JOURNAL OF CLINICAL MICROBIOLOGY,0095-1137/00/$04.0010

Oct. 2000, p. 3908–3909 Vol. 38, No. 10

Copyright © 2000, American Society for Microbiology. All Rights Reserved.

Bacteriocin Production by Lactobacillus salivarius of Animal Origin

Bacteriocins are secreted oligopeptides, proteins or proteincomplexes with antimicrobial activity against strains taxonom-ically related to the producer organism (2, 10). In recent years,numerous reports have been published on antimicrobial pep-tides or proteins produced by gram-positive bacteria, includinglactic acid bacteria (LAB) (4, 7), and bacteriocins for lactoba-cilli from different environments have been described (3).However, only two reports mention bacteriocin production inLactobacillus salivarius isolates, which were obtained from Jap-anese grass leaves (1) and from a human vaginal sample (8).The objective of this study was to detect bacteriocin productionin LAB isolated from animal fecal samples in order to deter-mine their spectrum of action, including human pathogens,and their significance in the ecology of the gastrointestinalenvironment.

Twenty-nine LAB isolates recovered from fecal samples of20 pigs and 9 pets (cats and dogs) were tested for antimicrobialactivity (one LAB isolate per fecal sample). These isolateswere the following: 18 Lactobacillus salivarius, 1 Lactobacillusfructivorans, 3 Lactobacillus fermentum, 1 Lactobacillus viride-scens, 1 Lactobacillus brevis, 2 Lactobacillus spp., 2 Pediococcuspentosaceus, and 1 Pediococcus acidilactici. Fifty-two bacterialisolates belonging to eight different genera and 26 differentspecies were used as indicators for bacteriocin production.Human pathogens and LAB of animal fecal origin were in-

cluded, among others, in the group of indicator strains (Table1). Bacteriocin production was evaluated by a method previ-ously described (5).

Bacteriocin activity was detected in 11 of 18 (61%) L. sali-varius isolates tested (all of them recovered from pigs) but notamong the other species of LAB studied. Table 1 shows thespectrum of antimicrobial activity of these 11 isolates againstthe 52 indicator isolates used. All bacteriocin-producing L.salivarius isolates strongly inhibited the growth of the threeclinical Staphylococcus aureus isolates tested as indicators (twomethicillin resistant and one methicillin susceptible), and forfive of them, antimicrobial activity was also detected againstclinical Staphylococcus epidermidis isolates (methicillin resis-tant and susceptible). None of these 11 bacteriocin-producingL. salivarius isolates showed growth inhibition activity againstEnterococcus spp., Bacillus spp., or E. coli. Two of these iso-lates (X13 and X61) showed the widest range of action, inhib-iting the growth of nine and seven different species of indica-tors tested, respectively. Curiously, both strains inhibited thegrowth of four different LAB species of animal intestinal ori-gin, and X13 also inhibited the growth of Listeria murrayi andMicrococcus luteus.

A negative PCR result was obtained with these 11 bacteri-ocin-producing L. salivarius isolates when the specific plnAprimers for plantaricine A were used (9). Bacteriocin activity

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

Indicator isolatea No. ofisolates

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 2Staphylococcus aureus METs 1 1 1 1 1 1 1Staphylococcus epidermidis METr 2 2 2 0 2 1 0Staphylococcus epidermidis METs 5 3 1 1 1 0 0Micrococcus luteus 1 1 0 0 0 0 0Listeria innocua 1 0 0 0 0 0 0Listeria monocytogenes 1 0 0 1 0 0 0Listeria grayi 1 0 0 0 0 0 0Listeria murrayi 1 1 0 0 0 0 0Lactobacillus salivarius 5 2 4 0 0 0 0Lactobacillus fermentum 2 1 1 0 0 0 0Lactobacillus paracasei 2 1 1 0 0 0 0Lactobacillus acidophylus 1 0 0 0 0 0 0Lactobacillus brevis 1 0 0 0 0 0 0Lactobacillus viridescens 1 0 0 0 0 0 0Lactobacillus plantarum 1 0 0 0 0 0 0Lactobacillus fructivorans 1 0 0 0 0 0 0Lactobacillus spp. 3 0 0 0 0 0 0Pediococcus acidilactici 1 1 1 0 0 0 0Pediococcus pentosaceus 2 1 1 0 0 0 0Enterococcus faecium 3 0 0 0 0 0 0Enterococcus faecalis 5 0 0 0 0 0 0Enterococcus durans 2 0 0 0 0 0 0Enterococcus gallinarum 1 0 0 0 0 0 0Enterococcus hirae 1 0 0 0 0 0 0Bacillus sphaericus 1 0 0 0 0 0 0Bacillus subtillis 1 0 0 0 0 0 0Bacillus thuringiensis 1 0 0 0 0 0 0Escherichia coli 2 0 0 0 0 0 0

a METr, methicillin resistant; METs, methicillin susceptible.

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of X13 and X61 could not be demonstrated after filtration(Millipore filter; 45-mm pore size), and this fact could be ex-plained by its adsorption to the cell membrane.

Therefore, antimicrobial peptides constitute a potent adap-tation advantage for those strains that dominate in a mediumand could play an important role in the ecology of the gastro-intestinal tract. In fact, L. salivarius is a species frequentlyfound in the intestinal tract of pigs (6), and this could beexplained by the production of bacteriocins in fecal L. salivariusisolates active against other animal fecal LAB. On the otherhand, the high inhibitory activity of these bacteriocins againstmethicillin-resistant and -susceptible staphylococci could be ofclinical interest.

We thank Carmen Tenorio for providing us with lactic acid bacteriafor this study.

This work was financed in part by a grant of the Fondo de Investi-gaciones Sanitarias (00/0545), and Beatriz Robredo has a fellowshipfrom the University of La Rioja, Logrono, Spain.

REFERENCES

1. Arihara, K., S. Ogihara, T. Mukai, M. Itoh, and Y. Kondo. 1996. Salivacin140, a novel bacteriocin from Lactobacillus salivarius subsp. salicinius T140active against pathogenic bacteria. Lett. Appl. Microbiol. 22:420–424.

2. Jack, R. W., J. R. Tag, and B. Ray. 1995. Bacteriocins of gram-positivebacteria. Microbiol. Rev. 59:171–200.

3. Klaenhammer, T. R. 1988. Bacteriocin of lactic acid bacteria. Biochimie70:337–340.

4. Klaenhammer, T. R. 1993. Genetics of bacteriocins produced by lactic acidbacteria. FEMS Microbiol. Rev. 12:39–86.

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. 88:44–51.

6. Nemcova, R., A. Laukova, S. Gancarcikova, and R. Kastel. 1997. In vitrostudies of porcine lactobacilli for possible probiotic use. Berl. Muench.Tieraerztl. Wochenschr. 110:413–417.

7. Nissen-Meyer, J., and I. F. Nes. 1997. Ribosomally synthesized antimicrobialpeptides: their function, structure, biogenesis, and mechanism of action.Arch. Microbiol. 167:67–77.

8. Ocana, V. S., A. A. Pesce de Ruiz Holgado, and M. E. Nader-Macıas. 1999.Characterization of a bacteriocin-like substance produced by a vaginal Lac-tobacillus salivarius strain. Appl. Environ. Microbiol. 65:5631–5635.

9. Remiger, A., M. A. Ehrmann, and R. F. Vogel. 1996. Identification of bac-teriocin genes in lactobacilli by polymerase chain reaction (PCR). Syst. Appl.Microbiol. 19:28–34.

10. Tagg, J. R., A. S. Dajani, and L. W. Wannamaker. 1976. Bacteriocins ofgram-positive bacteria. Bacteriol. Rev. 40:722–756.

Beatriz RobredoCarmen Torres*Area de Bioquimica y Biologia MolecularUniversidad de La Rioja26004 Logrono, Spain

*Phone: 34-941-299284Fax: 34-941-299274E-mail: carmen.torres@daa.unirioja.es

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