Optotracing for live selective fluorescence-based detection of Candida albicans biofilms

Kärkkäinen, Elina; Jakobsson, Saga G.; Edlund, Ulrica; Richter‐Dahlfors, Agneta; Choong, Ferdinand X.

doi:10.3389/fcimb.2022.981454

Cited by 6 publications

(12 citation statements)

References 30 publications

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“…Available EPS stainings are usually either toxic or destructive (not applicable for real-time studies) and target specific EPS structures, so ideally multiple dyes should be combined ( 47 ). Recently, optotracers are being used to monitor real-time EPS formation in a biofilm as these are non-toxic to cells ( 48 , 49 ). The optotracer EbbaBiolight 680 (Ebba Biotech) was evaluated but is not suitable for staining the EPS structures produced by the selected oral strains in our study.…”

Section: Discussionmentioning

confidence: 99%

Spatiotemporal monitoring of a periodontal multispecies biofilm model: demonstration of prebiotic treatment responses

Ghesquière,

Simoens,

Koos

et al. 2023

Appl Environ Microbiol

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Biofilms are complex polymicrobial communities which are often associated with human infections such as the oral disease periodontitis. Studying these complex communities under controlled conditions requires in vitro biofilm model systems that mimic the natural environment as close as possible. This study established a multispecies periodontal model in the drip flow biofilm reactor in order to mimic the continuous flow of nutrients at the air-liquid interface in the oral cavity. The design is engineered to enable real-time characterization. A community of five bacteria, Streptococcus gordonii -GFPmut3*, Streptococcus oralis -GFPmut3*, Streptococcus sanguinis -pVMCherry, Fusobacterium nucleatum , and Porphyromonas gingivalis -SNAP26 is visualized using two distinct fluorescent proteins and the SNAP-tag. The biofilm in the reactor develops into a heterogeneous, spatially uniform, dense, and metabolically active biofilm with relative cell abundances similar to those in a healthy individual. Metabolic activity, structural features, and bacterial composition of the biofilm remain stable from 3 to 6 days. As a proof of concept for our periodontal model, the 3 days developed biofilm is exposed to a prebiotic treatment with L-arginine. Multifaceted effects of L-arginine on the oral biofilm were validated by this model setup. L-arginine showed to inhibit growth and incorporation of the pathogenic species and to reduce biofilm thickness and volume. Additionally, L-arginine is metabolized by Streptococcus gordonii -GFPmut3* and Streptococcus sanguinis -pVMCherry, producing high levels of ornithine and ammonium in the biofilm. In conclusion, our drip flow reactor setup is promising in studying spatiotemporal behavior of a multispecies periodontal community. IMPORTANCE Periodontitis is a multifactorial chronic inflammatory disease in the oral cavity associated with the accumulation of microorganisms in a biofilm. Not the presence of the biofilm as such, but changes in the microbiota (i.e., dysbiosis) drive the development of periodontitis, resulting in the destruction of tooth-supporting tissues. In this respect, novel treatment approaches focus on maintaining the health-associated homeostasis of the resident oral microbiota. To get insight in dynamic biofilm responses, our research presents the establishment of a periodontal biofilm model including Streptococcus gordonii , Streptococcus oralis , Streptococcus sanguinis , Fusobacterium nucleatum , and Porphyromonas gingivalis . The added value of the model setup is the combination of simulating continuously changing natural mouth conditions with spatiotemporal biofilm profiling using non-destructive characterization tools. These applications are limited for periodontal biofilm research and would contribute in understanding treatment mechanisms, short- or long-term exposure effects, the adaptation potential of the biofilm and thus treatment strategies.

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Section: Discussionmentioning

confidence: 99%

Spatiotemporal monitoring of a periodontal multispecies biofilm model: demonstration of prebiotic treatment responses

Ghesquière,

Simoens,

Koos

et al. 2023

Appl Environ Microbiol

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“…Available EPS stainings are usually either toxic or destructive (not applicable for real-time studies) and target specific EPS structures, so ideally multiple dyes should be combined [60]. Recently, optotracers are being used to monitor real-time EPS formation in a biofilm as these are non-toxic to cells [61,62]. The optotracer EbbaBiolight 680 (Ebba Biotech) was evaluated, but is not suitable for staining the EPS structures produced by the selected oral strains in our study.…”

Section: L-arginine Exerts Multifaceted Effects On Periodontal Biofilmsmentioning

confidence: 99%

Spatiotemporal monitoring demonstrates prebiotic treatment responses in a periodontal multispecies biofilm model

Bernaerts

Ghesquière

Simoens

et al. 2023

Preprint

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In vitro periodontal model systems are required for further elucidating phenomena behind periodontal pathogenesis and treatment applications. However, most oral biofilm models apply static conditions and are restricted to end-point measurements. This study established a multispecies periodontal model in the drip flow biofilm reactor in order to mimic shear flow in the oral cavity. The design is re-engineered to enable real-time characterization. A community of five bacteria, Streptococcus gordonii-GFPmut3*, Streptococcus oralis-GFPmut3*, Streptococcus sanguinis-pVMCherry, Fusobacterium nucleatum, and Porphyromonas gingivalis-SNAP26 is visualized using two distinct fluorescent proteins and the SNAP-tag. The biofilm in the reactor develops into a spatially uniform, dense and metabolically active biofilm with relative cell abundances similar to those in a healthy individual. Metabolic activity, structural features and bacterial composition of the biofilm remain stable from 3 up to 6 days. As a proof of concept for our periodontal model, the 3-day developed biofilm is exposed to a prebiotic treatment with L-arginine. Multifaceted effects of L-arginine on the oral biofilm were validated by this model setup. L-arginine showed to inhibit growth and co-aggregations of the pathogenic species, and to reduce biofilm thickness and volume. Additionally, L-arginine is metabolized by Streptococcus gordonii-GFPmut3* and Streptococcus sanguinis-pVMCherry, producing high levels of ornithine in the biofilm. In conclusion, our drip flow reactor setup is promising in studying spatiotemporal behavior of a multispecies periodontal community.

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“…Irrespective of morphology, the main constituents of the cell wall of C. albicans are mannoproteins, chitins, and α- and β- linked glucans, whose relative amounts may vary between cell types ( Figure 6 ) [ 123 , 124 ]. Recognizing the similarities of glucans and chitin with cellulose, Kärkkäinen et al analyzed whether the fungal cell wall could act as a target for optotracers, and if so, enable detection of this pathogen [ 122 ]. Using purified extracts, productive interactions between the optotracer and commercially available preparations of chitin and β-1,3-glucans were found [ 122 ].…”

Section: Optotracing In Infection Research and Diagnosticsmentioning

confidence: 99%

“…Recognizing the similarities of glucans and chitin with cellulose, Kärkkäinen et al analyzed whether the fungal cell wall could act as a target for optotracers, and if so, enable detection of this pathogen [ 122 ]. Using purified extracts, productive interactions between the optotracer and commercially available preparations of chitin and β-1,3-glucans were found [ 122 ]. Analysis of the optotracer’s photophysical changes revealed the characteristic shift in absorption and emission patterns, alongside the on-switch of fluorescence [ 122 ].…”

Section: Optotracing In Infection Research and Diagnosticsmentioning

confidence: 99%

“…Using purified extracts, productive interactions between the optotracer and commercially available preparations of chitin and β-1,3-glucans were found [ 122 ]. Analysis of the optotracer’s photophysical changes revealed the characteristic shift in absorption and emission patterns, alongside the on-switch of fluorescence [ 122 ]. While the spectral signature of each individual polysaccharide was close to identical, the optotracer showed a stronger affinity for chitin over β-1,3-glucan when mixes of polysaccharides were used [ 122 ].…”

Section: Optotracing In Infection Research and Diagnosticsmentioning

confidence: 99%

See 1 more Smart Citation

Fluorescent optotracers for bacterial and biofilm detection and diagnostics

Richter‐Dahlfors

Kärkkäinen

Choong

2023

Science and Technology of Advanced Materials

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Effective treatment of bacterial infections requires methods that accurately and quickly identify which antibiotic should be prescribed. This review describes recent research on the development of optotracing methodologies for bacterial and biofilm detection and diagnostics. Optotracers are small, chemically well-defined, anionic fluorescent tracer molecules that detect peptide- and carbohydrate-based biopolymers. This class of organic molecules (luminescent conjugated oligothiophenes) show unique electronic, electrochemical and optical properties originating from the conjugated structure of the compounds. The photophysical properties are further improved as donor-acceptor-donor (D-A-D)-type motifs are incorporated in the conjugated backbone. Optotracers bind their biopolymeric target molecules via electrostatic interactions. Binding alters the optical properties of these tracer molecules, shown as altered absorption and emission spectra, as well as ON-like switch of fluorescence. As the optotracer provides a defined spectral signature for each binding partner, a fingerprint is generated that can be used for identification of the target biopolymer. Alongside their use for in situ experimentation, optotracers have demonstrated excellent use in studies of a number of clinically relevant microbial pathogens. These methods will find widespread use across a variety of communities engaged in reducing the effect of antibiotic resistance. This includes basic researchers studying molecular resistance mechanisms, academia and pharma developing new antimicrobials targeting biofilm infections and tests to diagnose biofilm infections, as well as those developing antibiotic susceptibility tests for biofilm infections (biofilm-AST). By iterating between the microbial world and that of plants, development of the optotracing technology has become a prime example of successful cross-feeding across the boundaries of disciplines. As optotracers offers a capacity to redefine the way we work with polysaccharides in the microbial world as well as with plant biomass, the technology is providing novel outputs desperately needed for global impact of the threat of antimicrobial resistance as well as our strive for a circular bioeconomy.

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Optotracing for live selective fluorescence-based detection of Candida albicans biofilms

Cited by 6 publications

References 30 publications

Spatiotemporal monitoring of a periodontal multispecies biofilm model: demonstration of prebiotic treatment responses

Spatiotemporal monitoring of a periodontal multispecies biofilm model: demonstration of prebiotic treatment responses

Spatiotemporal monitoring demonstrates prebiotic treatment responses in a periodontal multispecies biofilm model

Fluorescent optotracers for bacterial and biofilm detection and diagnostics

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