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

Abstract

BackgroundCaries 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.ConclusionsThis 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.

Heterologous Expression of sahH Reveals That Biofilm Formation Is Autoinducer-2-independent in Streptococcus sanguinis but Is Associated with an Intact Activated Methionine Cycle

“…These growth conditions proved to be optimal for biofilm formation (39). We quantified AI-2 under the selected conditions, monitored its release for 24 h, examined the A 600 , and determined the biofilm mass.…”

“…Bacteria were washed with PBS (pH 7.4) and adjusted to an A 600 of 0.6 to obtain 1 ϫ 10 8 cells/ml. Subsequently, each bacterial suspension was diluted 1:10 in a chemical defined medium (CDM) (38) supplemented with 50 mM sucrose (39) and inoculated in 96-Transwell polystyrene FIGURE 1. Schematic diagram of the AMC and the generation of the byproduct AI-2.…”

Antibacterial and antibiofilm activity of Ammi majus seed against Gram-positive bacteria

“…8 Some plants have been reported to be able to prevent the formation of biofilm in some bacteria such as Staphylococcus epidermidis, Staphylococcus aureus, Staphylococcus auricularis, Streptococcus mitis, Streptococcus salivarius, Streptococcus pneumoniae. [9][10][11] Among plants of the Apiaceae, family Ammi majus native to northern Africa, southern Europe, western Asia and India. The fruit laterally compressed, oblong, mericarps of the cremocarp separated by a carpophore.…”

Capacidade de dissolução do hipoclorito de sódio e da clorexidina sobre biofilme oral formado 'in situ'

“…Autoclave utilizada para esterilização dos blocos (C) A periodontite apical é a principal patologia associada com o fracasso do tratamento endodôntico, se encontrando nela uma vasta variedade de espécies bacterianas (SKUCAITE et al, 2010;ZIJNGE et al, 2010). No entanto, a maioria dos trabalhos analisa o poder antimicrobiano dos irrigantes endodônticos contra espécies bacterianas isoladas (JACINTO et al, 2003;EICK, SELTMANN, PFISTER, 2004;DUNAVANT et al, 2006;CLEGG et al, 2006;DUGGAN, SEDGLEY, 2007;GIARDINO et al, 2007;OZOK et al, 2007;SEDLACEK, WALKER, 2007;CAMARA et al, 2009;ARIAS-MOLIZ et al, 2010;CHAVEZ DE PAZ, BERGENHOLTZ, SVENSATER, 2010), não permitindo as interações bacterianas características dos biofilmes multi-espécie que os fazem mais resistentes (COOK, COSTERTON, LAMONT, 1998;STANDAR et al, 2010;ZIJNGE et al, 2010).…”

“…Os estudos in vitro são muito importantes para determinar as interações bacterianas que ocorrem em um biofilme (KOLENBRANDER et al, 1993;COOK;LAMONT, 1998;PALMER et al, 2001;DIAZ et al, 2006;STANDAR et al, 2010), mas não são capazes de simular de uma forma exata as condições de crescimento bacteriano na boca, como por exemplo, a diversidade de nutrientes, mudanças de humidade e pH, temperatura, saliva; etc.…”