Microbiological evaluation of LOCATOR® Legacy attachments: A cross‐sectional clinical study

Abstract Objective This retrospective cross‐sectional study aimed to evaluate quantitatively the oral microbiome in the tri‐lobe central cavity of Locator Legacy attachment and verify whether it harbors a different, potentially more pathogenic, bacterial spectrum than the adjacent edentulous ridge. Materials and Methods Edentulous patients rehabilitated with implant overdentures using Locator Legacy attachments were recruited for this study. The clinical examination comprised probing depths, mobility, peri‐implant, and periodontal health along with intraoral swabs for microbiological evaluation, polymerase chain reaction (PCR) testing, and candida culture. The swabs were collected from the trilobed cavity of the attachment and the adjacent edentulous ridge. PCR was performed to detect six specific bacteria, Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Tannerella forsythia, Treponema denticola, Prevotella intermedia, and Parvimonas micra. Statistical analyses were performed using McNemar's test and Wilcoxon's rank sum test with the significance set to p < .05. Results A total of 50 participants with a mean age of 71.5 ± 9.6 years participated in the study. No significant differences in the microbiome were found between samples from the ridge and the attachment. No significantly different numbers in the candida cultures were identified, and the presence of a removable prostheses did not demonstrate a significant association with the prevalence of candida. Conclusions Within the limits of this study and the investigated bacterial species, the trilobed cavity of the attachment does not seem to increase the bacterial load.

Most importantly, the LOCATOR® Legacy has been criticized because of its "nuisance factor" (Mackie et al., 2011). The trilobe central cavity present on the attachment head is a frequent site of debris accumulation, when left unremoved, and is a cause of great inconvenience to the elderly patient with compromised vision and manual dexterity. This debris accumulation in the central cavity may impede the insertion of the prosthesis and, especially when unilateral, may cause denture fracture. In addition, such noninsertion may severely impair the oral health-related quality of life of the patient. Moreover, accumulated debris and biofilm are a potential nidus for propagating oral or more distant general infections. This is of particular importance in the compromised elderly patient who is dependent for care as it could lead to potential complications such as aspiration pneumonia, especially when swallowing disorders are present (Daly et al., 2018;Iinuma et al., 2015;Müller, 2015;Pritchard, Crean, Olsen, & Singhrao, 2017;Yoneyama, Yoshida, Matsui, & Sasaki, 1999). Therefore, the aim of this cross-sectional study was to evaluate quantitatively the oral microbiome in the trilobe central cavity of the LOCATOR® Legacy attachment in order to verify if the central cavity harbors a different, potentially more pathogenic, bacterial spectrum than does the adjacent edentulous ridge. A secondary aim was to evaluate an association of the presence of Candida with the presence of removable prostheses. Therefore, the null hypotheses set for this study is that there is no differen ce in the quantity of the microflora present in the central cavity of the LOCATOR® Legacy attachment and the edentulous ridge and, that the presence of Candida is not related to the presence of a removable prostheses.

| MATERIALS AND METHODS
The study protocol was approved by the appropriate ethical committees in Geneva, Switzerland (CER No. 14-046

| Study design
The study was designed as a retrospective, single-center, crosssectional clinical study on human subjects.

| Study setting
The study was conducted in the removable prosthodontics clinics in a university-setting dental school. The participants were recruited and examined between May 2014 and November 2014. The participants were recruited according to the following inclusion criteria: • if they were treated at the university dental clinic and received either a removable partial or complete implant-retained overdenture using LOCATOR® attachments and the prostheses were present in situ for 12 months or longer; • if they were restored with microrough surface implants, which were loaded following a conventional loading protocol; and • if they were living independently.
The participants were excluded if they • presented with a history of repeated, unjustifiable missed appointments; • were unable to attend the appointment for health reasons or other causes; • presented with uncontrolled diabetes; • presented with a history or with a current oncological condition in the head and neck region; and • were not willing to participate and/or sign an informed consent.
All patients were clinically examined by two investigators (U. N. and C. G.). For each patient, the peri-implant health was evaluated by 6-point probing depth measurements using a standard periodontal probe, mobility (Miller, McEntire, Marlow, & Gellin, 2014), modified bleeding and plaque indices (Mombelli, van Oosten, Schurch, & Land, 1987). These were recorded for the natural teeth (if present) and for the implants with the LOCATOR® attachments.

| Polymerase chain reaction
A sterilized paper strip was dipped in the central cavity of the male part of the LOCATOR® abutment and subsequently enclosed in a sterile plastic 0.5-mL tube. A second sterilized paper strip was wiped on the adjacent edentulous ridge and was deposited in a second plastic 0.5-mL tube. Both tubes were then taken to the microbiology laboratory for analysis. Genomic DNA was extracted using the GenElute amplificates, 20 μL of PCR products was analyzed by gel electrophoresis on 0.8% standard agarose gels using 1DNA digested with HindIII as molecular mass standards. The bacterial species present were categorized as "absent," "limited presence," and "strong presence."

| Candidiasis test
A cotton swab designed to collect samples from the oral mucosa was rubbed on the tongue and on the insides of the cheek of the participant. The smear was applied to a chromogenic medium for the selective isolation of yeasts and the direct identification of Candida albicans. The different types of colonies were identified after incubation (48 hr at room temperature, without direct light), and the presence of C. albicans was quantified by means of an agar-type chromID™ Candida (CAN2) by bioMérieux (France).

| Study protocol
Patients were recruited from the university clinics of dental medicine.
An electronic search of the dental school's patient management software using the key word "LOCATOR®" was used to formulate an initial screening list of prospective patients. The selected patients were then sent a letter of invitation requesting them to participate in a clinical study. Following the letter, they were then contacted by telephone 10-14 days later to answer potential questions on the informed consent and, if they agreed, subsequently fix an appointment for consultation. After an initial screening, the willing participants signed the informed consent. As a first step, the patient's history and the relevant personal information were collected by the two investigators (U. N. and C. G.). The participants were then clinically examined, and all the information was duly recorded in the clinical record form.
The examination began with the candidiasis test, before removing the patients' partial or total prostheses. After removing the prostheses, swabs of the edentulous ridge and the LOCATOR® were made for the PCR analyses. The assessment of the periodontal and periimplant health parameters were then recorded. No treatment was performed in this study. If a treatment need was identified during the clinical examination, the study participants were informed and were then subsequently referred to their dentist or to a specialist clinician for the appropriate care.

| Statistical analysis
Potential quantitative differences between the microbiome of both locations (i.e., the edentulous ridge and the LOCATOR® Legacy attachment) were first verified for each bacterium using contingency tables. The association of the amount of bacteria present on the two

| RESULTS
A total of 50 patients (27 women, 23 men) with a mean age of 71.5 ± 9.6 years met the inclusion criteria and participated in the study. The detailed participant demographics has been described elsewhere (Guedat, Nagy, Schimmel, Muller, & Srinivasan, 2018). The details of the participant screening and recruitment process are shown in Figure 1. The incidence (number and frequency) of the studied bacteria on the ridge and on the attachment are listed in Table 2. No different frequencies were found between samples from the ridge and the LOCATOR®. All samples tested negative for Aa; therefore, this bacterium was not considered for the further analyses.
The amounts of Tf, Pi, Td, and Pm were similar in the samples from the ridge and from the LOCATOR® (Table 3) (Table 4; p = .20).
Patient characteristics, such as age and sex, were similar across these three patterns, on the ridge and on the LOCATOR®. The presence of a removable prostheses was not associated with a higher prevalence of Candida.

| DISCUSSION
The primary aim of the study was to compare detection frequencies and levels of six pathogenic microorganisms in the trilobe central cavity of the LOCATOR® Legacy attachment and the adjacent edentulous ridge. With the exception of one bacterium (Aa was absent in all samples), our results showed that both the ridge and the LOCA-TOR® were similarly colonized by the studied periodontal pathogens.

The most commonly detected bacteria in both locations were Tf and
Pg. To the best of our knowledge, this is the first study to compare the microflora on LOCATOR® attachments of elderly subjects wearing removable partial or complete implant-retained overdentures.
Several studies have shown that after complete loss of teeth, some of the above-mentioned target species still remain in the oral cavity Fernandes et al., 2010).
Therefore, not only teeth but also the oral soft tissues could act as important reservoirs of bacteria. Andjelkovic et al. (2017) aimed to compare the composition of oral microflora before and after rehabilitation by studying the changes in the prevalence of six common periodontal pathogens in elderly edentulous patients wearing complete dentures (Andjelkovic et al., 2017). Not only were the pathogens present before inserting the dentures, but their prevalence increased considerably during the 6 months that the dentures were worn. At the same time point, co-associations between bacteria were observed. It is important to emphasize that these bacteria were present in high amounts despite adequate oral hygiene and proper storage of the dentures.
C. albicans by its capability to adhere to mucosal surfaces has been shown to contribute to the pathogenesis of oral candidiasis (McIntyre, 2001). In our study, the high prevalence of C. albicans was not associated with the presence of a removable prosthesis. The study of Kilic et al. (2014) aimed to elucidate the difference between LOCATOR®and bar-retained overdentures in the prevalence of denture-related stomatitis and the colonization by Candida species (Kilic et al., 2014).
The authors reported higher colony forming unit values of Candida species in the bar-retained overdentures as compared to those retained by LOCATOR®. Furthermore, the presence of gingival inflammation and plaque increased the prevalence of denture-related stomatitis, emphasizing the importance for regular denture-and attachment-surface hygiene. In the same study, the authors observed that C. albicans was the most common species in both bar-retained and LOCATOR®-retained overdentures (81.3% vs. 38.1%), followed by Candida glabrata (37.5% vs. 23.8%, respectively).
With the increasing numbers of the old and very old patients receiving implant treatment, hygienic aspects of implant design become more important. Physiological aging includes impaired vision and tactile sensitivity, indicating a lower ability to notice biofilm on natural teeth, dental prostheses, and implant attachments (Boss & Seegmiller, 1981;Janssens, Pache, & Nicod, 1999;Weinstein & Anderson, 2010). Age-related impairment of manual dexterity precludes further, a meticulous removal of the biofilm that forms with time on any hard object in the oral cavity. Consequently, elderly patients often present with poor oral hygiene and a substantial bacterial load in the oral cavity (Andersson, Renvert, Sjogren, & Zimmerman, 2017;Pritchard et al., 2017). In younger persons, the morphology of a natural dentition is "self-cleaning," as the interproximal spaces are filled with gingival papillae and the gingival margin is located near the cemento-enamel junction. Young persons also rub the oral cavity clean during a meal by using the tongue and the cheeks. This muscle activity helps also in repositioning the food bolus on the oral cavity and/or pushing the food stuffs onto the tongue for a better taste sensation. With age, the forceful chewing and rubbing of the tongue and cheeks diminish substantially, as muscles atrophy and weaken with age and motor coordination becomes more erratic (Campbell, McComas, & Petito, 1973;Newton, Abel, Robertson, & Yemm, 1987;Newton, McManus, & Menhenick, 2004;Newton & Yemm, 1986;Newton, Yemm, Abel, & Menhinick, 1993;Roberts et al., 2016). These age-related changes explain the abundant presence of biofilm in the elderly persons' mouths and dentitions. Age-adequate dental restorations need to consider these age-related functional impairments and require a design, which facilitates the "self-cleaning." The central cavity in the LOCATOR® attachment is a functional necessity, as it allows insertion, tightening, and removal of the  attachment with the corresponding instrument. The nylon insert of the LOCATOR® does not fully engage into this central cavity, leaving some space, notably the circular undercut "empty." This volume presents a warm (37°), humid, and dark environment, which intuitively seems a favorable environment for bacterial growth. Clinical experience confirms that in nearly all LOCATOR® attachments a white biofilm is present, as the shape of the small cavity, with its circular undercut, is difficult to clean for the denture wearer. Hence, the aim of this study was to verify if the central cavity presents a particular risk for the accumulation of a-potentially more pathogenic-biofilm deposit, when compared to the adjacent edentulous ridge, where biofilm would be more easily cleared away by the action of the tongue and the saliva, where it could be more easily removed by a regular tooth brush. Bacterial load from the oral cavity, be it on the natural dentition or the dental prostheses, or dental implants, or even tongue coating, presents a risk of developing aspiration pneumonia (Abe, Ishihara, Adachi, & Okuda, 2008;Awano et al., 2008;Kageyama et al., 2018). The evinced risk factors include pocket depths of more than 5 mm, poor oral hygiene, nocturnal denture wearing, and the presence of swallowing disorders. The swallowing reflex requires a complex coordination of various motor patterns to assure a smooth transition from one phase of deglutition to the next. With motor coordination being affected by the aging process, the swallowing reflex more often "trips over" in the elderly, leading to the aspiration of saliva and food stuffs (Schmidt, Holas, Halvorson, & Reding, 1994). The prevalence of swallowing disorders increases from 6-9% in the adult population to 15-22% in persons aged 50 years or older and reaches 40-60% in institutionalized elders (Aslam & Vaezi, 2013).
Pneumonia is one of the major threats for the aged population with an estimated incidence of 33 to 114 cases for 1000 population per year for persons living in institutions (Janssens & Krause, 2004).
Pneumonia is the leading cause of all infections in nursing homes and the leading cause of death from infection in patients aged 65 years and older (El-Solh, 2011a;El-Solh, 2011b). Bacteria from the oral cavity, corresponding to the periodontal microbial flora, was identified from the bronchoalveolar sputum retrieved from the broncholavage in hospitalized elderly pneumonia patients (Imsand, Janssens, Auckenthaler, Mojon, & Budtz-Jorgensen, 2002;Quagliarello et al., 2005), confirming the contribution of the oral microbiome. Further evidence for a causal contribution of the periodontal bacteria arises from randomized controlled trials indicating a reduced incidence of pneumonia if weekly oral hygiene is practiced by dental personnel, such as hygienists or dentists (Andersson et al., 2017;Sjogren, Nilsson, Forsell, Johansson, & Hoogstraate, 2008;Sjogren, Wardh, Zimmerman, Almstahl, & Wikstrom, 2016). Even taking a removable prosthesis out during the night might reduce the microbiological burden and showed consequently a reduced risk for developing pneumonia, when compared with habitual nocturnal denture wearing (Iinuma et al., 2015).
Given the above-mentioned evidence on a frequently poor oral hygiene and its potential impact on an elderly person's well-being, it seems particularly important to verify if dental restorations, which are integrated into the oral cavity and coated with oral biofilm shortly after insertion, will not introduce a novel risk for bacterial load. The results from this present study confirm that the central cavity on the LOCATOR® attachment does not lead to a different bacterial spectrum quantitatively. Hence, using the LOCATOR® attachment does not present a risk for a changed/increased oral bacterial flora. However, the issue with the mechanical obstruction of the trilobe cavity with oral debris still remains. This might impede prosthesis insertion.
The problem can be prevented by filling the central cavity with a provisional composite restoration that can be easily removed on demand.
However, these fillings might also harbor microorganisms as they are not definitively bonded and hence do not provide a perfect marginal seal to prevent percolation of oral fluids and bacteria. Therefore, a design change would be a valid approach to reduce or eliminate this trilobed cavity to prevent complications.
Although this study has been conducted with sound methodology adhering to strict guidelines, certain weaknesses do exist.

| CONCLUSION
The results of the study confirm that the trilobed cavity present on the LOCATOR® Legacy attachment head does not seem to be introducing a novel bacterial spectrum or an increased bacterial load. However, this conclusion cannot be extrapolated beyond the investigated six bacterial species.