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

Abstract

Slackia exigua (SE) is a recently identified intestinal microbe, which recent oral surveys suggest may be associated with oral diseases including caries and periodontal disease. Based upon the lack of information regarding this organism, the primary objective of this study was to determine the oral prevalence of this microbe and any potential associations with patient characteristics such as age, sex, or the presence of orthodontic appliances. This retrospective study involved the screening of an existing saliva repository composed of previously collected unstimulated clinical saliva samples. More specifically, N = 266 were identified and screened using a spectrophotometer at absorbances of A260 and A280 nm to determine their DNA purity and concentration. qPCR screening of these samples revealed a higher prevalence of Slackia exigua positive samples among pediatric patients (63.1%) compared with adults (36.9%) in this clinic population, p = 0.0007. In addition, higher percentages of Slackia exigua were observed among orthodontic patients (71.2%) compared with non-orthodontic patients (28.8%), p = 0.0001. These results did not vary by sex with nearly equal percentages of Slackia exigua positive males and females among adult and pediatric patients, as well as orthodontic and non-orthodontic samples. These results suggest a strong potential for association between the prevalence of this organism with age as well as orthodontic status, given that younger patients and those with orthodontic brackets (regardless of age) were most likely to harbor this pathogen in sufficient levels to be detected in saliva. More research will be needed to determine any associations with specific outcomes, such as caries or periodontal disease, among Slackia exigua positive patients within these specific populations.

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“…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).…”

“…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 ].…”

“…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): Reverse primer SN-R1 5 -GCC TGC AAT CCG AAC TGA GA-3…”

Slackia exigua, an emerging anaerobic pathogen – isolation from a case of polymicrobial peritonitis and review of literature

“…Additionally, they demonstrated a significant correlation between the prevalence of this organism and orthodontic brackets. They are most closely associated with children who receive orthodontic care [17] .…”