archives of oral biology 60 (2015) 1410–1415
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Prevalence of Staphylococcus aureus and methicillin resistant Staphylococcus aureus (MRSA) in the oral cavity Georgios Koukos a,*, Dimitra Sakellari b, Minas Arsenakis c, Lazaros Tsalikis b, Theodora Slini d, Antonios Konstantinidis b a
251 General Air Force Hospital, Athens, Greece Department of Preventive Dentistry, Periodontology and Implant Biology, Dental School, Aristotle University of Thessaloniki, Greece c Department of Genetics and Molecular Biology, School of Biology, Aristotle University of Thessaloniki, Greece d Department of Mechanical Engineering, Aristotle University of Thessaloniki, Greece b
article info
abstract
Article history:
Objective: To assess the prevalence of Staphylococcus aureus and methicillin resistant Staph-
Accepted 13 June 2015
ylococcus aureus (MRSA) in plaque and tongue samples from systemically healthy subjects with periodontal health, gingivitis or chronic periodontitis.
Keywords:
Methods: After screening 720 potentially eligible subjects, 154 systemically healthy partici-
Staphylococcus aureus
pants were ultimately enrolled in the current study. Subgingival samples were taken from
Methicillin-resistant Staphylococcus
the first molars and the tongue and analyzed for the presence of S. aureus and MRSA by
aureus
polymerase chain reaction (PCR), using primers and conditions previously described in the
Molecular biology
literature. In addition, samples were taken from deep periodontal pockets of chronic
Polymerase chain reaction
periodontitis patients. Statistical analysis was performed by applying non-parametric tests
Antimicrobial drug resistance
(Kruskal–Wallis for clinical parameters, and z-test with Bonferroni corrections for distributions of assessed parameters). All comparisons were set at the 0.05 significance level. Results: S. aureus was detected in 18% of all participants and in 10% of the samples tested. No significant differences were found in its distribution among the three investigated groups (ztest for proportions with Bonferroni corrections, p > 0.05). The mecA gene was not present in any of the S. aureus found. Conclusions: S. aureus can be found in the oral environment regardless of the periodontal conditions and therefore should be considered as a member of the transient flora not participating in periodontal pathology. Subgingival sites and tongue surfaces seem to be an unusual habitat of MRSA. # 2015 Elsevier Ltd. All rights reserved.
1.
Introduction
The prevalence of Staphylococcus spp. in the oral cavity and their contribution to periodontal infections have not been
extensively investigated. Although they have been isolated in the oral cavity and are correlated with conditions such as suppurative parotitis, angular cheilitis, denture stomatitis and acute dentoalveolar infections, they are generally regarded as transient members of the oral flora.1–3 Recent studies also
* Corresponding author at: Department of Periodontology, 251 General Air Force Hospital, 11525 Athens, Greece. Tel.: +30 6983 520282. E-mail address: [email protected] (G. Koukos). http://dx.doi.org/10.1016/j.archoralbio.2015.06.009 0003–9969/# 2015 Elsevier Ltd. All rights reserved.
archives of oral biology 60 (2015) 1410–1415
suggest that Staphylococci can be frequently isolated from the oral cavity of specific patient groups such as newborns in hospital units, the elderly, patients with malignancies as well as subjects with immunocompromising systemic conditions such as rheumatoid arthritis.4–6 Last but not least they are considered as aetiological factors of prosthetic valve endocarditis.7 The relationship between periodontal disease and Staphylococci is currently not well understood. In a number of studies, using cultural techniques, they have been isolated from plaque samples, however a wide range for their prevalence (5.6–75%) has been reported. In studies including more than 500 subjects with periodontitis, it has been observed that approximately half harbour Staphylococcus spp. with S. epidermidis and to a lower extent S. aureus being the predominant species,1,8–11 but data comparing their prevalence with samples from periodontally healthy controls have not shown differences between these groups.3 Although usually a harmless colonizer of the skin and the nasopharynx in 25–35% of healthy individuals, on some occasions S. aureus is known to cause severe infections.12 One of the concerns with S. aureus is the current extent of the prevalence of methicillin resistant Staphylococcus aureus (MRSA) which was initially isolated 50 years ago, only two years after the introduction of methicillin in clinical practice and has developed into a major global health issue due to its pathogenic potential to cause bloodstream infections, pneumonia as well as surgical site infections.13 Resistance of S. aureus to b-lactam antibiotics is acquired by the exogenous mecA gene, which encodes a modified form of Penicillin Binding Protein (PBP2a), that does not allow proper binding and thus prevents the inhibition of cell wall synthesis that this class of antibiotics cause.14,15 Healthcare-associated MRSA (HA-MRSA) is related to prolonged length of hospital stay and is currently one of the most frequently identified pathogens in hospitals in many parts of the world.16 Furthermore, Community acquired MRSA (CA-MRSA) has demonstrated increasing trends, hence guidelines for prevention and surveillance have been issued by several healthcare officials. Thus, MRSA in the oral cavity could potentially be disseminated by carriers to the environment or to other individuals.17 The aim of the present study is to investigate the prevalence of S. aureus and the mecA gene encoding for MRSA in the oral cavity of Greek subjects with various periodontal conditions.
2.
Materials and methods
2.1.
Patient selection
or medications known to affect periodontal tissues, without infectious conditions (hepatitis, HIV) or pregnancy and lactation, who did not receive periodontal treatment or antibiotics within the last six months. They were also required to have at least 20 teeth present and meet the criteria of one of three periodontal conditions (healthy, gingivitis or periodontitis). Periodontally healthy subjects would have no periodontal pockets measuring more than 3 mm and bleeding on probing would be less than 10%. Gingivitis subjects had no periodontal pockets measuring more than 4 mm or attachment loss of more than 3 mm and bleeding on probing should exceed 20%, without radiographic bone loss. Moderate or advanced periodontitis subjects had 30% of teeth with proximal attachment loss 5 mm, and presented radiographic bone loss which exceeded 30%.18 Out of the 720 subjects initially screened, 156 fulfilled the inclusion criteria and 154 agreed to participate in the study. The study was conducted according to the protocol outlined by the Research Committee, Aristotle University of Thessaloniki, Greece and approved by the Ethical Committee of the School of Dentistry (#120), in compliance with the ethical principles of the World Medical Association Declaration of Helsinki. All patients read and signed an appropriate informed consent document prior to the participation in the study.
2.2.
Study design and clinical protocol
The present cross-sectional study, included three groups as follows: (a) periodontal health (n = 50), (b) gingivitis (n = 52), and (c) moderate or advanced chronic periodontitis (n = 52). Clinical recordings were performed at six points of all teeth present at the dentition (mesio-buccal, buccal, disto-buccal, mesio-lingual, lingual, distolingual). Recordings included the following parameters: Probing Pocket Depth (PPD), Recession, Clinical Attachment Level (CAL) and Bleeding on Probing (BOP). Recordings were performed by a calibrated examiner (GK) using an automated probe (Florida Probe, Florida Probe Corporation, Gainesville, FL, USA). All subjects were interviewed by one of the authors (DS) and completed a questionnaire regarding the following: smoking, frequency of antibiotic intake for medical and dental reasons, class of antibiotics used 6–12 months before the interview as well as within the 5-year period prior to the interview, whether they have ever obtained antibiotics without prescription, whether they have antibiotics available at home and whether they are aware of the phenomenon of antibiotic resistance. In order to avoid false reporting by participants, the class of antibiotics that they had used was recorded based on their personal National Health Record.
2.3. 720 subjects attending the Clinic of Periodontology at 251 Air Force Hospital, Athens, Greece and the Clinic of the Department of Preventive Dentistry, Periodontology and Implant Biology, Dental School, Aristotle University of Thessaloniki, Greece, were screened in order to be enrolled in the study from September 2011 to April 2014. Subjects were deemed eligible as long as they fitted the following criteria: age >30 years, absence of systemic diseases
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Sampling and analysis
Two clinical samples were collected from each patient: a pooled subgingival plaque sample from the mesiobuccal surface of the four first molars (or premolars when molars were missing) taken with sterile Gracey curettes, and a sample collected from the dorsal surface of the tongue with a sterile straight surgical bone curette, after applying three consecutive strokes. All samples were immediately placed in 200 ml of TE
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buffer (Tris–HCl 10 mM, EDTA 1 mM, pH 7.5) and stored at 20 8C, until assayed. In addition, chronic periodontitis patients contributed with one more sample from their deepest periodontal pocket. Analysis of samples was performed blindly (coded samples). All experiments were performed in the Department of Microbiology, School of Biology, Aristotle University of Thessaloniki, Greece. The protocol suggested by the National Food Institute of the Technical University of Denmark, for the identification of S. aureus (based on detection of the nuc gene, encoding the S. aureus-specific thermonuclease gene), followed by detection of the mecA gene.19–21 There is a detailed description of PCR conditions and primers available in the Supplementary Data section. DNA isolation was performed by using a commercial DNA isolation kit (QIAamp DNA Mini Kit, Qiagen, Hilden, Germany). Initially, PCR was performed for the detection of the 16S ribosomal RNA gene, in order to verify that the clinical samples contained identifiable bacterial DNA. The final volume of the reaction mixture for each PCR assay was 50 ml. A Peltier Thermal Cycler (PTC-100, Peltier Thermal Cycler, MJ Research) was used. For each set of samples analyzed by PCR a negative and a positive control were used. Sterile water for injection (Demo S.A. Pharmaceutical Industry) was used as negative control, replacing DNA template into the PCR reactions, while S. aureus strain 9518 (NCIMB: National Collection of Industrial, Food and Marine Bacteria, Scotland) and clinical samples from three sources (Papanikolaou Hospital, Thessaloniki, Greece, Microbiology Laboratory, Arta, Greece and the Department of General Microbiology, Veterinary School, Aristotle University Thessaloniki, Greece) positive for methicillin resistant S. aureus (MRSA), were used as positive controls. The products of the DNA amplification were electrophoresed for 45 min at 70 mA field, through a 2% agarose gel, stained with ethidium bromide (Sigma–Aldrich, St. Louis, USA), exposed under UV light (302 nm) and photographed. A 100 bp DNA ladder (Invitrogen, Camarillo, CA, USA) was also loaded on agarose gel as a molecular weight standard. The amplified fragment sizes were 886 bp for 16S rRNA, 255 bp for nuc and 527 bp for mecA. The electrophoresis for each PCR product was carried out twice in order to test the reproducibility of the method.
2.4.
Statistical analyses
The statistical analysis of the data was carried out with the statistical package SPSS (SPSS 20.0 version, IBM, Armonk, NY,
USA). The experiment was set to have at least 80% power to detect changes of 25% with a significance level (alpha) of 0.05 (two-tailed). A previous study provided the expected values of the primary outcome variable.22 As for the clinical parameters, indicators of descriptive statistics were used, such as mean and standard deviation for each group, with the patient being the observational unit. As far as differences in clinical parameters and age are concerned they were sought by applying the Kruskal–Wallis test. Likewise, differences regarding the distribution of participants according to gender and smoking were sought by applying the z-test for proportions adjusted with Bonferroni corrections. Finally, variations concerning the prevalence of the investigated genes and answers regarding the questionnaire were also sought among periodontally healthy, gingivitis and periodontitis subjects and within each group both for tongue and plaque samples, by applying the z-test for proportions adjusted with Bonferroni corrections.
3.
Results
Demographic data for participants are displayed in Table 1. Results regarding the prevalence and distribution of S. aureus and MRSA and comparisons among and within groups are presented in Table 2. The nuc gene, which encodes S. aureus specific thermonuclease was detected in 10% of all samples tested. 18% of the subjects (27 out of 154) had at least one sample positive for S. aureus. It is noteworthy to mention that the mecA gene was not detected in any of the 344 investigated samples. The results from the questionnaire filled by participants are presented in Table 3. According to the personal Health Record of participants during the last five years >90% have received a course of antibiotics for medical reasons and >56% for dental reasons, with b-lactams being the statistically significant predominant class (z-test for Bonferroni corrections p < 0.05). Antibiotic intake during the last 6–12 months was recorded for 25% of the participants, while pharmacists have delivered antibiotics without medical–dental prescription to >14% of the subject sample. Few differences were observed among groups. More specifically, tetracyclines have been more frequently prescribed for medical reasons to the periodontally healthy group compared to the chronic periodontitis group, and quite a lot of periodontally healthy subjects reported that they are aware of the phenomenon of antimicrobial resistance unlike participants with chronic periodontitis (z-test for proportions with Bonferroni corrections, p < 0.05).
Table 1 – Demographic data and clinical parameters of participants. Diagnosis
Periodontal health Gingivitis Chronic periodontitis
Total
Age (mean years SD)
Male (%)
Smokers (%)
Probing depth (mm) (mean SD)
Recession (mean SD)
Clinical attachment level (mm) (mean SD)
Bleeding on probing (%)
50 52 52
46 8 49 9 50 10
52 (a) 42 (b) 79 (a,b)
24 35 38
1.54 0.2 (a) 1.89 0.3 (b) 3.05 0.6 (a,b)
0.04 0.06 (a) 0.06 0.16 (b) 0.31 0.42 (a,b)
1.38 0.24 (a) 1.61 0.4 (b) 2.81 0.6 (a,b)
6 5% (a,b) 34 15% (a) 32 21% (b)
Statistically significant differences between groups are indicated by the same letter (Kruskal–Wallis test and z-test with Bonferroni corrections, p < 0.05).
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Table 2 – Prevalence of S. aureus in plaque and tongue samples according to periodontal conditions. The mecA gene encoding for methicillin resistant S. aureus (MRSA) was not detected on any of the 343 analyzed samples. Plaque % (positive/all) Periodontal health Gingivitis Chronic periodontitis Total presence in sites % (positive/all)
Tongue % (positive/all)
8% (4/50) 4% (2/52) 12% (6/52)
8% (4/50) 12% (6/52) 15% (8/52)
8% (12/154)
Deep pockets % (positive/all)
Total 8% (8/100) 8% (8/104) 14% (19/139)
14% (5/35)
12% (18/154)
Overall presence in 10% of all samples tested (35/343)
No statistically significant differences were observed among groups or within the same group (z-test for proportions with Bonferroni corrections, p < 0.05).
Table 3 – Consumption, attitude and knowledge about antibiotics of the subject sample. Periodontal health (%) n = 50
Gingivitis (%) n = 52
Chronic periodontitis Total (%) n = 52 (%) N = 154
Antibiotic use for medical reasons during the last 5 years (%)
1–2 times 3–4 times >5 times
38% (19/50) 26% (13/50) 36% (18/50) (a)
44% (23/52) 25% (13/52) 27% (14/52)
46% (24/52) 29% (15/52) 15% (8/52) (a)
43% 27% 26%
Antibiotic use for dental reasons during the last 5 years (%)
1–2 times 3–4 times >5 times
54% (27/50) 8% (4/50) 0%
42% (22/52) 10% (5/52) 4% (2/52)
42% (22/52) 8% (4/52) 6% (3/52)
46% 8% 3%
b-Lactams (%) Tetracyclines (%) Imidazoles (%)
Yes Yes Yes
89% (39/44) 31% (13/42) (a) 17% (7/41)
73% (37/51) 20% (10/51) 24% (12/51)
75% (38/51) 6% (3/47) (a) 13% (6/47)
78% 18% 18%
30% 2% 14% 28% 48%
33% 2% 25% 14% 29%
17% 0% 15% 33% 23%
27% 1% 18% 25% 33%
Available at home (%) Without prescription (%)
Yes Yes By a pharmacist Yes (b-lactams) Antibiotic intake last 6–12 months (%) Knowledge about antimicrobial resistance (%) Yes
(15/50) (1/50) (7/50) (14/50) (24/50) (a)
(17/52) (1/52) (13/52) (7/51) (a) (15/51)
(9/52) (0/52) (9/52) (17/52) (a) (12/52) (a)
Statistically significant differences among groups are indicated by the same letter. Statistically significant differences within each group are indicated by bold lettering (z-test for proportions with Bonferroni corrections, p < 0.05).
4.
Discussion
The aim of the current study was to determine the prevalence of S. aureus and MRSA in the oral cavity of Greek subjects. The prevalence was investigated by identifying the presence of the nuc and mecA genes in clinical samples from the tongue and gingival crevice of 154 individuals. Furthermore, the prevalence of these genes in pockets >5 mm in subjects with chronic periodontitis was also investigated. By comparing the prevalence of S. aureus in three different periodontal conditions (health, gingivitis and chronic periodontitis) we came up with interesting findings, which contribute to the question of whether this species is a transient member of the flora or a possible pathogen. The investigated prevalence of the mecA gene is also suggestive of the probability of dissemination of MRSA by carriers to other individuals or the environment, especially in a subject sample deriving from a country with documented high resistance rates to b-lactam antibiotics.23 The findings of this comprehensive study, using the described approach and methodology corroborate with previous reports in the literature demonstrating that in systemically healthy individuals S. aureus can be detected in all three investigated periodontal conditions and therefore does not
appear to participate in periodontal pathology. The range of the reported prevalence is within the one previously reported in the literature for plaque samples, although comparisons are difficult to be drawn, due to differences in study design and especially sampled sites, since most studies reporting on the detection of S. aureus in the oral cavity, involve cultured samples from the nares or oral rinses.3 S. aureus has also been recognized as an aetiological factor of infective endocarditis. Although Streptococcus viridans spp. have long been associated with this disease, an increasing percentage of cases is being attributed to S. aureus especially among intravenous drug users (IVDU).7 Therefore, the occurrence of S. aureus in subgingival plaque samples, especially in the presence of ulcerated epithelium as in the case of chronic periodontitis, could imply the possibility of bloodborn dissemination of this bacterial species, which could lead to infective endocarditis. The low prevalence of detection of S. aureus in the current subject sample is indicative of a low possibility for this route of dissemination. In addition, the mecA (MRSA encoding gene) was not detected in any of the 344 investigated samples. The presence of MRSA in the oral cavity can be an important factor in transmitting MRSA-related infections, since the transmission via the oropharynx is a well documented route. The presence
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of MRSA in the oral environment has been investigated, especially regarding the possibility of transmission from infected dental personnel to patients and vice versa. Early data in the literature have shown the transmission of MRSA from an infected dentist to two patients, but the report referred to an era when no standard hygiene precautions were established.24 More recent data have shown that in the USA, 21% of the 61 dental students included in the study, harbour MRSA in nasal swabs and therefore might be a source of infection for patients and the environment.25 According to the same study, the dental workspace might also be an important niche for dissemination of MRSA, since data have shown the presence of MRSA in 8.4% of investigated frequently touched clinic surfaces. The study suggested that further similar trials are needed, including more dental clinics and geographic locations, since MRSA contaminated surfaces and students, might pose a risk for further transmission. Data from 500 dental patients in a university clinic have shown the presence of S. aureus in the nares and/or pharynx of 42% of the subject sample, but an extremely low prevalence (2 patients) of MRSA.26 In the current study, the presence of MRSA has not been investigated in samples from the nasopharynx and was focused on oral environment habitats such as the tongue and the gingival crevice/periodontal pocket. The fact that the gene encoding for MRSA was not detected in the abovementioned habitats is especially encouraging, due to the fact that they derive from a subject pool known to be prone to antimicrobial resistance. Greece is, unfortunately, a European country with high rates of antibiotic consumption as reported by the 2013 European Centre for Disease Prevention and Control.27 To be more precise, antibiotic consumption in Greece is estimated to be 39.4 Defined Daily Doses or DDDs per 1000 inhabitants per day, with b-lactams being among the most frequently prescribed (21.8 DDDs of which 12.9 penicillins).23 Data have shown that, in 2012, 41% of the investigated hospital samples from Greece were S. aureus resistant to methicillin (MRSA), although a declining trend was observed between 2007 and 2010. In agreement with these reports, in the current study, according to results from the questionnaire and inspection of the personal National Health Records of participants, b-lactams were the predominant class of prescribed antibiotics in the investigated population. This antibiotic consumption trend might influence the occurrence of both S. aureus and MRSA.22 Taken collectively, data from the present study suggest that S. aureus should be considered a transient member of the tongue and the subgingival environment and that MRSA does not appear to be a frequent colonizer of the oral cavity.
Funding This research has been co-financed by the European Union (European Social Fund – ESF) and Greek national funds through the Operational Program ‘‘Education and Lifelong Learning’’ of the National Strategic Reference Framework (NSRF) – Research Funding Program: Heracletus II. Investing in knowledge society through the European Social Fund.
Competing interests None declared.
Ethical approval This study was approved by Aristotle University of Thessaloniki Ethical Committee of the School of Dentistry (#120), in compliance with the ethical principles of the World Medical Association Declaration of Helsinki.
Acknowledgments We thank all the patients who participated and the Microbiology Department of Papanikolaou Hospital, Thessaloniki, the Microbiology Laboratory in Arta and the Department of General Microbiology, Veterinary School, Aristotle University of Thessaloniki for kindly providing positive clinical samples.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j. archoralbio.2015.06.009.
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ScienceDirect journal homepage: http://www.elsevier.com/locate/aob
Prevalence of Staphylococcus aureus and methicillin resistant Staphylococcus aureus (MRSA) in the oral cavity Georgios Koukos a,*, Dimitra Sakellari b, Minas Arsenakis c, Lazaros Tsalikis b, Theodora Slini d, Antonios Konstantinidis b a
251 General Air Force Hospital, Athens, Greece Department of Preventive Dentistry, Periodontology and Implant Biology, Dental School, Aristotle University of Thessaloniki, Greece c Department of Genetics and Molecular Biology, School of Biology, Aristotle University of Thessaloniki, Greece d Department of Mechanical Engineering, Aristotle University of Thessaloniki, Greece b
article info
abstract
Article history:
Objective: To assess the prevalence of Staphylococcus aureus and methicillin resistant Staph-
Accepted 13 June 2015
ylococcus aureus (MRSA) in plaque and tongue samples from systemically healthy subjects with periodontal health, gingivitis or chronic periodontitis.
Keywords:
Methods: After screening 720 potentially eligible subjects, 154 systemically healthy partici-
Staphylococcus aureus
pants were ultimately enrolled in the current study. Subgingival samples were taken from
Methicillin-resistant Staphylococcus
the first molars and the tongue and analyzed for the presence of S. aureus and MRSA by
aureus
polymerase chain reaction (PCR), using primers and conditions previously described in the
Molecular biology
literature. In addition, samples were taken from deep periodontal pockets of chronic
Polymerase chain reaction
periodontitis patients. Statistical analysis was performed by applying non-parametric tests
Antimicrobial drug resistance
(Kruskal–Wallis for clinical parameters, and z-test with Bonferroni corrections for distributions of assessed parameters). All comparisons were set at the 0.05 significance level. Results: S. aureus was detected in 18% of all participants and in 10% of the samples tested. No significant differences were found in its distribution among the three investigated groups (ztest for proportions with Bonferroni corrections, p > 0.05). The mecA gene was not present in any of the S. aureus found. Conclusions: S. aureus can be found in the oral environment regardless of the periodontal conditions and therefore should be considered as a member of the transient flora not participating in periodontal pathology. Subgingival sites and tongue surfaces seem to be an unusual habitat of MRSA. # 2015 Elsevier Ltd. All rights reserved.
1.
Introduction
The prevalence of Staphylococcus spp. in the oral cavity and their contribution to periodontal infections have not been
extensively investigated. Although they have been isolated in the oral cavity and are correlated with conditions such as suppurative parotitis, angular cheilitis, denture stomatitis and acute dentoalveolar infections, they are generally regarded as transient members of the oral flora.1–3 Recent studies also
* Corresponding author at: Department of Periodontology, 251 General Air Force Hospital, 11525 Athens, Greece. Tel.: +30 6983 520282. E-mail address: [email protected] (G. Koukos). http://dx.doi.org/10.1016/j.archoralbio.2015.06.009 0003–9969/# 2015 Elsevier Ltd. All rights reserved.
archives of oral biology 60 (2015) 1410–1415
suggest that Staphylococci can be frequently isolated from the oral cavity of specific patient groups such as newborns in hospital units, the elderly, patients with malignancies as well as subjects with immunocompromising systemic conditions such as rheumatoid arthritis.4–6 Last but not least they are considered as aetiological factors of prosthetic valve endocarditis.7 The relationship between periodontal disease and Staphylococci is currently not well understood. In a number of studies, using cultural techniques, they have been isolated from plaque samples, however a wide range for their prevalence (5.6–75%) has been reported. In studies including more than 500 subjects with periodontitis, it has been observed that approximately half harbour Staphylococcus spp. with S. epidermidis and to a lower extent S. aureus being the predominant species,1,8–11 but data comparing their prevalence with samples from periodontally healthy controls have not shown differences between these groups.3 Although usually a harmless colonizer of the skin and the nasopharynx in 25–35% of healthy individuals, on some occasions S. aureus is known to cause severe infections.12 One of the concerns with S. aureus is the current extent of the prevalence of methicillin resistant Staphylococcus aureus (MRSA) which was initially isolated 50 years ago, only two years after the introduction of methicillin in clinical practice and has developed into a major global health issue due to its pathogenic potential to cause bloodstream infections, pneumonia as well as surgical site infections.13 Resistance of S. aureus to b-lactam antibiotics is acquired by the exogenous mecA gene, which encodes a modified form of Penicillin Binding Protein (PBP2a), that does not allow proper binding and thus prevents the inhibition of cell wall synthesis that this class of antibiotics cause.14,15 Healthcare-associated MRSA (HA-MRSA) is related to prolonged length of hospital stay and is currently one of the most frequently identified pathogens in hospitals in many parts of the world.16 Furthermore, Community acquired MRSA (CA-MRSA) has demonstrated increasing trends, hence guidelines for prevention and surveillance have been issued by several healthcare officials. Thus, MRSA in the oral cavity could potentially be disseminated by carriers to the environment or to other individuals.17 The aim of the present study is to investigate the prevalence of S. aureus and the mecA gene encoding for MRSA in the oral cavity of Greek subjects with various periodontal conditions.
2.
Materials and methods
2.1.
Patient selection
or medications known to affect periodontal tissues, without infectious conditions (hepatitis, HIV) or pregnancy and lactation, who did not receive periodontal treatment or antibiotics within the last six months. They were also required to have at least 20 teeth present and meet the criteria of one of three periodontal conditions (healthy, gingivitis or periodontitis). Periodontally healthy subjects would have no periodontal pockets measuring more than 3 mm and bleeding on probing would be less than 10%. Gingivitis subjects had no periodontal pockets measuring more than 4 mm or attachment loss of more than 3 mm and bleeding on probing should exceed 20%, without radiographic bone loss. Moderate or advanced periodontitis subjects had 30% of teeth with proximal attachment loss 5 mm, and presented radiographic bone loss which exceeded 30%.18 Out of the 720 subjects initially screened, 156 fulfilled the inclusion criteria and 154 agreed to participate in the study. The study was conducted according to the protocol outlined by the Research Committee, Aristotle University of Thessaloniki, Greece and approved by the Ethical Committee of the School of Dentistry (#120), in compliance with the ethical principles of the World Medical Association Declaration of Helsinki. All patients read and signed an appropriate informed consent document prior to the participation in the study.
2.2.
Study design and clinical protocol
The present cross-sectional study, included three groups as follows: (a) periodontal health (n = 50), (b) gingivitis (n = 52), and (c) moderate or advanced chronic periodontitis (n = 52). Clinical recordings were performed at six points of all teeth present at the dentition (mesio-buccal, buccal, disto-buccal, mesio-lingual, lingual, distolingual). Recordings included the following parameters: Probing Pocket Depth (PPD), Recession, Clinical Attachment Level (CAL) and Bleeding on Probing (BOP). Recordings were performed by a calibrated examiner (GK) using an automated probe (Florida Probe, Florida Probe Corporation, Gainesville, FL, USA). All subjects were interviewed by one of the authors (DS) and completed a questionnaire regarding the following: smoking, frequency of antibiotic intake for medical and dental reasons, class of antibiotics used 6–12 months before the interview as well as within the 5-year period prior to the interview, whether they have ever obtained antibiotics without prescription, whether they have antibiotics available at home and whether they are aware of the phenomenon of antibiotic resistance. In order to avoid false reporting by participants, the class of antibiotics that they had used was recorded based on their personal National Health Record.
2.3. 720 subjects attending the Clinic of Periodontology at 251 Air Force Hospital, Athens, Greece and the Clinic of the Department of Preventive Dentistry, Periodontology and Implant Biology, Dental School, Aristotle University of Thessaloniki, Greece, were screened in order to be enrolled in the study from September 2011 to April 2014. Subjects were deemed eligible as long as they fitted the following criteria: age >30 years, absence of systemic diseases
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Sampling and analysis
Two clinical samples were collected from each patient: a pooled subgingival plaque sample from the mesiobuccal surface of the four first molars (or premolars when molars were missing) taken with sterile Gracey curettes, and a sample collected from the dorsal surface of the tongue with a sterile straight surgical bone curette, after applying three consecutive strokes. All samples were immediately placed in 200 ml of TE
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buffer (Tris–HCl 10 mM, EDTA 1 mM, pH 7.5) and stored at 20 8C, until assayed. In addition, chronic periodontitis patients contributed with one more sample from their deepest periodontal pocket. Analysis of samples was performed blindly (coded samples). All experiments were performed in the Department of Microbiology, School of Biology, Aristotle University of Thessaloniki, Greece. The protocol suggested by the National Food Institute of the Technical University of Denmark, for the identification of S. aureus (based on detection of the nuc gene, encoding the S. aureus-specific thermonuclease gene), followed by detection of the mecA gene.19–21 There is a detailed description of PCR conditions and primers available in the Supplementary Data section. DNA isolation was performed by using a commercial DNA isolation kit (QIAamp DNA Mini Kit, Qiagen, Hilden, Germany). Initially, PCR was performed for the detection of the 16S ribosomal RNA gene, in order to verify that the clinical samples contained identifiable bacterial DNA. The final volume of the reaction mixture for each PCR assay was 50 ml. A Peltier Thermal Cycler (PTC-100, Peltier Thermal Cycler, MJ Research) was used. For each set of samples analyzed by PCR a negative and a positive control were used. Sterile water for injection (Demo S.A. Pharmaceutical Industry) was used as negative control, replacing DNA template into the PCR reactions, while S. aureus strain 9518 (NCIMB: National Collection of Industrial, Food and Marine Bacteria, Scotland) and clinical samples from three sources (Papanikolaou Hospital, Thessaloniki, Greece, Microbiology Laboratory, Arta, Greece and the Department of General Microbiology, Veterinary School, Aristotle University Thessaloniki, Greece) positive for methicillin resistant S. aureus (MRSA), were used as positive controls. The products of the DNA amplification were electrophoresed for 45 min at 70 mA field, through a 2% agarose gel, stained with ethidium bromide (Sigma–Aldrich, St. Louis, USA), exposed under UV light (302 nm) and photographed. A 100 bp DNA ladder (Invitrogen, Camarillo, CA, USA) was also loaded on agarose gel as a molecular weight standard. The amplified fragment sizes were 886 bp for 16S rRNA, 255 bp for nuc and 527 bp for mecA. The electrophoresis for each PCR product was carried out twice in order to test the reproducibility of the method.
2.4.
Statistical analyses
The statistical analysis of the data was carried out with the statistical package SPSS (SPSS 20.0 version, IBM, Armonk, NY,
USA). The experiment was set to have at least 80% power to detect changes of 25% with a significance level (alpha) of 0.05 (two-tailed). A previous study provided the expected values of the primary outcome variable.22 As for the clinical parameters, indicators of descriptive statistics were used, such as mean and standard deviation for each group, with the patient being the observational unit. As far as differences in clinical parameters and age are concerned they were sought by applying the Kruskal–Wallis test. Likewise, differences regarding the distribution of participants according to gender and smoking were sought by applying the z-test for proportions adjusted with Bonferroni corrections. Finally, variations concerning the prevalence of the investigated genes and answers regarding the questionnaire were also sought among periodontally healthy, gingivitis and periodontitis subjects and within each group both for tongue and plaque samples, by applying the z-test for proportions adjusted with Bonferroni corrections.
3.
Results
Demographic data for participants are displayed in Table 1. Results regarding the prevalence and distribution of S. aureus and MRSA and comparisons among and within groups are presented in Table 2. The nuc gene, which encodes S. aureus specific thermonuclease was detected in 10% of all samples tested. 18% of the subjects (27 out of 154) had at least one sample positive for S. aureus. It is noteworthy to mention that the mecA gene was not detected in any of the 344 investigated samples. The results from the questionnaire filled by participants are presented in Table 3. According to the personal Health Record of participants during the last five years >90% have received a course of antibiotics for medical reasons and >56% for dental reasons, with b-lactams being the statistically significant predominant class (z-test for Bonferroni corrections p < 0.05). Antibiotic intake during the last 6–12 months was recorded for 25% of the participants, while pharmacists have delivered antibiotics without medical–dental prescription to >14% of the subject sample. Few differences were observed among groups. More specifically, tetracyclines have been more frequently prescribed for medical reasons to the periodontally healthy group compared to the chronic periodontitis group, and quite a lot of periodontally healthy subjects reported that they are aware of the phenomenon of antimicrobial resistance unlike participants with chronic periodontitis (z-test for proportions with Bonferroni corrections, p < 0.05).
Table 1 – Demographic data and clinical parameters of participants. Diagnosis
Periodontal health Gingivitis Chronic periodontitis
Total
Age (mean years SD)
Male (%)
Smokers (%)
Probing depth (mm) (mean SD)
Recession (mean SD)
Clinical attachment level (mm) (mean SD)
Bleeding on probing (%)
50 52 52
46 8 49 9 50 10
52 (a) 42 (b) 79 (a,b)
24 35 38
1.54 0.2 (a) 1.89 0.3 (b) 3.05 0.6 (a,b)
0.04 0.06 (a) 0.06 0.16 (b) 0.31 0.42 (a,b)
1.38 0.24 (a) 1.61 0.4 (b) 2.81 0.6 (a,b)
6 5% (a,b) 34 15% (a) 32 21% (b)
Statistically significant differences between groups are indicated by the same letter (Kruskal–Wallis test and z-test with Bonferroni corrections, p < 0.05).
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Table 2 – Prevalence of S. aureus in plaque and tongue samples according to periodontal conditions. The mecA gene encoding for methicillin resistant S. aureus (MRSA) was not detected on any of the 343 analyzed samples. Plaque % (positive/all) Periodontal health Gingivitis Chronic periodontitis Total presence in sites % (positive/all)
Tongue % (positive/all)
8% (4/50) 4% (2/52) 12% (6/52)
8% (4/50) 12% (6/52) 15% (8/52)
8% (12/154)
Deep pockets % (positive/all)
Total 8% (8/100) 8% (8/104) 14% (19/139)
14% (5/35)
12% (18/154)
Overall presence in 10% of all samples tested (35/343)
No statistically significant differences were observed among groups or within the same group (z-test for proportions with Bonferroni corrections, p < 0.05).
Table 3 – Consumption, attitude and knowledge about antibiotics of the subject sample. Periodontal health (%) n = 50
Gingivitis (%) n = 52
Chronic periodontitis Total (%) n = 52 (%) N = 154
Antibiotic use for medical reasons during the last 5 years (%)
1–2 times 3–4 times >5 times
38% (19/50) 26% (13/50) 36% (18/50) (a)
44% (23/52) 25% (13/52) 27% (14/52)
46% (24/52) 29% (15/52) 15% (8/52) (a)
43% 27% 26%
Antibiotic use for dental reasons during the last 5 years (%)
1–2 times 3–4 times >5 times
54% (27/50) 8% (4/50) 0%
42% (22/52) 10% (5/52) 4% (2/52)
42% (22/52) 8% (4/52) 6% (3/52)
46% 8% 3%
b-Lactams (%) Tetracyclines (%) Imidazoles (%)
Yes Yes Yes
89% (39/44) 31% (13/42) (a) 17% (7/41)
73% (37/51) 20% (10/51) 24% (12/51)
75% (38/51) 6% (3/47) (a) 13% (6/47)
78% 18% 18%
30% 2% 14% 28% 48%
33% 2% 25% 14% 29%
17% 0% 15% 33% 23%
27% 1% 18% 25% 33%
Available at home (%) Without prescription (%)
Yes Yes By a pharmacist Yes (b-lactams) Antibiotic intake last 6–12 months (%) Knowledge about antimicrobial resistance (%) Yes
(15/50) (1/50) (7/50) (14/50) (24/50) (a)
(17/52) (1/52) (13/52) (7/51) (a) (15/51)
(9/52) (0/52) (9/52) (17/52) (a) (12/52) (a)
Statistically significant differences among groups are indicated by the same letter. Statistically significant differences within each group are indicated by bold lettering (z-test for proportions with Bonferroni corrections, p < 0.05).
4.
Discussion
The aim of the current study was to determine the prevalence of S. aureus and MRSA in the oral cavity of Greek subjects. The prevalence was investigated by identifying the presence of the nuc and mecA genes in clinical samples from the tongue and gingival crevice of 154 individuals. Furthermore, the prevalence of these genes in pockets >5 mm in subjects with chronic periodontitis was also investigated. By comparing the prevalence of S. aureus in three different periodontal conditions (health, gingivitis and chronic periodontitis) we came up with interesting findings, which contribute to the question of whether this species is a transient member of the flora or a possible pathogen. The investigated prevalence of the mecA gene is also suggestive of the probability of dissemination of MRSA by carriers to other individuals or the environment, especially in a subject sample deriving from a country with documented high resistance rates to b-lactam antibiotics.23 The findings of this comprehensive study, using the described approach and methodology corroborate with previous reports in the literature demonstrating that in systemically healthy individuals S. aureus can be detected in all three investigated periodontal conditions and therefore does not
appear to participate in periodontal pathology. The range of the reported prevalence is within the one previously reported in the literature for plaque samples, although comparisons are difficult to be drawn, due to differences in study design and especially sampled sites, since most studies reporting on the detection of S. aureus in the oral cavity, involve cultured samples from the nares or oral rinses.3 S. aureus has also been recognized as an aetiological factor of infective endocarditis. Although Streptococcus viridans spp. have long been associated with this disease, an increasing percentage of cases is being attributed to S. aureus especially among intravenous drug users (IVDU).7 Therefore, the occurrence of S. aureus in subgingival plaque samples, especially in the presence of ulcerated epithelium as in the case of chronic periodontitis, could imply the possibility of bloodborn dissemination of this bacterial species, which could lead to infective endocarditis. The low prevalence of detection of S. aureus in the current subject sample is indicative of a low possibility for this route of dissemination. In addition, the mecA (MRSA encoding gene) was not detected in any of the 344 investigated samples. The presence of MRSA in the oral cavity can be an important factor in transmitting MRSA-related infections, since the transmission via the oropharynx is a well documented route. The presence
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of MRSA in the oral environment has been investigated, especially regarding the possibility of transmission from infected dental personnel to patients and vice versa. Early data in the literature have shown the transmission of MRSA from an infected dentist to two patients, but the report referred to an era when no standard hygiene precautions were established.24 More recent data have shown that in the USA, 21% of the 61 dental students included in the study, harbour MRSA in nasal swabs and therefore might be a source of infection for patients and the environment.25 According to the same study, the dental workspace might also be an important niche for dissemination of MRSA, since data have shown the presence of MRSA in 8.4% of investigated frequently touched clinic surfaces. The study suggested that further similar trials are needed, including more dental clinics and geographic locations, since MRSA contaminated surfaces and students, might pose a risk for further transmission. Data from 500 dental patients in a university clinic have shown the presence of S. aureus in the nares and/or pharynx of 42% of the subject sample, but an extremely low prevalence (2 patients) of MRSA.26 In the current study, the presence of MRSA has not been investigated in samples from the nasopharynx and was focused on oral environment habitats such as the tongue and the gingival crevice/periodontal pocket. The fact that the gene encoding for MRSA was not detected in the abovementioned habitats is especially encouraging, due to the fact that they derive from a subject pool known to be prone to antimicrobial resistance. Greece is, unfortunately, a European country with high rates of antibiotic consumption as reported by the 2013 European Centre for Disease Prevention and Control.27 To be more precise, antibiotic consumption in Greece is estimated to be 39.4 Defined Daily Doses or DDDs per 1000 inhabitants per day, with b-lactams being among the most frequently prescribed (21.8 DDDs of which 12.9 penicillins).23 Data have shown that, in 2012, 41% of the investigated hospital samples from Greece were S. aureus resistant to methicillin (MRSA), although a declining trend was observed between 2007 and 2010. In agreement with these reports, in the current study, according to results from the questionnaire and inspection of the personal National Health Records of participants, b-lactams were the predominant class of prescribed antibiotics in the investigated population. This antibiotic consumption trend might influence the occurrence of both S. aureus and MRSA.22 Taken collectively, data from the present study suggest that S. aureus should be considered a transient member of the tongue and the subgingival environment and that MRSA does not appear to be a frequent colonizer of the oral cavity.
Funding This research has been co-financed by the European Union (European Social Fund – ESF) and Greek national funds through the Operational Program ‘‘Education and Lifelong Learning’’ of the National Strategic Reference Framework (NSRF) – Research Funding Program: Heracletus II. Investing in knowledge society through the European Social Fund.
Competing interests None declared.
Ethical approval This study was approved by Aristotle University of Thessaloniki Ethical Committee of the School of Dentistry (#120), in compliance with the ethical principles of the World Medical Association Declaration of Helsinki.
Acknowledgments We thank all the patients who participated and the Microbiology Department of Papanikolaou Hospital, Thessaloniki, the Microbiology Laboratory in Arta and the Department of General Microbiology, Veterinary School, Aristotle University of Thessaloniki for kindly providing positive clinical samples.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j. archoralbio.2015.06.009.
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