Robert C. Shields

Life after death: the critical role of extracellular DNA in microbial biofilms.

The death and lysis of microbial cells leads to the release of cytoplasmic contents, many of which are rapidly degraded by enzymes. However, some macromolecules survive intact and find new functions in the extracellular environment. There is now strong evidence that DNA released from cells during lysis, or sometimes by active secretion, becomes a key component of the macromolecular scaffold in many different biofilms. Enzymatic degradation of extracellular DNA can weaken the biofilm structure and release microbial cells from the surface. Many bacteria produce extracellular deoxyribonuclease (DNase) enzymes that are apparently tightly regulated to avoid excessive degradation of the biofilm matrix. Interfering with these control mechanisms, or adding exogenous DNases, could prove a potent strategy for controlling biofilm growth.

Efficacy of a Marine Bacterial Nuclease against Biofilm Forming Microorganisms Isolated from Chronic Rhinosinusitis

Background The persistent colonization of paranasal sinus mucosa by microbial biofilms is a major factor in the pathogenesis of chronic rhinosinusitis (CRS). Control of microorganisms within biofilms is hampered by the presence of viscous extracellular polymers of host or microbial origin, including nucleic acids. The aim of this study was to investigate the role of extracellular DNA in biofilm formation by bacteria associated with CRS. Methods/Principal Findings Obstructive mucin was collected from patients during functional endoscopic sinus surgery. Examination of the mucous by transmission electron microscopy revealed an acellular matrix punctuated occasionally with host cells in varying states of degradation. Bacteria were observed in biofilms on mucosal biopsies, and between two and six different species were isolated from each of 20 different patient samples. In total, 16 different bacterial genera were isolated, of which the most commonly identified organisms were coagulase-negative staphylococci, Staphylococcus aureus and α-haemolytic streptococci. Twenty-four fresh clinical isolates were selected for investigation of biofilm formation in vitro using a microplate model system. Biofilms formed by 14 strains, including all 9 extracellular nuclease-producing bacteria, were significantly disrupted by treatment with a novel bacterial deoxyribonuclease, NucB, isolated from a marine strain of Bacillus licheniformis. Extracellular biofilm matrix was observed in untreated samples but not in those treated with NucB and extracellular DNA was purified from in vitro biofilms. Conclusion/Significance Our data demonstrate that bacteria associated with CRS form robust biofilms which can be reduced by treatment with matrix-degrading enzymes such as NucB. The dispersal of bacterial biofilms with NucB may offer an additional therapeutic target for CRS sufferers.

Removal of biofilms from tracheoesophageal speech valves using a novel marine microbial deoxyribonuclease.

Objective The growth of biofilms on tracheoesophageal speech valves shortens their life span and produces a reservoir of pathogens that may infect the respiratory tract. The authors have discovered a novel nontoxic deoxyribonuclease, NucB, from a marine isolate of Bacillus licheniformis that is effective at dispersing a variety of mono and mixed-species bacterial biofilms. The aim of this preliminary study was to determine whether NucB could also disrupt and remove mixed-species biofilms from tracheoesophageal speech valves. Study Design Laboratory-based treatment and analysis of discarded tracheoesophageal speech valves. Setting University human biology laboratory and the Department of Speech and Language Therapy at a tertiary referral hospital. Subjects and Methods Seventeen ex vivo tracheoesophageal speech valves fouled with natural human biofilms were collected and divided into 2 equal parts. One half was treated with NucB and the other half with a control buffer solution. Biofilm removal was measured by microscopy and by culture of dispersed biofilm organisms on agar plates. Results Significantly more organisms were released from biofilms using NucB than with buffer solution alone. On nonselective medium, more organisms were cultured in 11 samples (65%, n = 17, P < .05). Using growth media favoring fungi, more organisms were cultured in 14 samples (82%, n = 17, P < .05). Conclusion The nontoxic deoxyribonuclease NucB was effective in releasing more microorganisms from biofilms on tracheoesophageal speech valves. This reflects its potential ability to break up and disperse these biofilms. Future studies will aim to develop NucB as a novel agent to prolong the life span of tracheoesophageal speech valves, thus reducing health care costs.

Bidirectional signaling in the competence regulatory pathway of Streptococcus mutans

Streptococcus mutans expresses comX (also known as sigX), which encodes a sigma factor that is required for development of genetic competence, in response to the peptide signals XIP and CSP and environmental factors. XIP (sigX inducing peptide) is derived from ComS and activates comX unimodally in chemically defined media via the ComRS system. CSP (competence stimulating peptide) activates comX bimodally in peptide-rich media through the ComDE two-component system. However, CSP-ComDE activation of comX is indirect and involves ComRS. Therefore, the bimodality of CSP-dependent activation of comX may arise from either ComRS or ComDE. Here we study, at the single-cell level, how genes in the CSP signaling pathway respond to CSP, XIP and media. Our data indicate that activation of comX stimulates expression of comE. In addition, activation of comE requires intact comR and comS genes. Therefore, not only does CSP-ComDE stimulate the ComRS pathway to activate comX expression, but ComRS activation of comX also stimulates expression of the CSP-ComDE pathway and its regulon. The results demonstrate the mutual interconnection of the signaling pathways that control bacteriocin expression (ComDE) and genetic competence (ComRS), both of which are linked to lytic and virulence behaviors.

Growth of Streptococcus mutans in Biofilms Alters Peptide Signaling at the Sub-population Level.

Streptococcus mutans activates multiple cellular processes in response to the formation of a complex between comX-inducing peptide (XIP) and the ComR transcriptional regulator. Bulk phase and microfluidic experiments previously revealed that ComR-dependent activation of comX is altered by pH and by carbohydrate source. Biofilm formation is a major factor in bacterial survival and virulence in the oral cavity. Here, we sought to determine the response of S. mutans biofilm cells to XIP during different stages of biofilm maturation. Using flow cytometry and confocal microscopy, we showed that exogenous addition of XIP to early biofilms resulted in robust comX activation. However, as the biofilms matured, increasing amounts of XIP were required to activate comX expression. Single-cell analysis demonstrated that the entire population was responding to XIP with activation of comX in early biofilms, but only a sub-population was responding in mature biofilms. The sub-population response of mature biofilms was retained when the cells were dispersed and then treated with XIP. The proportion and intensity of the bi-modal response of mature biofilm cells was altered in mutants lacking the Type II toxins MazF and RelE, or in a strain lacking the (p)ppGpp synthase/hydrolase RelA. Thus, competence signaling is markedly altered in cells growing in mature biofilms, and pathways that control cell death and growth/survival decisions modulate activation of comX expression in these sessile populations.

A Critical Role for Extracellular DNA in Dental Plaque Formation.

Extracellular DNA (eDNA) has been identified in the matrix of many different monospecies biofilms in vitro, including some of those produced by oral bacteria. In many cases, eDNA stabilizes the structure of monospecies biofilms. Here, the authors aimed to determine whether eDNA is an important component of natural, mixed-species oral biofilms, such as plaque on natural teeth or dental implants. To visualize eDNA in oral biofilms, approaches for fluorescently stained eDNA with either anti-DNA antibodies or an ultrasensitive cell-impermeant dye, YOYO-1, were first developed using Enterococcus faecalis, an organism that has previously been shown to produce extensive eDNA structures within biofilms. Oral biofilms were modelled as in vitro “microcosms” on glass coverslips inoculated with the natural microbial population of human saliva and cultured statically in artificial saliva medium. Using antibodies and YOYO-1, eDNA was found to be distributed throughout microcosm biofilms, and was particularly abundant in the immediate vicinity of cells. Similar arrangements of eDNA were detected in biofilms on crowns and overdenture abutments of dental implants that had been recovered from patients during the restorative phase of treatment, and in subgingival dental plaque of periodontitis patients, indicating that eDNA is a common component of natural oral biofilms. In model oral biofilms, treatment with a DNA-degrading enzyme, NucB from Bacillus licheniformis, strongly inhibited the accumulation of biofilms. The bacterial species diversity was significantly reduced by treatment with NucB and particularly strong reductions were observed in the abundance of anaerobic, proteolytic bacteria such as Peptostreptococcus, Porphyromonas and Prevotella. Preformed biofilms were not significantly reduced by NucB treatment, indicating that eDNA is more important or more exposed during the early stages of biofilm formation. Overall, these data demonstrate that dental plaque eDNA is potentially an important target for oral biofilm control.

Conserved and divergent functions of RcrRPQ in Streptococcus gordonii and S. mutans.

In the dental caries pathogen Streptococcus mutans, an MarR-like transcriptional regulator (RcrR), two ABC efflux pumps (RcrPQ) and two effector peptides encoded in the rcrRPQ operon provide molecular connections between stress tolerance, (p)ppGpp metabolism and genetic competence. Here, we examined the role of RcrRPQ in the oral commensal S. gordonii. Unlike in S. mutans, introduction of polar or non-polar rcrR mutations into S. gordonii elicited no significant changes in transformation efficiency. However, S. gordonii rcrR mutants were markedly impaired in their ability to grow in the presence of hydrogen peroxide, paraquat, low pH or elevated temperature. Sensitivity to paraquat could also be conferred by mutation of cysteine residues that are present in the RcrR protein of S. gordonii, but not in S. mutans RcrR. Thus, stress tolerance is a conserved function of RcrRPQ in a commensal and pathogenic streptococcus, but the study reveals additional differences in regulation of genetic competence development between S. mutans and S. gordonii.

A new arginine-based dental adhesive system: formulation, mechanical and anti-caries properties

Secondary caries at the margins of composite restorations has been attributed to adhesive failure and consequent accumulation of cariogenic biofilms. OBJECTIVES To develop and evaluate an etch-and-rinse adhesive system containing arginine for sustainable release and recharge without affecting its mechanical properties. Arginine metabolism by oral bacteria generates ammonia, which neutralizes glycolytic acids and creates a neutral environmental pH that is less favorable to the growth of caries pathogens, thus reducing the caries risk at the tooth-composite interface. METHODS Experimental adhesives were formulated with methacrylate monomers and arginine at 5%, 7%, and 10% or no arginine (control). Adhesives were tested for: (i) mechanical properties of true stress (FS and UTS), modulus of elasticity (E), degree of conversion (DC), Knoop hardness number (KHN) and dentin microtensile bond strength (μ-TBS), (ii) arginine release and recharge, and (iii) antibacterial activities. Data was analyzed by t-test, one-way ANOVA and Tukeys tests. RESULTS FS and UTS results showed no statistically significant differences between the 7% arginine-adhesive and control, while the results for E, DC, KHN and μ-TBS showed no difference among all groups. The 7% arginine-adhesive showed a high release rate of arginine (75.0μmol/cm2) at 2h, and a more sustainable, controlled release rate (up to 0.2μmol/cm2) at 30days. CONCLUSIONS Incorporation of 7% arginine did not affect the physical and mechanical properties of the adhesive. Arginine was released from the adhesive at a rate and concentration that exhibited antibacterial effects, regardless of shifts in biofilm conditions such as sugar availability and pH. CLINICAL SIGNIFICANCE Secondary caries is recognized as the main reason for failure of dental restorations. The development of an arginine-based adhesive system has the potential to dramatically reduce the incidence and severity of secondary caries in adhesive restorations in a very economical fashion.

Genome-Wide Screens Reveal New Gene Products That Influence Genetic Competence in Streptococcus mutans

A network of genes and at least two peptide signaling molecules tightly control when Streptococcus mutans becomes competent to take up DNA from its environment. Widespread changes in the expression of genes occur when S. mutans is presented with competence signal peptides in vitro, including the increased production of the alternative sigma factor, ComX, which activates late competence genes. Still, the way that gene products that are regulated by competence peptides influence DNA uptake and cellular physiology are not well understood. Here, we developed and employed comprehensive transposon mutagenesis of the S. mutans genome, with a screen to identify mutants that aberrantly expressed comX, coupled with transposon sequencing (Tn-seq) to gain a more thorough understanding of the factors modulating comX expression and progression to the competent state. The screens effectively identified genes known to affect competence, e.g., comR, comS, comD, comE, cipB, clpX, rcrR, and ciaH, but disclosed an additional 20 genes that were not previously competence associated. The competence phenotypes of mutants were characterized, including by fluorescence microscopy to determine at which stage the mutants were impaired for comX activation. Among the novel genes studied were those implicated in cell division, the sensing of cell envelope stress, cell envelope biogenesis, and RNA stability. Our results provide a platform for determining the specific chemical and physical cues that are required for genetic competence in S. mutans, while highlighting the effectiveness of using Tn-seq in S. mutans to discover and study novel biological processes.IMPORTANCE Streptococcus mutans acquires DNA from its environment by becoming genetically competent, a physiologic state triggered by cell-cell communication using secreted peptides. Competence is important for acquiring novel genetic traits and has a strong influence on the expression of virulence-associated traits of S. mutans Here, we used transposon mutagenesis and genomic technologies to identify novel genes involved in competence development. In addition to identifying genes previously known to be required for comX expression, 20 additional genes were identified and characterized. The findings create opportunities to diminish the pathogenic potential of S. mutans, while validating technologies that can rapidly advance our understanding of the physiology, biology, and genetics of S. mutans and related pathogens.

Genomewide Identification of Essential Genes and Fitness Determinants of Streptococcus mutans UA159

Tooth decay (dental caries) is a common cause of pain, impaired quality of life, and tooth loss in children and adults. It begins because of a compositional change in the microorganisms that colonize the tooth surface driven by repeated and sustained carbohydrate intake. Although several bacterial species are associated with tooth decay, Streptococcus mutans is the most common cause. Therefore, it is important to identify biological processes that contribute to the survival of S. mutans in the human mouth, with the aim of disrupting the processes with antimicrobial agents. We successfully applied Tn-seq to S. mutans, discovering genes that are required for survival, growth, and persistence, both in laboratory environments and in a mouse model of tooth decay. This work highlights new avenues for the control of an important human pathogen. ABSTRACT Transposon mutagenesis coupled with next-generation DNA sequencing (Tn-seq) is a powerful tool for discovering regions of the genome that are required for the survival of bacteria in different environments. We adapted this technique to the dental caries pathogen Streptococcus mutans UA159 and identified 11% of the genome as essential, with many genes encoding products required for replication, translation, lipid metabolism, and cell wall biogenesis. Comparison of the essential genome of S. mutans UA159 with those of selected other streptococci for which such information is available revealed several metabolic pathways and genes that are required in S. mutans, but not in some Streptococcus spp. We further identified genes that are essential for sustained growth in rich or defined medium, as well as for persistence in vivo in a rodent model of oral infection. Collectively, our results provide a novel and comprehensive view of the genes required for essential processes of S. mutans, many of which could represent potential targets for therapeutics. IMPORTANCE Tooth decay (dental caries) is a common cause of pain, impaired quality of life, and tooth loss in children and adults. It begins because of a compositional change in the microorganisms that colonize the tooth surface driven by repeated and sustained carbohydrate intake. Although several bacterial species are associated with tooth decay, Streptococcus mutans is the most common cause. Therefore, it is important to identify biological processes that contribute to the survival of S. mutans in the human mouth, with the aim of disrupting the processes with antimicrobial agents. We successfully applied Tn-seq to S. mutans, discovering genes that are required for survival, growth, and persistence, both in laboratory environments and in a mouse model of tooth decay. This work highlights new avenues for the control of an important human pathogen.