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.