Antimicrobial Resistance in Dental Bioaerosols: Insights from Microbiological Analysis of Face Shield MasksAbstract
Bioaerosols are airborne particles that may carry microorganisms and are frequently generated during dental procedures. This study aimed to analyze the microbiological profile of isolates found on face shields masks used by dental students during restorations with conventional restorative procedures (CRP) and atraumatic restorative treatment (ART), and to assess the antimicrobial susceptibility of the most frequent isolates. Samples were collected from the face shields of 30 students (15 CRP and 15 ART) using Rodac TSA contact plates after use. Plates were incubated at 36 ± 1 °C for 48 h, colonies counted, and microorganisms identified by MALDI-TOF mass spectrometry. The mean colony count was 35.9 CFU/plate for CRP and 28.6 CFU/plate for ART. In total, 49 microorganisms were isolated, predominantly Staphylococcus spp. (55.1%), including S. epidermidis, S. warneri, S. capitis, and S. saprophyticus. Among the 27 Staphylococcus isolates, non-susceptibility rates were highest for levofloxacin (100%), erythromycin (74.1%), and clindamycin (48.1%), while all were susceptible to chloramphenicol, linezolid, rifampicin, and sulfamethoxazole. Multidrug resistance was observed in 70.4% of isolates. Both procedures posed comparable risks of microbial dispersion through bioaerosol generation, including multidrug-resistant bacteria. These findings reinforce the need for strict biosafety and disinfection measures in dental practice to minimize infection risks.
Key words
Antimicrobial resistance; Bacteria; Bioaerosols; Dental restoration; Dentistry; Face shield masks
INTRODUCTION
Bioaerosols are airborne particles that may contain viable or non-viable microorganisms, microbial fragments, and viruses. These particles, generally smaller than 100 μm, can remain airborne for prolonged periods—even indefinitely under certain airflow and turbulence conditions—and may be transported over long distances, potentially many kilometers in the environment (George et al. 2022). During dental procedures, the generation of bioaerosols represents a significant contamination risk for dentists, assistants, patients, and the clinical environment (Gund et al. 2024). Moreover, the air quality in these settings can directly impact the spread of healthcare-associated infections (HAIs) (Stockwell et al. 2019, Khan et al. 2024).
Bioaerosols generated by dental procedures and equipment can carry microorganisms from the site of origin and promote cross-infections (Allison et al. 2024). Studies have shown that dispersed microorganisms can settle on surfaces or come into direct contact with the respiratory tract and ocular mucosa of professionals (Stockwell et al. 2019, Allison et al. 2024). Porphyromonas gingivalis, Streptococcus mutans, Streptococcus pneumoniae, papillomavirus, and HIV have been described in the dental environment, reinforcing the importance of strict biosafety measures during clinical procedures. Diseases such as hepatitis, herpes, tuberculosis, and other infections can also be transmitted in this clinical context (Allison et al. 2024). Therefore, the strict adoption of biosafety practices, including the appropriate use of personal protective equipment (PPE) and disinfection protocols, is essential to reduce contamination risks (Gama et al. 2020).
Antimicrobial resistance (AMR) adds an additional layer of concern to this scenario. Resistant microorganisms present in bioaerosols or on contaminated surfaces pose a higher risk of transmission and complicate potential infections (Binta & Patel 2016, Lee & Yoo 2022). Understanding the antimicrobial susceptibility profile of microorganisms dispersed in dental procedures supports safer therapeutic decision-making and strengthens recommendations for infection control in this clinical environment.
In this context, it is essential to understand the interaction between bioaerosols and the dental environment. Furthermore, prior knowledge of the bacterial susceptibility profile is crucial for more assertive therapeutic decisions. This profile is obtained through antimicrobial susceptibility testing, whose interpretation considers microbiological and pharmacodynamic aspects. This enables more accurate predictions of an antimicrobial’s effectiveness in treating specific infections (Allison et al. 2024).
Thus, identifying microorganisms present on PPE originating from bioaerosols generated in dental environments and their AMR profile is essential to prevent the dissemination of resistant strains. This is especially relevant in specialties such as endodontics, oral and maxillofacial surgery, and implantology, where infections caused by multidrug-resistant microorganisms pose a major clinical challenge (Vieira et al. 2011). Therefore, this study aimed to compare the total number of microorganisms isolated from face shields used by dental students after performing conventional restorative procedures (CRP) and atraumatic restorative treatment (ART), as an indicator of bioaerosol production, in addition to evaluating the antimicrobial susceptibility profile of the most frequently isolated microorganisms.
MATERIALS AND METHODS
Study design
This is a laboratory-based descriptive study, carried out through the analysis of face shields used by Dentistry students at the Federal University of Pernambuco (UFPE), as indicators of bioaerosol generation after performing conventional restorative procedures (CRP) or atraumatic restorative treatment (ART).
The study was conducted at the School Dental Clinic of the Federal University of Pernambuco (UFPE), an open clinical environment with multiple dental workstations operating simultaneously. The clinic provides free dental care to the surrounding community and predominantly serves children and adolescents from nearby neighborhoods.
Ethical considerations
The project was approved by the UFPE Research Ethics Committee under the opinion CAAE: 169134622.0.0000.5208. All steps followed current ethical standards for research involving human subjects.
Sample collection and processing
Samples were obtained from face shields routinely used by 30 students in the school clinic during restorative procedures (15 CRP and 15 ART) on patients with dental caries. This convenience sample included all eligible procedures performed during the data collection period; therefore, no formal sample size calculation was conducted, as the study had an exploratory and descriptive design. The average duration of restorative procedures was approximately 30 minutes, depending on the extent of the dental intervention. The face shields were made of transparent plastic material. Prior to each procedure, face shields were disinfected with 70% alcohol following the clinic’s standard biosafety protocol. Samples were collected immediately after the clinical procedure. Students were instructed not to touch the face shield at any point during the clinical procedure. A 4 cm² standardized area located at the central frontal region of each face shield was sampled using Rodac TSA (tryptic soy agar, KASVI) plates.
Collection was performed at room temperature by gently pressing the plate onto the mask surface for approximately 5 seconds to ensure full contact between the medium and the surface. The plates were then immediately sealed and transported to the Bacteriology and Molecular Biology Laboratory at UFPE. All samples were incubated exclusively under aerobic conditions at 36 ± 1 °C for 48 hours, after which colony-forming units (CFU) were counted using a manual colony counter.
Microbiological identification
The initial characterization of the isolated microorganisms was performed by analyzing cellular morphology and arrangement using light microscopy. Gram staining was used to differentiate Gram-positive and Gram-negative bacteria, and lactophenol cotton blue staining (Amann’s method) was used for preliminary identification of fungi (Koneman 2018).
From each culture plate, colonies (CFU) with distinct morphological and staining characteristics were selected, with one representative colony chosen for each pattern identified. This approach allowed for a broader analysis of microbial diversity in the samples.
Precise species-level identification was performed using Matrix-Assisted Laser Desorption/Ionization Time of Flight (MALDI-TOF) mass spectrometry, which allows for rapid and reliable analysis of microbial protein profiles (Jang & Kim 2018). Analyses were carried out at the Multiuser Laboratory for Research and Diagnosis in Tropical Diseases (LPPDT/UFPE) using equipment from Bruker Daltonics (Germany).
Antimicrobial Susceptibility Testing (AST)
AST was performed using the agar diffusion method and was restricted to Staphylococcus spp. due to its predominance among the recovered isolates and its clinical relevance. Interpretation followed criteria of the Brazilian Committee on Antimicrobial Susceptibility Testing (BrCAST 2025). The antimicrobial agents tested included: cefoxitin (30 µg), gentamicin (10 µg), levofloxacin (5 µg), sulfamethoxazole/trimethoprim (23.75–1.25 µg), penicillin (1 U), clindamycin (2 µg), tetracycline (30 µg), erythromycin (15 µg), rifampicin (5 µg), and linezolid (10 µg). For chloramphenicol, the breakpoint values established by the Clinical and Laboratory Standards Institute (CLSI 2025) were used. Isolates were classified as multidrug-resistant (MDR) when they exhibited resistance to three or more classes of antimicrobial agents (Magiorakos et al. 2012).
RESULTS
Quantification of microorganisms
A total of 30 restorative procedures were evaluated, comprising 15 CRP and 15 ART. Although CFU counts varied widely between samples, median values did not differ significantly between groups. The average CFU count on the plates collected after the restorative procedures was 35.9 CFU/plate in the CRP group and 28.6 CFU/plate in the ART group. The individual counts varied widely, ranging from 1 to 229 colonies per plate, with one plate showing no microbial growth (Table I).
Colony forming units (CFU) counting and microorganisms identified on the Rodac plates from face shield masks used by dental students during two different dental procedures. CRP: conventional restorative procedures; ART: atraumatic restorative treatment; CFU: colony forming units.
Macroscopic and microscopic characteristics of colonies
The bacterial colonies isolated presented diverse morphological features. The most common characteristics included white or pale yellow colonies, with smooth or rough surfaces, predominantly showing Gram-positive cocci and bacilli morphologies.
Fungal colonies exhibited a powdery appearance and black coloration, typical of filamentous fungi. These microorganisms were detected in two samples, one from the CRP group and one from the ART group.
Identification of microorganisms
Microbiological analysis of the face shield masks revealed the growth of 49 microorganisms, with a predominance of Gram-positive species. The genus Staphylococcus (S. epidermidis, S. warneri, S. capitis, and S. saprophyticus) was the most frequent (55.1%; 27/49), followed by Bacillus cereus-thuringiensis (18.4%; 9/49). Among Gram-negative microorganisms, Pseudomonas aeruginosa, Proteus spp., Klebsiella pneumoniae, and Moraxella spp. were identified (Table I).
Fungal colonies were observed on two Rodac plates—one from the CRP group and one from the ART group—with morphological characteristics compatible with filamentous fungi of the genus Aspergillus.
Antimicrobial susceptibility testing
Among the 27 Staphylococcus spp. isolates from face shield masks after restorative procedures, all exhibited non-susceptibility to levofloxacin. The highest frequencies of non-susceptibility were also observed for erythromycin (74.1%; 20/27), clindamycin (48.1%; 13/27), and tetracycline (33.3%; 9/27). In contrast, all isolates were susceptible to chloramphenicol, linezolid, rifampicin, and sulfamethoxazole/trimethoprim.
Species-specific analysis showed that S. epidermidis (n=20) had high frequencies of non-susceptibility to levofloxacin (100%), erythromycin (80%), and clindamycin (50%). Staphylococcus warneri (n=4) was also non-susceptible to levofloxacin (100%), erythromycin (75%), and clindamycin (50%). The single Staphylococcus capitis isolate was non-susceptible to levofloxacin, erythromycin, and clindamycin, while the two S. saprophyticus isolates were susceptible to all antimicrobial agents tested, except levofloxacin. Nineteen (70.4%) of the 27 Staphylococcus spp. isolates were classified as multidrug-resistant (MDR). Complete data on non-susceptibility by antimicrobial agent and species are detailed in Table II.
Frequency of non-susceptibility (resistant + intermediate) to antimicrobials among 27 samples of Staphylococcus spp. isolated from face shields after two different restorative procedure. ERI: erythromycin; CLI: clindamycin; OXA: oxacillin; PEN: penicillin; TET: tetracycline; LEV: levofloxacin; SXT: sulfamethoxazole/trimethoprim; CRP: conventional restorative procedures; ART: atraumatic restorative treatment.
Statistical analysis
The Shapiro-Wilk test indicated that the data did not follow a normal distribution for either group (CRP and ART), at a 95% confidence level. Therefore, the non-parametric Wilcoxon test was used to compare median values between groups. The results showed no statistically significant difference between the median CFU counts of the two groups, suggesting similar behavior in bioaerosol production between the restorative methods evaluated.
DISCUSSION
The data obtained in this study highlight the importance of using PPE, such as face shields, and of properly sanitizing dental surfaces and instruments. The presence of viable microorganisms in bioaerosols generated during dental care reinforces the risk of cross-contamination and HAIs, especially in clinical environments with high patient turnover. A recent study indicated that temporal trends in indoor bioaerosol concentrations, particularly in dental settings, can directly affect the exposure of professionals and patients, underscoring the need for continuous monitoring in these environments (Akhtar et al. 2025).
This work is innovative in comparing two types of restorative procedures—conventional and atraumatic restorative treatment—not only by quantifying microorganisms on face shields after procedures, which suggests aerosol dispersion, but also by performing bacterial identification, including MALDI-TOF mass spectrometry, and analyzing antimicrobial susceptibility profiles. This approach is particularly relevant given the increasing value placed on ART as a minimally invasive technique and its practical application in biosafety contexts, as discussed by Navarro et al. (2015).
Although the CRP group showed a numerically higher average CFU count than the ART group, the microbial loads isolated from face shields did not differ significantly between the procedures. This suggests that both techniques can lead to comparable levels of microbial dispersion on facial protective surfaces. Bioaerosols generated during clinical care—comprising saliva, blood, and oral microorganisms—can disseminate in the environment and contaminate personal protective equipment (PPE), such as masks and face shields. These findings underscore the importance of maintaining strict biosafety protocols and using physical barriers regardless of the restorative approach. Rautemaa et al. (2006) reported that rotary instruments can release high levels of bioaerosols (up to 970 CFU/m²/h), while Yang et al. (2023) highlighted the role of high-speed instruments in aerosol generation and the consequent risk of microbial spread, especially in hospital settings. Similarly, Han et al. (2021) demonstrated that aerosol contamination is widespread during routine dental procedures and affects various surfaces and PPE.
The predominance of Gram-positive bacteria, particularly of the genus Staphylococcus, on the face shields suggests that these microorganisms are among the main contaminants during restorative procedures. The frequency of S. epidermidis among the isolates is consistent with its known role as a common colonizer of skin and mucosa, but also as a potential opportunistic pathogen in clinical settings. Similar findings were reported by Gund et al. (2024), Santos et al. (2021) and De Castro Santos et al. (2021), who also detected Staphylococcus spp. on exposed surfaces during dental care. The detection of spore-forming Gram-positive bacilli as well as Gram-negative bacilli, such as Pseudomonas aeruginosa and Klebsiella pneumonia, reinforces the microbial diversity in the clinical environment. Furthermore, the growth of filamentous fungi compatible with Aspergillus spp. in samples from both groups indicates that exposure to bioaerosols may include not only bacteria, but also fungal structures with pathogenic potential, especially in immunocompromised individuals.
The antimicrobial resistance profile observed among Staphylococcus spp. isolates reinforces concerns about the presence of MDR strains in dental settings. This finding supports previous studies, such as Plum et al. (2018), which reported significant resistance to erythromycin and clindamycin in clinical dental isolates, and Mariano et al. (2024), who identified similar resistance patterns in primary endodontic infections. The detection of antimicrobial-resistant strains on personal protective equipment surfaces such as face shields highlights the potential of these devices to act as passive vectors for resistant microorganisms, especially during procedures that generate bioaerosols.
This situation is worsened by the widespread and often empirical use of antimicrobial agents in dental practice (Do Prado et al. 2021). The frequent prescription of drugs such as amoxicillin, metronidazole, and azithromycin without laboratory support may contribute to the selection and maintenance of antimicrobial-resistant strains in clinical environments. This unreasonable use, combined with constant exposure to contaminated aerosols, underscores the urgency of integrating microbiological surveillance into clinical practice to guide safer and more effective therapeutic decisions.
Our findings also align with those of Baudet et al. (2021), who reported the presence of antimicrobial-resistant bacteria on clinical surfaces in dental clinics, suggesting that environmental contamination control should be a priority in biosafety protocols. The presence of antimicrobial-resistant coagulase-negative staphylococci—often overlooked due to their commensal status—highlights their potential role as reservoirs of antimicrobial resistance genes and their relevance as opportunistic pathogens in invasive contexts.
We did not isolate Staphylococcus aureus, a species frequently reported as a relevant contaminant in dental environments (Yan et al. 2022), but S. epidermidis was predominant, which is consistent with a recent study (Gund et al. 2024). Although often underestimated due to their commensal role, coagulase-negative staphylococci like S. epidermidis have shown growing clinical relevance, especially in hospital and dental settings, where they act as opportunistic agents associated with device-related and invasive procedure infections (Heilmann et al. 2019). The high proportion of MDR strains among the Staphylococcus species isolated reinforces this concerning scenario, indicating the need for attention to both the pathogenic potential of these species and the risk of resistance dissemination in clinical environments.
The antimicrobial resistance observed among isolates reflects the microbial ecology of the clinical environment and highlights the relevance of monitoring resistant strains in dental care settings. These findings reinforce the need for continuous microbiological surveillance in environments where bioaerosols are frequently generated.
These findings not only emphasize the clinical importance of coagulase-negative staphylococci, but also reveal a significant gap in the national literature regarding antimicrobial resistance in microorganisms associated with dental bioaerosols. The scarcity of studies in this field limits the understanding of the microbiological risks faced by professionals and patients in dental clinics. Most investigations on bioaerosols focus on hospital environments, with a significantly smaller number of studies addressing the dental context (31 vs. 16), reinforcing the need to expand scientific production in this area and develop specific monitoring and control protocols (Zemouri et al. 2017).
Although the difference in microbial load between the CRP and ART groups did not reach statistical significance, the lower CFU count in the ART group suggests a possible microbiological benefit associated with less invasive approaches. This observation is relevant in the context of sustainable clinical practices, in which choosing procedures with lower microorganism dispersion potential may contribute to reducing occupational risk and the spread of resistant bacteria in dental environments. Studies such as those by Mirhoseini et al. (2021) and Lee & Yoo (2022) support this perspective by highlighting the importance of integrating biosafety, antimicrobial resistance control, and conscientious clinical practices.
Despite its contributions, some methodological aspects must be considered when interpreting the results. The sample size was limited, and the investigation was conducted at a single school clinic, which may limit the generalization of the findings to other dental contexts. Additionally, microbial exposure was not measured through direct air sampling, but rather through an indirect approach using face shields as sentinel surfaces to estimate microorganism dispersion during clinical procedures. The absence of anaerobic culturing may have limited the detection of anaerobic microorganisms potentially present in the samples. Even so, the study offers a viable, accessible, and reproducible strategy to monitor microbial load associated with different restorative techniques, contributing meaningfully to the discussion on biosafety and antimicrobial resistance in dental environments.
Given the presence of potentially pathogenic and multidrug-resistant microorganisms on face shields, strict PPE management is essential. Face shields should be cleaned with 70% alcohol between patients, inspected regularly for scratches or material degradation, and replaced when visibility becomes compromised. Importantly, face shields should complement—not replace—respiratory protection such as surgical masks or N95 respirators, especially during procedures known to generate aerosols.
CONCLUSIONS
Our findings demonstrate that, although the ART procedure showed lower median microbial counts on face shields compared to the CRP procedure, no statistically significant differences were observed between the two restorative techniques. Given the exploratory nature and sample size of this study, these findings should be interpreted with caution. Nonetheless, the presence of potentially pathogenic and antimicrobial-resistant microorganisms on PPE underscores the need for rigorous biosafety practices in dental environments.
The detection of potentially pathogenic microorganisms, such as S. epidermidis and K. pneumoniae, on the protective surfaces used by students reinforces the risk of cross-contamination in dental settings. Additionally, the high frequency of antimicrobial resistance, especially among Staphylococcus spp. strains that were resistant to quinolones, macrolides, and lincosamides—demonstrates the urgency of using antimicrobial agents judiciously and the importance of continuous microbiological surveillance in clinical contexts.
Thus, this study contributes to the understanding of microbiological risks associated with dental procedures and reinforces the need for effective biosafety policies and antimicrobial resistance control in oral healthcare environments.
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Handling editor
Vasco Azevedo
All data generated or analyzed during this study are included in this published article.
