Antibacterial effect of on planktonic cells and bio fi lms of Streptococcus mutans and Streptococcus parasanguinis

(1)

DOI 10.1007/s00044-017-1877-y

_RESEARCH

O R I G I N A L R E S E A R C H

Antibacterial effect of chalcogenoesters on planktonic cells and

bio

_ﬁ

lms of

Streptococcus mutans

and

Streptococcus parasanguinis

Leonardo Silva de Sousa1●Agena Camara-Leimbach1●

Mayron Alves de Vasconcelos1●Francisco Vassiliepe Sousa Arruda1●

Rafael Santos da Silva2●Luciano Dornelles2●Oscar Endrigo Dorneles Rodrigues2●

Edson Holanda Teixeira2

Received: 5 December 2016 / Accepted: 11 March 2017 / Published online: 23 March 2017 © Springer Science+Business Media New York 2017

Abstract The purpose of this work was to evaluate the

antibacterial and antibioﬁlm activity of ﬁve

chalcogenoe-sters synthetics on oral bacteria,Streptococcus mutans, and

Streptococcus parasanguinis. Five chalcogenoesters were synthesized, purified by chromatography in silica gel and its chemical structure determined by nuclear magnetic reso-nance. Antibacterial assays were performed using the microdilution methodology. The antibiofilm activity of chalcogenoesters was determined on biofilm formation and preformed biofilms by biofilm mass quantification (by crystal violet staining) and colony forming unit enumera-tion. Moreover, biofilms were also analyzed by scanning electron microscopy. In general, it was observed that the chalcogenolesters showed antibacterial activity against

Streptococcus mutans and Streptococcus parasanguinis. Regarding biofilm formation and preformed biofilm, all compounds reduced efficiently the biofilm mass and of

viable cells of Streptococcus parasanguinis and

Strepto-coccus mutans bioﬁlms. However, Streptococcus mutans

bioﬁlms showed greater resistance to action of chalco-genolesters. Scanning electron microscopy examination conﬁrmed the results, showing a reduction of the

extracellular polymeric substance and number of cells in bioﬁlm. Our result indicated that the chalcogenoesters tested here can be considered as promising molecules for prevention and control of oral bioﬁlms formed by

Streptococcus mutans andStreptococcus parasanguinis.

Keywords Chalcogenoesters ●Antibacterial●Bioﬁlm●

Streptococcus

Introduction

The human oral cavity is colonized by several bacterial

species (Cauﬁeld et al. 2015). Such cavity comprises

dif-ferent habitats, including teeth, gingival sulcus, tongue, cheeks, hard and soft palates, and tonsils, which can serve

as shelter for bacterial development (Dewhirst et al.2010).

More than 700 bacterial species or phylotypes have been detected in the oral cavity and some of these have been implicated in oral diseases such as dental caries and

periodontitis (Aas et al. 2005). In fact, dental caries is

considered the most prevalent human disease, affecting 80–90% of the world population (Simon-Soro and Mira

2015).

A key factor in the development of dental caries is the natural adhesion of certain bacteria to the tooth surface and subsequent formation of a dental plaque, which is a classic

example of bioﬁlm (Marsh2010). A bioﬁlm is an

assem-blage of microbial cells that is associated to a surface and enclosed in a self-produced matrix of hydrated extracellular

polymeric substances (EPS) (Donlan 2002; Flemming and

Wingender2010). Bacterial cells in bioﬁlms display a

dif-ferent phenotype from those growing planktonically, being

* Edson Holanda Teixeira [email protected]

1

Laboratório Integrado de Biomoléculas, Departamento de Patologia e Medicina Legal, Universidade Federal do Ceará, Fortaleza, CE, Brazil

2

NanoBio-LabSelen, Departamento de Química, Universidade Federal de Santa Maria, Santa Maria, RS, Brazil

(2)

resistant to several antibiotics and antimicrobial agents

(Marsh2004; Hall-Stoodley et al.2004).

Given the importance of bioﬁlms in human health as well as in industry, many studies have been carried out to develop new strategies to control its formation on either

biotic or abiotic surfaces (Simoes et al.2010). Therefore,

the search for new compounds able in inhibiting its for-mation or controlling its growth is of great importance.

Organochalcogens are a class of organic compounds that have aroused interest by the scientiﬁc community mainly due to their several biological properties, among them antioxidant, antitumor, and antimicrobial activities (Mugesh

et al.2001; Nogueira et al.2004; Sarma and Mugesh2008;

Das et al.2008; Bhabak and Mugesh2009; de Souza et al.

2015). Chalcogenoesters (selenol, thiol, and tellurol esters)

are useful synthetic intermediates that have been employed for several chemical transformations (Boger and Mathvink

1989; Boger and Mathvink 1992; Lucas and Schiesser

1996; Keck and Grier1999; Pattenden et al.2009; Rampon

et al. 2010). Additionally, some organoselenium

com-pounds have been screened regarding their antimicrobial properties showing some prominent responses (Vargas et al.

2012).

Therefore, this study was performed to evaluate the antimicrobial and antibioﬁlm activities of synthetic

chal-cogenoesters on oral bacteriaStreptococcus parasanguinis

American Type Culture Collection (ATCC) 903 and

Streptococcus mutans ATCC 25175, both involved in dental caries formation.

Materials and methods

Synthesis of chalcogenoesters

The chalcogenoesters were synthesized as previously

described. S501 and S503 (Ren et al. 2010), S502

(Nar-ayanaperumal et al.2011) andS505andS506(Wang et al.

2014). The chemical structures were conﬁrmed by1H and

13

C NMR.

Se-phenyl 4-methylbenzoselenoate (S501)

1

H NMR (CDCl3, 400 MHz),δ(ppm): 7.81 (d,J=8.31 Hz,

2H), 7.57 (m, 2H), 7.38 (m, 3H), 7.24 (d,J=8.31 Hz, 2H).

13

C NMR (CDCl3, 100 MHz),δ(ppm): 192.4, 144.8, 136.3,

136.2, 129.5, 129.2, 128.8, 127.4, 126.1, 21.6.

Se-phenyl 4-nitrobenzoselenoate (S502)

1

2H), 8.07 (d,J=9.04 Hz, 2H), 7.56 (m, 2H), 7.44 (m, 2H).

13

131.6, 129.6, 129.6, 129.1, 128.1, 124.2.

Se-p-tolyl 4-methylbenzoselenoate (S503)

1

2H), 7.44 (d,J=8.07, 2H), 7.26–7.15 (m, 4H), 2.40 (s, 3H),

2.38(s, 3H). 13C NMR (CDCl3, 100 MHz),δ(ppm): 193.1,

144.8, 139.01, 136.3, 130.2, 129.5, 127.4, 122.3, 21.7, 21.3.

S-4-chlorophenyl 4-methylbenzothioate (S505)

1

H NMR (CDCl3, 400 MHz), δ (ppm): 7.89 (d, J=8.31

Hz), 7.41–7.38 (m, 4H), 7.28 (d,J=_{8.31 Hz), 2.42 (s, 3H).}

13

129.2, 126.7, 21.7.

S-4-chlorophenyl 4-nitrobenzothioate (S506)

1

2H), 8.18 (d, J=_{9.04 Hz, 2H), 7.50–7.38 (m, 4H).} 13_C

NMR (CDCl3, 50 MHz), δ (ppm): 188.4, 150.7, 140.9,

136.6, 136.1, 129.8, 128.5, 124.5, 113.8.

Microorganisms

Microorganisms used in this study were S. mutans ATCC

25175 and S. parasanguinis ATCC 903, which were

obtained from the ATCC.

Culture conditions

Bacteria were grown in Brain Heart Infusion Agar (BHI

Agar; Himedia, India) by incubation at 37 °C in 5% CO2for

24 h. After growth on solid medium, some isolated colonies were removed and inoculated into 10 ml fresh BHI and

incubated again at 37 °C in 5% CO2for 24 h under constant

agitation. Prior to use, the cell density of each inoculum was

adjusted to 2×106cells/ml.

Antibacterial activity on planktonic cells

The effect of chalcogenoesters on planktonic cells was determined by the broth microdilution method described in

the guideline “Methods for Dilution Antimicrobial

Sus-ceptibility Tests for Bacteria that Grow Aerobically;

Approved Standard—Nineth Edition (CLSI document

M07-A9)”, with modiﬁcations. Brieﬂy, in 96-well

poly-styrene plates, the compoundsS501,S502,S503,S505, and

S506 were diluted in BHI broth containing 4% dimethyl

sulfoxide in concentrations ranging from 250 to 3.9µg/ml.

The assay was performed by the addition of 100μl of

(3)

with 1% sucrose and 100μl of each chalcogenoester at different concentrations. The microplates were then

incu-bated at 37 °C in 5% CO2for 24 h. The minimum inhibitory

concentration (MIC) was determined as the lowest chalco-genoester concentration showing a complete inhibition of visible bacterial growth.

Effect of chalcogenoesters on bio_ﬁlm formation

The methodology used to monitor the effects of chalco-genoesters on bioﬁlm formation was based on the microtiter

plate test developed by Stepanovic et al. (2000) with

some modifications. Briefly, sterile flat-bottom 96-well

polystyrene plates were prepared following a similar pro-cedure to that used to determine the effects on planktonic growth. However, the effect on bioﬁlm formation was

assessed by two distinct assays: biomass quantiﬁcation by

crystal violet staining and enumeration of bioﬁlm-entrapped viable cells.

Bioﬁlm mass quantiﬁcation

The biofilm mass was quantified by crystal violet staining. After biofilm development, the content of each well was

removed, and the wells were washed twice with 200µl of

ultrapure water to remove weakly adhered cells. Forﬁxation

of bioﬁlm mass, 200µl of 99% methanol were added to

each well, and after 15 min, the methanol was removed and the plates were allowed to dry at room temperature.

After-wards, 200µl of crystal violet stain (Merck, Germany) were

added to each well and after 5 min, the excess of crystal violet was removed. The plates were then washed twice

with water and after drying, 200µl of acetic acid (33%, v/v)

were added to wells to dissolve the bound crystal violet. The absorbance of each well was then measured at 590 nm

(OD590) using a microplate reader (SpectraMax® I3).

Enumeration of bioﬁlm-entrapped cells

In order to determine the effect of chalcogenoesters on the viability of bioﬁlm-entrapped cells the wells were washed

twice with 200µl of ultrapure water. Afterwards, the wells

were ﬁlled with 200µl of ultrapure water and the plates were placed in an ultrasonic bath for 10 min to release the cells entrapped within the bioﬁlm matrix. In order to quantify the viability, serial decimal dilutions from the obtained suspensions were plated on BHI agar and grown

for 24 h in 5 % CO2 at 37 °C. The number of colony

forming units (CFU) was determined and expressed as CFU

per ml (log10CFU/ml).

Effect of chalcogenoesters on pre-formed bio_ﬁlms

In order to evaluate the activity of chalcogenoesters on

bacterial mature bioﬁlms, cells suspensions (200µl of

106cells/ml) were added to each well and incubated for 24 h

at 37 °C in 5% CO2under constant agitation. After bioﬁlm

development the wells were washed twice with 200µl of

ultrapure water and 200µl of each chalcogenoester in BHI

(at concentrations ranging from 7.8 to 200µg/ml) was

added to wells. The plates were incubated for 24 h at 37 °C

in 5% CO2 under constant agitation. After of 24 h, the

medium was aspirated, and each well was washed twice with ultrapure water. Pre-formed biofilms were then eval-uated by biomass quantification and enumeration of biofilm viable cells as previously described.

Statistical analysis

Statistical analyses were performed by GraphPad Prism® version 5.0 from Microsoft Windows®. Data from all assays were compared using one-way analysis of variance, with Bonferroni post hoc test. Data were considered

sig-niﬁcant whenp<0.05.

Scanning electron microscopy

Bacterial bioﬁlms were grown in the presence of the

com-pound S501 at 250µg/ml and observed by scanning

elec-tron microscopy (SEM) in a Quanta 450 FEG (Fei, USA). The bioﬁlm formation assay was carried out in 24-well microplates. After bioﬁlm formation, the plates were washed with sterilized water, dehydrated with alcohol (70% ethanol for 10 min, 95% ethanol for 10 min, and 100% ethanol for 20 min), and allowed to dry prior to gold

coating as described by Vasconcelos et al. (2014).

Results

Chemical synthesis

Five analogs of chalcongenesters were properly synthesized

(S501,S502, S503, S505, and S506) using the respective

methodologies described previously in the literature.

Chemical structures were elucidated using 13C and 1H

nuclear magnetic resonance (NMR). The structures are

shown below (Fig.1).

Table1shows data acquired in experiments using NMR.

(4)

Minimal inhibitory concentration and minimal bactericidal concentration

The values of MIC of each compound onS. parasanguinis

andS. mutansare described in Table2.

Effect on bio_ﬁlm formation

Bioﬁlm mass

The bioﬁlm mass of S. parasanguinis was reduced when

bioﬁlms were grown in the presence of S503, S505, and

S506at different concentrations and fully inhibited by the

treatment with S501 and S502 at 250 and 31.25µg/ml,

respectively (Fig.2). Regarding S. mutans strain, the

bio-ﬁlm mass was reduced by the treatment withS501andS502

at most concentrations assayed, while S503, S505, and

S506were effective only at higher concentrations (Fig.2).

Viability of bioﬁlm-entrapped cells

The number of viable cells embedded within the matrix of bioﬁlms grown in the presence of chalcogenoesters was also determined. With few exceptions, all compounds reduced the viability of both strains in a dose-dependent manner

(Fig.3). In general,S. parasanguiniswas more susceptible

to all agents. The compoundsS501,S502,S503and S506

reduced the viability ofS. parasanguinis in all

concentra-tions tested, with reduction levels ranging from 2 to 4.5 log.

On the other hand, S505 was effective only at higher

concentrations.

Regarding S. mutans, the effect of compounds on its

viability was lower than that achieved onS. parasanguinis.

Curiously, the compounds S501, S502, S503, and S505

were less effective at higher concentrations, being the best effect achieved at lower concentration. On the other hand,

the compoundS506reduced the viability ofS. mutansin all

tested concentrations with reduction levels ranging from 1.9

to 3.7 log, which were achieved at higher concentrations

(Fig. 3).

Effect on preformed bio_ﬁlms

Bioﬁlm mass

The chalcogenoesters were also evaluated regarding their

effect on bioﬁlm mass of S. parasanguinis and S. mutans

preformed bioﬁlms. The results showed that all compounds

inhibited the biomass of S. parasanguinis (Fig. 4). The

compound S506 at 250µg/ml reduced S. parasanguinis

biomass at about 85%. Even in the lowest concentration

tested (7.8µg/ml), the same compound was effective on

biomass, reducing it in approximately 31%. The compounds S501,S502, andS505reduced biomass in 55, 63, and 56%, respectively.

The same results were not seen onS. mutans, since the

biomass was reduced only in higher concentrations

eval-uated (Fig. 4).

Viability of bioﬁlm-entrapped cells

The viability of bioﬁlm-entrapped cells of preformed

bio-ﬁlms was assessed on both strains after the treatment with

chalcogenoesters. All compounds reduced the viability of

S. parasanguinis. Moreover, this effect was seen mainly in

higher concentrations (Fig. 5). The best reduction was

achieved by the treatment with S502, which reduced the

viability at about 1.9, 1.2 and 1.1 log at concentrations of

250, 125 and 62.5µg/ml, respectively.

Regarding S. mutans, the viability was reduced mainly

by the treatment withS501,S502, andS503. Interestingly,

as previously reported (in the section Effect on preformed bioﬁlms), the best effect on preformed bioﬁlms was also

achieved when S. mutans bioﬁlms were treated with

chal-cogenoesters at lower concentrations (Fig. 5). The same

trend was not seen in bioﬁlms treated withS505andS506,

(5)

since the viability was better reduced by the treatment with higher concentrations of compounds.

Analysis of the bio_ﬁlms by SEM

The effect of S501 _{on bioﬁlm architecture of both}

strains was evaluated by SEM inspection (Fig. 6). The

images revealed morphological alterations in bioﬁlms of

both species. Regarding control groups,S. mutansbioﬁlms

showed a large number of cells and EPS accumulation

(Fig. 6a). On the other hand, S. parasanguinis showed a

lower bioﬁlm content in comparison with S. mutans

(Fig. 6b).

Bioﬁlms formed by both strains in the presence ofS501

showed an evident decrease in the number of cells as well as

EPS content (Fig.6c and d).

Discussion

This study evaluates for the ﬁrst time the effect of

ﬁve synthetic chalcogenoesters on planktonic cells and

bioﬁlms fromS. parasanguinisandS. mutans, two

impor-tant pathogens involved in dental plaque and caries development.

S. parasanguinis plays a key role in development of cariogenic bioﬁlms, since it express several genes related to adhesion proteins, such as Fap1 gene, which is responsible

for the production of ﬁmbriae, which mediates microbial

adhesion to dental surfaces and BapA1 gene, which is responsible for the direct link between initial and secondary colonizers, and thus contributing to the development of

bioﬁlms (Garnett et al. 2012; Geng et al. 2012). On the

other hand, S. mutansis widely known by its aciduric and

acidogenic properties, being considered one of the most important pathogen involved in dental caries (Liu and

Burne 2009). Oral bioﬁlms directly contribute to

develop-ment of dental caries. Thus, the discovery of substances able in inhibiting bioﬁlm formation by pathogens or even in destroying those already established is extremely important for human health.

The MIC found in this work indicates that each com-pound exerts a speciﬁc action against tested bacteria. This

fact can be observed by the effect of selenoestersS501and

Table 1 Chemical nomenclature and NMR data of synthesized chalcogenoesters Compound id Chemical nomenclature NMR data ( 1 H and 13 C) S501 Se-phenyl-4-methylselenobenzoate 1H NMR (CDCl 3 , 400 MHz): δ = 7.80 (d, J = 8.4 Hz, 2H), 7.58 – 7.55 (m, 2H), 7.38 – 7.36 (m, 3H), 7.20 (d, J = 8.0 Hz, 2H), 2.34 (s, 3H) 13 C NMR (CDCl 3 , 100 MHz) δ = 192.4,144.7, 136.1, 135.8, 129.4, 129.1, 128.7, 127.2, 125.8, 21.5 S502 Se-phenyl 4-nitroselenobenzoate 1H NMR (CDCl 3 , 400 MHz): δ = 8.35 – 8.33 (m, 2H), 8.17 – 8.00 (m, 2H), 7.66 – 7.40 (m, 2H), 7.30 – 7.23 (m, 3H) 13 C NMR (CDCl 3 , 100 MHz) δ = 192.5, 150.6, 143.0, 136.1, 131.5, 129.6, 128.1, 124.9, 124.2 S503 p -Toluil p -methylbenzoselenoate 1 H NMR (CDCl 3 , 400 MHz): δ = 7.80 (d, J = 8.07 Hz, 2H), 7.44 (d, J = 8.07 Hz, 2H), 7.26 – 7.15 (m, 4H), 2.40 (s, 3H), 2.38 (s, 3H) 13 C NMR (CDCl 3 , 100 MHz), δ = 193.13; 144.77; 139.09; 136.29; 130.17; 129.53; 127.42; 122.29; 21.71; 21.31 S505 p-Chlorophenyl p -methylbenzothioate 1 H NMR (CDCl 3 , 400 MHz), δ = 7.89 (d, J = 8.31 Hz), 7.41 – 7.38 (m, 4H), 7.28 (d, J = 8.31 Hz), 2.42 (s, 3H) 13 C NMR (CDCl 3 , 100 MHz), δ = 172.09, 144.57, 130.28, 129.19, 126.73, 21.67 S506 p -Chlorophenyl p -nitrobenzothioate 1 H NMR (CDCl 3 , 200 MHz), δ = 8,34 (d, J = 9.04 Hz, 2H), 8.18 (d, J = 9.04 Hz, 2H), 7.50 – 7.38 (m, 4H) 13 C NMR (CDCl 3 , 50 MHz), δ = 188.36, 150.72, 140.92, 136.59, 136.10, 129.77, 128.50, 124.54, 113.79

Table 2 Minimal inhibitory concentration of chalcogenesters determined after 24 h growth ofS. mutansandS. parasanguinis

Strain MIC (µg ml−1₎

S501 S502 S503 S505 S506

(6)

Fig. 2 Biofilm mass ofS. parasanguinisATCC 903aandS. mutans ATCC 25175bafter a 24 h growth in the presence of chalcogenesters. Biomass quantification was performed by staining biofilms with

crystal violet and reading the absorbance at 590 nm.Error bars indi-cate the standard deviation (SD). *=p<0.05 compared to the control group

Fig. 3 Viability of bioﬁlm-entrapped cells ofS. parasanguinisATCC 903aandS. mutansATCC 25175bafter a 24 h growth in the pre-sence of chalcogenoesters. The viability was determined by the

(7)

Fig. 4 Biofilm mass ofS. parasanguinisATCC 903aandS. mutans ATCC 25175bpreformed biofilms. The biofilms were treated with chalcogenoesters. Biomass quantification was performed by staining

bioﬁlms with crystal violet and reading the absorbance at 590 nm. Error barsindicate the standard deviation (SD). *p<0.05 compared with the control group

Fig. 5 Viability of biofilm-entrapped cells ofS. parasanguinisATCC 903aandS. mutansATCC 25175bpreformed biofilms. The biofilms were treated with chalcogenoesters. The viability was determined by

(8)

S502, which were more effective on S. parasanguinis

planktonic cells. When the structures of selenoesters are

compared, it is observed that S502displays a deactivator

group of the benzene ring. Such group is probably the

responsible for better actions exerted by S502 thanS501,

since the last presents an electron donating activator group

(–CH3), which probably generates a more stable radical.

Unlike selenoesters, the thioesters analogsS505and S506

were more effective on S. mutans planktonic cells, with

MIC values of 250 and 62.5µg/ml, respectively. The

dif-ferences in actions should be explained by the presence of

polar groups in benzene rings ofS506, which are able to

deactivate and remove electrons of such rings (–Cl and

–NO2). Such characteristic probably generates unstable ions

that can act as free radicals againstS. mutansand in a less

extent onS. parasanguinis.

Radhakrishna et al. (2010) showed that the

organosele-nium3-[(phenylcarbonyl)selenyl]propanoic acid and differ-ent ester derivatives from that compound showed potdiffer-ential

antibacterial activity against Staphylococcus aureus,

Sal-monella typhimurium, Escherichia coli and Bacillus

subtilis. Moreover, some chiral chalcogenoamines showed

antibacterial activity against Bacillus cereus, Listeria

monocytogenes and different species of Paenibacillus

(Vargas et al.2012).

Concerning the effects on bioﬁlm formation of both

strains, the results showed that all compounds were able in reducing signiﬁcantly the bioﬁlm formation. The viability

of cells embedded in EPS matrix of bioﬁlms grown in the

presence of compounds was also evaluated. All compounds

reduced the viability of S. parasanguinis and S. aureus.

Curiously, the effect onS. mutans was seen mainly by the

treatment withS501,S502,S503, andS505, which reduced

the number of viable cells as the concentration of com-pounds decreased. Interestingly, the bioﬁlm mass was higher in concentrations that reduced cell viability, sug-gesting that compounds initially stress bacterial cells, thus promoting high amounts of biomass. Afterwards, cells lose

their viability. The compoundS506was the most efﬁcient

in reducing viability ofS. mutans. Probably the two polar

groups and deactivators of the benzene ring were respon-sible for this phenomenon.

(9)

The effect of compounds on preformed biofilms of both strains was lower than that seen on biofilm formation. Both biofilm mass and cell viability were higher when compared to biofilm formation assay. This is not surprisingly, since bacterial growth in biofilms promotes protection against the action of antibiotics and environmental stressors mainly because the physical barrier formed by the extracellular matrix. In addition, when microbial cells are surrounded by EPS matrix, bacterial communication mechanisms stimulate the production of enzymes and proteins important in the

physiological adaptation of bioﬁlm (Corbin et al. 2011;

Soto2013).

Organochalcogens are lipophilic compounds that can interact with layers of polysaccharides, fatty acids, and phospholipids in bacterial membrane increasing its perme-ability and causing damage to the structures (Goldbeck et al.

2014). The mechanism of action of these compounds could

be explained by the disruption of the peptidoglycan layer, altering the cytoplasmic membrane, changing its perme-ability and releasing cellular constituents. Corroborating

with this hypothesis, Rosseti et al. (2015) showed that the

diphenyl diselenide [(PhSe)2] inhibited the growth and

bioﬁlm formation of Candida albicans by increasing the

membrane permeability.

SEM images corroborated with results obtained in the assays for quantiﬁcation of biomass and the number of

viable cells in bioﬁlms.S. mutans bioﬁlm has a thick EPS

matrix evolving from bacterial cells, which was clearly

reduced in the presence of S501 (Figs. 6a and b). EPS

matrix is an important component that promotes mechanical stability and protection to bioﬁlms (Flemming and

Win-gender 2010), antimicrobial compounds able in reduce or

promote changes in bioﬁlm matrix can be considered

pro-mising molecules. Regarding,S. parasangunis bioﬁlm, the

SEM images showed a strong decrease in the number of

bacterial cells (Fig. 6c and d). In fact, S501 reduced

abruptly the number of viable cells of S. parasanguinis

bioﬁlm (Fig.3a).

In general, the chalcogenoesters were more effective on

S. parasanguinis. Nevertheless,S. mutansstrain used is this study produces high amounts of bioﬁlms (Liu and Burne

2009), which may explain its increased resistance to

chalcogenoesters.

In conclusion, this study demonstrates, for theﬁrst time,

the antibacterial activity of chalcogenoesters on the plank-tonic growth, biofilm formation and disruption of the pre-formed biofilm of oral bacteria. Moreover, chalcogenoesters can be considered as promising molecules with potential for the treatment of some streptococcal biofilms, which play important roles in dental caries.

Acknowledgements The authors are grateful to the Fundação Cearense de Apoio ao Desenvolvimento Cientíﬁco e Tecnológico

(FUNCAP), Conselho Nacional de Desenvolvimento Cientíﬁco e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Central Analítica-UFC/CT-INFRA/ MCTI-SISNANO/Pró-Equipamentos CAPES. E. H. Teixeira is a member of the Brazilian Academy of Sciences.

Conﬂict of interest The authors declare that they have no conﬂict of interest.

References

Aas JA, Paster BJ, Stokes LN, Olsen I, Dewhirst FE (2005) Deﬁning the normal bacterialﬂora of the oral cavity. J Clin Microbiol 43 (11):5721–5732

Bhabak KP, Mugesh G (2009) Synthesis and structure–activity correlation studies of secondary- and tertiary-amine-based glutathione peroxidase mimics. Chem Eur J 15(22):9846–9854 Boger DL, Mathvink RJ (1989) Phenyl selenoesters as effective

pre-cursors of acyl radicals for use in intermolecular alkene addition reactions. J Org Chem 54(8):1777–1779

Boger DL, Mathvink RJ (1992) Acyl radicals: intermolecular and intramolecular alkene addition reactions. J Org Chem 57 (5):1429–1443

Cauﬁeld PW, Schön CN, Saraithong P, Li Y, Argimón S (2015) Oral lactobacilli and dental caries: a model for niche adaptation in humans. J Dent Res 94(2):110–118

Corbin A, Pitts B, Parker A, Stewart PS (2011) Antimicrobial pene-tration and efﬁcacy in an in vitro oral bioﬁlm model. J Anti-microb Chemother 55(7):3338–3344

Das D, Roy G, Mugesh GJ (2008) Antithyroid drug carbimazole and its analogues: synthesis and inhibition of peroxidase-catalyzed iodination of l-tyrosine. Med Chem 51(22):7313–7317

de Souza D, Mariano DOC, Nedel F, Schultze E, Campos VF, Seixas F, da Silva RS, Munchen TS, Ilha V, Dornelles L, Braga AL, Rocha JBT, Collares T, Rodrigues OED (2015) New orga-nochalcogen multitarget drug: synthesis and antioxidant and antitumoral activities of chalcogenozidovudine derivatives. J Med Chem 58(8):3329–3339

Dewhirst FE, Chen T, Izard J, Paster BJ, Tanner AC, Yu WH, Lakshmanan A, Wade WG (2010) The human oral microbiome. J Bacteriol 192(19):5002–5017

Donlan RM (2002) Bioﬁlms: microbial life on surfaces. Emerg Infect Dis 8(9):881–890

Flemming HC, Wingender J (2010) The bioﬁlm matrix. Nat Rev Microbiol 8(9):623–633

Garnett JA, Simpson PJ, Taylor J, Benjamin SV, Tagliaferri C, Cota E, Chen YY, Wu H, Matthews S (2012) Structural insight into the role of Streptococcus parasanguinis Fap1 within oral bioﬁlm formation. Biochem Biophys Res Commun 417(1):421–426 Geng J, Chiu CH, Tang P, Chen Y, Shieh HR, Hu S, Chen YY (2012)

Complete genome and transcriptomes of Streptococcus para-sanguinis FW213: phylogenic relations and potential virulence mechanisms. PLoS One 7:347–360

Goldbeck JC, Victoria FN, Motta A, Savegnago L, Jacob RG, Perin G, Lenardão EJ, Silva WP (2014) Bioactivity and morphological changes of bacterial cells after exposure to 3-(p-chlorophenyl)thio citronellal. Lwt- Food Sci Technol 59(2):813–819

Hall-Stoodley L, Costerton JW, Stoodley P (2004) Bacterial bioﬁlms: from the natural environment to infectious diseases. Nat Rev Microbiol 2:95–108

(10)

Liu Y, Burne RA (2009) Multiple two-component systems of Strep-tococcus mutans regulate agmatine deiminase gene expression and stress tolerance. J Bacteriol 191(23):7363–7366

Lucas MA, Schiesser CH (1996) (Aryltelluro)formates as precursors of alkyl radicals: thermolysis and photolysis of primary and secondary alkyl (aryltelluro)formats. J Org Chem 61(17): 5754–5761

Marsh PD (2004) Dental plaque as a microbial bioﬁlm. Caries Res 38 (3):204–211

Marsh PD (2010) Microbiology of dental plaque bioﬁlms and their role in oral health and caries. Dent Clin N Am 54 (3):441–454

Mugesh G, Du Mont WW, Sies H (2001) Chemistry of biologically important synthetic organoselenium compounds. Chem Rev 101 (7):2125–2179

Narayanaperumal S, Alberto EE, Gul K, Kawasoko CY, Dornelles L, Rodrigues OED, Braga AL (2011) Zn in ionic liquid: an efﬁcient reaction media for the synthesis of diorganyl chalcogenides and chalcogenoesters. Tetrahedron 67(25):4723–4730

Nogueira CW, Zeni G, Rocha JBT (2004) Organoselenium and organotellurium compounds: toxicology and pharmacology. Chem Rev 104(12):6255–6285

Pattenden G, Stoker DA, Winne JM (2009) A synthetic approach to C-nor-D-homosteroids based on a cascade of radical cyclisations from a vinylcyclopropane-substituted acyl radical precursor. Tetrahedron 65:5767–5775

Radhakrishna PM, Sharadamma KC, Vagdevi HM, Abhilekha PM, Rubeena Mubeen S, Nischal K (2010) Synthesis and antibacterial activity of novel organoselenium compounds. Int J Chem 2 (2):149–154

Rampon DS, Rodembusch FS, Goncalves PFB, Lourega RV, Merlo AA, Schneider PH (2010) An evaluation of the chalcogen atom effect on the mesomorphic and electronic properties in a new homologous series of chalcogeno esters. J Braz Chem Soc 21 (11):2100–2107

Ren K, Wang M, Liu P, Wang L (2010) Iron-catalyzed syhnthesis of selenoesters from diselenides and acyl chlorides or acid anhy-drides in the presence of magnesium dust. Synthesis (Mass) 7:1078–1082

Rosseti IB, Rocha JB, Costa MS (2015) Diphenyl diselenide (PhSe)2 inhibits bioﬁlm formation byCandida albicans, increasing both ROS production and membrane permeability. J Trace Elem Med Biol 29:289–295

Sarma BK, Mugesh G (2008) Thiol cofactors for selenoenzymes and their synthetic mimics. Org Biomol Chem 6:965–974

Simoes M, Simoes LC, Vieira MJ (2010) A review of current and emergent bioﬁlm control strategies. Lwt- Food Sci Technol 43 (4):573–583

Simon-Soro A, Mira A (2015) Solving the etiology of dental caries. Trends Microbiol 23(2):76–82

Soto SM (2013) Role of efﬂux pumps in the antibiotic resistance of bacteria embedded in a bioﬁlm. Virulence 4(3):32–41

Stepanovic S, Vukovic D, Dakic I, Savic B, Svabic-Vlahovic M (2000) A modified microtiter-plate test for quantification of staphylococcal biofilm formation. J Microbiol Methods 40 (2):175–179

Vargas J, Narayanaperumal S, Gul K, Ravanello BB, Dornelles L, Soares LC, Alves CFS, Schneider T, Vaucher RA, Santos RCV, Rodrigues OED (2012) Synthesis of chiralβ-chalcogen amine derivatives and Gram-positive bacteria activity. Tetrahedron 68:10444–10448

Vasconcelos MA, Arruda FVS, Santos HS, Rodrigues AS, Bandeira PN, Albuquerque MRJR, Cavada BS, Teixeira EH, Henriques M, Pereira MO (2014) Effect of a casbane diterpene isolated from Croton nepetaefoliuson the prevention and control of bioﬁlms formed by bacteria and Candida species. Ind Crops Prod 61:499–509