Key words: Anaerobic bacteria - full-mouth disinfection - Ion Torrent Personal Genome Machine
- localized aggressive periodontitis - nonsurgical treatment
INTRODUCTION
Localized and generalized aggressive periodontitis show different clinical features,[1 ],[2 ] being also characterized by slightly diverse bacterial profiles in the subgingival
microbiota.[3 ],[4 ] The etiology of both diseases is widely thought to be polymicrobial,[5 ],[6 ] but the role of individual species and their complex interactions with the host
have not been clearly defined yet. The first microbiological analyses on these pathologies
were conducted with conventional culture or immunohistochemical methods,[7 ],[8 ] followed by a generation of molecular detection methods such as hybridization assays
or polymerase chain reaction (PCR)[9 ],[10 ] and even using atomic force microscopy to indagate bacterial morphology and to examine
microbial interactions in subgingival biofilm.[11 ] The most recent advance has been cloning and sequencing of 16S rRNA genes using
the fast Ion Torrent Personal Genome Machine (PGM): this approach, up‑to‑date reported
only once in periodontology,[12 ] allows an open‑ended exploration of bacterial populations, potentially revealing
the presence of uncultivated species. The possibility of targeting the hypervariable
region V3 of the 16S rRNA gene could provide a complete analysis of subgingival plaque
samples, giving additional informations to evaluate the impact of different treatments
or to detect the presence of resistant forms of various bacteria species. The aim
of the present case report was to follow shifts in subgingival bacterial communities
after nonsurgical and pharmacological therapy in a case of localized aggressive periodontitis
using next generation 16S rRNA gene sequencing approach.[13 ]
CASE REPORT
A 16‑year‑old female Caucasian patient, smoker (10 cigarettes/day for 3 years), was
referred to our observation showing a localized aggressive form of periodontitis.[14 ] After retrieving general and dental anamneses, the patient underwent oral examination
to assess periodontal conditions and dental health: complete periodontal chart was
performed (full-mouth plaque score [FMPS]: 38%; full-mouth bleeding score [FMBS]:
28%), together with full‑mouth periapical radiographs. Then, after receiving oral
hygiene instructions and motivation, the patient underwent causal therapy according
to full‑mouth disinfection protocol,[15 ] together with the following pharmacological therapy: 875 mg amoxicillin + 125 mg
clavulanate twice a day and 250 mg metronidazole 3 times a day, for 10 days.[16 ],[17 ] The patient was reevaluated in 30 days and then recalled every 3 months for supportive
periodontal therapy, based on periodontal risk assessment.[18 ] New periapical radiographs and periodontal chart were collected after 1 year (FMPS:
16%; FMBS: 1%). No further pharmacological therapy was prescribed during the entire
follow‑up period and the patient did not quit smoking. Microbiological samples were
collected at baseline and at 30, 100, 365 days follow‑up from every pathological site
(probing depth [PD] ≥5 mm).
Supragingival plaque was gently removed; teeth were air‑dried and isolated with cotton
rolls. A sterile paper point (ISO45, Roeko Dental, Langenau, Germany) was inserted
for 10 s in each site, and all paper points were pooled in a sterile test tube.
Sample processing
16S rRNA gene amplification
DNA extraction was performed using the NucliSENS® easyMAG® system (Biomerieux, Gorman,
North Carolina, USA). A real‑time quantitative EvaGreen® dye (Fisher Molecular Biology,
Waltham, Massachusetts, USA) PCR was performed with the degenerated primer 27FYM to
better amplify several bacterial species, and the U534R primer, targeting the V1‑V3
region of 16S gene. A nested PCR was performed with the primers B338F_P1‑adaptor (B338F
5′‑ACTCCTACGGGAGGCAGC‑3′) and U534R_A_ barcode (U534R 5′‑ATTACCGCGGCTGCTGG‑3′), to
prepare a 200 base template for final sequencing, with a different barcode for each
sample, to amplify the bacterial V3 region.[19 ]
PCR sample processing
The correct size of the amplicon (260 bp) was assessed on a 2% agarose gel. Quantification
of the amount of DNA was assessed with a Qubit® 2.0 Fluorometer (Invitrogen, Carlsbad,
California, USA) and an equal quantity of PCR from each sample was used to produce
the pooled library.
Ion Torrent sequencing
The pooled library was diluted at a concentration of 26 pM. Template preparation was
performed using the Ion PGM Template OT2 200 kit on Ion OneTouch™ 2 System (Life Technologies,
Gran Island, New York, USA), after which the enrichment percentage was carried out
on Qubit® 2.0 Fluorometer. The templates were sequenced on the Ion PGM™ System machine,
using the Ion PGM sequencing 200 Kit V2 (Life Technologies, New York, USA).
Sequence data analysis
QIIME 1.8.0 (Knight Lab, Boulder, Colorado, USA) was used to process the sequence
data. High quality (Q > 25) sequences were demultiplexed and quality filtered using split_libraries_fastq.py
with default parameters, except for length parameter (150 bp). Sequences were clustered
into operational taxonomic units (OTU) using de novo OTU picking against the Greengenes 13_8 reference OTU database with a 94% and 97%
similarity threshold. Alpha diversity was calculated using Shannon metrics. Statistical
analysis was performed with SIMPER and ANOSIM tests.
RESULTS
At baseline, Capnocytophaga, Fusobacterium, Prevotella , and Treponema were the most represented genera in a composite microbiota (α diversity = 6). Nonsurgical
treatment following full‑mouth disinfection protocol together with systemic antibiotic
therapy drastically lowered the anaerobic species and only a limited presence of Capnocytophaga could be detected at 30 days after treatment (α diversity = 2). The recolonization
of the pockets by anaerobes started to be evident 100 days after treatment (α diversity
= 4). Among the detected genera, Porphyromonas, Prevotella, Dialister, Capnocytophaga , and Treponema showed a greater relative abundance at 365 days posttreatment comparing to the microbiota
composition at the baseline. Specifically, the presence of Tannerella was restored and Aggregatibacter was lowered; Fusobacterium was not present and Veillonella was detected. Conversely, commensal species such as Streptococcus, Leptotrichia, Eikenella, Neisseria , and Haemophilus were absent or lowered (α diversity = 5). The observed differences were not statistically
significant (assessed by Kruskal–Wallis test).
Figure 1: The genus‑level composition of the subgingival bacterial
DISCUSSION
In the present case [Figure 1 ], it appears clearly how the subgingival microbiota changed in quantity and quality
from pretreatment to posttreatment. As previously reported,[20 ],[21 ] recontamination of the affected sites was the main issue pushing researchers for
developing protocols such as “full‑mouth disinfection,” in all its updated versions.[15 ],[22 ],[23 ] Nevertheless, some authors consider the real advantages of this procedure as a questionable
and controversial matter.[24 ],[25 ] In this patient, shifts of subgingival microbiota determined both clinical and radiographical
improvement of the periodontal conditions [Figure 2 ]. FMPS and FMBS decreased along time as well as the probing depth of the pathological
pockets. However, the increasing of anaerobic pathogens appeared remarkable at 365
days after treatment, suggesting that the 3‑month recall program alone could be not
sufficient for maintaining stable periodontal conditions over time. It is interesting
to note that the bacterial shift toward a higher prevalence of pathogen species was
not accompanied by clinical signs of inflammation: microbiological analysis seems
to predict the necessity of further therapies before the clinical scenario becomes
evident.
Figure 2: Periapical radiographs comparing baseline condition (upper line) with the situation
1 year after nonsurgical and pharmacological therapy (lower line)
The main advantage of the Ion Torrent PGM platform is basically due to the high throughput
of the technique able to identify microbiological species missed by culture‑based
method. Differently from PCR and other microarray techniques, Ion Torrent performs
a quantitative analysis of the microbiota, irrespective of the searched species. The
metagenomic analysis applied in this case report could open possible new perspectives
in exploring differences of the subgingival microbiota in periodontal patients: changes
in community profiles and metrics could be diagnostically more predictive than the
detection of selected periodontal pathogens, such as in PCR or in culture‑based methods.
An accurate definition of the shifts in the bacterial community could help periodontal
researcher for a better understanding of the impact of different treatment approaches
or in intercepting nonresponsive conditions.
Financial support and sponsorship
Nil.
