Background: Casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) acts as a salivary biomimetic that provides bioavailable calcium and phosphate ions to augment fluoride-mediated remineralisation of early caries lesions. However, there are indications that it may also have beneficial ecological effects on the oral microbiome. Objective: This in vitro study investigated whether CPP-ACP could influence microbial counts, acidogenicity, and the relative abundance of specific caries- and health-associated bacterial -species in polymicrobial biofilms. Methods: Saliva-derived polymicrobial biofilms were grown for 96 h in a cariogenic environment and treated every 12 h with 2% CPP-ACP or vehicle control. Colony forming units (CFUs) and acidogenicity were estimated from the treated biofilms. Microbial ecological effects of CPP-ACP were assessed based on the relative abundance of 14 specific caries- and health-associated -bacterial species using a real-time quantitative PCR assay. -Results: CPP-ACP-treated biofilms showed relatively modest, but significant, reductions in microbial CFUs (21% reduction, p = 0.008) and acidogenicity (33% reduction, p < 0.001), compared to the control-treated biofilms. The CPP-ACP treated biofilms also exhibited significantly lower bacterial loads of cariogenic Scardovia wiggsiae (fold change 0.017, p < 0.001) and Prevotella denticola(fold change 0.005, p < 0.001), and higher bacterial loads of commensal Streptococcus sanguinis(fold change 30.22, p < 0.001), S. mitis/oralis(fold change 9.66, p = 0.012), and S.salivarius/thermophilus(fold change 89.35, p < 0.001) than the control-treated biofilms. Conclusions: The results indicate that CPP-ACP has virulence-attenuating attributes that can influence a beneficial microbial ecological change in the biofilm.

1.
Aas
JA
,
Griffen
AL
,
Dardis
SR
,
Lee
AM
,
Olsen
I
,
Dewhirst
FE
, et al.
Bacteria of dental caries in primary and permanent teeth in children and young adults
.
J Clin Microbiol
.
2008
Apr
;
46
(
4
):
1407
17
.
[PubMed]
0095-1137
2.
Adams
SE
,
Arnold
D
,
Murphy
B
,
Carroll
P
,
Green
AK
,
Smith
AM
, et al.
A randomised clinical study to determine the effect of a toothpaste containing enzymes and proteins on plaque oral microbiome ecology
.
Sci Rep
.
2017
Feb
;
7
(
1
):
43344
.
[PubMed]
2045-2322
3.
Aykut-Yetkiner
A
,
Kara
N
,
Ateş
M
,
Ersin
N
,
Ertuğrul
F
.
Does casein phosphopeptid amorphous calcium phosphate provide remineralization on white spot lesions and inhibition of Streptococcus mutans?
J Clin Pediatr Dent
.
2014
;
38
(
4
):
302
6
.
[PubMed]
1053-4628
4.
Becker
MR
,
Paster
BJ
,
Leys
EJ
,
Moeschberger
ML
,
Kenyon
SG
,
Galvin
JL
, et al.
Molecular analysis of bacterial species associated with childhood caries
.
J Clin Microbiol
.
2002
Mar
;
40
(
3
):
1001
9
.
[PubMed]
0095-1137
5.
Bowen
WH
,
Burne
RA
,
Wu
H
,
Koo
H
.
Oral biofilms: pathogens, matrix, and polymicrobial interactions in microenvironments
.
Trends Microbiol
.
2018
Mar
;
26
(
3
):
229
42
.
[PubMed]
0966-842X
6.
Burne
RA
.
Getting to know “The Known Unknowns”: heterogeneity in the oral microbiome
.
Adv Dent Res
.
2018
Feb
;
29
(
1
):
66
70
.
[PubMed]
0895-9374
7.
Burne
RA
,
Marquis
RE
.
Alkali production by oral bacteria and protection against dental caries
.
FEMS Microbiol Lett
.
2000
Dec
;
193
(
1
):
1
6
.
[PubMed]
0378-1097
8.
Burne
RA
,
Zeng
L
,
Ahn
SJ
,
Palmer
SR
,
Liu
Y
,
Lefebure
T
, et al.
Progress dissecting the oral microbiome in caries and health
.
Adv Dent Res
.
2012
Sep
;
24
(
2
):
77
80
.
[PubMed]
0895-9374
9.
Caruana
PC
,
Mulaify
SA
,
Moazzez
R
,
Bartlett
D
.
The effect of casein and calcium containing paste on plaque pH following a subsequent carbohydrate challenge
.
J Dent
.
2009
Jul
;
37
(
7
):
522
6
.
[PubMed]
0300-5712
10.
Chhour
KL
,
Nadkarni
MA
,
Byun
R
,
Martin
FE
,
Jacques
NA
,
Hunter
N
.
Molecular analysis of microbial diversity in advanced caries
.
J Clin Microbiol
.
2005
Feb
;
43
(
2
):
843
9
.
[PubMed]
0095-1137
11.
Dashper
SG
,
Shen
P
,
Sim
CP
,
Liu
SW
,
Butler
CA
,
Mitchell
HL
, et al.
CPP-ACP promotes SnF2 efficacy in a polymicrobial caries model
.
J Dent Res
.
2018
;
•••
:
22034518809088
.
[PubMed]
0022-0345
12.
DeSantis
TZ
,
Hugenholtz
P
,
Larsen
N
,
Rojas
M
,
Brodie
EL
,
Keller
K
, et al.
Greengenes, a chimera-checked 16S rRNA gene database and workbench compatible with ARB
.
Appl Environ Microbiol
.
2006
Jul
;
72
(
7
):
5069
72
.
[PubMed]
0099-2240
13.
Emamieh
S
,
Khaterizadeh
Y
,
Goudarzi
H
,
Ghasemi
A
,
Baghban
AA
,
Torabzadeh
H
.
The effect of two types chewing gum containing casein phosphopeptide-amorphous calcium phosphate and xylitol on salivary Streptococcus mutans
.
J Conserv Dent
.
2015
May-Jun
;
18
(
3
):
192
5
.
[PubMed]
0972-0707
14.
Erdem
AP
,
Sepet
E
,
Avshalom
T
,
Gutkin
V
,
Steinberg
D
.
Effect of CPP-ACP and APF on Streptococcus mutans biofilm: A laboratory study
.
Am J Dent
.
2011
Apr
;
24
(
2
):
119
23
.
[PubMed]
0894-8275
15.
Exterkate
RA
,
Crielaard
W
,
Ten Cate
JM
.
Different response to amine fluoride by Streptococcus mutans and polymicrobial biofilms in a novel high-throughput active attachment model
.
Caries Res
.
2010
;
44
(
4
):
372
9
.
[PubMed]
0008-6568
16.
Gross
EL
,
Beall
CJ
,
Kutsch
SR
,
Firestone
ND
,
Leys
EJ
,
Griffen
AL
.
Beyond Streptococcus mutans: dental caries onset linked to multiple species by 16S rRNA community analysis
.
PLoS One
.
2012
;
7
(
10
):
e47722
.
[PubMed]
1932-6203
17.
Heshmat
H
,
Banava
S
,
Abdian
H
,
Kharrazifard
MJ
: Effect of GC Tooth Mousse and MI Paste Plus on dental plaque acidity. J Dent Res
2014
a;92(Spec Iss B):82902.
18.
Heshmat
H
,
Banava
S
,
Mohammadi
E
,
Kharazifard
MJ
,
Mojtahedzadeh
F
.
The effect of recommending a CPP-ACPF product on salivary and plaque pH levels in orthodontic patients: a randomized cross-over clinical trial
.
Acta Odontol Scand
.
2014
b
Nov
;
72
(
8
):
903
7
.
[PubMed]
0001-6357
19.
Kianoush
N
,
Adler
CJ
,
Nguyen
KA
,
Browne
GV
,
Simonian
M
,
Hunter
N
.
Bacterial profile of dentine caries and the impact of pH on bacterial population diversity
.
PLoS One
.
2014
Mar
;
9
(
3
):
e92940
.
[PubMed]
1932-6203
20.
Koopman
JE
,
van der Kaaij
NC
,
Buijs
MJ
,
Elyassi
Y
,
van der Veen
MH
,
Crielaard
W
, et al.
The effect of fixed orthodontic appliances and fluoride mouthwash on the oral microbiome of adolescents - a randomized controlled clinical trial
.
PLoS One
.
2015
Sep
;
10
(
9
):
e0137318
.
[PubMed]
1932-6203
21.
Marchisio
O
,
Esposito
MR
,
Genovesi
A
.
Salivary pH level and bacterial plaque evaluation in orthodontic patients treated with Recaldent products
.
Int J Dent Hyg
.
2010
Aug
;
8
(
3
):
232
6
.
[PubMed]
1601-5037
22.
Marsh
PD
.
The commensal microbiota and the development of human disease - an introduction
.
J Oral Microbiol
.
2015
Sep
;
7
(
1
):
29128
.
[PubMed]
2000-2297
23.
Marsh
PD
.
In sickness and in health-what does the oral microbiome mean to us? An ecological perspective
.
Adv Dent Res
.
2018
Feb
;
29
(
1
):
60
5
.
[PubMed]
0895-9374
24.
Marsh
PD
,
Head
DA
,
Devine
DA
.
Ecological approaches to oral biofilms: control without killing
.
Caries Res
.
2015
;
49
Suppl 1
:
46
54
.
[PubMed]
0008-6568
25.
McBain
AJ
,
Sissons
C
,
Ledder
RG
,
Sreenivasan
PK
,
De Vizio
W
,
Gilbert
P
.
Development and characterization of a simple perfused oral microcosm
.
J Appl Microbiol
.
2005
;
98
(
3
):
624
34
.
[PubMed]
1364-5072
26.
Nascimento
MM
.
Potential uses of arginine in dentistry
.
Adv Dent Res
.
2018
Feb
;
29
(
1
):
98
103
.
[PubMed]
0895-9374
27.
Özdas
DO
,
Tuna
EB
,
Yilmaz
EY
,
Aytepe
Z
.
Casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) may be an alternative preventive therapy in children with cerebral palsy
.
Oral Health Prev Dent
.
2015
;
13
(
5
):
441
8
.
[PubMed]
1602-1622
28.
Peric
T
,
Markovic
D
,
Petrovic
B
,
Radojevic
V
,
Todorovic
T
,
Radicevic
BA
, et al.
Efficacy of pastes containing CPP-ACP and CPP-ACFP in patients with Sjögren’s syndrome
.
Clin Oral Investig
.
2015
Dec
;
19
(
9
):
2153
65
.
[PubMed]
1432-6981
29.
Philip
N
,
Bandara
N
,
Leishman
SJ
,
Walsh
LJ
.
Inhibitory effects of fruit berry extracts on Streptococcus mutans biofilms
.
Eur J Oral Sci
.
2018
a;
•••
:
[PubMed]
0909-8836
30.
Philip
N
,
Suneja
B
,
Walsh
LJ
.
Ecological approaches to dental caries prevention: paradigm shift or shibboleth?
Caries Res
.
2018
b;
52
(
1-2
):
153
65
.
[PubMed]
0008-6568
31.
Philip
N
,
Walsh
L
.
The potential ecological effects of casein phosphopeptide-amorphous calcium phosphate in dental caries prevention
.
Aust Dent J
.
2018
;
•••
:
[PubMed]
0045-0421
32.
Philip
N
.
State of the art enamel remineralization systems: the next frontier in caries management
.
Caries Res
.
2018
Oct
;
53
(
3
):
284
95
.
[PubMed]
1421-976X
33.
Plonka
KA
,
Pukallus
ML
,
Holcombe
TF
,
Barnett
AG
,
Walsh
LJ
,
Seow
WK
.
Randomized controlled trial: a randomized controlled clinical trial comparing a remineralizing paste with an antibacterial gel to prevent early childhood caries
.
Pediatr Dent
.
2013
Jan-Feb
;
35
(
1
):
8
12
.
[PubMed]
1942-5473
34.
Pukallus
ML
,
Plonka
KA
,
Holcombe
TF
,
Barnett
AG
,
Walsh
LJ
,
Seow
WK
.
A randomized controlled trial of a 10 percent CPP-ACP cream to reduce mutans streptococci colonization
.
Pediatr Dent
.
2013
;
35
(
7
):
550
5
.
[PubMed]
1942-5473
35.
Reilly
C
,
Goettl
M
,
Steinmetz
M
,
Nikrad
J
,
Jones
RS
.
Short-term effects of povidone iodine and sodium fluoride therapy on plaque levels and microbiome diversity
.
Oral Dis
.
2016
Mar
;
22
(
2
):
155
61
.
[PubMed]
1354-523X
36.
Reilly
C
,
Rasmussen
K
,
Selberg
T
,
Stevens
J
,
Jones
RS
.
Biofilm community diversity after exposure to 0·4% stannous fluoride gels
.
J Appl Microbiol
.
2014
Dec
;
117
(
6
):
1798
809
.
[PubMed]
1364-5072
37.
Reynolds
EC
.
The prevention of sub-surface demineralization of bovine enamel and change in plaque composition by casein in an intra-oral model
.
J Dent Res
.
1987
Jun
;
66
(
6
):
1120
7
.
[PubMed]
0022-0345
38.
Reynolds
EC
,
Cai
F
,
Shen
P
,
Walker
GD
.
Retention in plaque and remineralization of enamel lesions by various forms of calcium in a mouthrinse or sugar-free chewing gum
.
J Dent Res
.
2003
Mar
;
82
(
3
):
206
11
.
[PubMed]
0022-0345
39.
Reynolds
EC
,
Riley
PF
.
Protein dissimilation by human salivary-sediment bacteria
.
J Dent Res
.
1989
Feb
;
68
(
2
):
124
9
.
[PubMed]
0022-0345
40.
Reynolds
EC
,
Wong
A
.
Effect of adsorbed protein on hydroxyapatite zeta potential and Streptococcus mutans adherence
.
Infect Immun
.
1983
Mar
;
39
(
3
):
1285
90
.
[PubMed]
0019-9567
41.
Richards
VP
,
Alvarez
AJ
,
Luce
AR
,
Bedenbaugh
M
,
Mitchell
ML
,
Burne
RA
, et al.
Microbiomes of Site-Specific Dental Plaques from Children with Different Caries Status
.
Infect Immun
.
2017
Jul
;
85
(
8
):
85
.
[PubMed]
0019-9567
42.
Rose
RK
.
Binding characteristics of Streptococcus mutans for calcium and casein phosphopeptide
.
Caries Res
.
2000
b
Sep-Oct
;
34
(
5
):
427
31
.
[PubMed]
0008-6568
43.
Rose
RK
.
Effects of an anticariogenic casein phosphopeptide on calcium diffusion in streptococcal model dental plaques
.
Arch Oral Biol
.
2000
a
Jul
;
45
(
7
):
569
75
.
[PubMed]
0003-9969
44.
Schüpbach
P
,
Neeser
JR
,
Golliard
M
,
Rouvet
M
,
Guggenheim
B
.
Incorporation of caseinoglycomacropeptide and caseinophosphopeptide into the salivary pellicle inhibits adherence of mutans streptococci
.
J Dent Res
.
1996
Oct
;
75
(
10
):
1779
88
.
[PubMed]
0022-0345
45.
Simes
RJ
.
An improved Bonferroni procedure for multiple tests of significance
.
Biometrika
.
1986
;
73
(
3
):
751
4
. 0006-3444
46.
Simón-Soro
A
,
Mira
A
.
Solving the etiology of dental caries
.
Trends Microbiol
.
2015
Feb
;
23
(
2
):
76
82
.
[PubMed]
0966-842X
47.
Subramanian
P
,
Naidu
P
.
Effect of tooth mousse plus and cervitic gel on S. mutans
.
J Minim Interv Dent
.
2009
;
2
:
164
9
.
48.
Takahashi
N
.
Oral Microbiome Metabolism: From “Who Are They?” to “What Are They Doing?”
J Dent Res
.
2015
Dec
;
94
(
12
):
1628
37
.
[PubMed]
0022-0345
49.
Tanner
AC
,
Kent
RL
 Jr
,
Holgerson
PL
,
Hughes
CV
,
Loo
CY
,
Kanasi
E
, et al.
Microbiota of severe early childhood caries before and after therapy
.
J Dent Res
.
2011
Nov
;
90
(
11
):
1298
305
.
[PubMed]
0022-0345
50.
Tanner
AC
,
Kressirer
CA
,
Faller
LL
.
Understanding caries from the oral microbiome perspective
.
J Calif Dent Assoc
.
2016
Jul
;
44
(
7
):
437
46
.
[PubMed]
1043-2256
51.
Tanner
AC
,
Sonis
AL
,
Lif Holgerson
P
,
Starr
JR
,
Nunez
Y
,
Kressirer
CA
, et al.
White-spot lesions and gingivitis microbiotas in orthodontic patients
.
J Dent Res
.
2012
Sep
;
91
(
9
):
853
8
.
[PubMed]
0022-0345
52.
ten Cate
JM
.
Models and role models
.
Caries Res
.
2015
;
49
Suppl 1
:
3
10
.
[PubMed]
0008-6568
53.
Torlakovic
L
,
Klepac-Ceraj
V
,
Ogaard
B
,
Cotton
SL
,
Paster
BJ
,
Olsen
I
.
Microbial community succession on developing lesions on human enamel
.
J Oral Microbiol
.
2012
;
4
(
1
):
4
.
[PubMed]
2000-2297
54.
Vacca-Smith
AM
,
Van Wuyckhuyse
BC
,
Tabak
LA
,
Bowen
WH
.
The effect of milk and casein proteins on the adherence of Streptococcus mutans to saliva-coated hydroxyapatite
.
Arch Oral Biol
.
1994
Dec
;
39
(
12
):
1063
9
.
[PubMed]
0003-9969
55.
Yuan
JS
,
Reed
A
,
Chen
F
,
Stewart
CN
 Jr
.
Statistical analysis of real-time PCR data
.
BMC Bioinformatics
.
2006
Feb
;
7
(
1
):
85
.
[PubMed]
1471-2105
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