Antibacterial effects of nanopillar surfaces are mediated by cell impedance, penetration and induction of oxidative stress.

Some insects, such as dragonflies, have evolved nanoprotrusions on their wings that rupture bacteria on contact. This has inspired the design of antibacterial implant surfaces with insect-wing mimetic nanopillars made of synthetic materials. Here, we characterise the physiological and morphological effects of mimetic titanium nanopillars on bacteria. The nanopillars induce deformation and penetration of the Gram-positive and Gram-negative bacterial cell envelope, but do not rupture or lyse bacteria. They can also inhibit bacterial cell division, and trigger production of reactive oxygen species and increased abundance of oxidative stress proteins. Our results indicate that nanopillars' antibacterial activities may be mediated by oxidative stress, and do not necessarily require bacterial lysis.

Studies of Black Diamond as an antibacterial surface for Gram Negative bacteria: the interplay between chemical and mechanical bactericidal activity.

'Black silicon' (bSi) samples with surfaces covered in nanoneedles of length ~5 µm were fabricated using a plasma etching process and then coated with a conformal uniform layer of diamond using hot filament chemical vapour deposition to produce 'black diamond' (bD) nanostructures. The diamond needles were then chemically terminated with H, O, NH2 or F using plasma treatment, and the hydrophilicity of the resulting surfaces were assessed using water droplet contact-angle measurements, and scaled in the order O > H ≈NH2 >F, with the F-terminated surface being superhydrophobic. The effectiveness of these differently terminated bD needles in killing the Gram-negative bacterium E. coli was semi-quantified by Live/Dead staining and fluorescence microscopy, and visualised by environmental scanning electron microscopy. The total number of adhered bacteria was consistent for all the nanostructured bD surfaces at around 50% of the value for the flat diamond control. This, combined with a chemical bactericidal effect of 20-30%, shows that the nanostructured bD surfaces supported significantly fewer viable E. coli than flat surfaces. Moreover, the bD surfaces were particularly effective at preventing the establishment of bacterial aggregates - a precursor to biofilm formation. The percentage of dead bacteria also decreased as a function of hydrophilicity. These results are consistent with a predominantly mechanical mechanism for bacteria death based on the stretching and disruption of the cell membrane, combined with an additional effect from the chemical nature of the surface.

Studies of black silicon and black diamond as materials for antibacterial surfaces.

'Black silicon' (bSi) samples with surfaces covered in nanoneedles of varying length, areal density and sharpness, have been fabricated using a plasma etching process. These nanostructures were then coated with a conformal uniform layer of diamond using hot filament chemical vapour deposition to produce 'black diamond' (bD) surfaces. The effectiveness of these bSi and bD surfaces in killing Gram-negative (E. coli) and Gram-positive (S. gordonii) bacteria was investigated by culturing the bacteria on the surfaces for a set time and then measuring the live-to-dead ratio. All the nanostructured surfaces killed E. coli at a significantly higher rate than the respective flat Si or diamond control samples. The length of the needles was found to be less important than their separation, i.e. areal density. This is consistent with a model for mechanical bacteria death based on the stretching and disruption of the cell membrane, enhanced by the cells motility on the surfaces. In contrast, S. gordonii were unaffected by the nanostructured surfaces, possibly due to their smaller size, thicker cell membrane and/or their lack of motility.

Interspecies dynamics among bacteria associated with canine periodontal disease.

The etiology and pathogenic mechanisms associated with canine periodontal disease are less well understood than the disease in humans. In this study we have reconstructed defined consortia biofilms in vitro of microorganisms identified as prevalent in a same-breed cohort of dogs with or without periodontal disease. Frederiksenia canicola and Neisseria canis were selected as potential early colonizers of salivary pellicle, and Fusobacterium nucleatum and Porphyromonas gulae were included as high incidence canine oral bacteria. N. canis formed a biofilm substratum under aerobic conditions, but was unable to tolerate anaerobic conditions. Fr. canicola exhibited synergistic biofilm growth with Po. gulae under anaerobic conditions, but displayed an antagonistic relationship with Fu. nucleatum. However, strong co-adhesion between Fu. nucleatum and Po. gulae was able to overcome the inhibitory effects of Fr. canicola to facilitate three-species biofilm formation. Parvimonas micra, an anaerobic, asaccharolytic Gram-positive coccus found only under disease conditions in vivo, was able to form biofilms in conjunction with Fr. canicola and Po. gulae. Furthermore, the specific proteolytic activities of biofilms containing Fr. canicola and Po. gulae or Fu. nucleatum and Po. gulae were increased several-fold upon the addition of Pa. micra. This suggests that anaerobic cocci such as Pa. micra might provide a catalyst for progressive tissue destruction, inflammation and alveolar bone loss in canine periodontal disease, in keeping with the keystone-pathogen hypothesis.

Interactions between Streptococcus oralis, Actinomyces oris, and Candida albicans in the development of multispecies oral microbial biofilms on salivary pellicle.

The fungus Candida albicans is carried orally and causes a range of superficial infections that may become systemic. Oral bacteria Actinomyces oris and Streptococcus oralis are abundant in early dental plaque and on oral mucosa. The aims of this study were to determine the mechanisms by which S. oralis and A. oris interact with each other and with C. albicans in biofilm development. Spatial distribution of microorganisms was visualized by confocal laser scanning microscopy of biofilms labeled by differential fluorescence or by fluorescence in situ hybridization (FISH). Actinomyces oris and S. oralis formed robust dual-species biofilms, or three-species biofilms with C. albicans. The bacterial components tended to dominate the lower levels of the biofilms while C. albicans occupied the upper levels. Non-fimbriated A. oris was compromised in biofilm formation in the absence or presence of streptococci, but was incorporated into upper biofilm layers through binding to C. albicans. Biofilm growth and hyphal filament production by C. albicans was enhanced by S. oralis. It is suggested that the interkingdom biofilms are metabolically coordinated to house all three components, and this study demonstrates that adhesive interactions between them determine spatial distribution and biofilm architecture. The physical and chemical communication processes occurring in these communities potentially augment C. albicans persistence at multiple oral cavity sites.

Osteogenic and bactericidal surfaces from hydrothermal titania nanowires on titanium substrates.

Nanotopographical cues on Ti have been shown to elicit different cell responses such as cell differentiation and selective growth. Bone remodelling is a constant process requiring specific cues for optimal bone growth and implant fixation. Moreover, biofilm formation and the resulting infection on surgical implants is a major issue. Our aim is to identify nanopatterns on Ti surfaces that would be optimal for both bone remodelling and for reducing risk of bacterial infection. Primary human osteoblast/osteoclast co-cultures were seeded onto Ti substrates with TiO2 nanowires grown under alkaline conditions at 240 °C for different times (2, 2.5 or 3 h). Cell growth and behaviour was assessed by scanning electron microscopy (SEM), immunofluorescence microscopy, histochemistry and quantitative RT-PCR methods. Bacterial colonisation of the nanowire surfaces was also assessed by confocal microscopy and SEM. From the three surfaces tested the 2 h nanowire surface supported osteoblast and to a lesser extent osteoclast growth and differentiation. At the same time bacterial viability was reduced. Hence the 2 h surface provided optimal bone remodeling in vitro conditions while reducing infection risk, making it a favourable candidate for future implant surfaces.

Transcriptional landscape of trans-kingdom communication between Candida albicans and Streptococcus gordonii.

Recent studies have shown that the transcriptional landscape of the pleiomorphic fungus Candida albicans is highly dependent upon growth conditions. Here using a dual RNA-seq approach we identified 299 C. albicans and 72 Streptococcus gordonii genes that were either upregulated or downregulated specifically as a result of co-culturing these human oral cavity microorganisms. Seventy-five C. albicans genes involved in responses to chemical stimuli, regulation, homeostasis, protein modification and cell cycle were significantly (P ≤ 0.05) upregulated, whereas 36 genes mainly involved in transport and translation were downregulated. Upregulation of filamentation-associated TEC1 and FGR42 genes, and of ALS1 adhesin gene, concurred with previous evidence that the C. albicans yeast to hypha transition is promoted by S. gordonii. Increased expression of genes required for arginine biosynthesis in C. albicans was potentially indicative of a novel oxidative stress response. The transcriptional response of S. gordonii to C. albicans was less dramatic, with only eight S. gordonii genes significantly (P ≤ 0.05) upregulated at least two-fold (glpK, rplO, celB, rplN, rplB, rpsE, ciaR and gat). The expression patterns suggest that signals from S. gordonii cause a positive filamentation response in C. albicans, whereas S. gordonii appears to be transcriptionally less influenced by C. albicans.

Streptococcus gordonii DL1 adhesin SspB V-region mediates coaggregation via receptor polysaccharide of Actinomyces oris T14V.

Streptococcus gordonii SspA and SspB proteins, members of the antigen I/II (AgI/II) family of Streptococcus adhesins, mediate adherence to cysteine-rich scavenger glycoprotein gp340 and cells of other oral microbial species. In this article we investigated further the mechanism of coaggregation between S. gordonii DL1 and Actinomyces oris T14V. Previous mutational analysis of S. gordonii suggested that SspB was necessary for coaggregation with A. oris T14V. We have confirmed this by showing that Lactococcus lactis surrogate host cells expressing SspB coaggregated with A. oris T14V and PK606 cells, while L. lactis cells expressing SspA did not. Coaggregation occurred independently of expression of A. oris type 1 (FimP) or type 2 (FimA) fimbriae. Polysaccharide was prepared from cells of A. oris T14V and found to contain 1,4-, 4,6- and 3,4-linked glucose, 1,4-linked mannose, and 2,4-linked galactose residues. When immobilized onto plastic wells this polysaccharide supported binding of L. lactis expressing SspB, but not binding of L. lactis expressing other AgI/II family proteins. Purified recombinant NAVP region of SspB, comprising amino acid (aa) residues 41-847, bound A. oris polysaccharide but the C-domain (932-1470 aa residues) did not. A site-directed deletion of 29 aa residues (Δ691-718) close to the predicted binding cleft within the SspB V-region ablated binding of the NAVP region to polysaccharide. These results infer that the V-region head of SspB recognizes an actinomyces polysaccharide ligand, so further characterizing a lectin-like coaggregation mechanism occurring between two important primary colonizers.

Microbial interactions in building of communities.

Establishment of a community is considered to be essential for microbial growth and survival in the human oral cavity. Biofilm communities have increased resilience to physical forces, antimicrobial agents and nutritional variations. Specific cell-to-cell adherence processes, mediated by adhesin-receptor pairings on respective microbial surfaces, are able to direct community development. These interactions co-localize species in mutually beneficial relationships, such as streptococci, veillonellae, Porphyromonas gingivalis and Candida albicans. In transition from the planktonic mode of growth to a biofilm community, microorganisms undergo major transcriptional and proteomic changes. These occur in response to sensing of diffusible signals, such as autoinducer molecules, and to contact with host tissues or other microbial cells. Underpinning many of these processes are intracellular phosphorylation events that regulate a large number of microbial interactions relevant to community formation and development.

Stick to your gums: mechanisms of oral microbial adherence.

Studies on the adherence properties of oral bacteria have been a major focus in microbiology research for several decades. The ability of bacteria to adhere to the variety of surfaces present in the oral cavity, and to become integrated within the resident microbial communities, confers growth and survival properties. Molecular analyses have revealed several families of Gram-positive bacterial surface proteins, including serine-rich repeat, antigen I/II, and pilus families, that mediate adherence to a variety of salivary and oral bacterial receptors. In Gram-negative bacteria, pili, auto-transporters, and extracellular matrix-binding proteins provide components for host tissue recognition and building of complex microbial communities. Future studies will reveal in greater detail the binding pockets for these adhesin families and their receptors. This information will be crucial for the development of new inhibitors or vaccines that target the functional regions of bacterial proteins that are involved in colonization and pathogenesis.