Vol. 103 No.

5 May 2007

ORAL AND MAXILLOFACIAL SURGERY Editor: James R. Hupp

Identification of bacteria associated with spreading odontogenic

infections by 16S rRNA gene sequencing
Marcello P. Riggio, BSc, PhD,a Hiba Aga, BDS, MFDS RCPS,b
Colin A. Murray, PhD, BDS, FDS (Rest Dent), RCS,c Margaret S. Jackson, FIMBS,d
Alan Lennon,e Nicholas Hammersley, BDS, MBBS, FDS, RCS, FRCS, FDS RCPS,f and
Jeremy Bagg, BDS, PhD, FDS RCS, MRCPath, FDS RCPS, FRCPath, FFPH,g Glasgow, UK
and Airdrie, UK
UNIVERSITY OF GLASGOW DENTAL SCHOOL AND MONKLANDS HOSPITAL

Objective. To determine the bacterial species associated with spreading odontogenic infections (SOIs).
Study design. Pus samples from 4 cases of SOI were analyzed by microbiological culture methods for the presence of
bacteria, and by polymerase chain reaction (PCR) amplification, cloning, and sequencing of bacterial 16S rRNA genes.
Results. Culture methods identified species from the genera Prevotella, Streptococcus, and Fusobacterium, as well as
anaerobic streptococci. Molecular detection methods identified a far more diverse microflora. The predominant genus
detected was Prevotella, representing 102 (50.2%) of 203 clones analyzed. Prevotella oris was the most abundant
species identified, representing 45 (22.2%) of 203 clones analyzed. Twelve clones (5.9%) represented uncultivable
species, namely Prevotella PUS9.180, an uncultured Peptostreptococcus species, and an uncultured bacterium
belonging to the Bacteroidetes phylum.
Conclusions. Prevotella species may play an important role in SOIs, and further work to examine in more detail the
pathogenicity determinants of these organisms and associated host responses is warranted. (Oral Surg Oral Med Oral
Pathol Oral Radiol Endod 2007;103:610-7)

Spreading odontogenic infections (SOIs) are the most togenic infections represent the transformation from a
common type of serious orofacial infection encountered localized dentoalveolar infection, usually a periradicu-
by oral and maxillofacial surgeons.1 Spreading odon- lar abscess, to a destructive infection that can spread
rapidly through tissue planes, resulting in a significant
a
Senior Lecturer, Infection and Immunity Section, University of
incidence of mortality.2-4
Glasgow Dental School. Despite the serious nature of these infections, there
b
Dental Surgeon, Department of Oral and Maxillofacial Surgery, have been relatively few studies examining the contri-
Monklands Hospital. bution of host and environmental factors to the devel-
c
Clinical Senior Lecturer, Infection and Immunity Section, Univer-
opment of SOIs. The influence of local features such as
sity of Glasgow Dental School.
d
Chief Biomedical Scientist, Infection and Immunity Section, Uni- the anatomic location of teeth and the surrounding
versity of Glasgow Dental School. fascial planes5,6 is well understood, but the contribution
e
Research Technician, Infection and Immunity Section, University of of systemic factors to the development of SOI is less
Glasgow Dental School. well characterized. Several risk factors associated with
f
Consultant, Department of Oral and Maxillofacial Surgery,
Monklands Hospital. complications in odontogenic infection have been iden-
g
Professor, Infection and Immunity Section, University of Glasgow tified, including the presence of coexisting major sys-
Dental School. temic disease. Systemic conditions that interfere with
Received for publication Mar 20, 2006; returned for revision Jul 06, normal healing and hemostasis are significant contrib-
2006; accepted for publication Aug 11, 2006.
1079-2104/$ - see front matter
utors in host susceptibility to SOI. Examples include
© 2007 Mosby, Inc. All rights reserved. bleeding dyscrasias, diabetes mellitus, malignant dis-
doi:10.1016/j.tripleo.2006.08.009 ease, immune suppression, malnutrition, illicit intrave-

610
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Volume 103, Number 5 Riggio et al. 611

Table I. Clinical details of the 4 patients studied

Patient White cell count Neutrophil count
No. Age (y) Sex Clinical history Fascial spaces involved (⫻ 109/L)* (⫻ 109 / L)† CRP‡ Treatment
1 22 M Two-day history of Right buccal and 14.1 12.2 98 Incision and drainage of
right-sided facial submandibular right buccal space
swelling abscess and
extraction of 1, 3,
and 32
2 18 F Six-day history of Upper right buccal 11.8 7.6 NA Incision and drainage of
swelling right maxillary buccal
following root space abscess and
canal treatment apicectomy of 7
of 7
3 33 M Three-day history Right buccal and 15.6 12.9 111 Incision and drainage of
of pain, swelling submandibular right submandibular
and trismus swelling. Extraction
associated with of 2, 16, 30, and 31
30
4 20 M Four day history of Upper left buccal 13.3 10.5 114 Incision and drainage of
pain and swelling in left upper
swelling buccal space
associated with
10
Tooth numbering is according to the Universal/National System.
NA, not available.
*Reference range 4-11 ⫻ 109 / L.
†Reference range 2-8 ⫻ 109 / L.
‡Reference range 0-6.

nous drug use, and steroid therapy.2,6,7 Furthermore,
patients with preexisting medical conditions often suf-
fer more serious SOIs that require a longer period of
hospitalization.8,9 Social circumstances associated with
poverty are also implicated in host outcome to SOI.
Host determinants including age, systemic disease (par-
ticularly diabetes), malnourishment, alcohol abuse, to-
bacco use, and illicit drug use have been suggested as
contributing toward mortality from SOI.6,7
The polymicrobial nature of SOIs is well docu-
mented10-12 and shows similarity to that associated with
localized dentoalveolar infections.13 However, these
data have been generated by use of traditional micro-
biological culture techniques. The fastidious nature of
many of the organisms involved in purulent infections
of the head and neck region can result in significant
underdetection of key species. It is therefore highly
appropriate to apply molecular detection methods to
their analysis. Furthermore, since it has been estimated
that approximately 50% of the oral microflora is uncul-
tivable,14 it can be hypothesized that uncultivable or
perhaps even novel species contribute to the develop-
ment of SOI. Culture-independent, molecular-based
studies that have investigated the microbial flora in a
wide range of environmental and clinical samples have
identified a greater diversity of bacteria than culture
methods alone.15,16 It is therefore worthy to investigate
whether this situation exists in SOIs.
The purpose of this study was, therefore, to iden-
tify bacteria associated with SOIs by means of mod-
ern molecular detection methods. Four pus samples
were analyzed for the presence of bacteria by poly-
merase chain reaction (PCR) amplification, cloning,
and sequence analysis of bacterial 16S rRNA genes.
The results obtained were compared with data ob-
tained from microbiological culture of the same sam-
ples in the routine diagnostic microbiology labora-
tory.
MATERIAL AND METHODS
Ethical approval was obtained from the Research
Ethics Committee of Monklands Hospital, Ayrshire,
Scotland. All patients gave informed written consent
before enrollment into the study. Patients were selected
from those appearing as emergency cases at the Oral
and Maxillofacial Surgery Department at Monklands
Hospital, Ayrshire, Scotland. Patients with SOIs that
involved 1 or more fascial spaces were eligible. Demo-
graphic and clinical details were collected on a standard
proforma designed specifically for use in the study.
Demographic and clinical data for the 4 subjects are
shown in Table I. All of the patients had rapidly pro-
gressive SOIs that necessitated surgical drainage in a
hospital setting.

612 Riggio et al.
Pus sample collection
During the surgical management of the infection, pus
samples were obtained by aspiration. Before sampling,
the oral mucosa was disinfected with a solution of 0.2%
wt/vol chlorhexidine gluconate. Sampling was accom-
plished intraorally through intact oral mucosa by using
a sterile, disposable 5 mL syringe through a 21G nee-
dle.
Microbiological culture methods
A direct Gram-stained smear was prepared from each
aspirate. Each specimen was then inoculated onto Co-
lumbia blood agar for incubation in 5% CO2 at 37°C
and onto Fastidious Anaerobe Agar (BioConnections,
Wetherby, UK) supplemented with 7.5% wt/vol defi-
brinated horse blood for incubation in an anaerobic
chamber with an atmosphere of 85% N2, 10% CO2, and
5% H2 at 37°C. Plates were incubated for up to 7 days,
and morphologically distinct colonies were subcultured
to obtain pure cultures. Isolates were identified by
Gram staining, enzymatic activities, and sugar fermen-
tation characteristics (Rapid ID 32A; bioMerieux, Bas-
ingstoke, UK), and relevant antibiotic susceptibility
profiles were also determined using standard methods.
DNA extraction
Aliquots (50 ␮L) of each pus sample were diluted
10- to 100-fold in PCR diluent (10 mM Tris/HCl pH
8.0, 10 mM NaCl, 1 mM EDTA). Thirty microliters of
10% SDS and 3-␮L proteinase K (10 mg mL⫺1) were
added to 300-␮L-diluted pus and incubation was car-
ried out at 55°C for 3 hours. Lysed samples were
extracted twice with equal volumes of phenol/chloro-
form (1:1) and once with an equal volume of chloro-
form. DNA was precipitated by adding 0.1 volume 3 M
sodium acetate (pH 5.3) and 2 volumes 100% ethanol,
mixing and incubation at ⫺70°C for 30 minutes. Pre-
cipitated DNA was recovered by centrifugation, and the
pellet was resuspended in 100 ␮l sterile, molecular
biology-grade water.
Polymerase chain reaction
The primers used for amplification targeted con-
served regions of the 16S rRNA gene and were de-
signed to amplify DNA from most bacterial species.
The primers used were 5=-AGA GTT TGA TCM TGG
CTC AG-3= (27f; Escherichia coli nt 8-27) and 5=-
GGG CGG WGT GTA CAA GGC-3= (1387r; E coli nt
1387-1404) (MWG Biotech, Milton Keynes, UK),
where M ⫽ C ⫹ A and W ⫽ A ⫹ T, and give an
expected amplification product of approximately 1,400
bp.17 All PCR reactions were carried out in a total
volume of 50 ␮L, comprising 5 ␮L of extracted bac-
terial DNA and 45 ␮L of reaction mixture containing 1
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May 2007
⫻ PCR buffer (10mM Tris/HCl pH 9.0, 50 mM KCl,
1.5 mM MgCl2, 0.1% Triton X-100), 1.0 U Taq DNA
polymerase (Promega Corporation, Southampton, UK),
0.2 mM dNTPs (GE Health care, Little Chalfont, UK),
and each primer at a concentration of 0.2 ␮M. The PCR
cycling conditions comprised an initial denaturation
step at 94°C for 5 minutes, followed by 33 cycles of
denaturation at 94°C for 1 minute, annealing at 55°C
for 1.5 minutes and extension at 72°C for 1 minute, and
finally an extension step at 72°C for 10 minutes.
Polymerase chain reaction quality control
When carrying out PCR, stringent procedures were
employed to prevent contamination, as previously de-
scribed.18 Negative and positive controls were included
with each batch of samples being analyzed. The posi-
tive control had a standard PCR reaction mixture con-
taining 10 ng of E coli genomic DNA instead of sam-
ple, whereas the negative control contained sterile
water instead of sample. Ten microliters of each PCR
product was electrophoresed on a 2% agarose gel, and
amplified DNA was detected by staining with ethidium
bromide (0.5␮g mL⫺1) and visualization under ultravi-
olet light.
Cloning of 16S rRNA polymerase chain reaction
products
Polymerase chain reaction products were cloned into
pCR2.1-TOPO cloning vector by using the TOPO TA
Cloning Kit (Invitrogen, Paisley, UK) according to the
manufacturer’s instructions. Plasmid DNA from re-
combinant clones was purified with the QIAquick PCR
Purification Kit (QIAGEN, Crawley, UK).
Polymerase chain reaction amplification of 16S
rRNA gene inserts
Following cloning of the 16S rRNA gene product
amplified by PCR for each sample, 50 clones from each
generated library were randomly selected. The 16S
rRNA gene insert from each clone was amplified by
PCR by using the primer pair 5=- GCT ATT ACG CCA
GCT GGC GAA AGG GGG ATG TG-3= (M13FAP)
and 5=- CCC CAG GCT TTA CAC TTT ATG CTT
CCG GCA CG-3= (M13RAP). The M13FAP binding
site is located 52 base pairs downstream of the M13
forward primer-binding site, and the M13RAP binding
site is located 39 base pairs upstream of the M13
reverse primer-binding site, in the pCR2.1-TOPO vec-
tor.
Restriction fragment length polymorphism
analysis of clone inserts
Selected clones from the libraries generated from the
4 samples (203 clones in total) were subjected to re-
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Volume 103, Number 5 Riggio et al. 613

striction fragment length polymorphism (RFLP) analy- Table II. Bacterial species isolated by microbiological
sis with the restriction enzymes RsaI and MnlI. Ap- culture from pus aspirates obtained from 4 patients with
proximately 0.5 ␮g of each PCR product was digested SOI
in a total volume of 15 ␮L with 2.0 U MnlI (Helena Antimicrobial drug
Biosciences, Sunderland, UK) or 2.0 U RsaI (Promega) susceptibility
at 37°C for 3 hours. Restriction fragments were visu- Patient No. Bacteria isolated P A E C M
alized by the aforementioned agarose gel electrophore- 1 Fusobacterium nucleatum S S S S S
sis. For each library, clones were initially sorted into Prevotella buccae S S S S S
distinct groups on the basis of restriction profiles ob- Prevotella denticola S S S S S
tained with RsaI. Further discrimination was obtained 2 Streptococcus anginosus S S S S R
Anaerobic streptococci S S S S S
by digestion of clones with MnlI, a restriction enzyme 3 Prevotella loescheii S S R S S
that is highly efficient at generating unique bacterial Prevotella S S R S S
16S rRNA fingerprints, which resulted in the identifi- melaninogenica
cation of additional distinct RFLP groups. The 16S Streptococcus constellatus S S S S R
rRNA gene of a single, representative clone from each Streptococcus S S S S R
parasanguis
RFLP group identified by restriction enzyme analysis 4 Prevotella buccae S S S S S
was selected for sequencing analysis. Prevotella intermedia R R S S S
Prevotella loescheii R R S S S
DNA sequencing Streptococcus S S S S R
acidominimus
Sequencing reactions were performed with the Fer- Streptococcus intermedius S S S S R
mentas Life Sciences CycleReader Auto DNA Se-
quencing Kit (Helena Biosciences) and IRD800-la- P, penicillin; A, amoxicillin; E, erythromycin; C, cephalexin; M,
metronidazole; S, susceptible; R, resistant.
beled M13 universal (⫺21) primer on a Primus96 DNA
thermal cycler (MWG Biotech) by using the following
cycling parameters: (i) initial denaturation at 95°C for to penicillin, 2 were resistant to amoxicillin, and 2 were
30 seconds; (ii) 10 seconds at 95°C, 30 seconds at 57°C resistant to erythromycin, all 6 of which were Pre-
and 30 seconds at 70°C for 20 cycles; and (iii) 10 votella species. Five isolates (all Streptococcus species)
seconds at 95°C and 30 seconds at 70°C for 15 cycles. were resistant to metronidazole. No isolates were resis-
Six microliters of formamide loading dye was added to tant to cephalexin.
each reaction mixture following thermal cycling; 1.5
␮L of each denatured sequencing reaction mixture was Molecular analysis
run on a LI-COR Gene ReadIR 4200S automated DNA Two hundred three clones from 4 SOI samples were
sequencing system (MWG Biotech) according to the subjected to restriction enzyme analysis. Since many
manufacturer’s instructions. RFLP groups contained multiple clones with the same
Sequence data were compiled with LI-COR Base restriction profiles, a single representative clone from
ImagIR 4.0 software (LI-COR, Lincoln, NE, USA), each group was sequenced. A total of 109 clones were
converted to FASTA format, and compared with 16S sequenced. Generally, 500 to 800 bp of DNA sequence
rRNA gene sequences from public sequence databases were obtained for each clone.
Genbank and EMBL by using the advanced gapped
BLAST program, version 2.1.19 Species were classed Identification of bacterial genera
as potentially novel if the sequence identity of their 16S The genera identified across the 4 samples are shown
rRNA gene was less than 97.0%. in Table III. Prevotella was by far the most prevalent
genus, accounting for approximately half of the clones
RESULTS analyzed. Dialister accounted for 9.9% of clones ana-
Microbiological culture lyzed and Porphyromonas for 8.9% of clones analyzed.
The bacterial species isolated by microbiological cul- Other less predominant bacteria included bacteria from
ture for the 4 samples analyzed and their antimicrobial the genera Bulleidia, Fusobacterium, Peptostreptococ-
drug susceptibilities are indicated in Table II. The mi- cus, Solobacterium, and Lactobacillus, from the Bac-
croflora was limited to 3 genera. Prevotella species and teroidetes phylum, and from the Lachnospiraceae fam-
Streptococcus species were each present in 3 of the ily.
samples. Fusobacterium nucleatum was present in a
single sample. Fourteen isolates were obtained: Pre- Identification of bacterial species
votella (7), Streptococcus (5), anaerobic streptococci The bacterial species identified by molecular meth-
(1), and Fusobacterium (1). Two isolates were resistant ods in the 4 samples are shown in Table IV. P oris was
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614 Riggio et al. May 2007

Table III. Bacteria identified according to genera by Table IV. Bacteria identified according to species by
16S rRNA gene sequencing of 4 pus aspirates obtained 16S rRNA gene sequencing of 4 pus aspirates obtained
from patients with SOI from patients with SOI
No. of clones No. of clones No. of clones No. of clones
analyzed (%) sequenced (%) analyzed (%) sequenced (%)
Genus N ⫽ 203 n ⫽ 109 Bacterial species N ⫽ 203 n ⫽ 109
Prevotella 102 (50.2) 53 (48.6) Prevotella oris 45 (22.2) 25 (22.9)
Dialister 20 (9.9) 8 (7.3) Prevotella oral sp. 28 (13.8) 11 (10.1)
Porphyromonas 18 (8.9) 9 (8.3) Dialister pneumosintes 20 (9.9) 8 (7.3)
Bacteroidetes* 9 (4.4) 1 (0.9) Porphyromonas oral sp. 16 (7.9) 8 (7.3)
Bulleidia 8 (3.9) 6 (5.5) Prevotella denticola 13 (6.4) 4 (3.7)
Fusobacterium 8 (3.9) 4 (3.7) Uncultured Bacteroidetes* 9 (4.4) 1 (9.2)
Peptostreptococcus 8 (3.9) 7 (6.4) Prevotella multiformis 8 (3.9) 5 (4.6)
Lachnospiraceae# 7 (3.4) 7 (6.4) Lachnospiraceae oral sp. 7 (3.4) 7 (6.4)
Solobacterium 7 (3.4) 2 (1.8) Solobacterium oral sp. 7 (3.4) 2 (1.8)
Lactobacillus 6 (3.0) 4 (3.7) Lactobacillus catenaforme 6 (3.0) 4 (3.7)
Flexistipes 4 (2.0) 2 (1.8) Bulleidia moorei 6 (3.0) 4 (3.7)
Streptococcus 3 (1.5) 3 (2.8) Fusobacterium oral sp. 6 (3.0) 2 (1.8)
Megasphaera 1 (0.5) 1 (0.9) Peptostreptococcus micros 4 (2.0) 3 (2.8)
Unidentified oral bacterium 1 (0.5) 1 (0.9) Flexistipes oral sp. 4 (2.0) 2 (1.8)
Bacterium MDA4277 1 (0.5) 1 (0.9) Peptostreptococcus oral sp. 3 (1.5) 3 (2.8)
Prevotella tannerae 3 (1.5) 3 (2.8)
*Phylum.
Streptococcus anginosus 3 (1.5) 3 (2.8)
#Family.
Uncultured Prevotella sp. 2 (1.0) 2 (1.8)
Bulleidia extructa 2 (1.0) 2 (1.8)
Porphyromonas endodontalis 2 (1.0) 1 (0.9)
Prevotella melaninogenica 1 (0.5) 1 (0.9)
the most prevalent species, representing 22.2% of an- Prevotella genomosp. 1 (0.5) 1 (0.9)
alyzed clones, followed by Prevotella oral species Prevotella sp. 1 (0.5) 1 (0.9)
Uncultured Peptostreptococcus 1 (0.5) 1 (0.9)
(13.8%). The next most prevalent species were Dialis-
Fusobacterium nucleatum 1 (0.5) 1 (0.9)
ter pneumosintes (9.9%), Porphyromonas oral species Fusobacterium naviforme 1 (0.5) 1 (0.9)
(7.9%), and Prevotella denticola (6.4%). Megasphaera oral sp. 1 (0.5) 1 (0.9)
Unidentified oral bacterium 1 (0.5) 1 (0.9)
Uncultured species Bacterium MDA4277 1 (0.5) 1 (0.9)
Twelve clones (5.9%) represented uncultured species *Phylum.
(Table V). Nine clones were matched to an uncultured
bacterium clone from the Bacteroidetes phylum. Two
clones were identified as Prevotella PUS9.180, an un-
cultured species previously identified in dentoalveolar agents of induction and the role of participating host
abscesses.20 A single clone represented an uncultured cell populations associated with the perpetuation of
Peptostreptococcus species. periradicular disease, and factors associated with devel-
No potentially novel species were identified. opment of spreading odontogenic infections, are still
poorly understood.30
DISCUSSION Molecular microbiological techniques have revo-
Microorganisms have been established as the etio- lutionized diagnostic methods within several bran-
logic agents of odontogenic infections by studies dem- ches of medicine. These techniques are more sensi-
onstrating that in microbe-free conditions, periradicular tive, accurate, and efficient at microbial detection than
disease does not develop.21,22 These odontogenic conventional microbiological culture and are capable of
pathogens are responsible for instigating nonspecific detecting uncultivable bacteria. However, the identifi-
and antigen-specific host immune responses. Initially, cation of suspected etiologic pathogens responsible for
an inflammatory immune response occurs within the the initiation and perpetuation of intraradicular and
dental pulp, and subsequently, the periradicular tis- extraradicular odontogenic infection has only more re-
sues.23 Perpetuation of this chronic inflammatory reac- cently shifted from conventional culture techniques to
tion leads to the development of periradicular disease molecular identification methods. In many instances,
comprising periradicular granulomas, abscesses, and the extraradicular endodontic infection remains local-
periradicular bone cysts. Periradicular disease is com- ized within the periradicular tissues in the form of
mon, affecting 33% to 80% of the adult population.24-29 chronic periradicular cysts or granulomas adjacent to
It is therefore surprising that the specific etiologic the root apex and responds to endodontic treatment.
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Volume 103, Number 5
Table V. Details of clones sequenced representing uncultured species
Sample Sequenced bases Sequence identity
No.(clone) available for BLAST Matching bases % Accession No.
1 (06) 718 652/660 98.8
1 (50) 614 531/547 97.1
4 (02)* 549 532/539 98.7
4 (08) 524 493/503 98.0
*Nine clones possessed identical RFLP profiles.
Nevertheless, there is a risk of endodontic bacteria
becoming established within the periradicular tissues.
Dysregulation of the equilibrium between host immune
responses and microorganism-related factors has the
potential to lead to an acute and severe disseminating
infection. A rapidly spreading odontogenic infection
may lead to significant complications at distant sites,
including brain abscesses, cavernous sinus thrombosis,
septic shock, septicemia, venous septic emboli, osteo-
myelitis, and bacteremia. Furthermore, patients suffer-
ing from disseminating odontogenic infection are at
serious risk of airway compromise, systemic toxicity,
and multisystem organ failure, all of which may result
in mortality.2 Patients suffering from craniocervical
necrotizing fasciitis, deep neck infection, and descend-
ing necrotizing mediastinitis have significant mortality
rates, and the source of infection is most frequently
dental in origin.31
Patients with SOI usually respond to early aggressive
surgical intervention and concomitant antimicrobial
chemotherapy.4,32 Early diagnosis and management of
these infections is therefore essential to prevent or
minimize the development of potentially serious com-
plications such as airway obstruction and septicemia,5
which may prove fatal.32-34 Despite advances in com-
puted tomography, resuscitation procedures, and criti-
cal care techniques, survival rates of patients with SOI
have not improved.35 In a previous study, we observed
that 68% of patients with SOI had been administered
oral antibiotics by the time of hospital admission,36
highlighting the inappropriate use of antibiotics for the
initial management of odontogenic infections. The use
of molecular diagnostic methodology, which enables
the rapid identification of potential pathogens concom-
itantly with specific antibiotic susceptibility, would al-
low immediate and more appropriate patient care to
proceed, thereby avoiding extensive morbidity or mor-
tality. It is widely accepted that 16S rRNA gene se-
quencing is superior to conventional microbiological
methods for the identification of bacteria in clinical
samples, reducing the frequency of misidentification
associated with culture methods.37 Based upon the bac-
terial species identified, it would then be possible to
derive species-specific primers for use in direct PCR
Riggio et al. 615
Identified bacterial species
AJ012605 Uncultured Prevotella PUS9.180
AJ012605 Uncultured Prevotella PUS9.180
AY806532 Uncultured Bacteroidetes bacterium clone IS005B34
AY807200 Uncultured Peptostreptococcus sp. clone IS029B54
assays to rapidly and accurately detect the most prev-
alent bacteria present in a much larger number of SOI
samples. From our pilot study, it would be beneficial to
develop PCR primers targeting P oris, which accounted
for approximately 22% of the clones analyzed.
Previous studies have identified Actinomyces and
Propionibacterium species as the principal bacteria in-
volved in persisting localized extraradicular odonto-
genic infection.38 In contrast, acute orofacial odonto-
genic infections are primarily composed of Gram-
positive anaerobic cocci and anaerobic Gram-negative
rods.3,39 The most commonly isolated Gram-positive
cocci include Peptostreptococcus micros, Peptostrep-
tococcus anaerobius, Anaerococcus (formerly Pep-
tostreptococcus) prevotti, and the Streptococcus angi-
nosus group. The most frequently detected Gram-
negative rods within acute odontogenic infections
include Prevotella intermedia–like organisms, Porphy-
romonas endodontalis, Porphyromonas gingivalis,
Tannerella forsythensis, and F nucleatum. Generally,
2.2 to 6.1 bacterial isolates have been recovered from
pus specimens obtained from patients with SOI.39
Within this current study, the microbial species iso-
lated by culture from SOI-derived pus samples is in
agreement with others that have employed standard
laboratory culture techniques. Prevotella, Fusobacte-
rium, and bacteria from the S anginosus group were all
commonly detected isolates by conventional microbio-
logical culture. Cultural analysis also substantiated the
polymicrobial nature of SOI with 2 to 5 bacterial iso-
lates detectable within each pus specimen. Prevotella
and bacteria from the S anginosus group were the most
frequently cultured isolates from SOI samples. Interest-
ingly, Klebsiella pneumoniae was not detected in any
of the SOI specimens. This bacterial species has been
previously cultured from a significant number of pus
samples obtained from patients with deep neck infec-
tions, which were primarily odontogenic in origin.31
Although some correlation was found between the
species identified by culture and molecular detection
methods, anomalies did exist. For example, Streptococ-
cus species were identified in samples 2, 3, and 4 by
culture only. Conversely, S anginosus was detected in
sample 1 by molecular methods only. One possible

616 Riggio et al.
explanation for such an anomaly is primer bias, since
no consensus primers specific for bacterial 16S rRNA
genes will amplify all known bacterial species. One
may also question the accuracy of the phenotypic meth-
ods used to identify bacterial isolates, since although
Prevotella species were identified in most of the sam-
ples by culture, their identity did not generally correlate
with those identified by molecular methods. This prob-
lem could be overcome by identification of bacterial
isolates by 16S rRNA gene sequencing, which has been
shown to be a more reliable method than phenotypic
identification.37 However, this option was not available
in this study since the isolates were not retained by the
routine diagnostic microbiology laboratory carrying out
the cultural analysis.
The use of molecular detection methods in the anal-
ysis of endodontic infections40-42 and dentoalveolar
abscesses13 has shown the presence of a wide range of
fastidious, potentially novel, and uncultivable species.
Of particular significance in the current study was the
frequency of detection of bacteria of the Prevotella
genus, in particular P oris, from the pus aspirates.
Bacteria from this genus constituted approximately half
of all clones analyzed and sequenced. The precise role
that Prevotella species play in the exacerbation of acute
odontogenic infection is unknown. Further research is
necessary to establish the virulence factors responsible
for the pathogenicity of this bacterial genus and their
relationship with host immune responses.
In the current study, libraries generated by cloning of
PCR-amplified bacterial 16S rRNA were screened by
RFLP analysis with the restriction enzymes RsaI and
MnlI. This RFLP approach is often used to screen clone
libraries to avoid unnecessary sequencing of identical
clones.43,44
While primary treatment of SOI is surgical, the con-
comitant use of antibiotics is fundamental to the success-
ful outcome of treatment. The use of molecular techniques
would allow more rapid identification of microbial agents
within SOI, thereby enabling a quicker clinical diagnosis
of etiologic pathogens and guidance on use of antimicro-
bial agents. Furthermore, through the use of molecular
methodology, we also detected the presence of unculti-
vable bacteria within the SOI pus specimens. It is possible
that such species have a role in the development and
aggressive nature of SOI. Further work would be merited
to elucidate the significance of their involvement in dis-
ease progression.
Host factors including age, systemic disease (partic-
ularly diabetes), malnourishment,3 and abuse of alco-
hol, tobacco, and drugs have been suggested as con-
tributing toward mortality from SOI, as occurs in deep
neck infections.31,45 However, we hypothesize that pa-
tient susceptibility to these aggressive infections may
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May 2007
also be related to other, as yet, unidentified host re-
sponse factors. Clearly, further studies are required to
elucidate defects within the host’s immune response to
suspected SOI etiologic pathogens that may contribute
to an inability to confine and localize the odontogenic
infection.
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Reprint requests:
Marcello P Riggio, BSc, PhD
Level 9, Glasgow Dental Hospital & School
378 Sauchiehall Street
Glasgow G2 3JZ, Scotland, UK.
m.riggio@dental.gla.ac.uk