A novel small molecule, ZY354, inhibits dental caries-associated oral biofilms

Article abstract of DOI:10.1128/AAC.02414-18

Biofilm control is a critical approach to the better management of dental caries. Antimicrobial small molecules have shown their potential in the disruption of oral biofilm and control of dental caries. The objectives of this study were to examine the antimicrobial activity and cytotoxicity of a newly designed small-molecule compound, ZY354. ZY354 was synthesized, and its cytotoxicity was evaluated in human oral keratinocytes (HOK), human gingival epithelial cells (HGE), and macrophages (RAW) by CCK-8 assays. Minimal inhibitory concentrations (MICs), minimum bactericidal concentrations (MBCs), minimum biofilm inhibition concentrations (MBICs), and minimum biofilm reduction concentrations (MBRCs) of ZY354 against common oral streptococci (i.e., Streptococcus mutans, Streptococcus gordonii, and Streptococcus sanguinis) were determined by microdilution method. The exopolysaccharide (EPS)/bacterium ratio and the dead/live bacterium ratio in the ZY354-treated multispecies biofilms were determined by confocal laser scanning microscopy, and the microbial composition was visualized and quantified by fluorescent in situ hybridization and quantitative PCR (qPCR). The demineralizing activity of ZY354-treated biofilms was evaluated by transverse microradiography. The results showed that ZY354 exhibited low cytotoxicity in HOK, HGE, and RAW cells and exhibited potent antimicrobial activity against common oral streptococci. The EPS and the abundance of S. mutans were significantly reduced after ZY354 treatment, along with an increased dead/live microbial ratio in multispecies biofilms compared to the level with the nontreated control. The ZY354-treated multispecies biofilms exhibited reduced demineralizing activity at the biofilm/enamel interface. In conclusion, the small-molecule compound ZY354 exhibits low cytotoxicity and remarkable antimicrobial activity against oral streptococci, and it may have a great potential in anticaries clinical applications.

Full text of DOI:10.1128/AAC.02414-18

AAC Accepted Manuscript Posted Online 11 March 2019
Antimicrob. Agents Chemother. doi:10.1128/AAC.02414-18
Copyright © 2019 American Society for Microbiology. All Rights Reserved.
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A novel small molecule ZY354 inhibits dental caries-associated oral biofilms
a
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b, c
Chenzi Zhang , Xinyi Kuang , Yuanzheng Zhou , Xian Peng , Qiang Guo , Tao Yang
,
a
b#
a#
Xuedong Zhou , Youfu Luo and Xin Xu
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State Key Laboratory of Oral Diseases & Department of Operative Dentistry and
Endodontics, West China Hospital of Stomatology, Sichuan University, Chengdu, China.
b
State Key Laboratory of Biotherapy, West China Hospital, Sichuan University,
Chengdu, China.
c
Laboratory of Human Diseases and Immunotherapies, West China Hospital, Sichuan
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University, Chengdu, China.
Running Head: Effect of compound ZY354 on caries-associated biofilms
#Address correspondence to Youfu Luo, luo_youfu@scu.edu.cn, and Xin Xu,
xin.xu@scu.edu.cn,
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Abstract
Biofilm control is a critical approach to the better management of dental caries.
Antimicrobial small molecules have shown their potential in the disruption of oral biofilm
and control of dental caries. The objectives of this study were to examine the
antimicrobial activity and cytotoxicity of a newly designed small molecule compound
ZY354. ZY354 was synthesized and its cytotoxicity was evaluated in human oral
keratinocytes (HOK), human gingival epithelial cells (HGE) and macrophages (RAW) by
CCK-8 assays. Minimal inhibitory concentrations (MICs), minimum bactericidal
concentrations (MBCs), minimum biofilm inhibition concentrations (MBICs) and
minimum biofilm reduction concentrations (MBRCs) of ZY354 against common oral
streptococci (i.e. Streptococcus mutans, Streptococcus gordonii, Streptococcus sanguinis)
were determined by micro-dilution method. The exopolysaccharides (EPS)/bacteria and
the dead/live ratio in the ZY354-treated multispecies biofilms were determined by
confocal laser scanning microscopy, and the microbial composition was visualized and
quantified by fluorescent in situ hybridization and qPCR. The demineralizing activity of
ZY354-treated biofilms was evaluated by transverse microradiography. The results
showed that ZY354 exhibited low cytotoxicity against HOK, HGE and RAW, and
exhibited potent antimicrobial activity against common oral streptococci. The EPS and
the abundance of S. mutans were significantly reduced after ZY354 treatment, along with
an increased dead/live microbial ratio in multispecies biofilms compared with the
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non-treated control. The ZY354-treated multispecies biofilms exhibited a reduced
demineralizing activity at the biofilm/enamel interface. In conclusion, small molecule
compound ZY354 exhibits low cytotoxicity and remarkable antimicrobial activity against
oral streptococci, and it may have a great potential in the anti-caries clinical applications.
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Introduction
Dental caries is a multifactorial infectious disease dependent on diet, nutrition,
resident oral flora, and host response (1-3). Mechanical removal of dental plaque by tooth
brushing is utmost important to control dental caries. However, difficulties in maintaining
adequate plaque control, particularly at interproximal sites, necessitate the use of
chemotherapeutic agents for plaque control (4).
Among the chemotherapeutic agents used in mouthwashes, chlorhexidine (CHX) is
the ‘gold-standard’ or positive control in comparison with other substances due to its
proven efficiency (5-8). Though effective, it has certain side-effects including tooth
discoloration, oral mucosal erosion, and bitter taste. One possible drawback of CHX is its
cytotoxicity on alveolar bone cells and gingival epithelial cells (9, 10). Hence, there is a
need for alternative mouth rinse that could negate the side-effects of chlorhexidine, but
yet exhibit equivalent effectiveness.
A novel strategy to control oral biofilm is to disrupt its formation (11). Small
molecules are promising for controlling biofilm formation due to their good stability,
activity at low concentrations, and low toxicity (12). Previous studies showed that
molecules with nitroimidazole pharmacophore possessed biological activities against
various infectious diseases, especially antibacterial potency (13). Our previous work has
shown that 3-(substitutedmethylene)indolin-2-ones, such as compound 5c (Fig. 1),
exhibited excellent bactericidal activity against both clinical related Gram-positive and
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Gram-negative bacteria including methicillin-resistant Staphylococcus aureus (MRSA),
methicillin-sensitive Staphylococcus aureus (MSSA), Escherichia coli and Pseudomonas
aeruginosa (14). Antibiotics featuring nitrofuran, another antibacterial pharmacophore
with similar mode of action of nitroimidazole (13, 15), have shown inhibitory activities
against oral bacteria such as S. mutans and Enterococcus faecalis (16-18), implying the
good potential of this motif in the treatment of oral diseases such as dental caries. Hence,
we combined indole-2-one and nirtofuran motifs to get potent pharmacophore against
oral bacteria (Fig. 1). However, this type of hybrid usually has poor aqueous solubility,
which may limit its pharmaceutical characterization. Thus, in this study, we designed and
synthesized a novel water-soluble hybrid of indolin-2-one and nitrofuran ZY354 (Fig. 1),
in which a hydrophilic side chain, 3-(piperidin-1-yl) propenamide, was introduced at the
C-5 position.
The objectives of this study were to examine the cytotoxicity and antimicrobial
effects of a newly designed small molecule compound ZY354 against oral microbial
biofilms. We hypothesize that ZY354 exhibits low cytotoxicity but good antimicrobial
activity against oral streptococcal biofilms. In addition, ZY354 can alter the microbial
composition and consequently suppress the demineralizing activity of the oral biofilms.
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Results
Chemical Synthesis of compound ZY354
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As shown in Figure 1, the synthesis of the target compound ZY354 has been
accomplished in three steps: 1) a reduction of nitro group of the starting material,
5-nitroindolin-2-one,
was
performed
to
afford
intermediate
2.
2)
3-(piperidin-1-yl)propanoic acid was then attached to intermediate 2 through amide
condensation to afford intermediate 3. 3) finally, the target compound ZY354 was
obtained from an aldol condensation of intermediate 3 with 5-nitrofuran-2-carbaldehyde
(Fig. 1). Compound ZY354 exists as an E isomer exclusively (Fig. S1).
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ZY354 exhibits a good antimicrobial activity against oral streptococci
ZY354 was bactericidal against S. mutans, S. gordonii and S. sanguinis planktonic
cultures, with MICs ranging from 0.12 to 0.49 μg/mL and MBCs ranging from 0.24 to
1.95 μg/mL (Table 1). More importantly, ZY354 exhibited a good antibiofilm activity
against oral streptococcal biofilms, with MBICs against S. mutans, S. gordonii and S.
sanguinis biofilms ranging from 0.24 to 0.49 μg/mL, and MBRCs ranging from 0.12 to
31.25 μg/mL (Table 1).
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ZY354 exhibits low cytotoxicity against human oral cells
The cytotoxicity of ZY354 was evaluated by testing the viability of HOK, HGE and
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RAW after treatment. Both ZY354 and CHX exhibited low cytotoxicity against the tested
cells in an exposure duration of 5-min (IC50 > 62.5 μg/mL) (Fig. 2A, 2B and 2C). It is
noticeable that ZY354 was even less cytotoxic relative to CHX, as the viability of
ZY354-treated cells were significantly higher at the dose of 31.25 μg/mL and 62.50
μg/mL for HOK, 62.50 μg/mL for HGE, and ≥1.95 μg/mL for RAW compared to the
CHX-treated cells.
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ZY354 suppresses the development of oral streptococcal biofilms
The development of oral streptococcal biofilm is a concerted process of bacterial
accumulation and EPS generation. We further examined the effect of compound ZY354
on the bacterial cell viability and EPS production in a multispecies consortium. The
microarchitecture of the multispecies biofilms treated with ZY354 and CHX were
significantly disrupted as compared with the negative control (Fig. 3A). Either bacterial
viability or EPS production of the biofilms was significantly reduced after ZY354
treatment compared with the other two groups (P < 0.05; Fig. 3B). Specifically, the
dead/live fluorescent staining of the multispecies biofilms showed a comparable
inhibitory effect of ZY354 against the development of multispecies biofilms compared
with non-treated controls (Fig. 3D and 3E), and both ZY354 and CHX treatment did not
alter the EPS/bacteria ratio of the biofilms as compared to the negative control (Fig. 3C),
indicating that the reduction of EPS is parallel to the elimination of bacteria within the
biofilms.
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The effects of ZY354 on the microbial composition of multispecies biofilms were
further investigated with species-specific FISH and qPCR. Both ZY354 and CHX altered
the microbial composition of the oral streptococcal biofilms. The abundance of S. mutans
was significantly reduced after ZY354 treatment (Fig. 4A and 4B) compared to the
non-treated negative control. Conversely, the abundance of S. sanguinis and S. gordonii
increased after ZY354 treatment (Fig. 4A and 4B). Of note, although CHX treatment
suppressed S. mutans in the multispecies biofilms, it also depleted the commensal S.
sanguinis in the biofilms.
ZY354 halts the biofilm-mediated demineralization process of tooth enamel
The demineralizing activity of oral biofilm is closely related to its cariogenicity.
Hence, we developed saliva-derived biofilm on the tooth enamel and used transverse
microradiography to further evaluate the effect of ZY354 on the biofilm-mediated
demineralization process of tooth enamel. Either the depth of biofilm-induced lesion or
the mineral loss of the tooth enamel was significantly reduced after ZY354 treatment as
compared to the negative control. More importantly, the ZY354 treatment showed a
superior inhibitory effect against biofilm-mediated demineralization as compared to the
CHX treatment (Fig. 5A, 5B and 5C).
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Discussion
Dental caries has a polymicrobial infection etiology (3). Effective plaque biofilm
control is the utmost important measure for the management of dental caries. Along with
mechanical removal, chemotherapeutic is an indispensable supplementary to dental
plaque control (4, 19). Here we developed a small molecule compound ZY354, which
showed potent antimicrobial activity against streptococcal biofilms and low cytotoxicity
against human oral keratinocytes, human gingival epithelial cells and macrophages,
indicating its great translational potential in the clinical control of dental caries.
Our previous data have demonstrated that small molecules with nitroimidazole
pharmacophore (14) possesses biological activities against various infectious diseases
with good antibacterial potency (13). Antibiotics with nitrofuran (13, 15) also show
inhibitory activities against oral bacteria such as S. mutans and E. faecalis (16-18). Here
we designed and synthesized compound ZY354, a hybrid of indolin-2-one and nitrofuran.
We observed a good antimicrobial activity of this compound against oral streptococci in
both planktonic cultures and biofilms.
Oral biofilm formation is a well concerted process of bacterial adhesion, EPS
production and biomass accumulation. EPS produced by oral streptococci, particularly by
the cariogenic S. mutans, functions as scaffold for biofilm growth and maturation, and it
is also closely associated with biofilm properties including surface adhesion, spatial and
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chemical heterogeneities, synergistic/competitive interactions (20, 21, 36-38). Hence,
EPS has been well recognized as the key virulence factor of oral biofilm regarding caries
development, and to inhibit EPS generation is a promising approach to the disruption of
oral biofilms (20, 22, 39). In the current study, we found that compound ZY354 could
significantly reduce the EPS production by the oral biofilms, and this is likely attributed
to its great bactericidal effect against oral streptococci. The reduced live bacterial cells
within the oral biofilms after ZY354 treatment further led to a decreased production of
EPS, and ultimately disrupt the microarchitecture and formation of the matured biofilms.
Microbial equilibrium is a major determinant of biofilm cariogenicity (1-3, 23). An
oral biofilm with well-balanced acidogenic streptococci such as S. mutans and
peroxidase- and alkaline-generating competitors such as S. sanguinis is not cariogenic (1,
2, 24). The emergence of S. mutans in the multispecies biofilms usually imply an
enhanced acidogenicity and may consequently result in dental caries (25-27). Here we
developed a 3-species biofilm consisting of S. mutans, S. gordonii, and S. sanguinis, and
we found that ZY354 treatment could selectively inhibit S. mutans but enrich competing
streptococci within the biofilms. Interestingly, although exact mechanism still needs
further investigation, we found that as compared to CHX which suppressed both S.
mutans and alkaline-generating S. sanguinis, ZY354 treatment kept the abundance of S.
sanguinis intact and in the meantime enriched the peroxidase-generating S. gordonii
within the multispecies biofilms, leading to a microbial consortium with a better
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competitive edge against the opportunistic overgrowth of S. mutans, thus being beneficial
to caries management.
The biocompatibility of newly synthesized material is the bottle neck for its clinical
translation. This study tested the cytotoxicity of ZY354 against common human oral cells
that might be exposed to a mouth rinse, including HOK, HGE and RAW. Intriguingly,
although its exact activities on human cells are still unclear, the compound ZY354
exhibited lower cytotoxicity compared to CHX, one of the most commonly used
antimicrobial mouth rinse in the dental clinic (5-8), at the comparable tested
concentrations. These data demonstrate that short-duration exposure to ZY354 is not
cytotoxic to human oral cells, and this compound may have great clinical translational
potential with comparable anti-plaque efficacy as CHX.
In conclusion, this study investigated the antimicrobial effects and cytotoxicity of a
novel small molecule ZY354 against selected oral streptococci in planktonic cultures and
multispecies biofilms for the first time. The ZY354 has low cytotoxicity against common
human oral cells and possesses potent antimicrobial effect against oral biofilms. It can
also inhibit the key cariogenic factor of oral biofilm and alter the microbial composition,
leading to a biofilm with lessened demineralizing activity. The small molecule ZY354
may have a great potential in the development of novel anti-plaque and anti-caries oral
hygiene products.
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Materials and Methods
Synthesis of ZY354 and Specimen Preparation
Synthesis of intermediate 2. Activated carbon (1.0 g) and FeCl
3
(1.0 g) were added to
a suspension of 5-nitroindolin-2-one (5.0 g, 1.0 eq.) in EtOH (50 mL). The mixture was
heated to 78 °C and stirred for 10 minutes. Then 80% aqueous solution of hydrazine
hydrate (8.0 eq.) was added dropwise into the reaction mixture in 5 minutes, the resulting
mixture was stirred at 78 °C for 8-10 hours, then cooled to room temperature. The
mixture was filtered to remove residue of activated carbon, and the filtrate was
concentrated under vacuum to afford crude product that was purified by recrystallization
from EtOH (about 15 mL) to give 5-aminoindolin-2-one (intermediate 2) as a pale yellow
solid (yield 91.9%).
Synthesis
of
intermediate
3.
Triethylamine
(1.4
eq.)
and
1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium
3-oxide
hexafluorophosphate (HATU) (1.3 eq.) were added to
a
solution of
3-(piperidin-1-yl)propanoic acid (600 mg, 1.0 eq.) in dichloromethane (DCM) (7 mL).
The reaction mixture was stirred at room temperature for 20-30 minutes, then
5-aminoindolin-2-one (1.1 eq.) was added into the reaction mixture. The resulting
mixture was stirred at room temperature for 6 hours. After the reaction was finished, the
solvent was evaporated under vacuum to give the crude product which was purified by
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column chromatography to afford intermediate 3 as a colorless oil (yield 33.4%).
Synthesis of the target compound
(E)-N-(3-((5-nitrofuran-2-yl)methylene)-2-oxoindolin-5-yl)-3-(piperidin-1-yl)
ZY354,
propanamide. To a solution of the intermediate 3 (200 mg, 1.0 eq.) in MeOH (5 mL),
piperidine (1.5 eq.) and 5-nitrofuran-2-carbaldehyde (1.2 eq.) were added. The reaction
mixture was stirred at room temperature for 30 minutes. After the reaction was finished,
water (15 mL) was added to quench the reaction. The resulting mixture was extracted
with DCM (10 mL) for three times. The combined organic layer was evaporated under
vacuum, then purified by column chromatography to afford compound ZY354 as a dark
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brown solid. The product exists as an E isomer (yield 48.6 %). H NMR (400 MHz,
Pyr-d
7.56 (m, 1H), 7.10 (s, 1H), 7.02 (d, J = 7.6 Hz, 1H), 2.79 (m, 4H), 2.39 (m, 4H), 1.48 (m,
4H), 1.28 (m, 2H) (Fig. S2). 13C NMR (101 MHz, Pyr-d ) δ 170.89, 169.70, 152.92,
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) δ 12.03 (s, 1H), 10.86 (s, 1H), 8.98 (s, 1H), 7.84 (d, J = 6.0 Hz, 1H), 7.63 (s, 1H),
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152.56, 141.57, 133.90, 130.05, 126.04, 121.55, 120.72, 119.40, 117.19, 113.98, 110.20,
55.01, 53.98 (2C), 34.26, 26.14 (2C), 24.39 (Fig. S3). HRMS (Q-TOF): calculated for
C21H22N4O5 [M] :410.1590. Found [M+H]+ : 411.1665 (Fig. S4).
Bacterial Strains and Growth Media
Streptococcus mutans UA159, Streptococcus gordonii DL1, and Streptococcus
sanguinis ATCC 10556 were kindly provided by the State Key Laboratory of Oral
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Diseases (Sichuan University, Chengdu, China). S. mutans, S. gordonii and S. sanguinis
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were routinely grown at 37 C under aerobic condition (5% CO
broth (BHI; Difco, Sparks, MD).
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) in brain heart infusion
Bacteria Inoculation and Biofilm Formation
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Inoculum for the experiment was adjusted to 1×10 CFU/mL for S. mutans, S.
gordonii and S. sanguinis based on the OD600 nm versus CFU/mL graph of each
bacterium. When needed, medium was supplemented with 1% sucrose (designated BHIS),
or 1% sucrose and ZY354 at the same time, and the pH value was adjusted to 7.0 before
experiment. For multispecies biofilm formation, 300 ꢀL of bacterial inoculum consisting
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of S. mutans (1×10 CFUs/mL), S. gordonii (1×10 CFUs/mL), and S. sanguinis (1×10
CFUs/mL) in BHIS was added in the chemotaxis chamber (μ-slide 8 Well, 80826, Ibidi)
as described by Arthur et al (28). For single-species biofilm formation, bacteria were
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inoculated at a concentration of 1×10 CFUs/mL in 100 ꢀL of BHIS in 96-well plate. The
bacteria culture medium was changed every 24h.
Bacterial susceptibility assays
The minimum inhibitory concentration (MIC) and minimum bactericidal
concentration (MBC) of ZY354 against S. mutans, S. gordonii and S. sanguinis were
determined by a micro-dilution method as described previously (27) and by Nudera et al
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(29). Microtiter plates contained ZY354, their respective solvent controls, chlorhexidine
(CHX) as a positive control, cell control (test bacteria and growth medium) as a negative
control, blank well (sterile ddH
2
O), and blank medium (containing sterile ddH
2
O and
°
growth medium). All plates were then placed in aerobic condition (5% CO
2
) at 37.0 C for
24 hours for S. mutans, S. gordonii and S. sanguinis. OD600nm values were obtained with a
microplate reader (Power Wave 200 Microplate Scanning Spectrophotometer; Bio-TeK
Instruments Inc, Winooski, VT) and the Windows-based computer program KC4 Data
Analysis Software (Bio-TeK Instruments Inc). The MIC for test solutions was the
concentration of test solution allowing <0.05 A600 nm. The MBC of the test solutions
was determined by inoculating the solution mixture in wells representing the MIC and the
next 3 higher concentration wells onto the BHI agar plates (1.5% agar; Difco, Sparks,
°
MD). The inoculated agar plates were placed in an aerobic condition (5% CO
2
) at 37.0 C
for 24 hours, and the MBC was determined as the lowest concentration of test solution
that exhibited no growth.
Biofilm susceptibility assays
The effects of ZY354 on S. mutans, S. gordonii and S. sanguinis biofilm formation
were examined by the micro-dilution method as described by Wei et al (30). S. mutans
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UA159, S. gordonii or S. sanguinis (1×10 CFU/mL) was grown in BHIS and ZY354
°
(0.10 to 125 μg/mL) at 37 C for 24 h. The culture supernatant from each well was then
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decanted, and the adherent biofilm was washed three times with PBS, fixed with
methanol for 15 min, and stained with 0.1% (wt/vol) crystal violet (Sigma) for 5 min.
Subsequently, the wells were rinsed with deionized water until the blank wells appeared
colorless; 200 μl of 95% ethanol was added. The plates were shaken at room temperature
for 30 min, and the absorbance at 595 nm was recorded. The minimum biofilm inhibition
concentration (MBIC) was defined as the lowest ZY354 concentration that resulted in at
least 90% (MBIC90) inhibition of the formation of biofilms compared with that in the
untreated control. A parallel study was also performed with BHIS.
The effect of ZY354 on the removal of S. mutans, S. gordonii and S. sanguinis
biofilms was determined by the micro-dilution method modified from that of
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Ramage et al (31). A 200 μl of bacterial cell suspension (1 ×10 CFU/mL) in BHIS was
added to the wells of a 96-well microtiter plate for biofilm formation. After anaerobic
°
incubation at 37 C for 24 h, the growth medium was removed without disrupting the
integrity of the biofilms. The formed biofilms were then washed three times with PBS to
remove nonadherent cells. BHIS supplemented with ZY354 (0.10 to 125 μg/mL) was
°
added to wells containing biofilm and incubated at 37 C for 24 h. The control wells
contained BHIS without ZY354. The treated biofilms were then stained and quantified
with the method described above, and the minimum biofilm reduction concentration
(MBRC) was defined as the lowest ZY354 concentration that resulted in at least 90%
(MBRC90) reduction of biofilms compared with that in the untreated control.
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In vitro cytotoxicity/viability assay
The cell viability was determined colorimetrically with CCK-8 as described by Diab
et al (32) and Tsukatani et al (33). Cytotoxicity was evaluated in human oral
keratinocytes (HOK), human gingival epithelial cells (HGE) and macrophages (RAW).
3
HOK, HGE and RAW cells (50 × 10 cell/100 μl/ well in a 96 well plate) were grown in
medium for 24h, then treated with medium containing different concentrations of ZY354
(0.12 to 62.5 μg/mL) for 5min, and the positive control was treated with CHX at the same
time. After incubation, a volume of 10 μl of CCK-8 was added per well, and the plate was
2
incubated in the CO incubator for 3 h. The samples absorbance was measured at the
wavelength of 450 nm against blank which containing medium only. The cell viability
was calculated according to the following formula: (%) = (OD of sample - OD of blank)/
(OD of control - OD of blank) × 100%.
Biofilm Imaging
Oral multispecies biofilms were cultured in accordance with a previous study (28).
Biofilms were then exposed to PBS, 62.5 ꢀg/mL ZY354, 0.2% CHX for 3 days (5 min
three times per day).
For EPS staining, 2.5 ꢀM Alexa Fluor 647-labeled dextran conjugate (Molecular
Probes) was added at the beginning of biofilm formation and the bacteria were stained
with 2.5 ꢀM SYTO9 (Molecular Probes) for 15 min after biofilms formed (34). The
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biofilms were imaged with a Leica DMIRE2 confocal laser scanning microscope
equipped with a 60× oil immersion objective lens as dead/live imaging.
For dead/live imaging, biofilms were stained following the manufacturer’s
instruction (Invitrogen, Carlsbad, CA, USA). Briefly, the biofilms were stained with 2.5
ꢀM SYTO9 (Molecular Probes, Invitrogen) and propidium iodide (Molecular Probes) for
15 min. The labeled biofilms were imaged with a Leica DMIRE2 confocal laser scanning
microscope (Leica, Wetzlar, Germany) equipped with a 60× oil immersion objective lens
(34).
For fluorescent in situ hybridization imaging, biofilms were fixed in 4%
paraformaldehyde overnight and investigated by species-specific probes (34). The
biofilms were imaged with a Leica DMIRE2 confocal laser scanning microscope (Leica,
Wetzlar, Germany) equipped with a 60× oil immersion objective lens.
All 3-dimensional reconstructions of the biofilms were performed with Imaris 7.0.0
(Bitplane, Zürich, Switzerland), and the quantification of dead/live and EPS/bacteria
volume ratio was performed with Image-Pro Plus (Media Cybernetics, Silver Spring, MD,
USA) and COMSTAT (http://www.image-analysis.dk) (21), respectively.
DNA Isolation and Real-time Polymerase Chain Reaction
Total DNA of biofilms were isolated and purified using a TIANamp Bacteria DNA
kit (TIANGEN, Beijing, China) according to the manufacturer’s instructions. The
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bacteria were lysed using enzymatic lysis buffer (20 mM Tris-HCl, pH 8.0; 2 mM sodium
°
EDTA and 1.2% Triton X-100) containing 25 mg/mL of lysozyme at 37 C for 1.5 h. The
purity and concentration of DNA were detected by NanoDrop 2000 spectrophotometer
°
(Thermo Scientific, Waltham, MA, USA). The extracts were stored at −20 C until use.
TaqMan real-time polymerase chain reaction (Life Technologies, Carlsbad, CA, USA)
was used to quantify the absolute number of S. mutans, S. gordonii and S. sanguinis as
described by the manufacturer (Takara, Dalian, China).
Transverse Microradiography
This experiment was performed on extracted human teeth. Collection of extracted
human teeth was approved by the Ethics Committee of West China Hospital of
Stomatology, Sichuan University. All efforts were made to minimize suffering and ensure
the highest ethical and humane standards.
The labial dental crown of extracted tooth was cut into sections measuring 5 mm × 5
mm × 2 mm by using a diamond-coated band saw with continuous water cooling (Struers
Minitom; Struers, Copenhagen, Denmark). Enamel blocks were embedded in
polymethylmethacrylate and painted with two layers of acid-resistant nail varnish,
leaving a 4 mm × 4 mm window exposed on the labial enamel surface. These surfaces
were then ground flat with water-cooled carborundum discs of waterproof silicon carbide
paper (Struers) of various grits (1000, 1200, 2400, 3000 and 4000). All of the polished
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samples were individually sonicated in distilled water for 5 min to remove the residual
abrasives.
A 2 mL of bacterial inoculum consisting of S. mutans, S. gordonii, and S. sanguinis
7
(1×10 CFUs/mL for each bacterium) in BHIS was added to each well with enamel disc,
°
and then was cultured anaerobically in BHIS at 37 C (5% CO
2
). Then discs with biofilms
were exposed to PBS, 62.5 ꢀg/mL ZY354, 0.2% CHX for 5 min three times per day. To
minimize the variation in baseline mineral level, enamel discs obtained from the same
tooth were evenly distributed to each test group. The pH of all experimental solutions was
adjusted to 7.0 prior to treatment. After exposure, specimens were washed with PBS and
repositioned in the plate. BHIS was refreshed after the third exposure every day. After
5-day incubation, biofilms were detached by sonification and discs were repeatedly
washed by PBS. The result enamel discs were prepared as described by Eversole et al
(35). X-ray films of experimental lesions were acquired by an X-ray generator (Softex,
Japan) equipped with a microradiography camera, and then were further examined using
Zeiss AXIO Imager A2 microscope (Carl Zeiss, Germany). Quantitative data was
acquired by a calibrated analysis system TMR2006 (Inspektor Research Systems BV,
Netherlands). Data are obtained as the mean of 10 separate samples.
Statistical Analysis
All the experiments repeated at least 3 times independently. One-way analysis of
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variance was performed to detect the significant effects of variables, followed by the
Student-Newman-Keuls test. Differences were considered significant when P < 0.05.
Statistical analysis was performed with the SPSS software, version 16.0 (SPSS Inc.,
Chicago, IL, USA).
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Acknowledgments
This study was supported by the National Natural Science Foundation of China
(81670978 to X.Z., 81771099 to X.X., and 81602956 to T.Y.); a research grant from the
Science & Technology Department of Sichuan Province (2018SZ0121 to X.X.).
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Conflict of interest
The authors declare no competing financial interests.
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Figure legends
Figure 1.
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(A) Chemical structures of compound 5c and ZY354; (B) Synthesis of compound ZY354. Reagents
3
and conditions: (i) 80% aqueous solution of hydrazine hydrate, FeCl , activated carbon, EtOH, 78°C,
3
91.9 %; (ii) HATU, Et N, DCM, 25°C, 33.4 %; (iii) piperidine, MeOH, 25°C, 48.6 %.
Figure 2. Cytotoxicity of ZY354 on human oral keratinocytes, human gingival epithelial cells
and macrophages.
Viability of (A) HOK, (B) HGE, and (C) RAW treated with ZY354 was evaluated by CCK-8 assay.
Data are represented as mean % ± SD. HOK, human oral keratinocytes; HGE, human gingival
epithelial cells; RAW, macrophages; CHX, chlorhexidine.
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Figure 3. The antimicrobial effects of ZY354 against oral streptococcal multispecies biofilms.
(A) Representative images multispecies biofilms treated by ZY354. Green, bacteria (SYTO 9); red,
extracellular polysaccharides (EPS); (B) quantitative analysis of EPS and bacteria within the biofilms;
(C) the ratio of EPS/ bacteria within the biofilms; (D) Representative image of dead/live bacteria
within the multispecies biofilms after treatment; Green, live bacteria, red, dead bacteria; (E)
quantitative ratio of dead and live bacteria after treatment. Data are presented as mean ± standard
deviation. *P < 0.05.
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Figure 4. The composition shift of multispecies biofilms.
(A) Representative Fluorescent in situ hybridization images of multispecies biofilms; S. mutans (S. m,
green), S. gordonii (S. g, blue) and S. sanguinis (S. s, red); (B) The ratio of S. mutans, S. gordonii, and
S. sanguinis in multispecies biofilms quantified by qPCR. Data are presented as mean ± standard
deviation. *P < 0.05.
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Figure 5. The anti-demineralization effect of ZY354 against multispecies biofilms.
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(A) Representative transverse microradiography images of human enamel discs exposed to 5-day
biofilm-induced experimental demineralization. The high-density regions represent the sound enamel
tissues, while the low-density shadows indicate the caries-like lesions. (B) Lesion depth and (C)
mineral loss were calculated. Data are presented as mean ± standard deviation. *P < 0.05.
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Table 1. Antimicrobial effect of ZY354 against S. mutans, S. gordonii and S. sanguinis planktonic
cultures and biofilms in BHI medium.
Planktonic cells
Biofilm
Bacterial
Strain
MIC
MBC
MBI
MBRC
(ȝg/ml)
(ȝg/ml)
(ȝg/ml)
(ȝg/ml)
S.mutans
S.gordonii
S. sanguinis
0.24
0.49
0.12
1.95
0.98
0.24
0.24
0.24
0.49
31.25
15.63
0.12
MIC, minimum inhibitory concentration; MBC, minimum bactericidal concentration; MBIC,
minimum biofilm inhibition concentrations; MBRC, minimum biofilm reduction concentrations.