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. 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. 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.

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.

A recent genome-wide screen identified ~300 essential or growth-supporting genes in the dental caries pathogen Streptococcus mutans. To be able to study these genes, we built a CRISPR interference tool around the Cas9 nuclease (Cas9_Smu) encoded in the S. mutans UA159 genome. Using a xylose-inducible dead Cas9_Smu with a constitutively active single-guide RNA (sgRNA), we observed titratable repression of GFP fluorescence that compared favorably to that of Streptococcus pyogenes dCas9 (Cas9_Spy). We then investigated sgRNA specificity and proto-spacer adjacent motif (PAM) requirements. Interference by sgRNAs did not occur with double or triple base-pair mutations, or if single base-pair mutations were in the 3’ end of the sgRNA. Bioinformatic analysis of >450 S. mutans genomes allied with in vivo assays revealed a similar PAM recognition sequence as the Cas9_Spy. Next, we created a comprehensive library of sgRNA plasmids that were directed at essential and growth-supporting genes. We discovered growth defects for 77% of the CRISPRi strains expressing sgRNAs. Phenotypes of CRISPRi strains, across several biological pathways, were assessed using fluorescence microscopy. A variety of cell structure anomalies were observed, including segregational instability of the chromosome, enlarged cells, and ovococci-to-rod shape transitions. CRISPRi was also employed to observe how silencing of cell wall glycopolysaccharide biosynthesis (rhamnose-glucose polysaccharide, RGP) affected both cell division and pathogenesis in a wax worm model. The CRISPRi tool and sgRNA library are valuable resources for characterizing essential genes in S. mutans, some of which could prove to be promising therapeutic targets. Streptococcus species cause diverse infections in humans including pneumonia and meningitis. They are common inhabitants of the mouth and certain species are associated with the incredibly prevalent oral disease dental caries (tooth decay). Streptococcus mutans is the most studied and most often linked organism to the development and progression of dental caries. Recent studies have shown that the genome of this organism encodes a number of essential genes that are required for the survival of the bacteria. Many of these genes are conserved in other pathogenic streptococci, and some are unique to S. mutans. To facilitate the study of these genes in a high-throughput manner we developed a CRISPR interference gene-silencing system. This tool, which modifies the CRISPR-Cas9 system encoded by S. mutans, allowed us to study virtually all the essential genes encoded by the organism. We found that these genes are required for optimal growth of the organism. We also found that the morphological defects of these strains are diverse but also shared among genes related to similar molecular pathways. We used the CRISPRi tool to comprehensively assess the contribution of L-rhamnose glycopolymers to S. mutans viability, pathogenicity and coordination of cell division. Overall, these findings increase our understanding of Streptococcus essential genes, and may help in the development of new antimicrobial therapies.

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.

The ComRS system can function as a quorum sensing trigger for genetic competence in S. mutans . The signal peptide XIP, which is derived from the precursor ComS, enters the cell and interacts with the Rgg-type cytosolic receptor ComR to activate comX , which encodes the alternative sigma factor for the late competence genes. Previous studies have demonstrated intercellular signaling via ComRS, although release of the ComS or XIP peptide to the extracellular medium appears to require lysis of the producing cells. Here we tested the complementary hypothesis that ComRS can drive comX through a purely intracellular mechanism that does not depend on extracellular accumulation or import of ComS or XIP. By combining single-cell, coculture, and microfluidic approaches, we demonstrated that endogenously produced ComS can enable ComRS to activate comX without requiring processing, export, or import. These data provide insight into intracellular mechanisms that generate noise and heterogeneity in S. mutans competence.