November 2016⎪Vol. 26⎪No. 11
J. Microbiol. Biotechnol. (2016), 26(11), 1829–1835http://dx.doi.org/10.4014/jmb.1604.04008 Research Article jmbReview
Inhibitory Effect of Lactococcus lactis HY 449 on Cariogenic BiofilmYoung-Jae Kim1 and Sung-Hoon Lee2*
1Department of Pediatric Dentistry, School of Dentistry, Seoul National University, Seoul 03080, Republic of Korea2Department of Oral Microbiology and Immunology, College of Dentistry, Dankook University, Cheonan 31116, Republic of Korea
Introduction
Streptococcus mutans plays a potent role in the induction
of dental caries. This bacterium strongly produces lactic acid
and tolerates a low acidic environment [10]. Furthermore,
S. mutans produces glucan from sucrose by glucosyltransferases
such as GtfB, GtfC, and GtfD [9, 14] and uses sucrose as a
substrate for growth [8]. Oral biofilm consists of oral
bacteria, glucan, and debris. Among these components,
glucan is a key contributor to the development of biofilm
via formation of a thick barrier [11]. The biofilm of a
healthy person maintains a balanced composition of
bacterial species. However, when the conditions of the oral
biofilm are changed by a sugar-rich diet, low pH, and low
saliva flow, the proportion of S. mutans in the oral biofilm
increases compared with other streptococci [12]. Moreover,
continuous production of glucan and acid by S. mutans
reduces the pH level and leads to formation of mature
biofilm that ultimately induces dental caries. Therefore,
the glucosyltransferases (Gtfs) and acid production are
virulence factors of S. mutans. Cariogenic biofilm as oral
biofilm including S. mutans is considered to be a greater
risk factor for induction of dental caries than planktonic
S. mutans.
Probiotics have been widely used in the food industry
and in dairy products because of their beneficial effects.
These microorganisms have antibacterial activity against
pathogenic bacteria through production of bacteriocins [5].
Furthermore, probiotics slightly stimulate the immune
systems of hosts and inhibit the toxin activity of pathogenic
Received: April 5, 2016
Revised: June 4, 2016
Accepted: July 13, 2016
First published online
July 19, 2016
*Corresponding author
Phone: +82-41-550-1867;
Fax: +82-41-550-1859;
E-mail: [email protected]
pISSN 1017-7825, eISSN 1738-8872
Copyright© 2016 by
The Korean Society for Microbiology
and Biotechnology
Dental caries is caused by cariogenic biofilm, an oral biofilm including Streptococcus mutans.
Recently, the prevention of dental caries using various probiotics has been attempted.
Lactococcus lactis HY 449 is a probiotic bacterium. The aim of this study was to investigate the
effect of L. lactis HY 449 on cariogenic biofilm and to analyze its inhibitory mechanisms.
Cariogenic biofilm was formed in the presence or absence of L. lactis HY 449 and L. lactis
ATCC 19435, and analyzed with a confocal laser microscope. The formation of cariogenic
biofilm was reduced in cultures spiked with both L. lactis strains, and L. lactis HY 449 exhibited
more inhibitory effects than L. lactis ATCC 19435. In order to analyze and to compare the
inhibitory mechanisms, the antibacterial activity of the spent culture medium from both
L. lactis strains against S. mutans was investigated, and the expression of glucosyltransferases
(gtfs) of S. mutans was then analyzed by real-time RT-PCR. In addition, the sucrose
fermentation ability of both L. lactis strains was examined. Both L. lactis strains showed
antibacterial activity and inhibited the expression of gtfs, and the difference between both
strains did not show. In the case of sucrose-fermenting ability, L. lactis HY 449 fermented
sucrose but L. lactis ATCC 19435 did not. L. lactis HY 449 inhibited the uptake of sucrose and
the gtfs expression of S. mutans, whereby the development of cariogenic biofilm may be
inhibited. In conclusion, L. lactis HY 449 may be a useful probiotic for the prevention of dental
caries.
Keywords: Lactococcus lactis, probiotics, Streptococcus mutans, cariogenic biofilm, antibiofilm
effects
1830 Kim and Lee
J. Microbiol. Biotechnol.
bacteria [3, 17, 24]. Most studies of probiotics have been
focused on the bacterial ecology of the gut and gut immune
systems. Recently, probiotics have been investigated for
their effects on oral health. Lactobacillus rhamnosus GG
inhibits the growth and bioactivity of oral pathogens via
the production of small-molecular antimicrobial substances,
whereby it reduces the risks of dental caries and periodontitis
[13, 16, 21]. Moreover, L. reuteri and Bifidobacterium animalis
reduced the S. mutans amount in saliva [2, 20]. However,
the prevention of dental caries by these probiotics remains
controversial because they are aciduric bacteria, like
S. mutans [18]. Lactococcus lactis is a probiotic that has
antibacterial activity. It produces various bacteriocins such
as nisin and lactococcin, and diacetin [1, 7]. For these
reasons, L. lactis is widely used in fermented foods and
dairy products. Studies of L. lactis in oral health have
examined the inhibition of planktonic S. mutans growth
and mucosal immune stimulation [22, 23]. L. lactis HY 449
was isolated from contaminated milk products and
contained the same fatty acid profiles as L. lactis ATCC
19435 as a type strain. Moreover, their characteristics are to
ferment sucrose and salicine, unlike L. lactis ATCC 19435
[6]. The growth of L. lactis is inhibited at below pH 5 [4, 19].
Therefore, the current study investigated the effect of
L. lactis HY 449 on cariogenic biofilm and analyzed the
inhibitory mechanism of L. lactis HY 449 on the formation
of cariogenic biofilm.
Materials and Methods
Bacterial Strains and Culture Conditions
L. lactis HY449 was donated from Korea Yakult (Korea Yakult,
Korea), and L. lactis ATCC 19435 and S. mutans ATCC 25175 were
purchased from American Type Culture Collection (ATCC).
L. lactis ATCC 19435 and HY 449 were cultured in brain heart
infusion (BHI) broth (BD Bioscience, USA) at 37oC. S. mutans ATCC
25175 was cultivated in BHI broth including 1% sucrose at 37oC.
Preparation of Conditioning Plate for Biofilm Formation
Pooled saliva of six healthy donors was centrifuged at 7,000 ×g
for 10 min at 4oC. The supernatant was filtered through a
polyvinylidene fluoride (PVDF) membrane and diluted to 2-fold
with phosphate-buffered saline (PBS, pH 7.2). The prepared saliva
was added to wells on a 12-well polystyrene plate. The 12-well
plate was dried at 40oC in a drying oven and sterilized in an UV
sterilizer. These procedures were repeated three times.
Inhibitory Effect of L. lactis on Cariogenic Biofilm
Cariogenic biofilm was formed according to the method
described by Lee and Kim [9]. Unstimulated saliva was collected
from 10 healthy donors and pooled in equal proportions. The
pooled saliva was mixed with BHI broth containing 1% sucrose
and 1% mannose and centrifuged at 2,000 ×g for 10 min at 4oC to
remove debris. The supernatant was transferred into new tubes,
and S. mutans (1 × 106 cells) was added to form the cariogenic
biofilm. L. lactis HY 449 and L. lactis ATCC 19435 (5 × 105 and 1 ×
106 cells) were inoculated into each tube containing salivary
bacteria. The preparation was vortexed for 10 sec, and 1 ml or
400 µl of the mixtures was then inoculated into a conditioned 12-
well plate or 8-well glass chamber, respectively. The plates were
incubated at 37oC for 72 h, and the media were changed daily by
replacing with fresh BHI containing 1% sucrose and 1% mannose.
The biofilm-formed 12-well plate was washed three times with
PBS to remove planktonic bacteria, and the biofilm was disrupted
mechanically with a scraper. The suspensions were diluted
serially and plated on BHI agar plate and mitis-salivarius
bacitracin (MSB) agar plates to count total bacteria and S. mutans
in the biofilm, respectively. For the analysis of biofilm formation,
an 8-well glass chamber was washed three times with PBS and
stained with SYTO 9 dye (Invitrogen, USA) according to the
manufacturer’s instructions. The biofilm was visualized by a
confocal laser scanning microscope (Carl-Zeiss, Germany) using
z-stack scans from 0 to 30 µm.
Comparison of Antibacterial Activity of L. lactis against S. mutans
In order to analyze the inhibitory mechanism of L. lactis HY 449
on formation of cariogenic biofilm, a susceptibility assay of
S. mutans for the spent culture medium of L. lactis was performed
according to the methods recommended by the Clinical and
Laboratory Standards Institute (CLSI) [15]. The spent culture
medium of L. lactis HY 449 and L. lactis ATCC 19435 was collected
by centrifugation at 7,000 ×g for 10 min at 4oC and filtered
through a PVDF membrane with a pore size of 0.22 µm. The spent
culture medium was dispensed from 20 to 180 µl into the column
wells of a 96-well polystyrene plate (SPL Life Sciences, Korea),
and fresh BHI broth was added to bring the final volume up to
180 µl in the dispensed well of the spent culture medium.
S. mutans was counted in a Petroff-Hasser bacteria counting
chamber (Hausser Scientific, USA) and diluted to 1 × 106 cells/ml
with BHI broth. The diluted suspension of S. mutans (20 µl) was
inoculated into each well. The plate was incubated at 37oC for
36 h, and the optical density was measured at 600 nm by a
spectrophotometer.
Real-Time RT-PCR for the Analysis of Glucan Expression of
S. mutans
Glucan production by S. mutans was indirectly analyzed by
comparison of the expression levels of gtfs. S. mutans was cultured
in BHI including 1% sucrose with or without the spent medium of
both L. lactis at a non-killing concentration (20%) for S. mutans for
6 h. Total RNA was extracted with a TRIzol Max bacterial RNA
isolation kit (Invitrogen Life Tech, USA) according to the
L. lactis HY 449 as a Probiotic for Dental Caries 1831
November 2016⎪Vol. 26⎪No. 11
manufacturer’s instruction. cDNA was synthesized by Maxime
RT Premix (random primer; iNtRON, Korea) in a 20 µl reaction
volume, and the mixture was incubated at 45oC for 1 h. cDNA was
mixed with 10 µl of SYBR Premix Ex Taq and 0.4 µM of each
primer pair in a 50 µl final volume and was then subjected to 40
PCR cycles (94oC for 15 sec, 60oC for 10 sec, and 72oC for 33 sec) by
ABI 7500 real-time PCR (Applied Biosystems, USA). 16S rRNA,
which is a housekeeping gene, was used as a reference to
normalize the expression levels and to quantify changes in
expression levels of gtfs between cultures that were untreated and
treated with the spent culture medium. The sequences of primers
for real-time RT-PCR were as follows : 5’-AGC AAT GCA GCC
AAT CTA CAA AT-3’ and 5’-ACG AAC TTT GCC GTT ATT GTC
A-3’ for the gtfB gene; 5’-CTC AAC CAA CCG CCA CTG TT-3’
and 5’-GGT TAA CGT CAA AAT TAG CTG TAT TAG C-3’ for the
gtfC gene; 5’-CAC AGG CAA AAG CTG AAT TAA CA-3’ and 5’-
AAT GGC CGC TAA GTC AAC AG-3’ for the gftD gene; and 5’-
GAA AGT CTG GAG TAA AAG GCT A-3’ and 5’-GTT AGC TCC
GGC ACT AAG CC-3’ for the 16S rRNA gene.
Analysis of Sucrose Fermentation by L. lactis
In order to investigate the sucrose-fermenting ability of L. lactis
HY 449 and L. lactis ATCC 19435, the bacteria were harvested by
centrifugation at 4,000 ×g for 10 min at 4oC and washed with fresh
BHI broth. The bacteria were resuspended with fresh BHI broth,
and the concentration was adjusted to 1 × 107 cells/ml by using a
bacterial counting chamber. Ten milliliters of BHI was dispensed
into new conical tubes with or without sucrose and prewarmed at
37oC until inoculation. The bacterial suspension (1 ml) was inoculated
into 15 tubes and the level of pH was measured every hour.
Fig. 1. Inhibition of formation of cariogenic biofilm by L. lactis.
The cariogenic biofilm was formed using salivary bacteria and S. mutans in the presence or absence of L. lactis HY 449 and L. lactis ATCC 19435.
The biofilms were then washed with PBS, stained with SYTO 9, and analyzed by a confocal laser scanning microscope (A-C). To count bacteria in
the biofilm, the biofilm was disrupted by a scraper and resuspended with BHI. After plating on BHI agar plates for total bacteria (D) and MSB
agar plates for S. mutans (E), the agar plates were incubated for 36 and 48 h, respectively. The colonies on the agar plates were then counted. Data
are represented as the mean ± SD from six total experiments. * Statistically significant difference compared with untreated L. lactis (p < 0.001).
1832 Kim and Lee
J. Microbiol. Biotechnol.
Statistical Analysis
Statistically significant differences were analyzed by the
Kruskal-Wallis and Mann-Whitney tests using IBM SPSS Statistics
21 software (IBM, USA). P-values less than 0.05 were considered
statistically significant.
Results
Inhibitory Effect of L. lactis on Formation of Cariogenic
Biofilm
Salivary biofilm containing S. mutans as cariogenic biofilm
is more related to dental caries than S. mutans biofilm.
Therefore, the effect of L. lactis on cariogenic biofilm was
investigated. L. lactis HY 449 and L. lactis ATCC 19435
inhibited formation of cariogenic biofilm (Figs. 1A-1C).
The level of total bacteria in cariogenic biofilm was
decreased when the growth medium was spiked with
L. lactis (Fig. 1D). The level of S. mutans in the biofilm was
also decreased in the presence of L. lactis in a dose-
dependent manner (Fig. 1E). The peculiar point is that the
anti-biofilm activity of L. lactis HY 449 and L. lactis ATCC
19435 was different. L. lactis HY 449 showed more
inhibitory effect on formation of cariogenic biofilm than
did L. lactis ATCC 19435.
Antimicrobial Activity of L. lactis against S. mutans
In order to investigate the difference in the inhibitory
effects, the antibacterial activity of both L. lactis strains was
compared. Although the growth of S. mutans was
significantly decreased in media containing a greater than
40% concentration of the spent culture medium of both
L. lactis strains, there was no difference in the antimicrobial
activity of L. lactis HY 449 and L. lactis ATCC 19435 (Fig. 2).
Reduction of Glucosyltransferase Expression by the Spent
Culture Medium of L. lactis
The formation and development of S. mutans biofilm are
related to the presence of soluble and insoluble glucan, and
glucan is a key contributor to the development of biofilm
[9]. When S. mutans was cultivated in the presence or
absence of the spent culture medium of both L. lactis strains
at a non-killing concentration for S. mutans, the spent
medium of both L. lactis strains was found to reduce
expression of the three gtf genes (Fig. 3). However, there
was no difference in the inhibitory effect of L. lactis HY 449
and L. lactis ATCC 19435.
Sucrose Fermentation Ability of L. lactis
Sucrose is a substrate for synthesis of glucan, and the
Fig. 2. Susceptibility of S. mutans to the spent culture medium
of L. lactis.
S. mutans was cultivated with or without the spent culture medium of
L. lactis HY 449 or L. lactis ATCC 19435 at various concentrations on
96-well polystyrene plates. The growth of S. mutans was measured by
a microplate reader at 600 nm. The experiments were carried out
three times in duplicates, and data are presented as the mean ± SD. *
Statistically significant difference compared with cultures not treated
with the spent culture medium (p < 0.05).
Fig. 3. Effect of the spent culture medium of L. lactis on
expression of glucosyltransferases of S. mutans.
S. mutans was cultivated in the presence or absence of the spent
culture medium of L. lactis HY 449 or L. lactis ATCC 19435 at a non-
killing concentration for S. mutans for 12 h. Total RNA was isolated,
and cDNA was synthesized. The expression levels of gtfB, gtfC, and
gtfD were analyzed by real-time PCR. Data are presented as the
mean ± SD from six total experiments. * Statistically significant
difference compared with cultures not treated with the spent culture
medium (p < 0.001).
L. lactis HY 449 as a Probiotic for Dental Caries 1833
November 2016⎪Vol. 26⎪No. 11
glucan synthesis of S. mutans depends on the concentration
of sucrose [8]. Therefore, the sucrose-fermenting ability of
L. lactis was investigated. The acid production of L. lactis
HY 449 and L. lactis ATCC 19435 with or without sucrose
was compared. The pH level of L. lactis culture media was
determined to be near pH 5.5, even after 5 h in the presence
or absence of sucrose. Interestingly, L. lactis HY 449 was
found to produce a higher level of acid in the presence of
sucrose than in the absence of sucrose, and the pH level of
L. lactis ATCC 19435 culture media was not significantly
different between BHI broth with or without sucrose
(Fig. 4).
Discussion
Dental caries is associated with oral biofilm including
S. mutans. When the proportion of S. mutans in the oral
biofilm increases compared with other streptococci by
changing oral conditions such as sugar-rich diet, low pH,
and low saliva flow, the biofilm is called cariogenic biofilm
and induces caries lesions on the tooth surface. Therefore,
S. mutans is considered to be a greater risk factor for
induction of dental caries than planktonic S. mutans.
L. rhamnosus GG interferes with the growth and bioactivity
of cariogenic bacteria, whereby it may reduce the risks of
dental caries [13, 16]. Moreover, L. reuteri and B. animalis
reduced the S. mutans amount in saliva [2, 20]. However,
the prevention of dental caries by these probiotics remains
controversial because they are aciduric bacteria, like
S. mutans [18]. However, L. lactis is not an aciduric
bacterium because its growth is inhibited at below pH 5 [4,
19]. Therefore, the current study investigated the effects of
L. lactis HY 449 on cariogenic biofilm.
L. lactis significantly inhibited formation of cariogenic
biofilm as well as the growth of total bacteria and S. mutans
in biofilm. This report is the first to investigate the effect of
L. lactis on a cariogenic biofilm model using salivary
bacteria and S. mutans. The peculiar point is that L. lactis
HY 449 strongly inhibited formation of cariogenic biofilm
compared with L. lactis ATCC 19435. Therefore, the
characteristics of both L. lactis strains were compared and
analyzed. When the susceptibility test of S. mutans was
examined by the spent culture medium of L. lactis HY 449
and L. lactis ATCC 19435, the growth of S. mutans was
significantly decreased in media containing a greater than
40% concentration of the spent culture medium of both
L. lactis strains. However, there were no differences of the
antimicrobial activity between L. lactis HY 449 and L. lactis
ATCC 19435. Therefore, the correlation between the
antibacterial activity and inhibitory difference of the
biofilm was excluded.
Next, since glucan plays a key role in the development
and formation of cariogenic biofilm [8], we investigated the
effect of the spent culture medium of both L. lactis strains
on the expression of glucosyltransferases. When S. mutans
was cultivated with the spent culture medium of both
L. lactis strains at a non-killing concentration for S. mutans,
the expression levels of the three glycosyltransferases gtfB,
gtfC, and gtfD were significantly reduced compared with
single-cultured S. mutans. However, this experiment did
not exhibit the difference between L. lactis HY 449 and
L. lactis ATCC 19435 like the antibacterial experiment.
Finally, sucrose as a biofilm formation-related factor was
investigated. The difference between L. lactis HY 449 and
L. lactis ATCC 19435 is in their sucrose-fermenting ability.
L. lactis HY 449 ferments sucrose, unlike L. lactis ATCC
19435 [6]. Both L. lactis strains were cultivated in BHI broth
with or without sucrose, and the pH level of the culture
medium of each bacterium was measured. The pH level of
culture medium of L. lactis HY 449 quickly decreased in the
presence of sucrose compared with in the absence of
sucrose. However, the decrease of pH level of L. lactis
ATCC 19435 culture medium was not different in the two
conditions. From the S. mutans standpoint, L. lactis HY 449
is a sucrose competitor. Kreth et al. [8] reported that when
Fig. 4. pH curve of bacterial growth culture media.
L. lactis HY 449 and L. lactis ATCC 19435 were cultivated in BHI broth
with or without sucrose. Initial pH was measured immediately after
inoculating both bacteria cultures, and the pH level of the culture
medium was measured at hourly intervals. The experiments were
carried out three times in duplicates. * Statistically significant
difference compared with sucrose-free condition (p < 0.05).
1834 Kim and Lee
J. Microbiol. Biotechnol.
S. mutans is with other streptococcal competitors during
oral biofilm formation and uses sucrose for a substrate,
the antimicrobial susceptibility of S. mutans decreases.
Moreover, the glucan synthesis of S. mutans depends on the
concentration of sucrose [8]. Tong et al. [22] showed an
antagonizing effect of L. lactis on S. mutans biofilm through
production of a bacteriocin, nisin. On the basis of these
studies, L. lactis HY 449 consumes sucrose for glucan
synthesis and metabolism, whereby S. mutans may be
inhibited to take up sucrose for glucan synthesis, and the
resistance of oral bacteria in cariogenic biofilm may
decrease for the antimicrobial peptide of L. lactis HY 449.
Recently, various groups have studied to apply probiotics
for oral health, and sought candidate probiotics. However,
most proposed probiotics are controversial because of their
aciduric characteristic. In the current study, the culture
medium of L. lactis was determined to be near pH 5.5, like
oral commensal bacteria. S. mutans is a key contributor to
the development of biofilm via glucan synthesis. L. lactis
HY 449 is a competitor to S. mutans in the uptake of sucrose
and inhibited glucan synthesis by S. mutans. Therefore,
L. lactis HY 449 inhibited the formation of cariogenic
biofilm. Furthermore, the antibacterial activity of L. lactis
HY 449 inhibited the growth of S. mutans in immature
biofilm. Eventually, L. lactis HY 449 might effectively
inhibit the formation of cariogenic biofilm compared with
L. lactis ATCC 19435.
In this study, we showed that L. lactis has an inhibitory
effect on the formation of cariogenic biofilm by antimicrobial
activity, inhibition of expression of Gtfs, and consumption
of sucrose. Moreover, this bacterium is not aciduric. For
these reasons, L. lactis HY 449 may be a potential probiotic
for use in the prevention of dental caries.
Acknowledgments
The present research was supported by a research fund
of Dankook University in 2016.
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