Spiramycin resistance in human periodontitis microbiota
Spiramycin resistance in human periodontitis microbiota
Thomas E. Rams Sebastien Dujardin Jacqueline D. Sautter , John E. Degener ,Arie J. van Winkelhoff ,
a Department of Periodontology and Oral Implantology, and Oral Microbiology Testing Service Laboratory, Temple University School of Dentistry, 3223 North Broad Street,Philadelphia, PA 19140, USAb Department of Microbiology and Immunology, Temple University School of Medicine, 3500 North Broad Street, Philadelphia, PA 19140, USAc Center for Dentistry and Oral Hygiene, Department of Oral Microbiology, University Medical Center Groningen, University of Groningen, Antonius Deusinglaan 1,9713 AV Groningen, The Netherlandsd Department of Medical Microbiology, University Medical Center Groningen, University of Groningen, Hanzeplein 1, 9713 GZ Groningen, The Netherlands
Purpose: The occurrence of in vitro resistance to therapeutic concentrations of spiramycin, amoxicillin,
and metronidazole was determined for putative periodontal pathogens isolated in the United States.
Materials and methods: Subgingival plaque specimens from 37 consecutive adults with untreated severe
periodontitis were anaerobically cultured, and isolated putative periodontal pathogens were identiﬁed to
a species level. In vitro resistance to spiramycin at 4 mg/ml, amoxicillin at 8 mg/ml, and/or metronidazole
at 16 mg/ml was noted when putative periodontal pathogen growth was noted on the respectiveantibiotic-supplemented primary culture plates.
Results: A total of 18 (48.7%) subjects yielded antibiotic-resistant putative periodontal pathogens with
spiramycin at 4 mg/ml in drug-supplemented primary isolation plates, as compared to 23 (62.2%) subjects
with amoxicillin at 8 mg/ml, and 10 (27.0%) subjects with metronidazole at 16 mg/ml. Spiramycin in vitro
resistance occurred among species of Fusobacterium nucleatum (44.4% of organism-positive subjects),
Prevotella intermedia/nigrescens (11.1%), Parvimonas micra (10.8%), Streptococcus constellatus (10%),
Streptococcus intermedius (10%), Porphyromonas gingivalis (6.7%), and Tannerella forsythia (5.3%). Amox-icillin in vitro resistance was found in P. intermedia/nigrescens (55.5%), T. forsythia (15.8%), S. constellatus(10%), F. nucleatum (5.6%), and P. micra (2.7%). Only S. constellatus (70%) and S. intermedius (40%) exhibitedin vitro resistance to metronidazole. When subject-based resistance data for spiramycin and metroni-dazole were jointly considered, all isolated putative periodontal pathogens were inhibited in vitro by oneor the other of the antibiotic concentrations, except for one strain each of S. constellatus andS. intermedius from one study subject. Similarly, either amoxicillin or metronidazole at the drugconcentrations tested inhibited in vitro all recovered putative periodontal pathogens, exceptS. constellatus in one subject.
Conclusions: In vitro spiramycin resistance among putative periodontal pathogens of United States originoccurred in approximately one-half of severe periodontitis patients evaluated, particularly among sub-gingival F. nucleatum species. In vitro resistance patterns also suggest that therapeutic concentrations ofspiramycin plus metronidazole may have potential antimicrobial efﬁcacy in non-Aggregatibacter acti-nomycetemcomitans-associated periodontitis similar to amoxicillin plus metronidazole, which may bebeneﬁcial, where spiramycin is clinically available, for patients hypersensitive to amoxicillin or otherbeta-lactam antibiotics.
Ó 2011 Elsevier Ltd. All rights reserved.
Spiramycin is a medium-spectrum, 16-membered, macrolide
antibiotic widely used in the treatment of respiratory infections ,
* Corresponding author. Department of Periodontology and Oral Implantology,
except in the United States, where it is available by special
Temple University School of Dentistry, 3223 North Broad Street, Philadelphia, PA
permission for treatment of toxoplasmosis in women during the
19140, USA. Tel.: þ1 215 707 2941; fax: þ1 215 707 4223.
ﬁrst trimester of pregnancy. Since the drug enters the oral cavity
1075-9964/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved.
T.E. Rams et al. / Anaerobe 17 (2011) 201e205
through gingival crevicular ﬂuid and saliva, and persists for
supplemented with either spiramycin at 4 mg/ml, amoxicillin at
extended periods at potentially therapeutic concentrations ,
8 mg/ml, or metronidazole at 16 mg/ml (all antimicrobials were
spiramycin is also prescribed in some countries for various odon-
obtained as pure powder from SigmaeAldrich, St. Louis, MO). These
togenic infections, including endodontic abscesses, pericoronitis,
antimicrobial concentrations represent non-susceptible/resistant
and destructive forms of periodontal disease (periodontitis)
breakpoint concentrations for anaerobic bacteria recommended
A number of studies have assessed the antimicrobial activity of
by the French Society for Microbiology for spiramycin, and the
spiramycin on putative periodontopathic bacteria in subgingival
Clinical and Laboratory Standards Institute (CLSI) for amoxi-
plaque bioﬁlms eExcept for ones completed over 25 years
cillin and metronidazole. All EBBA and Hammond’s selective
ago these studies focused on clinical isolates from perio-
medium plates were incubated at 35 C for seven days in a Coy
dontitis patients located outside of the United States. Since
anaerobic chamber (Coy Laboratory Products, Ann Arbor, MI) con-
geographic differences , and emergence of drug-resistant
taining 85% N2-10% H2-5% CO2, and TSBV plates were incubated at
strains over time may markedly alter antibiotic susceptibility
35 C for three days in 5% CO2-95% air.
patterns of subgingival bacterial species, the present-day antimi-crobial effects of spiramycin on putative periodontal pathogens in
the United States remain speculative.
The present study examined, using fresh subgingival isolates
On non-selective EBBA plates examined with a ring-light
from periodontitis patients in the United States, the occurrence of
magnifying loupe and a dissecting stereomicroscope, the presence
in vitro resistance among putative periodontal pathogens to ther-
and levels of total anaerobic viable counts, Porphyromonas gingivalis,
apeutic concentrations of spiramycin, as well as amoxicillin and
Prevotella intermedia/nigrescens, Parvimonas micra, staphylococci
and enterococci were determined using presumptive phenotypicmethods previously described ; Tannerella forsythia was
identiﬁed as gram-negative, non-motile, anaerobic rods exhibitinggrey-pink speckled, convex, pinpoint colonies seen with a stereo-
microscope, lack of long-wave ultraviolet light autoﬂuorescence,and a positive CAAM test for trypsin-like activity ; Fusobacterium
A total of 37 adults (17 males, 20 females; aged 35e85 years;
nucleatum was identiﬁed as long-wave ultraviolet light auto-
mean 57.5 Æ 12.0 (SD) years), diagnosed with severe periodontitis
ﬂuorescent charteuse-positive gray, iridescent colonies of
by periodontists in United States private dental practices, were
gram-negative, ﬁlamentous, spindle-shaped, non-motile rods;
consecutively included in the present study as their microbiological
Streptococcus intermedius was recognized as gram-positive, lactose
samples were received by the testing laboratory. Persons identiﬁed
MUG-test positive , non-motile, facultative cocci exhibiting
with aggressive periodontitis, or antibiotic use within the past six
small dry, white, raised colonies with wrinkled edges; and Strepto-
months, were excluded. Only 4 (10.8%) of the study subjects were
coccus constellatus was deﬁned as gram-positive, lactose MUG-test
negative, non-motile, facultative cocci demonstrating small white,opaque, circular, beta-hemolytic, surface colonies with irregular
edges, and positive for a-D-glucosidase enzyme activity, with orwithout b-D-fucosidase, b-D-glucosidase positive reactions, as
Subgingival plaque specimens were obtained by each study
determined with the Fluo-Card Milleri test kit (Key Scientiﬁc Prod-
subject’s periodontist, following a standardized sampling protocol,
ucts Co., Stamford, TX) Campylobacter rectus was quantitated on
prior to treatment from the 3-5 deepest periodontal pockets (mean
Hammond’s medium, and Aggregatibacter actinomycetemcomitans,
7.5 Æ 0.3 (SE) mm) per subject which exhibited bleeding on probing.
gram-negative enteric rods, pseudomonads, and Candida species on
In brief, after isolation with cotton rolls, and removal of saliva and
TSBV agar, as previously described . Proportional subject
supragingival deposits, one to two sterile, absorbent paper points
recovery of the various test putative periodontal pathogens was
(Johnson & Johnson, East Windsor, NJ) were advanced into each
calculated as the percent recovery of the test species colony forming
selected periodontal site for approximately 10 s. Upon removal, all
units (CFU) among the total subgingival anaerobic viable CFU count
paper points per study subject were pooled into a glass vial con-
as determined on non-selective EBBA plates.
taining 6-8 small glass beads and 2.0 ml of anaerobically preparedand stored VMGA III transport medium which possesses a high
2.5. In vitro antibiotic resistance testing
preservation capability for oral microorganisms during post-sampling transit to the laboratory The subgingival samples
In vitro resistance to the antibiotic breakpoint concentrations of
were then transported within 24 h to the Oral Microbiology Testing
spiramycin (4 mg/ml), amoxicillin (8 mg/ml), or metronidazole (16 mg/
Service (OMTS) Laboratory at Temple University School of Dentistry,
ml) was recorded per subject when test putative periodontal path-
which is licensed for high complexity bacteriological analysis by the
ogen growth was noted on antibiotic-supplemented and non-
supplemented primary isolation EBBA plates Bacteroidesthetaiotaomicron ATCC 29741, Clostridium perfringens ATCC 13124, and
a multi-antibiotic-resistant clinical periodontal isolate of F. nucleatumwere employed as positive and negative quality controls for all anti-
At the OMTS Laboratory, the specimen vials were warmed to
biotic resistance testing on drug-supplemented EBBA plates.
35 C to liquefy the VMGA III transport medium, and sampledmicroorganisms were mechanically dispersed from the paper
points with a Vortex mixer at the maximal setting for 45 s. Serial10-fold dilutions of dispersed bacteria were prepared in Möller’s
Recovered test putative periodontal pathogens were grouped
VMG I anaerobic dispersion solution . Appropriate dilution
for reporting purposes into subgingival bacterial clusters (i.e., red
aliquots were plated onto non-selective enriched Brucella blood
complex, orange complex, and other species) described by Soc-
agar (EBBA) , Hammond’s selective Campylobacter medium
ransky et al. Descriptive analysis was used to calculate the
, and TSBV agar . Aliquots were also plated onto EBBA
subject occurrence and proportional cultivable recovery of test
T.E. Rams et al. / Anaerobe 17 (2011) 201e205
putative periodontal pathogens on non-selective EBBA, and the
in primary isolation plates (. Between 10 and 11% of subject
subject occurrence of in vitro test putative periodontal pathogen
strains each of P. intermedia/nigrescens, P. micra, and S. intermedius
antibiotic resistance. In vitro data was also combined post-hoc for
demonstrated in vitro spiramycin resistance, and one subject strain
spiramycin and metronidazole, and for amoxicillin and metroni-
each of P. gingivalis and T. forsythia were resistant in vitro to non-
dazole, to determine the number and proportion of organism-
susceptible breakpoint concentrations of spiramycin ).
positive study subjects where test putative periodontal pathogens
Amoxicillin at 8 mg/ml in primary isolation plates inhibited all
exhibited in vitro resistance to both antibiotics at the employed
subject strains of P. gingivalis, C. rectus, and S. intermedius. In
non-susceptible breakpoint concentrations.
contrast, 55.5% of P. intermedia/nigrescens subject strains, and2.7e15.8% of T. forsythia, S. constellatus, F. nucleatum, and P. micra
subject strains displayed in vitro resistance to 8 mg/ml of amoxicillinin primary isolation plates
All microbiological procedures were performed on a standard-
All subject strains of P. gingivalis, T. forsythia, P. intermedia/
ized, blinded basis without knowledge of the clinical status of the
nigrescens, P. micra, C. rectus, and F. nucleatum were inhibited in vitro
study subjects, or their inclusion in the present analysis. Approval
by 16 mg/ml of metronidazole in primary isolation plates. However,
for the study was provided by the Temple University Human
70% of S. constellatus, and 40% of S. intermedius subject strains,
Subjects Institutional Review Board.
revealed in vitro resistance to 16 mg/ml of metronidazole (
When subject-based in vitro resistance data for 4 mg/ml of spi-
ramycin and 16 mg/ml of metronidazole were jointly considered
post-hoc, all isolated test periodontal pathogens were inhibitedin vitro by one or the other of the antibiotic concentrations, except
3.1. Total cultivable counts and putative periodontal pathogen
for one strain each of S. constellatus and S. intermedius in one (2.7%)
study subject. Similarly, either 8 mg/ml of amoxicillin or 16 mg/ml ofmetronidazole inhibited in vitro all recovered test periodontal
Total subgingival anaerobic viable counts on non-antibiotic-
pathogens, except S. constellatus in one (2.7%) subject ().
supplemented EBBA plates averaged 7.32 Â 107 Æ 1.6 Â 107 (SE)organisms/ml of sample in the study subjects (range ¼ 2 Â 106 to
lists the distribution of recovered subgingival test
The present study ﬁndings represent the ﬁrst data in over 25
putative periodontal pathogens in the 37 study subjects.
years on in vitro spiramycin resistance in putative periodontal
Among red complex bacterial species, P. gingivalis was isolated
pathogens in the United States. In vitro resistance among recovered
from 15 (40.5%) subjects, and T. forsythia from 19 (51.4%) subjects, at
putative periodontal pathogens to spiramycin at 4 mg/ml was found
mean subgingival proportions of 14.4% and 1.4%, respectively, in
in 48.7% of severe periodontitis patients evaluated, which was less
culture-positive subjects. Among orange complex and other
in vitro resistance than occurred with amoxicillin at 8 mg/ml (62.2%
species, P. micra was isolated from all 37 subjects, P. intermedia/
of subjects), but more than was found with metronidazole at 16 mg/
nigrescens and F. nucleatum were each found in 36 (97.3%) subjects,
ml (27.0%). These ﬁndings highlight the considerable subject vari-
C. rectus was recovered from 20 (54.1%) subjects, and S. constellatus
ation seen across single antibiotic drugs in their antimicrobial
and S. intermedius were each present in 10 (27.0%) subjects
effects against putative periodontal pathogens, which can mark-
Cultivable subgingival A. actinomycetemcomitans, gram-
edly inﬂuence the selection and efﬁcacy of periodontal antibiotic
negative enteric rods, pseudomonads, staphylococci, enterococci
and Candida species were not detected in any of the study subjects.
Among individual microbial species, most red and orange
complex organisms, which are associated with increasingly more
3.2. In vitro antibiotic resistance testing
severe forms of periodontitis were inhibited in vitro by spi-ramycin at 4 mg/ml in primary isolation plates ). However,
A total of 18 (48.7%) subjects yielded antibiotic-resistant puta-
F. nucleatum was frequently resistant in vitro to spiramycin (44.4% of
tive periodontal pathogens with spiramycin at 4 mg/ml in drug-
subject strains), consistent with previous reports on periodontitis
supplemented primary isolation plates, as compared to 23 (62.2%)
subjects with amoxicillin at 8 mg/ml, and 10 (27.0%) subjects with
Some subject strains of P. gingivalis, T. forsythia,
P. intermedia/nigrescens and P. micra also exhibited in vitro resistance
F. nucleatum most frequently exhibited in vitro spiramycin resis-
to spiramycin, but at lower proportions than homologous species
tance, with 44.4% of subject strains resistant to 4 mg/ml of spiramycin
strains previously isolated and tested in France . For reasons tobe determined, all C. rectus in the present study were inhibited
Occurrence and proportional subgingival recovery of selected putative periodontal
bacterial pathogens in 37 adults with severe periodontitis.
compared to 80% C. rectus resistance to spiramycin at the samein vitro concentration in France
No. (%) of culture- Mean % Æ SE recovery Range %
Since there is negligible spiramycin use in the general United
States population, it is unlikely that the spiramycin resistance
detected in the present study subjects is the result of prior spi-
ramycin drug exposures promoting selection of resistant microbial
species, in contrast to persons in countries where spiramycin is
extensively prescribed. Instead, the increasing dissemination
among gram-negative bacteria, via mobile plasmids and/or con-
jugative transposons, of various drug resistance genes active
against all macrolide-class antibiotics may account for in vitro
spiramycin resistance among periodontal microorganisms not
previously exposed to spiramycin However, the molecular
T.E. Rams et al. / Anaerobe 17 (2011) 201e205
Table 2Occurrence of in vitro resistance among subgingival test putative periodontal pathogens to non-susceptible antibiotic breakpoint concentrations in primary isolation plates.
a No. (%) of species-positive subjects with strains resistant in vitro to non-susceptible antibiotic breakpoint concentrations in primary isolation plates.
b Joint post-hoc consideration of in vitro resistance data for each antibiotic.
basis for the in vitro spiramycin drug resistance detected in the
in some previous studies . However, these favorable
present study remains to be determined.
microbiological effects are consistent with the results of a double-
In vitro resistance to amoxicillin included most P. intermedia/
blind clinical trial ﬁnding systemic spiramycin plus metronidazole
nigrescens subject strains, and a smaller subset of T. forsythia,
to signiﬁcantly enhance gains of clinical periodontal attachment on
S. constellatus, F. nucleatum, and P. micra, with metronidazole resis-
initially deep periodontal pockets at 6 months post-treatment on
tance found only among most S. constellatus and some S. intermedius
clinical isolates, similar to previous reports
A similar post-hoc joint analysis also found either amoxicillin at
The direct plating method used in this study to detect antibiotic
8 mg/ml or metronidazole at 16 mg/ml in primary isolation plates
resistance on primary culture plates has been previously used in
inhibited all recovered putative periodontal pathogens, except
a number of periodontal microbiology studies , and
S. constellatus in one (2.7%) subject, which is also consistent with
shown to highly correlate (r2 ¼ 0.99) with the CLSI-approved
clinical trials demonstrating enhanced therapeutic beneﬁts of
multiple agar dilution assay in successfully identifying antibiotic-
systemic amoxicillin plus metronidazole in aggressive and chronic
resistant periodontal microorganisms Similarly, Lewis et al.
reported a 94% agreement between primary versus secondary
While the periodontists who performed clinical examinations in
(subculture) antibiotic susceptibility plate testing of acute den-
the present study were not formally calibrated, support for their
toalveolar abscess bacterial isolates. The relatively high viable
severe periodontitis diagnoses was evidenced by their identiﬁca-
anaerobic counts (on average in excess of 107 organisms/ml)
tion in the study subjects of three or more periodontal sites with
recovered on non-selective EBBA media in the present study
deep probing depths with bleeding on probing (mean 7.5 mm at
suggests that an adequate subgingival sample inocula was spread
microbiologically-sampled periodontal sites), which strongly
on antibiotic-supplemented primary isolation plates examined for
correlates (94.1% positive predictive value) with the presence of
resistant species growth. However, the inability to determine exact
severe periodontal attachment loss in adult patients
minimal inhibitory concentration (MIC) values of antimicrobials
Overall, these ﬁndings suggest that the in vitro effectiveness of
tested against microbial species is a shortcoming of this approach,
spiramycin against putative periodontal pathogens can be enhanced
since only non-susceptible/resistant breakpoint concentrations, as
by broadening its antimicrobial spectrum with metronidazole, and
recommended by the French Society for Microbiology for
that the joint in vitro antimicrobial effects of spiramycin plus
spiramycin, and the Clinical and Laboratory Standards Institute
metronidazole against a wide range of putative periodontal patho-
for amoxicillin and metronidazole, were employed per antibiotic in
gens appears to be similar to that seen with amoxicillin plus
the in vitro resistance testing. Nevertheless, identiﬁcation of
metronidazole. Thus, a clinical periodontitis treatment strategy
in vitro periodontal pathogen resistance to therapeutic concen-
involving systemic spiramycin plus metronidazole, but not spi-
trations of antimicrobials contemplated for patient care is highly
ramycin alone, may have potential therapeutic efﬁcacy similar to the
relevant and clinically practical to dentists striving to avoid
combination of amoxicillin plus metronidazole, and may be
prescribing antibiotics ineffective against drug-resistant pathogens.
particularly beneﬁcial as an alternative for periodontitis patients
Since increasing interest has developed on use of combination
hypersensitive to amoxicillin or other beta-lactam antibiotics, and
systemic antibiotic regimens in periodontitis treatment, where two
not colonized by A. actinomycetemcomitans strains resistant to spi-
antibiotics with complementary antibacterial activity are adminis-
tered to broaden the spectrum of antimicrobial effects against sub-gingival and soft tissue-invading bacterial pathogens the
present study also jointly considered subject-based resistance datapost-hoc for spiramycin at 4 mg/ml plus metronidazole at 16 mg/ml.
In vitro spiramycin resistance among putative periodontal path-
Interestingly, all isolated putative periodontal pathogens were
ogens of United States origin was found in approximately one-half of
inhibited in vitro by one or the other of the antibiotic concentrations
in primary isolation plates, except for one strain each of
F. nucleatum species. In vitro resistance patterns also suggest that
S. constellatus and S. intermedius from one (2.7%) study subject. Since
therapeutic concentrations of spiramycin plus metronidazole may
the two antibiotic concentrations were not incorporated together
have potential antimicrobial efﬁcacy in non-A. actinomycete-
into antibiotic-supplemented primary isolation plates, no conclu-
mcomitans-associated periodontitis similar to amoxicillin plus
sions can be drawn about synergistic drug effects of spiramycin plus
metronidazole, which may be beneﬁcial, where spiramycin is clini-
metronidazole against oral bacterial species, which have been noted
cally available, for patients hypersensitive to amoxicillin or other
T.E. Rams et al. / Anaerobe 17 (2011) 201e205
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J o u r n é e s « L i e n s o c i a l » Organisées par le LEMNA Laboratoire de gestion et de management de Nantes Atlantique Coordination scientifique Fabien TRIPIER Professeur d’économie à l’université de Nantes 16 - 17 mai 2011 Résumés des communications Maison des sciences de l’homme Ange-Guépin SOMMAIRE Frédéric Allaire La taxe de risque systémique sur
The Food Information Regulation (“the Regulation”) was finally published in December 2011. The Regulation is to be binding in its entirety and directly applicable to all Member states. It applies to food business operators at all stages of the food chain, where their activities concern the provision of food information to consumers, including food intended for supply to mass caterers. It is