediococcus pentosaceus Enterococcus faecium Enterococcus faecalis Enterococcus durans Enterococcus gallinarum Enterococcus hirae Bacillus sphaericus Bacillus subtillis Bacillus thuringiensis Escherichia coli
a

2 1 2 5 1 1 1 1 1 5 2 2 1 1 1 1 1 3 1 2 3 5 2 1 1 1 1 1 2

2 1 2 3 1 0 0 0 1 2 1 1 0 0 0 0 0 0 1 1 0 0 0 0 0 0 0 0 0

2 1 2 1 0 0 0 0 0 4 1 1 0 0 0 0 0 0 1 1 0 0 0 0 0 0 0 0 0

2 1 0 1 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

2 1 2 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

2 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

2 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

METr, methicillin resistant; METs, methicillin susceptible.

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LETTERS TO THE EDITOR

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of X13 and X61 could not be demonstrated after ltration (Millipore lter; 45- m 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.
We thank Carmen Tenorio for providing us with lactic acid bacteria for this study. This work was nanced 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, Logrono, Spain.
REFERENCES 1. Arihara, K., S. Ogihara, T. Mukai, M. Itoh, and Y. Kondo. 1996. Salivacin 140, a novel bacteriocin from Lactobacillus salivarius subsp. salicinius T140 active against pathogenic bacteria. Lett. Appl. Microbiol. 22:420424. 2. Jack, R. W., J. R. Tag, and B. Ray. 1995. Bacteriocins of gram-positive bacteria. Microbiol. Rev. 59:171200.

3. Klaenhammer, T. R. 1988. Bacteriocin of lactic acid bacteria. Biochimie 70:337340. 4. Klaenhammer, T. R. 1993. Genetics of bacteriocins produced by lactic acid bacteria. FEMS Microbiol. Rev. 12:3986. 5. Navarro, L., M. Zarazaga, J. Saenz, F. Ruiz-Larrea, and C. Torres. 2000. Bacteriocin production by lactic acid bacteria isolated from Rioja red wines. J. Appl. Microbiol. 88:4451. 6. Nemcova, R., A. Laukova, S. Gancarcikova, and R. Kastel. 1997. In vitro studies of porcine lactobacilli for possible probiotic use. Berl. Muench. Tieraerztl. Wochenschr. 110:413417. 7. Nissen-Meyer, J., and I. F. Nes. 1997. Ribosomally synthesized antimicrobial peptides: their function, structure, biogenesis, and mechanism of action. Arch. Microbiol. 167:6777. 8. Ocana, V. S., A. A. Pesce de Ruiz Holgado, and M. E. Nader-Macas. 1999. Characterization of a bacteriocin-like substance produced by a vaginal Lactobacillus salivarius strain. Appl. Environ. Microbiol. 65:56315635. 9. Remiger, A., M. A. Ehrmann, and R. F. Vogel. 1996. Identication of bacteriocin genes in lactobacilli by polymerase chain reaction (PCR). Syst. Appl. Microbiol. 19:2834. 10. Tagg, J. R., A. S. Dajani, and L. W. Wannamaker. 1976. Bacteriocins of gram-positive bacteria. Bacteriol. Rev. 40:722756.

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Beatriz Robredo Carmen Torres* Area de Bioquimica y Biologia Molecular Universidad de La Rioja 26004 Logrono, Spain *Phone: 34-941-299284 Fax: 34-941-299274 E-mail: carmen.torres@daa.unirioja.es