Understanding the Matrix:The Role of Extracellular DNA in Oral Biofilms

Dental plaque is the key etiological agent in caries formation and the development of the prevalent chronic oral inflammatory disease, periodontitis. The dental plaque biofilm comprises a diverse range of microbial species encased within a rich extracellular matrix, of which extracellular DNA (eDNA) has been identified as an important component. The molecular mechanisms of eDNA release and the structure of eDNA have yet to be fully characterized. Nonetheless, key functions that have been proposed for eDNA include maintaining biofilm structural integrity, initiating adhesion to dental surfaces, acting as a nutrient source, and facilitating horizontal gene transfer. Thus, eDNA is a potential therapeutic target for the management of oral disease–associated biofilm. This review aims to summarize advances in the understanding of the mechanisms of eDNA release from oral microorganisms and in the methods of eDNA detection and quantification within oral biofilms

Critical roles of arginine in growth and biofilm development by Streptococcus gordonii

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/112199/1/mmi13023.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/112199/2/mmi13023-sup-0001-si.pd

Hierarchical rose-petal surfaces delay the early-stage bacterial biofilm growth

A variety of natural surfaces exhibit antibacterial properties; as a result significant efforts in the past decade have been dedicated towards fabrication of biomimetic surfaces that can help control biofilm growth. Examples of such surfaces include rose petals, which possess hierarchical structures like the micro-papillae measuring tens of microns and nano-folds that range in the size of 700 ±100 nm. We duplicated the natural structures on rose-petal surfaces via a simple UV-curable nanocasting technique, and tested the efficacy of these artificial surfaces in preventing biofilm growth using clinically relevant bacteria strains. The rose-petal structured surfaces exhibited hydrophobicity (contact angle~130.8º ±4.3º) and high contact angle hysteresis (~91.0° ±4.9°). Water droplets on rose-petal replicas evaporated following the constant contact line mode, indicating the likely coexistence of both Cassie and Wenzel states (Cassie-Baxter impregnating wetting state). Fluorescent microscopy and image analysis revealed the significantly lower attachment of Staphylococcus epidermidis (86.1± 6.2% less) and Pseudomonas aeruginosa (85.9 ±3.2% less) on the rose-petal structured surfaces, compared with flat surfaces over a period of 2 hours. Extensive biofilm matrix was observed in biofilms formed by both species on flat surfaces after prolonged growth (several days), but was less apparent on rose-petal biomimetic surfaces. In addition, the biomass of S. epidermidis (63.2 ±9.4% less) and P. aeruginosa (76.0 ±10.0% less) biofilms were significantly reduced on the rose-petal structured surfaces, in comparison to the flat surfaces. By comparing P. aeruginosa growth on representative unitary nano-pillars, we demonstrated that hierarchical structures are more effective in delaying biofilm growth. The mechanisms are two-fold: 1) the nano-folds across the hemispherical micro-papillae restrict initial attachment of bacterial cells and delay the direct contacts of cells via cell alignment, and 2) the hemispherical micro-papillae arrays isolate bacterial clusters and inhibit the formation of a fibrous network. The hierarchical features on rose petal surfaces may be useful for developing strategies to control biofilm formation in medical and industrial contexts

Resistance Toward Chlorhexidine in Oral Bacteria – Is There Cause for Concern?

The threat of antibiotic resistance has attracted strong interest during the last two decades, thus stimulating stewardship programs and research on alternative antimicrobial therapies. Conversely, much less attention has been given to the directly related problem of resistance toward antiseptics and biocides. While bacterial resistances toward triclosan or quaternary ammonium compounds have been considered in this context, the bis-biguanide chlorhexidine (CHX) has been put into focus only very recently when its use was associated with emergence of stable resistance to the last-resort antibiotic colistin. The antimicrobial effect of CHX is based on damaging the bacterial cytoplasmic membrane and subsequent leakage of cytoplasmic material. Consequently, mechanisms conferring resistance toward CHX include multidrug efflux pumps and cell membrane changes. For instance, in staphylococci it has been shown that plasmid-borne qac (“quaternary ammonium compound”) genes encode Qac efflux proteins that recognize cationic antiseptics as substrates. In Pseudomonas stutzeri, changes in the outer membrane protein and lipopolysaccharide profiles have been implicated in CHX resistance. However, little is known about the risk of resistance toward CHX in oral bacteria and potential mechanisms conferring this resistance or even cross-resistances toward antibiotics. Interestingly, there is also little awareness about the risk of CHX resistance in the dental community even though CHX has been widely used in dental practice as the gold-standard antiseptic for more than 40 years and is also included in a wide range of oral care consumer products. This review provides an overview of general resistance mechanisms toward CHX and the evidence for CHX resistance in oral bacteria. Furthermore, this work aims to raise awareness among the dental community about the risk of resistance toward CHX and accompanying cross-resistance to antibiotics. We propose new research directions related to the effects of CHX on bacteria in oral biofilms

Slippery Liquid-Like Solid Surfaces with Promising Antibiofilm Performance in both Static and Flow Conditions

[Image: see text] Biofilms are central to some of the most urgent global challenges across diverse fields of application, from medicine to industries to the environment, and exert considerable economic and social impact. A fundamental assumption in anti-biofilms has been that the coating on a substrate surface is solid. The invention of slippery liquid-infused porous surfaces—a continuously wet lubricating coating retained on a solid surface by capillary forces—has led to this being challenged. However, in situations where flow occurs, shear stress may deplete the lubricant and affect the anti-biofilm performance. Here, we report on the use of slippery omniphobic covalently attached liquid (SOCAL) surfaces, which provide a surface coating with short (ca. 4 nm) non-cross-linked polydimethylsiloxane (PDMS) chains retaining liquid–surface properties, as an antibiofilm strategy stable under shear stress from flow. This surface reduced biofilm formation of the key biofilm-forming pathogens Staphylococcus epidermidis and Pseudomonas aeruginosa by three–four orders of magnitude compared to the widely used medical implant material PDMS after 7 days under static and dynamic culture conditions. Throughout the entire dynamic culture period of P. aeruginosa, SOCAL significantly outperformed a typical antibiofilm slippery surface [i.e., swollen PDMS in silicone oil (S-PDMS)]. We have revealed that significant oil loss occurred after 2–7 day flow for S-PDMS, which correlated to increased contact angle hysteresis (CAH), indicating a degradation of the slippery surface properties, and biofilm formation, while SOCAL has stable CAH and sustainable antibiofilm performance after 7 day flow. The significance of this correlation is to provide a useful easy-to-measure physical parameter as an indicator for long-term antibiofilm performance. This biofilm-resistant liquid-like solid surface offers a new antibiofilm strategy for applications in medical devices and other areas where biofilm development is problematic

Multiple adhesin proteins on the cell surface of Streptococcus gordonii are involved in adhesion to human fibronectin

Adhesion of bacterial cells to fibronectin (FN) is thought to be a pivotal step in the pathogenesis of invasive infectious diseases. Viridans group streptococci such as Streptococcus gordonii are considered commensal members of the oral microflora, but are important pathogens in infective endocarditis. S. gordonii expresses a battery of cell-surface adhesins that act alone or in concert to bind host receptors. Here, we employed molecular genetic approaches to determine the relative contributions of five known S. gordonii surface proteins to adherence to human FN. Binding levels to FN by isogenic mutants lacking Hsa glycoprotein were reduced by 70 %, while mutants lacking CshA and CshB fibrillar proteins showed approximately 30 % reduced binding. By contrast, disruption of antigen I/II adhesin genes sspA and sspB in a wild-type background did not result in reduced FN binding. Enzymic removal of sialic acids from FN led to reduced S. gordonii DL1 adhesion (>50 %), but did not affect binding by the hsa mutant, indicating that Hsa interacts with sialic acid moieties on FN. Conversely, desialylation of FN did not affect adherence levels of Lactococcus lactis cells expressing SspA or SspB polypeptides. Complementation of the hsa mutant partially restored adhesion to FN. A model is proposed for FN binding by S. gordonii in which Hsa and CshA/CshB are primary adhesins, and SspA or SspB play secondary roles. Understanding the basis of oral streptococcal interactions with FN will provide a foundation for development of new strategies to control infective endocarditis

Antiwetting and Antifouling Performances of Different Lubricant-Infused Slippery Surfaces

The concept of slippery lubricant-infused surfaces has shown promising potential in antifouling for controlling detrimental biofilm growth. In this study, nontoxic silicone oil was either impregnated into porous surface nanostructures, referred to as liquid-infused surfaces (LIS), or diffused into a polydimethylsiloxane (PDMS) matrix, referred to as a swollen PDMS (S-PDMS), making two kinds of slippery surfaces. The slippery lubricant layers have extremely low contact angle hysteresis, and both slippery surfaces showed superior antiwetting performances with droplets bouncing off or rolling transiently after impacting the surfaces. We further demonstrated that water droplets can remove dust from the slippery surfaces, thus showing a “cleaning effect”. Moreover, “coffee-ring” effects were inhibited on these slippery surfaces after droplet evaporation, and deposits could be easily removed. The clinically biofilm-forming species P. aeruginosa (as a model system) was used to further evaluate the antifouling potential of the slippery surfaces. The dried biofilm stains could still be easily removed from the slippery surfaces. Additionally, both slippery surfaces prevented around 90% of bacterial biofilm growth after 6 days compared to the unmodified control PDMS surfaces. This investigation also extended across another clinical pathogen, S. epidermidis, and showed similar results. The antiwetting and antifouling analysis in this study will facilitate the development of more efficient slippery platforms for controlling biofouling

Interspecies competition in oral biofilms mediated by Streptococcus gordonii extracellular deoxyribonuclease SsnA

Abstract Extracellular DNA (eDNA) is a key component of many microbial biofilms including dental plaque. However, the roles of extracellular deoxyribonuclease (DNase) enzymes within biofilms are poorly understood. Streptococcus gordonii is a pioneer colonizer of dental plaque. Here, we identified and characterised SsnA, a cell wall-associated protein responsible for extracellular DNase activity of S. gordonii. The SsnA-mediated extracellular DNase activity of S. gordonii was suppressed following growth in sugars. SsnA was purified as a recombinant protein and shown to be inactive below pH 6.5. SsnA inhibited biofilm formation by Streptococcus mutans in a pH-dependent manner. Further, SsnA inhibited the growth of oral microcosm biofilms in human saliva. However, inhibition was ameliorated by the addition of sucrose. Together, these data indicate that S. gordonii SsnA plays a key role in interspecies competition within oral biofilms. Acidification of the medium through sugar catabolism could be a strategy for cariogenic species such as S. mutans to prevent SsnA-mediated exclusion from biofilms

Biofilms in the potable water distribution network

The roles of vegetative dormancy and attachment to surfaces in the survival and growth of bacteria in potable water systems were investigated. Species present in the water were identified following isolation or direct observation of static batch enrichment cultures. Using the latter approach, many prosthecate and other stalked bacteria were found. Prosthecate bacteria undergo bi- or poly-phasic life cycles involving asymmetric division to produce reproductive cells and dormant swarmer cells and their presence in tap water supports the theory that vegetative dormancy is an important survival mechanism in this environment. A continuous flow model was established to analyse the metabolic activity of planktonic and attached bacteria in potable water. A physiological dye, 5-cyano-2,3-ditolyl tetrazolium chloride (CTC), was shown to stain active cells specifically in batch cultures of a Sphingomonas sp. and Caulobacter crescentus. Unsuccessful attempts were made to identify cellular proteins of Sphingomonas sp. cells that were specific to the attached or planktonic phenotype. By comparing the total bacterial counts in potable water with the total viable counts it was shown that a large proportion of the microflora was not cultivable on heterotrophic media. However, a proportion of these cells became culturable following enrichment with peptone. After ceasing the exogenous nutrient addition cellular aggregation occurred, presumably reflecting physiological changes in response to nutrient depletion. No clear trend in the activity of attached cells during biofilm development was detected. However, firmly attached cells were buffered against changes in the chemistry of the water. Growth within biofilms and release into the water column elevated the concentration of bacteria in the water. Attached cells were resistant to 0.3 mg free chlorine 1-1 added for 3 hrs, although this did weaken the architecture of the biofilm. Long term biofilms (one year-old) were almost devoid of bacteria - an observation that could not be adequately explained