ORIGINAL ARTICLE

A pattern of microbiological colonization

of orthodontic miniscrew implants
Gyanda Mishra,a Om Prakash Kharbanda,a Rama Chaudhry,b and Ritu Duggala
New Delhi, India

Introduction: Current orthodontic literature reveals a lack of studies on bacterial colonization of orthodontic
miniscrew implants (MSI) and their role in the stability of MSI. This study aimed to determine the pattern of micro-
biological colonization of miniscrew implants in 2 major age groups, to compare it with the microbial flora of
gingival sulci in the same group of patients and to compare microbial flora in successful and failed miniscrews.
Methods: The study involved 102 MSI placed in 32 orthodontic subjects in 2 age groups: (1) aged #14 years and
(2) aged .14 years. Gingival and peri-mini implant crevicular fluid samples were collected using sterile paper
points (International Organization for Standardization no. 35) .3 months and processed by conventional
microbiologic culture and biochemical techniques. A microbiologist characterized and identified the bacteria,
and the results were subjected to statistical analysis. Results: Initial colonization was reported within 24 hours,
with Streptococci being the dominant colonizer. The relative proportion of anaerobic bacteria over aerobic bac-
teria increased over time in peri-mini implant crevicular fluid. Group 1 had greater Citrobacter (P 5 0.036) and
Parvimonas micra (P 5 0.016) colonizing MSI than group 2. Failed MSI showed a significantly higher presence
of Parvimonas micra (P 5 0.008) in group 1 and Staphylococci (P 5 0.008), Enterococci (P 5 0.011), and
Parvimonas micra (P \0.001) in group 2. Conclusions: Microbial colonization around MSI is established within
24 hours. Compared to gingival crevicular fluid, peri-mini implant crevicular fluid is colonized by a higher
proportion of Staphylococci, facultative enteric commensals and anaerobic cocci. The failed miniscrews
showed a higher proportion of Staphylococci, Enterobacter, and Parvimonas micra, suggesting their possible
role in the stability of MSI. The bacterial profile of MSI varies with age. (Am J Orthod Dentofacial Orthop
2023;164:554-66)

S
uccessful osseointegration of dental implants pio-
neered by Branemark and colleagues encouraged
orthodontists to use implants for anchorage
reinforcement.1,2 Kanomi3 first described in 1997 the
successful intrusion of mandibular incisors with a
mini-implant. Since then, orthodontic miniscrew im-
plants (MSI) have undergone significant design and pro-
tocol refinement for useful clinical application.4 MSIs
have enabled difficult clinical solutions in orthodontic
therapy, previously deemed inconceivable with tradi-
tional anchorage modalities.5
a
Division of Orthodontics and Dentofacial Deformities, Centre for Dental Educa-
tion and Research, All India Institute of Medical Sciences, New Delhi, India.
b
Department of Microbiology, All India Institute of Medical Sciences, New Delhi,
India.
All authors have completed and submitted the ICMJE Form for Disclosure of Po-
tential Conflicts of Interest, and none were reported.
Address correspondence to: Dr. Om Prakash Kharbanda, Ramaiah University of
Applied Sciences, University House, New BEL Road, MSR Nagar, Bengaluru,
560054 India; e-mail, dr.opk15@gmail.com.
Submitted, July 2022; revised and accepted, February 2023.
0889-5406/$36.00
Ó 2023 by the American Association of Orthodontists. All rights reserved.
https://doi.org/10.1016/j.ajodo.2023.02.023
MSIs must remain stable to anchor until the desired
tooth movements are achieved. Unfortunately, the fail-
ure rate of MSIs (10%-20%) has been reported to be
higher than that of dental implants (3%) and other skel-
etal anchorage devices (2.6%-7.3%).6-11 Many factors
affect MSI’s stability, essentially grouped as host-
related, implant-related, and operator-related.6-9,11-14
A complex interplay of the host response to implant
material, size, age, jaw, sex, design and the operator’s
experience and technique has been observed to affect
the stability of the MSI.6-14
Oral hygiene is critical to healthy peri-implant tissues.
Peri-implantitis is a major cause of the failure of dental
implants.8,15 Healthy peri-implant tissues act as a bio-
logical barrier to the ingress of microorganisms in the
peri-implant sulcus.16 Unlike a tooth-gingival relation-
ship, a microscopic gap, devoid of periodontal fibers, is
present between the gingival collar of the MSI and the
surrounding soft tissue.17 Because of their transgingival
placement, when the MSI is subjected to mechanical or
microbial challenge, a weak implant-soft tissue
interface may lead to bacterial ingress and breakdown
of underlying tissues leading to MSI failure.18 In dental

554
Mishra et al
implants, bacterial penetration occurs along the
implant-abutment interface and is independent of the
type of connection used.19-22 An analysis of the
surface elemental composition of retrieved MSI found
a more significant proportion of iron in the failed MSIs
than in the successful MSIs.23 It is conceivable that
increased iron in failed MSIs is derived from increased
blood flow caused by inflammation precipitated by bac-
terial invasion.
Studies have established differences in the microflora
of successful and failed dental implants. Coccoid cells
(Streptococci, Enterococci) predominate in peri-
implant crevices of healthy and successful implants,
along with a few non-motile rods (Klebsiella, Citro-
bacter, Enterobacter) and a tiny percentage of spiro-
chaetes and fusiform bacteria. In contrast,
unsuccessful implant sites are reported to harbor com-
plex microbiota with fewer coccoids (mainly Staphylo-
cocci) and a more significant proportion of enteric
gram-negative rods (Klebsiella, Enterobacter, Pseudo-
monas, Actinobacillus, Porphyromonas, Proteus),
black-pigmented Bacteroides, Fusobacterium, Peptos-
treptococcus and yeasts.24,25
Literature on microbial colonization of orthodontic
MSIs is scant, and even fewer studies are available on
the temporal evolution of peri-MSI microbiota. Apel
et al26 using conventional and molecular tools, found
no difference in the total amount or species (spp.)
composition between successful and failed mini-
implants. However, they found Actinomyces viscosus
and Campylobacter gracilis in stable controls but not
failed mini-implants. Another polymerase chain reaction
(PCR) based study found no significant association be-
tween the mobility of the mini-implants and 3 periodon-
topathogens (Prevotella intermedia, Actinobacillus
actinomycetemcomitans, and Porphyromonas gingiva-
lis).27 de Freitas et al28 evaluated microbial colonization
of non-specific Streptococcus, Lactobacillus casei and
Candida spp. using cell growth techniques and 16S ribo-
somal DNA-directed PCR for Porphyromonas gingivalis
in the mini-implant region from baseline to three
months. Of these, only Streptococcus spp. showed sig-
nificant initial colonization within the first 24 hours.
With limited information on the evolution of micro-
bial colonization and their spectrum in the peri-MSI sul-
cus, we aimed to determine the chronological pattern of
establishment of the microbial colonization process in
orthodontic MSIs during various stages of orthodontic
treatment in 2 different major age groups. We also at-
tempted to compare the pattern of microbial coloniza-
tion of successful and failed MSIs using conventional
techniques.
American Journal of Orthodontics and Dentofacial Orthopedics
555
MATERIALS AND METHODS
A prospective study was conducted on 32 adolescent
and young adult patients (age 12 years to 27 years; mean
age 16.6 6 3.9 years) irrespective of sex (7 males, 25 fe-
males) undergoing orthodontic treatment at a postgrad-
uate orthodontic department. The sample included 19
Class I bimaxillary protrusion and 13 Class II Division 1
patients requiring fixed mechanotherapy with maximum
anchorage and extraction of first premolars in either
maxilla or both maxilla and mandible. One hundred
and two MSIs (64 maxillary, 38 mandibular) were placed
with the same protocol. As there is evidence that gingival
inflammation and total bacterial count increase at the
onset of puberty and decrease after the age of 14 years,
thereby affecting the microbiological profile of the oral
cavity, the sample was divided into 2 groups on the basis
of age: group 1 (aged #14 years; 4 males, 8 females) and
group 2 (aged .14 years; 3 males, 17 females).29-32
Only the subjects free from systemic diseases, hormon-
al imbalance, drug intake and periodontal disease were
included in the study. Patients with local or systemic con-
ditions affecting bone metabolism, a history of orthodon-
tic treatment and poor oral hygiene were excluded from
the study. Study models, intraoral and extraoral photo-
graphs, and panoramic and lateral cephalograms were
prepared for each patient. Figure 1 gives a brief overview
of the methodology followed in this study.
MSI (8 mm long, 1.5 mm diameter, Tomas; Dentau-
rum, Ispringen, Germany) were placed buccally under
local anesthesia between the second premolar and first
molar as anchorage units for en-masse retraction. As
part of a standard protocol for MSI patients, each patient
was asked to rinse twice daily with 10 mL of 0.12%
chlorhexidine gluconate solution throughout the study
period. Three weeks after placement, MSIs were directly
loaded with a 9.0 mm nickel-titanium closed coil spring
for en-masse retraction providing approximately 200 g
of retracting force.
Different time intervals for sample collection were
earmarked as follows: immediately before MSI place-
ment (T0), 24 hours after MSI placement (T1), immedi-
ately before loading MSI at 3 weeks (T2), 24 hours
after loading MSI (T3), 4 weeks after the placement of
MSI (T4), and 12 weeks after the placement of MSI
(T5). Gingival crevicular fluid (GCF) was collected at
T0, T1, T2, T3, T4 and T5 in each patient. Peri-mini-
implant crevicular fluid (PMICF) was collected at T1,
T2, T3, T4 and T5.
The patients were asked to gargle vigorously with a
glass of sterile water, cheek retractors were placed, and
implant sites were isolated and dried using cotton rolls.
Sterile International Organization for Standardization
October 2023  Vol 164  Issue 4
556 Mishra et al

Fig 1. A brief overview of methodology.

no. 35 paper points were carefully inserted into the sul-
cus formed between the transmucosal neck of the MSI
and the attached gingiva and left in situ for 1 minute
to obtain PMICF as reported by Hartroth et al33 (Fig 2,
A). GCF was collected similarly from the maxillary sec-
ond premolar gingival sulcus (Fig 2, B). The sampled pa-
per points were inoculated in Robertson’s cooked meat
broth and immediately transferred to an incubator as
per microbiological methods described by Cruick-
shank.34 The failed MSIs were removed under sterile
conditions and directly transferred to Robertson’s
cooked meat broth for incubation. A total of 430 PMICF
samples and 192 GCF samples were collected.
Samples were inoculated into blood agar (5%) (BA)
and Macconkey agar (MA) plates for the isolation of aer-
obic organisms. The specimens were plated into brain
heart infusion agar with 5% sheep blood supplemented
with haemin and vitamin K for anaerobic cultures. The
MA plates were incubated at 37 C aerobically, and BA
plates were \5% carbon dioxide and examined at 24

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Mishra et al
Fig 2. A and B showing collection of PMICF and GCF sample from MSI and gingival sulcus respec-
tively, using sterile International Organization for Standardization no. 35 absorbent paper points under
isolation. PMICF, peri-mini-implant crevicular fluid; GCF, gingival crevicular fluid; MSI, miniscrew
implants.
and 48 hours. The brain heart infusion agar plates were
incubated anaerobically at 37 C in an anaerobic glove
box in an atmosphere of 80% nitrogen, 10% hydrogen
and 10% carbon dioxide for 48 hours.34-36
The plates were examined after 48 hours for colony
morphology and fluorescence. Metronidazole-sensitive
colonies were further subcultured aerobically and anaer-
obically. In the second subculture, colonies that grew
anaerobically but not aerobically were further processed
per standard procedures and confirmed by Vitek 2 Sys-
tems (version 07.01; bioM
erieux, Marcy l’Etoile, France)
when indicated. Clinical parameters examined included
assessment of implant mobility using Periotest (Medi-
zintechnik Gulden, Bensheim, Germany) and clinical
implant mobility scale and assessment of peri-implant
inflammation by modified Loe and Silness index.33,37,38
A Periotest value of .10 and clinical implant mobility
scale grade 2 and above implied mobility and clinical
failure of MSI. The MSIs were evaluated only for the
duration of this study.
RESULTS
A total of 192 GCF and 430 PMICF samples were
collected and cultured. The type of bacterial growth
was categorized as aerobic, anaerobic and mixed
(Fig 3). The data from the 2 groups were statistically
analyzed using the SPSS software (version 23, IBM, Ar-
monk, NY) to compare the aerobic and anaerobic bacte-
rial populations in GCF and PMICF over different time
intervals and to compare the individual bacterial popu-
lation in successful and failed MSIs.
In PMICF, Streptococci formed the predominant
isolate at T1. Streptococcal colonization at T1 was signif-
icantly higher than at T2-T5 for all samples in group 2, but
there was no significant difference in group 1. Other bac-
teria isolated at T1 included enteric facultative anaerobic
American Journal of Orthodontics and Dentofacial Orthopedics
557
gram-negative rods (Klebsiella, Enterobacter, Pseudo-
monas, Acinetobacter, Citrobacter), Staphylococci, Par-
vimonas micra and Veillonella.
Eight hundred fifty-nine isolates (pure cultured
strains) were obtained from the 430 PMICF samples,
and 322 isolates were obtained from the 192 GCF sam-
ples across both age groups. The spectrum of aerobic
and anaerobic bacteria isolated from GCF and PMICF
is shown in Tables I and II, respectively. Figures 4 and
5 present the temporal distribution of the bacterial spec-
trum across the sampling intervals.
Predominant isolates were Streptococci in both GCF
and PMICF, accounting for more than half of the iso-
lates. Table I demonstrates that Staphylococci colonized
MSI 5 times more frequently in group 1 and 3 times more
frequently in group 2 compared to gingival sulci. Facul-
tative anaerobic gram-negative bacteria and anaerobic
gram-negative cocci were also more predominant in
PMICF than the GCF.
The relative proportions of aerobic and anaerobic
bacteria in GCF did not change significantly. In GCF
samples from group 1, the aerobes decreased from
78.1% at T0 to 66.7% at T5, whereas the anaerobes
increased from 21.8% at T0 to 33.3% at T5. In group
2 GCF samples, aerobic isolates decreased from 70.7%
at T0 to 60.8% at T5, whereas the anaerobes increased
from 29.3% at T0 to 39.1% at T5. However, in PMICF,
the aerobic population decreased from 95.6% at T1 to
61.5% at T5 in group 1 and from 79.8% at T1 to
59.1% at T5 in group 2. The anaerobic population
increased from 4.3% at T1 to 38.5% at T5 in group 1
and from 20.8% at T1 to 40.2% at T5 in group 2
(Fig 6 and 7). Independent t test did not reveal any sig-
nificant change in the number of aerobic and anaerobic
bacterial isolates obtained at different time intervals in
either group.
October 2023  Vol 164  Issue 4
558 Mishra et al

Fig 3. A and B illustrating the type of bacterial growth in GCF and PMICF in group 1 (aged #14 years)
and group 2 (aged .14 years) respectively. Most samples in both groups showed mixed (both aerobic
and anaerobic) bacterial growth followed by aerobic and obligate anaerobic bacterial growth. GCF,
gingival crevicular fluid; PMICF, peri-mini-implant crevicular fluid.

Table I. Aerobic bacterial spectrum in GCF and PMICF: type and frequency
Group 1 Group 2

GCF (n 5 88) PMICF (n 5 156) GCF (n 5 104) PMICF (n 5 274)

Aerobic No. of isolates No. of isolates No. of isolates No. of isolates

microorganisms obtained Frequency obtained Frequency obtained Frequency obtained Frequency
Gram (1) Cocci
Streptococcus 57 64.8 93 59.6 89 85.6 176 64.2
Staphylococcus 5 5.7 44 28.2 17 16.3 113 41.2
Enterococcus 0 0 2 1.3 0 0 2 0.7
Gram ( ) cocci
Neisseria 4 4.6 4 2.6 0 0 0 0
Gram (1) Bacilli
Bacillus 7 7.9 11 7.1 8 7.7 12 4.4
Gram ( ) Bacilli
Klebsiella 5 5.7 12 7.7 7 6.7 23 8.4
Acinetobacter 11 12.5 0 0 6 5.8 19 6.9
Enterobacter 2 2.2 0 0 5 4.8 9 3.3
Proteus 0 0 0 0 1 0.9 1 0.3
Pseudomonas 0 0 0 0 0 0 13 4.7
Citrobacter 0 0 1 1.1 0 0 2 0.7
Total 91 – 167 – 133 – 370 –
Isolates, pure cultured strains; Frequency, % of total samples cultures; PMICF, peri-mini-implant crevicular fluid; GCF, gingival crevicular fluid.

Eight maxillary and 13 mandibular MSIs (n 5 21)
from 8 patients failed in this study. Of these, 7 MSIs (1
maxillary, 6 mandibular) belonged to 3 patients from
group 1, and the remaining 14 belonged to 5 patients
from group 2. Seventeen of these were found to be mo-
bile before loading, and the other 4 failed within 24
hours of loading. The age of patients showing failure
of MSI ranged from 12y to 24y (mean, 15.62 6 3.58).
The chi-square test to compare the success and failure
rates in the 2 groups did not show any significant
association with either age (P 5 1.000) or gender
(P 5 0.625).
Independent t test was applied to assess differences
in aerobic, obligate anaerobic and total isolate counts
in PMICF between subjects with successful and failed
MSI. A significant difference was observed in total iso-
lates (group 1, P 5 0.015, group 2, P 5 0.024) and aer-
obic isolate counts (group 1, P 5 0.006; group 2, P 5
0.001) between the successful and failed MSI in both
groups. However, no significant differences in obligate
anaerobe isolate counts were observed in either group
(group 1, P 5 0.166; group 2, P 5 0.133).
To test the respective proportions of various bacteria
in successful and failed MSIs, an independent t test was

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Mishra et al 559

Table II. Anaerobic bacterial spectrum in GCF and PMICF: type and frequency
Group 1 Group 2

GCF (n 5 88) PMICF (n 5 156) GCF (n 5 104) PMICF (n 5 274)

Anaerobic No. of Isolates No. of Isolates No. of Isolates No. of Isolates

microorganism obtained Frequency obtained Frequency obtained Frequency obtained Frequency
Gram (1) Cocci
Parvimonas micra 12 13.6 36 23.1 32 30.8 71 25.9
Gram ( ) Cocci
Veillonella 6 6.8 24 15.4 5 4.8 50 18.2
Gram (1) Bacillus
Lactobacillus 20 22.7 33 21.2 13 12.5 71 25.9
Clostridia 2 2.3 0 0 2 1.9 8 2.9
Gram ( ) Bacilli
Prevotella 1 1.1 0 0 0 0 23 8.4
Porphyromonas 5 5.7 6 3.8 0 0 0 0
gingivalis
Total 46 – 99 – 52 – 223 –

Isolates, pure cultured strains; Frequency, % of total samples cultures; PMICF, peri-mini-implant crevicular fluid; GCF, gingival crevicular fluid.

Fig 4. Shows the distribution of different species in GCF over a 3-month period at designated time in-
tervals T0-T5 in group 1 (aged #14 years) and group 2 (aged .14 years). AGNB, Aerobic Gram Nega-
tive Bacteria; GCF, gingival crevicular fluid.

applied. Compared with successful MSI, failed MSI in
group 1 showed a significantly higher association with
Staphylococci (P 5 0.008), Enterococci (P 5 0.011)
and Parvimonas micra (P \0.001), whereas failed MSI
in group 2 showed a significantly higher association
with Parvimonas micra (P 5 0.008) (Table III).
The chi-square test was applied to test the proportion
of bacteria in subjects \14 years compared with
subjects .14 years. The proportions of Citrobacter
(P 5 0.036) and Parvimonas micra (P 5 0.016) were
significantly higher in group 1 than in group 2.
DISCUSSION
A vast body of literature supports that bacterial infec-
tion is a major cause of peri-implantitis in dental
implants leading to mobility, bone loss and implant

American Journal of Orthodontics and Dentofacial Orthopedics October 2023  Vol 164  Issue 4
560 Mishra et al

Fig 5. Shows the distribution of different species in PMICF over a 3-month period at designated time
intervals T0-T5 in group 1 (aged #14 years) and group 2 (aged .14 years). AGNB, Aerobic Gram
Negative Bacteria; PMICF, peri-mini-implant crevicular fluid.

Fig 6. Illustrates the changes in relative proportions of aerobic and anaerobic isolates in GCF and
PMICF at different time intervals over a 3-month period in group 1 (aged #14 years). PMICF,
peri-mini-implant crevicular fluid; GCF, gingival crevicular fluid.

failure.15,16,22,24,25,39-41 In contrast, limited literature is pathogens or attempted to differentiate the microflora

available on the impact of microbial colonization on the of retrieved failed implants from the stable ones in
stability of orthodontic MSI. The existing studies have situ.26-28,42 Using closed-ended molecular techniques tar-
adopted a targeted approach toward known periodontal geting bacterial identification based on prior knowledge

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Mishra et al 561

Fig 7. Illustrates the changes in relative proportions of aerobic and anaerobic isolates in GCF and
PMICF at different time intervals over a 3-month period in group 2 (aged .14 years). PMICF,
peri-mini-implant crevicular fluid; GCF, gingival crevicular fluid.

Table III. Comparison of bacterial spectrum in failed and successful MSI

Group 1 (aged #14 y) (n 5 12, 36 MSI) Group 2 (aged .14 y) (n 5 20, 66 MSI)

Successful MSI Failed MSI Successful MSI Failed MSI

Bacteria (n 5 9, 29 MSI) (n 5 3, 7 MSI) P value (n 5 15, 52 MSI) (n 5 5, 14 MSI) P value
Streptococcus 86.67 6 17.32 72.22 6 25.46 0.286 76.00 6 27.46 52.00 6 50.20 0.187
Staphylococcus 35.56 6 13.33 72.22 6 25.46 0.008* 57.33 6 23.74 70.00 6 44.72 0.420
Enterococcus 2.22 6 6.67 50.00 6 50.00 0.011* 1.33 6 5.16 0.00 6 0.00 0.578
Klebsiella 22.22 6 21.08 16.67 6 28.87 0.723 16.00 6 25.30 4.00 6 8.94 0.320
Pseudomonas 0.00 6 0.00 0.00 6 0.00 – 6.67 6 16.33 0.00 6 0.00 0.382
Acinetobacter 2.22 6 6.67 0.00 6 0.00 0.588 10.67 6 21.20 20.00 6 44.72 0.529
Enterobacter 2.22 6 6.67 33.33 6 57.74 0.109 6.67 6 20.93 0.00 6 0.00 0.493
Proteus 0.00 6 0.00 0.00 6 0.00 – 1.33 6 5.16 0.00 6 0.00 0.578
Citrobacter 4.44 6 8.82 0.00 6 0.00 0.418 1.33 6 5.16 0.00 6 0.00 0.578
Escherichia 0.00 6 0.00 0.00 6 0.00 – 0.00 6 0.00 4.00 6 8.94 0.083
Bacillus 15.56 6 31.27 0.00 6 0.00 0.424 6.67 6 14.47 8.00 6 17.89 0.868
Parvimonas micra 6.70 6 20.00 100.00 6 0.00 \0.001* 29.33 6 34.53 80.00 6 27.39 0.008*
Veillonella 24.40 6 37.12 0.00 6 0.00 0.295 25.33 6 30.67 0.00 6 0.00 0.086
Lactobacilli 40.00 6 26.46 38.89 6 34.69 0.954 38.67 6 30.67 36.00 6 49.80 0.887
Clostridia 2.22 6 6.67 22.22 6 38.49 0.131 0.00 6 0.00 0.00 6 0.00 –
Prevotella 0.00 6 0.00 0.00 6 0.00 – 13.33 6 30.34 0.00 6 0.00 0.347
Porphyromonas gingivalis 8.90 6 26.67 0.00 6 0.00 0.588 0.00 6 0.00 0.00 6 0.00 –

MSI, miniscrew implants.

*Significant difference at P #0.05 (Independent t test).

of periodontitis creates a selection bias and does not
permit the establishment of the full diversity of the micro-
bial profile of MSI.43 Furthermore, it is not yet established
whether the microbial flora of MSI is similar to healthy
gingival sulci, periodontal pockets, or infected MSI and
whether it is affected by the age of the subject.
Our study using nontargeted conventional microbio-
logic techniques to isolate and identify the microorgan-
isms in the peri-MSI sulcus and GCF supported the
previous findings by de Freitas et al28 that microbial
colonization around MSI is established within 24 hours
of their placement in the oral cavity. Streptococcus

American Journal of Orthodontics and Dentofacial Orthopedics October 2023  Vol 164  Issue 4
562
spp. was the predominant pioneering colonizer. Other
early colonizers included enteric facultative anaerobic
gram-negative rods (Klebsiella, Enterobacter, Pseudo-
monas, Acinetobacter, Citrobacter), Staphylococci,
Parvimonas micra and Veillonella. Streptococci were
also the most common isolates in both GCF and PMICF
in group 1 (aged #14 years) and group 2 (aged .14
years). This is consistent with Maestre et al,44 who re-
ported that Streptococcus spp. accounted for nearly
half the isolates cultivated from subgingival plaque sam-
ples in subjects with periodontitis.
Staphylococci were isolated from 11% of all GCF
samples (group 1, 5.7%; group 2, 16.3%). Although
Staphylococcus spp. have frequently been isolated
from supragingival and subgingival plaques, Smith
et al45 and McCormack et al46 argue whether Staphylo-
coccus spp. play a role in the healthy ecology of the oral
cavity. They suggest a reappraisal of the role of Staphy-
lococcus aureus in the oral cavity in both health and dis-
ease. The preferential attachment of Staphylococci to
the titanium implant surfaces was also demonstrated
in this study by their relatively higher proportion in
PMICF (total, 36.5%; group 1, 28.2%; group 2, 41.2%)
compared with GCF (total, 11.5%; group 1, 5.7%; group
2, 16.3%).
GCF and PMICF samples showed many enteric com-
mensals, including Klebsiella, Acinetobacter, Entero-
bacter, Proteus, Pseudomonas and Citrobacter.
Enterobacteriaceae are not generally considered essen-
tial members of the oral microbiota.47 However, we iso-
lated members of this family in 16 out of 32 patients
(50%), constituting 19.2% of total aerobic isolates in
GCF (group 1, 20%; group 2, 18.2%) and 17.2% aerobic
isolates in PMICF (group 1, 8.3%; group 2, 24.5%). The
presence of fixed orthodontic appliances increases the
carriage of enteric commensals in the oral cavity and
could explain their increased presence in GCF and
PMICF.48,49 These confounders must be considered
while establishing the microbial colonization pattern of
MSI and their role in causing the failure of MSI.
Among the anaerobic cocci, Parvimonas micra was
isolated from 22.9% GCF samples (group 1, 13.6%;
group 2, 30.8%). This was comparable with Riggio
et al,50 who detected the species in 19 of 68 (28%) sub-
gingival plaque samples and 20 of 28 (71%) periodontal
pus samples analyzed by PCR. In contrast, Veillonella
was isolated from only 5.8% of GCF samples (group 1,
6.8%; group 2, 4.8%). This agrees with the findings of
Mashima and Nakazawa, who reported a significantly
higher frequency of Veillonella in the periodontal
pockets than in the healthy gingival sulcus.51
Both Parvimonas micra and Veillonella presented
with greater frequency in PMICF compared to GCF
October 2023  Vol 164  Issue 4 American Journal of Orthodontics and Dentofacial Orthopedics
Mishra et al
(24.9% and 17.2%, respectively). Parvimonas micra is
a gram-positive anaerobic coccus known as a
commensal in the oral cavity.52 The bacterium has also
been frequently isolated in patients with systemic infec-
tions, implantitis and periodontitis.53 Veillonella spp.
are recognized as early colonizers in oral biofilms. They
promote mutualistic community development through
interbacterial interactions and the production of vitamin
K, which stimulates the growth of the typical periodontal
pathogen Porphyromonas gingivalis.54
Although Lactobacilli showed a higher isolation fre-
quency in PMICF (group 1, 21.2%; group 2, 25.9%)
compared with GCF (group 1, 22.7%; group 2, 12.5%),
they are reportedly benign and do not contribute to
either periodontitis or peri-implantitis, although the
insertion of mechanical appliances such as orthodontic
bands and dentures can dramatically increase the
numbers of oral lactobacilli.55,56
The putative periodontal pathogens were rarely iso-
lated in this study. Prevotella was isolated from the
GCF in only 1 patient and from the PMICF in 3 patients,
whereas Porphyromonas gingivalis was isolated from
GCF and PMICF in only 1 patient. Aggregatibacter
actinomycetemcomitans, Campylobacter, Fusospiro-
chaetes, and Treponema, were not isolated in this study
from either GCF or PMICF samples. Maeda et al57 re-
ported that Prevotella intermedia is isolated more
frequently from periodontally diseased sites than from
periodontally healthy sites, although Prevotella inter-
media and Prevotella nigrescens might occur simulta-
neously in the oral cavity. Although Prevotella spp is
implicated in periodontitis, it may be compatible with
peri-implant health by achieving a symbiotic equilibrium
in the peri-implant niche.43
According to Rudney et al,58 Porphyromonas gingi-
valis can behave as a commensal or pathogen, and its
presence is not a predictor of disease. They also reported
that Porphyromonas gingivalis, Tannerella forsythia,
and Aggregatibacter actinomycetemcomitans are
concentrated in buccal epithelial cells in healthy mouths,
providing a reservoir for these periodontal pathogens
enabling subgingival recolonization in periodontitis pa-
tients.59 If true, localizing the pathogenic microflora
could pose a challenge in overcoming these periopatho-
gens. The possibility of host immune response on the
pathogenicity of Porphyromonas intermedia and Por-
phyromonas gingivalis and its significance in peri-MSI
sulcus needs to be explored in further studies.
A comparison of individual bacterial proportions in
PMICF showed a significantly higher proportion of Cit-
robacter and Parvimonas micra in group 1 compared
to group 2, indicating a possible difference in bacterial
population in the 2 age groups. No studies were
Mishra et al
available in the literature for comparison. However, the
oral ecosystem in the prepubertal period harbors a
significantly higher proportion of anaerobes and enteric
commensals, which could be attributed to changes
related to the eruption of permanent teeth as well as pu-
bertal hormones.32,60
Failed MSIs in group 1 were found to have a signifi-
cantly higher proportion of Staphylococci, Enterococci
and Parvimonas micra. Analysis of bacterial proportions
in failed MSIs in group 2 revealed a significantly higher
proportion of Parvimonas micra. Total and aerobic bac-
terial counts between the successful and failed MSI
differed significantly in both groups, but there was no
difference in the obligate anaerobic counts. The differ-
ence in the age of the patients in the 2 groups, the diver-
sity of resident oral microflora, the depth of the peri-MSI
sulcus and oxygen tension in the peri-MSI sulcus may
account for this variation.
Although oral carriage of Staphylococcus aureus
varies between 24%-84% depending on the population
studied, their role in oral health and disease remains
contentious.46 S. aureus has been known to cause oral
infections (eg, angular cheilitis, parotitis, staphylococcal
mucositis) and most indwelling medical device-related
infections.46 Staphylococcus aureus and Coagulase
Negative Staphylococci are the most common bacteria
responsible for prosthesis-related infections.46,61,62 Its
ability to colonize implant surface is derived from
possessing several cell-surface adhesion molecules,
including microbial surface components recognizing ad-
hesive matrix molecules, that interact with and facilitate
adhesion to extracellular matrix proteins deposited on
the biomaterial surface.63 A higher prevalence of Staph-
ylococci in PMICF and failed MSI points towards the
possibility of its contribution to peri-MSI inflammation
and subsequent failure. Further work in this area may
improve understanding of Staphylococci in oral health
and disease and reduce MSI failures.
Facultative anaerobic enteric commensals like Entero-
cocci and Enterobacter are transient and opportunistic,
and their prevalence in the oral cavity increases after
the placement of dental appliances.47,64 Such a shift in
bacterial community dynamics, owing to complex inter-
actions among microorganisms and host tissues, cause
opportunistic mucosal infections in medically compro-
mised elderly and periodontitis patients.39,65 Some of
these species are also quite resistant to various forms of
antimicrobial treatment and pose a challenge in manag-
ing infections caused by them.66 Given that large propor-
tions of enteric commensals have been reported from
failing titanium implants, our findings indicate their
possible role in the failure of orthodontic MSI. The
increased carriage of these enteric commensals may
American Journal of Orthodontics and Dentofacial Orthopedics
563
precipitate an inflammatory response within the host
leading to the loosening of the MSI. Future studies will
be required to evaluate this hypothesis.
Parvimonas micra (formerly Peptostreptococcus mi-
cros) is a gram-positive anaerobic coccus known as a
commensal in the oral cavity and implicated in many in-
traoral and extraoral polymicrobial infections, such as
periodontitis, dentoalveolar infections, endodontic in-
fections as well as intracranial abscesses, pericarditis
and necrotizing fasciitis.52,53 It is not only an established
early colonizer (˂2 weeks) of titanium implant surfaces
but has also been associated with failing dental im-
plants.25,67 Its role in oral disease is less well-defined
and presents an opportunity for future research.
The relatively low isolation of anaerobic species in the
gingival crevices and peri-implant sulci compared to
periodontal pockets in this study may be attributed to
many factors, such as geographical and dietary differ-
ences of the Indian population compared to Caucasians
and Europeans.68 Dietary differences evolve from differ-
ences in the variety and proportion of vegetarian foods,
meat-based foods and refined sugars. In addition, the
smaller sulcus depth around orthodontic MSI may be
nonconducive to the establishment and proliferation
of fastidious anaerobic bacteria implicated in periodon-
titis and peri-implantitis.
The oral cavity is a highly heterogeneous ecological
system with different microbial communities showing
site-specific niches and variation with aging and state
of dentition (deciduous or permanent; dentulous or
edentulous).60,69 Thus, the patient’s age may also affect
the composition of microflora colonizing around the
MSI. Mombelli et al30 reported the changes in subgingi-
val microbiota in children aged 11-15 years over 4 years.
They found increased total bacterial counts and preva-
lence of Actinomyces odontolyticus, Capnocytophaga
and black-pigmented Bacteroides (now Prevotella in-
termedia and Prevotella melaninogenica) at the onset
of puberty. These counts decreased after the age of 14
years. Because of their findings and the results of this
study, it may be surmised that the age of the patients
may influence the microbial community in GCF and
PMICF. Further studies may be planned to assess the mi-
crobial profiles of preadolescents and postadolescents
and their prevalence in the peri-MSI crevice.
Although not statistically significant, it would be
pertinent to note the absolute absence of Veillonella
in failed MSI. The role of Veillonella in health and dis-
ease is still being determined, with conflicting reports
in the literature. Although Mashima and Nakazawa re-
ported that Veillonella parvula was predominantly iso-
lated from the periodontal pockets in subjects with
chronic periodontitis, Kumar isolated Veillonella in
October 2023  Vol 164  Issue 4
564
higher numbers from healthy samples than from peri-
odontitis samples, similar to the outcome of this
study.51,70 Further studies are required to investigate
the role of this species in peri-MSI inflammation.
Peri-implant lesions harbor not only periodontal
pathogens (Porphyromonas gingivalis, Prevotella
intermedia or Prevotella nigrescens and Actinobacillus
actinomycescomitans) but also demonstrate higher
proportions of microorganisms not usually associated
with periodontitis or dental abscesses such as Staphylo-
cocci, Enterobacteriaceae and Candida spp.71 Stable or-
thodontic MSI in this study also showed greater
colonization of gram-positive aerobes (Staphylococci),
aerobic gram-negative bacteria (Klebsiella, Acineto-
bacter, Enterobacter, Citrobacter) and anaerobic cocci
(Parvimonas micra). Failed MSIs were associated with
significantly higher proportions of Staphylococci,
enteric commensals, and Parvimonas micra.
However, one must acknowledge that an unambigu-
ous assignment to health or disease has proven difficult
for many taxa.72 Many of these are common commensals
of the oral cavity and develop pathogenicity in susceptible
hosts because of changes in the oral environment,
lowering of host responses, environmental selection
through nutrient and atmospheric gradients, or the mi-
crobial community itself through the complex phenome-
non of cohesion and communication between species.40
Thus, the establishment and maturation of a micro-
biological community depend on many factors yet to
be fully understood, such as a favorable ecological envi-
ronment influenced by host factors, age, diet, state of
dentition, and possible intraoral bacterial reser-
voirs.43,60,73 Metallurgical composition, hydrophobicity,
surface roughness, surface chemistry and surface free
energy, and implant design features such as length,
form, and surface modifications also play a role in micro-
bial colonization.73 That being so, the predictability and
clinical success of any antimicrobial therapy would be
limited clinically with current levels of knowledge. A
more systematic and detailed analysis of the microbio-
logical factors, treatment outcomes and formulation of
biomedical engineered materials for MSI is suggested
to successfully prevent peri-MSI infection.
The lack of an objective periodontal evaluation based
on the Loe and Silness periodontal index as part of the
selection criteria is a flaw in the design of this study.
Bearing that oral carriage of some bacteria is increased
by the presence of fixed orthodontic appliances, a micro-
biological profile of patients could have been performed
before beginning orthodontic treatment. The confound-
ing effect of hormones associated with puberty on the
oral microbial profile has not been excluded in this study
and presents an opportunity for future studies.
October 2023  Vol 164  Issue 4 American Journal of Orthodontics and Dentofacial Orthopedics
Mishra et al
The findings of this study should be interpreted with
a caveat that the use of chlorhexidine gluconate mouth-
wash (0.12%) could have influenced the bacterial
composition of our samples. The use of chlorhexidine
mouthwash (0.12%) is an established protocol for ortho-
dontic MSI patients, and it would not be justified to
eliminate its use in this study.
Using conventional techniques to isolate and identify
microorganisms has brought to light a newer under-
standing of microorganisms in the peri-MSI sulcus and
their role in the success and failure of MSI. This area
further needs to be explored with molecular techniques
to characterize the nature of pathogens and their pur-
ported significance.
The findings of this study may prove instrumental in
developing novel surface modification techniques to
inhibit or disrupt bacterial adhesion to implant surfaces,
which would be significant not only for the clinical sta-
bility of orthodontic MSI but also for helping reduce in-
fections related to prosthetic implants and in-situ
medical devices.
CONCLUSIONS
1. The microbial colonization of orthodontic MSI was
established within 24 hours of placement. The rela-
tive proportion of anaerobes over aerobes in the
PMICF increased over 3 months, whereas it re-
mained relatively constant in the GCF in patients
aged #14 years and those aged .14 years.
2. PMICF samples presented a higher proportion of
Staphylococci, facultative anaerobic enteric com-
mensals (Klebsiella, Acinetobacter, Enterobacter,
Pseudomonas, Citrobacter) and anaerobic cocci
(Parvimonas micra, Veillonella) in both age
groups.
3. Failed MSIs (group 1, n 5 7; group 2, n 5 14) were
characterized by a higher proportion of Staphylo-
cocci, facultative anaerobic enteric commensal
Enterobacter, and obligate anaerobe Parvimonas
micra.
4. Patients aged #14 years had a significantly higher
proportion of enteric commensal Citrobacter and
obligate anaerobe Parvimonas micra indicating
possible variations in microbial colonization around
MSI in younger age.
5. From a clinical perspective, therapeutic strategies
must be designed to reduce MSI failure because of
microbial infection, including unique tools to facil-
itate peri-MSI plaque removal. The use of local
drugs or surface coatings carries the risk of devel-
oping resistant strains, but the findings of this study
suggest research on modifying implant surfaces in
Mishra et al
the neck and emerging profile region to inhibit or
disrupt microbial attachment to the titanium
biomaterial.
AUTHOR CREDIT STATEMENT
Gyanda Mishra contributed to investigation, data cu-
ration, and manuscript preparation; Om Prakash Khar-
banda contributed to conceptualization, supervision,
and manuscript review and editing; Rama Chaudhry
contributed to methodology, investigation, resources,
and data curation; and Ritu Duggal contributed to su-
pervision and manuscript review and editing.
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