Development of a Multispecies Biofilm Community by Four Root Canal Bacteria

doi:10.1016/j.joen.2011.11.008

Journal of Endodontics

Volume 38, Issue 3, March 2012, Pages 318-323

https://doi.org/10.1016/j.joen.2011.11.008 Get rights and content

Abstract

Introduction

The development of multispecies biofilm models are needed to explain the interactions that take place in root canal biofilms during apical periodontitis. The aim of this study was to investigate the ability of 4 root canal bacteria to establish a multispecies biofilm community and to characterize the main structural, compositional, and physiological features of this community.

Methods

Four clinical isolates isolated from infected root canals, Actinomyces naeslundii, Lactobacillus salivarius, Streptococcus gordonii, and Enterococcus faecalis, were grown together in a miniflow cell system. Simultaneous detection of the 4 species in the biofilm communities was achieved by fluorescence in situ hybridization in combination with confocal microscopy at different time points. The LIVE/DEAD BacLight technique (Molecular Probes, Carlsbad, CA) was used to assess cell viability and to calculate 3-dimensional architectural parameters such as biovolume (μm³). Redox fluorescence dye 5-cyano-2,3-ditolyl tetrazolium chloride was used to assess the metabolic activity of biofilm bacteria.

Results

The 4 species tested were able to form stable and reproducible biofilm communities. The biofilms formed in rich medium generally showed continuous growth over time, however, in the absence of glucose biofilms showed significantly smaller biovolumes. A high proportion of viable cells (>90%) were generally observed, and biofilm growth was correlated with high metabolic activity of cells. The community structure of biofilms formed in rich medium did not change considerably over the 120-hour period, during which E. faecalis, L. salivarius, and S. gordonii were most abundant.

Conclusions

The ability of 4 root canal bacteria to form multispecies biofilm communities shown in this study give insights into assessing the community lifestyle of these microorganisms in vivo. This multispecies model could be useful for further research simulating stresses representative of in vivo conditions.

Section snippets

Culture Conditions and Formation of Multispecies Biofilms

A miniflow cell system was set up to characterize the formation and growth of the multispecies biofilm community analyzed in this study (Fig. 1). The miniflow system allowed for the simultaneous coculture of 4 species of root canal organisms under identical growth conditions. The mixed species inoculum was prepared by growing planktonic cultures of 4 clinical isolates from infected root canals (7), A. naeslundii, E. faecalis (GUL6), L. salivarius (GUL1), and S. gordonii, in the liquid growth

Detection of 4 Species in Biofilm Communities by FISH

The 4 root canal organisms, A. naeslundii, E. faecalis, L. salivarius, and S. gordonii, were able to grow together and form a stable multispecies biofilm community that could be detected by FISH. All the oligonucleotide probes used in this study presented satisfactory levels of specificity and sensitivity with a standard hybridization protocol (Fig. 2A). Probe STR405, labeled with a green fluorochrome, allowed the identification of S. gordonii cells (Fig. 2B and C). Probes LAC722 (red) and

Discussion

In vitro multispecies biofilm experiments conducted under controlled conditions are required in order to address the community lifestyle of root canal bacteria and to assess the effects of stresses. In this study, a root canal biofilm microbial community was established, which included A. naeslundii, E. faecalis, L. salivarius, and S. gordonii, isolated from teeth with persistent infections. This multispecies system provided biofilm samples with a reduced variability that were readily subjected

Acknowledgments

The author thanks Dr Blythe Kaufman and Dr Larz Spångberg for valuable criticism.

The author denies any conflicts of interest related to this study.

References (29)

P.N. Nair et al.
Intraradicular bacteria and fungi in root-filled, asymptomatic human teeth with therapy-resistant periapical lesions: a long-term light and electron microscopic follow-up study
J Endod
(1990)
D. Ricucci et al.
Biofilms and apical periodontitis: study of prevalence and association with clinical and histopathologic findings
J Endod
(2010)
G. Sundqvist et al.
Microbiologic analysis of teeth with failed endodontic treatment and the outcome of conservative re-treatment
Oral Surg Oral Med Oral Pathol Oral Radiol Endod
(1998)
I.N. Rocas et al.
Polymerase chain reaction identification of microorganisms in previously root-filled teeth in a South Korean population
J Endod
(2004)
M.T. Arias-Moliz et al.
Enterococcus faecalis biofilms eradication by root canal irrigants
J Endod
(2009)
M.T. Arias-Moliz et al.
Eradication of Enterococcus faecalis biofilms by cetrimide and chlorhexidine
J Endod
(2010)
T.R. Dunavant et al.
Comparative evaluation of endodontic irrigants against Enterococcus faecalis biofilms
J Endod
(2006)
K.C. Lima et al.
Susceptibilities of Enterococcus faecalis biofilms to some antimicrobial medications
J Endod
(2001)
Y. Shen et al.
Bacterial viability in starved and revitalized biofilms: comparison of viability staining and direct culture
J Endod
(2010)
Y. Shen et al.
Antimicrobial efficacy of chlorhexidine against bacteria in biofilms at different stages of development
J Endod
(2011)

K. Sakai et al.

Fluorescent in situ hybridization analysis of open lactic acid fermentation of kitchen refuse using rRNA-targeted oligonucleotide probes

J Biosci Bioeng

(2004)

M.E. Stiles et al.

Lactic acid bacteria of foods and their current taxonomy

Int J Food Microbiol

(1997)

J. Dworkin et al.

Developmental commitment in a bacterium

Cell

(2005)

L.E. Chavez de Paz

Redefining the persistent infection in root canals: possible role of biofilm communities

J Endod

(2007)

Cited by (34)

Methods to Grow and Measure In Vitro Static Biofilms
2022, Encyclopedia of Infection and Immunity
Biofilms are microbial communities that adhere to one another or to surfaces and are composed of exopolysaccharides, extracellular nucleic acids, lipids, and proteins. They pose a major health threat because of their ability to protect pathogenic bacteria from host immune responses and antibiotic therapy. There are many methods to investigate biofilm growth of bacteria. In vitro static biofilm growth models, such as the colony biofilm model, the air-liquid interface model, and the microtiter plate model, are closed systems that have been extensively applied by many researchers. Various methods, from basic agar plate counting to advanced microscopic assays, including confocal laser scanning microscopy and many others, have been used to detect and study biofilms. There is no standard method to detect bacterial biofilms; each assay has advantages and disadvantages. This article will discuss in vitro static biofilm growth models and methods of biofilm detection and characterization.
Plasminogen coating increases initial adhesion of oral bacteria in vitro
2016, Microbial Pathogenesis
Citation Excerpt :
Clinical root canal isolates of Enterococcus faecalis, Lactobacillus salivarius, Actinomyces naeslundii, and Streptococcus gordonii and three other oral streptococci, S. oralis, S. anginosus, and S. sanguinis, were grown anaerobically in a mini-flow chamber system coated with human plasminogen. The bacterial strains used in the present study, E. faecalis, L. salivarius, A. naeslundii, and S. gordonii, were isolated from dental root canals, undergoing endodontic treatment, that presented with persistent infections [6,7]. Three additional strains of oral streptococci were also used; S. oralis, S. anginosus, and S. sanguinis.
Plasminogen is a major plasma protein and the zymogen of the broad spectrum protease plasmin. Plasmin activity leads to tissue degradation, direct and through activation of metalloproteinases. Infected tooth root canals, as a consequence of the inflammatory response and eventual necrosis, contain tissue fluid and blood components. These will coat the root canal walls and act as conditioning films that allow bacterial biofilms to grow and be a potential source of hematogenously spreading bacteria. We investigated the effect of in vitro surface conditioning with human plasminogen on the initial adhesion of bacteria. Four bacterial species, L. salivarius, E. faecalis, A. naeslundii, and S. gordonii, isolated from dental root canals, and three other oral streptococci, S. oralis, S. anginosus, and S. sanguinis, were grown in albumin- or plasminogen-coated flow chambers and studied by confocal laser scanning microscopy using the cell viability staining LIVE/DEAD and 16S rRNA fluorescence in situ hybridization (FISH). A. naeslundii, L. salivarius and in particular S. gordonii showed a higher initial adhesion to the plasminogen-coated surfaces. E. faecalis did not show any preference for plasminogen. Four-species biofilms cultured for 96 h showed that streptococci increased their proportion with time. Further experiments aimed at studying different streptococcal strains. All these adhered more to plasminogen-coated surfaces than to albumin-coated control surfaces. The specificity of the binding to plasminogen was verified by blocking lysine-binding sites with epsilon-aminocaproic acid. Plasminogen is thus an important plasma component for the initial adhesion of oral bacteria, in particular streptococci. This binding may contribute to their spread locally as well as to distant organs or tissues.
The Starvation Resistance and Biofilm Formation of Enterococcus faecalis in Coexistence with Candida albicans, Streptococcus gordonii, Actinomyces viscosus, or Lactobacillus acidophilus
2016, Journal of Endodontics
Citation Excerpt :
This speculation needs to be examined further. Multispecies biofilms of root canal bacteria have been described in previous studies (28, 29). E. faecalis biofilms on the biotic and abiotic surface are usually unstable and easily disturbed.
Enterococcus faecalis is the most frequently detected species in root canal–treated teeth, and it is able to survive under starvation conditions. However, persistent periapical disease is often caused by multispecies. The aim of this study was to explore the survival of E. faecalis in starvation conditions and biofilm formation with the 4 common pathogenic species.
A dual-species model of Candida albicans, Streptococcus gordonii, Actinomyces viscosus, or Lactobacillus acidophilus in combination with E. faecalis was established and allowed to grow in phosphate-buffered saline for the examination of starvation survival. Cefuroxime sodium and vancomycin at a concentration of 100 mg/L were added into brain-heart infusion plate agar to count the 2 bacteria separately in the dual species. Scanning electron microscopy was used to observe the dual species and multiple species on the root canal dentin of bovine teeth for 48 hours. A confocal laser scanning microscope was used to show the 4 groups of dual-species biofilms on substrates with glass bottoms for 48 hours.
E. faecalis was more resistant to starvation in coexistence with C. albicans, S. gordonii, A. viscosus, or L. acidophilus, and S. gordonii was completely inhibited in coexistence with E. faecalis. The dual-species biofilm showed that E. faecalis formed thicker and denser biofilms on the root canal dentin and glass slides in coexistence with S. gordonii and A. viscosus than C. albicans and L. acidophilus.
The multispecies community is conducive to the resistance to starvation of E. faecalis and biofilm formation in root canals.
Antibiofilm efficacy of photosensitizer-functionalized bioactive nanoparticles on multispecies biofilm
2014, Journal of Endodontics
Citation Excerpt :
The multispecies biofilms could be used as a routine model to test newer antimicrobial agents. Biofilm models using specific strains and dental plaque have been reported in the literature (24, 30). The bacterial species used in the present study have been shown to be isolated from root canals of infected teeth (31, 32).
Newer disinfection strategies based on antibacterial nanoparticles and photodynamic therapy (PDT) aim to eliminate residual biofilm bacteria during root canal treatment. The aim of the current study was to test the newly developed rose bengal–functionalized chitosan nanoparticles (CSRBnps) for their interaction/uptake with monospecies bacteria/biofilm and assess their antibiofilm efficacy on a multispecies biofilm model in vitro.
The interaction of CSRBnps with bacterial cells was conducted using atomic force microscopy. Their membrane-damaging effect was determined by measuring the absorbance at 260 nm (OD_260nm) using Enterococcus faecalis. The penetration of CSRBnps into E. faecalis biofilms was evaluated using confocal laser scanning microscopy (CLSM). Multispecies biofilms of Streptococcus oralis, Prevotella intermedia, and Actinomyces naeslundii were grown on dentin sections for 21 days to assess the antibiofilm efficacy. The biofilms were subjected to PDT (60 J/cm²) using CSRBnps and rose bengal. The treated/untreated biofilms were examined under scanning electron microscopy and CLSM.
The CSRBnps synthesized were 60 ± 20 nm and showed absorption spectra similar to rose bengal. Atomic force microscopy showed adherence of CSRBnps to bacteria, roughening of cell surface, and cell disruption after PDT. CSRBnp treatment resulted in significantly increased bacterial membrane damage (P < .05). CSRBnps exhibited deeper penetration into the biofilm structure. Scanning electron microscopy and CLSM confirmed the complete disruption of multispecies biofilm with a reduction in viable bacteria and biofilm thickness (P < .05).
These novel photosensitizer functionalized bioactive nanoparticles with increased affinity to bacterial cell membrane, higher penetration into biofilm structure, and enhanced ability to eliminate clinically relevant multispecies bacterial biofilm present a potential antibiofilm agent for root canal disinfection.
A new approach to the simulation of microbial biofilms by a theory of fluid-like pressure-restricted finite growth
2014, Computer Methods in Applied Mechanics and Engineering
In general, the term ’growth’ characterises the process by which a living body increases in size by addition of mass. Living matter grows in various different ways, triggered by genetic and biological factors. In addition, the configuration of the grown body in space depends on its interaction with the environment at the boundaries. In this paper, we deal with mechanical constraints on growth at the boundary of the body. Particularly, we present a model for growth such that residual stresses resulting from an isotropic deposition of new material are continuously relieved and that depends on the hydrostatic pressure acting on the material. As an example for this pressure-restricted fluid-like type of growth, we consider microbial biofilms growing between rigid obstacles in geometrically confined environments. The presented concept unites two classical constitutive formulations of large strain viscoelasticity and finite growth. The model was implemented into a finite element framework to illustrate its performance in several benchmark problems.
Microbial composition and diversity in intraradicular biofilm formed in situ: New concepts based on next-generation sequencing
2024, Molecular Oral Microbiology

View all citing articles on Scopus

View full text

Basic ResearchDevelopment of a Multispecies Biofilm Community by Four Root Canal Bacteria

Abstract

Introduction

Methods

Results

Conclusions

Section snippets

Culture Conditions and Formation of Multispecies Biofilms

Detection of 4 Species in Biofilm Communities by FISH

Discussion

Acknowledgments

J Endod

J Endod

Oral Surg Oral Med Oral Pathol Oral Radiol Endod

J Endod

J Endod

J Endod

J Endod

J Endod

J Endod

J Endod

J Biosci Bioeng

Int J Food Microbiol

Cell

Redefining the persistent infection in root canals: possible role of biofilm communities

J Endod

Basic Research
Development of a Multispecies Biofilm Community by Four Root Canal Bacteria