Fluoride-resistant Strain Research Articles

Ecological influence by colonization of fluoride-resistant Streptococcus mutans in oral biofilm.

Dental caries is one of the oldest and most common infections in humans. Improved oral hygiene practices and the presence of fluoride in dentifrices and mouth rinses have greatly reduced the prevalence of dental caries. However, increased fluoride resistance in microbial communities is concerning. Here, we studied the effect of fluoride-resistant Streptococcus mutans (S. mutans) on oral microbial ecology and compare it with wild-type S. mutans in vitro. Biofilm was evaluated for its polysaccharide content, scanning electron microscopy (SEM) imaging, acid-producing ability, and related lactic dehydrogenase (LDH), arginine deiminase (ADS), and urease enzymatic activity determination. Fluorescence in situ hybridization (FISH) and quantitative real-time polymerase chain reaction (qRT-PCR) were used to evaluate the S. mutans ratio within the biofilm. It was followed by 16S rRNA sequencing to define the oral microbial community. Fluoride-resistant S. mutans produced increased polysaccharides in presence of NaF (P < 0.05). The enzymatic activities related to both acid and base generation were less affected by the fluoride. In presence of 275 ppm NaF, the pH in the fluoride-resistant strain sample was lower than the wild type. We observed that with the biofilm development and accumulative fluoride concentration, the fluoride-resistant strain had positive relationships with other bacteria within the oral microbial community, which enhanced its colonization and survival. Compared to the wild type, fluoride-resistant strain significantly increased the diversity and difference of oral microbial community at the initial stage of biofilm formation (4 and 24 h) and at a low fluoride environment (0 and 275 ppm NaF) (P < 0.05). Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that fluoride-resistant strain enhanced the metabolic pathways and glucose transfer. Fluoride-resistant S. mutans affected the microecological balance of oral biofilm and its cariogenic properties in vitro, indicating its negative impact on fluoride's caries prevention effect.

Effects of Streptococcus mutans and its fluoride resistant strains on the adhesion of CAD/CAM ceramics to teeth and resin.

The similar elastic modulus of resin-matrix ceramics to dentin has resulted in their recent widespread application clinically. Nevertheless, the bacterial environment of oral cavity can degrade the resin composite. The objective was to analyse the effect of S. mutans and its fluoride-resistant strains on the adhesion of three CAD/CAM ceramics. S. mutans UA159 (UA) was identified, and its fluoride-resistant strain (FR) was induced. For crack observation, three kinds of CAD/CAM ceramics (IPS Empress, Lava Ultimate and Vita Enamic) were bonded with tooth complex (enamel, dentin and flowable resin) through adhesive. For micro-tensile test, ceramics were bonded with flowable resin, and cut into strip test pieces. Then specimens were immersed into the UA, FR and the control solution (BHI) separately for 14 d. Ceramic-adhesive interface and adhesive-tooth complex interface were observed and analyzed through electron microscope and stereomicroscope. Micro-tensile test was conducted. Specimens in bacterial solutions had more cracks and comparatively weaker micro-tensile strength than those in BHI. In ceramic-adhesive interface, Lava Ultimate produced the most cracks. In adhesive-tooth complex interface, adhesive-dentin produced the most cracks. Meanwhile, IPS Empress had the strongest micro-tensile strength when bonded with resin. S. mutans and its fluoride resistant strain can cause cracks in the bonding of ceramics and dental tissue, especially resin-matrix ceramic and dentin, and weaken the bonding strength between ceramics and resin.

Influence of Fluoride-Resistant Streptococcus mutans Within Antagonistic Dual-Species Biofilms Under Fluoride In Vitro.

The widespread application of fluoride, an extremely effective caries prevention agent, induces the generation of fluoride-resistant strains of opportunistic cariogenic bacteria such as fluoride-resistant Streptococcus mutans (S. mutans). However, the influence of this fluoride-resistant strain on oral microecological homeostasis under fluoride remains unknown. In this study, an antagonistic dual-species biofilm model composed of S. mutans and Streptococcus sanguinis (S. sanguinis) was used to investigate the influence of fluoride-resistant S. mutans on dual-species biofilm formation and pre-formed biofilms under fluoride to further elucidate whether fluoride-resistant strains would influence the anti-caries effect of fluoride from the point of biofilm control. The ratio of bacteria within dual-species biofilms was investigated using quantitative real-time PCR and fluorescence in situ hybridization. Cristal violet staining, scanning electron microscopy imaging, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide assay were used to evaluate biofilm biomass, biofilm structure, and metabolic activity, respectively. Biofilm acidogenicity was determined using lactic acid and pH measurements. The anthrone method and exopolysaccharide (EPS) staining were used to study the EPS production of biofilms. We found that, in biofilm formation, fluoride-resistant S. mutans occupied an overwhelming advantage in dual-species biofilms under fluoride, thus showing more biofilm biomass, more robust biofilm structure, and stronger metabolic activity (except for 0.275 g/L sodium fluoride [NaF]), EPS production, and acidogenicity within dual-species biofilms. However, in pre-formed biofilms, the advantage of fluoride-resistant S. mutans could not be fully highlighted for biofilm formation. Therefore, fluoride-resistant S. mutans could influence the anti-caries effect of fluoride on antagonistic dual-species biofilm formation while being heavily discounted in pre-formed biofilms from the perspective of biofilm control.

Genetic Mutations That Confer Fluoride Resistance Modify Gene Expression and Virulence Traits of Streptococcus mutans.

Fluoride is an inorganic monatomic anion that is widely used as an anti-cariogenic agent for the control of caries development. The aims of this study were to identify the mutated genes that give rise to fluoride-resistant (FR) strains of the cariogenic pathogen Streptococcus mutans and explore how genetic alterations in the genome of an S. mutans FR strain optimize the metabolism(s) implicated in the expression of virulence-associated traits. Here, we derived an S. mutans FR strain from a wild-type UA159 strain by continuous shifts to a medium supplemented with increasing concentrations of fluoride. The FR strain exhibited a slow growth rate and low yield under aerobic and oxidative stress conditions and was highly sensitive to acid stress. Notably, microscopy observation displayed morphological changes in which the FR strain had a slightly shorter cell length. Next, using the sequencing analyses, we found six mutations in the FR genome, which decreased the gene expression of the phosphoenolpyruvate-dependent phosphotransferase system (PTS). Indeed, the ability to intake carbohydrates was relatively reduced in the FR strain. Collectively, our results provide evidence that the genetic mutations in the genome of the FR strain modulate the expression of gene(s) for carbon metabolism(s) and cellular processes, leading to diminished fitness with respect to virulence and persistence.

Genetic Loci Associated With Fluoride Resistance in Streptococcus mutans.

The prolonged exposure of the cariogenic bacterial species Streptococcus mutans to high concentrations of fluoride leads to the development of fluoride resistance in this species. Previous studies confirmed the involvement of a mutation in a single chromosomal region in the occurrence of fluoride resistance. The involvement of multiple genomic mutations has not been verified. The aim of this study is to identify multiple genetic loci associated with fluoride resistance in S. mutans. The previously published whole genome sequences of two fluoride-resistant S. mutans strains (UA159-FR and C180-2FR) and their corresponding wild-type strains (UA159 and C180-2) were analyzed to locate shared chromosomal mutations in fluoride-resistant strains. Both fluoride-resistant strains were isolated in laboratory by culturing their mother strains in media with high concentrations of fluoride. The corresponding gene expression and enzyme activities were accordingly validated. Mutations were identified in two glycolytic enzymes, namely pyruvate kinase and enolase. Pyruvate kinase was deactivated in fluoride-resistant strain C180-2FR. Enolase was less inhibited by fluoride in fluoride-resistant strain UA159-FR than in its wild-type strain. Mutations in the promoter mutp constitutively increased the promoter activity and up-regulated the expression of the downstream fluoride antiporters in fluoride-resistant strains. Mutations in the intergenic region glpFp led to lower expression of glpF, encoding a glycerol uptake facilitator protein, in fluoride-resistant strains than in wild-type strains. Our results revealed that there is overlap of chromosomal regions with mutations among different fluoride-resistant S. mutans strains. They provide novel candidates for the study of the mechanisms of fluoride resistance.

The Fitness Cost of Fluoride Resistance for Different Streptococcus mutans Strains in Biofilms.

The cariogenic bacterium Streptococcus mutans can develop stable resistance to fluoride through chromosomal mutations in vitro. Fluoride-resistant S. mutans has seldom been isolated in clinical settings, despite the wide application of fluoride in oral-care products. One explanation is that the fluoride-resistant S. mutans strains have decreased fitness. However, so far, there has been no conclusive evidence to support this idea. The aim of this study was to investigate the fitness cost of 48-h biofilms of two fluoride-resistant S. mutans strains, UF35 and UA159-FR (UAFR), using the wild-type fluoride-sensitive strain UA159 as a reference. The engineered UF35 strain contains one point mutation, whereas UAFR, selected from NaF-containing agar plates, has multiple chromosomal mutations. All biofilms were formed for 48 h under a constantly neutral pH or a pH-cycling (8 h of neutral pH and 16 h of pH 5.5) condition in the absence of fluoride. The biomass of the biofilms was quantified with a crystal violet assay. The biofilms were also treated with chlorhexidine or solutions at pH 3.0, after which their lactic acid production was quantified. Compared to the UF35 and UA159 biofilms, the biomass of UAFR biofilms was two–four fold higher, and the UAFR biofilms were more resistant to chlorhexidine and low pH in terms of lactic acid production. No difference in biomass and lactic acid production was detected between UF35 and UA159 biofilms. The fluoride resistance of UAFR and UF35 strains in biofilms was further confirmed by treating the biofilms with NaF solutions. The level of NaF resistance of the three biofilms is generally ranked as follows: UAFR > UF35 > UA159. In conclusion, there is indeed a fitness consequence in UAFR, but surprisingly, this fluoride-resistant strain performs better than UF35 and UA159 under the described conditions. In addition, UF35 did not display a reduced fitness; it performed as well as the wild-type fluoride-sensitive strain.

Identification of an operon involved in fluoride resistance in Enterobacter cloacae FRM

Fluorine is ubiquitous and the most active non-metal element in nature. While many microorganisms have developed fluoride resistance as a result of the widespread and prolonged application of oral hygiene products, the mechanisms used by these organisms to overcome fluoride toxicity are incompletely understood. In this study, a fluoride-resistant strain, Enterobacter cloacae FRM, was identified which could grow well at a fluoride concentration of 4,000 mg/L. According to comparative genomics, transcriptome under fluoride stress, and sequence analyses of two fluoride-resistant fosmid clones, the genomic island GI3 was found to be important for fluoride resistance. The result of quantitative RT-PCR indicated that six genes on GI3, ppaC, uspA, eno, gpmA, crcB, and orf5249, which encode a fluoride transporter, fluoride-inhibited enzymes, and a universal stress protein, reside in an operon and are transcribed into two mRNAs activated by fluoride with a fluoride riboswitch. The results of knockout and complementation experiments indicated that these genes work together to provide high fluoride resistance to E. cloacae FRM. This study clarified the resistance mechanism of this high fluoride-resistant organism and has expanded our understanding of the biological effects of fluoride.

Intestinal microecology associated with fluoride resistance capability of the silkworm (Bombyx mori L.).

The silkworm (Bombyx mori L.) is an ideal model of Lepidoptera. However, the diversity and function of the intestinal microbiota in the gut of silkworm remain largely unknown. Changes in the intestinal microecology in fluoride-resistant strain T6 and fluoride-susceptible strain 734 of the silkworm in response to fluoride exposure were investigated. T6 and 734 were treated with 200mg/kg fluoride (designated as T6-T and 734-T groups) and deionized water (designated as T6-C and 734-C groups). Culture-dependent approach revealed that the numbers of intestinal bacteria in the 734-T group significantly decreased compared with that in the 734-C group (4.8 ± 0.6 × 10(7)CFU/mL vs. 7.5 ± 0.7 × 10(7)CFU/mL; P < 0.05). Analyses of the intestinal content pH showed that the pH decreased in the 734-T group only. Additionally, SCFA concentrations significantly decreased in both treatment groups compared with the control groups. High-throughput sequencing indicated that the intestinal microbiota in the 734-T group was significantly more diverse than those in the other groups. The bacterial community was composed of two dominant groups (Firmicutes and Proteobacteria). Principal component analyses revealed a significant difference in the composition of the intestinal microbiota in the 734-T group compared with those in the other groups. Thaumarchaeota and Euryarchaeota were more abundant in the 734-T group, but they were less abundant in the other groups. This study enhances our understanding about the diversity and function of silkworm intestinal microbiota in response to fluoride exposure among silkworm strains with diverse resistance.

Identification and functional analysis of genome mutations in a fluoride-resistant Streptococcus mutans strain.

It is known that fluoride-resistant microorganisms are different from fluoride-sensitive ones in growth, adherence and metabolic activity. It was hypothesized that these phenotypic differences were due to stable genotypic changes in the fluoride-resistant strains. However, until now, no studies have reported these genotypic changes. The aim of this study is to identify such changes in a fluoride-resistant Streptococcus mutans strain (C180-2FR) using whole-genome shotgun (WGS) sequencing and to examine the potential function of the identified mutations by comparing gene expression between the fluoride-sensitive (C180-2) and C180-2FR strains. We performed 50 bp paired-end Illumina shotgun sequencing for both strains. Through extensive bioinformatic analysis, we were able to identify 8 single nucleotide polymorphisms (SNPs) in the genome of C180-2FR, which were further confirmed by Sanger sequencing. Expression of the genes containing or in proximity to the SNPs in C180-2 and C180-2FR was then quantified by real-time PCR. A gene cluster containing genes coding for fluoride antiporters was up-regulated 10-fold in C180-2FR when compared to that in C180-2, independent of growth phase. Two SNPs are located in this gene cluster, one in its promoter region and the other in its protein-coding region. In addition, one gene, which codes for a putative glycerol uptake facilitator protein, was found to be down-regulated by 60% in C180-2FR at an early growth phase. The promoter region of this gene contained a SNP. No difference in expression was found for the other SNP-containing genes. In summary, using WGS sequencing, we were able to uncover genetic changes in the genome of a fluoride-resistant strain. These findings can provide new insights into the mechanism of microbial fluoride resistance.

Differential proteomics analysis on fluoride-resistant Streptococcus mutans by label-free quantitation

To figure out the changed biological characteristics of a fluoride-resistant strain compared with those of the parent sensitive strain of Streptococcus mutans(S. mutans) with the help of differential proteomics analysis, sixty-seven proteins with obvious differential expression were identified in fluoride-resistant strain. Among them are the important virulence factors causing caries, including glucosyltransferase-S, glucosyltransferase-I, glucosyltransferase-Si, major cell-surface adhesin Pac, F-ATPase β-subunit, Ffh, molecular chaperone DnaK, chaperonin GroEL, and the lactate dehydrogenase. We obtained the differential proteomics expression profiles of the fluoride-resistant strain(UA159-FR) and its parental strain(UA159) of S. mutans. Several important virulence factors bringing about caries were identified to have differential expressions, among which the expression of lactate dehydrogenase in fluoride-resistant strain was enhanced compared to that in its parent strain. The results could provide the basis for analysing the functions of proteins encoded by caries-related genes from the fluoride-resistant S. mutans strain.

Fatty-acid profiles and expression of the fabM gene in a fluoride-resistant strain of Streptococcus mutans

ObjectiveThe aim of this study was to compare the membrane fatty acid profile and expression of the fabM gene in a fluoride-resistant Streptococcus mutans strain with those of its wild-type counterpart. Additionally, we aimed to investigate whether mutations were present in the fabM gene of the fluoride-resistant strain. DesignA fluoride-resistant strain of S. mutans was obtained by step-by-step induction in vitro. The fluoride-resistant strain and its wild-type counterpart were grown anaerobically in a brain heart infusion broth, harvested and resuspended in a salt solution for an acid survival assay and a pH-drop experiment. The membrane fatty acid profile was determined by gas chromatography–mass spectrometry. Expression of the fabM gene was quantified by real-time PCR. The fabM gene was also sequenced. ResultsThe ability of the fluoride-resistant strain to resist acid stress was greater than that of the wild-type strain. A significant difference in the amount of long-chain monounsaturated fatty acids between the fluoride-resistant strain and the wild-type strain was detected in acidic condition (P<0.01). In addition, the level of fabM mRNA in the fluoride-resistant strain was significantly higher than that of the wild-type strain in the acidic condition as well (P<0.01). However, the sequence of the fabM gene from the fluoride-resistant strain was 100% homologous with that from the wild-type strain. ConclusionsThe membrane fatty acid profile and expression of the fabM gene in the fluoride-resistant strain were altered compared to the wild-type strain in acidic conditions, but no differences were found in the sequence of the fabM gene.

Effects of Different Kinds of Fluorides on Enolase and ATPase Activity of a Fluoride-Sensitive and Fluoride-Resistant Streptococcus mutans Strain

Enolase and ATPase are sensitive to fluoride. It is unclear whether this sensitivity differs for F-sensitive and F-resistant cells or for different types of fluoride. Permeabilized cells of the fluoride-sensitive strain Streptococcus mutans C180-2 and its fluoride-resistant mutant strain C180-2 FR were preincubated at pH 7 or 4 with NaF, the amine fluorides Olaflur and Dectaflur and amine chloride controls. After preincubations, enolase and ATPase activities of the cells were assessed. Enolase activity was more inhibited after preincubation at pH 7 with NaF than with Olaflur. Amine chloride stimulated, although not with statistical significance, the enolase activity of both strains. After preincubation at pH 4 the enolases were strongly inactivated, but the fluoride-resistant strain’s enolase to a lesser extent. The results suggested that amine acts to protect enolase activity against the detrimental low pH effect. Gene sequencing showed that the enolase genes of the fluoride-resistant and fluoride-sensitive strain were identical. ATPase activity was not reduced after NaF preincubation at either pH 7 or pH 4. The amine fluorides and their chloride controls in the preincubation mixture reduced the ATPase activity significantly at both pH values. In conclusion, our results showed that preincubation with amine fluoride did not inhibit enolase activity more effectively than NaF. The amine part of the molecule may protect enolase activity against preincubations at low pH. ATPase activity was not inhibited by NaF preincubation but was significantly inhibited after preincubation with amine fluorides and amine chlorides.

In vitro induced fluoride resistance of Streptococcus mutans and dental caries in rats.

The aim of this investigation was to study the effect of an in vitro acquired, stable, fluoride-resistant strain of Streptococcus mutans on dental caries in rats. Three groups (P0, P5 and P10) of SPF Osborne-Mendel rats were inoculated with the fluoride-sensitive S. mutans C180-2. Three other groups (FR0, FR5 and FR10) were inoculated with cultures of the fluoride-resistant S. mutans C180-2FR. Diet SSP 20/5 and tap water (less than 0.2 ppm fluoride) were available ad libitum to all groups. The drinking water of groups P5 and FR5 was supplemented with 5 mg F-/1 and the drinking water of groups P10 and FR10 with 10 mg F-/1. At the end of the caries experiment (days 58/59) there were no significant differences in the percentages and in the numbers of S. mutans in the total maxillary plaques of groups P0 to P10 and of groups FR0 to FR10, respectively. The fluoride tolerance of S. mutans cells re-isolated from rats, inoculated with the fluoride-sensitive strain, showed an unstable decrease compared to before inoculation. S. mutans cells re-isolated from rats, inoculated with the fluoride-resistant strain, showed both unstable and stable adaptation towards fluoride sensitivity. The animals superinfected with the fluoride-resistant strain developed less dentinal/fissure caries. Fluoride given in the drinking water at concentrations of 5 and 10 mg/l was effective in reducing caries irrespective of whether the animals were superinfected with the fluoride-sensitive or the fluoride-resistant strain.(ABSTRACT TRUNCATED AT 250 WORDS)

Development of a fluoride-resistant strain of L cells: membrane and metabolic characteristics.

ARTICLESDevelopment of a fluoride-resistant strain of L cells: membrane and metabolic characteristicsDO Quissell, and JW SuttieDO Quissell, and JW SuttiePublished Online:01 Sep 1972https://doi.org/10.1152/ajplegacy.1972.223.3.596MoreSectionsPDF (2 MB)Download PDF ToolsExport citationAdd to favoritesGet permissionsTrack citations ShareShare onFacebookTwitterLinkedInWeChat Previous Back to Top Next Download PDF FiguresReferencesRelatedInformation More from this issue > Volume 223Issue 3September 1972Pages 596-603 Copyright & PermissionsCopyright © 1972 by American Physiological Societyhttps://doi.org/10.1152/ajplegacy.1972.223.3.596PubMed5066349History Published online 1 September 1972 Published in print 1 September 1972 Metrics