Discussion
We utilized PDMS microfluidic devices to observe microbial mono- and
co-cultures of oral isolates in nanoliter volumes. The gas permeability
of PDMS facilitates loading of the dead-end chamber through a single
channel by applying vacuum, while the coverslip provides optical clarity
for imaging approaches and a physical surface on which the microbes can
grow. In Actinomyces and Streptococcus co-cultures, we observed
formation of a physical “exclusion zones” bordering Actinomyces
graevenitzii microcolonies with certain species ofStreptococcus . Interestingly, exclusion zones were also observed
with a GFP-expressing strain of Staphylococcus aureus , allowing
us to perform detailed analysis of exclusion zone formation using
automated imaging approaches. These studies supported a model in which
exclusion zone formation is triggered by interaction of specific
species.
While it appears that the exclusion zone represents a physical space
containing no living bacteria, it is unclear whether this is due to
physical exclusion for matrix deposition, suppression of proliferation
in this area by quorum signaling, or active killing of invading cells by
a toxic metabolite. While the formation of exclusion zones only occurred
between Actinomyces graevenitzii with S. cristatus, S.
salivarius and S. aureus , it is unlikely that the formation of a
simple physical barrier explains the interactions. Additionally, the
kinetics of S. aureus coverage in co-culture with A.
graevenitzii , which appear to show an initial increase followed by a
decrease, suggest that formation of the exclusion zone involves death of
existing S. aureus cells in that area. Macroscopic co-culture on
solid media did not result in any visible cross-inhibition betweenA. graevenitizii and S. aureus colonies, highlighting the
importance of small volume culture and high-resolution analysis for
identification of such interactions. One limitation of the microfluidic
chamber design used is the inability to rapidly fix and stain cells to
gain a more detailed understanding of their structure, due to the
excessive time taken for fixatives and labelling compounds to diffuse
through the connecting channel. Either way, this phenomenon provides
clear visualization of an antagonistic relationship between competing
bacterial species and may provide some insight regarding community
structures in the oral cavity.
These interactions are particularly interesting in the context of
well-known opportunistic pathogens such as S. aureus . Modulation
of inflammatory responses in these experiments may be a direct response
to compounds released by the bacteria during co-culture, or may simply
follow from suppression of S. aureus proliferation, which we
previously demonstrated to be important for effective neutrophil
recruitment[17].
In cases where S. aureus is identified in oral lesions, it is
often isolated in the company of other opportunistic pathogens such asCandida albicans[25] , infections with which are generally
associated with loss of microbiome stability. Thus, exclusion ofS. aureus by A. graevenitzii builds on the concept that
established and stable commensal communities are important to prevent
colonization of a niche by pathogens.
Dissecting the complexity of microbial community structure and its role
in the health and disease in the oral cavity remains a focus of ongoing
research. Physical and metabolic characteristics facilitate successful
colonization of diverse oral surfaces and ongoing survival in this
complex and dynamic environment[6]. Many oral microbes have proven
challenging to culture, often because they require the presence of one
or more speicies in consortia to process specific metabolites[28].
In addition to such relationships, microbial community structure is also
dictated by antagonism driven by competition for space and
nutrients[29].
Overall, low-volume techniques such as microchambers and droplet-based
microfluidics may enhance quorum sensing and competition for
nutrients[9], better mimicking in vivo conditions. The microfluidic
technique overcome the limiations of traditional bulk suspension
co-culture approaches, which provide limited spatiotemporal information
regarding interspecies interactions and often simply result in
domination by the faster-growing species.
Data availablility: All data presented will be made available
by the authors upon request.
Author Contributions: Fatemeh Jalali: Data curation
(lead), Investigation (lead), Methodology (equal), Visualization
(equal), Writing – Original Draft Preparation (supporting).Felix Ellett: Formal Analysis (lead), Investigation
(supporting), Methodology (equal), Visualization (equal), Supervision
(supporting), Writing – Original Draft Preparation (lead), Writing –
Review and Editing (equal). Pooja Balani: Resources
(supporting). Margaret Duncan: Conceptualization (equal),
Resources (equal), Writing – Review and Editing (equal). Floyd
Dewhirst: Conceptualization (equal) , Resources (equal), Writing –
Review and Editing (equal). Gary Borisy: Conceptualization
(equal), Resources (equal), Writing – Review and Editing (equal).Daniel Irimia: Conceptualization (equal), Project
Administration (lead), Supervision (lead), Writing – Review and Editing
(equal).
Acknowledgements: This work was supported by a grant from the
National Institute of Dental and Craniofacial Research (DE024468).
Microfabrication was conducted at the BioMEMS Resource Center at
Massachusetts General Hospital, supported by a grant from the National
Institute of Biomedical Imaging and Bioengineering (EB002503). Dr. Felix
Ellett was supported by a fellowship from the Executive Committee for
Research at Massachusetts General Hospital.
Conflict of Intererest: None declared
Ethics Statement: None required