To date, the paucity of investigation into the role of oral microbes in the pathogenesis of BRONJ has hindered accurate disease characterization, treatment, and prognostication. Furthermore, research on the infectious aspects of jaw diseases in general has focused on the internal development and the pathogenicity of dental biofilms, and relatively little attention has been given to the source of the biofilm microorganisms, such as saliva.28
Oral bacteria exist in a planktonic state in saliva before colonizing oral and dental surfaces as biofilms, such as exposed bone and soft tissue surfaces in patients with BRONJ. Therefore, a better understanding of salivary pathogens and saliva-derived biofilms could lead to new approaches for diagnosing and managing oral infectious diseases.28
Human viruses are also frequent inhabitants of the mouth, yet until now no information existed on the presence or the role that viruses may play in BRONJ despite the fact that viruses are known to heavily influence gene transfer among microbes, virulence, and the population pathobiology of microbial biofilms.
In this preliminary study we found that individuals with BRONJ harbored different microbial assemblages than nonaffected patients and that ambient viral abundance increased substantially with biofilm formation. This trend is often observed in other natural environments.29
The finding of Proteobacteria, Firmicutes, and Actinobacteria being the dominant phylotypes found in patients is consistent with recent in vivo molecular findings of affected bone and soft tissue specimens in BRONJ patients.30,31
This suggests that saliva may be one source for bone colonization and infection in patients with BRONJ in addition to other possible sources such as odontogenic or periradicular infections.32
Further, planktonic salivary diversity may be influenced by the microbial niche of the dentition. We found that BRONJ patients had fewer teeth on average and some were edentate compared with the control group. Because patients with teeth have a different microbial flora than edentulous individuals, this could influence microbial findings. However, the small size of the patient population, the nature, and the scope of the present study does not allow for any statistical analysis or meaningful conclusions in this respect.
Our results indicate that the majority of microbes in affected patients are facultative anaerobes, making them ideal organisms for surviving in oxygen-depleted areas of necrotic bone that lack adequate blood supply, which is a hallmark of BRONJ. These findings provide insight into targeted antimicrobial therapy and indicate that qualitative rather than quantitative changes in community composition are most related to disease or disease-free status, and that fundamental changes in microbial composition, community subsets, and viral assemblages occur when host cells shift from a primarily planktonic (saliva) to a surface (biofilm) lifestyle.
A possible limitation of our study is the number of 16S reads that were obtained, averaging ~3,000 per sample. As a reference, the National Institutes of Health’s Human Genome Project has been generating similar reads per samples from various human micro-biome samples, including the oral cavity, and investigators have shown that 1,000 sequences per sample is sufficient to address the taxonomy question.33
Additionally, metagenomic identification of bacteria in general determines the presence of specific DNA but not viability; because bacterial DNA can remain PCR-detectable for up to 1 year after cell death, metagenomics may overestimate current bacterial load.34
Further, a single microbiologic examination can not determine if specific bacteria are permanent or transient residents or are primary pathogens or merely bystanders to disease. Laboratory contamination or contamination by nonhuman bacteria is also a potential problem in studies such as this one, which we sought to minimize by our methodology. Our results of the types of bacteria present suggest low contamination because they are consistent with recent reports of identified oral species in a similar context.30,31
However, the lack of detection of any obligate anaerobes in our study, in contrast to those earlier studies, is a limitation and is likely due to the nature of our study, which involved analysis of salivary pathogens and not BRONJ lesions directly. Finally, our results showing differences in microbial and viral abundance or morphotypes between samples or groups may be due to inherent differences in adherence or biofilm formation potential of organisms. Such differences should not be considered to be solely due to other covariates such as disease state.
Metagenomic approaches applied in this study allowed us to identify clinically relevant microbes in patients with BRONJ. Microbial cultures in patients with BRONJ traditionally have not been helpful in directing therapy, because specific pathogens have not been identified and most of the associated biofilm organisms are noncultivable35
—thus the need for metagenomic approaches. Importantly, viruses had not been identified or described in this condition before the present study. Their presence may account for some of the virulence and pathobiology of biofilm microbes. The presence of viruses existing as prophage could also account for some of the antibiotic resistance that has been reported in BRONJ-related microbes.31
Collectively, this translational research has overcome critical barriers to progress in this field and informed our understanding of the pathogenesis of BRONJ. Future investigations with larger populations conducted prospectively to correlate metagenomic findings with disease progression and treatment response over time may allow for more accurate diagnostics and prognostication. Continuing in this line of investigation will be critical in the future for informing clinical approaches to disease intervention and the realization of targeted antimicrobial therapeutics for jaw osteonecrosis.