Role of the Clp system in stress tolerance, biofilm formation, and intracellular invasion in Porphyromonas gingivalis

Clp proteases and chaperones are ubiquitous among prokaryotes and eukaryotes, and in many pathogenic bacteria the Clp stress response system is also involved in regulation of virulence properties. In this study, the roles of ClpB, ClpC, and ClpXP in stress resistance, homotypic and heterotypic biofilm formation, and intracellular invasion in the oral opportunistic pathogen Porphyromonas gingivalis were investigated. Absence of ClpC and ClpXP, but not ClpB, resulted in diminished tolerance to high temperatures. Response to oxidative stress was not affected by the loss of any of the Clp proteins. The clpC and clpXP mutants demonstrated elevated monospecies biofilm formation, and the absence of ClpXP also enhanced heterotypic P. gingivalis-Streptococcus gordonii biofilm formation. All clp mutants adhered to gingival epithelial cells to the same level as the wild type; however, ClpC and ClpXP were found to be necessary for entry into host epithelial cells. ClpB did not play a role in entry but was required for intracellular replication and survival. ClpXP negatively regulated the surface exposure of the minor fimbrial (Mfa) protein subunit of P. gingivalis, which stimulates biofilm formation but interferes with epithelial cell entry. Collectively, these results show that the Clp protease complex and chaperones control several processes that are important for the colonization and survival of P. gingivalis in the oral cavity.     

Oral Community Interactions of Filifactor alocis In Vitro

Filifactor alocis is a gram positive anaerobe that is emerging as an important periodontal pathogen. In the oral cavity F. alocis colonizes polymicrobial biofilm communities; however, little is known regarding the nature of the interactions between F. alocis and other oral biofilm bacteria. Here we investigate the community interactions of two strains of F. alocis with Streptococcus gordonii, Fusobacterium nucleatum, Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans, organisms with differing pathogenic potential in the oral cavity. In an in vitro community development model, S. gordonii was antagonistic to the accumulation of F. alocis into a dual species community. In contrast, F. nucleatum and the type strain of F. alocis formed a synergistic partnership. Accumulation of a low passage isolate of F. alocis was also enhanced by F. nucleatum. In three species communities of S. gordonii, F. nucleatum and F. alocis, the antagonistic effects of S. gordonii superseded the synergistic effects of F. nucleatum toward F. alocis. The interaction between A. actinomycetemcomitans and F. alocis was strain specific and A. actinomycetemcomitans could either stimulate F. alocis accumulation or have no effect depending on the strain. P. gingivalis and F. alocis formed heterotypic communities with the amount of P. gingivalis greater than in the absence of F. alocis. However, while P. gingivalis benefited from the relationship, levels of F. alocis in the dual species community were lower compared to F. alocis alone. The inhibitory effect of P. gingivalis toward F. alocis was dependent, at least partially, on the presence of the Mfa1 fimbrial subunit. In addition, AI-2 production by P. gingivalis helped maintain levels of F. alocis. Collectively, these results show that the pattern of F. alocis colonization will be dictated by the spatial composition of microbial microenvironments, and that the organism may preferentially accumulate at sites rich in F. nucleatum.     

Regulation of olfactomedin 4 by Porphyromonas gingivalis in a community context

At mucosal barriers, the virulence of microbial communities reflects the outcome of both dysbiotic and eubiotic interactions with the host, with commensal species mitigating or potentiating the action of pathogens. We examined epithelial responses to the oral pathogen Porphyromonas gingivalis as a monoinfection and in association with a community partner, Streptococcus gordonii. RNA-Seq of oral epithelial cells showed that the Notch signaling pathway, including the downstream effector olfactomedin 4 (OLFM4), was differentially regulated by P. gingivalis alone; however, regulation was overridden by S. gordonii. OLFM4 was required for epithelial cell migratory, proliferative and inflammatory responses to P. gingivalis. Activation of Notch signaling was induced through increased expression of the Notch1 receptor and the Jagged1 (Jag1) agonist. In addition, Jag1 was released in response to P. gingivalis, leading to paracrine activation. Following Jag1-Notch1 engagement, the Notch1 extracellular domain was cleaved by P. gingivalis gingipain proteases. Antagonism by S. gordonii involved inhibition of gingipain activity by secreted hydrogen peroxide. The results establish a novel mechanism by which P. gingivalis modulates epithelial cell function which is dependent on community context. These interrelationships have relevance for innate inflammatory responses and epithelial cell fate decisions in oral health and disease.Subject terms: Microbial ecology, Microbial ecology     

Interaction between Streptococcus spp. and Veillonella tobetsuensis in the Early Stages of Oral Biofilm Formation

Dental plaque is a multispecies oral biofilm, the development of which is initiated by adherence of the pioneer Streptococcus spp. Oral Veillonella spp., including V. atypica, V. denticariosi, V. dispar, V. parvula, V. rogosae, and V. tobetsuensis, are known as early colonizers in oral biofilm formation. These species have been reported to coaggregate with Streptococcus spp. in a metabolic cooperation-dependent manner to form biofilms in human oral cavities, especially in the early stages of biofilm formation. However, in our previous study, Streptococcus gordonii showed biofilm formation to the greatest extent in the presence of V. tobetsuensis, without coaggregation between species. These results suggest that V. tobetsuensis produces signaling molecules that promote the proliferation of S. gordonii in biofilm formation. It is well known in many bacterial species that the quorum-sensing (QS) system regulates diverse functions such as biofilm formation. However, little is known about the QS system with autoinducers (AIs) with respect to Veillonella and Streptococcus spp. Recently, autoinducer 1 (AI-1) and AI-2 were detected and identified in the culture supernatants of V. tobetsuensis as strong signaling molecules in biofilm formation with S. gordonii. In particular, the supernatant from V. tobetsuensis showed the highest AI-2 activity among 6 oral Veillonella species, indicating that AIs, mainly AI-2, produced by V. tobetsuensis may be important factors and may facilitate biofilm formation of S. gordonii. Clarifying the mechanism that underlies the QS system between S. gordonii and V. tobetsuensis may lead to the development of novel methods for the prevention of oral infectious diseases caused by oral biofilms.     

Mutualistic Biofilm Communities Develop with Porphyromonas gingivalis and Initial,Early, and Late Colonizers of Enamel

Porphyromonas gingivalis is present in dental plaque as early as 4 h after tooth cleaning, but it is also associated with periodontal disease, a late-developing event in the microbial successions that characterize daily plaque development. We report here that P. gingivalis ATCC 33277 is remarkable in its ability to interact with a variety of initial, early, middle, and late colonizers growing solely on saliva. Integration of P. gingivalis into multispecies communities was investigated by using two in vitro biofilm models. In flow cells, bacterial growth was quantified using fluorescently conjugated antibodies against each species, and static biofilm growth on saliva-submerged polystyrene pegs was analyzed by quantitative real-time PCR using species-specific primers. P. gingivalis could not grow as a single species or together with initial colonizer Streptococcus oralis but showed mutualistic growth when paired with two other initial colonizers, Streptococcus gordonii and Actinomyces oris, as well as with Veillonella sp. (early colonizer), Fusobacterium nucleatum (middle colonizer), and Aggregatibacter actinomycetemcomitans (late colonizer). In three-species flow cells, P. gingivalis grew with Veillonella sp. and A. actinomycetemcomitans but not with S. oralis and A. actinomycetemcomitans. Also, it grew with Veillonella sp. and F. nucleatum but not with S. oralis and F. nucleatum, indicating that P. gingivalis and S. oralis are not compatible. However, P. gingivalis grew in combination with S. gordonii and S. oralis, demonstrating its ability to overcome the incompatibility when cultured with a second initially colonizing species. Collectively, these data help explain the observed presence of P. gingivalis at all stages of dental plaque development.Removal of dental plaque by routine oral hygiene procedures is followed by a repetition of a species succession that starts with initially colonizing streptococci and actinomyces (5, 16). Other species follow as early, middle, and late colonizers, which establishes the following developmental process: successive attachment of saliva-suspended species to already attached bacteria and formation of multispecies communities.Attachment is a critical event essential to preventing the bacteria from being swallowed by salivary flow. Initial colonizers bind to host-derived receptors in the salivary pellicle coating of the tooth enamel. The remainder of typical plaque development occurs by accretion of saliva-suspended species and growth of attached bacteria, thereby increasing the microbial diversity. Adherence of suspended single cells to attached cells is called coadhesion (1). Some suspended cells are already coaggregated and adhere to attached cells as coaggregates; coaggregation is defined as the specific cell-to-cell recognition and adherence of genetically distinct cell types (8). All human oral bacterial species exhibit coaggregation. For example, Streptococcus oralis coaggregates with Streptococcus gordonii (intrageneric coaggregation). Both species pair with Actinomyces oris (intergeneric coaggregation), and all of them coaggregate with Fusobacterium nucleatum (multigeneric coaggregation). Multispecies communities composed of coaggregating species characterize dental plaque biofilms in vivo (3, 17, 18).To increase our understanding of interactions among species, we have employed two in vitro model systems and are testing numerous combinations of seven species for their ability to grow on saliva as their sole nutritional source (20, 21). First, we reported that F. nucleatum (middle colonizer) failed to grow when paired with S. oralis but grew well when A. oris was included in the three-species biofilm (20), indicating specificity by F. nucleatum for the presence of a particular initial colonizer. Recently, we showed that Aggregatibacter actinomycetemcomitans (late colonizer and periodontopathogen) exhibited mutualistic relationships with F. nucleatum and Veillonella sp. (early colonizer and commensal organism), illustrating the ability of commensals and pathogens to grow together (21).Porphyromonas gingivalis, another periodontopathogen, forms three-species communities with F. nucleatum and S. gordonii (11). Proteomics of P. gingivalis in this three-species community revealed a broad increase in proteins involved in protein synthesis, suggesting that a multispecies relationship is advantageous for the porphyromonad (11). This research group had previously reported the presence of differentially regulated porphyromonad genes when P. gingivalis and S. gordonii were together in biofilms (22). Thus, P. gingivalis responds to the presence of other oral species.P. gingivalis is detected in dental plaque samples within 6 h after professional tooth cleaning (5, 13), and its numbers increase in periodontally diseased sites (15). It forms biofilms with S. gordonii but not with Streptococcus mutans (12) or Streptococcus cristatus (23). P. gingivalis required a preformed streptococcal substratum for its incorporation into a biofilm (12). Partner specificity was also noted among four fresh isolates of P. gingivalis, which showed no coaggregation with a variety of oral actinomyces, aggregatibacteria, capnocytophagae, and streptococci (9) but coaggregated with F. nucleatum (7, 10). We show here that P. gingivalis exhibits widespread mutualism with initial, early, middle, and late colonizers but also shows specificity with initially colonizing streptococci, which could help explain its early appearance in the development of dental plaque biofilms. The relationship of porphyromonads with initial, early, middle, and later colonizers during biofilm growth on saliva as a sole nutritional source has not been explored previously. We hypothesize that the ability of P. gingivalis to coaggregate with S. gordonii and A. oris (initial colonizers), Veillonella sp. (early colonizer), F. nucleatum (middle colonizer), and A. actinomycetemcomitans (late colonizer) allows these bacteria to form multispecies biofilm communities.     

Antimicrobial Effects of Peptides from Human Beta-Defensin-3 on Planktonic and Biofilm States of Streptococci

Human beta-defensin-3 (hBD3) acts as a first line of defense against both Gram-positive and Gram-negative bacteria infection. Streptococci are the significant cause for oral biofilm associated diseases. We synthesized three fragments (hBD3-1, hBD3-2, hBD3-3) from the hBD3 and evaluated the antibacterial efficacy on oral streptococci. All of the three fragments from hBD3 had good estimated solubility and hBD3-3 had a higher net positive charge than others. Structure analysis showed that the three fragments shared stable β-sheet structure, but tyrosine were not found in hBD3-2 and hBD3-3 by using Raman and circular dichroism spectroscopy. The inhibition ability of the peptides was examined on the bioactivity of Streptococcus oralis (S.oralis), Streptococcus sanguinis (S. sanguinis) and Streptococcus gordonii (S. gordonii) by minimal inhibitory concentration, minimum bactericidal concentration and anti-biofilm formation test. Three fragments had antimicrobial activity on planktonic state of streptococci, and S. oralis had much more sensitive to the three peptides. Results of antibiofilm experiment showed that streptococci biofilm formation was more sensitive to hBD3-3. Confocal laser scanning microscopy and scanning electron microscopy showed the decrease of biomass and bacterial morphology destruction, which indicated that the antimicrobial mechanism of hBD3-3 might involve an electrostatic charge-based impact on membrane permeability. In conclusion, hBD3-3 possessed the potential capacity for depressing the growth of bacteria, especially first colonizers during the development of oral biofilm. Powerful, endogenous antimicrobial peptide provides the potential to interfere with biofilm by disorganizing early biofilm formation and thereby inhibiting biofilm-associated diseases.     

LuxS-Based Signaling Affects Streptococcus mutans Biofilm Formation

Streptococcus mutans is implicated as a major etiological agent in human dental caries, and one of the important virulence properties of this organism is its ability to form biofilms (dental plaque) on tooth surfaces. We examined the role of autoinducer-2 (AI-2) on S. mutans biofilm formation by constructing a GS-5 luxS-null mutant. Biofilm formation by the luxS mutant in 0.5% sucrose defined medium was found to be markedly attenuated compared to the wild type. Scanning electron microscopy also revealed that biofilms of the luxS mutant formed larger clumps in sucrose medium compared to the parental strain. Therefore, the expression of glucosyltransferase genes was examined and the gtfB and gtfC genes, but not the gtfD gene, in the luxS mutant were upregulated in the mid-log growth phase. Furthermore, we developed a novel two-compartment system to monitor AI-2 production by oral streptococci and periodontopathic bacteria. The biofilm defect of the luxS mutant was complemented by strains of S. gordonii, S. sobrinus, and S. anginosus; however, it was not complemented by S. oralis, S. salivarius, or S. sanguinis. Biofilm formation by the luxS mutant was also complemented by Porphyromonas gingivalis 381 and Actinobacillus actinomycetemcomitans Y4 but not by a P. gingivalis luxS mutant. These results suggest that the regulation of the glucosyltransferase genes required for sucrose-dependent biofilm formation is regulated by AI-2. Furthermore, these results provide further confirmation of previous proposals that quorum sensing via AI-2 may play a significant role in oral biofilm formation.     

Porphyromonas gingivalis and Treponema denticola Synergistic Polymicrobial Biofilm Development

Chronic periodontitis has a polymicrobial biofilm aetiology and interactions between key bacterial species are strongly implicated as contributing to disease progression. Porphyromonas gingivalis, Treponema denticola and Tannerella forsythia have all been implicated as playing roles in disease progression. P. gingivalis cell-surface-located protease/adhesins, the gingipains, have been suggested to be involved in its interactions with several other bacterial species. The aims of this study were to determine polymicrobial biofilm formation by P. gingivalis, T. denticola and T. forsythia, as well as the role of P. gingivalis gingipains in biofilm formation by using a gingipain null triple mutant. To determine homotypic and polymicrobial biofilm formation a flow cell system was employed and the biofilms imaged and quantified by fluorescent in situ hybridization using DNA species-specific probes and confocal scanning laser microscopy imaging. Of the three species, only P. gingivalis and T. denticola formed mature, homotypic biofilms, and a strong synergy was observed between P. gingivalis and T. denticola in polymicrobial biofilm formation. This synergy was demonstrated by significant increases in biovolume, average biofilm thickness and maximum biofilm thickness of both species. In addition there was a morphological change of T. denticola in polymicrobial biofilms when compared with homotypic biofilms, suggesting reduced motility in homotypic biofilms. P. gingivalis gingipains were shown to play an essential role in synergistic polymicrobial biofilm formation with T. denticola.     

Antibacterial effect of bestatin during periodontitis

Periodontitis is a polymicrobial disease inciting inflammatory destruction of the tooth-supporting tissues, i.e., periodontium. The initiation of this infectious disease is ascribed to the formation of subgingival biofilms. These biofilms cause stimulation of myriad of chronic inflammatory reactions by the affected tissue. The Gram-negative anaerobe Porphyromonas gingivalis is commonly found as part of the microbiota of subgingival biofilms, and is involved in the occurrence of the disease. P. gingivalis possesses numerous virulence factors supporting its survival, regulating its communication with other species in the biofilm, degrading host tissues. Fusobacterium nucleatum is pivotal for formation of biofilm and promotes growth and invasion properties of P. gingivalis. Bestatin is an aminopeptide inhibitor, produced by actinomycetes. It possesses antibacterial properties against P. gingivalis and F. nucleatum. The following review focuses on action of bestatin on the mentioned bacteria.     

Strain-specific colonization patterns and serum modulation of multi-species oral biofilm development

Periodontitis results from an ecological shift in the composition of subgingival biofilms. Subgingival community maturation is modulated by inter-organismal interactions and the relationship of communities with the host. In an effort to better understand this process, we evaluated biofilm formation, with oral commensal species, by three strains of the subgingivally prevalent microorganism Fusobacterium nucleatum and four strains of the periodontopathogen Porphyromonas gingivalis. We also tested the effect of serum, which resembles gingival exudates, on subgingival biofilms. Biofilms were allowed to develop in flow cells using salivary medium. We found that although not all strains of F. nucleatum were able to grow in mono-species biofilms, forming a community with health-associated partners Actinomyces oris and Veillonella parvula promoted biofilm growth of all F. nucleatum strains. Strains of P. gingivalis also showed variable ability to form mono-species biofilms. P. gingivalis W50 and W83 did not form biofilms, while ATCC 33277 and 381 formed biofilm structures, but only strain ATCC 33277 grew over time. Unlike the enhanced growth of F. nucleatum with the two health-associated species, no strain of P. gingivalis grew in three-species communities with A. oris and V. parvula. However, addition of F. nucleatum facilitated growth of P. gingivalis ATCC 33277 with health-associated partners. Importantly, serum negatively affected the adhesion of F. nucleatum, while it favored biofilm growth by P. gingivalis. This work highlights strain specificity in subgingival biofilm formation. Environmental factors such as serum alter the colonization patterns of oral microorganisms and could impact subgingival biofilms by selectively promoting pathogenic species.     

Setup of an In Vitro Test System for Basic Studies on Biofilm Behavior of Mixed-Species Cultures with Dental and Periodontal Pathogens

Background

Caries and periodontitis are important human diseases associated with formation of multi-species biofilms. The involved bacteria are intensively studied to understand the molecular basis of the interactions in such biofilms. This study established a basic in vitro single and mixed-species culture model for oral bacteria combining three complimentary methods. The setup allows a rapid screening for effects in the mutual species interaction. Furthermore, it is easy to handle, inexpensive, and reproducible.

Methods

Streptococcus mitis, S. salivarius and S. sanguinis, typical inhabitants of the healthy oral cavity, S. mutans as main carriogenic species, and Porphyromonas gingivalis, Fusobacterium nucleatum, Parvimonas micra, S. intermedius and Aggregatibacter actinomycetemcomitans as periodontitis-associated bacteria, were investigated for their biofilm forming ability. Different liquid growth media were evaluated. Safranin-staining allowed monitoring of biofilm formation under the chosen conditions. Viable counts and microscopy permitted investigation of biofilm behavior in mixed-species and transwell setups.

Findings

S. mitis, F. nucleatum, P. gingivalis and P. micra failed to form biofilm structures. S. mutans, S. sanguinis, S. intermedius and S. salivarius established abundant biofilm masses in CDM/sucrose. A. actinomycetemcomitans formed patchy monolayers. For in depth analysis S. mitis, S. mutans and A. actinomycetemcomitans were chosen, because i) they are representatives of the physiological-, cariogenic and periodontitis-associated bacterial flora, respectively and ii) their difference in their biofilm forming ability. Microscopic analysis confirmed the results of safranin staining. Investigation of two species combinations of S. mitis with either S. mutans or A. actinomycetemcomitans revealed bacterial interactions influencing biofilm mass, biofilm structure and cell viability.

Conclusions

This setup shows safranin staining, microscopic analysis and viable counts together are crucial for basic examination and evaluation of biofilms. Our experiment generated meaningful results, exemplified by the noted S. mitis influence, and allows a fast decision about the most important bacterial interactions which should be investigated in depth.     

Reduced Glutathione Mediates Resistance to H2S Toxicity in Oral Streptococci

Periodontal disease is associated with changes in the composition of the oral microflora, where health-associated oral streptococci decrease while Gram-negative anaerobes predominate in disease. A key feature of periodontal disease-associated anaerobes is their ability to produce hydrogen sulfide (H₂S) abundantly as a by-product of anaerobic metabolism. So far, H₂S has been reported to be either cytoprotective or cytotoxic by modulating bacterial antioxidant defense systems. Although oral anaerobes produce large amounts of H₂S, the potential effects of H₂S on oral streptococci are currently unknown. The aim of this study was to determine the effects of H₂S on the survival and biofilm formation of oral streptococci. The growth and biofilm formation of Streptococcus mitis and Streptococcus oralis were inhibited by H₂S. However, H₂S did not significantly affect the growth of Streptococcus gordonii or Streptococcus sanguinis. The differential susceptibility of oral streptococci to H₂S was attributed to differences in the intracellular concentrations of reduced glutathione (GSH). In the absence of GSH, H₂S elicited its toxicity through an iron-dependent mechanism. Collectively, our results showed that H₂S exerts antimicrobial effects on certain oral streptococci, potentially contributing to the decrease in health-associated plaque microflora.     

Proteomics of Porphyromonas gingivalis within a model oral microbial community

Background  

Porphyromonas gingivalis is a periodontal pathogen that resides in a complex multispecies microbial biofilm community known as dental plaque. Confocal laser scanning microscopy showed that P. gingivalis can assemble into communities in vitro with Streptococcus gordonii and Fusobacterium nucleatum, common constituents of dental plaque. Whole cell quantitative proteomics, along with mutant construction and analysis, were conducted to investigate how P. gingivalis adapts to this three species community.     

The synthetic human beta-defensin-3 C15 peptide exhibits antimicrobial activity against <Emphasis Type="Italic">Streptococcus mutans</Emphasis>, both alone and in combination with dental disinfectants

Streptococcus mutans is a major etiologic agent of human dental caries that forms biofilms on hard tissues in the human oral cavity, such as tooth and dentinal surfaces. Human β-defensin-3 (HBD3) is a 45-amino-acid natural antimicrobial peptide that has broad spectrum antimicrobial activity against bacteria and fungi. A synthetic peptide consisting of the C-terminal 15 amino acids of HBD3 (HBD3-C15) was recently shown to be sufficient for its antimicrobial activity. Thus, clinical applications of this peptide have garnered attention. In this study, we investigated whether HBD3-C15 inhibits the growth of the representative cariogenic pathogen Streptococcus mutans and its biofilm formation. HBD3-C15 inhibited bacterial growth, exhibited bactericidal activity, and attenuated bacterial biofilm formation in a dose-dependent manner. HBD3-C15 potentiated the bactericidal and anti-biofilm activity of calcium hydroxide (CH) and chlorhexidine digluconate (CHX), which are representative disinfectants used in dental clinics, against S. mutans. Moreover, HBD3-C15 showed antimicrobial activity by inhibiting biofilm formation by S. mutans and other dentinophilic bacteria such as Enterococcus faecalis and Streptococcus gordonii, which are associated with dental caries and endodontic infection, on human dentin slices. These effects were observed for HBD3-C15 alone and for HBD3-C15 in combination with CH or CHX. Therefore, we suggest that HBD3-C15 is a potential alternative or additive disinfectant that can be used for the treatment of oral infectious diseases, including dental caries and endodontic infections.     

Expression of Green Fluorescent Protein in Streptococcus gordonii DL1 and Its Use as a Species-Specific Marker in Coadhesion with Streptococcus oralis 34 in Saliva-Conditioned Biofilms In Vitro

Streptococcus gordonii is one of the predominant streptococci in the biofilm ecology of the oral cavity. It interacts with other bacteria through receptor-adhesin complexes formed between cognate molecules on the surfaces of the partner cells. To study the spatial organization of S. gordonii DL1 in oral biofilms, we used green fluorescent protein (GFP) as a species-specific marker to identify S. gordonii in a two-species in vitro oral biofilm flowcell system. To drive expression of gfp, we isolated and characterized an endogenous S. gordonii promoter, PhppA, which is situated upstream of the chromosomal hppA gene encoding an oligopeptide-binding lipoprotein. A chromosomal chloramphenicol acetyltransferase (cat) gene fusion with PhppA was constructed and used to demonstrate that PhppA was highly active throughout the growth of bacteria in batch culture. A promoterless 0.8-kb gfp (′gfp) cassette was PCR amplified from pBJ169 and subcloned to replace the cat cassette downstream of the S. gordonii-derived PhppA in pMH109-HPP, generating pMA1. Subsequently, the PhppA-′gfp cassette was PCR amplified from pMA1 and subcloned into pDL277 and pVA838 to generate the Escherichia coli-S. gordonii shuttle vectors pMA2 and pMA3, respectively. Each vector was transformed into S. gordonii DL1 aerobically to ensure GFP expression. Flow cytometric analyses of aerobically grown transformant cultures were performed over a 24-h period, and results showed that GFP could be successfully expressed in S. gordonii DL1 from PhppA and that S. gordonii DL1 transformed with the PhppA-′gfp fusion plasmid stably maintained the fluorescent phenotype. Fluorescent S. gordonii DL1 transformants were used to elucidate the spatial arrangement of S. gordonii DL1 alone in biofilms or with the coadhesion partner Streptococcus oralis 34 in two-species biofilms in a saliva-conditioned in vitro flowcell system. These results show for the first time that GFP expression in oral streptococci can be used as a species-specific marker in model oral biofilms.     

Antimicrobial and antibiofilm activity of polyurethane/Hypericum perforatum extract (PHPE) composite

Microbial accumulation in materials used in sectors such as medical, textile and food can lead to serious diseases, infections and uncontrollable problems. Many of the materials used in the above-mentioned industries have highly sensitive surfaces for microorganisms and cause colonization and biofilm formation. Colonization and biofilm formation threaten human health and they cause many diseases that result in death every year. Antimicrobial materials have an important role in combating pathogens. This article is about a new material with antibiofilm and antimicrobial properties combining polyurethane and Hypericum perforatum extract (PHPE) together. Antimicrobial effect of H. perforatum extract was determined against three clinical pathogens; C. albicans, E. coli and S. aureus. The highest antimicrobial activity of H. perforatum extract was found against S. aureus strain. Antibiofilm analysis results revealed that H. perforatum was also inhibited by the biofilm formation of S. aureus by 56.85%. The combination of polyurethane material and H. perforatum extract (PHPE) resulted in 92.85% decrease in S. aureus biofilm compared to control group. The reduction of S. aureus after H. perforatum incorporation was revealed by Scanning Electron Microscopy (SEM) study. The results show that the polyurethane material combined with H. perforatum extract inhibits the formation of S. aureus biofilm.