Caries pathology is a microbial condition, mainly bacterial, which nowadays is causing gangrene in our populations. Ranked 4th globally, it comes after cancer, diabetes and AIDS (Acquired Immuno-Deficiency Syndrome)
[1]
. In fact, 80% of the world population, 60% to 90% of whom are school-aged children, and 100% adults suffer from this disease. In Ivory Coast this condition rate is 60% and constitutes a public health problem
[2]
.
Antibiotics are natural substances (produced by microorganisms) or synthetic (Laboratory), with antimicrobial activity relatively at low concentrations. Their property is to eliminate or inhibit the growth of microorganisms . However, in oral medicine, antibiotics remain particularly useful, because the treatment of an infectious source is most often non-drug. Antibiotics are useful when they can help the host defenses to eliminate residual microorganisms not destroyed by appropriate local treatment . Despite the instructions for the use of antibiotics in endodontics, prescriptions are only increasing. Thus, Beta lactams are sufficiently prescribed with 65.2% consumption
[4]
. Also, it is remarkable that the prescriptions are not adapted
[5]
, which consequently indicates an insufficiency in antibiotic prophylaxis and in the choice of antibiotic therapy
[6]
. Furthermore, in Ivory Coast, street drugs, which are widely used, represent potential health hazards. Indeed, less than 20% of national pharmaceutical coverage for the sale of drugs is available. This situation, which undoubtedly remains a key factor in the emergence of bacterial resistance, it seemed important to us to evaluate the resistance of caries strains to current antibiotics. The objective of this work is to determine the resistance profile of bacterial isolates from carious teeth from the dental office of the GON COULIBALY Péléforo University.
2. Materials and Methods
This work took place during the period from July 7 to September 30, 2024. The samples of carious teeth were taken at the dental office of Péléforo GON COULIBALY University. The identification and antibiogram were carried out at the Institut Pasteur de Cote d’Ivoire (IPCI).
2.1. Materials
Caries teeth were extracted from patients whose treatment requires tooth extraction. Patient ages range from 10 to over 50 and of all genders. Nutrient and ordinary agar as well as Muller Hinton culture medium were used. The MALDI TOF device and antibiotic blotting discs were also used. Finally, all the technical equipment of the Microbiology Laboratory was used.
2.2. Methods
After consulting the patient, the dentist assesses whether there is a need to extract the diseased tooth or not. If yes, then the tooth is extracted with all the Kit available to the dentist. Once extracted, the carious tooth is preserved in deep agar in Eppendorf tubes prepared for this purpose, then stored at office laboratory temperature.
The bacterial sample called the calibrating strain (E. coli) is placed on the plate beforehand. Using a platinum loop, an isolated colony is taken and deposited on the microplates in the form of a smear which corresponds to the positive control (T+). In the same column and in the second line, a second spot called negative control (T−) is placed and each spot is covered with 1 µL of saturated matrix α-cyanohydroxnamic acid (ACHC). This operation is repeated for each colony to be identified. Then the MALDI TOF target is left to dry on the bench for 15 min. The plates are scanned on the computer and this step constitutes the information phase of the preparation station (PRE-STAT). Thus, the information concerning each colony isolated on the plate are represented on the computer. A second computer constituting the Sample Acquisition or Analysis Monitoring Station (ACQ-STAT) allows the MALDI TOF device to be launched by command. The plate carrier is removed, and the microplates are inserted inside. Each plate is scanned in order to record the information on the second computer. Once this operation is completed, the device closes automatically. After a few hours of analysis, the MS (Mass Spectrometer) software displays the results in the “MYLATM” program and indicates the percentage of identification by measuring the very abundant proteins in the bacteria
[7]
. Indeed, the characteristic patterns of these very abundant proteins have been used to reliably and very precisely identify bacterial strains. Thus, the results of a target are validated if and only if (T+) corresponds to E. coli at 99.9% and (T−) corresponds to a score without identification lower than 99.99%.
To determine the resistance profile of the different strains isolated, a standardized antibiogram in agar media in several stages is carried out according to the recommendations of the Antibiogram Committee of the French Society of Microbiology
[8]
.
1) Preparation of the bacterial inoculum
From a young culture of 18 to 24 hours grown on ordinary agar, a bacterial suspension is prepared. A pure bacterial colony is taken using a swab and suspended in a hemolysis tube containing 3 mL of 0.85% NaCl saline solution or physiological water (Api® Biomérieur®). The suspension is then homogenized using a vortex. The density of the mixture is adjusted using a Densichek® to obtain 0.5 Mc Ferland or 5.105 CFU/mL
[8]
. The inoculum can be adjusted by adding either a bacterial culture if it is weak, or physiological water (NaCl) if it is strong. Inoculation is done after 15 minutes following preparation of the inoculum.
2) Seeding on Mueller-Hinton (MH) agar for pseudomonas
1 µl of the bacterial inoculum is introduced into a hemolysis tube containing 3 ml of saline solution (NaCl), then homogenized well using a vortex and finally inoculated over the entire surface of the Mueller-Hinton agar in tight streaks, rotating the petri dish each time
[8]
.
3) Seeding on MH for staphylococcus
1000 µl of the bacterial inoculum are introduced into a hemolysis tube containing 3 mL of physiological water then homogenized using a vortex and finally inoculated over the entire surface of the Mueller-Hinton agar in tight streaks, rotating the petri dish each time
[8]
.
4) Seeding on MH for Enterobacteria and other families
100 µl of the bacterial inoculum is introduced into a hemolysis tube containing 3 mL of physiological water and homogenized well and finally inoculated on the entire surface of the Muller-Hinton agar in tight streaks, rotating the petri dish each time
[8]
.
5) Application of antibiotic discs
The antibiotic disks are brought to room temperature, then using a manual applicator placed on the agar 30 mm from each other and from the edge of the petri dish. After application of the discs, the seeded petri dishes are left at room temperature (25˚C ± 2˚C) on the bench for approximately 15 minutes to allow pre-diffusion of the antibiotic discs. Incubation takes place at 35˚C ± 2˚C, aerobically, for 20 ± 4 h
[8]
.
6) Interpretation of results
The inhibition diameters are measured using a caliper and the inhibition values are obtained in (mm). Using the different classes of strains: (S) for sensitive ones, (I) for intermediate ones and (R) for resistant ones, in accordance with the standards
[8]
, the distribution is thus made.
3. Results
During the study period, a total of 56 patients were consulted with 25 dental extractions. The age of people who have undergone tooth extraction ranges from 08 to 60 years. The different extraction rates depending on age are given as shown in the diagram below (
Figure 1
).
The distribution of patients who had at least one tooth extracted during the survey period according to sex or gender is represented by the diagram below with 60% female and 40% male (
Figure 2
).
As a result of the identification by the MALDI-TOF method the MALDI-TOF method made it possible to identify 25 species of different strains corresponding to the 25 samples taken, with an accuracy of 99.9% of almost all the strains analyzed except 5 strains which displayed a purity of 50% (
Table 1
).
Analysis of the isolates from the samples using MALDI TOF revealed 25 different
<xref ref-type="bibr" rid="scirp.145350-"></xref>Figure 1. Diagram representing the rate of extraction of carious teeth as a function of the age of the patients.
<xref ref-type="bibr" rid="scirp.145350-"></xref>Figure 2. Diagram representing the rate of gender of patients having extracted at least one tooth during the survey period.
<xref ref-type="bibr" rid="scirp.145350-"></xref>Table 1. Table representing the nature of the strain contained in different isolates, identification by MALDI TOF from the UPGC dental office.
Order number
Strain number
Strain name
Confidence level (%)
1
P1KO-1
Klebsiella pneumoniae
99.9
2
P2KO-1
Citrobacter koseri
99.9
3
P11KO-1
Pluralibacter gergoviae
99.9
4
P8KO-1
Klebsiella pneumoniae
99.9
5
P9KO-1
Pseudomonas stutzeri
99.9
6
P10KO-1
Pseudomonas putida
99.5
7
P19KO-1
Enterococcus faecalis
99.9
8
P17KO-1
Staphylococcus epidermidis
99.9
9
P14KO-1
Stenotrophomonas maltophilia
99.9
10
P15KO-1
Pseudomonas aeruginosa
99.9
11
P16KO-1
Pseudomonas aeruginosa
99.9
12
P12KO-1
Staphylococcus saprophyticus
99.9
13
P13KO-1
Klebsiella pneumoniae
99.9
14
P20KO-1
Klebsiella pneumoniae
99.9
15
P23KO-1
Klebsiella pneumoniae
99.9
16
P25KO-1
Pseudomonas aeruginosa
99.9
17
P3KO-1
Enterobacter hormaechei
83.1
18
P7KO61
Staphylococcus saprophyticus
99.9
19
P4KO-1
Staphylococcus arlettae
99.9
20
P6KO6-1
Klebsiella pneumoniae
92.8
21
P18KO-1
Enterobacter cloacae
50.0
22
P22KO-1
Enterobacter asburia
50.0
23
P21KO-1
Enterobacter cloacae
50.0
24
P24KO-1
Enterobacter asburiae
50.0
25
P5KO-1
Enterobacter asburiae
50.0
<xref ref-type="bibr" rid="scirp.145350-"></xref>Figure 3. Diagram representing the dominance rate of the families of the isolated strains.
strains divided into 5 families as shown in the diagram below (
Figure 3
). Only the Enterobacteriaceae family was the majority (56%) of the different families represented.
Antibiogram of the different strains isolated from the carious teeth of the patients. The different strains isolated from the carious teeth were the subject of a sensitivity study to the different antibiotic disks. Thus, for the Staphylococcus strains 7 antibiotic disks were used on 04 strains from patients P4, P7, P12, and P17. It emerges from this analysis that the Staphylococcus strains are resistant to tetracycline (TET) with inhibition diameters less than 18 mm. The same is true for tobramycin (TOB) except for the strain from patient P7 which is sensitive to (TOB) with a diameter equal to 28 greater than 18. As for the antibiotics levofloxacin (LVX) and Ciprofloxacin (CIP), they remain of intermediate sensitivity to all strains of Staphylococcus studied (
Table 2
).
The sensitivity of Pseudomonas strains required 06 antibiotic disks for 05 strains from patients P9, P10, P15, P16 and P25. It appears from this analysis that all the strains subjected to the study showed resistance to piperacillin-tazobactam (TZP) with inhibition diameters less than 20 mm. However, Ceftazidine (CAZ) and piperacillin (PIR) were of intermediate sensitivity to contact with all Pseudomonas strains (
Table 3
).
<xref ref-type="bibr" rid="scirp.145350-"></xref>Table 2. antibiogram of the isolated Staphylococcus strains.
<xref ref-type="bibr" rid="scirp.145350-"></xref>Table 3. Antibiogram of isolated Pseudomonas strains.
ATB Patients
TZP
LVX
MEM
AK
CAZ
PIR
P9
06
28
23
17
20
20
P10
06
20
25
18
22
22
P15
06
27
35
19
22
24
P16
06
24
28
22
23
22
P25
06
23
33
20
23
20
Concerning Enterobacteria, out of 14 antibiotic disks submitted to the study, only 02 antibiotics (Amoxicillin plus clavulanic acid (AMC) and piperacillin-tazobactam (TZP)) were subject to absolute resistance to the 15 strains tested with inhibition diameters lower than 16 and 20 mm respectively (
Table 4
). But as for piperacillin (PIP) and fosfomycin (FOS), they were of relative sensitivity. The other antibiotics except these 04 mentioned, were almost sensitive to all the strains studied as indicated in the table below with diameters greater than 22 mm.
Of the 25 strains studied, 06 strains show resistance expression, i.e. 24%, and 19 strains show sensitivity expression, i.e. 76% (
Figure 4
).
4. Discussion
The prospective study of the resistance of cariogenic bacteria allowed us to identify 25 distinct strains distributed according to age, sex and by family of bacteria. Thus, the patients with carious teeth from which the strains came were those whose ages ranged from 10 to 20 years and from 21 to 30 years. This result is like those of
[9]
, who determined during their work on the evaluation of antibiotic prescriptions for patients suffering from dental pain, an age group of 25 to 34 years more represented. This representation could be explained by the fact that it
<xref ref-type="bibr" rid="scirp.145350-"></xref>Table 4. Antibiogram of isolated enterobacteria strains.
ATB Patients
AMC
LVX
AK
PIP
FOX
CRO
MEM
FOS
TZP
PEF
GMN
ETP
CTX
CZD
P1
06
28
17
06
22
07
27
18
06
25
21
28
06
08
P2
06
32
22
16
23
30
27
27
06
29
21
32
24
27
P3
06
26
17
14
22
27
24
17
06
23
19
10
22
23
P5
06
39
25
26
35
30
22
27
06
26
21
36
24
30
P6
06
27
20
16
19
26
25
15
06
24
19
29
28
24
P8
06
18
20
15
24
30
26
20
06
13
19
30
29
24
P11
06
30
22
20
35
30
25
20
06
29
22
35
22
31
P13
06
29
24
24
29
28
25
22
06
25
20
30
23
23
P18
06
29
25
25
29
27
24
15
06
23
20
29
22
22
P20
06
24
20
18
06
26
26
16
06
21
18
27
22
24
P21
06
37
22
27
30
25
19
15
06
32
30
34
31
32
P22
06
37
22
27
30
25
19
16
06
30
29
33
29
30
P23
06
38
22
20
28
28
26
20
06
25
20
29
24
25
P24
06
37
20
20
25
28
25
15
06
24
20
29
24
23
<xref ref-type="bibr" rid="scirp.145350-"></xref>Figure 4. Summary diagram of sensibility of isolated strains.
is indeed in this age group that we eat everything without much control, and believe in the strength of teeth like iron. Regarding sex, it is the female sex that was more represented with a sex ratio equal to 1.5. However, this result differs from those obtained by
[9]
. Indeed, out of a total of 200 decayed teeth of all sexes, 128 were male versus 72 females with a sex ratio of 1.7. Which consequently suggests that dental caries is not linked to sex.
Furthermore, the strains were identified for the most part with a confidence level of 99.9%, thus indicating the purity of the strains studied, except for six strains whose confidence level fluctuates between 50 and 85. Also, this identification made it possible to divide into 5 families with greater repetitiveness in Enterobacteriaceae followed by Pseudomonas and then Staphylococcus. The majority presence of Enterobacteriaceae in carious teeth would be due to the hygiene conditions not respected because these bacteria are generally of intestinal origin. Pseudomonas and Staphylococcus are found there due to an environment conducive to their growth. Analysis of the antibiotic resistance profile showed that all strains studied showed resistance to some antibiotics and multi-resistance to others. Regarding the Pseudomonas and Enterobacteriaceae families, all strains showed resistance to piperacillin tazobactam (TZP) and amoxicillin (AMC).
For the Staphylococcus family, all strains were resistant to tetracycline (TET). However, only the Staphylococcus and Enterobacteriaceae families showed multiresistance to the antibiotics tested, including piperacillin tazobactam (TZP), amoxicillin (AMC), tetracycline (TET), tobramycin (TOB) and erythromycin (ERY). This observation suggests that piperacillin tazobactam (TZP), amoxicillin (AMC), tetracycline (TET) would be the molecules most prescribed by dentists and clinicians in antibiotic therapy. In addition, this multiresistance could be attributed to the fact that the large family of beta-lactams, which include penicillins and cephalosporins, is the most used in first-line anti-infective therapy because it is the most diverse and the least toxic
[10]
. These results would agree with those of
[11]
who showed a prescription of nearly 98.20% of beta-lactams as first-line during their work on antibiotics. The same is true of the work of
[12]
and
[13]
respectively in the United States and Brazil who made the same observations. It seems obvious that this strong prescription of antibiotics is often without necessity the fundamental cause of this resistance and bacterial multiresistance of these strains studied. It should be remembered that the prescription of antibiotics to be prophylactic should not be systematic in patients suffering from general pathology. This is inappropriate. Indeed, only in patients at high risk of infective endocarditis or any dental procedure involving manipulation of the gum, the periapical region of the tooth and in the event of extraction
[14]
. In view of this, the fight against bacterial resistance or multiresistance would not be a magic fact. It is and remains above all a paradigm shifts both at the level of patients and at the level of practitioners in our various health centers. We need intense awareness now to raise awareness about the use of antibiotics. Thus, human shipwrecks could be avoided for antibiotic therapy for future generations.
5. Conclusion
At the end of this study, it should be noted that dental pathology is not linked to age or gender. It is a disease that can be contracted at any age. The bacterial germs involved in this study are mainly Enterobacteria, Pseudomonas and Staphylococcus. The antibiogram carried out revealed resistance on the one hand and multiresistance on the other hand on the different strains identified. This observation is therefore a challenge not only for practitioners in dental practice but also for patients who must be careful about the use of antibiotics. It should be remembered here that the fight against the phenomenon of bacterial resistance or even multiresistance is a real fact and concerns everyone at whatever level. In view of these convincing results, this study could be extended to other hospital centers such as school medical centers in the region, in order to increase the size of the sample to be analyzed and provide statistics on a regional scale.
Source of Funding
This study was supported by Department of Biochemistry-Genetics/UFR Biological Sciences of the UPGC.
Acknowledgments
The authors thank participants and field workers in the study.
References
Djossou, D., Nancy, J., Houinato, D. and Lanchoessi, D. (2015) Prévalence of Dental Caries in School in the City of Ouidah en 2013. Odonto-Stomatologie Tropicale, 38, 15-22.
World Health Organization (2014) Global Oral Health Database. WHO Collaborating Centre for Education, Training and Research in Oral Health. >https://www.mah.se/CAPP
Bourguignon, C. (2015) Us et abus des antibiotiques en endodontie. Infodent International, No. 11, 16-19.
Katie, A.L., Bakayoko-Ly, R. and Kacou, D.H. (1996) Affections odontologiques des enfants de 12 ans. Enquête épidémiologique en milieu scolaire (résultats partiels sur 342 enfants). 3e congrès du COSA-CMF.
Souaga, K., Adou, A., Kouamé, P., Sahouré, K., Aka-Adouko, J. and Aangoh, Y. (2007) Prescriptions d’antibiotiques en odontologie: enquête auprès des praticiens de la région d’Abidjan. Revue Edition Universitaire de Cote d’Ivoire, 1, 18-23.
Azodo, C.C. and Ojehanon, P.I. (2014) Antibiotics Prescription in Nigerian Dental Healthcare Services. OdontoStomatol, 37, 34-42.
Karas, M. and Hillenkamp, F. (2010) Laser Desorption Ionization of Protiens with Molecular Masses Exceeding 10,000 Daltons. Analytical Chemistry, 60, 2299-2301. >https://doi.org/10.1021/ac00171a028
EUCAST-CA-SFM (2024) Comité de l’antibiogramme de la société Française de Microbiologie. Recommandations. >https://www.eucast.org
Camara, S.A., Togo, A.K., Kané, A.S.T., Camara, M. and Traore, N. (2021) Eavaluation de la prescription des antibiotiques en pratique endodontique à l’hopital national de donka de CONAKRY. EDUCI, rev Col Odonto stomatol afr Chi Maxillo fac, Revue Sciences de la Santé, 1, 18-23.
Descroix, V. (2006) Savoir prescrire en endodontie. Les 10 points clés en endodontie. Editions CDP, Paris, 90-98.
Krah-Sinan, A., Adou-Assoumou, M., Traoré Dossahoua, K., Adou, J. and Mansilla, E. (2018) Utilisation des antibiotiques en endodontie: Enquête sur les connaissances et pratiques des chirurgiens den-tistes d’Abidjan. Rev Col Odonto-Stomatol Afr Chir Maxillo-Fac, 1, 18-25.
Germack, M., Sedgley, C.M., Sabbah, W. and Whit-Ten, B. (2017) Antibiotic Use by Members of the American Association of Endodontists: Report of a National Survey. Journal of Endodontics, 25, 662-663.
Bolfoni, M., Pappen, F.G., Pereira-Cenci, T. and Ja-Cinto, R.C. (2017) Antibiotic Prescription for Endodontic Infections: A Survey of Brazilian Endodontists. International Endodontic Journal, 51, 148-156.
AFSSAPS (Agence française de sécurité sanitaire des produits de santé) (2011) Prescription des antibiotiques en pratique buccodentaire. Recommandations. >https://ansm.sante.fr/uploads/2021/02/04/reco-prescriptiondes-antibiotiques-en-pratique-buccodentaire