Cuaderno de odontología. Revista científica               

Vol. 3(2), 1-20, 2025

https://doi.org/10.62574/cap48829

1

Evaluation of microbial colonisation at the attachment interface in fixed

orthodontic treatments

Evaluación de la colonización microbiana en la interfase aditamento diente

en tratamientos de ortod oncia fija

María Elizabeth Llivichuzca-I llescas

maría.llivic huzca.37@est.ucacue.edu.ec

Catholic Universi ty of Cuenca, Azogues, Azuay,               Ecuador

https://orcid.org/0000 -0002-1966-3846

Magaly Noemí Jimenez-Romero

mjimenezr@ucacue.edu.ec

Catholic Universi ty of Cuenca, Azogues, Azuay,               Ecuador               

https://orcid.org/0000 -0002-0736-6959

Jess ica María Sarmiento-Ordóñez

sarmientoo@ucacue.edu.ec

Catholic Universi ty of Cuenca, Cuenca, Azuay, Ecuador

https://orcid.org/0000 -0003-4159-9286

Carlos Alberto  Flores-Cárdenas

odontflores@hotmail.com

Catholic Universi ty of Cuenca, Cuenca, Azuay, Ecuador

https://orcid.org/0009 -0008-5714-4351

ABSTRACT

Objective:  To  determine  the  presence  of  oral  m              icroorganisms  in  patients  with  fixed  orthodontic

appliances,  con sidering  that  adhesion  and  colonisation  may  b              e  influenced  by  several  factors.

Materials and methods: Ob servational and cross-se              ctional study co              nducted in 67 patients. Samples

were  co llected  with  Citoswab ®  swabs  from  three  anatomical  sites  in  the  u              pper  r              ight  quadrant              .

Results: Fifteen microorganisms were isolated, with Streptococcus mutans being the most frequent,

followed  by  Can dida  albicans.               No               significant               association               was               found               with               cement               type,               age               or               sex;

however,  statistically  significant               differences (              p =               0.0              17)               were               obta              ined               with               treatment               time,               in               which

the  count  of  Candida  albicans  increased  in  the  tre              atment  ranges  of  five  to  twelve  m              onths.

Conclusions:  A  significant  correlation  was  o              bserved  between  orth              odontic  treatment  time  and

increased microbial colonisation. This suggests that prolonged treatment favours th              e development of

biofilms in the attachment -tooth interface.

Keywords: bacteria; dental biofilm; fixed orthodontic appliances. (DeCS).  

RESUMEN

Objetivo:   Determinar  la  presencia  de  microorganismos  orales  en                pacientes  con  aparatología

ortodóntica fija, considerando que la ad              herencia y colon              ización pueden estar influenciadas por varios

factores.  Materiales y métodos: Estudio observacional y t              ransversal realizado en 67 pacientes. Se

recolectaron  m uestras  con  hisopos  Citoswab®  en  tr              es  sitios  ana              tómicos  del  cuadrante  superior

derecho.  Resultados: Se aislaron quince microorganismos, siendo el Stre              ptococcus mutans el más

frecuente,  seguido  de   Candida  a lbicans.  No  se  encontró  asociación  significativa  con  el  tipo  de

cemento,  edad  o  sexo ;  sin  e              mbargo,               se                             o              btuvieron               diferencias               estad              ísticamente               sign              ificativas               (p               =

0.017) con el tiempo de tratamiento, en el cual au              mento el conteo de Candida albicans, en los rangos

de tratamiento de cinco a d oce meses. Conclusiones: Se observó u              na correlación sign              ificativa entre

el  tiempo  del  tratamiento  ortodóntico               y               el               aumento               en               la               colonización               microbiana.               Esto               sugiere               que

la  prolongación  del  tratamiento  favorece  el               desar              rollo               de  biopelículas  en  la  interfase  aditamento

diente.

Descriptores : bacterias; biopelícula dental; aparatos ortodóncicos fijos. (DeCS).  

Received: 17/07/2025. Reviewed : 28/07/2025. Approve              d: 14/08/2025. Published: 20/08/2025.

Original a rticle  

Cuaderno de odontología. Revista científica  

Vol. 3(2), 1-20, 2025

Evaluación de la colonización microbiana en la interfase aditamento diente en

tratamientos de ortodoncia fija

Evaluation of microbial colonisation at the attachment interface i              n fixed orthodontic treatments               

María Elizabeth Llivichuzca-Illescas

Magaly Noemí Jimenez-Romero

Jessica María Sarmiento-Ordóñez

Carlos Alberto Flores-Cárdenas

2

INTRODUCTION  

Orthodontic  treatments  have               become               the               treatment               of               choice               when               solvin              g               dental

problems  suc h  as  malocclusion  and  facial  an              d  d              ental  aesthetics,  contributing

significantly to the proper functioning o              f the stomatognathic system.              (1) Howe              ver, this

type  of  treatment  involves  different                attachments  such  as  brackets,  arches,

elastomeric chains, metal ligatures, (2) etc., which create retention sites and promote

the  accumulation  of  dental  biofilm,  causing  alterations  in  the  oral  micr              obiota  by

promoting bacterial colonisation. (3)  

The  oral  microbiota  contains               a               wide               v              ariety               of               species               found               in               both               soft               and               hard

tissues. I t is estimated that there are at               least 700 species, including bacteria, viruses

and  fungi,(4)  which  contribute               to               the               overall               health               of               the               host               when               in               a               state               of

equilibrium (eubiosis). Oral hyg              iene th              erefore plays a fundamental role in preventing

the  colonisation  and  proliferation  of  microorganisms  in               th              e               interface  between               the

tooth and fixed orthodontic appliances              .(3)

However,  disruption of t his balance (dysbiosis) ca              n contribute to the accumul              ation of

biofilm in this ar ea, facilitating the development of exogenous bacteria a              nd fu              ngi that

can  colonise  the  appliances  due               to  their  ability  to               adhere               to  surfaces               by               f              orming

resistant  biofilm,  causing  disease               and               affecting               overall               health.              (5)               St              udies               report               an

increase in the gram-positive microbial population, especially Streptococcus mutans

(SM),  Lactobacillus  spp  and  Actino              micetes  spp,  which  are  responsible  for  t              he

development  of  white  spot               lesions,               dental               caries               and               periodontal               disease               after               the

start  of  treatment  with  fixed               orthodontic               appliances.              (6)               There               is               also               an               increase               in

the  colonisation  of  fungi  such  as  Candida  spp,  Cand              ida  a              lbicans  (CA)  being  a

common opportunistic  pathogen               of th              e oral c              avity,               functioning               as a               reservoir               for the

spread of microorganisms, with the ability to colonise dental tissues.               (7,8)

Cuaderno de odontología. Revista científica  

Vol. 3(2), 1-20, 2025

Evaluación de la colonización microbiana en la interfase aditamento diente en

tratamientos de ortodoncia fija

Evaluation of microbial colonisation at the attachment interface i              n fixed orthodontic treatments               

María Elizabeth Llivichuzca-Illescas

Magaly Noemí Jimenez-Romero

Jessica María Sarmiento-Ordóñez

Carlos Alberto Flores-Cárdenas

3

The adhesion  and co lonisation of               these microorganisms               during               orthodontic t              herapy

may  be  influenced  by  various  factors,  such  as  the  type  of  bracket,  orthodontic

cement,  duration  of  treatment               and               age               of               the               patient.               (2,9)               Oral               hygiene               is               also               a

factor, as the presence of orthodontic appliances can hinder effective cleaning.               (10)

To  date,  scientific  evi dence               supporting               the               influence               of               t              hese               f              actors               is               limit              ed.               For

this  reason,  the  present  study  ai              med  to  d              etermine  the  presence  of  oral

microorganisms  in  patients               with               fixed               orthodontic               appliances,               considering v              ariables

such as treatment time, type of orthodontic cement, sex, and patient age.               

METHOD  

A  descriptive,  observational,  cross-sectional,               prospective               study               was               conducted               on

patients  tre ated  at  the  postgraduate               o              rthodontic               c              linic               of               a               pr              ivate               university               in               the

city of Azogues, Ecuador. 

Samples were taken during mo              nthly orthodontic treatment check              -ups. P              atient              s were

informed  of  the  req uirements               prior               to               sampling:               no               antibiotics               in               the               last               month,               no

food consumption, and no tooth brushing one hour before the appointment.                

The inclusion criteria were patients in good general health, aged between 12 and 42

years,  and  undergoing  conventional  fixed  orthodontic  treatment.  T              he  exclusion

criteria  were  patients  with  systemic  diseases,  pregnant  women,  and  participants

undergoing treatment with oral contraceptives or a              ntibiotics.  

Participants were selected from the specialist training clinic at a private university in

the  city  of  Azogues,  where  orthodontic  treatment               was  being               c              arried               out.  Patients

undergoing  orthodontic  f ollow-up  were  contacted               a              nd               invite              d               to  participate  in  the

study,   and  instructions  were  provide              d  on  the  importance  of  brushing  their  teeth

before attending  the consultation. Al              l participants               signed an               informed co              nsent               form,

and  in  t he  case  of  minors,               the  corresponding               consent               was  obtained.               A              n               intraoral

clinical examination was performed to verify the selection criteria of the participants.

Cuaderno de odontología. Revista científica  

Vol. 3(2), 1-20, 2025

Evaluación de la colonización microbiana en la interfase aditamento diente en

tratamientos de ortodoncia fija

Evaluation of microbial colonisation at the attachment interface i              n fixed orthodontic treatments               

María Elizabeth Llivichuzca-Illescas

Magaly Noemí Jimenez-Romero

Jessica María Sarmiento-Ordóñez

Carlos Alberto Flores-Cárdenas

4

Finally, the sample was collected.               

The  dependent  variables  i ncluded  the  presence  a              nd               d              iversity  of  microbes  in               the

bracket-tooth interface. The independent variables were the age of the parti              cipants,

categorised  into  three  gro ups:  1              2–18                years,  19              –26  y              ears  and  27–              42  years;                the

treatment time was divided into 1–4 months, 5–8 months and 9–12 months; and the

gender  of  the  participants,               which               was               categorised               as               female               and               male.               The type               of

cement  us ed  was  grouped               into               five               c              ategories:               GoTo®,               Orthobond®,               Orthocem®,

Enlight®  and  Ortholink®.  5–8               months               and               9–12               months;               female               an              d               male               gender;

and  the  type  of  cement  used,  which  was  grouped  into  five  categories:  GoTo®,

Orthobond®, Orthocem®, Enlight® and Ortholink®.               

For  sample  collection,  a  mouth               guard               was               placed               t              o               prevent               cross              -contamination

and facilitate access  to the  examination               areas. Prior to               sampling, active orthodontic

elements were removed. In accordance with biosafety measures, swabs were taken

using  Citoswab®  swa bs  applied  to  t              he  vestibular  s              urface  of  the  br              acket              -tooth

interface at three anatomical sites in the upper right quadrant, as shown in Figure 1.                

These  samples  were  st ored               in               transport               boxes               with               cooling               gel               at               4              –8°C,               ensuring

their preservation until laboratory analysis.               

To minimise bias, each participant was informed of the requirements prior to sample

collection  and  the  collection  proto              col  was  standardised.  Microbiological  and

molecular  b iology  techniques  considered  to  be  the  g              old  sta              ndard  were  used  to

identify  microorganisms.  The  analyses  were  performed  by  an  independent                team,

blind to demographic and clinical variables.               

Cuaderno de odontología. Revista científica  

Vol. 3(2), 1-20, 2025

Evaluación de la colonización microbiana en la interfase aditamento diente en

tratamientos de ortodoncia fija

Evaluation of microbial colonisation at the attachment interface i              n fixed orthodontic treatments               

María Elizabeth Llivichuzca-Illescas

Magaly Noemí Jimenez-Romero

Jessica María Sarmiento-Ordóñez

Carlos Alberto Flores-Cárdenas

5

Figure 1.  Smear from the bracket-toot              h interface of the right lateral incisor.               

Microbiological analysis

Each  sample  was  cultured               on               blood               a              gar               (BA),               Mitis               Salivarius Agar               (MSA),                             Eosin-

Methylene Blue Agar (EMBA), and Chr              omoagar for Candida (C              C), which were left at

35ºC  for  48  hours.  T he  samples  s              eeded  i              n               AMS  were  placed  in  microaerophilic

conditions  with  10%  carbon  dioxide  (CO2).               After  this  t              ime,  the  isolated  bacterial

colonies were taken and a second               isolation was performed, with t              he               aim of growing

pure colonies  for  identification               by               the               Polymerase               Chain               Reaction               (PCR)               technique

and by Matrix-Assist ed Laser Desorpti              on/Ionisation Time of Flight (MALDITOF).(              9)

Extraction  o f  deoxyribonu cleic  acid  (DNA)  for  SM  and  Aggregatibacter

actinomycetemcomitans (Aa) 

DNA  extraction  was  performed               using               the               chemical               method               of               1%               sodium               dodecyl

sulphate  (SDS)  in  0.5N  Na(OH).               SM               and Aa               colonies               were               placed               in               a               1.5               mL               tube

Cuaderno de odontología. Revista científica  

Vol. 3(2), 1-20, 2025

Evaluación de la colonización microbiana en la interfase aditamento diente en

tratamientos de ortodoncia fija

Evaluation of microbial colonisation at the attachment interface i              n fixed orthodontic treatments               

María Elizabeth Llivichuzca-Illescas

Magaly Noemí Jimenez-Romero

Jessica María Sarmiento-Ordóñez

Carlos Alberto Flores-Cárdenas

6

in  1  mL  of  distilled  water,  centrifuged  f              or  10  mi              nutes  at  10,000  RPM  after

homogenisation  in  a  vortex,               the               supernatant               was               disc              arded               and               50               uL               of  SDS               in               

Na(OH)  was  added,  brought               to               t              he               boil               i              n               a               thermoblock               at               98               °C               for               10               minutes,

then 450 µL of  ultrapure w ater was               added, the tube               was centrifuged for 30 seconds

at 3000 RPM and stored frozen until use. (              11,12)

PCR for SM and Aa

The primers used were GTFB F: 5'-ACTACACTTT              CGGGTGGCTTGG-3' and GTFB

R:  5'-CAGTATAAGCGCCAGTTTCATC-3'  for  SM  and  Aa  F:                5'-

ATTGGGGTTTAGCCCTGGTG-3'               and               Con               R:               5'              -ACGTCATCCCCACCTTC              CTC-3'

for Aa.  PCR was performed               using the               SimpliAmp               thermocycler from               Invitrogen.               Ten

microlitres  of  GoTaq  Green               Master               Mix               2X               from               Promega,               6               microlitres of               ultrapure

water, 1.5 microlitres of each primer and 1 microlitre of DNA were used, giving               a final

reaction  volume  of  20  microlitres.  The  thermocycler  protocol  used  was  a              n  initial               

denaturation  at  98  °C,  30               cycles               of               denaturation               at               9              5               °C,               hybridisation               at               53               °C,

extension at 72 °C for 1 minute and a final extension at 72 °C for 10 minutes. (              11)

MALDITOF

The  isolated  colonies  are  exposed  t              o  laser               pulses  that  generate               ions,  which  are

analysed  by  the  spectrometer. T              he data               obtained               are               compared               with               a               database               of

protein  profiles  of  known               micr              oorganisms,               allowing               identification               in               minutes.                             Each

bacterium  has  a  s pecific  "molecular  fingerprint"  that  can  be  visualised  by  t              he

formation  of  a  pattern  of               peaks               specific               to               each               species,               which               are               comp              ared               with

the  database.  The  most  accurate               match,  i.e.               99.9%,               corresponds               to               the               bacterial

identification. 

The  data  were  obtained  thro              ugh  dire              ct  collection  of  microbiological  samples  and

analysis  in  the  laboratory  belonging  to  t              he  Centre  for  Research,  Innovation  and

Technology Transfer ( CITT).               The               study               protocol               was               developed               in               accordance               with

Cuaderno de odontología. Revista científica  

Vol. 3(2), 1-20, 2025

Evaluación de la colonización microbiana en la interfase aditamento diente en

tratamientos de ortodoncia fija

Evaluation of microbial colonisation at the attachment interface i              n fixed orthodontic treatments               

María Elizabeth Llivichuzca-Illescas

Magaly Noemí Jimenez-Romero

Jessica María Sarmiento-Ordóñez

Carlos Alberto Flores-Cárdenas

7

the Helsinki guidelines and was approv              ed by the Human Research Et              hics Committee

of the Catholic University of Cuenca (CEISH-               UCACUE- 2024-065-15/07/2024).

Statistical methods

Frequency  and  contingency               tables               were               presented.               Pearson's               chi              -square               test               was

used  to  analyse  the  association  bet              ween  m              icroorganisms  and  dem              ographic  an              d

clinical  variables,  and  Cramer's               V               was               used               t              o               establish               the               association               b              etween

microorganisms  a nd  ceme nt  type.  Kendall's  tau              -c  test  was  used  to  establish  the

association  between  the  presence  of  microorganisms  and  treatment  time.  The

significance  level  was  0.05%,  and  the  Statistical  Package  for  Social  Sciences

(SPSS) version 25 was used.  

RESULTS 

Of  a  t otal  of  158  patients  treated  at               the  postgraduate               ortho              dontic               clinic,  91  were

excluded  because  they  did  not               me              et               the               inclusion               criteria.               The               stu              dy               evaluated               the

presence  of  oral  microorganisms               in               67               patients               with               fixed               orthodontic               appliances.

Most participants  were  between 12               and 18               years old (55.2%), with               a predominance

of  females  (65.7%). The  most               comm              on               treatment               time               was               1               t              o               4               months               (50.7              %),

and  the  most  commonly  used               cement               was               GoTo®               (58.2%). A               higher               pres              ence               of

microorganisms  w as  observed  in  t              hese  groups,  which  can  be  attributed  to  their

predominant representation in the sample (Table 1).               

Cuaderno de odontología. Revista científica  

Vol. 3(2), 1-20, 2025

Evaluación de la colonización microbiana en la interfase aditamento diente en

tratamientos de ortodoncia fija

Evaluation of microbial colonisation at the attachment interface i              n fixed orthodontic treatments               

María Elizabeth Llivichuzca-Illescas

Magaly Noemí Jimenez-Romero

Jessica María Sarmiento-Ordóñez

Carlos Alberto Flores-Cárdenas

8

Table 1 Demographic distribution of the sample

No statistically significant association was observed b              etween the type of cement and

the presence of  microorganisms, as               the absence of               these was               high for               all cements,

reflecting  a  low  level  of  colonisation  in  ge              neral,  wh              ich  indicates  that  the  type  of

cement does  not  have a  relevant               impact on               the               presence               of microorganisms               in t              his

study;  however,  GoTo®  cement               had  slightly               higher  percentages               compared  to  the

other cements: Gram-positive 12%, Gram-negative 11.9              % and yeast 6% (Table 2).               

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Table 2.  Association between microorganisms and cement type.  

Table  3  reveals  a  statistically               significant               association               between               the               duration               o              f               fixed

orthodontic  treatment  and  the               pr              esence               of               yeasts,               specifically               CA               (p               =               0.0              17). An

increase  in  CA  colonisation was               observed               in the               treatment               ranges of               5 to               8               months

and 9 to 12 months, reaching a maximum of 3%.               

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Table 3. Association between microorganisms and time.  

Figure  2  shows  the  distribution  of  microorganisms  identified  in  the  oral  cavity  of

patients  with  fixed  orthodontic  appliances.  SM  was  the  most  prevalent

microorganism,  isolated  in               8               patients,               representing               its               high               cariogenic               capacity               and

affinity  for  retentive  surfaces  such  as  t              he  br              acket-tooth  interface.  This  finding

reinforces  its  central  role  in  the  development  of  white  spot  lesio              ns  and  caries  in               

patients with orthodontic appliances.               

Next  was  CA ,  detected  in               5               p              atients.               This               opportunistic               yeast               has               been               associated

with  imb alances  in  the               ora              l               microbiot              a,               especially               in               conditions               of               poor               hygiene               or

the  presence  of  foreign  b              odies,               such  as  brackets,               which               favour               its               adhesion  and

persistence.

Thirdly,  Pseudomonas fluorescens and Pseudomonas fragi stand out, each present

in  4  patients.  Although  they  are  not                typical  oral  pathogens,  their  ability  to  form

biofilms  and  their  antimicrobial               resistance               may               represent               a               potential               risk               in               patients

with gingival inflammation or pre-ex              isting periodontitis.

The  remaining  microorganisms  identified,  Achromobacter  sp.              ,  St              aphylococcus

aureus,  Pantoea  agglomerans,               an              d               E              nterobact              er               cloacae,               wer              e               f              ound               in               a               single

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patient  e ach. Although  rare  in               t              he               oral               cavity,               these               bacteria               can               act               as               secondary

colonisers,  especially  in  modified  environments  such  as  thos              e  in              duced  b              y  fixed

appliances.

Figure 2.  Distribution of patients according to the presence of the microorga              nism              .

DISCUSSION

The  results  obtained  sh ow  that  t              he  highest  proportion  of  colonisation  by

microorganisms occ urs in  patients with orthodontic               treatment               lasting 1               to 4               months,

compared  to  lower  proportions  in  the  other  groups.  More  than  half  of  the  study

participants were in the 1 to 4 month group, which could have directly influenced the

higher frequency of cases with the presence of microorganisms in this interval.                

8

5

4

4

2

1

1

1

1

1

1

1

1

1

1

0246810

Streptococcus mutans

Candida albicans

Pseudomonas fragi

Pseudomon as fluorescens

Aggregatibacter actinomycetemcomitans

Achromobacter denitrificans

Enterobacter  hormaechei

Pantoe ag glomerans

Raultella terrigena

Stenotrophomonas maltophilia

Enterobacter cloacae

Enterococcus  faecalis

Staphylococc us saprophyticus

Enterococcus faecium

Staphylococcus aureus

I sol ated   mi c roo rgan              is              ms

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The  results  showed  that,  although               so              me               cements               such               as               G              oTo®               were               used               more

frequently,  t heir  greater  representation               also               led               to               a  higher               n              umber               of               cas              es               with

both the presence (2 3.9%) and absence (34.3%) of microorganisms, which could be

explained by  the distribution of               the sa              mple rather               than by               a differentia              l acti              on of               the

material  itself.  Furthermore,  the  presence  or  absence  of  microorganisms  is  not

significantly  associated with               the               type of               orthodontic               cement us              ed (p              -value               = 0.326).

This  indicates  that  bacterial               colonisation               is               statistically               in              dependent               of               t              he               brand               of

cement used in the study patients.                

From  a  clinical  and  microbiological               perspective,               several               studies               have               shown               t              hat,

although  the  chemical  com position               of               orthodontic               materials               can               influence               b              acterial

plaque  retention,  this  i nfluence  is  lim              ited  compared  to  oth              er  factors  such  as  the

patient's oral hygiene, the surface of the bracket or the cem              entation tech              nique used.

In  general,  current  orthodontic  cements  are  d              esigned  with  basic  or  neutral

antimicrobial  properties,  but  do  not  show  significant  differences  in  their  a              bility  to

prevent or promote micro bial growth under real clinical conditions (13,14).               

This result coincides with recent studies indicating that oral microbiota tends to adapt

quickly  to  the  materials  present,               and               that               the               greatest               influence on               its               development

is  associated  with  exposure  time,  plaque  accumulation               and  the               p              atient's  hygiene

habits,  rather  t han  the  specific  type  of  adhesive               used  (1              5).               Several               studi              es               have

shown  that  t he  use  of  f ixed  appliances  can  temporarily  alter  the  oral  microbiota,

affecting both its qu antity and quality (16) ,especially if not accompanied by adequate

oral hy giene (1,3, 9). This imbalance favours the proliferation of microorganis              ms such

as SM and CA,  both identified as predominant in the present study (              17,18).

In this study, a statistically significant relationship was observed between orthodontic

treatment time and the presence of               CA (p = 0.017), showing a progressive increase

in  colonisation  as  t reatment               prolonged.               This               finding               is               in               line               with               that               reported               by

Perkowski et al.,(19) who detected a higher presence of bacteria and fu              ngi, including

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CA,  in patients with  fixed appliances               compared to               those wit              h removable app              liances

or without orthodontic treatment.               

However,  some st udies have shown               different results              . Sanz-Orrio-Soler et               al.(8)               and

Torlakovic  et  al.(20)  found  no               significa              nt               association               b              etween               fixed               appliances               and

increased CA.  It is important to note that these studies differ in their methodological

design;  for  example,  Torlakovic               et               al.               (20)               t              ook               samples               o              nly               from               central               i              ncisors,

which are easy to clean, while th              is st              udy also i              ncluded posterior teeth, where               access

to cleaning is more limited and therefore more prone to biofilm accumulation.               

Similarly,  studies  such  as               t              hose               by               Saloom               et               al.               (21)               and               Brusca               et               al.               (22)               have

evaluated  th e  ad hesion  of  CA  to  different  types  of  brackets.  Althoug              h  t              heir

conclusio ns  differ,  some  indicate               greater               adhesion               to               metal               brackets               and               others               to

ceramic  or  composite  brackets,               both               agree               th              at               the               materials               used               in               orth              odontics

influence  the  colonisation  pattern.  Given  t              hat  all  patients  in  our  study  wore

conventional  meta l  brackets,               it               is               pos              sible               that               this               type               of               surface,               combined               with

poor oral hygiene, facilitated the growth of               CA over time.

SM  is  one  of  t he  most               prevalent               cariogenic               bacteria               in               the               oral               cavity               an              d               plays               a

key  role  i n  the  formation               of               white               spot               lesions               during               fixed               orthodontic               treatment.

These  lesions  represent  the               first               clinical               manifestation               of               enamel               demineralisation

and can progress to caries if not properly treated               (23).

The presence of fixed orthodontic appliances creates retentive niches that favour the

accumulation  of  bacterial  p laque,  especially  around  brack              ets.  This  accumulation

hinders oral hygiene and promotes an acidic environment due to the ferment              ation of

carbohydrates  by bacteria  such               as SM,               which produces               organic               acids,               mainly lactic

acid, lowering the local pH and causing enamel demineralisation (2              4).

Enamel  demineralisation  weakens  t              he  tooth  structur              e,  which  can  com              promise

bracket  adhesion  and  increase  the  risk  of  fractures  or  detachment  during  the

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application of orthodontic forces (24).  Rigorous oral hygiene, the use of high-f              luoride

toothpastes  and  antibacterial  mouthwashes  are  recommended  to  reduce  the  SM

load.  In  addition,  patient  education               on               proper               brushing               techniques               and               reduction               of

fermentable sugar consumption is recommended.               

In  the  present  study,  species  of  the  genus  Pseudomonas  were  also  ide              ntified,

specifically Ps eudomonas  fragi               and P              seudomon              as fluorescens. Although th              ese are

not  traditionally  considered               oral               pat              hogens,               their               presence               in               the               oral               environment               

has b een documented, especially in conditions th              at alter th              e h              omeostasis of the oral

microbiota during the use of fixed orthodontic appliances               (25).

Pseudomonas  fluorescens  is  an               environmental               bacterium               commonly               found               in               soil,

water,  and  moist  surfaces. Although               rarely               associated               with               human               inf              ections,               it               has

a  remarkable  ability  to  form  biofilms                and  exhibit  i              ntrinsic  r              esistance  t              o  multiple

classes  of  antibiotics,  making  it  a  clinically  relevant  microorganism  (26).  St              udies

have shown t hat t his species ca              n adh              ere to               non-dental               surfaces an              d even               colonise

the  oral cavity  when  there               are               devices               that               interrupt               regular               cleaning,               such               as               fixed

orthodontic appliances (2 7).

The antimicrobial resistance of               Pseudomonas spp. represents a potential risk in the

periodontal  field.  If  these  bacteria               integrate               i              nto               an               existing               su              bgingival               biofilm,               for

example  in patients  with periodontitis,               they               can               aggravate               the               disease               by               hindering

the action of conventional antibiotic treatments and enhancing inflammation (27,28).

Its presence in the oral cavity of patients with fixed orthodontic appliances               i              ndicates

the  importance  of  enhanced               hygiene               strategies,               regular               microbiological               mo              nitoring

and preventive measures during treatment.               

The  presence  of  Aa  in  this               study               is               particularly               important               given               its               ability               to               adhere

to  dental  surfaces  and  orthodontic  materials,  f              orming  resistant  biofilms  t              hat  can

exacerbate  gingival  i nflammation  and  compromise  periodo              ntal  stability                during

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orthodontic  treatment. T he  surface               of               brackets,               especially               if               there               are               micro              defects

or excess  cement, can  act  as               an ec              ological               niche for               Aa,               facilitating               its colo              nisation

and  persiste nce  over  time.  This  is  especially  cr              itical  in  p              atients  with  poor  oral

hygiene,  as  subgingival  biofilm  can  harbour  this  microorganism  in  hard-to-reach

areas,  such  as  t he  cement              -bracket-tooth               junction               and               g              ingival               pock              ets               induced               by               

chronic inflammation (2 9).

Studies h ave also shown that Aa h              as the ability to induce a hypoxic and inflammatory

environment that  can alter t he bone               balance necessary               for proper tooth               movement,

leading to  complications s uch as               root               resorption,               loss of               attachment, and               persistent

gingival  bleeding  (30).  This  poses  a  risk  to  the  efficacy  and  normal  course  of

orthodontic treatment in patients colonised with this microorganism.               

The clinical relevance of this research lies in the information provided on changes in

the  oral  microbiota  during  orthod              ontic  treatment,  allowing  us  to  prevent

complications.  Th e  accumulation               of               microorganisms               could               favour               the               development

of caries  and peri odontal  diseases,               so               early               detection would h              elp to minimise               these

risks.

Likewise,  kn owing  th e  diversity  and  presence  of  m              icroorganisms  e              nables  the

establishment  of  personalised  oral  h              ygiene  protocols,  thus  optimising  treatment

results. By identifying the factors that               can cause changes in the microbiota,               specific

preventive  strategies  can  be               developed,               such               as               stren              gthening               oral               education               and

raising  patient  awareness  of               the               importance               of               maintaining               proper               hygiene               before,

during  and  after  treatment,               as               we              ll               as               incorporating               the               use               of               antimicrobial               agents

to promote a healthy oral environm              ent throughout the process.               

CONCLUSION  

Fifteen  types  of  microorganisms  were  identified  in  patients  with  fixed               orthodontic

appliances.  The  most  common  were  Streptococcus  mutans,  Candida  albicans,

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Evaluation of microbial colonisation at the attachment interface i              n fixed orthodontic treatments               

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Carlos Alberto Flores-Cárdenas

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Pseudomonas  fragi,  Pseudomonas  fluorescens  and  Aggrega              tibacter

actinomycetemcomitans.  The               rest               of               the               microorganisms               were               found               at               a               very               low

frequency.

No  statistically  significant  ass              ociation  was  found  b              etween  the  prese              nce  of

microorganisms  and  variables  such  as  cement  type,  age  or  gender.  However,

significant  differences  were  observed  in  relation  t              o  treatment  time  for  yeasts,

specifically  C andida  Al bicans               in               t              he               5  to               12               month               treatment               ranges,               showing               an

increase in count, which suggests that their prevalence increases as treatment time

progresses.

These results  highlight  the importance               of providing               clear guidelines o              n oral hygiene

and preventive strategies during fixed orthodontic treatment in order to minimise the

risk of complications and optimise treatment outcomes.               

FUNDING  

Non-monetary  

CONFLICT OF INTEREST

There is no conflict of interest with individuals or institutions linked to the research.                

ACKNOWLEDGEMENTS  

The authors w ould like to thank the Centre for Research, Innovation and Technology

Transfer (CITT) for its  full support during the research process.                

Contributions of the authors:

Conceptualisation:  Elizabeth Llivichuzca, Magaly Jiménez.               

Formal analysis: Elizabeth Llivichuzca, Magaly Jiménez, Carlos Flores.               

Research:  Elizabeth  Llivichuzca,  Magaly  Jiménez,  Carlos  Flores,  Jessica

Sarmiento. 

Methodology:  Elizabeth  Llivichuzca,  Magaly  Jiménez,  Carlos  F              lores,  Jessica

Sarmiento. 

Statistical analysis: Eliz abeth Llivichuzca, Carlos Flores, Jessica Sarmiento              .

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Evaluación de la colonización microbiana en la interfase aditamento diente en

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Evaluation of microbial colonisation at the attachment interface i              n fixed orthodontic treatments               

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Magaly Noemí Jimenez-Romero

Jessica María Sarmiento-Ordóñez

Carlos Alberto Flores-Cárdenas

17

Supervision: Magaly Jiménez, Jessica Sarmiento.               

Writing, review, and editing: Elizabeth Llivichuzca, Magaly Jiménez, Carlos Flores,

Jessica Sarmiento.

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1. Partouche AJ D, Castro F, Baptista AS, Costa LG, Fernandes JCH, F              ernandes

GV  de  O.  Effects  of  multibracket               orth              odontic               treatment               v              ersus cl              ear               aligners

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