In Vitro Analysis of Cross-Contamination and Disinfection Methods of Prosthetic Components Coming from Laboratories

Abstract

The customization and handling of implant abutments in prosthetic laboratories can lead to microbial contamination, requiring disinfection before clinical use. This study evaluated cross-contamination in abutments from three prosthetic laboratories, identifying microorganisms and assessing the disinfection efficacy of 2% chlorhexidine digluconate (2% CL) and ultraviolet C (UV-C) radiation. Sixty abutments were analyzed, with contamination detected in 83%, predominantly Enterococcus faecalis (34.2%) and Staphylococcus epidermidis (17.8%). Post-disinfection, CFU reduction was 92% (UV-C) and 93% (CL), confirmed by the Wilcoxon test (Z = −4.373; p < 0.001). A comparative analysis using the Kruskal–Wallis test showed no significant difference between the two methods (p > 0.05). These findings confirm that both UV-C and CL effectively reduce microbial load, providing reliable disinfection protocols for clinical practice.

1. Introduction

The need to customize abutments, as well as their handling to make crowns, can generate residue and the colonization of microorganisms, requiring cleaning prior to insertion into the patient’s mouth [1]. Dentists, assistants, hygienists, and prosthetists are constantly exposed to saliva/blood, as well as other potentially contaminated materials, and are more vulnerable to cross-contamination. A significant number of laboratory technicians, unaware of the disinfection processes and their importance, do not always use adequate protection when handling prosthetic work, sending them to dentists without any type of prior disinfection. As a result, prosthetic laboratories can serve as vectors for transmission, as microorganisms from dental offices may circulate within these environments [2,3,4,5,6,7,8,9].

Misinformation or negligence among dentists, dental technicians, and staff can lead to inadequate preventive measures, thereby increasing the risk of cross-contamination. Microorganisms that can be found in laboratories include Staphylococcus, Streptococcus, Lactobacillus, Actinomyces, and some species of Candida, as well as opportunistic microorganisms such as Acinetobacter calcoaceticus. These microorganisms were isolated in laboratories where prosthetic abutments, intended for installation in patients’ oral cavities, are handled. Consequently, these abutments can act as carriers for microorganisms and potentially contribute to serious systemic diseases, particularly in vulnerable hosts [2,7,8,10,11]. In addition, contaminated prosthetic components can also contribute to the stimulation and progression of peri-implantitis [4].

Peri-implant soft tissues are hypovascular and hypocellular, with a lower immunological capacity compared to periodontal tissues around natural teeth, making them less resistant to bacterial infection [12]. Breaking the cycle of infection with safety measures and control standards is essential to reduce the risk of cross-contamination, avoiding the transmission of pathogenic microorganisms [2]. To break this cycle, easy-to-use antimicrobial agents that can prevent the survival or proliferation of pathogenic microorganisms have been the subject of several studies. Among them, 2% chlorhexidine (2% CL) and ultraviolet C (UV-C) radiation have proven to be effective in this process [13,14].

Recent evidence suggests that the use of UV-C rays on impression materials can promote complete disinfection without the need for chemicals or heat. Its action has also been demonstrated on dental implant surfaces, demonstrated by a reduction in bacterial adhesion and biofilm formation, suppressing bacterial growth [15,16,17]. The use of chlorhexidine (CHX) may be useful in reducing bone loss during oral rehabilitation since it limits the formation of bacterial biofilm at the abutment/implant interface [18]. The disinfectant capacity of CHX was also reported by Sinjari et al. ref. [19] suggesting that the use of CHX, in the phases preceding the definitive placement of the prosthesis, can contribute to the reduction of peri-implant bone loss, which reinforces the importance of disinfection before placing the prosthesis in the mouth of patients.

One of the greatest challenges in dentistry is preventing cross-contamination, a responsibility shared by dentists, prosthetists, and the entire dental team. Effective infection control is crucial for maintaining patient safety [2,15,17]. However, limited data exist on microbial contamination in implant abutments from prosthetic laboratories, and the efficacy of different disinfection protocols remains unclear. Therefore, the objective of this study was to evaluate cross-contamination in implant abutments received from prosthetic laboratories, identify the microorganisms present, and assess the disinfection efficacy of two antimicrobial agents. By addressing these aspects, this study aims to provide valuable insights into improving infection control protocols in dental practice.

2. Materials and Methods

2.1. Study Design

The present cross-sectional study using in vitro laboratory components was approved by the Ethics and Research Committee (CEP-ULBRA No. 5,776,590) in 2023. Three reference prosthetic laboratories for the university were selected, each registered with the regional dentistry council and with a large volume of daily work.

The present research analyzed 60 prosthetic abutments that had no prior contact with the oral cavity, only with the prosthetic laboratories.

In this study, three prosthetic laboratories were selected based on their expertise in dental restoration and experience with the manufacturing of implant abutments. These laboratories were chosen to ensure the reliability of the study’s outcome and were located across different regions. All laboratories followed standardized protocols for processing the abutments to ensure consistency. Furthermore, quality control procedures were implemented to minimize variability in the handling and treatment of the samples. The selection of these laboratories was based on their established track record in dental prosthetics and their capability to meet the research requirements.

Each laboratory manipulated 20 abutments, totaling 60 abutments. These abutments served as a base for the manufacture of single prosthetic crowns. The collection of possible microorganisms was carried out prior to sending the sample from the laboratory to the dentist. This collection of material and counting of microorganism colonies followed the technique known as “spread plate” [20]. Because the abutments are small pieces and may contain little material for analysis, it was decided to use the Brain Heart Infusion (BHI) culture medium in broth and agar which allows the isolation of microorganisms from clinical samples or surfaces that have a low quantity of material.

2.2. Microbial Collection and Seeding Process

To collect possible contaminating microorganisms, BHI broth was prepared and dispensed into 60 test tubes up to a limit of 10 mL of broth in each tube [21]. To perform the seeding, the tubes were taken to each prosthetic laboratory where the abutments, after being manipulated by the laboratory, were seeded in the selected culture medium. Initially, the supposedly contaminated abutments were completely submerged in the test tubes with the BHI broth and shaken for 1 min (vortex).

Next, the abutments were removed from the test tubes and dried with an air jet. Ten abutments from each laboratory were placed for 10 min in a sterile universal collection bottle containing 30 mL of 2% CL (RHIOEX 2%—aqueous solution—Vic Pharma^®, Taquaritinga, Brazil) [22]. The abutments, now supposedly disinfected, were inserted into another tube containing BHI broth. These procedures were repeated in the three selected laboratories.

The other 10 abutments from the same laboratory received UV-C radiation (Surface UV-C from MMO^®MMOptics, São Carlos, Brazil) for one minute at a distance of 1 cm from the abutments following the manufacturer’s recommendations. To allow UV-C action across the entire surface of the abutments, these were previously marked so that their position could be modified, and the radiation action could reach the entire abutment. These procedures were repeated in the three selected laboratories.

Thus, after the disinfection process, two groups were formed: Group 1 (G1)-2% CL—possibly infected and disinfected with 2% CL; Group 2 (G2)-UV-C—possibly infected and disinfected with UV-C radiation.

Subsequently, with the aid of a micropipette calibrated at 0.1 mL, a triplicate transfer was performed on Petri dishes containing BHI agar with the aim of seeding microorganisms and allowing the number of colonies forming units (CFUs) to be counted [23]. Using a Drigalsky spatula, the broth was spread over the plates, and these were taken to bacteriological incubators at a temperature of 37 °C for 72 h to promote the growth of colonies of microorganisms. They were identified according to the group to which they belonged.

After cultivation, the plates were removed from the greenhouse and, using a magnifying glass, the colony-forming units per milliliter (CFU/mL) counts were performed. There was no dilution of the initial sample. As a control, test tubes containing the broth without contamination were placed in bacteriological incubators at 37 °C together with the plates used to observe whether they were cloudy, which would indicate possible contamination. To identify microorganisms, the Matrix-associated laser desorption-ionization–time of flight (MALDI-TOF) test was used, which is very efficient and accurate in identifying bacteria, viruses, and other rare microorganisms [24].

2.3. Statistical Analysis

First, the normality test was performed to determine whether the sampled data set was modeled by a normal distribution or not. To compare the data from the groups before and after disinfection, the paired Wilcoxon test was used, verifying the disinfection capacity of the two groups according to the disinfectant agent. To compare contamination and decontamination in the UV-C and 2% CL Groups, the Kruskal–Wallis test was used. A significance level of 5% (p = 0.05) was adopted. In order to evaluate the presence of microorganisms on the abutments, the frequency of occurrence in the samples and the percentage of microorganisms found on the prosthesis were also presented.

3. Results

Among the 60 abutments analyzed, a contamination rate of 83% was found (Figure 1). When analyzing the disinfection percentage per group, the UV-C obtained 92% and the 2% CL obtained 93%.

In Figure 2, the incidence of contamination in the UV-C and 2% CL groups can be observed (

Table 1. Number of CFUs in the contaminated and decontaminated test specimens in the UV-C and 2% CL groups with their respective efficacy rates.

ID	Contaminated G2	Decontaminated G2	Efficacy Rate G2	Contaminated G1	Decontaminated G1	Reduction Rate G1
cp1	300	0	100.0	700	0	100
cp2	340	80	76.5	530	0	100
cp3	980	70	92.9	240	0	100
cp4	360	120	66.7	570	0	100
cp5	0	0		670	0	100
cp6	1120	0	100.0	970	0	100
cp7	120	0	100.0	280	0	100
cp8	660	0	100.0	0	0	0
cp9	160	0	100.0	140	0	100
cp10	950	0	100.0	100	0	100
cp11	110	0	100.0	1700	250	85.3
cp12	900	0	100.0	0	0	0
cp13	410	0	100.0	100	0	100
cp14	0	0		0	0	0
cp15	120	0	100.0	1200	150	87.5
cp16	1060	130	87.7	1400	0	100
cp17	870	0	100.0	7600	0	100
cp18	0	0		1110	0	100
cp19	0	0		1300	340	73.8
cp20	1830	390	78.7	950	0	100
cp21	620	0	100.0	2509	0	100
cp22	980	0	100.0	420	0	100
cp23	240	0	100.0	1350	140	89.6
cp24	0	0		0	0	0
cp25	230	0	100.0	1010	0	100
cp26	890	380	57.3	2200	900	59.1
cp27	1500	130	91.3	0	0	0
cp28	530	0	100.0	370	0	100
cp29	310	40	87.1	1210	220	81.8
cp30	430	0	100.0	350	0	100
TOTAL	16,020	1340	91.6	28,979	2000	93.0984506

). Both groups (UV-C and 2% CL groups) had five cases without contamination. The number of CFU/mL ranged from 120 to 1830 in the UV-C group, while it ranged from 100 to 7600 in the 2% CL group.

Figure 3 shows the difference between the contaminated UV-C and 2% CL groups.

The paired Wilcoxon test showed that the number of microorganisms found before decontamination with the UV-C and 2% CL was higher than the number of microorganisms found after decontamination (p < 0.001). Both results presented show that there was a significant efficacy rate when comparing the number of contaminated and decontaminated CFU/mL. Therefore, there was a reduction in the number of colonies of microorganisms that were growing in the Petri dish after decontamination, significantly with the application of UV-C rays and 2% CL. The UV-C and 2% CL groups were also comparatively analyzed in relation to the contamination and decontamination process, with no significant difference between the groups. The result demonstrates that there is no significant difference in the decontamination of the prosthetic abutments between the UV-C and 2% CL groups (Table 1). These findings highlight the importance of implementing a disinfection protocol before inserting abutments into the patient’s oral cavity, as a significant proportion of abutments received from prosthetic laboratories exhibited microbial contamination. Given the comparable efficacy of both methods, UV-C disinfection may serve as an alternative to chemical disinfection, particularly in cases where concerns regarding chemical residues exist.

The microorganisms present on the prosthetic abutments were identified and 17 different species were found, the most frequent being Enterococcus faecalis with 34.2% of occurrences, followed by Staphylococcus epidermidis with 17.8% (

Table 2. Number of microorganisms found in the prostheses sampled in the study.

Microrganisms	Frequency	Percentage
Acinetobacter iwoffii	4	5.5
Acinetobacter radioresistens	4	5.5
Aerococus viridans	2	2.7
Bacillus licheniformis	1	1.4
Candida albicans	2	2.7
Enterobacter cloacae	4	5.5
Enterococcus faecalis	25	34.2
Enterococcus sp.	1	1.4
Kocuria marina	1	1.4
Paenibacillus lactis	2	2.7
Pichia kudriavzerli	5	6.8
Pseudomonas sp.	2	2.8
Rhodotorula mucilaginosa	1	1.4
Staphylococcus aureus	4	5.5
Staphylococcus epidermidis	13	17.8
Staphylococcus succinus	1	1.4
Staphylococcus warneri	1	1.4
Total	73	100.0

).

4. Discussion

The presence of numerous species of bacteria, including some nosocomial ones, in the prosthetic abutments was demonstrated in the present study. The variability of bacteria found and the amount of contamination from laboratories are quite worrying. A contamination rate of 83% is notably high, especially considering that these components have not yet come in contact with patients. Regarding microorganisms, the most frequently detected were Enterococcus faecalis and Staphylococcus epidermidis. It is worth remembering that Badillo et al. ref. [25] found 95% of the contaminated parts, identifying cocci, bacilli and streptococci. Although the study by Badillo et al. evaluated complete prosthesis, the important issue remains that dental prosthesis laboratories do not follow disinfection protocols, generating cross-contamination.

Likewise, Cotrim, Santos, and Jorge ref. [7] found high percentages of contamination in 20 laboratories, with pumice stone being the most contaminated item affecting 100% of the laboratories. Fungi, mutans streptococci group, enterobacteria, and pseudomonas were identified. Given that many of these microorganisms were the same as those found on the prosthetic abutments evaluated in the present study, in addition to the fact that they were sterile before handling, it is likely that this contamination was due to bacteria circulating in the laboratories.

Other authors corroborate these findings demonstrating high contamination rates together with low adherence both by dentists and prosthetists to biosafety practices [7,8,9,25]. A non-disinfected impression can contaminate the entire laboratory area, allowing microorganisms to be transported to the clinical area [26,27].

Kim et al. ref. [1], when evaluating prosthetic abutments made by computer-aided design/computer-aided manufacturing (CAD/CAM), identified 40% of the samples contaminated with bacilli and staphylococci, and, among them, Staphylococcus warneri, an opportunistic pathogen that can cause bacteremia, and this bacterium was also identified in the present study.

This high percentage of contamination may be related to the possibility of prosthetics laboratories functioning as a disseminator of pathogenic agents given the absence of disinfection protocols [3,4,6,9,28,29]. Laboratories receive work from various dentists who often also neglect the importance of disinfecting impressions and models [7].

Due to the neglect of disinfection, microorganisms begin to circulate with the potential to contaminate both the team and other work carried out in the laboratories [2,6,7,8,9].

Given the close contact of the abutments with peri-implant tissues, the presence of microorganisms found in the present study may lead the host to an inflammatory response that delays healing [18]. It is known that peri-implant tissues have a lower anti-inflammatory response capacity and that their contamination, together with solid micro-waste, could make healing even more difficult [10].

Nasser et al. ref. [11], when evaluating microleakage at the implant–abutment interface, observed the growth of microorganisms regardless of the type of connection between them. Such microorganisms may have been inoculated externally when the abutments were installed on the implants. According to the authors, this may interfere with the healing process of the peri-implant tissue. Additionally, it is clinically relevant that among the microorganisms identified in the present study, some are opportunistic nosocomial pathogens with the potential to trigger serious diseases such as pneumonia and infective endocarditis, among others [30].

The great difficulty of both laboratories and dentists in adhering to disinfection protocols can lead to situations whose consequences and aftereffects can take days, weeks, or even months after infection to identify and treat possible diseases originating from these microorganisms [31,32].

The literature offers general recommendations for disinfection, primarily focusing on impressions [2,7,33]. However, few address the disinfection of work coming from laboratories and even fewer prosthetic abutments. Therefore, techniques are suggested and range from sterilization to disinfection with chemical and physical agents [34], and these must be efficient, easy to use, and low cost. Among these, the following stand out: CHX, ultraviolet radiation, glutaraldehyde, alcohol, and ultrasonic tanks.

In the present study, 2% CL and UV-C radiation were used, and it was possible to evaluate the efficacy of the high number of bacterial colonies eliminated after the use of these two agents. These results are in line with the findings of other authors [29,35,36,37,38,39].

It is important to emphasize that the microbiological universe is broad and that some microorganisms may not have been detected. The presence of viruses, for example, is not always easy to identify, and there is a possibility that they are circulating in prosthetic laboratories and could be transmitted to the work team and other patients [40].

Based on the results obtained, most of the microorganisms cultivated in the Petri dishes were successfully identified, demonstrating the disinfection efficacy of these agents in eliminating nearly all identified microorganisms—a finding consistent with other studies [14,15,16,17,22].

Given that the culture medium was not selective, meaning that several species could grow, the MALDI-TOF identification method was chosen. This method allows the identification of different species based on their molecular mass and has a high degree of precision [24,40].

Among the limitations of the present study are the challenges in isolating all microorganisms present—since some may not have been detected by the culture media used—and the inability to identify microorganisms resistant to disinfection, which can survive in adverse conditions; these factors may lead to an underestimation of the true level of contamination in prosthetic laboratories and an incomplete assessment of disinfection efficacy, potentially overlooking opportunistic pathogens that could contribute to cross-contamination and compromise patient health, thus emphasizing the urgent need for enhanced and comprehensive disinfection protocols. Despite these limitations, the study reinforces evidence from the scientific literature and calls for increased awareness among all dental professionals—from laboratory technicians to dental surgeons—regarding the critical importance of biosafety practices in preventing cross-contamination and ensuring improved clinical outcomes.

Future studies utilizing a larger and more diverse sample of laboratories would provide enhanced statistical power.

Awareness can prevent cross-contamination, protecting patient health and improving practices in dental offices.

5. Conclusions

While the study confirms that prosthetic abutments in crown production laboratories exhibit high contamination rates and that both UV-C radiation and 2% CL digluconate are effective disinfection methods with comparable efficacy, these findings have broader implications. They underscore the urgent need for rigorous, standardized disinfection protocols in dental laboratories to prevent cross-contamination and enhance patient safety. The predominance of Enterococcus faecalis and Staphylococcus epidermidis suggests that these bacteria could pose significant risks if not adequately controlled. Future research should investigate the long-term clinical outcomes of these disinfection strategies, assess the potential emergence of disinfection-resistant strains, and explore the scalability of these protocols across a larger and more diverse set of laboratories.