Presence of egc-positive major clones ST 45, 30 and 22 among methicillin-resistant and methicillin-susceptible oral Staphylococcusaureus strains

The oral cavity may comprise a significant reservoir for Staphylococcus aureus but the data on molecular epidemiology and clonal distribution of oral strains are really scarce. This study aimed to evaluate the clonal relatedness in S. aureus isolated from oral cavity and their relationship with carriage of virulence genes, and antimicrobial resistance profiles. A total of 139 oral S. aureus isolates were obtained from 2327 analysed oral samples of dental patients. Antimicrobial susceptibility testing was performed. Isolates were characterized using protein A gene (spa) typing, spa-CC clonal complexes, toxin genes and SCCmec typing for MRSA. High resistance rates for penicillin, tetracycline and gentamicin were detected, respectively 58.3%, 42.4%, and 35.2%. Twelve (8.6%) S. aureus isolates were identified as MRSA. All of MRSA isolates were mecA-positive and mecC-negative. SCCmec IV was the most common type (66.7%), which was typical for community-acquired MRSA (CA-MRSA). Overall, the enterotoxin gene cluster (egc) was the most frequent detected virulence factor (44.9%), both in MSSA and MRSA isolates. Presence of genes encoding for the enterotoxins (sea, seb, sec, seh, sek), exfoliative toxin A (eta), and toxic shock syndrome toxin-1 (tst) was also observed. Strains carrying lukS-PV/lukF-PV genes belonged to SCCmecV- spa type t437. The most prevalent spa types were t091, t015, t084, t002, t571, and t026 among all 57 identified. Spa types, including 3 new ones, grouped in 6 different spa-CC clonal complexes, with four major dominated; CC45, CC30, CC5, and CC15. This study demonstrated that both methicillin-susceptible and methicillin-resistant major European clones of S. aureus could be isolated from the oral cavity of dental patients, with the emergence of PVL-positive CA-MRSA strains. The oral cavity should be considered as a possible source of toxigenic egc-positive S. aureus strains, in terms of potential risk of cross-infection and dissemination to other body sites.


Results
Prevalence of S. aureus isolates. A total 139 S. aureus were isolated from 2327 oral samples of 750 (18.5%) dental patients with symptoms of infection. S. aureus were detected among 128 patients, 71 females and 57 males aged between 17 and 82 years (mean 56 years). The number of collected samples from each patients varied, from one sample per patients (118 patients) to two (9 patients) or three samples (1 patient). Sixty seven isolates derived from dorsum of the tongue swabs, 38 isolates from buccal mucosa swabs, 19 isolates from denture swabs and 15 isolates from the corners of the mouth swabs.
The identified spa types were clustered into six spa-clonal complexes (spa-CCs) by BURP repeat analysis; spa-CC 571/1451, spa-CC 021, spa-CC 084, spa-CC 888, spa-CC 015, and spa-CC 267. Forty-six (33.1%) isolates represented 19 spa types and belonged to singletons, ten (7.2%) isolates were excluded from BURP cluster analysis due to presence of less than four spa repeats. The newly described spa types were distributed across various clusters and singletons (Fig. 1, Table 3).
Twelve MRSA isolates were assigned to three clonal complexes (spa-CC 084, spa-CC 888 and spa-CC 021) and five singletons, comprising of 9 spa types (t012, t091, t156, t189, t437, t888, t5644, t13670, t18953). Two isolates were excluded due to the low number of repeats. Particular attention should be given to two isolates of the spa type t437 having lukS-PV/lukF-PV genes as well as seb and sek (Table 3).
Most of isolates belonging to major MLST clonal complexes (CC45, 30, 5, and 22) were egc-positive. The differences in prevalence of egc-positive isolates were statistically significant (p < 0.001), as shown in Table 5.

Discussion
The epidemiology of S. aureus infections constantly changes, with novel clones emerging in various geographical regions 19,27,32 . This warrants continuous surveillance of staphylococcal isolates from different sources, especially from the oral cavity. Recent evidence suggests that the latter constitutes a significant yet underrated reservoir of S. aureus 10 .
Staphylococcus aureus isolation rates differ considerably depending on the population. In healthy adults, oral carriage rates vary from 12 to 36%, with more frequent isolation (17-48%) among students 4,33 . The carriage rate documented in our study (18.5%) was similar as reported by other authors [34][35][36] , and remained within a typical range for European and American adult dental patients. It was, however, lower than in dentistry students examined by Ohara-Nemoto (46.6%) 7 , and in patients with periodontitis included in another study 6 . Acrylic denture wearers also seem to be predisposed to the oral carriage 37 , with S. aureus isolation rates of 27-48% 2,38 .
Nearly 9% of isolates included in this study were resistant to methicillin. The prevalence of MRSA in the oral cavity of adult dental patients is known to be relatively low, 5-12% 2,5,36,39 . Smith found MRSA strains among 6% Table 3. spa types, clonal complexes, genetic profile and antimicrobial resistance of oral S. aureus isolates. spa-CC spa clonal complex, MLST multi-locus sequence typing, NT non-typeable, FOX cefoxitin, P penicillin, AMC amoxicillin/clavulanic acid, E erythromycin, CC clindamycin, CCind clindamycin inducible resistance (iMLS B ), CIP ciprofloxacin, GM gentamicin, TE tetracycline, C chloramphenicol, SXT trimethoprimsulfamethoxazole.  (1) www.nature.com/scientificreports/ of studied patients, more often in those over 70 years 39 . Long-term MRSA carriage was also reported in 6 out of 10 complete denture wearers participating in a Finnish study, and 10.3% of Asian denture wearers 38,40 . MRSA are typically isolated in a hospital setting; such hospital-acquired MRSA (HA-MRSA) are believed to spread primarily via human-to-human transmission 41 . However, a growing number of infections caused by non-nosocomial MRSA, referred to as community-acquired MRSA (CA-MRSA) was also observed in the last decade 42,43 . CA-MRSA genetically differ from HA-MRSA, are less resistant to non-β-lactam antibiotics and carry a smaller version of staphylococcal cassette chromosome mec (SCCmec) 44,45 . All MRSA isolates in this study harbored SCCmec type IV or V and were more susceptible to non-β-lactam antimicrobials, which suggested their community origin (CA-MRSA). None of the MRSA were resistant to ciprofloxacin, chloramphenicol or vancomycin. The proportions of strains resistant to gentamycin, clindamycin, and erythromycin were similar as in other types of staphylococcal infections 27,46 . The analyzed MRSA showed multidrug resistance (MDR) more often than MSSA, which is consistent with the characteristics of MRSA strains 47,48 .

No of cluster
spa typing demonstrated a broad genetic diversity of our staphylococcal isolates. We did not find any published report about the clonal variety of oral S. aureus. Nevertheless, the heterogeneity observed in our series corresponds well with recent European and American data about MRSA involved in other infections 49,50 . Rather than showing clustering, a typical feature of MRSA, most of our methicillin-susceptible isolates displayed extensive genetic diversity. Our MSSA corresponded to major clones widespread in Poland and other European countries 19,49,[51][52][53][54] . In contrast, different predominant clonal complexes were reported in Africa and Asia, indicating geographical variation 53,55,56 .
The most toxigenic clonal complexes in our series were CC45, CC30 and CC22, which included the largest proportion of staphylococci testing positively for toxin genes in egc locus. The enterotoxin gene cluster (egc) encodes up to six enterotoxins (SEG, SEI, SEM, SEN, SEO, and SEU) being superantigens. The superantigens induce T lymphocytes and antigen-presenting cells causing massive cytokine production, with lethal effects dependent on direct toxic and cytokine effects on the cardiovascular system 57 . Recent studies showed that egcclustered enterotoxins are the most prevalent virulence factors in S. aureus isolated nowadays 27,46,58 . However, longitudinal studies are needed to better elucidate the role of locus egc in oral S. aureus strains.
The predominant clonal complex in our material was CC45, with the most common spa type being spa-CC t015. CC45 are widely distributed among both nasal colonization and bloodstream infections strains in Europe 19,59 , and recent results suggest that the nasal isolates carry the potential to cause an invasive disease 59 . However, no reports on severe infections caused by oral CC45 strains have been published to date. Bonnet observed predominance of spaCC t015 among S. aureus associated with infective endocarditis 60 , and Deasy pointed to this spa type as an emerging etiological factor of bloodstream infections 19 . Other authors reported on infective endocarditis after dental extraction and treatment 61,62 . These findings imply that peroral spread of endogenous S. aureus should be considered in at least some instances. Our findings suggest that spa-CC t015 seems to be particularly prone to the acquisition of virulence factors, including superantigens genes, sec, sel, sej and egc. These findings are consistent with the data from a report on bloodstream infections 63 .
The second most common clonal complex in our series was CC30. Many previous studies analyzed CC30 strains and their link with endocarditis 64 ; and some authors demonstrated their role in the development of an invasive disease 19,20 . Our spa-CC 021 belonging to CC30 clone characterized high proportion of egc-, sea-and tst-positive strains, especially CC30-t012, which is consistent with the results of previous studies 28,52,65 . Also, according to an American report, CC30 strains from patients with infective endocarditis were significantly more Table 4. Prevalence of toxin genes among six spa-clonal complexes (spa-CC) of oral S. aureus isolates.  Table 5. Prevalence of egc-positive isolates among five major MLST clonal complexes.  www.nature.com/scientificreports/ likely to contain these enterotoxin genes and had the potential to cause hematogenous complications 64 .The high resistance rates to penicillin and tetracycline was observed among our CC30 strains. To this date, resistance to tetracycline was not reported in S. aureus from the oral cavity but in the strains from other human or animal sources [66][67][68][69][70] . However, tetracycline is also used in the treatment of some oral infections in humans, especially in patients with periodontitis 71 . Thus, our observation on the potential emergence of tetracycline-resistant oral strains warrants further investigation. To the best of our knowledge, present study was the first to demonstrate the prevalence of spa type t437 SCCmec-V-pvl-positive S. aureus strains in dental patients. Panton-Valentine leukocidin (PVL) genes are considered a stable genetic marker for CA-MRSA strains carrying SCCmec type IV or V 72,73 . More than half (66.6%) of strains identified in this study were assigned to SCCmec IV, whereas the strains represented spa t437 harbored SCCmec V. Similar strains were isolated in Germany and Taiwan, and according to a recent Polish report, t437 SCCmec-IV-pvl-positive strains predominated in specimens from diabetic patients [74][75][76] . The PVL -positive strains were associated with purulent skin infections, necrotizing pneumonia, pyomyositis and other S. aureus infections 74,[77][78][79] . PV leukocidin is considered a potent inducer of inflammation and cytotoxicity but its role in oral infections is still little unknown 80,81 .
Our study has several limitations. First, the proportion of MRSA strains was small in comparison with number of MSSA strains. It should be taken account in the interpretation of the results. Second, we did not analyze clinical data of the swabbed patients, such as type of oral infection and its manifestations. However, even considering these drawbacks, the results of our study add to current knowledge about oral S. aureus strains.
In conclusion, this study demonstrated that both methicillin-susceptible and methicillin-resistant S. aureus strains major European clones could be isolated from the oral cavity of dental patients, with the emergence of PVL-positive CA-MRSA strains. The oral cavity should be considered as a possible source of toxigenic egcpositive S. aureus strains, in terms of potential risk of cross-infection and dissemination to other body sites.

Materials and methods
Isolation and identification of S. aureus. The study included oral S. aureus isolated from all 2327 oral microbiological samples analysed consecutively at the Laboratory of Department of Oral Microbiology of the Medical University of Gdansk during routine clinical laboratory procedures, over a period of three years. The samples were obtained with sterile cotton swabs from the oral mucosa, the dorsal surface of the tongue, denture surface and angular cheilitis lesions. The analysed S. aureus were not specifically isolated for this research, they were part of the diagnostic laboratory procedure and no humans were involved in the experiments.
All samples were plated onto Columbia blood agar (GrasoBiotech, Starogard Gd., Poland) and mannitol salt agar (bioMérieux, Marcy l'Etoile, France) and were incubated 18-24 h at 37 °C. Suspected staphylococcal colonies were identified by standard methods, on the basis of colony characteristics, pigment production, Gram-staining, haemolysis and Pastorex StaphPlus latex agglutination kit (Bio-Rad, Marnes la Coquette, France). Further, all isolates eventually identified as S. aureus based on PCR amplification of species-specific thermostable nuclease gene (nuc) 82 .
After final identification, the isolates were stored at − 80 °C in Trypticase Soy Broth (Becton Dickinson, Franklin Lakes, NJ, USA) supplemented with 20% glycerol.
Antimicrobial susceptibility testing. The antimicrobial susceptibility was determined on Mueller-Hinton agar plates (Becton Dickinson, Franklin Lakes, NJ, USA) by the disk diffusion method and interpreted according to the EUCAST 83 . The following antimicrobial agents were tested: oxacillin, cefoxitin, gentamicin, erythromycin, clindamycin, tetracycline, chloramphenicol, ciprofloxacin, amoxicillin/clavulanic acid, trimethoprim/sulfamethoxazole (Bio-Rad, Marnes la Coquette, France) and penicillin G (Oxoid, Basingstoke, England). The inducible resistance to macrolide-lincosamide-streptogramin B (MLS B ) was detected by disk diffusion method with use the clindamycin (2 μg) and erythromycin (15 μg) disks positioned 15-26 mm apart 83 . Resistance to gentamicin was verified by using E-tests (bioMérieux, Marcy l'Etoile, France), an isolate with MIC value > 1 µg/ml was considered as a resistant 83 . Vancomycin susceptibility was determined with E-test strips (bioMerieux, Marcy-l'Etoile, France), in line with the manufacturer's instruction. Multidrug resistance (MDR) was defined as a resistance to three or more classes of antimicrobials. Resistance to methicillin was first identified using cefoxitin (30 µg) and oxacillin (1 µg) disks, and then confirmed by the detection of PBP2a protein (Latex Agglutination Test Kit, Oxoid, Basingstoke, England), and verified by the detection of the mecA gene according to Khairalla et al. 25 and mecC according to Stegger et al. 84 . S. aureus ATCC25923 (methicillin-susceptible) and S. aureus ATCC43300 (MDR) were used as the reference strains.

Molecular characterization. Isolation of staphylococcal DNA. Genomic Micro AX Staphylococcus
Gravity kit (A&A Biotechnology, Gdynia, Poland) was used to isolate genomic DNA from bacteria by gravity according to the manufacturer's instructions.
SCCmec typing. Typing of the five (I-V) major staphylococcal chromosomal cassette mec (SCCmec) in MRSA strains was determined by PCR as described by Oliveira et al. 88 and by Milheiriço et al. 89 . The SCCmec type was determined on the basis of the band pattern profiles obtained.

Scientific Reports
| (2020) 10:18889 | https://doi.org/10.1038/s41598-020-76009-1 www.nature.com/scientificreports/ spa typing. The spa typing, based on amplification of the variable X region of protein A gene, was performed as described previously 90 . The spa types were assigned using the Ridom StaphType software version 2.2.1 (https ://www.ridom .de/ Ridom GmbH, Wurzburg, Germany) and the Ridom SpaServer database (https ://spase rver.ridom .de/). The predicted MLST were assigned based on Ridom SpaServer. The based upon repeat pattern (BURP) algorithm was used to calculate spa clonal complexes (spa-CCs) with following parameters: (I) exclude spa types shorter than 5 repeats; (II) cost less or equal to 4; (III) cluster composed of 2 or more related spa types was regarded as CC; (IV) a spa type that was not grouped into a CC was considered as singleton.
The significance of between-group differences in the percentages of positive isolates was verified with Pearson chi-squared test or Fisher exact test. The threshold of statistical significance was set at p ≤ 0.05, with Bonferroni correction applied whenever multiple comparisons had to be carried out.