Biofilm forming ability of Actinomyces naeslundii strains obtained from children with aggressive
periodontitis
Miyai Yukiko*, Naka Shuhei, Matsumoto-Nakano Michiyo
Department of Pediatric Dentistry, Okayama University Graduate School of Medicine, Dentistry andPharmaceutical Sciences.
- Objectives
We have encountered cases of aggressive periodontitis with premature loss of deciduous teeth due torapid alveolar bone destruction. In our previous study, a significantly higher percentage of suchpatients were found to possess Actinomyces naeslundii, a pathogenic bacterium associated withbiofilm formation. The present study was conducted to investigate the biofilm formation capability ofA. naeslundii strains isolated from those patients.
- Materials and Methods
Four A. naeslundii strains isolated from four patients as well as A. naeslundii JCM8350 were used.
Each was grown overnight at 37°C, then inoculated into Trypticase soy broth. Growth curves weredetermined by measuring changes in optical density at 550 nm at one-hour intervals at 37°C under ananaerobic condition. Next, biofilms formed by each strain were stained with 1% crystal violet andabsorbance at 570 nm was measured using a microplate reader. Furthermore, biofilm formed by thebacterial cells was labeled with 10 mM hexidium iodide and examined, with observations wereperformed using a confocal scanning laser microscope.
- Results
The growth rate of an A. naeslundii strains isolated form a patient with severe alveolar bone resorption
was significantly lower as compared to that of the strains isolated from the other patients as well as
JCM8350, while the quantity of biofilm it formed was also tended to be lower. Additionally, thebiofilm associated with that strain showed a non-uniform three-dimensional structure with interlinked
bacteria, while biofilms formed by the other strains were found to be comprised of the two parts, rigidand loose.
- Conclusion
These results suggest that A. naeslundii strains have varied biofilm forming abilities related to thepathogenicity of aggressive periodontitis.

Antimicrobial resistance of Streptococcus mutans organisms isolated from children
undergoing hematopoietic stem cell transplantation
Hikari Ogawa, Kana Goto, Keiko Hirano, Yuko Morikawa, Michiyo Matsumoto-Nakano
Department of Pediatric Dentistry, Okayama University Graduate School of Medicine,Dentistry and Pharmaceutical Sciences, JapanObjectives
For pediatric patients undergoing hematopoietic stem cell transplantation (HSCT), highdose chemotherapy and radiation therapy are given as a conditioning regimen, along withcontinuous antimicrobial agent administration for the resulting immunocompromisedstate. Following HSCT, oral conditions and flora generally worsen, leading to dental
caries development. Using samples isolated from children undergoing HSCT, the present
study investigated the antimicrobial resistance of Streptococcus mutans, a cariogenic
bacterium, in those isolates.
Materials and Methods
The Okayama University Ethics Review Committee approved this study (Lab 1508-016).
Saliva samples were collected from pediatric patients before, and one and three months
after undergoing HSCT, then plated in Mitis Salivarius agar including bacitracin to isolate
S. mutans. Minimum inhibitory concentration (MIC) values were determined using anantibacterial susceptibility test. RNA sequencing analysis was also performed to confirm
the expression status of all antimicrobial resistant S. mutans genes. Furthermore, usingfluorescent probes, the plasma membrane fluidity of antimicrobial-resistant S. mutansorganisms was determined and compared to that of S. mutans MT8148.
Results
In samples obtained one month after HSCT, all S. mutans organisms showed resistanceto various antimicrobial agents. The MIC of the organisms for showing resistance to βlactams, penicillin, cephems, and carbapenems at one month was higher as compared tobefore as well as three months after treatment. Furthermore, 94 genes demonstrated
increased expression in S. mutans organisms with as compared to without antimicrobial
resistance, including genes related to ABC transporter and permease drugs. All
antimicrobial-resistant S. mutans had lower levels of plasma membrane fluidity than that
noted for MT8148.
Conclusion
S. mutans was found to acquire antimicrobial resistance following HSCT, thus reducing
the effects of extracellular antimicrobials.

Analysis of ABC-transporter related genes in Streptococcus mutans associated withbiofilm formation
Sakuya Matsuura, Haruka Asaumi, Kana Goto, and Michiyo Matsumoto-Nakano
Department of Pediatric Dentistry, Okayama University Graduate School of Medicine,Dentistry and Pharmaceutical Sciences, Okayama, Japan
Objectives: Streptococcus mutans has been implicated as a primary causative agent ofdental caries in humans. One of the important virulence properties of S. mutans organisms
is their ability to form biofilm, resulting in dental plaque on tooth surfaces. In general,
biofilm formation is initiated by interactions between planktonic bacteria and the surface
in response to environmental stress factors, with the mechanism likely related to ATPbinding cassette (ABC) transporters. However, details related to of this mechanism
remain unclear. The present study examined gene expressions in biofilm-forming bacteria
and extracted SMU922, an ABC transporter-related gene, then, biological functions wereinvestigated using SMU922-deficient mutant strains.
Materials and Methods: The DNA fragment encoding SMU922 was amplified by PCR
using specific primer sets and ligated into a pGEM-T Easy Vector. Next, the resultant
plasmid was digested with EcoRI and inserted into an erythromycin resistance cassette,linearized with SalⅠ, and used to transform S. mutans MT8148, which was termed
SMU922 deletion mutant strain (Δ922). To examine bacterial growth, MT8148 and Δ922
were grown for 18 hours at 37°C, then inoculated into Todd Hewitt (TH) broth at 37°C.
Cell density was determined by measuring he optical density (OD) at 600 nm. In addition,
MT8148 and Δ922 were inoculated into 100 μl of TH broth containing 0.5% or 1%
sucrose in 96-well polystyrene microtiter plates. Following incubation for 48 hours,
crystal violet staining was performed and biofilm formation quantified by measuring
OD570.
Results: The growth rate of Δ922 was similar to that of MT8148. On the other hand, thequantities of biofilm formed by Δ922 were significantly reduced as compared to thoseformed by MT8148 in broth containing 0.5% or 1% sucrose.
Conclusion: These results suggest that SMU922 is associated with biofilm formation.