ORIGINAL ARTICLE

Molecular detection of Aggregatibacter

actinomycetemcomitans on metallic brackets
by the checkerboard DNA-DNA hybridization
technique
~o Andrucioli,c Magda Feres,d
Paulo Nelson-Filho,a Karla Orfelina Carpio-Horta,b Marcela Cristina Damia
Raquel Assed Bezerra da Silva,e Francisco Wanderley Garcia Paula-Silva,f and Fabio Lourenço Romanoe
S~ao Paulo and Guarulhos, Brazil

Introduction: The purpose of this randomized clinical study was to evaluate the presence of the periodontal
pathogen Aggregatibacter actinomycetemcomitans on metallic brackets and the effectiveness of a 0.12% chlo-
rhexidine digluconate mouthwash in inhibiting this microorganism. Methods: The study involved 35 patients of
both sexes having orthodontic treatment with fixed appliances between the ages of 14 and 22 years, randomized
into 2 groups: experimental (n 5 17) and control (n 5 18). Two new metallic brackets were placed on the patients'
premolars, and the subjects rinsed with a solution of 0.12% chlorhexidine digluconate or a placebo solution twice
a week for 30 days. After that, the brackets were removed and underwent microbiologic analysis with the check-
erboard DNA-DNA hybridization technique. Data were analyzed by using the Student t, Fisher exact, and Mann-
Whitney tests at the significance level of 5%. Results: The results showed that A actinomycetemcomitans was
present in all brackets from the subjects in the control group vs 83% of the subjects who rinsed with chlorhexidine
digluconate (P \0.0001). There were also significantly lower levels of this species in the chlorhexidine digluco-
nate group compared with the control group (P 5 0.0003). Conclusions: We concluded that 0.12% chlorhex-
idine digluconate rinsing, twice a week for 30 days during orthodontic treatment, is effective in reducing the
presence and levels of A actinomycetemcomitans on metallic brackets. (Am J Orthod Dentofacial Orthop
2012;142:481-6)

a
Chairman and professor, Department of Pediatric Clinics, Preventive and Social
Dentistry, School of Dentistry of Ribeir~ao Preto, University of S~ao Paulo, Ribeir~ao
Preto, S~ao Paulo, Brazil.
b
Resident, Department of Orthodontics, Department of Pediatric Clinics, School
of Dentistry of Ribeir~ao Preto, University of S~ao Paulo, Ribeir~ao Preto, S~ao Paulo,
Brazil.
c
Postgraduate student, Department of Pediatric Clinics, Preventive and Social
Dentistry, School of Dentistry of Ribeir~ao Preto, University of S~ao Paulo, Ribeir~ao
Preto, S~ao Paulo, Brazil.
d
Professor, Dental Research Division, Department of Periodontology, University
of Guarulhos, Guarulhos, Brazil.
e
Professor, Department of Pediatric Clinics, Preventive and Social Dentistry,
School of Dentistry of Ribeir~ao Preto, University of S~ao Paulo, Ribeir~ao Preto,
S~ao Paulo, Brazil.
f
Pedodontist, Department of Pediatric Clinics, Preventive and Social Dentistry,
School of Dentistry of Ribeir~ao Preto, University of S~ao Paulo, Ribeir~ao Preto,
S~ao Paulo, Brazil.
The authors report no commercial, proprietary, or financial interest in the prod-
ucts or companies described in this article.
Financial support from the National Council for Scientific and Technological De-
velopment (CNPq) (481894/2007-1 and 304793/2007-8).
Reprint requests to: Paulo Nelson-Filho, Departamento de Clınica Infantil, Odon-
tologia Preventiva e Social, Faculdade de Odontologia de Ribeir~ao Preto, USP,
Avenida do Cafe, s/n Ribeir~ao Preto, S~ao Paulo, Brazil 14040-904; e-mail,
nelson@forp.usp.br.
Submitted, October 2011; revised and accepted, April 2012.
0889-5406/$36.00
Copyright Ó 2012 by the American Association of Orthodontists.
http://dx.doi.org/10.1016/j.ajodo.2012.04.021
O
rthodontic appliances create a favorable environ-
ment for the accumulation of microbial biofilm,1
which can lead to adverse effects on the perio-
dontium.2,3 The periodontal pathogen, Aggregatibacter
actinomycetemcomitans,4-6 is a gram-negative cocoba-
cillus,7,8 considered a main pathogen of inflammatory
periodontal disease, and has been related to aggressive
periodontitis.9 It is also associated with certain systemic
infections such as endocarditis, meningitis, osteomyeli-
tis, and brain abscesses.10-12
Periodontitis is difficult to control by mechanical
removal of the subgingival biofilm alone,13,14 and
studies have indicated that this kind of treatment is not
effective in eliminating A actinomycetemcomitans.15,16
Therefore, there is great interest about the effects of
systemic antibiotics and other antimicrobial agents
in controlling this periodontal pathogen.17-19 Several
commercially available mouthwashes are used to
improve oral hygiene, although their efficacy is
controversial. To improve the knowledge on this
matter, antimicrobial studies have been conducted;
however, most of them were tested against cariogenic
481
482
bacteria.20-25 Chlorhexidine is a topical antimicrobial
agent that has positive effects in inhibiting plaque
formation and reducing the bacteria in the oral
cavity.26-30
Microbial diagnostic tests with genomic DNA probes
have been widely used in the detection and quantification
of pathogenic microorganisms,31-33 because they are
faster and more reliable than conventional microbiologic
culture techniques.34,35 The checkerboard DNA-DNA hy-
bridization technique allows rapid identification of several
bacterial species in a large number of oral samples.36,37
This technique has been used in the dental areas of
periodontology, endodontics, implantology, pediatric
dentistry, and cariology.24,30,38-42 In orthodontics, this
technique was used to compare the total bacterial
counts on ceramic and metallic brackets, and it has
been shown that metallic brackets can be highly
contaminated with A actinomycetemcomitans.1
Therefore, the aim of this pilot clinical study was to in-
vestigate, in vivo, the contamination of metallic brackets
by the periodontal pathogen A actinomycetemcomitans
by using the checkerboard DNA-DNA hybridization
technique and the efficacy of a 0.12% chlorhexidine
mouthwash in controlling this microorganism.
MATERIAL AND METHODS
Eligible participants were selected from patients of
both sexes in orthodontic corrective treatment for less
than 16 months at the orthodontic clinic of the School
of Dentistry of Ribeir~ao Preto, University of S~ao Paulo,
in Brazil. They had good general health and had not
used antibiotics or antimicrobial mouthwashes within
3 months before the study. Thirty-five 14- to 22-year-
old patients who met these inclusion criteria were
enrolled as participants. The research protocol was
reviewed and approved by the institutional research
ethics committee (process 2008.1.163.58.8).
One week before the beginning of the study, each pa-
tient's plaque index was determined by 1 operator
(M.C.D.A.) according to the method of Silness and
Loe43 to confirm that all patients had similar amounts
of dental biofilm. A biofilm disclosing agent was used,
and scores were attributed to the 4 sides of each tooth
as follows: 0, no biofilm on tooth surface; 1, biofilm
was invisible to the naked eye but could be collected
with a probe; 2, biofilm was visible at the gingival mar-
gin; and 3, biofilm was visible at the gingival margin and
covered a significant portion of the tooth surface. The
average score for a tooth was equal to the sum of the
scores for all sides divided by 4, and the plaque index
for each patient was equal to the sum of the average
scores for all teeth divided by the number of teeth.
October 2012  Vol 142  Issue 4 American Journal of Orthodontics and Dentofacial Orthopedics
Nelson-Filho et al
Next, dental biofilm deposits were cleaned by using non-
fluoridated pumice and water. The patients were in-
structed to brush their teeth at least 3 times a day
after meals using a toothbrush (Professional; Colgate-
Palmolive Ind ustria e Comercio, S~ao Paulo, S~ao Paulo,
Brazil) and fluoride-containing dentifrice (Maxima
Proteç~ao Anticaries; Colgate-Palmolive Ind ustria e
Comercio) supplied by the researchers throughout the
experimental period. In this study, all patients had sim-
ilar baseline levels of dental plaque (range, 0.5-1.5) and
were periodontally healthy.
The 35 patients were randomized to 2 groups (exper-
imental and control) by using statistical software (version
9.1.3 for Windows; SAS Institute, Cary, NC). In all pa-
tients, 2 new sterile edgewise metallic orthodontic
brackets (0.022 3 0.028-in slot) (Generus; GAC Interna-
tional, Bohemia, NY) were bonded with orthodontic
light-cured composite (Transbond XT; 3M Unitek, Mon-
rovia, Calif) to 2 premolars (maxillary right or left and
mandibular right or left) selected randomly with the
SAS software. Seventeen patients used 0.12% chlorhex-
idine mouthwash (Periogard; Kollynos do Brasil, Osasco,
S~ao Paulo, Brazil) (experimental) as an antimicrobial
agent, and 18 patients used a placebo mouthwash
(Erva Doce Pharmacy, Ribeir~ao Preto, S~ao Paulo, Brazil)
with color, taste, and composition similar to those of
Periogard (control). In both groups of patients, oral rinses
were done with 10 mL of the test solution for 30 seconds
twice a week (Tuesdays and Fridays) for 30 days. On
Tuesdays, mouth rinsing was performed at the dental
school under the researchers' supervision; on Fridays,
mouth rinsing was done at the patients' homes at night,
1 hour after toothbrushing. Both solutions were stored in
120-mL plastic bottles and were given to the subjects
every week after prophylaxis. The patients were blinded
to which mouthwash was being used.
After 30 days, the brackets were removed with pliers
(How 110; 3M Unitek) by a trained, calibrated operator
(M.C.D.A.) (orthodontist) in a blinded fashion and sent
for analysis. Two brackets from each patient of both
groups were placed in individual labeled plastic tubes
(Eppendorf AG Barkhausenweg, Hamburg, Germany),
containing 150 mL of Tris-EDTA buffer solution (pH
7.6) and 100 mL of 0.5 M sodium hydroxide, and were
agitated for microbial desorption. Next, the brackets
were removed with sterile clinical pliers, and the plastic
tubes containing the bacterial suspension were stored
frozen at –20
C for further analysis with the checker-
board DNA-DNA hybridization technique.31 The pa-
tients received new brackets, and corrective
orthodontic treatment proceeded as planned.
The DNA probe was prepared by using whole geno-
mic DNA from A actinomycetemcomitans (strains
Nelson-Filho et al 483

43718 and 29523). The collected samples were boiled

Table I. Distribution of subjects according to age, sex,
for 10 minutes to produce cell lysis and DNA denatur-
and plaque index
ation. The DNA was then fixed in individual lanes on
a positively charged nylon membrane (Boehringer Man- Control group Experimental group
nheim, Indianapolis, Ind) by using a checkerboard slot (n 5 18) (n 5 17) P
blot device (Minislot 30; Immunetics, Cambridge, Age (y) 15.3 6 1.3 20.6 6 1.7 0.02*
Sex
Mass). The digoxigenin-labeled genomic DNA probe Female 44.5% 58.8% 0.06y
(Roche Applied Science, Indianapolis, Ind) was then hy- Male 55.5% 41.2%
bridized perpendicularly to the lanes of the clinical sam- Plaque index
ples by using a Miniblotter 45 apparatus (Immunetics). Baseline 0.92 6 0.30 1.04 6 0.24 0.19*
Bound probes were detected with phosphatase- 30 days 0.88 6 0.37 0.51 6 0.15 0.0006*
P 0.2358z \ 0.0001z
conjugated antibody to digoxigenin (Roche Applied Sci-
ence). After incubation in a solution containing the Values are shown as averages and standard deviations.
*P value for the Student t test; yP value for the Fisher exact test; zP
CDP-Star substratum (Amersham Pharmacia Biotech value for the paired Student t test, comparing baseline vs 30 days.
Inc, Piscataway, NJ), the membranes were placed in an
autoradiography cassette under a radiographic film
(X-Omat; Kodak, Rochester, NY), developed for chemilu-
minescence signal detection. Signals were evaluated vi-
sually by comparing with the standards at 105 and 106
bacterial cells of the test species on the last 2 lanes of
the same membrane. This provided the approximate
number of bacterial cells per sample for the bacterial
strain evaluated; this was equal to the sum of the values
obtained in the 2 brackets removed from each patient.
The data were read twice by a blinded examiner
(M.C.D.A.) (kappa, .0.8). The sensitivity of this assay
was settled to allow detection of 104 cells. To permit
semiquantitative examination of chemiluminescence
signals for A actinomycetemcomitans in each sample,
the intensity of the signals (ie, contamination of the
brackets) was evaluated at the following levels: 0 (not
Fig. Percentages of the scores representing the
detected), 1 3 104, 1 3 105, 5 3 105, 1 3 106, and 1
amounts of A actinomycetemcomitans detected on the
3 107. Data were analyzed by using the Student t, Fisher metallic brackets of the experimental and control groups.
exact, and Mann-Whitney tests at the significance level
of 5%.
group and in 82.3% of the brackets in the experimental
group (P \0.0001). In addition, it was observed that al-
RESULTS most 40% of the brackets in the control group had
The 35 patients selected participated for the entire higher levels of A actinomycetemcomitans (5 3 105)
study period. The results of the randomization analysis vs less than 10% in the chlorhexidine group. Conversely,
of the subjects in the control and experimental groups about 5% of the brackets in the control group and more
are described in Table I. There was no statistical differ- than 50% of those in the experimental group had low
ence in the sex of the members of the 2 groups levels of this species (1 3 104) (Fig).
(P .0.05). However, the subjects in the experimental When the total levels were considered, the experimen-
group had a higher average age than did those in the tal group, in which the patients used the chlorhexidine
control group (P 5 0.02). mouthwash, showed a significantly lower level of this
At baseline, all patients had similar levels of dental pathogen compared with the controls (P 5 0.0003)
plaque (P 5 0.19), whereas after using the mouthwash (Table II).
with chlorhexidene, the experimental group had less
dental plaque accumulation compared with the control DISCUSSION
group (P 5 0.0006) (Table I). For this study, we chose to examine the presence and
In terms of prevalence, A actinomycetemcomitans levels of A actinomycetemcomitans bacteria on metallic
were detected in 100% of the brackets in the control brackets because prior evidence indicated that this

American Journal of Orthodontics and Dentofacial Orthopedics October 2012  Vol 142  Issue 4
484
Table II. Comparison between the experimental and control groups for the mean numbers of bacterial cells of A ac-
tinomycetemcomitans on metallic brackets
Control group (n 5 18)
A actinomycetemcomitans 300,000 (100,000-500,000)
Values are expressed as M (Q1-Q3), where M is the median, Q1 is the first quartile, and Q3 is the third quartile.
*P value for the Mann Whitney test.
species might be highly associated with gingival inflam-
mation and periodontal destruction.44 The checker-
board DNA-DNA hybridization is widely used to assess
oral microbiota38 and to determine the effect of antimi-
crobial therapies, mainly in periodontics.44,45 However,
to date, no published orthodontic studies have
evaluated the clinical effectiveness of an antimicrobial
agent against A actinomycetemcomitans with this
technique.
We found that the brackets of all patients in the con-
trol group were colonized by A actinomycetemcomi-
tans, most of them by high levels of these pathogens.
In addition, most brackets in the experimental group
(83%) were also colonized by this species, although in
lower levels. These data indicate that A actinomycetem-
comitans can colonize orthodontic brackets, in agree-
ment with a previous study that also used the same
microbiologic technique.1 These data might have direct
clinical implications, since this microorganism has
been associated with the onset and progression of peri-
odontal diseases, especially in young patients.46
Although the average ages in the control and exper-
imental groups were different, the microbiota of an ad-
olescent with a complete permanent dentition was the
same as that of an adult with a complete permanent
dentition, and this microbiota starts changing only
with dental loss.47 Also, the lowest (14 years) and the
highest (22 years) ages of the participants correspond
to ages at which persons have adequate motor skills
for the mechanical control of bacterial biofilm. Even
though the influence of individual compliance in
toothbrushing might be pointed out, similar levels of
dental plaque at baseline indicate that dental hygiene
performance was not an issue for the groups in this
study.
Antimicrobial agents might be needed to completely
remove microorganisms from areas that are difficult to
be reached by toothbrushing.15,16 Chlorhexidine
(0.12%) was chosen as the rinsing solution in this
study because it is considered to be the most effective,
and it is the most widely used antimicrobial agent in
dentistry.17 In addition, it has recently been shown
that 0.12% chlorhexidine can reduce the levels of A
actinomycetemcomitans cells cultivated in an in-vitro
hydrodynamic biofilm model.48 Those in-vitro findings
October 2012  Vol 142  Issue 4 American Journal of Orthodontics and Dentofacial Orthopedics
Nelson-Filho et al
Experimental group (n 5 17) P
10,000 (5,000-77,500) 0.0003*
are corroborated by our results, which demonstrate
in vivo a reduction in the counts of this bacterium in pa-
tients who used a mouthwash containing chlorhexidine.
Also, chlorhexidine treatment reduced dental plaque ac-
cumulation compared with the control group.
Chlorhexidine has reversible local side effects such as
staining of teeth, impaired sense of taste, increased
formation of supragingival calculus, sialolithiasis, and
occasionally mucous membrane irritation and desqua-
mation.49-51 Although in this study the subjects were
instructed to use the mouthwash only twice a week,
which is a much lower frequency than in other studies
(usually twice a day), the side effects of chlorhexidine
were not evaluated. Therefore, further studies are
necessary to confirm whether this protocol can
minimize its side effects.
This study demonstrated that, even when used less
frequently, for 30 days, chlorhexidine mouthwash re-
duces the presence and levels of A actinomycetemcomi-
tans on orthodontic brackets without causing
undesirable side effects. However, further studies should
be conducted to evaluate this product's effectiveness on
other microorganisms that might also be found on or-
thodontic brackets.
CONCLUSIONS
In vivo, metallic brackets had elevated levels of the
periodontal pathogen A actinomycetemcomitans. The
total levels of A actinomycetemcomitans, however,
were significantly lower in subjects rinsing twice
a week with a solution of 0.12% chlorhexidine for 30
days. Thus, the use of a mouthwash containing chlo-
rhexidine might be an option in clinical practice for pa-
tients having orthodontic corrective appliances to
reduce the contamination by this important periodontal
pathogen.
We thank Dr Luciene Figueiredo from the University
of Guarulhos for helpful assistance with the checker-
board DNA-DNA hybridization technique.
REFERENCES
1. Anhoury P, Nathanson D, Hughes CV, Socransky S, Feres M,
Chou LL. Microbial profile on metallic and ceramic bracket mate-
rials. Angle Orthod 2002;72:338-43.
Nelson-Filho et al
2. Ash MM, Gitlin BN, Smith WA. Correlation between plaque and
gingivitis. J Periodontol 1964;35:424-9.
3. Kloehn JS, Pfeifer JS. The effect of orthodontic treatment on the
periodontium. Angle Orthod 1974;44:127-34.
4. Sallum EJ, Nouer DF, Klein MI, Gonçalves RB, Machion L, Sallum AW,
et al. Clinical and microbiologic changes after removal of orthodontic
appliances. Am J Orthod Dentofacial Orthop 2004;126:363-6.
5. Paolantonio M, Festa F, di Placido G, D'Attilio M, Catamo G,
Piccolomini R. Site-specific subgingival colonization by Actinoba-
cillus actinomycetemcomitans in orthodontic patients. Am J Or-
thod Dentofacial Orthop 1999;115:423-8.
6. Lundstr€ om F, Hamp SE, Nyman S. Systematic plaque control in
children undergoing long-term orthodontic treatment. Eur J Or-
thod 1980;2:27-39.
7. Mandel RL, Socransky SS. A selective medium for Actinobacillus
actinomycetemcomitans and the incidence of the organism in ju-
venile periodontitis. J Periodontol 1981;52:593-8.
8. Slots J. Update on Actinobacillus actinomycetemcomitans and
Porphyromonas gingivalis in human periodontal disease. J Int
Acad Periodontol 1999;1:121-6.
9. Tonetti MS, Mombelli A. Early-onset periodontitis. Ann Periodon-
tol 1999;4:39-53.
10. Overholt BF. Actinobacillus actinomycetemcomitans endocardi-
tis. Arch Intern Med 1966;117:99-102.
11. Muhle I, Rau J, Ruskin J. Vertebral osteomyelitis due to Actinoba-
cillus actinomycetemcomitans. JAMA 1979;241:1824-5.
12. Martin BF, Derby BM, Budzilovich GN, Ransohoff J. Brain abscess
due to Actinobacillus actinomycetemcomitans. Neurology 1967;
17:833-7.
13. Mombelli A, Gm€ ur R, Gobbi C, Lang NP. Actinobacillus actinomy-
cetemcomitans in adult periodontitis. II. Characterization of iso-
lated strains and effect of mechanical periodontal treatment. J
Periodontol 1994;65:827-34.
14. Takamatsu N, Yano K, He T, Umeda M, Ishikawa I. Effect of initial
periodontal therapy on the frequency of detecting Bacteroides
forsythus, Porphyromonas gingivalis, and Actinobacillus actino-
mycetemcomitans. J Periodontol 1999;70:574-80.
15. Cugini MA, Haffajee AD, Smith C, Kent RL Jr, Socransky SS. The
effect of scaling and root planning on the clinical and microbio-
logical parameters of periodontal diseases: 12-month results. J
Clin Periodontol 2000;27:30-6.
16. Renvert S, Wilkstr€ om M, Dahlen G, Slots J, Egelberg J. On the in-
ability of root debridement on periodontal surgery to eliminate
Actinobacillus actinomycetemcomitans from periodontal
pockets. J Clin Periodontol 1990;17:351-5.
17. Mandel ID. Antimicrobial mouthrinses: overview and update. J Am
Dent Assoc 1994;125(Suppl 2):2S-10S.
18. Van der Ouderaa FJG. Anti-plaque agents. Rationale and prospects
for the prevention of gingivitis and periodontal disease. J Clin Pe-
riodontol 1991;18:447-54.
19. van Winkelhoff AJ, Rodenburg JP, Goene RJ, Abbas F, Winkel EG,
de Graaff J. Metronidazole plus amoxicillin in the treatment of Ac-
tinobacillus actinomycetemcomitans associated periodontitis. J
Clin Periodontol 1989;16:128-31.
20. Ahn SJ, Lee SJ, Lim BS, Nahm DS. Quantitative determination of
adhesion patterns of cariogenic streptococci to various orthodon-
tic brackets. Am J Orthod Dentofacial Orthop 2007;132:815-21.
21. Ahn SJ, Lim BS, Lee SJ. Prevalence of cariogenic streptococci on
incisor brackets detected by polymerase chain reaction. Am J Or-
thod Dentofacial Orthop 2007;131:736-41.
22. Faltermeier A, Burgers R, Rosentritt M. Bacterial adhesion of
Streptococcus mutans to esthetic bracket materials. Am J Orthod
Dentofacial Orthop 2008;133(Suppl):S99-103.
American Journal of Orthodontics and Dentofacial Orthopedics
485
23. Sari E, Birinci I. Microbiological evaluation of 0.2% chlorhexidine
gluconate mouth rinse in orthodontic patients. Angle Orthod
2007;77:881-4.
24. Nelson-Filho P, Olmedo LY, Andrucioli MC, Saraiva Mda C,
Matsumoto MA, de Queiroz AM, et al. Use of the checkerboard
DNA-DNA hybridisation technique for in vivo detection of cario-
genic microorganisms on metallic brackets, with or without use
of an antimicrobial agent. J Dent 2011;39:513-7.
25. Dogan AA, Cetin ES, H€ ussein E, Adiloglu AK. Microbiological eval-
uation of octenidine dihydrochloride mouth rinse after 5 days' use
in orthodontic patients. Angle Orthod 2009;79:766-72.
26. Addy M. Chlorhexidine compared with other locally delivered an-
timicrobials. A short review. J Clin Periodontol 1986;13:957-64.
27. Gjermo P. Chlorhexidine in dental practice. J Clin Periodontol
1974;1:143-52.
28. Rijkom H, Truin G, Van't Hof M. A meta-analysis of clinical studies
on the caries-inhibiting effect of chlorhexidine treatment. J Dent
Res 1996;75:790-5.
29. Ousehal L, Lazrak L, Es-Said R, Hamdoune H, Elquars F,
Khadija A. Evaluation of dental plaque control in patients wearing
fixed orthodontic appliances: a clinical study. Int Orthod 2011;9:
140-55.
30. Nelson-Filho P, Valdez RM, Andrucioli MC, Saraiva MC, Feres M,
Sorgi CA, et al. Gram-negative periodontal pathogens and bacte-
rial endotoxin in metallic orthodontic brackets with or without an
antimicrobial agent: an in-vivo study. Am J Orthod Dentofacial Or-
thop 2011;140:e281-7.
31. Gracias KS, McKillip JL. A review of conventional detection and
enumeration methods for pathogenic bacteria in food. Can J Mi-
crobiol 2004;50:883-90.
32. Mastronardi CC, Ramirez-Arcos S. Quantitative PCR for detection
and discrimination of the bloodborne pathogen Staphylococcus
epidermidis in platelet preparations using divIVA and icaA as tar-
get genes. Can J Microbiol 2007;53:1222-31.
33. Baddour MM, Alkhalifa DH. Evaluation of three polymerase chain
reaction techniques for detection of Brucella DNA in peripheral
human blood. Can J Microbiol 2008;54:352-7.
34. Colombo AP, Teles RP, Torres MC, Rosalem W, Mendes MC,
Souto RM, et al. Effects of non-surgical mechanical therapy
on the subgingival microbiota of Brazilians with untreated
chronic periodontitis: 9-month results. J Periodontol 2005;76:
778-84.
35. Haffajee AD, Japlit M, Bogren A, Kent RL, Goodson JM,
Socransky SS. Differences in the subgingival microbiota of
Swedish and USA subjects who were periodontally healthy or ex-
hibited minimal periodontal disease. J Clin Periodontol 2005;32:
33-9.
36. Socransky SS, Smith C, Martin L, Paster BJ, Dewhirst FE, Levin AE.
“Checkerboard” DNA-DNA hybridization. Biotechniques 1994;17:
788-92.
37. Socransky SS, Haffajee AD, Smith C, Martin L, Haffajee JA,
Uzel NG, et al. Use of checkerboard DNA-DNA hybridization to
study complex microbial ecosystems. Oral Microbiol Immunol
2004;19:352-62.
38. Matarazzo F, Figueiredo LC, Cruz SE, Faveri M, Feres M. Clinical
and microbiological benefits of systemic metronidazole and amox-
icillin in the treatment of smokers with chronic periodontitis: a ran-
domized placebo-controlled study. J Clin Periodontol 2008;35:
885-96.
39. Siqueira JF Jr, Rocas IN. The microbiota of acute apical abscesses. J
Dent Res 2009;88:61-5.
40. Nascimento C, Barbosa RE, Issa JP, Watanabe E, Ito IY, de Albu-
querque Junior RF. Use of checkerboard DNA-DNA hybridization
October 2012  Vol 142  Issue 4
486
to evaluate the internal contamination of dental implants and
comparison of bacterial leakage with cast or pre-machined abut-
ments. Clin Oral Implants Res 2009;20:571-7.
41. Ruviere DB, Leonardo MR, da Silva LA, Ito IY, Nelson-Filho P. As-
sessment of the microbiota in root canals of human primary teeth
by checkerboard DNA-DNA hybridization. J Dent Child 2007;74:
118-23.
42. Filoche SK, Soma D, van Bekkum M, Sissons CH. Plaques from dif-
ferent individuals yield different microbiota responses to oral-
antiseptic treatment. FEMS Immunol Med Microbiol 2008;54:
27-36.
43. Silness J, Loe H. Periodontal disease in pregnancy. II. Correlation
between oral hygiene and periodontal condition. Acta Odontol
Scand 1964;22:121-35.
44. Shaddox L, Andia DC, Casati MZ, Nociti FH Jr, Sallum EA,
Gollwitzer J, et al. Microbiologic changes following administration
of locally delivered doxycycline in smokers: a 15-month follow-up.
J Periodontol 2007;78:2143-9.
October 2012  Vol 142  Issue 4 American Journal of Orthodontics and Dentofacial Orthopedics
Nelson-Filho et al
45. Ximenez-Fyvie LA, Haffajee AD, Socransky SS. Comparison of the
microbiota of supra- and subgingival plaque in health and peri-
odontitis. J Clin Periodontol 2000;27:648-57.
46. Faveri M, Figueiredo LC, Duarte PM, Mestnik MJ, Mayer MPA,
Feres M. Microbiological profile of untreated subjects with local-
ized aggressive periodontitis. J Clin Periodontol 2009;36:739-49.
47. Socranky SS, Manganiello SD. The oral microbiota of man from
birth to senility. J Periodontol 1971;42:485-96.
48. Sliepen I, Van Essche M, Quirynen M, Teughels W. Effect of
mouthrinses on Aggregatibacter actinomycetemcomitans biofilms
in a hydrodynamic model. Clin Oral Investig 2010;14:241-50.
49. Mandel ID. Chemotherapeutic agents for controlling plaque and
gingivitis. J Clin Periodontol 1988;15:488-98.
50. Addy M, Willis L, Moran J. The effect of toothpaste and chlorhex-
idine rinses on plaque accumulation during 4-day period. J Clin
Periodontol 1983;10:89-98.
51. Greene JC, Vermillion J. The simplified oral hygiene index. J Am
Dent Assoc 1964;68:7-13.