Ratiometric Imaging of Extracellular pH in Bacterial Biofilms with C-SNARF-4

pH in the extracellular matrix of bacterial biofilms is of central importance for microbial metabolism. Biofilms possess a complex three-dimensional architecture characterized by chemically different microenvironments in close proximity. For decades, pH measurements in biofilms have been limited to monitoring bulk pH with electrodes. Although pH microelectrodes with a better spatial resolution have been developed, they do not permit the monitoring of horizontal pH gradients in biofilms in real time. Quantitative fluorescence microscopy can overcome these problems, but none of the hitherto employed methods differentiated accurately between extracellular and intracellular microbial pH and visualized extracellular pH in all areas of the biofilms. Here, we developed a method to reliably monitor extracellular biofilm pH microscopically with the ratiometric pH-sensitive dye C-SNARF-4, choosing dental biofilms as an example. Fluorescent emissions of C-SNARF-4 can be used to calculate extracellular pH irrespective of the dye concentration. We showed that at pH values of <6, C-SNARF-4 stained 15 bacterial species frequently isolated from dental biofilm and visualized the entire bacterial biomass in in vivo-grown dental biofilms with unknown species composition. We then employed digital image analysis to remove the bacterial biomass from the microscopic images and adequately calculate extracellular pH values. As a proof of concept, we monitored the extracellular pH drop in in vivo-grown dental biofilms fermenting glucose. The combination of pH ratiometry with C-SNARF-4 and digital image analysis allows the accurate monitoring of extracellular pH in bacterial biofilms in three dimensions in real time and represents a significant improvement to previously employed methods of biofilm pH measurement.     

Osteopontin reduces biofilm formation in a multi-species model of dental biofilm

Background

Combating dental biofilm formation is the most effective means for the prevention of caries, one of the most widespread human diseases. Among the chemical supplements to mechanical tooth cleaning procedures, non-bactericidal adjuncts that target the mechanisms of bacterial biofilm formation have gained increasing interest in recent years. Milk proteins, such as lactoferrin, have been shown to interfere with bacterial colonization of saliva-coated surfaces. We here study the effect of bovine milk osteopontin (OPN), a highly phosphorylated whey glycoprotein, on a multispecies in vitro model of dental biofilm. While considerable research effort focuses on the interaction of OPN with mammalian cells, there are no data investigating the influence of OPN on bacterial biofilms.

Methodology/Principal Findings

Biofilms consisting of Streptococcus oralis, Actinomyces naeslundii, Streptococcus mitis, Streptococcus downei and Streptococcus sanguinis were grown in a flow cell system that permitted in situ microscopic analysis. Crystal violet staining showed significantly less biofilm formation in the presence of OPN, as compared to biofilms grown without OPN or biofilms grown in the presence of caseinoglycomacropeptide, another phosphorylated milk protein. Confocal microscopy revealed that OPN bound to the surface of bacterial cells and reduced mechanical stability of the biofilms without affecting cell viability. The bacterial composition of the biofilms, determined by fluorescence in situ hybridization, changed considerably in the presence of OPN. In particular, colonization of S. mitis, the best biofilm former in the model, was reduced dramatically.

Conclusions/Significance

OPN strongly reduces the amount of biofilm formed in a well-defined laboratory model of acidogenic dental biofilm. If a similar effect can be observed in vivo, OPN might serve as a valuable adjunct to mechanical tooth cleaning procedures.     

Ratiometric Imaging of Extracellular pH in Dental Biofilms

The pH in bacterial biofilms on teeth is of central importance for dental caries, a disease with a high worldwide prevalence. Nutrients and metabolites are not distributed evenly in dental biofilms. A complex interplay of sorption to and reaction with organic matter in the biofilm reduces the diffusion paths of solutes and creates steep gradients of reactive molecules, including organic acids, across the biofilm. Quantitative fluorescent microscopic methods, such as fluorescence life time imaging or pH ratiometry, can be employed to visualize pH in different microenvironments of dental biofilms. pH ratiometry exploits a pH-dependent shift in the fluorescent emission of pH-sensitive dyes. Calculation of the emission ratio at two different wavelengths allows determining local pH in microscopic images, irrespective of the concentration of the dye. Contrary to microelectrodes the technique allows monitoring both vertical and horizontal pH gradients in real-time without mechanically disturbing the biofilm. However, care must be taken to differentiate accurately between extra- and intracellular compartments of the biofilm. Here, the ratiometric dye, seminaphthorhodafluor-4F 5-(and-6) carboxylic acid (C-SNARF-4) is employed to monitor extracellular pH in in vivo grown dental biofilms of unknown species composition. Upon exposure to glucose the dye is up-concentrated inside all bacterial cells in the biofilms; it is thus used both as a universal bacterial stain and as a marker of extracellular pH. After confocal microscopic image acquisition, the bacterial biomass is removed from all pictures using digital image analysis software, which permits to exclusively calculate extracellular pH. pH ratiometry with the ratiometric dye is well-suited to study extracellular pH in thin biofilms of up to 75 µm thickness, but is limited to the pH range between 4.5 and 7.0.     

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.     

Application of a pH-Sensitive Fluoroprobe (C-SNARF-4) for pH Microenvironment Analysis in Pseudomonas aeruginosa Biofilms

An important feature of microbial biofilms is the development of four-dimensional physical and chemical gradients in space and time. There is need for novel approaches to probe these so-called microenvironments to determine their effect on biofilm-specific processes. In this study, we describe the use of seminaphthorhodafluor-4F 5-(and-6) carboxylic acid (C-SNARF-4) for pH microenvironment analysis in Pseudomonas aeruginosa biofilms. C-SNARF-4 is a fluorescent ratiometric probe that allows pH quantification independent of probe concentration and/or laser intensity. By confocal scanning laser microscopy, C-SNARF-4 revealed pH heterogeneity throughout the biofilm in both the x,y and x,z planes, with values ranging from pH 5.6 (within the biofilm) to pH 7.0 (bulk fluid). pH values were typically remarkably different than those just a few micrometers away. Although this probe has been successfully used in a number of eukaryotic systems, problems have been reported which describe spectral emission changes as a result of macromolecular interactions with the fluorophore. To assess how the biofilm environment may influence fluorescent properties of the dye, fluorescence of C-SNARF-4 was quantified via spectrofluorometry while the probe was suspended in various concentrations of representative biofilm matrix components (i.e., proteins, polysaccharides, and bacterial cells) and growth medium. Surprisingly, our data demonstrate that few changes in emission spectra occur as a result of matrix interactions below pH 7. These studies suggest that C-SNARF-4 can be used as a reliable indicator of pH microenvironments, which may help elucidate their influence on the medical and geobiological roles of natural biofilms.     

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.     

Nanoparticle-Encapsulated Chlorhexidine against Oral Bacterial Biofilms

Background

Chlorhexidine (CHX) is a widely used antimicrobial agent in dentistry. Herein, we report the synthesis of a novel mesoporous silica nanoparticle-encapsulated pure CHX (Nano-CHX), and its mechanical profile and antimicrobial properties against oral biofilms.

Methodology/Principal Findings

The release of CHX from the Nano-CHX was characterized by UV/visible absorption spectroscopy. The antimicrobial properties of Nano-CHX were evaluated in both planktonic and biofilm modes of representative oral pathogenic bacteria. The Nano-CHX demonstrated potent antibacterial effects on planktonic bacteria and mono-species biofilms at the concentrations of 50–200 µg/mL against Streptococcus mutans, Streptococcus sobrinus, Fusobacterium nucleatum, Aggregatibacter actinomycetemcomitans and Enterococccus faecalis. Moreover, Nano-CHX effectively suppressed multi-species biofilms such as S. mutans, F. nucleatum, A. actinomycetemcomitans and Porphyromonas gingivalis up to 72 h.

Conclusions/Significance

This pioneering study demonstrates the potent antibacterial effects of the Nano-CHX on oral biofilms, and it may be developed as a novel and promising anti-biofilm agent for clinical use.     

Crenarchaeal biofilm formation under extreme conditions

Background

Biofilm formation has been studied in much detail for a variety of bacterial species, as it plays a major role in the pathogenicity of bacteria. However, only limited information is available for the development of archaeal communities that are frequently found in many natural environments.

Methodology

We have analyzed biofilm formation in three closely related hyperthermophilic crenarchaeotes: Sulfolobus acidocaldarius, S. solfataricus and S. tokodaii. We established a microtitre plate assay adapted to high temperatures to determine how pH and temperature influence biofilm formation in these organisms. Biofilm analysis by confocal laser scanning microscopy demonstrated that the three strains form very different communities ranging from simple carpet-like structures in S. solfataricus to high density tower-like structures in S. acidocaldarius in static systems. Lectin staining indicated that all three strains produced extracellular polysaccharides containing glucose, galactose, mannose and N-acetylglucosamine once biofilm formation was initiated. While flagella mutants had no phenotype in two days old static biofilms of S. solfataricus, a UV-induced pili deletion mutant showed decreased attachment of cells.

Conclusion

The study gives first insights into formation and development of crenarchaeal biofilms in extreme environments.     

dATP/ATP, a multifunctional nucleotide, stimulates bacterial cell lysis, extracellular DNA release and biofilm development

Background

Signaling by extracellular adenosine 5′-triphosphase (eATP) is very common for cell-to-cell communication in many basic patho-physiological development processes. Rapid release of ATP into the extracellular environment from distressed or injured eukaryotic cells due to pathogens or other etiological factors can serve as a “danger signal”, activating host innate immunity. However, little is known about how or whether pathogenic bacteria respond to this “danger signal”.

Methods and Principal Findings

Here we report that extracellular dATP/ATP can stimulate bacterial adhesion and biofilm formation via increased cell lysis and extracellular DNA (eDNA) release. We demonstrate that extracellular dATP/ATP also stimulates bacterial adherence in vitro to human bronchial epithelial cells.

Conclusions and Significance

These data suggest that bacteria may sense extracellular dATP/ATP as a signal of “danger” and form biofilms to protect them from host innate immunity. This study reveals a very important and unrecognized phenomenon that both bacteria and host cells could respond to a common important signal molecule in a race to adapt to the presence of one another. We propose that extracellular dATP/ATP functions as an “inter-domain” warning signal that serves to induce protective measures in both Bacterial and Eukaryotic cells.     

Development of a toxR-based loop-mediated isothermal amplification assay for detecting Vibrio parahaemolyticus

Background

Microbial cell-cell interactions in the oral flora are believed to play an integral role in the development of dental plaque and ultimately, its pathogenicity. The effects of other species of oral bacteria on biofilm formation and virulence gene expression by Streptococcus mutans, the primary etiologic agent of dental caries, were evaluated using a dual-species biofilm model and RealTime-PCR analysis.

Results

As compared to mono-species biofilms, biofilm formation by S. mutans was significantly decreased when grown with Streptococcus sanguinis, but was modestly increased when co-cultivated with Lactobacillus casei. Co-cultivation with S. mutans significantly enhanced biofilm formation by Streptococcus oralis and L. casei, as compared to the respective mono-species biofilms. RealTime-PCR analysis showed that expression of spaP (for multi-functional adhesin SpaP, a surface-associated protein that S. mutans uses to bind to the tooth surface in the absence of sucrose), gtfB (for glucosyltransferase B that synthesizes α1,6-linked glucan polymers from sucrose and starch carbohydrates) and gbpB (for surface-associated protein GbpB, which binds to the glucan polymers) was decreased significantly when S. mutans were co-cultivated with L. casei. Similar results were also found with expression of spaP and gbpB, but not gtfB, when S. mutans was grown in biofilms with S. oralis. Compared to mono-species biofilms, the expression of luxS in S. mutans co-cultivated with S. oralis or L. casei was also significantly decreased. No significant differences were observed in expression of the selected genes when S. mutans was co-cultivated with S. sanguinis.

Conclusions

These results suggest that the presence of specific oral bacteria differentially affects biofilm formation and virulence gene expression by S. mutans.     

Glycerol monolaurate antibacterial activity in broth and biofilm cultures

Background

Glycerol monolaurate (GML) is an antimicrobial agent that has potent activity against gram-positive bacteria. This study examines GML antibacterial activity in comparison to lauric acid, in broth cultures compared to biofilm cultures, and against a wide range of gram-positive, gram-negative, and non-gram staining bacteria.

Methodology/Principal Findings

GML is ≥200 times more effective than lauric acid in bactericidal activity, defined as a ≥3 log reduction in colony-forming units (CFU)/ml, against Staphylococcus aureus and Streptococcus pyogenes in broth cultures. Both molecules inhibit superantigen production by these organisms at concentrations that are not bactericidal. GML prevents biofilm formation by Staphylococcus aureus and Haemophilus influenzae, as representative gram-positive and gram-negative organisms, tested in 96 well microtiter plates, and simultaneously is bactericidal for both organisms in mature biofilms. GML is bactericidal for a wide range of potential bacterial pathogens, except for Pseudomonas aeruginosa and Enterobacteriaceae. In the presence of acidic pH and the cation chelator ethylene diamine tetraacetic acid, GML has greatly enhanced bactericidal activity for Pseudomonas aeruginosa and Enterobacteriaceae. Solubilization of GML in a nonaqueous delivery vehicle (related to K-Y Warming®) enhances its bactericidal activity against S. aureus. Both R and S, and 1 and 2 position lauric acid derivatives of GML exhibit bactericidal activity. Despite year-long passage of Staphylococcus aureus on sub-growth inhibitory concentrations of GML (0.5 x minimum bactericidal concentration), resistance to GML did not develop.

Conclusions/Significance

GML may be useful as a broad-spectrum human or animal topical microbicide and may be useful as an environmental surface microbicide for management of bacterial infections and contamination.     

Molecular analysis of microbial communities in endotracheal tube biofilms

Background

Ventilator-associated pneumonia is the most prevalent acquired infection of patients on intensive care units and is associated with considerable morbidity and mortality. Evidence suggests that an improved understanding of the composition of the biofilm communities that form on endotracheal tubes may result in the development of improved preventative strategies for ventilator-associated pneumonia.

Methodology/Principal Findings

The aim of this study was to characterise microbial biofilms on the inner luminal surface of extubated endotracheal tubes from ICU patients using PCR and molecular profiling. Twenty-four endotracheal tubes were obtained from twenty mechanically ventilated patients. Denaturing gradient gel electrophoresis (DGGE) profiling of 16S rRNA gene amplicons was used to assess the diversity of the bacterial population, together with species specific PCR of key marker oral microorganisms and a quantitative assessment of culturable aerobic bacteria. Analysis of culturable aerobic bacteria revealed a range of colonisation from no growth to 2.1×10⁸ colony forming units (cfu)/cm² of endotracheal tube (mean 1.4×10⁷ cfu/cm²). PCR targeting of specific bacterial species detected the oral bacteria Streptococcus mutans (n = 5) and Porphyromonas gingivalis (n = 5). DGGE profiling of the endotracheal biofilms revealed complex banding patterns containing between 3 and 22 (mean 6) bands per tube, thus demonstrating the marked complexity of the constituent biofilms. Significant inter-patient diversity was evident. The number of DGGE bands detected was not related to total viable microbial counts or the duration of intubation.

Conclusions/Significance

Molecular profiling using DGGE demonstrated considerable biofilm compositional complexity and inter-patient diversity and provides a rapid method for the further study of biofilm composition in longitudinal and interventional studies. The presence of oral microorganisms in endotracheal tube biofilms suggests that these may be important in biofilm development and may provide a therapeutic target for the prevention of ventilator-associated pneumonia.     

Atmospheric pressure plasma: a high-performance tool for the efficient removal of biofilms

Introduction

The medical use of non-thermal physical plasmas is intensively investigated for sterilization and surface modification of biomedical materials. A further promising application is the removal or etching of organic substances, e.g., biofilms, from surfaces, because remnants of biofilms after conventional cleaning procedures are capable to entertain inflammatory processes in the adjacent tissues. In general, contamination of surfaces by micro-organisms is a major source of problems in health care. Especially biofilms are the most common type of microbial growth in the human body and therefore, the complete removal of pathogens is mandatory for the prevention of inflammatory infiltrate. Physical plasmas offer a huge potential to inactivate micro-organisms and to remove organic materials through plasma-generated highly reactive agents.

Method

In this study a Candida albicans biofilm, formed on polystyrene (PS) wafers, as a prototypic biofilm was used to verify the etching capability of the atmospheric pressure plasma jet operating with two different process gases (argon and argon/oxygen mixture). The capability of plasma-assisted biofilm removal was assessed by microscopic imaging.

Results

The Candida albicans biofilm, with a thickness of 10 to 20 µm, was removed within 300 s plasma treatment when oxygen was added to the argon gas discharge, whereas argon plasma alone was practically not sufficient in biofilm removal. The impact of plasma etching on biofilms is localized due to the limited presence of reactive plasma species validated by optical emission spectroscopy.     

Real-Time Microsensor Measurement of Local Metabolic Activities in Ex Vivo Dental Biofilms Exposed to Sucrose and Treated with Chlorhexidine

Dental biofilms are characterized by structural and functional heterogeneity. Due to bacterial metabolism, gradients develop and diverse ecological microniches exist. The aims of this study were (i) to determine the metabolic activity of microorganisms in naturally grown dental biofilms ex vivo by measuring dissolved oxygen (DO) and pH profiles with microelectrodes with high spatial resolution and (ii) to analyze the impact of an antimicrobial chlorhexidine (CHX) treatment on microbial physiology during stimulation by sucrose in real time. Biofilms were cultivated on standardized human enamel surfaces in vivo. DO and pH profiles were measured in a flow cell system in sterile human saliva, after sucrose addition (10%), again after alternative treatment of the sucrose exposed biofilms with CHX (0.2%) for 1 or 10 min or after being killed with paraformaldehyde (4%). Biofilm structure was visualized by vitality staining with confocal microscopy. With saliva as the sole nutrient source oxygen consumption was high within the superficial biofilm layers rendering deeper layers (>220 μm) anoxic. Sucrose addition induced the thickness of the anaerobic zone to increase with a concurrent decrease in pH (7.1 to 4.4). CHX exposure reduced metabolic activity and microbial viability at the biofilm surface and drove metabolic activity deeper into the biofilm. CHX treatment led to a reduced viability at the biofilm surface with minor influence on overall biofilm physiology after 1 min; even after 10 min there was measurable respiration and fermentation inside the biofilm. However, the local microenvironment was more aerated, less acidogenic, and presumably less pathogenic.Biofilms are complex, surface-associated, microbiological communities (7) that are characterized by microscale spatial, structural, and functional heterogeneity (40). The biofilm consists of microorganisms that are embedded in an extracellular slime matrix consisting of biopolymers of microbial origin such as polysaccharides, proteins, and DNA (16). This extracellular polymeric slime is highly hydrated and influences both the structure and the diffusion behavior within the biofilm (39). Bacterial metabolism results in the development of chemical and physiologic/metabolic gradients within the biofilm (17). Due to different concentrations of oxygen, nutrients, and microbial metabolic by-products, local microecological niches are created, allowing the coexistence of microorganisms with different growth requirements in close proximity (30). For example, the growth of anaerobic microorganisms within a generally aerobic environment within the oral cavity is possible. Carbohydrates and sugar are the most important energy sources for microorganisms in dental plaque (23) and, in the case of a lacking external substrate supply, they are able to metabolize salivary glycoproteins (5). Nutrient depletion causes the microorganisms to either grow very slowly or to stop growing completely, entering a dormant-like state.Changes in the ecologic balance of the oral microflora and in dental biofilms are a causative factor for the development of dental caries (43), gingivitis, and periodontitis (1); thus, these diseases can be considered as biofilm mediated. Fundamental factors that may lead to a shift in the microflora and the predominance of pathogens are the local pH value, the redox potential, and the availability of nutrients and/or carbohydrates (30). Caries, for instance, is a multifactorial disease. However, its main cause is the bacterial carbohydrate catabolism and the release of organic acids by acidogenic bacteria in the biofilm. This promotes the predominance of cariogenic pathogens such as Streptococcus mutans, Streptococcus sobrinus, and other acidogenic microorganisms (28, 43). Consequently, this results in further acid production and a decreasing pH. Associated with this is the demineralization and lesion development of dental hard substance (54).Next to individual improvement of mechanical oral hygiene (i.e., mechanical and manual brushing, as well as flossing), prevention and therapy of oral disease is achieved by adjunctive oral hygiene products containing antimicrobial agents (29, 47). A concentration of 0.2% chlorhexidine (CHX) in oral mouth rinses showed the best efficacy in clinical studies and is still considered a “gold standard” (45). The antiplaque effect of CHX is based on a broad antibacterial spectrum. During application it immediately shows bactericidal effect and continuous bacteriostatic effect due to its high substantivity in the oral cavity (20). Electron microscopic examinations showed that CHX binds to and damages bacterial cell membranes and leads to structural changes and leakage of cytoplasm (3). Furthermore, contraction of in vitro grown biofilms after CHX exposure was shown in addition to cell damage (19, 44), which could cause changes in the diffusion behavior by changes to extracellular polymeric slime density. However, in deep layers of oral biofilms not all bacteria were reached (49). Direct visualization by fluorescence microscopy of the CHX effect was described by Takenaka et al. (44) for a three-species oral biofilm grown in vitro. Cell damage started from the periphery of bacterial aggregates and slowly continued into the depths. Other authors have reported the inability of CHX to completely kill all bacteria in different in vitro biofilm systems (15, 19, 32) and in vivo studies (48, 55) when a normally used clinical concentration was applied at usual exposure times. The killing efficacy was dependent on the age of the biofilm and thus on its thickness and composition.Detection of concentration gradients in oxygen, pH, and metabolites in undisturbed biofilms in situ requires a microsensor technique. Microelectrodes with a tip diameter of a few micrometers offer precisely localized measurements on the microscale in three dimensions under real-time conditions (9, 35). Thus far, microelectrodes have been applied for measuring plaque pH in dental research in a groove model ex vivo (53, 54) and in in vitro-grown S. mutans biofilms (13, 42). However, there is no information on direct measurements of oxygen distribution and consumption in dental biofilms grown in vivo in the human oral cavity. In the field of environmental microbiology, analysis of the microenvironment, local activities, and gradients by microelectrodes is correlated with microscopic examination of the biofilm structure, thus allowing a relationship to be made between biofilm physiology and structure (18, 37). We decided to take a similar approach to characterize the effects of sucrose and CHX on ex vivo plaque biofilm physiology. The goal of the present study was to examine the metabolic activity (oxygen consumption and acid formation) and viability and the effect of antimicrobial CHX treatment on the physiology of the dental biofilms during exposure to sucrose. The spatial distribution of live and dead biofilm cells was examined by confocal laser scanning microscopy (CLSM), as well as by microbiological culture.     

Influence of microbial biofilms on the preservation of primary soft tissue in fossil and extant archosaurs

Background

Mineralized and permineralized bone is the most common form of fossilization in the vertebrate record. Preservation of gross soft tissues is extremely rare, but recent studies have suggested that primary soft tissues and biomolecules are more commonly preserved within preserved bones than had been presumed. Some of these claims have been challenged, with presentation of evidence suggesting that some of the structures are microbial artifacts, not primary soft tissues. The identification of biomolecules in fossil vertebrate extracts from a specimen of Brachylophosaurus canadensis has shown the interpretation of preserved organic remains as microbial biofilm to be highly unlikely. These discussions also propose a variety of potential mechanisms that would permit the preservation of soft-tissues in vertebrate fossils over geologic time.

Methodology/Principal Findings

This study experimentally examines the role of microbial biofilms in soft-tissue preservation in vertebrate fossils by quantitatively establishing the growth and morphology of biofilms on extant archosaur bone. These results are microscopically and morphologically compared with soft-tissue extracts from vertebrate fossils from the Hell Creek Formation of southeastern Montana (Latest Maastrichtian) in order to investigate the potential role of microbial biofilms on the preservation of fossil bone and bound organic matter in a variety of taphonomic settings. Based on these analyses, we highlight a mechanism whereby this bound organic matter may be preserved.

Conclusions/Significance

Results of the study indicate that the crystallization of microbial biofilms on decomposing organic matter within vertebrate bone in early taphonomic stages may contribute to the preservation of primary soft tissues deeper in the bone structure.