Molecular investigation of macrolide and Tetracyclineresistances in oral bacteria isolated from Tunisian children

Bochra Kouidhi a,*, Tarek Zmantar a, Hajer Hentati c, Fayrouz Najjari c,Kacem Mahdouni b, Amina Bakhrouf a

a Laboratoire d’Analyses, Traitement et Valorisation des Polluants de l’Environnement et des Produits, Faculte de Pharmacie de Monastir,

Biologie Clinique, Rue Avicenne, 5000 Monastir, Tunisieb Laboratoire de Biologie moleculaire, Hopital Regionale de Kairouan, TunisiecService de Medecine et chirurgie buccales Clinique hospitalo-universitaire d’Odontologie, Monastir, Tunisie

a r c h i v e s o f o r a l b i o l o g y 5 6 ( 2 0 1 1 ) 1 2 7 – 1 3 5

a r t i c l e i n f o

Article history:

Accepted 13 September 2010

Keywords:

Caries

Streptococci

Antibiotic susceptibility

Resistance genes

a b s t r a c t

Objective: This study aims to investigate the antibiotic susceptibility of strains isolated from

the oral cavity of Tunisian children.

Design: Strains were isolated from the oral cavity of Tunisian children (60 caries-actives and

30 caries-free). Molecular characterization was assessed by PCR assay to detect erythromy-

cin methylase gene (ermB), macrolide efflux (mefI) and tetracycline resistance genes (tetM

and tetO).

Results: A total of 21 species were isolated and identified. Antimicrobial susceptibility

revealed that the resistance rate to antibiotics was as follow: erythromycin (22%), tetracy-

cline (15.6%), cefotaxim, (7.3%), trimethoprim-sulfamethoxazol (37.6%), nitrofurantoine

(2.8%), pristinamycin (17.4%), quinupristin-dalfopristin (15.6%), and rifampicin (3.7%). The

majority of mefI positive strains (31.2%) were isolated from the carious children (n = 34) in

comparison with 8.25% from the control group (n = 9). In addition, frequency of strains

caring resistance genes were as follow: 12.84% for ermB, 9.17% for tetM and 27.52% for tetO

from the carious children in comparison to 0.092%, 3.67% and 3.67% from the caries free

group respectively.

Conclusion: Multi-resistance strains towards macrolides and tetracycline were recorded.

The majority of strains carrying antibiotics resistance genes were isolated from the caries

active children. The presence of multi-resistant bacteria in the oral cavity can be the major

cause of antibiotic prophylaxis failure in dental practise.

# 2010 Elsevier Ltd. All rights reserved.

avai lab le at www.sc iencedi rec t .com

journal homepage: http://www.elsevier.com/locate/aob

1. Introduction

Dental caries is one of the most important causes of oral

infections not only in dentistry but also in medicine. Oral

streptococci associated with dental caries have long been

considered as significant pathogenic agents in dental caries,1

for which they represent the most frequent causative

agent.2,3 Indeed, their clinical importance includes acting

* Corresponding author. Tel.: +216 99225866.E-mail address: bochrak@yahoo.fr (B. Kouidhi).

0003–9969/$ – see front matter # 2010 Elsevier Ltd. All rights reservedoi:10.1016/j.archoralbio.2010.09.010

as causative agents in infective endocarditis, cardiovascular

diseases and bacterial pneumonia which often take place

after some sort of dental procedure.4 More importantly,

oral infections can lead with the introduction of oral

microorganisms into the bloodstream system.5 In addition,

oral streptococci play a significant role as a reservoir of

antimicrobial resistance genes transferable to more patho-

genic organisms.6

d.

a r c h i v e s o f o r a l b i o l o g y 5 6 ( 2 0 1 1 ) 1 2 7 – 1 3 5128

The antibiotic prophylaxis contributes in the spread of

resistant streptococci. The most frequent antibiotic prescribed

for dental and oral infection was penicillin V (60% of total

prescriptions); erythromycin (14%), amoxicillin (12%) and

metronidazole (8%). Tetracycline are used infrequently in

dental practise because of the side-effects associated with this

family of drugs, which can affect tooth colour.7 However,

association of tetracycline resistance with penicillin and

erythromycin resistance makes it potentially dangerous and

may facilitate the dissemination of other resistance determi-

nants.8

Resistance to erythromycin is most commonly due to the

acquisition of erm genes which codes for rRNA methylases.

Other mechanisms by which bacteria express macrolide

resistance include drug inactivation by an enzyme encoded

by mph, and efflux of macrolides by an ATP-binding

transporter encoded by msrA.9 Low-level macrolide resistance

in the oral flora may also be associated with the expression of

genes in the mef family, encoding another efflux pump.10

Two mechanisms of resistance against tetracycline have

been described in enterococci: an efflux-mediated mechanism

encoded by tet(K) or tet(L) genes and ribosomal protection

mediated by tet(M), tet(O), or tet(S) genes.11 The erm(B) gene is

frequently linked with the tet(M) gene on the highly mobile

conjugative transposon Tn1545, which predominates in

clinically important Gramme-positive bacteria.12,13 Macrolide

and glycopeptide resistance genes have also been described on

the same transferable genetic element in Enterococcus faecium

from pigs and humans.14

Cells within a biofilm has been shown to be conducive to

gene transfer.15 The most extensively studied group of

conjugative transposons are those of the Tn916 family,16

which usually encode tetracycline resistance which is

transferable in vitro biofilm models 17 and in vivo mouse

gastrointestinal tracts.18 In a recent study, it has been shown

that Veillonella dispar transfers in oral biofilms Tn916 to

Streptococcus spp. Furthermore, its DNA can transform Strepto-

coccus mitis to tetracycline resistance strains.19

The different patterns of susceptibility to a number of

antibiotics 20 make it necessary to increase our knowledge of

the activity of antibiotics on oral streptococci.

The objective of this study was to determine the prevalence

of antibiotic susceptibility and the distribution of erythromy-

cin resistance gene (ermB), the macrolide efflux (mefI) and

tetracycline resistance genes (tetM, tetO) in oral bacteria

including streptococci and enterococci isolated from Tunisian

children.

2. Material and methods

2.1. Patients and bacterial strains

The study was done on 90 children (60 subjects and 30

controls) from dental clinic of Monastir, Tunisia from July 2008

to January 2009. The age group selected for the present study

was about 4–12 years. Ethical clearance was taken prior to the

commencement of study. Written informed consent was

obtained from the parents of all participants. All clinical

procedures were approved by the Ethical Committee of the

Faculty of Medicine, University of Monastir, Tunisia. A detailed

medical and dental history was obtained from each parent.

The criteria for inclusion were: no antibiotic treatment

during the 4 weeks previous to sampling, no use of mouth

rinses or any other preventive measure that might involve

exposure to antimicrobial agents and no systemic disease. The

dental caries was determined by a calibrated dentist using

WHO criteria.

Samples were taken from the oral cavity of each patient

(one of dental caries, and one of plaque) with a steril swab.

After incubation in brain heart infusion (BHI) during 24 h the

swab were plated on 5% defibrinated sheep’s blood agar plates.

Plates were incubated for 48 h at 37 8C in an atmosphere

containing 10% CO2. Identification of streptococci was based

on their colony morphology and confirmed by biochemical

tests.

Isolated strains were identified with Api 20 Strep strips

(bioMerieux, France) according to the manufacturer’s recom-

mendations. The results were read with a microbiological

mini-Api automate (Biomerieux, Fance).

2.2. Antimicrobial susceptibility testing

The inoculum was standardized to 0.5 McFarland with a

densimat (Bio-Merieux, Ltd., France). Antimicrobial suscepti-

bility was tested using the ATB Strep strips (Bio-Merieux, Ltd.,

France) which contain a range of 18 antibiotics: penicillin

(PEN), amoxicillin (AMO), ampicillin enterococcus (AMPE),

cefotaxim, (CTX), kanamycin HC (KAH), gentamycin HC (GEH),

tetracycline (TET), trimethoprim-sulfamethoxazol (TSU),

erythromycin (ERY), nitrofurantoine (FUR), telithromycin

(Tel), pristinamycin (PRI), quinupristin-dalfopristin (QDA),

levofloxacin streptococcus (LVXS), vancomycin (VAN), teico-

planin (TEC), rifampicin (RFA), and linezolid (LNZ).

The results were interpreted with a mini-Api automate

(Bio-Merieux, Ltd., France) according to the published guide-

lines of the manufactures.

2.3. Detection of resistance genotypes by PCR

The presence of genes encoding antibiotics resistance (ermB,

mef, tetM, and tetO), was examined in all isolated strains using

specific primers as shown in Table 1.

We used a duplex PCR technique to amplify ermB, and mefI21 and a PCR assay to amplify tetM and tetO genes in the tested

strains as described by Perez-Trallero et al. 22

Chromosomal DNA was extracted using a Wizard Genomic

purification Kit (Promega, USA), 60 ml of lysozyme at 0.1 mg/l

(Sigma) was added to the cell lysate. Negative PCR control

without the bacterial DNA was included with each PCR reaction.

Duplex PCR assays were performed in 25 ml PCR containing

1 U of GO Taq DNA polymerase (Promega, Lyon, France), 5 ml

green Go Taq buffer (5�), 25 pM each forward and reverse

primers of ermB and mefI gene, 100 mM concentrations (each)

of the four dNTPs and DNA template (50 ng). The PCR mixtures

were subjected to thermal cycling conditions presented in

Table 1 with a Gene Amp PCR System 9700 (Applied

Biosystems Int, USA).

In the PCR assay, mixture was prepared as previously

described and 25 pM of forward and reverse primers of tetM or

Table 1 – List of primers used for the detection of antibiotics resistances genes.

Genes Primers 5–3 Product size (bp) PCR conditions References

ermB GAAAAGGTACTCAACCAAATA 639 94 8C, 30S; 52 8C, 30S; 72 8C, 60S 21

AGTAACGGTACTTAAATTGTTTAC

mefI ATGGAAAAATACAACAATTGGAAA 263

CCAGCTGCTGCGATAATTAA

tetM AGTTTTAGCTCATGTTGATG 1862 94 8C, 30S 22

TCCGACTATTTGGACGACGG 58 8C, 45S

72 8C, 45S

tetO GCGGAACATTGCATTTGAGGG 538 94 8C, 30S

CTCTATGGACAACCCGACAGAAG 53 8C, 30S

72 8C, 30S

a r c h i v e s o f o r a l b i o l o g y 5 6 ( 2 0 1 1 ) 1 2 7 – 1 3 5 129

tetO genes were used separately. Ten microliter of PCR product

were resolved on a 2% agarose gel containing ethidium

bromide (0.5 mg/ml) in Tris-borate-EDTA buffer (89 mM Tris,

89 mM boric acid, 2 mM EDTA) at 90 V for 1 h which was

visualized under UV transillumination and photographed

using Gel Doc XR apparatus (Bio-rad, USA).

Table 2 – Correlation between erythromycin susceptibility tesantibiotic resistance genes (ermB and mefI) in oral streptococc

Species identification (number of strains) Erya

Streptococcus mutans (n = 13) I

R

S

Streptococcus oralis (n = 13) I

R

S

Enterococcus faecalis (n = 12) I

R

S

Streptococcus constellatus (n = 11) R

S

Streptococcus mitis (n = 10) S

Streptococcus salivarius ssp. salivarius (n = 9) R

S

Streptococcus pyogenes (n = 5) R

S

Gemella morbillorum (n = 5) I

S

Enterococcus faecium (n = 5) R

S

Gemella haemolysans (n = 4) I

S

Streptococcus anginosus (n = 4) R

S

Streptococcus sanguis (n = 3) S

Lactococcus lactis ssp. cremoris (n = 3) S

Streptococcus uberis (n = 2) R

Streptococcus bovis (n = 2) S

Aerococcus viridans (n = 2) S

Lactococcus lactis ssp lactis (n = 2) S

Leuconostoc spp. (n = 1) S

Streptococcus pneumoniae (n = 1) R

Streptococcus equinus (n = 1) S

Enterococcus avium (n = 1) S

(–) Absence of gene.a Erythromycin susceptibility (ATB Strep).b Numbers of strains.c Numbers of strains carrying the ermB or the mefI gene.

3. Statistical analysis

Statistical analysis was performed on SPSS v.17.0 statistics

software. Pearson’s chi-square x2 test was used to assess inter-

group significance. In addition Statistical significance was set

at P < 0.05.

ting (ATB Strep) and the presence of clinically relevanti.

Nb ermBc mefIc

1 – –

2 2 –

10 2 4

1 – 1

4 1 3

8 1 4

1 – 1

2 – –

9 – –

1 – 1

10 3 3

10 2 6

2 1 2

7 – 2

3 – 1

2 – 1

1 – 1

4 – 2

4 – 1

1 – –

2 – 2

2 – 1

3 2 2

1 – 1

3 – 1

3 – 2

2 – 1

2 1 –

2 – –

2 – –

1 – –

1 – –

1 – –

1 – –

a r c h i v e s o f o r a l b i o l o g y 5 6 ( 2 0 1 1 ) 1 2 7 – 1 3 5130

4. Results

4.1. Biochemical characterization and antibioticsusceptibility

109 strains were isolated and identified by the Api 20 Strep

system. As presented in Table 2, the most isolated strains were

Streptococcus mutans (n = 13), Streptococcus oralis (n = 13), Entero-

coccus faecalis (n = 12), Streptococcus constellatus (n = 11), S. mitis

(n = 10) and Streptococcus salivarius ssp. salivarius (n = 9). In

addition 15 various species which may be implicated in dental

caries such as Streptococcus sanguis (n = 3), Lactococcus lactis ssp.

lactis (n = 2), Lactococcus lactis ssp. cremoris (n = 3) were identified.

The antibiotic susceptibility test revealed the presence of

multi-resistant strains towards the 18 previously cited anti-

biotics. Amongst the 109 strains, resistance was detected to

erythromycin (22%), tetracycline (15.6%), cefotaxim, (7.3%),

trimethoprim-sulfamethoxazol (37.6%), nitrofurantoine

(2.8%), pristinamycin (17.4%), quinupristin-dalfopristin

(15.6%), and rifampicin (3.7%). In addition we noted that 21

out of 24 erythromycin resistant strains were isolated from the

children group with carious teeth.[()TD$FIG]

Fig. 1 – (a) Agarose gel electrophoresis of polymerase chain reacti

DNA molecular size marker (Invitrogen, USA); lane 2, negative co

amplification of oral bacteria; lane 3, B198; lane 4, B509; lane 5, B6

B484; lane 11, B403; lane 12, B496; lane 13, B580; lane 14, B155; l

electrophoresis of polymerase chain reaction (PCR) amplification

(Invitrogen, USA); lane 2, negative control; lanes 3–8, PCR amplico

B560; lane 4, B619; lane 5, B9; lane 6, B480; lane 7, B585; lane8, B

reaction (PCR) amplification of tetO gene. Lane 1, 100 bp DNA Ladd

amplicons obtained with DNA amplification of oral bacteria; lane

Furthermore, 96.3% of intermediate susceptibility was

recorded in the case of kanamycin HC and gentamycin HC.

The intermediate susceptibility to the other antibiotics was as

follow: penicillin (38.5%), amoxicillin (18.3%), pristinamycin

(4.6%), ampicillin enterococcus (2.1%), cefotaxim (16.5%),

erythromycin (5.5%), tetracycline (2.8%), quinupristin-dalfo-

pristin (13.8%), levofloxacin streptococcus (7.3%), vancomycin

(3.7%), rifampicin (13.8%) and linezolid (15.6%). We noted also

that all the tested strains were susceptible to ampicillin

enterococcus, telithromycin and teicoplanin.

4.2. Detection by PCR of resistance genes

Erythromycin-resistance genes (ermB and mefI) were identi-

fied by duplex PCR (Fig. 1a), 15 out of 109 (13.8%) were ermB

positive (Table 2). The majority of these strains were isolated

from caries-actives children. However mefI gene was present

in 43 of 109 strains (39.4%) amongst them eight carried the

ermB gene.

Tetracycline resistance genes were also detected by PCR

(Fig. 1a and b), 14 out of 109 (12.8%) were tetM+ and 34 (31.2%)

were tetO+. We noted also the presence of six strains which

on (PCR) amplification of ermB and mefI gene. Lane 1, 100 bp

ntrol; lanes 3–17, PCR amplicons obtained with DNA

1; lane 6, B160; lane 7, B736; lane 8, B9; lane 9, B860; lane 10,

ane 15, B448; lane 16, B673; lane 17, B215. (b) Agarose gel

of tetM gene. Lanes 1, 100 bp DNA molecular size marker

ns obtained with DNA amplification of oral bacteria; lane 3,

155. (c) Agarose gel electrophoresis of polymerase chain

er (Promega, France); lane 2, negative control; lanes 3–7, PCR

3, B100; lane 4, B76; lane 5, B677; lane 6, B634; lane 7, B496.

[()TD$FIG]

Fig. 3 – Correlation between tetracycline susceptibility and the presence of tetracycline resistance genes (tetM and tetO).

[()TD$FIG]

Fig. 2 – Correlation between erythromycin susceptibility strains and the presence of erythromycin resistance gene (ermB)

and the macrolide efflux (mefI).

a r c h i v e s o f o r a l b i o l o g y 5 6 ( 2 0 1 1 ) 1 2 7 – 1 3 5 131

were tetM+ and tetO+ (Table 4). However in seven tetracycline

resistant strains the tetM and tetO were not detected.

As presented in Fig. 2, three ermB and mefI positive were

isolated from caries-actives children and were erythromycin

resistant. In addition only one ermB and mefI positive strain

was isolated from the control group and was erythromycin

intermediate susceptible. The three erythromycin resistant

isolated from the caries-free children were ermB negative and

mefI positive (Fig. 2). Two strains (one from carious children

and one from control) were tetM and tetO positive and

tetracycline resistant. In addition five tetracycline resistances

strains isolated from caries actives children were tetM and

tetO negatives (Fig. 3).

5. Discussion

Early childhood caries continue to be a serious public health

problem in many areas of the world.23 Cariogenic diet and oral

hygiene contribute to the development of caries.

Table 3 – Association between species identification and carie formation.

Strains Numbers of strains (%) P-valuesa Odds ratio (95%CI)b

Streptococcus mutans 13 (11.9%) 0.034 7.250 (0.895, 58.699)

Streptococcus oralis 13 (11.9%) 0.034 7.250 (0.895, 58.699)

Enterococcus faecalis 12 (11%) 0.048 6.510 (0.799, 53.056)

Streptococcus constellatus 11 (10.1%) 0.820 0.858 (0.230, 3.198)

Streptococcus mitis 10 (9.2%) 0.018 –c

Streptococcus salivarius ssp. salivarius 9 (8.3%) 0.456 1.849 (0.360, 9.502)

Gemella morbillorum 5 (4.6%) 0.745 0.737 (0.116, 4.665)

Enterococcus faecium 5 (4.6%) 0.515 2.071 (0.221, 19.394)

Streptococcus pyogenes 5 (4.6%) 0.745 0.737 (0.116, 4.665)

Streptococcus anginosus 4 (3.7%) 0.148 –

Gemella haemolysans 4 (3.7%) 0.148 –

Streptococcus sanguis 3 (2.8%) 0.213 –

Lactococcus lactis ssp. cremoris 3 (2.8%) 1 1 (0.087, 11.490)

Lactococcus lactis ssp. lactis 2 (1.8%) 0.613 0.492 (0.030, 8.142)

Aerococcus viridans 2 (1.8%) 0.613 0.492 (0.030, 8.142)

Streptococcus bovis 2 (1.8%) 0.312 –

Streptococcus uberis 2 (1.8%) 0.312 –

Enterococcus avium 1 (0.9%) 0.477 –

Streptococcus equinus 1 (0.9%) 0.155 –

Streptococcus pneumoniae 1 (0.9%) 0.477 –

Leuconostoc spp. 1 (0.9%) 0.477 –

a Calculated using Pearson’s chi-square tests.b Odds of being a children with caries active associated with species identification.c Non applicable.

a r c h i v e s o f o r a l b i o l o g y 5 6 ( 2 0 1 1 ) 1 2 7 – 1 3 5132

In this study, 109 Streptococci were isolated from the oral

cavity of Tunisian children (Table 2). The most isolated strains

were S. mutans and S. oralis (n = 13) which were isolated

essentially from the caries-active children group. Dental caries

is closely associated with mutans streptococci colonizing the

oral cavity.24 In our investigation, only 12% of S. mutans were

isolated and identified. We noted also the presence of 15

various species which may be implicated in dental caries such

as Streptococcus anginosus, S. sanguis, Lactococcus lactis ssp. Lactis

and L. lactis ssp. cremoris.

As presented in Table 3, statistical analysis revealed that

there is a correlation between the presence of S. mutans

(P = 0.034), S. oralis (P = 0.034), E. faecalis (P = 0.034), S. mitis

(P = 0.034), and caries formation in the studied population.

It has been reported a higher isolation frequencies of S.

mutans and lactobacilli in Dundee, Scotland, 1-year old infants

with caries compared to those who were clinically caries-

free.25 Moreover, earlier studies reported that the most species

frequently detected in school children were S. mutans,

Streptococcus sanguinis, S. salivarius and S. anginosus.26

The failure of antibiotic prophylaxis in dental practise is the

major cause of bacteremia after dental procedures. The ability

of antibiotic therapy to reduce the frequency of bacteremia

associated with dental procedure is controversial.27 The

antibiotic resistance amongst oral bacteria must be investi-

gated before dental practise. This study was focused on the

susceptibility testing of oral bacteria since these microorgan-

isms are frequently isolated from the oral cavity and play a

significant role as a reservoir of antimicrobial resistance

genes.

The susceptibility analysis showed a 19.26% rate of

resistance to erythromycin and 12.84% to tetracycline in

caries-active group compared to 2.75% in the caries-free one.

Our result was contradictory with another study 28 which

reported no high resistance to tetracycline in the healthy

group. Ono et al. 29 indicated much higher resistance rate to

erythromycin for S. mitis, S. oralis and S. sanguis isolated from

oral infections. We noted also that all the isolated strains were

susceptible to telithromycin. This result was in agreement

with other study which found that telithromycin have a higher

in vitro activity.30

Macrolide resistance rates of clinical Streptococci pyogenes

isolates has been recognized in many parts of the world.31 In

this study only one S. pyogenes (B205) was erythromycin and

tetracycline resistant. Examination of macrolide-resistance

genes is very important to understand the erythromycin

susceptibility state.32 Transformation of DNA is a major

contributor to horizontal gene transfer in many species of

bacteria inhabiting the oral cavity.19

The most prevalent mechanisms of macrolide resistance in

streptococci are target modification due to ribosomal methyl-

ation associated with the erm(B) or the erm(A) gene and a

macrolide-specific efflux mechanism encoded by mef genes.33

MLS antibiotics are frequently used as first-line treatment for

streptococcal infections in children.34 Acquired resistance

against these antibiotics has frequently been described in

enterococci.35 A strong association of the erm(B) and tet(M)

genes with the mobile transposon Tn1545-related elements

was found in human oral streptococci.36 Lancaster et al.37

suggested that the high prevalence of tetracycline and

macrolide resistance in oral bacteria may be due to acquisition

of resistance genes from food products.

In this study we found that 15 out of 109 (13.8%) strains

were ermB positive (Table 2), amongst them 14 strains were

isolated from caries-actives children. This result is contradic-

tory with a recent study of Liu et al. 38 who reported that the

ermB gene was frequently present in streptococci. Other

Japanese studies showed that the ermB gene was detected in

Table 4 – Correlation between tetracycline susceptibility testing (ATB Strep) and the presence of clinically relevantantibiotic resistance genes (tetM and tetO) in oral streptococci.

Species identification (number of strains) Teta Nb tetMc tetOc

Streptococcus mutans (n = 13) I 3 1 3

R 1 1 1

S 9 – 5

Streptococcus oralis (n = 13) R 1 – 1

S 12 2 2

Enterococcus faecalis (n = 12) R 1 – 1

S 11 – 6

Streptococcus constellatus (n = 11) R 1 – –

S 10 2 1

Streptococcus mitis (n = 10) R 1 – 1

S 9 – 2

Streptococcus salivarius ssp. salivarius (n = 9) R 2 1 –

S 7 1 –

Streptococcus pyogenes (n = 5) R 3 – –

S 2 – –

Gemella morbillorum (n = 5) S 5 – 1

Enterococcus faecium (n = 5) S 5 1 3

Gemella haemolysans (n = 4) S 4 – 1

Streptococcus anginosus (n = 4) R 2 2 –

S 2 1 1

Streptococcus sanguis (n = 3) R 3 1 –

Lactococcus lactis ssp. cremoris (n = 3) R 1 – 1

S 2 – –

Streptococcus uberis (n = 2) S 2 – 1

Streptococcus bovis (n = 2) R 1 – 1

S 1 – 1

Aerococcus viridans (n = 2) S 2 1 –

Lactococcus lactis ssp. lactis (n = 2) S 2 – 1

Leuconostoc spp. (n = 1) S 1 – –

Streptococcus pneumoniae (n = 1) S 1 – –

Streptococcus equinus (n = 1) S 1 – –

Enterococcus avium (n = 1) S 1 – –

(–) Absence of gene.a Tetracycline susceptibility (ATB Strep).b Numbers of strains.c Numbers of strains carrying the tetM or the tetO gene

a r c h i v e s o f o r a l b i o l o g y 5 6 ( 2 0 1 1 ) 1 2 7 – 1 3 5 133

57.1% 39 and in 50% of the erythromycin-non-susceptible

isolates.21 mefI gene was present in 39.4% strains. We noted

also that only three ermB and mefI positive strains were

isolated from carious children and were erythromycin resis-

tant (Fig. 2).

In addition ten strains isolated from the carious children

were erythromycin resistant but they do not carry the ermB

and mefI genes (Fig. 2). Statistical analysis showed that the

presence of ermB gene in oral streptococci is not correlated

with erythromycin resistance obtained with ATB Strep

(P = 0.143). This discordance may be explained by the presence

of other erythromycin resistant genes as previously described

in streptococci.40

Tetracycline is a broad-spectrum antibiotic used in the

treatment of oral infection. Tetracycline-resistant streptococ-

ci are frequently isolated from the oral cavity of humans,41 and

resistance is most commonly conferred by tetM, a ribosomal

protection protein often associated with the conjugative

transposon Tn916.42 Transferable Tn916-like elements have

been found in many oral streptococci.43 It has been shown in a

recent study that tet(M) was the most prevalent tetracycline

resistance genes found in the oral metagenomes extracted

from saliva samples.44 Additionally, tetO have been shown to

be common.45

As presented in Fig. 3, the susceptibility analysis showed a

12.84% rate of resistance to tetracycline in caries-active group

compared to 2.75% in the control group. In addition six strains

were tetM and tetO positives (Table 4). The presence of these

two genes was previously demonstrated but was not detected

in other European studies.46 The tetO gene has been found in

different species from the oral and respiratory tracts.41 As

shown in Fig. 3, eight Streptococcus strains were susceptible to

tetracycline but they carry the tetM gene. As presented in

Table 3, statistical analysis showed no correlation between the

tetracycline resistance and the presence of tetM (P = 0.039) or

tetO genes (P = 0.028).

In conclusion, this study revealed that the most isolated

strains were S. mutans, S. oralis, E. faecalis and S. constellatus.

Multi-resistance strains towards macrolides and tetracycline

were also recorded. A discordance between the phenotypic

and genotypic analysis of drugs resistances was noted. The

emergence of antibiotic-resistant bacteria within the oral flora

will have an impact on the prescription of antibiotics in

dentistry. The presence of resistant bacteria in the oral cavity

a r c h i v e s o f o r a l b i o l o g y 5 6 ( 2 0 1 1 ) 1 2 7 – 1 3 5134

can be the major cause of dental antibiotic prophylaxis failure.

Particular attention should be paid to antibiotics that are most

frequently used in dental practise.

Funding: ‘‘Ministere Tunisien de l’Enseignement Superieur,

de la Recherche Scientifique’’ through the ‘‘Laboratoire

d’Analyses, Traitement et Valorisation des Polluants de

l’Environnement et des Produits, Faculte de Pharmacie, rue

Avicenne 5000 Monastir (Tunisie)’’.

Conflict of interest: Not declared.

Ethical approval: All clinical procedures were approved by

the Ethical Committee of the Faculty of Medicine, University of

Monastir, Tunisia.

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