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Confocal Raman microscope. Schematic overview of confocal Raman microscope; see text for details. Abbreviations: BPF, bandpass filter; SF, spatial filter; NF, notch filter.
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A new method of mapping multiple species of oral bacteria in intact biofilms has been developed, using the optical technique of confocal Raman microscopy. A species classification algorithm, developed on dried biofilms, was used to analyze spectra of hydrated biofilms containing two microbial species central to dental health: Streptococcus sanguini...
Citations
... Combined with this information, it can be used for qualitative and structural analysis of the measured substance. Since biofilms comprise EPS-containing proteins, polysaccharides, etc., this analysis method can identify the composition and structure of substances adhering to the sample surface, and thus determine the present substances [40][41][42][43][44][45][46]. Although quantitative analysis is also possible based on comparison of peak values obtained by Raman spectroscopy, in this study, Raman was measured by randomly selecting areas where biofilm formation was confirmed by microscopy, so the results are used only as one qualitative result representing the presence and composition, not the entire sample surface. ...
Biofilms, mainly comprised of bacteria, form on materials’ surfaces due to bacterial activity. They are generally composed of water, extracellular polymeric substances (polysaccharides, proteins, nucleic acids, and lipids), and bacteria. Some bacteria that form biofilms cause periodontal disease, corrosion of the metal materials that make up drains, and slippage. Inside of a biofilm is an environment conducive to the growth and propagation of bacteria. Problems with biofilms include the inability of disinfectants and antibiotics to act on them. Therefore, we have investigated the potential application of alternating electromagnetic fields for biofilm control. We obtained exciting results using various materials’ specimens and frequency conditions. Through these studies, we gradually understood that the combination of the type of bacteria, the kind of material, and the application of an electromagnetic field with various low frequencies (4 kHz–12 kHz) changes the circumstances of the onset of the biofilm suppression effect. In this study, relatively high frequencies (20 and 30 kHz) were applied to biofilms caused by Escherichia coli (E. coli) and Staphylococcus epidermidis (S. epidermidis), and quantitative evaluation was performed using staining methods. The sample surfaces were analyzed by Raman spectroscopy using a Laser Raman spectrometer to confirm the presence of biofilms on the surface.
... In this study, corresponding characteristic regions were identified for different biofilm components within the spectra, and detailed chemical information about these matrix components could be provided, without, however, focusing on the characterization of the microbial species in the biofilm themselves. In 2012, Beier et al. studied two-species bacterial hydrated biofilms with microbial species of importance for dental health: Streptococcus sanguinis and Streptococcus mutans [16]. They developed an algorithm based on data from hydrated biofilms dried on surfaces and made predictions about species in hydrated biofilms [16]. ...
... In 2012, Beier et al. studied two-species bacterial hydrated biofilms with microbial species of importance for dental health: Streptococcus sanguinis and Streptococcus mutans [16]. They developed an algorithm based on data from hydrated biofilms dried on surfaces and made predictions about species in hydrated biofilms [16]. ...
Moist/hydrated biofilms have been well-studied in the medical area, and their association with infections is widely recognized. In contrast, dry-surface biofilms (DSBs) on environmental surfaces in healthcare settings have received less attention. DSBs have been shown to be widespread on commonly used items in hospitals and to harbor bacterial pathogens that are known to cause healthcare-acquired infections (HAI). DSBs cannot be detected by routine surface swabbing or contact plates, and studies have shown DSBs to be less susceptible to cleaning/disinfection products. As DSBs are increasingly reported in the medical field, and there is a likelihood they also occur in food production and manufacturing areas, there is a growing demand for the rapid in situ detection of DSBs and the identification of pathogens within DSBs. Raman microspectroscopy allows users to obtain spatially resolved information about the chemical composition of biofilms, and to identify microbial species. In this study, we investigated Staphylococcus aureus mono-species DSB on polyvinylchloride blanks and stainless steel coupons, and dual-species (S. aureus/Bacillus licheniformis) DSB on steel coupons. We demonstrated that Raman microspectroscopy is not only suitable for identifying specific species, but it also enables the differentiation of vegetative cells from their sporulated form. Our findings provide the first step towards the rapid identification and characterization of the distribution and composition of DSBs on different surface areas.
... Thus any functional group has its own characteristic vibrational frequency. Spectral characteristics of a compound vary with solvent Raman microspectroscopy [102] environment, binding angles, binding forces, conformation or association with other compounds. Monitoring these spectral alterations w.r.t peak intensity or peak position can thus be used to identify the compound under observation. ...
Microorganisms adhering to biotic or abiotic surfaces depend on each other for their survival in extreme environmental stress conditions such as the lack of nutrient supply, presence of antimicrobial agents, alterations in temperature and pH of the surroundings, etc. These adherent cells known to form biofilms are enclosed within a matrix of extracellular polymeric substance (EPS) comprising of polysaccharides, nucleic acids, proteins, and lipids that furnishes a suitable environment for the survival and exchange of genetic material among neighbouring cells. EPS is also the first line of defence against various antimicrobial substances as it prevents the diffusion of these components into the indwelling cells. Thus, understanding the mode of biofilm formation and survival strategy of sessile microbial species is needed for developing new therapeutic agents against these pathogenic strains. So far traditional morphological and biochemical methods are being used to identify the genotypic and phenotypic features of the microbes. However, these methods are time consuming and require expensive reagents to perform. Thus, vibrational spectroscopic techniques such as Raman and Fourier Transform Infrared (FTIR) spectroscopy have been used as an alternate method to unravel the mysteries of biofilm formation and spread of pathogenesis. They provide a rapid and accurate identification of microbial species by providing “whole organism fingerprinting” through display of spectral features characteristic to the biochemical constituents of bacterial cell. Altogether, improvement in screening efficiency mainly in clinical microbiology would greatly help in identifying the source and cause of an infection.
... This study used Raman spectroscopy to detect biochemical differences between planktonic and biofilm bacteria 53,54 . Since media residues were washed off the planktonic bacteria, the Raman spectra obtained from them can be used as a reference to observe the chemical changes that occurred during biofilm formation. ...
Psychrotrophic Pseudomonas species are the key spoilage bacteria of aerobically stored chilled meat. These organisms readily form biofilms on meat under refrigerated conditions leading to consumer rejection and associated economic losses. Limited information is available on the matrix composition of the biofilms formed by these bacteria. We quantified and characterized the main components of the matrix of mono-species biofilms of selected Pseudomonas fragi and Pseudomonas lundensis strains using chemical analysis and Raman spectroscopy. The biofilms were grown at 10 °C and 25 °C on nitro-cellulose membranes placed on surface sterilized beef cuts. Extra-cellular polymeric substances of the matrix were extracted in soluble and bound forms and were chemically assessed for total carbohydrates, proteins and extra-cellular DNA. Both Pseudomonas species showed a significant increase in total carbohydrates and total proteins when grown at 10 °C as compared to 25 °C. Extra-cellular DNA did not show a strong correlation with growth temperature. Raman spectra were obtained from planktonic bacteria and membrane grown biofilms at 10 °C and 25 °C. Higher levels of guanine were detected in planktonic cells as compared to biofilm cells. This study suggests that psychrotrophic Pseudomonas species may respond to cold stress by increasing extra-cellular polymer secretions.
... However, the acquired signals from these biomedical components are highly complex [19][20][21]. Therefore, differentiation based on Raman spectra has significant limitations so far [16,[22][23][24][25]. To the best of our knowledge, only a few studies employed the use of CRM for environmental biofilms [26,27], but there has been very limited information in the field of oral biofilm mapping [23,28]. ...
... Because bacteria in subgingival biofilms live in the same habitat, they show similar chemical compositions. While differences in spectral fingerprint patterns of oral bacteria have been shown to be minor, they still allow differentiation between species to be made [22,31]. Given these differences it is therefore important to apply statistical models to spectral data to discriminate between species. ...
The study of oral disease progression, in relation to the accumulation of subgingival biofilm in gingivitis and periodontitis is limited, due to either the ability to monitor plaque in vitro. When compared, optical spectroscopic techniques offer advantages over traditional destructive or biofilm staining approaches, making it a suitable alternative for the analysis and continued development of three-dimensional structures. In this work, we have developed a confocal Raman spectroscopy analysis approach towards in vitro subgingival plaque models. The main objective of this study was to develop a method for differentiating multiple oral subgingival bacterial species in planktonic and biofilm conditions, using confocal Raman microscopy. Five common subgingival bacteria (Fusobacterium nucleatum, Streptococcus mutans, Veillonella dispar, Actinomyces naeslundii and Prevotella nigrescens) were used and differentiated using a 2-way orthogonal Partial Least Square with Discriminant Analysis (O2PLS-DA) for the collected spectral data. In addition to planktonic growth, mono-species biofilms cultured using the ‘Zürich Model’ were also analyzed. The developed method was successfully used to predict planktonic and mono-species biofilm species in a cross validation setup. The results show differences in the presence and absence of chemical bands within the Raman spectra. The O2PLS-DA model was able to successfully predict 100% of all tested planktonic samples and 90% of all mono-species biofilm samples. Using this approach we have shown that Confocal Raman microscopy can analyse and predict the identity of planktonic and mono-species biofilm species, thus enabling its potential as a technique to map oral multi-species biofilm models.
... The advantage of this technique is that it is applicable both in laboratory and under industrial conditions and it does not need any sample preparation. The usefulness of this method was proven by several research groups [98][99][100]. ...
The microbiologically/microbially influenced corrosion (MIC) is a special type of corrosion; in this case the microorganisms by their presence and aggressive metabolites alter the processes on solid surfaces via electrochemical and chemical reactions. When microorganisms are present in most cases the degradation of metals or alloys happens by microbes embedded in biofilms and by their excreted metabolites (e.g. acids), macromolecules (with complexing ability) and by other molecules that can form insoluble precipitates; all these reactions increase the deterioration. The paper summarizes the most important characteristics of the MIC, mainly the so-called biocorrosion of metals and alloys. Not only the chemical and electrochemical processes, but the roles of the corrosion relevant microorganisms in the deteriorating processes, as well as the information about the mechanisms of the MIC worked out in the past and in the very last period are discussed. The most important (aerobic, anaerobic, slime former, acid producer etc.) microorganisms, their nutrient requirements and the formation and role of biofilms are presented, characterized and discussed, as well as the influence of biofilms on the MIC is also demonstrated. The impact of metals on the MIC is also discussed. The history of the research on MIC from its discovery till the 21 th century will demonstrate the enormous work that allowed the understanding of this special type of corrosion as well as its mechanism and the role of the biofilm in MIC. The paper will expose the reactions that go on between the slimy layer (that surrounds the microorganisms even in planktonic form) and the metal surface. The mostly used techniques to visualize what on the surface happens and to measure the change in the current density/corrosion potential and in the corrosion rate due to microbial action are also summarized and in all cases the advantages and disadvantages of all methods are discussed.
... Therefore, the process of interpretation of the obtained spectroscopic data is extremely difficult even in the case of a healthy dentin. The development of the carious process and penetration of microorganisms into the dentin tissues [27], as well as interaction of products of their vital activity with a native tissue of the tooth, further complicate the problem of decoding the obtained information [28][29][30]. Therefore, determination of the demineralization degree of dentin and composition of mineral phases in it, as well as identification of products of vital activity of pathogenous bacteria and determination of agents of the natural response of dentin to the carious process, is a complex interdisciplinary problem. ...
Using laser-induced fluorescence and Raman spectromicroscopy methods, an analysis of microregions of the human tooth dentin tissue affected by caries was performed within the frameworks of the integrated research technique (λ = 514.5 nm). A simultaneous screening by two methods of microregions close to the interface between the intact and infected dentin allowed us to observe an intensive response in both the Raman spectrum and range of induced fluorescence. The data analysis showed that vibrational modes registered by the method of the Raman spectromicroscopy are related to the amino acids of DNA/RNA of cariogenic bacteria and porphyrins, which are a consequence of their vital activity. This is confirmed by the data of laser-induced fluorescence. The revealed spectral features can be successfully used in stomatology when diagnosing carious lesions of different level of formation.
... Raman spectroscopy has been previously exploited in this area only superficially, despite its considerable potential. It was applied in the analysis of carious lesions in enamel [16], analysis of enamel fluorosis at healthy and affected samples [17], study of dentine [18], or analysis of microorganism present in oral cavity [19,20]. The potential of Raman spectroscopy was considerably increased with a discovery of surface-enhanced Raman scattering (SERS) by Fleishman [21]. ...
Endodontic treatment of immature permanent teeth with necrotic pulp poses several clinical challenges and is one of the most demanding interventions in endodontics. Recently, with new discoveries in the field of tissue engineering, novel treatment protocols have been established. The most promising treatment modality is revascularization, whose integral part is the exposure of collagen matrix and embedded growth factors. However, optimization of the treatment protocol requires a development of analytical procedures able to analyze growth factors directly on the sample surface. In this work, method based on surface-enhanced Raman spectroscopy (SERS) was developed to investigate the influence of the time of the medical treatment using EDTA on exposure and accessibility of the growth factors, namely TGF-ß1, BMP-2, and bFGF on the dentine surface. The nanotags, which consist of magnetic Fe3O4@Ag nanocomposite covalently functionalized by tagged antibodies (anti-TGF-ß1-Cy3, anti-BMP-2-Cy5, and anti-bFGF-Cy7), were employed as a SERS substrate. Each antibody was coupled with a unique label allowing us to perform a parallel analysis of all three growth factors within one analytical run. Developed methodology presents an interesting alternative to a fluorescence microscopy and in contrary allows evaluating a chemical composition and thus minimizing possible false-positive results.
... Knowing the species from which a sample derives can be highly informative of the environment, whether this is at the microscopic or macroscopic scale. In the case of microorganisms this can be important to understand processes of infection [1][2][3] and/or decay [4,5], whereas in the case of animals it can be important for understanding the effects of climate change or human impacts on biodiversity [6][7][8], or targeted at wildlife crime [9,10]. For reasons relating to either difficulties in identification or practicalities of analysing high numbers of samples, molecular methods are often preferred over morphological approaches, the most common being those that utilise DNA [11]. ...
... ML differs from clustering methods in the way that it is intrinsically selective towards certain markers since majority voting almost always favours some markers over others, whereas clustering methods usually treat markers with equal weights. Higher performance of ML indicates that using differential weights on markers could be important for distinguishing low level taxonomic groups, which agrees with previous work on keratin for species identification [3]. ...
Background:
Biomolecular methods for species identification are increasingly being utilised in the study of changing environments, both at the microscopic and macroscopic levels. High-throughput peptide mass fingerprinting has been largely applied to bacterial identification, but increasingly used to identify archaeological and palaeontological skeletal material to yield information on past environments and human-animal interaction. However, as applications move away from predominantly domesticate and the more abundant wild fauna to a much wider range of less common taxa that do not yet have genetically-derived sequence information, robust methods of species identification and biomarker selection need to be determined.
Results:
Here we developed a supervised machine learning algorithm for classifying the species of ancient remains based on collagen fingerprinting. The aim was to minimise requirements on prior knowledge of known species while yielding satisfactory sensitivity and specificity. The algorithm uses iterations of a modified random forest classifier with a similarity scoring system to expand its identified samples. We tested it on a set of 6805 spectra and found that a high level of accuracy can be achieved with a training set of five identified specimens per taxon.
Conclusions:
This method consistently achieves higher accuracy than two-dimensional principal component analysis and similar accuracy with hierarchical clustering using optimised parameters, which greatly reduces requirements for human input. Within the vertebrata, we demonstrate that this method was able to achieve the taxonomic resolution of family or sub-family level whereas the genus- or species-level identification may require manual interpretation or further experiments. In addition, it also identifies additional species biomarkers than those previously published.
... Although there were subtle differences between the spectra of the two strains, the principal component analysis (PCA) of the spectra clearly discriminated the two strains and showed the effects of the biofilm matrix ingredients on the spectra, especially that of polysaccharide inter-cellular adhesions (PIA). Beier et al. [33] discriminated two oral bacteria species, Streptococcus sanguinis and Streptococcus mutans, in their hydrated biofilm mixtures using a classification algorithm that was previously developed by using their dried biofilms. ...
Biofilms are a communal way of living for microorganisms in which microorganism cells
are surrounded by extracellular polymeric substances (EPS). Most microorganisms can live in biofilm
form. Since microorganisms are everywhere, understanding biofilm structure and composition is
crucial for making the world a better place to live, not only for humans but also for other living
creatures. Raman spectroscopy is a nondestructive technique and provides fingerprint information
about an analyte of interest. Surface-enhanced Raman spectroscopy is a form of this technique
and provides enhanced scattering of the analyte that is in close vicinity of a nanostructured noble
metal surface such as silver or gold. In this review, the applications of both techniques and their
combination with other biofilm analysis techniques for characterization of composition and structure
of biofilms are discussed.
