To the authors’ knowledge, there have been no previous characterizations of an equine blood microbiome, either in health or disease. The present study is the first to provide information about the equine blood microbiome in adult horses before and after exodontia. 16S rRNA gene profiling has consistently yielded greater microbial diversity in samples with an anticipated low microbial biomass (such as amniotic fluid and blood) than appreciated based on culture-dependent methods [4]. We adopted an approach that had been successfully employed to improve 16S rRNA sequencing in several types of samples, including murine blood [37]. The method entailed increasing the PCR cycle number during library preparation from 25 to 40 cycles and was highly effective in the present study, yielding detection of many ASVs in blood of horses both before and after exodontia. While the requisite reagent controls yielded greater coverage than many of the blood samples, the marked increases observed in a subset of post-exodontia samples, along with the compositional similarity to oral microbiota in those same samples, demonstrate the utility of increased cycle number for similar low microbial biomass samples.
Post-exodontia bacteremic showering has been well documented in non-equine species and is associated with various potential health complications [4–6, 8, 10, 13, 15, 17, 31, 38, 39]. Results of earlier studies have varied based on the specific surgical treatment undertaken, method used for bacterial identification, immune system responsiveness, and whether antimicrobial drugs were present in sampled blood at the time of collection [5]. In most instances, distant site bacterial infections (such as bacterial endocarditis) were attributed to bacteria originating from the oral microbiome (including Streptococcus mitis and Streptococcus oralis in the human medical context) [36]. Although there have been few reports of distant site infections associated with post-exodontia bacterial showering in horses, implicated pathogenic bacteria were also likely derived from oral cavity microbiota [25, 26]. Results of the present study show that the 16S rRNA signatures of bacteria present at the gingiva in proximity to an extracted (diseased) tooth are similar to those detected in the blood following exodontia. Moreover, the results of the present study indicate that bacteremic showering by oral commensal bacteria is still evident one hour following exodontia.
Whereas the human oral microbiome (reportedly the most extensively studied human microflora) has been extensively characterized [36, 40], only a few descriptions of the equine oral microbiome have been published [21, 33, 41, 42]. Approximately 600 prevalent bacterial species have been identified in the human oral cavity based on bacteriological culturing [36]. However, using culture-independent 16S rRNA gene clonal analyses, a majority of bacterial species present in the oral cavity are uncultivable [40, 43, 44]. Earlier investigations of the equine oral cavity microbiota using bacteriological culturing methods showed that Gram positive cocci (mainly Streptococci, Micrococci, and starch hydrolysers) represent prevalent colonizers in healthy horses [33, 45, 46]. Both Gemella spp. and Actinobacillus spp. are also frequently associated with periodontal health in horses [31, 33, 42]. Corynebacterium spp. and Moraxella spp. have also been identified in the oral cavity of healthy horses [33]. In another study, Actinobacillus spp. and an unclassified Pasteurellaceae sp. were the most abundant taxa present in healthy subgingival plaque samples from horses [41]. In that study, Gammaproteobacteria, Firmicutes, and Bacteroidetes (with Treponema, Tannerella, and Porphyromonas species detected at low levels) represented the predominant bacterial phyla identified in the healthy equine subgingival microbiome [21, 41].
16S rRNA gene sequencing was used to show that periodontitis is associated with disruption of the oral cavity microbiota (dysbiosis) in horses [21]. Whereas bacteria in the healthy oral cavity included Prevotella spp., Veillonella spp., Gemella spp., and Actinobacillus spp., both Tannerella and Treponema genera were significantly increased when periodontitis was identified [21]. 16S rRNA PCR was also used to show that acidogenic and aciduric bacteria, including Streptococcus species, are associated with peripheral caries in horses, as has been reported in other species [32]. Novel red complex bacteria, Treponema and Tannerella species, were also identified through their DNA signatures from the gingiva of EOTRH-affected horses [42]. In another study, 18 of 20 horses developed positive blood cultures following exodontia and, in some of those horses, gingival elevation alone resulted in bacteremia [31]. The most commonly identified bacteria on blood culture in that study were Streptococcus spp., Actinomyces spp., Fusobacterium spp., and Prevotella spp.; bacterial genera isolated from swab samples of extracted teeth were similar to those detected in the blood, emphasizing that bacteremia resulted from translocation of oral cavity bacteria [31]. However, it should also be noted that results of bacteriological culturing underestimate the extent of bacteremic showering because most bacteria are uncultivable [34].
Collectively, these studies demonstrate commonalities in oral microbiota composition between diverse species (human, canine, and feline) and that the equine oral microbiome appears to be broadly similar at the taxonomic level of genus and higher [21, 41]. Consistent with previous publications, predominant genera that were identified in the oral cavity of horses in the present study included Actinobacillus, Fusobacterium, Leptotrichia, Porphyromonas, Prevotella, Streptococcus, and Veillonella. Moreover, these same taxa were identified in post-exodontia blood samples the five blood samples that yielded unexpectedly deep coverage (prolonged bacterial DNA presence). Four of those horses were also affected with sinusitis, suggesting that post-exodontia bacteremia may be more significant when exodontia is undertaken in horses with comorbid sinusitis.
The use of 16S rRNA gene cloning and sequencing methods has led to the discovery that many diverse bacterial phyla that were previously unrecognized or considered unimportant do play a significant role in some diseases [35]. It is becoming increasingly evident that uncultivable commensal bacteria from the oral cavity microbiome are important in the pathogenesis of post-exodontia complications in people following dental surgery [10, 34, 38, 40, 44]. Although 16S rRNA gene cloning and sequencing methods do not differentiate living bacteria from residual bacterial nucleic acid, even residual microbial DNA (in the absence of viable bacterial cells) can serve as an inflammatory signal via innate immune mechanisms including various Toll-like receptors [47]. In light of the fact that a majority of identified bacteria are uncultivable, it is not possible to conclude which, if any, of the identified bacteria are playing a clinically important role in the pathogenesis of exodontia-associated disease based on 16S rRNA signatures [34].
It has long been recognized that bacteremic showering resulting from either dental infection or dental surgery can lead to distant infection (such as bacterial endocarditis), especially in immunocompromised individuals [6, 14, 22–24, 30]. Disruption of the gingival-blood barrier as a result of disease or surgical intervention potentially facilitates translocation of bacteria and bacterial products into the circulation, potentially leading to systemic diseases [1]. Moreover, there is emerging realization that uncultivable anaerobic commensal bacteria from the oral cavity might, given access to the circulation, play a role in the pathogenesis of a remarkable and diverse inventory of extra-oral diseases. Various (human) diseases that have been attributed to this phenomenon include diabetes mellitus, respiratory disease, cardiovascular disease, and atheroma [3, 48, 49]. Of special interest in this regard is Fusobacterium nucleatum, which has been associated with dental disease, various adverse pregnancy outcomes (chorioamnionitis, preterm birth, stillbirth, neonatal sepsis, and preeclampsia), neoplastic and inflammatory gastrointestinal diseases, and various other infections in human patients [3]. Although it remains to be seen whether uncultivable oral cavity commensals might contribute to systemic disease in a hitherto unrecognized manner in horses, the fact that periodontal disease is very common in aging horses and that Fusobacteria were prominently identified in post-exodontia blood in the present study suggests that parallel equine studies should be undertaken [50].
The extent to which post-procedural bacteremic showering persists has not been extensively reported. In one (human) investigation it was reported that viridans group streptococci were rapidly (within 10 minutes) eliminated from 42 of 46 patients undergoing various oral surgical procedures [5]. In one equine study, two blood samples yielded positive cultures following exodontia (samples obtained 10 minutes after the termination of surgery), providing evidence for short term persistence of bacteremia [31]. Those authors speculated that persistence of bacteremic showering could have resulted from a greater number of bacteria (quantitative bacterial counts were not performed) or a result of immune function variations between individual horses (two horses in that study were bacteremic prior to the surgical procedure) [31]. Results of earlier work in other species suggests that intravascular bacteria are rapidly cleared from the circulation by the reticuloendothelial system (within 10–20 minutes) [51]. Our results show that significant post-exodontia bacteremia is still evident at 60 minutes following conclusion of surgery in some horses. The immune status of the horses in this study was not examined, but future investigations could incorporate an evaluation of the immune system for horses receiving exodontia surgery. Further studies might also evaluate additional time points beyond one hour for evidence of longer-persisting bacteremia.
The use of prophylactic antimicrobials peri-operatively is restricted to more invasive dental procedures in human dentistry, especially for individuals affected with immunocompromising comorbidities or those with orthopedic implants [52]. Antimicrobials are used under the assumption that they do not prevent bacteremia but inhibit bacterial propagation and bacterial adherence to tissues/implants [52]. Specific guidelines for antimicrobial use in horses receiving exodontia have not been published. Results of the present study showing marked post-exodontia bacteremia persisting for at least one hour suggest that antimicrobial use might be important in this setting, especially for immunocompromised horses.
Using only a solitary time point for blood sampling post-exodontia (one hour post-operatively) was a limitation of this study and the results imply significant post-procedural bacteremia may persist beyond this timeframe and is deserving of further investigation. Although time expended with each exodontia was not measured, it is reasonable to assume that difficult extractions requiring more time could be associated with increased post-procedural bacteremic showering when compared with more expeditiously concluded procedures. Other limitations include the limited number of cases and the lack of age-matched controls. Blood microbiome results do not necessarily reflect a normal population as all recruited horses were affected with dental disease necessitating exodontia and pre-exodontia blood microbiomes may have been influenced by the presence of dental infection. It should be emphasized that 16S rRNA gene sequencing results are relative, meaning that the actual quantity of bacteria in a given sample is uncertain [53]. It is also possible that each 16S rRNA gene may not amplify with equal efficiency during PCR reactions due to differential primer affinity and GC content and taxonomy assignment is conditional upon the completeness of reference databases [53].
The results of this study affirm that bacteremic showering by oral cavity commensals occurs in horses following dental extraction. Additionally, post-exodontia bacteremia is still evident in some individuals for up to one-hour, which is much longer than had been previously documented. These results include the first extensive documentation of a blood microbiome based on 16S rRNA gene sequencing in adult horses. The extent of post-exodontia bacteremic showering, especially as pertains to uncultivable commensal bacteria and their propensity to contribute to extra-oral disease, is deserving of further investigation in horses.