Oral Prevalence of Akkermansia muciniphila Differs among Pediatric and Adult Orthodontic and Non-Orthodontic Patients

Abstract

Akkermansia muciniphila (AM) is one of many highly abundant intestinal microbes that influences homeostasis and metabolic disorders and may also play a role in oral disorders. However, there is little evidence regarding the oral prevalence of this organism. Based upon this lack of evidence, the primary goal of this project is to survey an existing saliva repository to determine the overall prevalence of this organism and any associations with demographic or patient characteristics (age, sex, body mass index, race/ethnicity, orthodontic therapy). Using an approved protocol,, a total n = 141 pediatric samples from an existing saliva repository were screened using qPCR revealing 29.8% harbored AM with nearly equal distribution among males and females, p = 0.8347. Significantly higher percentages of pediatric, non-orthodontic patients were positive for AM (42.3%) compared with age-matched orthodontic patients (14.3%)—which were equally distributed among non-orthodontic males (42.1%) and non-orthodontic females (42.5%). In addition, analysis of the adult samples revealed that nearly equal percentages of males (18.2%) and females (16.7%) harbored detectable levels of salivary AM, p = 0.2035. However, a higher proportion of non-orthodontic adult samples harbored AM (21.3%) compared to orthodontic samples (12.8%, p = 0.0001), which was equally distributed among males and females. These results suggest that both age and the presence of orthodontic brackets may influence microbial composition and, more specifically, are associated with reduction in AM among both pediatric and adult populations from their baseline levels.

Administration of Clinical COVID-19 Mouthwashing Protocol and Potential Modulation of Pediatric Oral Bacterial Prevalence of Selenomonas noxia: A Pilot Study

“…To facilitate the molecular screening, saliva samples were first thawed and processed to isolate the DNA using the TRIzol reagent from Invitrogen (Waltham, MA, USA), as previously described in [ 26 , 27 ]. In brief, samples were thawed at room temperature and then vortexed.…”

“…To this mixture, 200 µL of molecular biology grade Chloroform from Fisher Scientific (Waltham, MA, USA) was added and incubated on ice for ten minutes prior to centrifugation at 12,000× g relative centrifugal force (RCF) at 4 °C in a refrigerated microcentrifuge from Eppendorf—Model 5425 (Hamburg, Germany). Following centrifugation and separation, the upper aqueous phase (approximately 300 to 400 µL) was transferred to a new sterile microcentrifuge tube and an equal volume of molecular biology grade Isopropanol from Fisher Scientific (Waltham, MA, USA) was added to precipitate the DNA from each sample [ 26 , 27 ]. All samples were then centrifuged again for ten minutes at 12,000× g RCF at 4 °C in a refrigerated microcentrifuge from Eppendorf—Model 5425 (Hamburg, Germany).…”

“…In brief, sample DNA was screened using the Fast SYBR green master mix from Fisher Scientific (Waltham, MA, USA) involving 12.5 µL of 2X SYBR green master mix, 6.0 µL of nuclease-free water from Fisher Scientific (Waltham, MA, USA), 1.75 µL of forward and reverse primer diluted to a standard concentration of 10 µM, and 2.0 µL of sample DNA. Cycle settings were derived from the manufacturer protocol, involving a 20 s activation set at 95 °C followed by 40 cycles of denaturation at 95 °C for five seconds and extension at 60 °C for thirty seconds, as previously described [ 25 , 26 , 27 ]. Validated primers synthesized by Eurofins Scientific (Louisville, KY, USA) included ( Table 1 ):…”

“…Finally, for the purpose of calculating the sample size minimum that would be appropriate for this type of salivary-based qPCR microbial screening, the DNA extraction recovery rate (sample-limiting step) of 90–95% was used to determine the maximum expected experimental difference of 10% or 0.10. Using this information with a Power (p) of 0.90 and a significance level of alpha = 0.95, the minimum total sample size of N = 50 was calculated for this study [ 26 , 27 ].…”

Prevalence of Oral Pathogen Slackia exigua among Clinical Orthodontic and Non-Orthodontic Saliva Samples

“…Other studies from this group have analyzed the changes in oral microbial prevalence and changes to specific microbial populations, including Scardovia wiggsiae, Selenomonas noxia, Streptococcus mutans, and Porphyomonas gingivalis, which may be elevated among orthodontic patient populations in particular [23][24][25]. In addition, another study from this group recently found that the commensal bacterium Akkermansia muciniphila (which also exhibits anti-inflammatory properties) is decreased among adults versus pediatric patients, as well as among orthodontic versus non-orthodontic patients regardless of age within this clinic population [26]. In contrast, the results from the current study appear to suggest that the oral prevalence of Slackia exigua may be higher among orthodontic patients than non-orthodontic patients, which holds true regardless of age.…”