Transcriptomic response in planktonic and biofilm cells of S. mutans treated with sublethal concentrations of chlorhexidine

Transcriptomic response in planktonic and biofilm cells of S. mutans treated with sublethal concentrations of chlorhexidine | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Transcriptomic response in planktonic and biofilm cells of S. mutans treated with sublethal concentrations of chlorhexidine Sara Arbulu, Thomas F. Oftedal, Morten Kjos This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5834130/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Chlorhexidine, an antimicrobial with a broad inhibitory spectrum, is commonly used to treat oral infections as an active ingredient in mouthwash. While typically used at high concentrations (1–2 mg/ml), oral bacteria can be exposed to sublethal concentrations due to the bioavailability and protective barrier of biofilms (dental plaques). Sublethal concentrations can cause transcriptional remodelling of bacteria such as Streptococcus mutans , a key player in dental caries. Using an RNA-seq approach, this report provides a compendium on the effect of sublethal concentrations of chlorhexidine on the transcriptome of S. mutans as planktonic cells and in biofilm states. S. mutans showed major transcriptional remodelling between planktonic and biofilm states. The transcriptional response towards chlorhexidine was more pronounced in planktonic cells compared to sessile cells. However, the response observed for biofilm-associated cells was not specific to chlorhexidine, as biofilms exposed to the β-lactam amoxicillin showed similar responses. Furthermore, we found that S. mutans modulates transcription of a multitude of ABC transporters both in planktonic and biofilm-associated cells upon exposure to these antimicrobials. Bacteriology Bioinformatics General Microbiology Infectious Diseases oral biofilms Streptococcus mutans RNA-seq compendium transcriptome sublethal antibiotics chlorhexidine amoxicillin Figures Figure 1 Figure 2 Figure 3 Figure 4 1. Introduction Dental caries is the gradual loss and demineralisation of the hard tissues of teeth caused by acid production from biofilm-associated bacteria in the oral cavity (Pitts et al. 2017 ). If untreated, caries can lead to severe infection and require antibiotic treatment. Dental caries can be prevented by frequent disruption of biofilms in the oral cavity by brushing and flossing, and by the use of antiseptic mouthwashes. Clinically relevant concentrations of these antimicrobials are likely lead to sublethal concentrations at certain target sites due to decreased bioavailability and accessibility (Delacher et al. 2000 ; Liu, Prentice and Webber 2024 ). This is particularly relevant for biofilm-related conditions such as dental caries, the most prevalent microbe-related health condition worldwide (Ward and Goldie 2024 ). Chlorhexidine and amoxicillin are commonly used to control oral infections with different clinical indications (Brookes et al. 2020 ; Abdullah et al. 2024 ). Chlorhexidine is often used as a mouthwash (containing 0.1–0.2% chlorhexidine digluconate) by dental clinicians during pre-surgical preparation to reduce the bacterial load, and after surgery as a preventative measure (Brookes et al. 2020 ; Poppolo Deus and Ouanounou 2022 ). Furthermore, in many regions chlorhexidine is available as over-the-counter mouthwash and used to manage early gum disease (gingivitis) or used as a prophylactic and adjunct to brushing to prevent plaque formation.(Brookes et al. 2020 ). Chlorhexidine is a cationic agent that interacts with the negatively charged bacterial membranes altering their osmoregulation and leading to leakage of ions and cellular components that eventually leads to cell lysis (Lim and Kam 2008 ). Chlorhexidine is a broad-spectrum antiseptic that is active against bacteria, viruses and fungi and can also disrupt biofilms (Alvendal et al., 2020 ; Bonez et al., 2013 ; Karpiński and Szkaradkiewicz, 2015 ; Poppolo Deus and Ouanounou, 2022 ). On the other hand, orally administered amoxicillin is the most common choice for systemic tooth infection treatment (Akhavan, Khanna and Vijhani 2024). Amoxicillin is a β-lactam that targets peptidoglycan synthesis in actively dividing cells and it displays activity against gram-positive bacteria, including Streptococcus , and some gram-negative bacteria such as Escherichia coli (Castle 2007 ). Sublethal concentrations of antibacterials affect the physiology of the cells and can select for resistant bacteria, promote genetic variability and function as signalling molecules affecting virulence, biofilm formation and communication mechanisms (Andersson and Hughes 2014 ; Silva et al. 2014 ; Waack and Nicholson 2018 ; Liu et al. 2020 ; Penesyan et al. 2020 ; Guo et al. 2021 ; Byun et al. 2022 ). Understanding those effects as part of the antibiotic response can help design optimised therapies and gain an increased understanding of the ecology of these bacteria. While most research on the effect of antimicrobials has been conducted on planktonic cells, pathogens such as the oral bacterium Streptococcus mutans often form biofilms during or as part of their infection cycle. S. mutans is key to dental caries development and oral biofilm formation (Lemos et al. 2019 ). By metabolising carbohydrates in the oral cavity, S. mutans produces glucans that contribute to forming the biofilm extracellular matrix. As a by-product, organic acids that lower the pH are produced leading to tooth decay. However, this process is only part of the explanation for the complex, multifactorial, and multispecies aetiology of dental caries (Cai and Kim 2023 ). Biofilm-associated cells differ from planktonic cells in their response to antibiotics (Shree et al. 2023 ) and generally display greater heterogeneity (Obando and Serra 2024 ). Bacteria embedded in biofilm matrices are more resilient to antibiotic treatment due to limited diffusion of antibiotic and slower growth rates of the cells, (Cozens et al. 1986 ) (Lebeaux, Ghigo and Beloin 2014 ). Consequently, antibiotic concentrations effective against planktonic cells become subinhibitory for biofilm-associated cells in dental caries. In this work, we challenged S. mutans UA159 planktonic and biofilm cells with a sublethal concentration of chlorhexidine for a short duration to mimic exposure from the use of mouthwash. We then used RNA-seq to analyse and compare differential gene expression changes. Amoxicillin was also applied to the biofilm cultures for comparison purposes. The results showed an overall differential regulation of planktonic and biofilms, with extensive downregulation of chlorhexidine and amoxicillin-treated biofilm metabolism. 2. Materials and methods 2.1. Bacterial strains and antibiotics used S. mutans UA159 was cultured in Brain Heart Infusion (BHI, Difco) for planktonic growth in broth and in BHI supplemented with 1% sucrose (BHIS) for the biofilm experiments. The strain was grown at 37°C in anaerobiosis (10% CO 2 ) or airtight tubes, unless otherwise stated. Chlorhexidine digluconate 20% (Sigma) was prepared and diluted to the desired concentration in sterile Milli-Q water. For amoxicillin, stocks at 10 mg/ml were prepared by dissolving amoxicillin trihydrate in an equal volume of 0.1 M NaOH and phosphate-buffered saline (pH 7.2) and further diluted in Milli-Q water to the desired concentration. 2.2. Antibiotic susceptibility tests to amoxicillin and chlorhexidine 2.2.1 Minimum Inhibitory Concentration (MIC) determination The experiments were set up in 96-well microtiter plates with a total volume of 300 µl using the broth microdilution method. Two-fold dilution series of chlorhexidine (starting concentration 100 µg/ml) and amoxicillin (starting concentration 100 ng/ml) were prepared in BHI, with each well containing 150 µl of the diluted solutions. Subsequently, 150 µl of S. mutans UA159 culture at an OD 600 0.05 were added to each well. The cultures were incubated at 37°C, and the plate was shaken for 5 sec before measurements of OD 600 were taken every 10 min throughout the experiment using a Hidex Sense (Hidex Oy) plate reader. The experiments were repeated three times. MIC were established as the lowest concentration of antimicrobial that inhibited bacterial growth. 2.2.3. Tolerance to chlorhexidine during growth S. mutans UA159 culture at OD 600 0.05 was incubated at 37°C until it reached an OD 600 of 0.3–0.4. At this point, chlorhexidine was added. The same two-fold dilution series used for the MIC assays was tested (100, 50, 25, 12.5, 6.25, 3.125, 1.5, 0.78, 0 µg/ml). Growth was monitored in a plate reader as described above. 2.2.4. Biofilm-Oriented-Antimicrobials Test (BOAT) The metabolic activity of S. mutans UA159 biofilm-cells treated with chlorhexidine and amoxicillin was determined by the Biofilm-Oriented Antimicrobial Test (BOAT) (Grønseth et al. 2017 ; Kranjec et al. 2020 ). S. mutans UA159 culture at an OD 600 of 0.5 was diluted 1:1000 in BHIS and 100 µl was added to the wells of a 96-well plate and allowed to form biofilms for 24 h at 37°C in anaerobiosis. Different concentrations of chlorhexidine (800, 775, 750, 725, 700, 650, 600, 120, 100, 71, 50, 42, 24, 14, 8, 6, 5, 3, 2 µg/ml) and amoxicillin (5000, 2500, 1250, 625, 313, 156, 78, 39, 20, 10, 5, 2, 1 µg/ml) were prepared in BHI to a volume of 175 µl. These concentrations were selected after several rounds of BOAT assays to show the effect of a wide range of antibiotic concentrations on S. mutans UA159 biofilms. Biofilms were washed twice with 100 µl of 0.9% NaCl and 150 µl of the antibiotic dilutions were transferred to the biofilm plate. Antibiotic treatment was applied for 5 min, 30 min and 24 h while incubating at 37°C in anaerobiosis. The biofilms were washed three times with 0.9% NaCl and 100 µl of 0.025% triphenyl-tetrazolium chloride (TTC, Sigma) dissolved in BHI was added to each well and further incubated at 37°C for 5 h. The presence of red colour was used as a measure of cellular respiration. TTC was removed and 200 µl ethanol:acetone (70:30) were added per well and incubated overnight to extract the red dye. The metabolic activity was then measured at 492 nm in a plate reader (Fluostar Optima, BMG, LabTech). Biofilms without antibiotic treatment were used as controls. Three replicates per condition were performed. 2.5. RNA isolation and sequencing RNA was isolated from planktonic-broth and biofilm cultures of S. mutans UA159 challenged with sublethal concentrations of amoxicillin and chlorhexidine for 5 min at 37°C in anaerobiosis. In broth, chlorhexidine was used at a final concentration of 6.25 ng/ml. In biofilm experiments, chlorhexidine and amoxicillin were used at 70 µg/ml and 5 mg/ml, respectively. Concentrations used were based on MIC and BOAT assays. The initial broth culture of S. mutans UA159 was prepared by inoculating 50 ml BHI with 0.5 ml of a starting inoculum with an OD 600 of 0.4–0.5. Growth was monitored until an OD 600 of 0.3 (early-mid exponential phase). Aliquots of 10 ml were used for chlorhexidine treatment and as a negative control (treated with BHI). The cells were harvested by centrifugation at 6,000 x g for 1 min at 4°C. The pelleted cells were immediately frozen in liquid nitrogen and stored at -80°C. This experiment was repeated three times (biological replicates). For the biofilm cultures of S. mutans UA159, 20 µl of a starting culture with an OD 600 of 0.4–0.5 were inoculated in 20 ml BHIS. The biofilm was grown in 48-well plates with 400 µl of culture per well and allowed to grow for 24 h at 37°C in anaerobiosis. Planktonic cells were removed by aspiration, attached cells/biofilms were washed once with 400 µl 0.9% NaCl and then 400 µl of amoxicillin or chlorhexidine at the concentrations mentioned above. The control with no antibiotic was treated with BHIS. Four wells per condition were used. After the antibiotic treatment, the biofilms were washed with 400 µl sterile RNAse-free water (Invitrogen). Then 400 µl of RNAprotect (Qiagen) were added to the wells and the biofilms were scraped off the surface using a pipette tip (Kragh et al. 2019 ). The suspended biofilms from the 4 wells for each condition was transferred to 15 ml tubes and centrifuged at 6,000 x g for 1 min at 4°C. The pelleted cells were immediately frozen in liquid nitrogen and stored at -80°C. The experiment was repeated three times (biological replicates). RNA was extracted using the RNeasy Mini Kit (Qiagen), followed by DNase treatment and phenol-chloroform extraction, as described by Stamsås et al. (Stamsås et al. 2018 ). Library preparation, quality assessment, and sequencing were conducted by Novogene (Germany). In summary, rRNA was removed from the total RNA, followed by ethanol precipitation. Second-strand cDNA synthesis incorporated dUTPs instead of dTTPs to generate a directional (stranded) library. Library quantification was performed using Qubit and real-time PCR, while size distribution was assessed with a Bioanalyzer. The quantified libraries were pooled and sequenced on an Illumina instrument. The raw sequencing reads were processed to remove adapters, reads with > 10% ambiguous bases (N), and low-quality reads (Qscore ≤ 5). The reads were then aligned against S. mutans UA159 (Genbank accession number: AE014133.2) and differentially expressed genes (DEGs) between planktonic, biofilm, treated and untreated samples were calculated using DESeq2 (Love, Huber and Anders 2014 ) on 3 independent biological replicates for each tested condition using the Bioconductor R package. See Table 1 for the conditions being analysed and compared. An additional rRNA removal step was done bioinformatically by filtering out ribosomal locus tags based on the reference genome. Genes with an adjusted p -value ≤ 0.05 were used for further analysis. DEGs were scored as upregulated if they had a log 2 fold change ≥ 1.0 and downregulated if they had a log 2 fold change ≤ -1.0. Visualizations of DEGs were done as volcano plots and a heatmap using the EnhancedVolcano and heatmap R packages, respectively. A Venn diagram was also constructed to identify the common DEGs obtained from the different compared conditions using the gvenn R package. Transcripts Per Million (TPM) values were calculated based on the normalized counts provided by DESeq2. Bray-Curtis dissimilarity was used to compute distance matrices based on TPM values, and Principal Coordinates Analysis (PCoA) was performed to visualize sample clustering. Additionally, a heatmap was calculated using the pheatmap R package to visualize expression patterns across samples, and a correlation matrix showed associations between samples using the corrplot R package. 2.6. Functional annotations and pathway analysis To obtain insights into the biological meaning of the DEGs the clusterProfiler R package v4.13.0 (Yu et al. 2012 ) was used for gene set enrichment analysis (GSEA) using the Kyoto Encyclopedia of Genes and Genomes database via the functions enrichKEGG and compareCluster setting a p -value cut-off < 0.05. Note that the global maps and the overview maps are a special class of metabolic pathway maps within KEGG (Kanehisa et al. 2017 ). These categories represent an integrated picture of the metabolism connecting different pathways present in the dataset. Sequencing data availability The raw FASTQ data are accessible at https://www.ebi.ac.uk/ena/browser/home with accession number PRJEB83273. 3. Results and discussion 3.1. Selection of sublethal chlorhexidine and amoxicillin concentrations applied on planktonic and biofilm cultures of S. mutans UA159 To select a relevant sublethal concentration of chlorhexidine that could exert changes at the transcriptome level, we first established the sensitivity of planktonic S. mutans UA159 to chlorhexidine by MIC assays (Fig. 1A and 1B). Complete inhibition of growth was observed at 1.56 µg/ml Fig. 1A). This is in line with other studies, which have found the MIC of S. mutans for chlorhexidine to be below 1 µg/ml (Järvinen, Tenovuo and Huovinen 1993 ; Mohammed Ghilan et al. 2023 ). The same concentration range of 0.3–100 µg/ml was used to assay if S. mutans UA159 grown to early-mid exponential phase could tolerate the presence of chlorhexidine (Fig. 1B). At this phase of growth, S. mutans UA159 was only affected by chlorhexidine concentrations above 6.25 µg/ml. A concentration of 6.25 µg/ml was therefore used for the chlorhexidine transcriptomic stress response analysis in broth. The MIC for amoxicillin was determined to be 50 ng/ml which is in line with other studies (Fig. 1A) (Kwon and Lee 2020 ; Maisonneuve et al. 2020 ) We then examined the effect of chlorhexidine on S. mutans UA159 biofilms (Fig. 1C). Chlorhexidine is reported to disrupt S. mutans biofilms in a dose-dependent manner (Ccahuana-Vásquez and Cury 2010 ; Silva et al. 2014 ; Lee et al. 2016 ) favouring S. mutans biofilm detachment (Liu et al. 2012 ). To determine the concentration needed to disrupt S. mutans biofilms, we used the BOAT assay (Kranjec et al. 2020 ). Using this assay, we were able to quantify the remaining metabolic activity of 24 h-old biofilms treated with chlorhexidine for 5 min, 30 min and 24 h (Fig. 1C). The metabolic activity was reduced according to concentration and treatment duration. For the 5 min treatment, a chlorhexidine concentration of 600 µg/ml was needed to fully reduce the metabolic activity, indicating a complete disruption of the formed biofilm. The corresponding concentrations for the 30 min and 24 h treatments were 50 µg/ml and 24 µg/ml chlorhexidine, respectively (Fig. 1C). The highest concentration of chlorhexidine that did not appreciably affect metabolic activity after a 5 min treatment was determined to be 70 µg/ml, which we chose as the sublethal concentration for the transcriptomic experiments. We also attempted to determine a corresponding concentration for amoxicillin against biofilms, however, no effect was observed on the metabolic activity even with the highest concentration tested (5 mg/ml) (Fig. 1C). This is most probably due to the nature of the cells in the biofilms and highlights the difference in physiology between cells in biofilms compared to planktonic growth. Amoxicillin and other β-lactams inhibit bacterial cell-wall synthesis due to binding to the penicillin-binding proteins, thereby inhibiting transpeptidation of peptidoglycan. However, most cells in a biofilm are not actively dividing and relatively mature S. mutans biofilms allowed to establish for at least 24 h contain mostly inactive non-dividing cells (Lewis 2005 ). 3.2. Overview of S. mutans RNA-seq analysis To investigate the transcriptomic response of planktonic and biofilm cultures of S. mutans UA159 upon 5-min treatment with sub-inhibitory concentrations of chlorhexidine and amoxicillin, an RNA-seq analysis was conducted. We selected a 5-min treatment duration due to the short contact time that mouthwashes typically have with their targets. A total of 247,746,462 raw reads were generated, of which 98.10% were clean reads, with an average GC content of 39.96%, Q > 30 93.77%, and an underlying error of 0.03%, indicating the high quality of the sequencing data (Table S1). TPM values were used to compare the proportion of reads mapped to a gene in each sample (Table S2). Principal component analysis (PCoA) of the gene data showed grouping of the biological replicates, with a clear separation between planktonic and biofilm samples and further separation between non-treated and amoxicillin and chlorhexidine-treated samples (Figure S1). A heatmap of all genes and a correlation plot further confirmed the expected clustering of the samples (Figure S2). The distribution of differentially expressed genes (DEGs; |log 2 FC| > 1, P adj < 0.05) between the compared conditions (Table 1) was visualised using volcano plots (Fig. 2). A list of all DEGs can be found in the Supplementary material (Table S3). 3.3. Biofilm transcriptional behaviour differs from planktonic cells of S. mutans UA159 Gene regulation of planktonic and biofilm cultures can differ significantly reflecting the two different microbial lifestyles (Shemesh, Tam and Steinberg 2007 ; Lo et al. 2009 ; Charlebois, Jacques and Archambault 2016a ; Castro et al. 2017 ; Sánchez et al. 2019a ; Zheng et al. 2022 ). Indeed, with a threshold of |log 2 FC| > 1 and P adj < 0.05, a total of 879 genes (412 upregulated, 467 downregulated genes, 43% of genes in total) were found to be differentially expressed in biofilm relative to planktonic cultures of S. mutans UA159 (Fig. 2A, Fig. 3A). Previous DNA-microarray analyses showed about 12% of S. mutans UA159 genes to be differentially expressed in biofilms (Shemesh, Tam and Steinberg 2007 ) whereas studies in other species showed broader variability, from 1% differential expression in Pseudomonas aeruginosa (Whiteley et al. 2001 ), 4.8% in Porphyromonas gingivalis (Sánchez et al. 2019b ) or 25.7% in Clostridium perfringes (Charlebois, Jacques and Archambault 2016) biofilms highlighting important differences in cell metabolism and the techniques used. To get an overview of the physiological processes affected in the S. mutans biofilms compared to planktonic cells, the expression patterns were examined by KEGG pathway enrichment analysis revealing several significantly up- and downregulated pathways (Fig. 4A, Table S4). Fatty acid biosynthesis, phosphotransferase systems, starch and glucose metabolism, methane metabolism, β-lactam resistance and ABC transporters were upregulated in the wild-type biofilm compared to the planktonic cells (Fig. 4A). On the other hand, purine metabolism and biosynthesis of secondary metabolites, metabolic pathways and 2-oxocarboxylic acid metabolism within the general global and overview maps category were downregulated (Fig. 4A). The global biofilm gene expression pattern, representing the average across the heterogeneous biofilm cell population, showed a significant number of differentially regulated genes compared to the planktonic cells, indicating that biofilms exhibit distinct metabolic and physiological adaptations. 3.4. The transcriptomic response to chlorhexidine treatment is dependent on the lifestyle of S. mutans Chlorhexidine is the gold-standard oral antiseptic widely used in dental practice and as an over-the-counter mouthwash. Swallowing, expectoration, or insufficient contact time can lead to sub-inhibitory concentrations of chlorhexidine at target sites known to promote biofilm formation (Ebrahimi et al. 2014 ) and alter the metabolism and microbial composition of the oral microbiota (Chatzigiannidou et al. 2020 ). Planktonic and biofilm cells were treated with sub-inhibitory concentrations of chlorhexidine to evaluate its impact on the S. mutans UA159 transcriptome. Planktonic chlorhexidine-treated S. mutans cultures showed a total of 499 DEGs (225 upregulated, 274 downregulated genes, 24.4% of genes in total) while chlorhexidine-treated biofilms showed 100 DEGs (25 upregulated and 75 downregulated, 4.9%) (Fig. 2B) compared to the non-treated strain. Thus, as expected, there was a greater degree of differential regulation in chlorhexidine-exposed planktonic cells compared to chlorhexidine-exposed biofilms. This is illustrated in the heatmaps of the DEG distribution (Fig. 3A). These results are consistent with observations across different species showing that biofilm cells are less metabolically active than their planktonic counterparts (Wan et al. 2018 ; Sadiq et al. 2020 ; Wang et al. 2023 ). Moreover, the 24 h mature biofilms tested in this study were likely in a state of nutrient limitation and therefore at a slow growth rate leading to a reduced metabolism. Only 35 genes were found to be common between the planktonic and biofilm chlorhexidine-treated cells (Fig. 3B, Table S5). Those genes included ABC transporters, membrane proteins, the gene comX1 , which is a key regulator of the natural competence system in streptococci (Aspiras, Ellen and Cvitkovitch 2004 ; J et al. 2015), and mostly hypothetical genes with unknown functions. 3.4.1. Transcriptomic response in planktonic chlorhexidine-treated cultures For the planktonic-chlorhexidine treated cultures, the global KEGG analysis of the enriched pathways revealed no upregulated pathways (Fig. 4B-C; Table S4), while quorum-sensing systems, two-component systems and ABC-transporters were downregulated, along with pyruvate metabolism, Val/Leu/Ile biosynthesis and 2-oxocarboxylic acid metabolism. Specifically, the regulatory genes included ciaR-ciaH , encoding the highly conserved streptococcal CiaRH regulatory system, which has been shown to be involved in natural competence, biofilm formation, bacteriocin production and cell wall biosynthesis and autolysis (He et al. 2021 ). The htrA gene, encoding a CiaRH-regulated protease which takes part in oxidative stress tolerance was also downregulated (Sebert et al. 2002 ; Ibrahim et al. 2004 ; He et al. 2021 ). Likewise, the gene comE is part of the ComCDE system, one of the natural competence pathways in S. mutans also involved in bacteriocin production (van der Ploeg 2005 ). Although the direct connection between these systems and chlorhexidine is unknown, one could speculate that membrane targeting agents such as chlorhexidine shift S. mutans metabolism to survival mechanisms not related to the CiaRH and ComCDE functions. The genes with highest fold-changes (|log 2 FC| > 5 and P adj < 0.05, Table 2) included genes encoding hypothetical proteins and ABC transporters, such as the yet unstudied operon SMU_1550 - SMU_1554, which represent prime candidates for further studies of factors affecting chlorhexidine sensitivity. 3.5. The biofilm transcriptomic response is not specific to chlorhexidine Chlorhexidine-treated biofilms showed no upregulated pathways compared to the wild-type biofilms, with only carbohydrate metabolism (galactose, starch and sucrose) being downregulated (Fig. 4C). In a similar study testing the effect of inhibitory concentrations of curcumin and chlorhexidine on S. mutans biofilms, carbohydrate metabolism, quorum sensing, and two-component transduction systems were found downregulated (Li et al. 2018 ). We were also interested in understanding whether the transcriptional remodeling in biofilms, was specific to chlorhexidine. Amoxicillin is another relevant antibiotic in oral infection treatment. It is given as prophylaxis before oral intervention or for tooth infection treatment administered orally with a systemic effect that is expected to kill sessile bacteria prior to attachment and biofilm formation in the oral cavity. Amoxicillin was not effective in disrupting S. mutans UA159 biofilms with the used concentrations (up to 100,000-fold MIC), as observed in the BOAT assays (Fig. 1A and C). However, amoxicillin-treated biofilms still showed 149 DEGs (65 upregulated, 84 downregulated genes; 7.3% of genes in total) (Fig. 2C), compared to 100 DEGs for the chlorhexidine treatment. When directly comparing the transcriptomes of chlorhexidine-treated with the amoxicillin-treated biofilms, only 31 DEGs (3 upregulated, 28 downregulated; 1.5% of the total genes) were detected between the conditions (Fig. 2C), suggesting that the majority of the transcriptional responses to the individual agents in biofilms were not significantly different between the chlorhexidine and amoxicillin treatments, and rather represent general responses in the biofilm setting. Interestingly, 68 commonly regulated genes were found between chlorhexidine- and amoxicillin-treated biofilms. The most downregulated ones were the same after chlorhexidine and amoxicillin treatments and included the phosphotransferase system-related genes ptcA, ptsG and mtlA1 , the pdh operon (pyruvate dehydrogenase) important during glucose starvation (Busuioc, Buttaro and Piggot 2010 ) and naoX (noX) that encodes the main enzyme in oxygen metabolism in S. mutans (Yamamoto et al. 2000 ; Derr et al. 2012 ) (Fig. 3B,Table S3, Table S5). Notably, the clpB gene (SMU_1425) exhibited the lowest log2FC value of -5.07 and − 5.21 under both conditions (Table S3, Table 2 ). ClpB is a molecular chaperone part of the Clp ATPase family involved in homeostasis and stress tolerance (Lemos and Burne 2002 ; Frees et al. 2007 ) which might be reduced in biofilms compared to planktonic cells due to physiological adaptations (Stewart and Franklin 2008 ). Furthermore, amoxicillin-treated biofilms showed downregulation of carbohydrate metabolism (galactose, starch and sucrose) by the KEGG pathway analysis (Fig. 4D), similar to what was observed in chlorhexidine-treated biofilms (Fig. 4C). However, on the amoxicillin-treated biofilms upregulated pathways that were not significantly different in the chlorhexidine treatment were detected (Fig. 4D, Table S4). These included fatty acid metabolism and biosynthesis, carbon fixation, and branched-amino acids metabolism. Additionally, biosynthesis of secondary metabolites, propanoate metabolism, metabolic factors and biosynthesis of cofactors were detected within the Global and overview maps category. Overall, these observations suggest that there may be a common nonspecific biofilm response to chlorhexidine and amoxicillin. 3.6. ABC transporters were differentially regulated across experimental conditions in S. mutans ABC transporters play important roles in the active transport of molecules across the membrane for maintenance of cellular nutrient supply and integrity (Davidson et al. 2008 ). A large number of different ABC transporters were significantly regulated across the compared conditions tested here (Table S6 and S7). Upon subinhibitory chlorhexidine exposure, 20% of the differentially regulated genes in broth cultures (10 out of 499) and 50% of those genes in biofilms (50 out of 100) were annotated as ABC transporter proteins. Considering that ABC transporter proteins in the annotated S. mutans UA159 reference genome are only 6.9% of the genes (141 out of 2043), ABC transporter genes were overrepresented among the regulated genes. Among those whose functions are known, the oppADF genes, part of the opp operon responsible for oligopeptide uptake (Nepomuceno et al. 2007 ) and msmFGK and malFX genes involved in disaccharide uptake (Webb, Homer and Hosie 2008 ) were upregulated in S. mutans UA159 biofilm compared to its planktonic form and downregulated in chlorhexidine ( malX ) and amoxicillin-treated biofilms ( malX , malF ), whereas the opu genes opuBa , opuBc , opuCd that regulate osmotic stress (Abranches, Lemos and Burne 2006 ) and the mutF , part of the MutEFG transporter that has been linked with nisin resistance in S. mutans (Le, Kawada-Matsuo and Komatsuzawa) were downregulated in wild-type biofilms and after CHX-treated planktonic S. mutans UA159. Numerous transcriptomic studies report differential expression of ABC transporters in different bacterial species (Allan et al., 2014; Guo et al., 2022; Rahman et al., 2022; Rice et al., 2017; Zhu et al., 2008), highlighting their ubiquity and multifaceted nature. Up and downregulation of ABC transporters might be a response to counteract the stress induced by chlorhexidine and amoxicillin. In this sense, ABC transporters are known to be involved in the resistance and transport of antimicrobials (Abbood, Hijazi and Gould 2023 ) and can function as antibiotic efflux pumps (Costa et al. 2013 ; Nagayama et al. 2014 ). Other functions of ABC transporters such as transport of diverse molecules (Biswas and Biswas 2011 ; Kim et al. 2012 ; Lemos et al. 2019 ) or nutrient uptake (McLaughlin and Ferretti 1996 ; Kilic, Honeyman and Tao 2007 ; Webb, Homer and Hosie 2008 ) might as well be relevant in the response of S. mutans to chlorhexidine and amoxicillin. 3.8. Conclusions The results provided here provide a comprehensive overview of the transcriptomic response of S. mutans after exposure to subinhibitory concentrations of chlorhexidine and amoxicillin. Notably, subinhibitory concentrations of chlorhexidine and amoxicillin exert a significant transcriptomic impact on planktonic and biofilm cultures of the oral commensal S. mutans , pointing to yet unexplored effects these antimicrobials. This compendium thus serves as a resource for further gene-targeted analysis to elucidate the roles of S. mutans genes that are differentially regulated under these settings. Declarations Conflict of Interest The authors declare no conflict of interest. Acknowledgements This work was supported by Marie Skłodowska-Curie Actions H2020-MSCA-EF-ST-2020 grant #101029099. References Abbood HM, Hijazi K, Gould IM. Chlorhexidine resistance or cross-resistance, that is the question. Antibiotics 2023; 12 :798. Abdullah FM, Hatim QY, Oraibi AI et al. 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Transcriptome sequencing reveals the difference in the expression of biofilm and planktonic cells between two strains of Salmonella Typhimurium. Biofilm 2022; 4 :100086. Additional Declarations The authors declare no competing interests. Supplementary Files Supplementary.docx Supplementary figures and Table S6 TableS1.Statsu2.xlsx Table S1 TableS2.TPMu4.xlsx Table S2 TableS3.AllDEGsu.xlsx Table S3 TableS4.enrichKEGGgenesu.xlsx Table S4 TableS5.Venndiagramsgenesu.xlsx Table S5 TableS7.ABCPTSu.xlsx Table S7 Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5834130","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":439514804,"identity":"d8bb52d1-d38d-480a-8138-d6fbcb26bae7","order_by":0,"name":"Sara Arbulu","email":"","orcid":"https://orcid.org/0000-0001-9889-3692","institution":"Norwegian University of Life Sciences","correspondingAuthor":false,"prefix":"","firstName":"Sara","middleName":"","lastName":"Arbulu","suffix":""},{"id":439515755,"identity":"03fcd27b-4889-4421-9934-f64a402b048f","order_by":1,"name":"Thomas F. Oftedal","email":"","orcid":"https://orcid.org/0000-0003-4182-3831","institution":"Norwegian University of Life Sciences","correspondingAuthor":false,"prefix":"","firstName":"Thomas","middleName":"F.","lastName":"Oftedal","suffix":""},{"id":439515756,"identity":"82d61adc-e218-4a1f-85af-46408b6d7a3b","order_by":2,"name":"Morten Kjos","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzUlEQVRIiWNgGAWjYBADA8YGBsYHDAwHiFINVAzRwmxAmhYgZpMgSotue+/zBx93MBgzt7c/qy7MuZPYIN2MX5/ZmeOGjTPPMJgx9pwxuz1z27PEBpljCfi13EhjbOZtY7BhnJHDdpt32+HEBokcA8Ja/oK1pD8rJl4LYxvQYTMSzJiJ03LmGOPM3jYJY6BfjKV5tz0zbpNII+CX420MH3622RhubG9/+Jl32x3ZfonkA3i1QIEEg2EDlMlGjHowkCda5SgYBaNgFIw4AADRWkkvgTZ0BgAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0003-4448-9082","institution":"Norwegian University of Life Sciences","correspondingAuthor":true,"prefix":"","firstName":"Morten","middleName":"","lastName":"Kjos","suffix":""}],"badges":[],"createdAt":"2025-01-15 11:51:12","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-5834130/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5834130/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":80133064,"identity":"400bed70-94b4-4410-a4c5-7b3a9b491a92","added_by":"auto","created_at":"2025-04-08 09:36:30","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":518542,"visible":true,"origin":"","legend":"\u003cp\u003eDetermination of the sensitivity of planktonic and biofilm cultures of \u003cem\u003eS. mutans\u003c/em\u003e UA159 to amoxicillin and chlorhexidine. (A) Minimal Inhibitory Concentration assays, (B) Tolerance to growth with chlorhexidine and amoxicillin, (C) Biofilm Oriented Antiseptic Test assays. The boxplots show the remaining metabolic activity after antibiotic treatment at different concentrations at treatment durations (5 min, 30 min, 24 h). The median distribution is shown as a cross of the boxplots and the degree of variability (interquartile region) is represented as the height of the boxes. The whiskers indicate the maximum and minimum values observed.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5834130/v1/68945bcb3404ab13c4da329e.jpg"},{"id":80132976,"identity":"30e803cf-2183-45ca-bff3-d0a0f283faf7","added_by":"auto","created_at":"2025-04-08 09:28:30","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":581941,"visible":true,"origin":"","legend":"\u003cp\u003eVolcano plots showing differentially expressed genes between planktonic and biofilm cultures of \u003cem\u003eS. mutans\u003c/em\u003e UA159 treated with sublethal concentrations of amoxicillin and chlorhexidine. The x-axis represents the fold-change in gene expression between different conditions, and the y-axis represents the statistical significance of the found differences. Significantly up and down-regulated genes are filtered (|log2FC| \u0026gt; 1, Padj \u0026lt; 0.05) and highlighted in yellow and blue dots, respectively. Genes that are not differentially expressed are shown in black. Abbreviations: CHX, chlorhexidine, AMOX, amoxicillin.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5834130/v1/47e0f06c7d07a68c1a83613e.jpg"},{"id":80132982,"identity":"ea891a64-b50b-4f3f-8935-816676fcbdaf","added_by":"auto","created_at":"2025-04-08 09:28:30","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":569790,"visible":true,"origin":"","legend":"\u003cp\u003eDifferentially expressed genes (DEGs) representations across compared conditions. (A) Heatmap of DEGs. (B) Venn diagrams comparing different conditions. Abbreviations: CHX, chlorhexidine, AMOX, amoxicillin.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5834130/v1/acba12a7ff04feda0acaf692.jpg"},{"id":80132984,"identity":"0c52907c-9dc0-452a-b722-3eb36877fdd4","added_by":"auto","created_at":"2025-04-08 09:28:31","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":679916,"visible":true,"origin":"","legend":"\u003cp\u003eUp- and downregulated KEGG pathways enrichment results of the analysed conditions. The gene ratio represents the proportion of genes in a given pathway relative to the total number of genes in that pathway (\u003cem\u003ep\u003c/em\u003e-value \u0026lt; 0.05). The size of the dots represents the count of genes coloured based on the adjusted \u003cem\u003ep\u003c/em\u003e-value. Abbreviations: CHX, chlorhexidine, AMOX, amoxicillin.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5834130/v1/c742b15adf0741b4fa12ebe7.jpg"},{"id":80134336,"identity":"0c44649a-7106-4489-995d-dc613b333f1d","added_by":"auto","created_at":"2025-04-08 09:52:31","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3498250,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5834130/v1/6b616574-db8e-49da-9f06-e8e2f988c260.pdf"},{"id":80132978,"identity":"0a845718-2662-453e-aca1-220aa8e7f9c2","added_by":"auto","created_at":"2025-04-08 09:28:30","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":593765,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary figures and Table S6\u003c/p\u003e","description":"","filename":"Supplementary.docx","url":"https://assets-eu.researchsquare.com/files/rs-5834130/v1/75e8d671be2cc5e2547ed661.docx"},{"id":80132975,"identity":"3d0c12e0-fc37-4c46-8a10-916aa2053ab6","added_by":"auto","created_at":"2025-04-08 09:28:30","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":13391,"visible":true,"origin":"","legend":"\u003cp\u003eTable S1\u003c/p\u003e","description":"","filename":"TableS1.Statsu2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5834130/v1/931e35916d9242b8bf05de91.xlsx"},{"id":80132989,"identity":"ea3dd81c-1e85-4a97-8d4f-40a4871bc338","added_by":"auto","created_at":"2025-04-08 09:28:31","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":263382,"visible":true,"origin":"","legend":"\u003cp\u003eTable S2\u003c/p\u003e","description":"","filename":"TableS2.TPMu4.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5834130/v1/88c94b27747d6e96398583a9.xlsx"},{"id":80132986,"identity":"bca3b6a7-15ea-41c5-904e-231ca0c980ae","added_by":"auto","created_at":"2025-04-08 09:28:31","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":215149,"visible":true,"origin":"","legend":"\u003cp\u003eTable S3\u003c/p\u003e","description":"","filename":"TableS3.AllDEGsu.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5834130/v1/fba1d5088a1152cc19009976.xlsx"},{"id":80134007,"identity":"f2f39c1d-9074-40a5-804e-7a77b50a6c38","added_by":"auto","created_at":"2025-04-08 09:44:31","extension":"xlsx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":34714,"visible":true,"origin":"","legend":"\u003cp\u003eTable S4\u003c/p\u003e","description":"","filename":"TableS4.enrichKEGGgenesu.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5834130/v1/f8e9a398f36b856cb56d3cba.xlsx"},{"id":80133070,"identity":"0f4c788c-a34a-4030-ac10-e01930a3a39c","added_by":"auto","created_at":"2025-04-08 09:36:31","extension":"xlsx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":44147,"visible":true,"origin":"","legend":"\u003cp\u003eTable S5\u003c/p\u003e","description":"","filename":"TableS5.Venndiagramsgenesu.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5834130/v1/4f294a7656ae3d8f21e203dc.xlsx"},{"id":80134006,"identity":"1c88d71b-4f4f-4cc8-94d1-8ca15cea784f","added_by":"auto","created_at":"2025-04-08 09:44:30","extension":"xlsx","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":23685,"visible":true,"origin":"","legend":"\u003cp\u003eTable S7\u003c/p\u003e","description":"","filename":"TableS7.ABCPTSu.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5834130/v1/536f60a6005ef651ba4a3f48.xlsx"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eTranscriptomic response in planktonic and biofilm cells of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eS. mutans\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e treated with sublethal concentrations of chlorhexidine\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eDental caries is the gradual loss and demineralisation of the hard tissues of teeth caused by acid production from biofilm-associated bacteria in the oral cavity (Pitts et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). If untreated, caries can lead to severe infection and require antibiotic treatment. Dental caries can be prevented by frequent disruption of biofilms in the oral cavity by brushing and flossing, and by the use of antiseptic mouthwashes. Clinically relevant concentrations of these antimicrobials are likely lead to sublethal concentrations at certain target sites due to decreased bioavailability and accessibility (Delacher et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Liu, Prentice and Webber \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). This is particularly relevant for biofilm-related conditions such as dental caries, the most prevalent microbe-related health condition worldwide (Ward and Goldie \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eChlorhexidine and amoxicillin are commonly used to control oral infections with different clinical indications (Brookes et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Abdullah et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Chlorhexidine is often used as a mouthwash (containing 0.1\u0026ndash;0.2% chlorhexidine digluconate) by dental clinicians during pre-surgical preparation to reduce the bacterial load, and after surgery as a preventative measure (Brookes et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Poppolo Deus and Ouanounou \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Furthermore, in many regions chlorhexidine is available as over-the-counter mouthwash and used to manage early gum disease (gingivitis) or used as a prophylactic and adjunct to brushing to prevent plaque formation.(Brookes et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Chlorhexidine is a cationic agent that interacts with the negatively charged bacterial membranes altering their osmoregulation and leading to leakage of ions and cellular components that eventually leads to cell lysis (Lim and Kam \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Chlorhexidine is a broad-spectrum antiseptic that is active against bacteria, viruses and fungi and can also disrupt biofilms (Alvendal et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Bonez et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Karpiński and Szkaradkiewicz, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Poppolo Deus and Ouanounou, \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). On the other hand, orally administered amoxicillin is the most common choice for systemic tooth infection treatment (Akhavan, Khanna and Vijhani 2024). Amoxicillin is a β-lactam that targets peptidoglycan synthesis in actively dividing cells and it displays activity against gram-positive bacteria, including \u003cem\u003eStreptococcus\u003c/em\u003e, and some gram-negative bacteria such as \u003cem\u003eEscherichia coli\u003c/em\u003e (Castle \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2007\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSublethal concentrations of antibacterials affect the physiology of the cells and can select for resistant bacteria, promote genetic variability and function as signalling molecules affecting virulence, biofilm formation and communication mechanisms (Andersson and Hughes \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Silva et al. \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Waack and Nicholson \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Liu et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Penesyan et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Guo et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Byun et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Understanding those effects as part of the antibiotic response can help design optimised therapies and gain an increased understanding of the ecology of these bacteria.\u003c/p\u003e \u003cp\u003eWhile most research on the effect of antimicrobials has been conducted on planktonic cells, pathogens such as the oral bacterium \u003cem\u003eStreptococcus mutans\u003c/em\u003e often form biofilms during or as part of their infection cycle. \u003cem\u003eS. mutans\u003c/em\u003e is key to dental caries development and oral biofilm formation (Lemos et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). By metabolising carbohydrates in the oral cavity, \u003cem\u003eS. mutans\u003c/em\u003e produces glucans that contribute to forming the biofilm extracellular matrix. As a by-product, organic acids that lower the pH are produced leading to tooth decay. However, this process is only part of the explanation for the complex, multifactorial, and multispecies aetiology of dental caries (Cai and Kim \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBiofilm-associated cells differ from planktonic cells in their response to antibiotics (Shree et al. \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) and generally display greater heterogeneity (Obando and Serra \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Bacteria embedded in biofilm matrices are more resilient to antibiotic treatment due to limited diffusion of antibiotic and slower growth rates of the cells, (Cozens et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e1986\u003c/span\u003e) (Lebeaux, Ghigo and Beloin \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Consequently, antibiotic concentrations effective against planktonic cells become subinhibitory for biofilm-associated cells in dental caries.\u003c/p\u003e \u003cp\u003eIn this work, we challenged \u003cem\u003eS. mutans\u003c/em\u003e UA159 planktonic and biofilm cells with a sublethal concentration of chlorhexidine for a short duration to mimic exposure from the use of mouthwash. We then used RNA-seq to analyse and compare differential gene expression changes. Amoxicillin was also applied to the biofilm cultures for comparison purposes. The results showed an overall differential regulation of planktonic and biofilms, with extensive downregulation of chlorhexidine and amoxicillin-treated biofilm metabolism.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Bacterial strains and antibiotics used\u003c/h2\u003e \u003cp\u003e \u003cem\u003eS. mutans\u003c/em\u003e UA159 was cultured in Brain Heart Infusion (BHI, Difco) for planktonic growth in broth and in BHI supplemented with 1% sucrose (BHIS) for the biofilm experiments. The strain was grown at 37\u0026deg;C in anaerobiosis (10% CO\u003csub\u003e2\u003c/sub\u003e) or airtight tubes, unless otherwise stated.\u003c/p\u003e \u003cp\u003eChlorhexidine digluconate 20% (Sigma) was prepared and diluted to the desired concentration in sterile Milli-Q water. For amoxicillin, stocks at 10 mg/ml were prepared by dissolving amoxicillin trihydrate in an equal volume of 0.1 M NaOH and phosphate-buffered saline (pH 7.2) and further diluted in Milli-Q water to the desired concentration.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Antibiotic susceptibility tests to amoxicillin and chlorhexidine\u003c/h2\u003e \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e \u003ch2\u003e2.2.1 Minimum Inhibitory Concentration (MIC) determination\u003c/h2\u003e \u003cp\u003eThe experiments were set up in 96-well microtiter plates with a total volume of 300 \u0026micro;l using the broth microdilution method. Two-fold dilution series of chlorhexidine (starting concentration 100 \u0026micro;g/ml) and amoxicillin (starting concentration 100 ng/ml) were prepared in BHI, with each well containing 150 \u0026micro;l of the diluted solutions. Subsequently, 150 \u0026micro;l of \u003cem\u003eS. mutans\u003c/em\u003e UA159 culture at an OD\u003csub\u003e600\u003c/sub\u003e 0.05 were added to each well. The cultures were incubated at 37\u0026deg;C, and the plate was shaken for 5 sec before measurements of OD\u003csub\u003e600\u003c/sub\u003e were taken every 10 min throughout the experiment using a Hidex Sense (Hidex Oy) plate reader. The experiments were repeated three times. MIC were established as the lowest concentration of antimicrobial that inhibited bacterial growth.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003e2.2.3. Tolerance to chlorhexidine during growth\u003c/h2\u003e \u003cp\u003e \u003cem\u003eS. mutans\u003c/em\u003e UA159 culture at OD\u003csub\u003e600\u003c/sub\u003e 0.05 was incubated at 37\u0026deg;C until it reached an OD\u003csub\u003e600\u003c/sub\u003e of 0.3\u0026ndash;0.4. At this point, chlorhexidine was added. The same two-fold dilution series used for the MIC assays was tested (100, 50, 25, 12.5, 6.25, 3.125, 1.5, 0.78, 0 \u0026micro;g/ml). Growth was monitored in a plate reader as described above.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003e2.2.4. Biofilm-Oriented-Antimicrobials Test (BOAT)\u003c/h2\u003e \u003cp\u003eThe metabolic activity of \u003cem\u003eS. mutans\u003c/em\u003e UA159 biofilm-cells treated with chlorhexidine and amoxicillin was determined by the Biofilm-Oriented Antimicrobial Test (BOAT) (Gr\u0026oslash;nseth et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Kranjec et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). \u003cem\u003eS. mutans\u003c/em\u003e UA159 culture at an OD\u003csub\u003e600\u003c/sub\u003e of 0.5 was diluted 1:1000 in BHIS and 100 \u0026micro;l was added to the wells of a 96-well plate and allowed to form biofilms for 24 h at 37\u0026deg;C in anaerobiosis. Different concentrations of chlorhexidine (800, 775, 750, 725, 700, 650, 600, 120, 100, 71, 50, 42, 24, 14, 8, 6, 5, 3, 2 \u0026micro;g/ml) and amoxicillin (5000, 2500, 1250, 625, 313, 156, 78, 39, 20, 10, 5, 2, 1 \u0026micro;g/ml) were prepared in BHI to a volume of 175 \u0026micro;l. These concentrations were selected after several rounds of BOAT assays to show the effect of a wide range of antibiotic concentrations on \u003cem\u003eS. mutans\u003c/em\u003e UA159 biofilms. Biofilms were washed twice with 100 \u0026micro;l of 0.9% NaCl and 150 \u0026micro;l of the antibiotic dilutions were transferred to the biofilm plate. Antibiotic treatment was applied for 5 min, 30 min and 24 h while incubating at 37\u0026deg;C in anaerobiosis. The biofilms were washed three times with 0.9% NaCl and 100 \u0026micro;l of 0.025% triphenyl-tetrazolium chloride (TTC, Sigma) dissolved in BHI was added to each well and further incubated at 37\u0026deg;C for 5 h. The presence of red colour was used as a measure of cellular respiration. TTC was removed and 200 \u0026micro;l ethanol:acetone (70:30) were added per well and incubated overnight to extract the red dye. The metabolic activity was then measured at 492 nm in a plate reader (Fluostar Optima, BMG, LabTech). Biofilms without antibiotic treatment were used as controls. Three replicates per condition were performed.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.5. RNA isolation and sequencing\u003c/h2\u003e \u003cp\u003eRNA was isolated from planktonic-broth and biofilm cultures of \u003cem\u003eS. mutans\u003c/em\u003e UA159 challenged with sublethal concentrations of amoxicillin and chlorhexidine for 5 min at 37\u0026deg;C in anaerobiosis. In broth, chlorhexidine was used at a final concentration of 6.25 ng/ml. In biofilm experiments, chlorhexidine and amoxicillin were used at 70 \u0026micro;g/ml and 5 mg/ml, respectively. Concentrations used were based on MIC and BOAT assays.\u003c/p\u003e \u003cp\u003eThe initial broth culture of \u003cem\u003eS. mutans\u003c/em\u003e UA159 was prepared by inoculating 50 ml BHI with 0.5 ml of a starting inoculum with an OD\u003csub\u003e600\u003c/sub\u003e of 0.4\u0026ndash;0.5. Growth was monitored until an OD\u003csub\u003e600\u003c/sub\u003e of 0.3 (early-mid exponential phase). Aliquots of 10 ml were used for chlorhexidine treatment and as a negative control (treated with BHI). The cells were harvested by centrifugation at 6,000 x g for 1 min at 4\u0026deg;C. The pelleted cells were immediately frozen in liquid nitrogen and stored at -80\u0026deg;C. This experiment was repeated three times (biological replicates).\u003c/p\u003e \u003cp\u003eFor the biofilm cultures of \u003cem\u003eS. mutans\u003c/em\u003e UA159, 20 \u0026micro;l of a starting culture with an OD\u003csub\u003e600\u003c/sub\u003e of 0.4\u0026ndash;0.5 were inoculated in 20 ml BHIS. The biofilm was grown in 48-well plates with 400 \u0026micro;l of culture per well and allowed to grow for 24 h at 37\u0026deg;C in anaerobiosis. Planktonic cells were removed by aspiration, attached cells/biofilms were washed once with 400 \u0026micro;l 0.9% NaCl and then 400 \u0026micro;l of amoxicillin or chlorhexidine at the concentrations mentioned above. The control with no antibiotic was treated with BHIS. Four wells per condition were used. After the antibiotic treatment, the biofilms were washed with 400 \u0026micro;l sterile RNAse-free water (Invitrogen). Then 400 \u0026micro;l of RNAprotect (Qiagen) were added to the wells and the biofilms were scraped off the surface using a pipette tip (Kragh et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The suspended biofilms from the 4 wells for each condition was transferred to 15 ml tubes and centrifuged at 6,000 x g for 1 min at 4\u0026deg;C. The pelleted cells were immediately frozen in liquid nitrogen and stored at -80\u0026deg;C. The experiment was repeated three times (biological replicates).\u003c/p\u003e \u003cp\u003eRNA was extracted using the RNeasy Mini Kit (Qiagen), followed by DNase treatment and phenol-chloroform extraction, as described by Stams\u0026aring;s et al. (Stams\u0026aring;s et al. \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Library preparation, quality assessment, and sequencing were conducted by Novogene (Germany). In summary, rRNA was removed from the total RNA, followed by ethanol precipitation. Second-strand cDNA synthesis incorporated dUTPs instead of dTTPs to generate a directional (stranded) library. Library quantification was performed using Qubit and real-time PCR, while size distribution was assessed with a Bioanalyzer. The quantified libraries were pooled and sequenced on an Illumina instrument. The raw sequencing reads were processed to remove adapters, reads with \u0026gt;\u0026thinsp;10% ambiguous bases (N), and low-quality reads (Qscore\u0026thinsp;\u0026le;\u0026thinsp;5).\u003c/p\u003e \u003cp\u003eThe reads were then aligned against \u003cem\u003eS. mutans\u003c/em\u003e UA159 (Genbank accession number: AE014133.2) and differentially expressed genes (DEGs) between planktonic, biofilm, treated and untreated samples were calculated using DESeq2 (Love, Huber and Anders \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) on 3 independent biological replicates for each tested condition using the Bioconductor R package. See Table\u0026nbsp;1 for the conditions being analysed and compared. An additional rRNA removal step was done bioinformatically by filtering out ribosomal locus tags based on the reference genome. Genes with an adjusted \u003cem\u003ep\u003c/em\u003e-value\u0026thinsp;\u0026le;\u0026thinsp;0.05 were used for further analysis. DEGs were scored as upregulated if they had a log\u003csub\u003e2\u003c/sub\u003efold change\u0026thinsp;\u0026ge;\u0026thinsp;1.0 and downregulated if they had a log\u003csub\u003e2\u003c/sub\u003efold change \u0026le; -1.0. Visualizations of DEGs were done as volcano plots and a heatmap using the EnhancedVolcano and heatmap R packages, respectively. A Venn diagram was also constructed to identify the common DEGs obtained from the different compared conditions using the gvenn R package.\u003c/p\u003e \u003cp\u003eTranscripts Per Million (TPM) values were calculated based on the normalized counts provided by DESeq2. Bray-Curtis dissimilarity was used to compute distance matrices based on TPM values, and Principal Coordinates Analysis (PCoA) was performed to visualize sample clustering. Additionally, a heatmap was calculated using the \u003cem\u003epheatmap\u003c/em\u003e R package to visualize expression patterns across samples, and a correlation matrix showed associations between samples using the \u003cem\u003ecorrplot\u003c/em\u003e R package.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Functional annotations and pathway analysis\u003c/h2\u003e \u003cp\u003eTo obtain insights into the biological meaning of the DEGs the clusterProfiler R package v4.13.0 (Yu et al. \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) was used for gene set enrichment analysis (GSEA) using the Kyoto Encyclopedia of Genes and Genomes database via the functions \u003cem\u003eenrichKEGG\u003c/em\u003e and \u003cem\u003ecompareCluster\u003c/em\u003e setting a \u003cem\u003ep\u003c/em\u003e-value cut-off \u0026lt;\u0026thinsp;0.05. Note that the global maps and the overview maps are a special class of metabolic pathway maps within KEGG (Kanehisa et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). These categories represent an integrated picture of the metabolism connecting different pathways present in the dataset.\u003c/p\u003e \u003cp\u003e \u003cb\u003eSequencing data availability\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe raw FASTQ data are accessible at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ebi.ac.uk/ena/browser/home\u003c/span\u003e\u003cspan address=\"https://www.ebi.ac.uk/ena/browser/home\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e with accession number PRJEB83273.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results and discussion","content":"\u003cp\u003e \u003cb\u003e3.1. Selection of sublethal chlorhexidine and amoxicillin concentrations applied on planktonic and biofilm cultures of\u003c/b\u003e \u003cb\u003eS. mutans\u003c/b\u003e \u003cb\u003eUA159\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo select a relevant sublethal concentration of chlorhexidine that could exert changes at the transcriptome level, we first established the sensitivity of planktonic \u003cem\u003eS. mutans\u003c/em\u003e UA159 to chlorhexidine by MIC assays (Fig.\u0026nbsp;1A and 1B). Complete inhibition of growth was observed at 1.56 \u0026micro;g/ml Fig.\u0026nbsp;1A). This is in line with other studies, which have found the MIC of \u003cem\u003eS. mutans\u003c/em\u003e for chlorhexidine to be below 1 \u0026micro;g/ml (J\u0026auml;rvinen, Tenovuo and Huovinen \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e1993\u003c/span\u003e; Mohammed Ghilan et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The same concentration range of 0.3\u0026ndash;100 \u0026micro;g/ml was used to assay if \u003cem\u003eS. mutans\u003c/em\u003e UA159 grown to early-mid exponential phase could tolerate the presence of chlorhexidine (Fig.\u0026nbsp;1B). At this phase of growth, \u003cem\u003eS. mutans\u003c/em\u003e UA159 was only affected by chlorhexidine concentrations above 6.25 \u0026micro;g/ml. A concentration of 6.25 \u0026micro;g/ml was therefore used for the chlorhexidine transcriptomic stress response analysis in broth. The MIC for amoxicillin was determined to be 50 ng/ml which is in line with other studies (Fig.\u0026nbsp;1A) (Kwon and Lee \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Maisonneuve et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2020\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eWe then examined the effect of chlorhexidine on \u003cem\u003eS. mutans\u003c/em\u003e UA159 biofilms (Fig.\u0026nbsp;1C). Chlorhexidine is reported to disrupt \u003cem\u003eS. mutans\u003c/em\u003e biofilms in a dose-dependent manner (Ccahuana-V\u0026aacute;squez and Cury \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Silva et al. \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Lee et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) favouring \u003cem\u003eS. mutans\u003c/em\u003e biofilm detachment (Liu et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). To determine the concentration needed to disrupt \u003cem\u003eS. mutans\u003c/em\u003e biofilms, we used the BOAT assay (Kranjec et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Using this assay, we were able to quantify the remaining metabolic activity of 24 h-old biofilms treated with chlorhexidine for 5 min, 30 min and 24 h (Fig.\u0026nbsp;1C). The metabolic activity was reduced according to concentration and treatment duration. For the 5 min treatment, a chlorhexidine concentration of 600 \u0026micro;g/ml was needed to fully reduce the metabolic activity, indicating a complete disruption of the formed biofilm. The corresponding concentrations for the 30 min and 24 h treatments were 50 \u0026micro;g/ml and 24 \u0026micro;g/ml chlorhexidine, respectively (Fig.\u0026nbsp;1C). The highest concentration of chlorhexidine that did not appreciably affect metabolic activity after a 5 min treatment was determined to be 70 \u0026micro;g/ml, which we chose as the sublethal concentration for the transcriptomic experiments.\u003c/p\u003e \u003cp\u003eWe also attempted to determine a corresponding concentration for amoxicillin against biofilms, however, no effect was observed on the metabolic activity even with the highest concentration tested (5 mg/ml) (Fig.\u0026nbsp;1C). This is most probably due to the nature of the cells in the biofilms and highlights the difference in physiology between cells in biofilms compared to planktonic growth. Amoxicillin and other β-lactams inhibit bacterial cell-wall synthesis due to binding to the penicillin-binding proteins, thereby inhibiting transpeptidation of peptidoglycan. However, most cells in a biofilm are not actively dividing and relatively mature \u003cem\u003eS. mutans\u003c/em\u003e biofilms allowed to establish for at least 24 h contain mostly inactive non-dividing cells (Lewis \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2005\u003c/span\u003e).\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Overview of \u003cem\u003eS. mutans\u003c/em\u003e RNA-seq analysis\u003c/h2\u003e \u003cp\u003eTo investigate the transcriptomic response of planktonic and biofilm cultures of \u003cem\u003eS. mutans\u003c/em\u003e UA159 upon 5-min treatment with sub-inhibitory concentrations of chlorhexidine and amoxicillin, an RNA-seq analysis was conducted. We selected a 5-min treatment duration due to the short contact time that mouthwashes typically have with their targets.\u003c/p\u003e \u003cp\u003eA total of 247,746,462 raw reads were generated, of which 98.10% were clean reads, with an average GC content of 39.96%, Q\u0026thinsp;\u0026gt;\u0026thinsp;30 93.77%, and an underlying error of 0.03%, indicating the high quality of the sequencing data (Table S1). TPM values were used to compare the proportion of reads mapped to a gene in each sample (Table S2). Principal component analysis (PCoA) of the gene data showed grouping of the biological replicates, with a clear separation between planktonic and biofilm samples and further separation between non-treated and amoxicillin and chlorhexidine-treated samples (Figure S1). A heatmap of all genes and a correlation plot further confirmed the expected clustering of the samples (Figure S2). The distribution of differentially expressed genes (DEGs; |log\u003csub\u003e2\u003c/sub\u003eFC| \u0026gt; 1, P\u003csub\u003eadj\u003c/sub\u003e \u0026lt; 0.05) between the compared conditions (Table\u0026nbsp;1) was visualised using volcano plots (Fig.\u0026nbsp;2). A list of all DEGs can be found in the Supplementary material (Table S3).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Biofilm transcriptional behaviour differs from planktonic cells of \u003cem\u003eS. mutans\u003c/em\u003e UA159\u003c/h2\u003e \u003cp\u003eGene regulation of planktonic and biofilm cultures can differ significantly reflecting the two different microbial lifestyles (Shemesh, Tam and Steinberg \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Lo et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Charlebois, Jacques and Archambault \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2016a\u003c/span\u003e; Castro et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; S\u0026aacute;nchez et al. \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2019a\u003c/span\u003e; Zheng et al. \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Indeed, with a threshold of |log\u003csub\u003e2\u003c/sub\u003eFC| \u0026gt; 1 and P\u003csub\u003eadj\u003c/sub\u003e \u0026lt; 0.05, a total of 879 genes (412 upregulated, 467 downregulated genes, 43% of genes in total) were found to be differentially expressed in biofilm relative to planktonic cultures of \u003cem\u003eS. mutans\u003c/em\u003e UA159 (Fig.\u0026nbsp;2A, Fig.\u0026nbsp;3A). Previous DNA-microarray analyses showed about 12% of \u003cem\u003eS. mutans\u003c/em\u003e UA159 genes to be differentially expressed in biofilms (Shemesh, Tam and Steinberg \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2007\u003c/span\u003e) whereas studies in other species showed broader variability, from 1% differential expression in \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e (Whiteley et al. \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e2001\u003c/span\u003e), 4.8% in \u003cem\u003ePorphyromonas gingivalis\u003c/em\u003e (S\u0026aacute;nchez et al. \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2019b\u003c/span\u003e) or 25.7% in \u003cem\u003eClostridium perfringes\u003c/em\u003e (Charlebois, Jacques and Archambault 2016) biofilms highlighting important differences in cell metabolism and the techniques used.\u003c/p\u003e \u003cp\u003eTo get an overview of the physiological processes affected in the \u003cem\u003eS. mutans\u003c/em\u003e biofilms compared to planktonic cells, the expression patterns were examined by KEGG pathway enrichment analysis revealing several significantly up- and downregulated pathways (Fig.\u0026nbsp;4A, Table S4). Fatty acid biosynthesis, phosphotransferase systems, starch and glucose metabolism, methane metabolism, β-lactam resistance and ABC transporters were upregulated in the wild-type biofilm compared to the planktonic cells (Fig.\u0026nbsp;4A). On the other hand, purine metabolism and biosynthesis of secondary metabolites, metabolic pathways and 2-oxocarboxylic acid metabolism within the general global and overview maps category were downregulated (Fig.\u0026nbsp;4A). The global biofilm gene expression pattern, representing the average across the heterogeneous biofilm cell population, showed a significant number of differentially regulated genes compared to the planktonic cells, indicating that biofilms exhibit distinct metabolic and physiological adaptations.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.4. The transcriptomic response to chlorhexidine treatment is dependent on the lifestyle of \u003cem\u003eS. mutans\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eChlorhexidine is the gold-standard oral antiseptic widely used in dental practice and as an over-the-counter mouthwash. Swallowing, expectoration, or insufficient contact time can lead to sub-inhibitory concentrations of chlorhexidine at target sites known to promote biofilm formation (Ebrahimi et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) and alter the metabolism and microbial composition of the oral microbiota (Chatzigiannidou et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003ePlanktonic and biofilm cells were treated with sub-inhibitory concentrations of chlorhexidine to evaluate its impact on the \u003cem\u003eS. mutans\u003c/em\u003e UA159 transcriptome. Planktonic chlorhexidine-treated \u003cem\u003eS. mutans\u003c/em\u003e cultures showed a total of 499 DEGs (225 upregulated, 274 downregulated genes, 24.4% of genes in total) while chlorhexidine-treated biofilms showed 100 DEGs (25 upregulated and 75 downregulated, 4.9%) (Fig.\u0026nbsp;2B) compared to the non-treated strain. Thus, as expected, there was a greater degree of differential regulation in chlorhexidine-exposed planktonic cells compared to chlorhexidine-exposed biofilms. This is illustrated in the heatmaps of the DEG distribution (Fig.\u0026nbsp;3A). These results are consistent with observations across different species showing that biofilm cells are less metabolically active than their planktonic counterparts (Wan et al. \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Sadiq et al. \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Moreover, the 24 h mature biofilms tested in this study were likely in a state of nutrient limitation and therefore at a slow growth rate leading to a reduced metabolism.\u003c/p\u003e \u003cp\u003eOnly 35 genes were found to be common between the planktonic and biofilm chlorhexidine-treated cells (Fig.\u0026nbsp;3B, Table S5). Those genes included ABC transporters, membrane proteins, the gene \u003cem\u003ecomX1\u003c/em\u003e, which is a key regulator of the natural competence system in streptococci (Aspiras, Ellen and Cvitkovitch \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; J \u003cem\u003eet al.\u003c/em\u003e 2015), and mostly hypothetical genes with unknown functions.\u003c/p\u003e \u003cdiv id=\"Sec14\" class=\"Section3\"\u003e \u003ch2\u003e3.4.1. Transcriptomic response in planktonic chlorhexidine-treated cultures\u003c/h2\u003e \u003cp\u003eFor the planktonic-chlorhexidine treated cultures, the global KEGG analysis of the enriched pathways revealed no upregulated pathways (Fig.\u0026nbsp;4B-C; Table S4), while quorum-sensing systems, two-component systems and ABC-transporters were downregulated, along with pyruvate metabolism, Val/Leu/Ile biosynthesis and 2-oxocarboxylic acid metabolism. Specifically, the regulatory genes included \u003cem\u003eciaR-ciaH\u003c/em\u003e, encoding the highly conserved streptococcal CiaRH regulatory system, which has been shown to be involved in natural competence, biofilm formation, bacteriocin production and cell wall biosynthesis and autolysis (He et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The \u003cem\u003ehtrA\u003c/em\u003e gene, encoding a CiaRH-regulated protease which takes part in oxidative stress tolerance was also downregulated (Sebert et al. \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Ibrahim et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; He et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Likewise, the gene \u003cem\u003ecomE\u003c/em\u003e is part of the ComCDE system, one of the natural competence pathways in \u003cem\u003eS. mutans\u003c/em\u003e also involved in bacteriocin production (van der Ploeg \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Although the direct connection between these systems and chlorhexidine is unknown, one could speculate that membrane targeting agents such as chlorhexidine shift \u003cem\u003eS. mutans\u003c/em\u003e metabolism to survival mechanisms not related to the CiaRH and ComCDE functions.\u003c/p\u003e \u003cp\u003eThe genes with highest fold-changes (|log\u003csub\u003e2\u003c/sub\u003eFC| \u0026gt; 5 and P\u003csub\u003eadj\u003c/sub\u003e \u0026lt; 0.05, Table\u0026nbsp;2) included genes encoding hypothetical proteins and ABC transporters, such as the yet unstudied operon SMU_1550 - SMU_1554, which represent prime candidates for further studies of factors affecting chlorhexidine sensitivity.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.5. The biofilm transcriptomic response is not specific to chlorhexidine\u003c/h2\u003e \u003cp\u003eChlorhexidine-treated biofilms showed no upregulated pathways compared to the wild-type biofilms, with only carbohydrate metabolism (galactose, starch and sucrose) being downregulated (Fig.\u0026nbsp;4C). In a similar study testing the effect of inhibitory concentrations of curcumin and chlorhexidine on \u003cem\u003eS. mutans\u003c/em\u003e biofilms, carbohydrate metabolism, quorum sensing, and two-component transduction systems were found downregulated (Li et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWe were also interested in understanding whether the transcriptional remodeling in biofilms, was specific to chlorhexidine. Amoxicillin is another relevant antibiotic in oral infection treatment. It is given as prophylaxis before oral intervention or for tooth infection treatment administered orally with a systemic effect that is expected to kill sessile bacteria prior to attachment and biofilm formation in the oral cavity. Amoxicillin was not effective in disrupting \u003cem\u003eS. mutans\u003c/em\u003e UA159 biofilms with the used concentrations (up to 100,000-fold MIC), as observed in the BOAT assays (Fig.\u0026nbsp;1A and C). However, amoxicillin-treated biofilms still showed 149 DEGs (65 upregulated, 84 downregulated genes; 7.3% of genes in total) (Fig.\u0026nbsp;2C), compared to 100 DEGs for the chlorhexidine treatment. When directly comparing the transcriptomes of chlorhexidine-treated with the amoxicillin-treated biofilms, only 31 DEGs (3 upregulated, 28 downregulated; 1.5% of the total genes) were detected between the conditions (Fig.\u0026nbsp;2C), suggesting that the majority of the transcriptional responses to the individual agents in biofilms were not significantly different between the chlorhexidine and amoxicillin treatments, and rather represent general responses in the biofilm setting.\u003c/p\u003e \u003cp\u003eInterestingly, 68 commonly regulated genes were found between chlorhexidine- and amoxicillin-treated biofilms. The most downregulated ones were the same after chlorhexidine and amoxicillin treatments and included the phosphotransferase system-related genes \u003cem\u003eptcA, ptsG and mtlA1\u003c/em\u003e, the \u003cem\u003epdh\u003c/em\u003e operon (pyruvate dehydrogenase) important during glucose starvation (Busuioc, Buttaro and Piggot \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) and \u003cem\u003enaoX (noX)\u003c/em\u003e that encodes the main enzyme in oxygen metabolism in \u003cem\u003eS. mutans\u003c/em\u003e (Yamamoto et al. \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Derr et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) (Fig.\u0026nbsp;3B,Table S3, Table S5). Notably, the \u003cem\u003eclpB\u003c/em\u003e gene (SMU_1425) exhibited the lowest log2FC value of -5.07 and \u0026minus;\u0026thinsp;5.21 under both conditions (Table S3, Table\u0026nbsp;2 ). ClpB is a molecular chaperone part of the Clp ATPase family involved in homeostasis and stress tolerance (Lemos and Burne \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Frees et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2007\u003c/span\u003e) which might be reduced in biofilms compared to planktonic cells due to physiological adaptations (Stewart and Franklin \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2008\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFurthermore, amoxicillin-treated biofilms showed downregulation of carbohydrate metabolism (galactose, starch and sucrose) by the KEGG pathway analysis (Fig.\u0026nbsp;4D), similar to what was observed in chlorhexidine-treated biofilms (Fig.\u0026nbsp;4C). However, on the amoxicillin-treated biofilms upregulated pathways that were not significantly different in the chlorhexidine treatment were detected (Fig.\u0026nbsp;4D, Table S4). These included fatty acid metabolism and biosynthesis, carbon fixation, and branched-amino acids metabolism. Additionally, biosynthesis of secondary metabolites, propanoate metabolism, metabolic factors and biosynthesis of cofactors were detected within the Global and overview maps category.\u003c/p\u003e \u003cp\u003eOverall, these observations suggest that there may be a common nonspecific biofilm response to chlorhexidine and amoxicillin.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.6. ABC transporters were differentially regulated across experimental conditions in \u003cem\u003eS. mutans\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eABC transporters play important roles in the active transport of molecules across the membrane for maintenance of cellular nutrient supply and integrity (Davidson et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). A large number of different ABC transporters were significantly regulated across the compared conditions tested here (Table S6 and S7). Upon subinhibitory chlorhexidine exposure, 20% of the differentially regulated genes in broth cultures (10 out of 499) and 50% of those genes in biofilms (50 out of 100) were annotated as ABC transporter proteins. Considering that ABC transporter proteins in the annotated \u003cem\u003eS. mutans\u003c/em\u003e UA159 reference genome are only 6.9% of the genes (141 out of 2043), ABC transporter genes were overrepresented among the regulated genes.\u003c/p\u003e \u003cp\u003eAmong those whose functions are known, the \u003cem\u003eoppADF\u003c/em\u003e genes, part of the \u003cem\u003eopp\u003c/em\u003e operon responsible for oligopeptide uptake (Nepomuceno et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2007\u003c/span\u003e) and \u003cem\u003emsmFGK\u003c/em\u003e and \u003cem\u003emalFX\u003c/em\u003e genes involved in disaccharide uptake (Webb, Homer and Hosie \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e2008\u003c/span\u003e) were upregulated in \u003cem\u003eS. mutans\u003c/em\u003e UA159 biofilm compared to its planktonic form and downregulated in chlorhexidine (\u003cem\u003emalX\u003c/em\u003e) and amoxicillin-treated biofilms (\u003cem\u003emalX\u003c/em\u003e, \u003cem\u003emalF\u003c/em\u003e), whereas the \u003cem\u003eopu\u003c/em\u003e genes \u003cem\u003eopuBa\u003c/em\u003e, \u003cem\u003eopuBc\u003c/em\u003e, \u003cem\u003eopuCd\u003c/em\u003e that regulate osmotic stress (Abranches, Lemos and Burne \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2006\u003c/span\u003e) and the \u003cem\u003emutF\u003c/em\u003e, part of the MutEFG transporter that has been linked with nisin resistance in \u003cem\u003eS. mutans\u003c/em\u003e (Le, Kawada-Matsuo and Komatsuzawa) were downregulated in wild-type biofilms and after CHX-treated planktonic \u003cem\u003eS. mutans\u003c/em\u003e UA159. Numerous transcriptomic studies report differential expression of ABC transporters in different bacterial species (Allan et al., 2014; Guo et al., 2022; Rahman et al., 2022; Rice et al., 2017; Zhu et al., 2008), highlighting their ubiquity and multifaceted nature. Up and downregulation of ABC transporters might be a response to counteract the stress induced by chlorhexidine and amoxicillin. In this sense, ABC transporters are known to be involved in the resistance and transport of antimicrobials (Abbood, Hijazi and Gould \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) and can function as antibiotic efflux pumps (Costa et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Nagayama et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Other functions of ABC transporters such as transport of diverse molecules (Biswas and Biswas \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Kim et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Lemos et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) or nutrient uptake (McLaughlin and Ferretti \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Kilic, Honeyman and Tao \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Webb, Homer and Hosie \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e2008\u003c/span\u003e) might as well be relevant in the response of \u003cem\u003eS. mutans\u003c/em\u003e to chlorhexidine and amoxicillin.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.8. Conclusions\u003c/h2\u003e \u003cp\u003eThe results provided here provide a comprehensive overview of the transcriptomic response of \u003cem\u003eS. mutans\u003c/em\u003e after exposure to subinhibitory concentrations of chlorhexidine and amoxicillin. Notably, subinhibitory concentrations of chlorhexidine and amoxicillin exert a significant transcriptomic impact on planktonic and biofilm cultures of the oral commensal \u003cem\u003eS. mutans\u003c/em\u003e, pointing to yet unexplored effects these antimicrobials. This compendium thus serves as a resource for further gene-targeted analysis to elucidate the roles of \u003cem\u003eS. mutans\u003c/em\u003e genes that are differentially regulated under these settings.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConflict of Interest\u003c/h2\u003e \u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eThis work was supported by Marie Skłodowska-Curie Actions H2020-MSCA-EF-ST-2020 grant #101029099.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAbbood HM, Hijazi K, Gould IM. 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While typically used at high concentrations (1\u0026ndash;2 mg/ml), oral bacteria can be exposed to sublethal concentrations due to the bioavailability and protective barrier of biofilms (dental plaques). Sublethal concentrations can cause transcriptional remodelling of bacteria such as \u003cem\u003eStreptococcus mutans\u003c/em\u003e, a key player in dental caries. Using an RNA-seq approach, this report provides a compendium on the effect of sublethal concentrations of chlorhexidine on the transcriptome of \u003cem\u003eS. mutans\u003c/em\u003e as planktonic cells and in biofilm states. \u003cem\u003eS. mutans\u003c/em\u003e showed major transcriptional remodelling between planktonic and biofilm states. The transcriptional response towards chlorhexidine was more pronounced in planktonic cells compared to sessile cells. However, the response observed for biofilm-associated cells was not specific to chlorhexidine, as biofilms exposed to the β-lactam amoxicillin showed similar responses. Furthermore, we found that \u003cem\u003eS. mutans\u003c/em\u003e modulates transcription of a multitude of ABC transporters both in planktonic and biofilm-associated cells upon exposure to these antimicrobials.\u003c/p\u003e","manuscriptTitle":"Transcriptomic response in planktonic and biofilm cells of S. mutans treated with sublethal concentrations of chlorhexidine","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-08 09:28:26","doi":"10.21203/rs.3.rs-5834130/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f391a3e5-5383-4d6e-880c-04e3adcccdd4","owner":[],"postedDate":"April 8th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":46790735,"name":"Bacteriology"},{"id":46790736,"name":"Bioinformatics"},{"id":46790737,"name":"General Microbiology"},{"id":46790738,"name":"Infectious Diseases"}],"tags":[],"updatedAt":"2025-04-08T09:28:26+00:00","versionOfRecord":[],"versionCreatedAt":"2025-04-08 09:28:26","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5834130","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5834130","identity":"rs-5834130","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}