Quantitation of biofilm and planktonic life forms of coexisting periodontal species.
BACKGROUND
Complexity of oral polymicrobial communities has prompted a need for developing in vitro models to study behavior of coexisting bacteria. Little knowledge is available of in vitro co-growth of several periodontitis-associated species without early colonizers of dental plaque.


OBJECTIVE
was to determine temporal changes in the quantities of six periodontal species in an in vitro biofilm model in comparison with parallel planktonic cultures.


METHODS
Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Prevotella intermedia, Parvimonas micra, Campylobacter rectus and Fusobacterium nucleatum were anaerobically grown as multispecies and monospecies biofilms and parallel planktonic cultures using cell culture plates and microfuge tubes, respectively. After incubating 2, 4, 6, 8 days, biofilms and planktonic cultures were harvested, DNA extracted and the target species quantified using qPCR with species-specific 16S rDNA primers. Biofilm growth as monocultures was visualized at day 2 and 8 with confocal microscopy and crystal violet staining.


RESULTS
The six species were found throughout the test period in all culture conditions, except that P. gingivalis and F. nucleatum were not detected in multispecies planktonic cultures at day 8. In multispecies biofilm, P. gingivalis qPCR counts (cells/ml) increased (P<0.05) from day 2-8 and were then higher (P<0.05) than those of A. actinomycetemcomitans and C. rectus, whereas in monospecies biofilm, P. gingivalis counts were lower (P<0.05) than those of the other species, except A. actinomycetemcomitans. When multi- and monospecies biofilm cultures were compared, P. gingivalis counts were higher (P<0.05) but those of the other species, except P. intermedia, lower (P<0.05) in multispecies biofilm. Comparison between planktonic and biofilm cultures showed that A. actinomycetemcomitans, P. micra and C. rectus had higher (P<0.05) counts in planktonic cultures no matter whether grown in mono- or multispecies environment.


CONCLUSIONS
Six periodontal species were able to form multispecies biofilm up to 8 days in vitro without pioneer plaque bacteria. P. gingivalis seemed to prefer multispecies biofilm environment whereas P. micra and A. actinomycetemcomitans planktonic culture.
Oral Microbiology, General Facility Laboratory, Faculty of Dentistry, Kuwait University, Kuwait.
Maribasappa
M
Karched
Oral Microbiology, General Facility Laboratory, Faculty of Dentistry, Kuwait University, Kuwait.
Radhika G
RG
Bhardwaj
Oral Microbiology, General Facility Laboratory, Faculty of Dentistry, Kuwait University, Kuwait.
Anandavalli
A
Inbamani
Oral Microbiology, General Facility Laboratory, Faculty of Dentistry, Kuwait University, Kuwait. Electronic address: sirkka.asikainen@hsc.edu.kw.
Sirkka
S
Asikainen
D016976:Aggregatibacter actinomycetemcomitans / Q000235:genetics / Q000254:growth & development / Q000502:physiology; D018441:Biofilms; D044883:Campylobacter rectus / Q000235:genetics / Q000254:growth & development / Q000502:physiology; D003773:Dental Plaque / Q000382:microbiology*; D000068536:Bacillota / Q000235:genetics / Q000254:growth & development / Q000502:physiology; D016967:Fusobacterium nucleatum / Q000235:genetics / Q000254:growth & development / Q000502:physiology; D010519:Periodontium / Q000382:microbiology*; D010933:Plankton / Q000235:genetics / Q000254:growth & development / Q000502:physiology*; D016966:Porphyromonas gingivalis / Q000235:genetics / Q000254:growth & development / Q000502:physiology; D018713:Prevotella intermedia / Q000235:genetics / Q000254:growth & development / Q000502:physiology
Biofilm; Confocal microscopy; Crystal violet; Late plaque colonizers; Periodontitis; Planktonic; Quantitative real-time PCR