Unveiling the Relevance of the Oral Cavity as a Staphylococcus aureus Colonization Site and Potential Source of Antimicrobial Resistance

Staphylococcus aureus is both a human commensal and a pathogen, that causes serious nosocomial and community-acquired infections. Despite nostrils being considered its preferred host habitat, the oral cavity has been demonstrated to be an ideal starting point for auto-infection and transmission. The antibiotic resistance assessment of S. aureus is a priority and is often reported in clinical settings. This study aimed to explore the prevalence and antimicrobial susceptibility of S. aureus in the oral and nasal cavities of healthy individuals. The participants (n = 101) were subjected to a demographic and clinical background survey, a caries evaluation, and to oral and nasal swabbing. Swabs were cultured in differential/selective media and S. aureus isolates were identified (MALDI-TOF MS) and tested for antibiotic susceptibility (EUCAST/CLSI). Similar S. aureus prevalence was found exclusively on nasal (13.9%) or oral (12.0%) habitats, whereas 9.9% of the population were simultaneous nasal and oral carriers. In oro-nasal cavities, similar antibiotic resistance rates (83.3–81.5%), including MDR (20.8–29.6%), were observed. Notably, 60% (6/10) of the simultaneous nasal and oral carriers exhibited different antibiotic resistance profiles between cavities. This study demonstrates the relevance of the oral cavity as an independent colonization site for S. aureus and as a potential source of antimicrobial resistance, a role which has been widely neglected so far.


Introduction
The Staphylococcus genus consists of 70 species and subspecies, from which the majority are common colonizers of the human skin and mucous membranes [1]. Although several Staphylococcus species are major pathogens in humans, Staphylococcus aureus stands out as the most relevant one, considered a High Priority Pathogen by the World Health Organization (WHO) due to causing severe infections worldwide and to the rise and emergence of strains resistant to clinically relevant antibiotics [2]. In particular, methicillin-resistant Staphylococcus aureus (MRSA), a term used to describe strains of S. aureus that have acquired resistance to β-lactam antibiotics (such as methicillin, amoxicillin, and cephalosporins, except fifth generation), is a major cause of concern, with an estimated 150,000 infections occurring every year in European countries [3,4]. In fact, infections by multidrug-resistant Pathogens 2023, 12, 765 2 of 10 pathogens have become so prevalent that, in 2019, the WHO included antimicrobial resistance as one of the top ten threats to global health [5].
The nostrils are considered the preferred host site of S. aureus, with a crucial role in the dissemination of S. aureus diseases [6]. S. aureus nasal carriages can be divided into persistent, intermittent (or transient), and resistant (non-carriers). Persistent carriers generally carry one single strain at high levels of colonization over time, while intermittent carriers carry varying strains at lower levels of colonization over time [7]. Persistent carriers possess higher counts of S. aureus and are as frequent as non-carriers, both having a prevalence of around 30% in the general population, although some studies point to a prevalence of 12-30% in persistent healthy carriers [7][8][9]. Although specific host characteristics, such as the composition of the nasal microbiota, seem to be determinant in S. aureus nasal colonization, the mechanisms leading to this process are multifactorial [8]. Vulnerable groups, such as hospitalised, diabetic, obese, allergic, and immunosuppressed patients, have an increased risk for S. aureus nasal carriage and, consequently, an increased risk for developing life-threatening infections [8,[10][11][12]. According to the Human Oral Microbiome Database (eHOMD) online platform, S. aureus colonization is associated with the nasal cavity and the palatine tonsils, (http://www.homd.org, accessed on 1 April 2023), but several other host sites (e.g., skin, rectum, vagina, gastrointestinal and urinary tracts, and oropharynx) can act as reservoirs of this species, playing a role either in the transmission to others or in the transition of this microorganism to other body sites within the host [6,8].
The colonization of the oral cavity by opportunistic/pathogenic microorganisms or the imbalanced oral microbiota are associated with several risks, such as the development of oral diseases (e.g., dental caries, halitosis, periodontitis, oral cancer, systemic infections), and may even impact the progression of multiple systemic diseases (e.g., osteoporosis, atherosclerosis, diabetes, cardiovascular diseases, ischemic cardiomyopathy) [13]. In addition to the alteration from a symbiotic to a dysbiotic microbial community, several other factors are known to have a relevant impact on oral health, such as age, general health, lifestyle, and nutritional status [14]. Particularly regarding the oral carriage of S. aureus, evidence is still severely lacking in comparison to nasal colonization. Reported oral carriage rates vary from 24% to 84%, depending on the studied population, yet S. aureus's status as a member of the oral microbiota remains unclear, since it is still mostly regarded as a transient member in this host site [15][16][17]. So far, the S. aureus oral carriage has most frequently been associated with specific groups of patients where carriage rates are higher than normal, such as in children, the elderly, terminally ill patients, in individuals with decreased salivary secretion, with carious lesions, removable dentures, fixed prosthetic restorations, periodontitis, rheumatoid arthritis, and with haematological malignancies [15,18]. Nevertheless, some reports have demonstrated the oral cavity to be an important reservoir of S. aureus, indicating it to be an ideal starting point for diffusion within the host through external path or through an hematogenous route, via daily dental hygiene routines or during invasive dental procedures [19][20][21]. As S. aureus carriage is a risk factor for infection, screening of the nares of vulnerable populations is already carried out for this species, and particularly for MRSA, typically followed by decolonization with mupirocin to prevent infection and/or transmission [21][22][23][24]. The role of the oral cavity as a S. aureus reservoir has, however, remained wildly neglected. Oral screening is not yet routinely carried out in clinical settings. Notably, evidence suggests this practice may be useful in the detection of carriers missed by nasal sampling [18,19]. Moreover, the widespread use of prophylactic antibiotics in dentistry has been associated with the emergence of antibiotic resistance in several commensal microorganisms, including staphylococci [25], and MRSA has been previously isolated from the oral cavity, particularly in dentures-wearers and patients with oral infections [26,27]. For these reasons, understanding the role of the oral cavity as a reservoir of colonization and antibiotic resistance becomes a public health necessity.
Accordingly, this study aimed to evaluate the prevalence and the antimicrobial susceptibility profile of S. aureus isolates in the oral cavity of a population of healthy adults and compare the results to the nasal cavity, in order to obtain a better understanding of the role of the oral cavity as a S. aureus reservoir and as a potential source of antimicrobial resistance.

Study Design and Population
In this study, a total of 101 healthy adults were invited to participate from February to April of 2020. All participants were previously informed about the aim and procedures of the research and gave their informed consent. Each participant filled out a self-report questionnaire to collect demographic and clinical background information (e.g., age, gender, pregnancy history, smoking habits, hormonal contraception history, atopic dermatitis, obesity, systemic diseases, gingivitis, periodontitis, fixed or removable prostheses) (Figure 1). The WHO decayed, missing, and filled teeth (DMFT) index, conducted by a dental professional, was used to assess and quantify the severity of dental caries history in the participants. The exclusion criteria for this study included the inability to give informed consent, pregnancy, recent history of infection, and antibiotic therapy (<3 months).
Pathogens 2023, 12, x FOR PEER REVIEW 3 of 11 Accordingly, this study aimed to evaluate the prevalence and the antimicrobial susceptibility profile of S. aureus isolates in the oral cavity of a population of healthy adults and compare the results to the nasal cavity, in order to obtain a be er understanding of the role of the oral cavity as a S. aureus reservoir and as a potential source of antimicrobial resistance.

Study Design and Population
In this study, a total of 101 healthy adults were invited to participate from February to April of 2020. All participants were previously informed about the aim and procedures of the research and gave their informed consent. Each participant filled out a self-report questionnaire to collect demographic and clinical background information (e.g., age, gender, pregnancy history, smoking habits, hormonal contraception history, atopic dermatitis, obesity, systemic diseases, gingivitis, periodontitis, fixed or removable prostheses) ( Figure 1). The WHO decayed, missing, and filled teeth (DMFT) index, conducted by a dental professional, was used to assess and quantify the severity of dental caries history in the participants. The exclusion criteria for this study included the inability to give informed consent, pregnancy, recent history of infection, and antibiotic therapy (<3 months). Figure 1. Schematic representation of the methodology used in this study, including participant recruitment, sample collection, and data analysis. MSA, mannitol salt agar; BHI, brain heart infusion; and MALDI-TOF MS, matrix-assisted laser desorption/ionization desorption ionizationtime of flight mass spectrometry.

Oral and Nasal Bacteria Isolation and Identification
The samples were collected aseptically from oral and nasal cavities. Sterile swabs were inserted into both nostrils and gently turned for about 30 s in each. To collect samples from the oral cavity, the swabs were brushed against the inner cheeks, tongue, teeth, and gums for about a minute. The swabs were immediately processed by streaking the surface of selective and differential culture medium Mannitol Salt Agar (MSA, VWR Chemicals BDH, Leuven, Belgium) and incubated at 37 °C for 48 h ( Figure 1).
All colonies with distinct appearances (3 to 6 isolates per sample) were selected and re-isolated in MSA ( Figure 1). The selected isolates (n = 462) were sub-cultured in Brain Heart Infusion (BHI) agar (Biolab Inc., Budapest, Hungary) and incubated at 37 °C for 18-20 h for species identification by Matrix Assisted Laser Desorption/Ionization Matrix-Assisted Laser Desorption Ionization-Time Of Flight Mass Spectrometry (MALDI-TOF MS, Bruker, Mannheim, Germany), according to the manufacturer's instructions ( Figure   Figure 1. Schematic representation of the methodology used in this study, including participant recruitment, sample collection, and data analysis. MSA, mannitol salt agar; BHI, brain heart infusion; and MALDI-TOF MS, matrix-assisted laser desorption/ionization desorption ionization-time of flight mass spectrometry.

Oral and Nasal Bacteria Isolation and Identification
The samples were collected aseptically from oral and nasal cavities. Sterile swabs were inserted into both nostrils and gently turned for about 30 s in each. To collect samples from the oral cavity, the swabs were brushed against the inner cheeks, tongue, teeth, and gums for about a minute. The swabs were immediately processed by streaking the surface of selective and differential culture medium Mannitol Salt Agar (MSA, VWR Chemicals BDH, Leuven, Belgium) and incubated at 37 • C for 48 h (Figure 1).
All colonies with distinct appearances (3 to 6 isolates per sample) were selected and re-isolated in MSA (Figure 1). The selected isolates (n = 462) were sub-cultured in Brain Heart Infusion (BHI) agar (Biolab Inc., Budapest, Hungary) and incubated at 37 • C for 18-20 h for species identification by Matrix Assisted Laser Desorption/Ionization Matrix-Assisted Laser Desorption Ionization-Time Of Flight Mass Spectrometry (MALDI-TOF MS, Bruker, Mannheim, Germany), according to the manufacturer's instructions ( Figure 1). All isolates were stored at −80 • C in BHI broth (BHI, Biolab Inc., Budapest, Hungary) with 10% glycerol until being used for further studies (Figure 1).

Data Analysis
The results were subjected to statistical analysis ( Figure 1) using the Statistical Package for the Social Sciences (IBM ® SPSS ® Statistics, SPSS Inc., Chicago, IL, USA, 26.0 version). The categorical variables were described through relative frequencies (%) and analyzed by the Chi-square test applying continuity correction, and Fisher's Exact Test when cells had expected counts less than 5. The non-categorical variable was analyzed by t-test and described using mean ± standard deviation (SD). For each test, the statistical significance was set at a α of 5%.

Population Characterization
The clinical and demographic information of the 101 participants is included in Table 1. The studied population exhibited a mean age of 21.8 ± 3.5 years old, ranging from 18 to 45 years, and was mainly composed of female subjects (82.1%). Overall, for the different factors analyzed, no significant differences were observed. In addition, no cases of periodontitis or removable prosthesis were reported. Regarding systemic diseases, one case of Hashimoto's thyroiditis, one of psoriasis, and three of chronic gastritis were reported. Results are shown in prevalence (%) or as mean ± SD. a Female intake percentage. DMFT, decayed, missing, and filled teeth index.

Prevalence of S. aureus
A total of 51 S. aureus isolates (24 oral and 27 nasal) were observed in 35.6% of the population (n = 36 carriers). S. aureus prevalence, as depicted in Figure 1, was similar between exclusive oral (12.0%) and nasal (13.9%) carriers (p = 0.248). The simultaneous nasal and oral carriers included 9.9% of the participants (Figure 2).
A total of 51 S. aureus isolates (24 oral and 27 nasal) were observed in 35.6% of the population (n = 36 carriers). S. aureus prevalence, as depicted in Figure 1, was similar between exclusive oral (12.0%) and nasal (13.9%) carriers (p = 0.248). The simultaneous nasal and oral carriers included 9.9% of the participants (Figure 2).

S. aureus Carriage and Its Correlation with Clinical and Demographic Factors
The analysis of S. aureus prevalence in the different colonization sites (oral and/or nasal) in relation to different factors, such as gender, is depicted in Table 2. No statistically significant differences (p > 0.05) were observed regarding S. aureus colonization and gender, hormonal contraception intake, or DMFT index. Additional correlations between S. aureus carriage and other specific factors, such as smoking habits, obesity, gingivitis, fixed prostheses, asthma and allergies, and atopic dermatitis, were not carried out due to the very limited number of participants with these conditions.

S. aureus Carriage and Its Correlation with Clinical and Demographic Factors
The analysis of S. aureus prevalence in the different colonization sites (oral and/or nasal) in relation to different factors, such as gender, is depicted in Table 2. No statistically significant differences (p > 0.05) were observed regarding S. aureus colonization and gender, hormonal contraception intake, or DMFT index. Additional correlations between S. aureus carriage and other specific factors, such as smoking habits, obesity, gingivitis, fixed prostheses, asthma and allergies, and atopic dermatitis, were not carried out due to the very limited number of participants with these conditions.

Discussion
The results of our study highlight the relevance of the oral cavity as a habitat of equal importance to the nasal cavity regarding S. aureus colonization and antibiotic resistance, and alert the healthcare community to the need for the simultaneous screening of nasal and oral S. aureus when surveillance and effectiveness of infection control are required.
Regarding the nasal cavity, our results (23.8% nasal carriers) are in accordance with previous works showing similar S. aureus carriage rates (21.6-43.8%) among healthy adults, including healthcare workers [30][31][32][33][34]. Around half of S. aureus carriers exhibited simultaneous oral and nasal carriage, emphasising the possibility of trafficking of this microorganism between the oral and nasal cavities. The other half (~10% of the studied population) exhibited exclusive oral or nasal S. aureus colonization, which suggests and reinforces the possibility raised by some authors of S. aureus colonizing the oral cavity without necessarily being present in the nares of the same individual [16,17]. These results demonstrate the importance and the independence of the oral cavity as a reservoir of S. aureus, further Pathogens 2023, 12, 765 7 of 10 highlighting the necessity for oral screening in order to detect non-nasal carriers and effectively prevent infection and transmission. Moreover, even in the participants of our study with simultaneous oral and nasal carriage of S. aureus, more than half presented different antibiotic resistance profiles between cavities, further reinforcing the possibility of the oral cavity as an independent reservoir of S. aureus.
However, it remains controversial whether S. aureus belongs to the oral microbiota or if it is just a transient member due to the close anatomic connection with the nasal cavity. For example, in the eHOMD platform, S. aureus appears as exclusively of nasal origin, with slight colonization of the palatine tonsils (http://www.homd.org, accessed on 1 April 2023). Thereby, studying the oral and nasal carriage over a large span of time will be necessary to deepen the knowledge on this matter. Nevertheless, as previously mentioned, the prevalence of S. aureus in the oral and nasal cavities in this study was similar (21.8% and 23.8%, respectively), which strongly suggests its possible integration in the human oral microbial community, as already raised in previous works [16].
Regarding the correlation between S. aureus carriage and clinical and demographic factors, the discrepancies of S. aureus carriage rates were not statistically significant in any habitat. Moreover, no statistically significant differences (p > 0.05) were observed regarding gender, hormonal contraception intake, or DMFT index ( Table 2). As previously mentioned, a correlation between S. aureus carriage and other factors (smoking habits, obesity, oral issues, asthma and allergies, and atopic dermatitis) was not carried out due to the very limited number of participants with these conditions. In order to better understand the prevalence of S. aureus in this population, it would be interesting to discriminate persistent carriers from intermittent carriers. Hence, the sample collection should be carried out two or more times, at least one week apart, and the evaluation of S. aureus should be quantitative, since persistent carries have higher loads of S. aureus and are consequently at a greater risk of acquiring S. aureus infections [6].
Considering antibiotic resistance, the oral isolates of S. aureus exhibited similar resistance rates and profiles to the nasal cavity. The resistance rates (to at least one antibiotic) of the oral isolates exceeded 80% and MDR surpassed 20%, both values being similar to the ones observed in the nasal cavity (81.5% and 29.6%, respectively, p > 0.05). In particular, high resistance rates were observed for the several antibiotics (gentamycin, amoxicillin, erythromycin, and clindamycin) ( Table 3) most commonly used in dentistry, but also used as alternatives to treat nosocomial staphylococci infections, including methicillin-sensitive S. aureus (MSSA) infections [35]. Indeed, these antibiotic resistances have been frequently associated with mechanisms known to be easily acquired by other commensal and/or pathogenic staphylococci by the horizontal gene transfer of mobile genetic elements, therefore favoring their dissemination [36]. Additionally, high resistance and carriage rates of oral S. aureus have been previously reported, with carriage rates in the oral cavity varying greatly from study to study, from 24% to values as high as 84%, even in healthy individuals [15][16][17]33,34]. Interestingly, Kearney et al. reported a carriage of oral MSSA of 31.3% in healthcare workers across nine inpatient wards over a two-year period [37]. Compared to the literature, the rate of oral carriage observed in the present study (22%) seems to be on the lower end of the spectrum of what has been reported. Antibiotic resistance in oral S. aureus has been observed more frequently in recent years, with MRSA oral carriage being particularly reported [25][26][27]37]. Although MRSA isolates were not detected in this study, resistance rates to other antibiotics are still relevant. A recent study by Garbacz et al. assessed antibiotic resistance in oral staphylococci and obtained high percentages of S. aureus resistance to several antibiotics (44% resistance to gentamicin, 35% to tetracycline, 19% to erythromycin, 18% to clindamycin, 12% to cefoxitin, 3% to trimethoprim-sulfamethoxazole, and 1% to ciprofloxacin) and a percentage of MDR of 23% [25]. In this study the isolates also exhibited high rates of resistance to gentamicin (~67%), erythromycin, and clindamycin (~31% each), although the resistance to other antibiotics was lower (Table 3). Furthermore, a similar percentage of MDR isolates, of around 26%, was observed. Considering that this study portrays healthy individuals with no history of concomitant systemic diseases or recurrent antibiotic use, the results are alarming and may even suggest the need for new dentistry therapeutic options, as the use of prophylactic antibiotics in dentistry has been associated with the emergence of antimicrobial resistance in recent years [25,26].
Remarkably, multiple resistance profiles were detected in this study (Table 4), with the phenotypes observed among oral isolates being similar to those of nasal isolates, which reinforces that the oral cavity, in parallel with the nasal cavity, is most likely to be an important reservoir of resistant staphylococci. Nevertheless, different profiles between both cavities were observed in the same subjects, suggesting that both cavities are colonized by different S. aureus strains, which highlights the role of oral cavity as an independent reservoir of S. aureus, including antibiotic-resistant S. aureus. Therefore, this study provides evidence that the oral cavity can represent an important reservoir and, consequently, a potential source of resistant bacteria, both to other people through cross-infection and to other regions of the human body by auto-infection. In fact, the oral cavity may be a privileged site of endogenous dissemination within the host, given that daily routines of oral hygiene and invasive dental procedures may promote oral microbial translocation to circulation, potentially causing infection in vulnerable populations [20].
As already mentioned, one drawback of this study is the fact that persistent and intermittent carriers were not distinguished; this distinction would be important in order to further clarify our results regarding nasal and oral carriage. Moreover, further longitudinal studies with larger cohorts of participants would be helpful in assessing how the additional demographic and clinical factors not explored in this study may influence S. aureus carriage. It is important to note that participants self-reported their clinical and demographic information, which could introduce bias in the data. In the future, strain-level analysis studies will be crucial (e.g., whole-genome sequencing). This analysis will allow us to reinforce our understanding of the role of the oral cavity as an independent colonization site for S. aureus. What is more, it will give relevant information regarding antibiotic resistance genes, and their location (to assess the potential of horizontal gene transfer), virulence factors (to assess the potential of pathogenicity), and clonal linages.
Nonetheless, this study reinforces the idea that the detection of S. aureus in the oral cavity, not only in the nasal cavity, is crucial in properly detecting this potential pathogen and avoiding further infection. In fact, exclusive nasal screening is not enough, as demonstrated by the fact that some of our participants were S. aureus oral carriers while testing culture negative for nasal carriage. In fact, Donkor and Kotey suggested that oral S. aureus colonization could partly explain why decolonization programs targeting only the nasal cavity are often met with failure [26]. As such, we urge the scientific and medical communities to recognize the oral cavity as a relevant staphylococci and antibiotic resistance reservoir, and to include it in screening programs alongside the nasal cavity.
In summary, the present study stresses the relevance of the oral cavity as an independent colonization site for S. aureus and as a potential source of antimicrobial resistance to commonly prescribed antibiotics, a role which has been widely neglected so far. Consequently, these findings highlight the need for the oral cavity to be included in surveillance and decolonization programs among healthcare workers and vulnerable patients, in order to prevent the transmission and infection of S. aureus.