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Applied and Environmental Microbiology, December 1999, p. 5631-5635, Vol. 65, No. 12
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Characterization of a Bacteriocin-Like Substance
Produced by a Vaginal Lactobacillus salivarius
Strain
Virginia S.
Ocaña,1
Aída A.
Pesce de Ruiz
Holgado,1,2 and
María Elena
Nader-Macías1,2,*
CERELA1 and
Facultad de Bioquímica, Química y Farmacia,
Universidad Nacional de Tucumán,2
Tucumán, Argentina
Received 30 April 1999/Accepted 1 October 1999
 |
ABSTRACT |
A novel bacteriocin-like substance produced by vaginal
Lactobacillus salivarius subsp. salivarius CRL
1328 with activity against Enterococcus faecalis,
Enterococcus faecium, and Neisseria gonorrhoeae was characterized. The highest level of production of this
heat-resistant peptide or protein occurred during the late exponential
phase. Its mode of action was shown to be bactericidal. L. salivarius subsp. salivarius CRL 1328 could be used
for the design of a probiotic to prevent urogenital infections.
| |
TEXT |
Lactobacilli are the dominant
microorganisms isolated from the vaginas of healthy premenopausal women
(11, 19). They interfere with the colonization of pathogens
by different mechanisms, such as the production of organic acids,
H2O2, and bacteriocins (2, 6, 9,
10-12). Bacteriocins are proteinaceous, bactericidal substances
synthesized by bacteria and usually have a narrow spectrum of activity
(7). The term bacteriocin-like substance is applied to
antagonistic substances which are not completely defined or do not fit
the typical criteria of bacteriocins. They have been reported to
inhibit a wide range of both gram-positive and gram-negative bacteria
as well as fungi (11). Although bacteriocin production has
been described as an antagonistic mechanism that could be exerted by
lactobacilli in the vaginal tract, no bacteriocin has been
characterized to date for Lactobacillus strains isolated from the vagina.
At present, bacteriocin-producing lactic acid bacteria are widely used
for the elaboration of probiotics for the gastrointestinal tract
(4, 5, 13, 17). To select probiotic strains for local
application in the human vagina, isolation, identification, and certain
characteristics (e.g., adhesion and production of H2O2) of vaginal Lactobacillus
strains were previously reported (14-16). In the
present paper, the screening of bacteriocin production in 134 human
vaginal lactobacilli is reported. Characterization, production
kinetics, and mode of action of a bacteriocin produced by
Lactobacillus salivarius subsp. salivarius CRL
1328 are described in more detail.
Strains and culture conditions.
Lactobacillus strains
were isolated from vaginal swabs as described before (16).
The other microorganisms employed are indicated in Table
1.
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TABLE 1.
Sensitive and resistant microorganisms tested for
inhibition by a bacteriocin-like substance produced by L. salivarius subsp. salivarius CRL 1328
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Detection of antimicrobial activity.
Inhibitory substances
from Lactobacillus culture supernatants were studied
according to standard methods (7). Lactobacilli were grown
in Laptg broth (18), and supernatants were separated, neutralized, and filter sterilized. The pathogens tested were vaginal
Staphylococcus aureus, Enterococcus faecalis, and
group B Streptococcus spp. Agar plates were sown with the
pathogens, and Lactobacillus supernatant aliquots were
assayed on them. After incubation for 24 h at 37°C, the
diameters of the inhibition halos were measured.
L. salivarius subsp.
salivarius CRL 1328 was
selected because it was able to inhibit the growth of
E. faecalis. Although
L. salivarius subsp.
salivarius CRL 1328 is not the predominant species
isolated
from the vagina, its isolation from this environment
was reported
previously (
3). The same method was used to test
its
inhibitory activity against other microorganisms. Different
Enterococcus strains,
Neisseria gonorrhoeae, and
Lactobacillus paracasei subsp.
paracasei CRL 1289 were inhibited (Table
1).
Characterization of antimicrobial substance.
Aliquots of
supernatant were treated with catalase (1,000 U ml
1),
proteases (1 mg ml1), 
-amylase (0.1 M), sodium
metaperiodate (10 mM) and lipase (10 g ml1). Activity was
lost after treatment with chymotrypsin and proteinase K, but the
substance was resistant to catalase, trypsin, pepsin, type II protease,
type XV protease, lipase, and -amylase. The effect of sodium
periodate could not be evaluated because the reagent control was positive.
Effects of temperature, lyophilization, and pH on antimicrobial
activity.
Heat resistance was studied after the supernatants were
heated to 60, 80, and 100°C for 10 min each and also after
autoclaving (121°C; 15 min). They were cooled and tested for
activity. The activity was also evaluated by modifying the pH (between
2 and 8) of the supernatants with 2 N NaOH. Furthermore, resistance to
lyophilization was studied. The supernatant fluid of a 12-h L. salivarius subsp. salivarius CRL 1328 culture was
filter sterilized. Fractions of 10 ml each were lyophilized and later
resuspended in the same volume of Laptg broth. The bacteriocin activity
of the resuspended lyophilized powder was determined.
The substance responsible for the inhibition of
E. faecalis
retained activity after heating and even after autoclaving.
Antagonistic
activity was observed at all tested pH levels.
Lyophilization
did not alter the
activity.
Kinetics of bacteriocin production.
Laptg broth was inoculated
with a 2% concentration of 12-h L. salivarius subsp.
salivarius CRL 1328 culture and incubated at 37°C. Samples
were taken every 3 h to determine the titer of the bacteriocin and
the number of CFU per milliliter. For the titration, supernatants were
filter sterilized and serially diluted in Laptg broth. Twenty-five
microliters of each dilution was poured into the holes of agar plates
containing vaginal E. faecalis. The titer was expressed in
arbitrary units (AU), defined as the inverse of the dilution in
milliliters. The number of CFU per milliliter was determined by using
Laptg agar plates.
Activity was detected after 3 h of incubation. The highest
concentration was obtained between 9 and 12 h of incubation, with
titers of 1,280 AU ml
1 of supernatant. The production
kinetics are shown in Fig.
1.
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FIG. 1.
Kinetics of bacteriocin-like substance production during
the growth of Lactobacillus salivarius subsp.
salivarius CRL 1328. Growth of L. salivarius
subsp. salivarius CRL 1328 was determined by the plate count
method ( ) and culture pH ( ); bacteriocin concentration is
expressed as arbitrary units per milliliter ( ) and per log CFU
(×).
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Mixed cultures of L. salivarius subsp.
salivarius CRL 1328 and vaginal E. faecalis.
The
effect of L. salivarius subsp. salivarius CRL
1328 on E. faecalis growth in mixed cultures in Laptg broth
at 37°C was studied. The initial inocula were 106 CFU
ml1 for lactobacilli and 105 or
107 CFU ml1 for enterococci. The number of
CFU was determined with selective media, Lactobacillus
selective medium, and Streptococcus faecalis medium agar
plates. The plates were incubated for 48 h at 37°C.
L. salivarius subsp.
salivarius CRL 1328 inhibited
E. faecalis, depending on the initial inoculum of
the pathogen. At an inoculum
of 10
7 CFU ml
1,
the growth rate was 4.74 log units lower than that in the pure
culture
(Fig.
2A). The decrease was observed in a
12-h culture
when the bacteriocin concentration was the highest (1,280 AU ml
1). With an inoculum of 10
5 CFU
ml
1, the growth rate was 7.02 log units lower (Fig.
2B).
L. salivarius subsp.
salivarius CRL 1328 growth
was not affected in mixed cultures
and no antagonistic substances from
E. faecalis against lactobacilli
were found (data not
shown). Even though complete inhibition of
the pathogen growth was not
obtained, the number of viable cells
decreased significantly. Control
of the pathogen overgrowth by
L. salivarius subsp.
salivarius CRL 1328 would be of interest
in the prevention
of genitourinary infections. Reid et al. (
20)
have suggested
that the dominance of inhibitor-producing lactobacilli
on the
urogenital epithelium and the ability of these organisms
to interact
closely with uropathogens would constitute an important
host defense
mechanism against infection.
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FIG. 2.
Pure and mixed cultures of L. salivarius
subsp. salivarius CRL 1328 and E. faecalis.
Lactobacilli were inoculated at 107 CFU ml1;
enterococci were inoculated at 107 (A) and 105
(B) CFU ml1. Lactobacilli in pure ( ) and mixed ( )
cultures and enterococci in pure ( ) and mixed ( ) cultures are
shown.
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Mode of action.
Five milliliters of supernatant of a 12-h
L. salivarius subsp. salivarius CRL 1328 culture
was lyophilized, resuspended in the same volume of Laptg broth,
inoculated with E. faecalis, and incubated at 37°C.
Samples were taken at different times, and the number of CFU per
milliliter was determined by using S. faecalis medium agar
plates. The bacteriocin-like substance was shown to be bactericidal
when 103 CFU ml1 of E. faecalis
was inoculated in Lactobacillus spent supernatant with 1,280 AU ml1 of bacteriocin during 120 h of culture (Fig.
3A). With larger inocula (105
and 107 CFU ml1), a bacteriostatic effect
during the first 24 h of growth was observed and was then
reestablished (Fig. 3B). Another set of experiments was performed with
a twofold concentration of bacteriocin. Larger amounts of the E. faecalis inoculum were completely inhibited after incubation times
were increased (Fig. 4), thus indicating a bactericidal mode of action.
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FIG. 3.
Mode of action of a L. salivarius subsp.
salivarius CRL 1328 bacteriocin-like substance against
E. faecalis. (A) E. faecalis inoculated at
103 CFU ml1 without () and with ()
bacteriocin. (B) E. faecalis inoculated at 107
CFU ml1 without bacteriocin () and inoculated at
107 () and 105 () CFU ml1
with bacteriocin.
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FIG. 4.
Mode of action of an L. salivarius subsp.
salivarius CRL 1328 bacteriocin-like substance against
E. faecalis. E. faecalis was inoculated at 103
(A), 105 (B), and 107 (C) CFU ml1
without bacteriocin () and with a twofold bacteriocin concentration
().
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Electron microscopy.
E. faecalis harvested from an
early-stationary-phase culture (108 CFU ml1)
was incubated for 12 h at 37°C in the reconstituted supernatant of L. salivarius subsp. salivarius CRL 1328 with
a bacteriocin titer of 2,560 AU ml1. After incubation,
the microorganisms were separated and prepared for transmission
electron microscopy. E. faecalis showed vesiculization of
protoplasm, formation of pores, and complete disintegration of cells
(Fig. 5).
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FIG. 5.
Electron photomicrographs of the effect of L. salivarius subsp. salivarius CRL 1328 bacteriocin-like
substance on E. faecalis. The Enterococcus
control cell (A), vesiculization of protoplasm (B), vesiculization of
protoplasm and a damaged cell wall (C), pore formation in the cell wall
(D), a disintegrated cell with loss of the protoplasmic material
through a cell wall pore (E), and a disintegrated cell (F) are shown.
The method is described in the text.
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Characterization and purification studies of the bacteriocin-like
substance are still in progress. Sodium dodecyl sulfate-polyacrylamide
gel electrophoresis was performed with a high-titer bacteriocin
extract
(
21), showing that only one band was responsible for
the
inhibitory effect (data not shown). These results would indicate
a
single compound
substance.
Many bacteriocins for lactobacilli from different environments have
been described (
8). However, no bacteriocin for an
L. salivarius strain has been reported, except Salivacin 140,
which
is produced by a
Lactobacillus salivarius subsp.
salicinius strain isolated from Japanese grass leaves
(
1).
The properties of
L. salivarius subsp.
salivarius
CRL 1328 allow us to further study its probable application in a
probiotic
for the prevention of urogenital infections. The inhibition
of
Enterococcus would prevent the recurrence of urinary
tract infections
(of significant incidence in elderly women) while the
effect on
N. gonorrhoeae suggests its potential use in
keeping gonorrhea
under control. Thermotolerance and retention of
activity after
lyophilization are desired characteristics if its use as
an additive
in a probiotic is to be considered. The activity of the
substance
at low pH is also important because the effect would be
exerted
in the vagina, where the pH is between 3.8 and 4.5.
| |
ACKNOWLEDGMENTS |
This paper was supported by CONICET (Consejo Nacional de
Investigaciones Científicas y Técnicas, Buenos Aires,
Argentina) (PID 0359-1998-2000) and CIUNT (Consejo de
Investigación de la Universidad Nacional de Tucumán,
Tucumán, Argentina) (D-128) grants.
We kindly thank Clara Silva and Olga Aulet for providing the pathogenic
strains from the Culture Collection of the Instituto de
Microbiología Luis C. Verna from the Universidad Nacional de
Tucumán. We also thank Beatriz Winik for her technical assistance in the electron microscopy studies and Raúl Raya for critical review of the manuscript.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: CERELA,
Chacabuco 145, 4000 Tucumán, Argentina. Phone: (381) 431-1720. Fax: (381) 431-0465. E-mail: mmacias{at}cerela.org.ar.
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Applied and Environmental Microbiology, December 1999, p. 5631-5635, Vol. 65, No. 12
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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