Introduction
The complexity of bacterial communities that make up our microbiome mirrors the complexity of niches within human body. Of these niches, the oral cavity is perhaps one of the most diverse, presenting extremes of tissue stiffness, surface topography, transient temperature shifts, and nutrient flux[1]. Although the accessibility of the oral cavity has made it a focus of research into microbial community structure and diversity, our understanding of interspecies relationships and their role in health and disease remains limited.
A wide range of co-culture strategies have been developed to facilitate characterization of interspecies relationships, largely focusing on metabolic compatibility and coaggregation of species that form oral biofilms and plaque[2-4]. Efforts to culture previously “unculturable” species have focused on identifying co-culture partners that provide complementory metabolic functions to compensate for lack of specific metabolic pathways[5]. Physical distances and culture volume play key roles in metabolic symbiosis, interspecies communication, and cell-cell adherence[6]. Thus, multiple recent studies have used microfluidic approaches to achieve small-volume co-culture and to engineer co-culture devices with defined physical constraints[7-9].
Two bacterial genera commonly associated with oral biofilm formation areActinomyces [10] and Streptococcus [11]. The genusActinomyces has recently been subdivided with the creation of the genus Schaalia [12] with both Actinomyces and Schaaliabeing members of the family Actinomycetacea e. The bacteriaA. odontolyticus and Streptococcal spp. are considered early colonizers, adhering directly to the salivary pellicle coating the tooth surface. This facilitates the secondary adherence of intermediate colonizers, such as Actinomyces spp., followed by late colonizers such as Fusobacterium nucleatum and Porphyromonas gingivalis during formation of dental plaque[6]. Sequential adherence of different bacterial species depends on their co-adhesion compatibility, which is often species-specific[13], while co-aggregation of bacterial species in suspension has been shown to directly influence gene expression to induce metabolic outputs to the benefit of both species[14]. Some bacterial species are incompatible for co-culture, leading to domination by one species at the expense of the other, often in a nutrient-dependent manner[15, 16].
Here, we utilized a previously-developed microfluidic device[17] to perform low-volume co-culture of multiple actinomyces and streptococcal species. Detailed microscopy revealed formation of defined “exclusion zones” surrounding A. graevenitzii microcolonies when co-cultured with S. cristatus or S. salivarius but notS. oralis or S. mitis . Additionally, exclusion zones were not observed with S. odontolytica or A. naeslundiiin co-culture with any Streptococcal species tested, suggesting that the phenomenon exhibits a degree of species-specificity.
Interestingly, formation of exclusion zones around A graevenitziimicrocolonies was also observed in co-culture with ofStaphylococcus aureus. S. aureus is a common commensal present on the skin and upper respiratory tract of up to 50% of healthy individuals[18, 19]. It is also considered an important and dangerous opportunistic pathogen, due to high infection rates and the emergence of many antibiotic-resistant strains[20].
Innate immune cells, especially neutrophils, are the first cellular line of defense against S. aureus infection once physical barriers are breached[21]. As such, S. aureus infections induce a robust inflammatory response, which can lead to conditions such as cellulitis in the skin[22], more severe arthritic conditions following infections of the bones and joints[23], and sepsis. Studies have identified S. aureus in between 17-48% of healthy oral samples, with even higher rates of up to 64% present in young children[24]. Despite this, S. aureus is not considered a significant oral pathogen, and infections in the oral cavity are usually limited to inflammatory conditions such as angular cheilitis[25], along with rarer cases of jaw cysts[26] and oral mucosal lesions[27]. Systemic dissemination of S. aureus originating from the oral cavity remains a relatively unexplored topic.
Here, we characterized the interactions of several species of Actinomyces and Streptococcus in nanoliter confinement and observed the formation of exclusion zones between colonies, which were not observable in traditional co-cultures. Moreover, using a GFP-expressing strain ofS. aureus , we observed that the innate immune responses toS. aureus -A. graevenitzii co-cultures were significantly dampened compared to S. aureus mono-cultures.