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RESEARCH ARTICLE
| MICROBIOLOGY
OPEN ACCESS
Interkingdom assemblages in human saliva display group-level
surface mobility and disease-promoting emergent functions
Zhi Rena,b,c,d,1 , Hannah Jeckele,f,1 , Aurea Simon-Soroa,b,c,2 , Zhenting Xianga,b,c
Nyi-Nyi Tini , Anderson Harai , Knut Drescherf,4 , and Hyun Kooa,b,c,d,4
, Yuan Liua,b,c,g, Indira M. Cavalcantia,b,c,3
, Jin Xiaoh,
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Edited by Edward DeLong, University of Hawaii at Manoa, Honolulu, HI; received June 10, 2022; accepted August 31, 2022
Fungi and bacteria often engage in complex interactions, such as the formation of
multicellular biofilms within the human body. Knowledge about how interkingdom
biofilms initiate and coalesce into higher-level communities and which functions the
different species carry out during biofilm formation remain limited. We found nativestate assemblages of Candida albicans (fungi) and Streptococcus mutans (bacteria) with
highly structured arrangement in saliva from diseased patients with childhood tooth
decay. Further analyses revealed that bacterial clusters are attached within a network of
fungal yeasts, hyphae, and exopolysaccharides, which bind to surfaces as a preassembled
cell group. The interkingdom assemblages exhibit emergent functions, including
enhanced surface colonization and growth rate, stronger tolerance to antimicrobials,
and improved shear resistance, compared to either species alone. Notably, we discovered
that the interkingdom assemblages display a unique form of migratory spatial mobility
that enables fast spreading of biofilms across surfaces and causes enhanced, more extensive tooth decay. Using mutants, selective inactivation of species, and selective matrix
removal, we demonstrate that the enhanced stress resistance and surface mobility arise
from the exopolymeric matrix and require the presence of both species in the assemblage. The mobility is directed by fungal filamentation as hyphae extend and contact
the surface, lifting the assemblage with a “forward-leaping motion.” Bacterial cell clusters can “hitchhike” on this mobile unit while continuously growing, to spread across
the surface three-dimensionally and merge with other assemblages, promoting community expansion. Together, our results reveal an interkingdom assemblage in human
saliva that behaves like a supraorganism, with disease-causing emergent functionalities
that cannot be achieved without coassembly.
Significance
Fungi and bacteria form
multicellular biofilms causing
many human infections. How such
distinctive microbes act in concert
spatiotemporally to coordinate
disease-promoting functionality
remains understudied. Using
multiscale real-time microscopy
and computational analysis, we
investigate the dynamics of fungal
and bacterial interactions in
human saliva and their biofilm
development on tooth surfaces.
We discovered structured
interkingdom assemblages
displaying emergent functionalities
to enhance collective surface
colonization, survival, and growth.
Further analyses revealed an
unexpected group-level surface
mobility with coordinated “leapinglike” and “walking-like” motions
while continuously growing. These
mobile groups of growing cells
promote rapid spatial spreading of
both species across surfaces,
causing more extensive tooth
decay. Our findings show
multicellular interkingdom
assemblages acting like
supraorganisms with
functionalities that cannot be
achieved without coassembly.
interkingdom interaction j microbial mobility j spatial structure j supraorganism j oral biofilm
The microbial life on Earth often resides on surfaces, where cells form multicellular structures known as biofilms (1). Extensive efforts have been devoted to understanding the
biofilm formation process and the mechanisms underlying the biofilm lifestyle (1–3).
While most studies have focused on bacteria, eukaryotic microbes also frequently form
biofilms. Furthermore, previous studies have revealed that biofilms composed of bacteria
and fungi are highly abundant in nature, establishing complex interkingdom interactions
(4–7). Such bacterial–fungal biofilms can display enhanced virulence and survival, which
is achieved through tight cell–cell cohesion, metabolite exchange, and extracellular polymeric matrices within established communities (4–6). How interkingdom biofilms initiate and develop on the surface, and which functions the different species carry out during
this process, remains unclear.
In the human oral cavity, biofilms formed by bacteria and fungi have a major impact
on health (7, 8). For example, patients affected by severe childhood caries (tooth
decay), a widespread and costly infectious disease affecting toddlers worldwide (9),
display high carriage of the bacterium Streptococcus mutans and the fungus Candida
albicans, both in saliva and in biofilms formed on teeth (dental plaque) (10). Previous
studies have shown that these distinct microbes form interkingdom biofilms with
enhanced virulence under sugar-rich conditions (11, 12). However, interactions of
these two species in saliva have not been characterized, and the extent to which the
interactions between S. mutans and C. albicans influence the dynamics of biofilm
formation and its functional properties is unknown.
In this study, we investigated the interactions between S. mutans and C. albicans during colonization and biofilm formation in human saliva, and made several unexpected
discoveries with implications for disease. We observed that in saliva of toddlers affected
by severe tooth decay, S. mutans and C. albicans formed highly structured interkingdom assemblages. Using real-time multiscale imaging and computational analysis, we
studied the organization of such interkingdom assemblages and assessed their role
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This article is a PNAS Direct Submission.
Copyright © 2022 the Author(s). Published by PNAS.
This open access article is distributed under Creative
Commons Attribution License 4.0 (CC BY).
1
Z.R. and H.J. contributed equally to this work.
2
Present address: Department of Stomatology, School
of Dentistry, University of Seville, 41004 Seville, Spain.
3
Present address: Department of AI Development and
Performance, Relu, 3001 Leuven, Belgium.
4
To whom correspondence may be addressed. Email:
knut.drescher@unibas.ch or koohy@upenn.edu.
This article contains supporting information online at
http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.
2209699119/-/DCSupplemental.
Published October 3, 2022.
https://doi.org/10.1073/pnas.2209699119
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during biofilm formation spatiotemporally. These experiments
showed that bacterial clusters attached to yeast and hyphal
complexes to form assemblages that display emergent properties, including enhanced surface colonization, a higher growth
rate, and a stronger tolerance to shear stress and antimicrobials,
which are not observed in either bacteria or fungi alone.
Surprisingly, when individually tracked, these interkingdom
assemblages display a unique mode of migratory group-level
mobility, enabled by fungal filamentation across surfaces, which
is used by the attached bacterial clusters for “hitchhiking.”
Through this mobility, the interkingdom assemblages rapidly
proliferate across the surface and expand three-dimensionally,
leading to biofilm superstructures and extensive enamel decay
on ex vivo tooth surfaces that cannot be achieved by each
species alone. Hence, our data reveal an interkingdom assemblage found in human saliva that efficiently colonizes, displays
emergent properties, and enhances surface spreading through a
group-level mobility mechanism that propels clusters of otherwise
nonmotile bacteria and fungi across the surface, to ultimately promote community spatial expansion and disease-causing activity.
Results
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Interkingdom Microbial Assemblages Occur in Human Saliva
from Childhood Caries Patients. We collected saliva from
healthy (caries-free) children and children with severe childhood caries and analyzed the intact, naturally present microbial
content by fluorescence in situ hybridization (FISH) and super
resolution confocal imaging (Fig. 1A). We found that saliva
from diseased patients was enriched with assemblages of fungal
and bacterial cells. In these assemblages, bacterial clusters were
physically associated with fungal cells (yeasts and hyphae/
pseudohyphae) forming a multicellular structure (Fig. 1 A,
Right). In contrast, saliva from healthy (caries-free) children
contained mostly single-cell bacteria or bacterial aggregates
(Fig. 1 A, Left). To determine the species composition of the
assemblage, we performed FISH imaging using species-specific
probes, noting that early childhood caries patients typically
harbor high levels of C. albicans and S. mutans in saliva (9, 10).
We found that interkingdom assemblages in saliva from
early childhood caries patients were comprised primarily of
C. albicans and S. mutans (SI Appendix, Fig. S1). We also detected
α-glucans, an extracellular polysaccharide (EPS) associated with
tooth-decay (13). α-Glucans are produced predominantly by
S. mutans-derived exoenzymes termed glucosyltransferases, or
Gtfs (14). We observed α-glucans on the bacterial and fungal
cell surfaces within the interkingdom assemblage in saliva from
the diseased patients (SI Appendix, Fig. S2A). We assessed Gtf
activity using radiolabeling and scintillation counting and
found higher Gtf activity levels in the saliva from diseased
children (vs. healthy children) (SI Appendix, Fig. S2B). In the
diseased plaque biofilm, both C. albicans and S. mutans were
found in high levels (Fig. 1B), but significantly less in healthy
samples, suggesting a dynamic interaction of S. mutans and
C. albicans as they transition from the fluid phase to an
apatitic surface.
These results from patient samples show that interkingdom
assemblages in saliva constitute a complex biostructure of bacteria, fungi, and EPS α-glucans, which cooccurs with early childhood caries. The influence of these interkingdom assemblages
on tooth surface colonization and biofilm formation, and the
properties of these assemblages, are investigated in this study, as
described below.
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Experimental Model for Interkingdom Assemblage and Surface
Colonization in Saliva. To investigate the assemblages of
S. mutans and C. albicans in more detail, we sought to recreate
such interkingdom assemblages in the laboratory. We therefore
developed an experimental model using C. albicans and
S. mutans incubated in human saliva at 37 °C, and hydroxyapatite as a tooth-mimetic surface (Fig. 1C). In this model system,
we found that planktonic C. albicans and S. mutans can coassemble to form assemblages with similar structural features to
those naturally present in the patients’ saliva, which are characterized by yeast and hyphal forms intertwined with S. mutans
clusters and EPS α-glucans (SI Appendix, Fig. S3). We then
assessed whether these bacterial-fungal assemblages can bind to
saliva-coated hydroxyapatite (sHA) surfaces. We found that
these assemblages, formed in the saliva prior to surface contact,
attached to the sHA surface as a cell group (SI Appendix, Fig.
S4A). These findings were further corroborated by culturing
the viable cells recovered from sHA surfaces, which revealed
higher counts of S. mutans and C. albicans when they were
incubated together in saliva (vs. each alone) (SI Appendix, Fig.
S4B), indicating that coassembly may benefit both species for
enhanced surface binding. Three-dimensional (3D) reconstruction of the confocal fluorescence images showed a network of
C. albicans yeast cells and hyphae harboring S. mutans within
the assemblage structure, which attaches to the sHA surface as
a group (Fig. 1D; individual fluorescence channels shown in SI
Appendix, Fig. S5A). The 3D images showed fungal hyphae
located at the periphery adhering to the surface in a pillar-like
arrangement, whereas most of the bacterial cells were clustered
and attached onto the fungal surface like “cargo” (SI Appendix,
Fig. S5).
Next, we employed computational image analysis (15) to
investigate the composition and the spatial structuring of the
microbial and EPS components within the interkingdom
assemblages. We found that the core of the assemblage, in the
vicinity of the center-of-mass (referred to as “centroid”), harbored a mix of C. albicans and S. mutans, whereas the periphery
of the assemblage was predominantly comprised of C. albicans
hyphae (close-up shown in Fig. 1 E, Left; quantification in Fig.
1F; schematic diagram in Fig. 1G). A schematic diagram
depicting the location of the centroid and periphery of the
assemblage as well as the surface is shown in Fig. 1G. Notably,
fungal cells localized across the entire structure including the
surface-contacting areas (Fig. 1 H, Right). In contrast, bacteria
localized mostly around the inner core, close to the centroid,
which is significantly above the surface (Fig. 1 H, Left). These
results for the bacterial and fungal organization in the interkingdom assemblages are summarized in Fig. 1G. Matching the
location of the bacterial clusters, EPS α-glucans were detected
both in the core of the assemblage and also at peripheral locations
across the hyphae surface (Fig. 1 E, Center and Right, and Fig. 1 H,
Center). This is consistent with previous reports indicating that
Gtf exoenzymes (which produce α-glucans) can bind to both
bacterial and fungal cell membranes to mediate bacterial clustering and in situ glucans synthesis on the C. albicans surface (16).
Given the spatial location of hyphal cells and EPS, we hypothesized that fungal hyphal formation, adhesins, and streptococcal
Gtfs (12, 17) are key factors for the interkingdom assemblage and
colonization. To test this hypothesis, we initially used C. albicans
mutants in which core transcriptional regulators associated with
hyphal formation were deleted (18), to determine their impact on
the multicellular structure (SI Appendix, Fig. S6). Among Candida
mutants, we found that the efg1 knockout strain, a master regulator
of C. albicans hyphal formation, was most disruptive to
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Fig. 1. Interkingdom microbial assemblages in saliva attach to surfaces as structured cell groups. (A) Using fluorescent staining and confocal microscopy,
native fungal–bacterial assemblages are found in saliva of patients with early childhood caries, but not in healthy individuals. (B) S. mutans and C. albicans
were found in high levels in the diseased plaque. Data are presented as median with interquartile range. *P < 0.05 by Mann–Whitney U test. (C) Interkingdom assemblages and surface colonization is recapitulated using an in vitro model based on human saliva and hydroxyapatite surfaces, to mimic the tooth
enamel. (D) Spatially structured S. mutans and C. albicans assemblage on the tooth-mimetic surface. (E) Surface-colonized assemblage using different fluorescent markers, as indicated underneath the images. EPS: extracellular α-glucan matrix produced by S. mutans. Asterisk: inner core harboring a mix of C. albicans and S. mutans. Arrowheads: peripheral areas containing mostly Candida hypha covered with bacterial-derived EPS. (F) Spatial distribution of S. mutans
and C. albicans within the assemblage. Lines correspond to mean and shaded region to SD of n = 4 independent replicates. (G) Schematic diagram describing the spatial arrangement of the two species inside the assemblage, based on the computational image analysis results from F and H. (H) Spatial organization of the fungal and bacterial species relative to the surface. Lines correspond to mean and shaded area to SD of n = 4 independent replicates. (I) Confocal
images of initial surface colonizers, for WT–WT assemblages and different WT–mutant assemblages (using C. albicans ΔΔefg1 or S. mutans ΔgtfBC mutants).
(Scale bars, 10 μm.)
assemblage formation, and resulted in only a sparse colonization of mostly single cells of bacteria and fungi on the sHA surface (vs. C. albicans WT strain) (Fig. 1I and SI Appendix, Fig.
S6A). We next analyzed C. albicans Efg1-regulated adhesins
expressed on the hyphal cell wall using homozygous knockout
strains, including ΔΔals1/ΔΔals3, ΔΔhwp2, ΔΔhyr1, and
ΔΔeap1 (SI Appendix, Fig. S6A). Since Als1 and Als3 have a
substantial overlap in functions (19), we used a double mutant
with a disruption of both genes. Similar to the efg1 knockout,
the ΔΔals1/ΔΔals3 deletion caused a severe reduction of coassembly, whereas ΔΔhwp2, ΔΔhyr1, and ΔΔeap1 did not show
a significant impact (SI Appendix, Fig. S6A). Furthermore,
ΔΔals1/ΔΔals3 also led to a significant disruption of surface
colonization, resulting in few cells adhered on the surface (SI
Appendix, Fig. S6A). Conversely, we investigated whether the
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Gtf-derived EPS contributes to this process. Using a S. mutans
double-knockout of gtfB and gtfC, we found that interkingdom
assembly was also disrupted harboring mostly single-chain bacterial cells (Fig. 1I and SI Appendix, Fig. S6B). This finding was
further confirmed by adding glucanohydrolases (20) to the saliva
exogenously, which specifically break down the α-glucans produced by S. mutans GtfB and GtfC (SI Appendix, Fig. S6B).
Moreover, in the absence of sucrose (the substrate for EPS
α-glucans synthesis by Gtf enzymes), the ability of C. albicans
and S. mutans to colonize as structured interkingdom assemblages was impaired (SI Appendix, Fig. S7). These results indicate
that Efg1-regulated Candida hyphal formation, hypha-specific
Als adhesins, and streptococcal Gtf-derived α-glucans play important roles for the formation of interkingdom assemblages and for
the surface colonization by these multicellular biostructures.
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Altogether, the experiments in our saliva-based biofilm model
showed that C. albicans and S. mutans can coassemble in saliva
into a structured interkingdom assemblage, which enables
enhanced colonization of both species on tooth-mimetic surfaces.
Analysis of the spatial organization of the interkingdom assemblages revealed three features: 1) C. albicans hyphae are located at
the periphery and hyphal surface contacts may serve as anchors;
2) Surface colonization of S. mutans clusters is promoted by
attachment to the fungal network, which carries the bacterial
clusters like cargo; and 3) hyphae formation and the presence of
EPS α-glucans are both critical for the assemblage formation and
surface colonization.
Interkingdom Assemblages Display Enhanced Mechanical
Resistance and Antimicrobial Tolerance. The coassembled fungi
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and bacteria can colonize the surface as a highly structured
group, which may confer additional advantages under various
challenges. We examined whether these interkingdom assemblages withstand mechanical shear stress generated by fluid flow,
and their susceptibility to antimicrobial treatment.
After attachment of the interkingdom assemblages to sHA
surfaces, we applied increasing shear stress ranging from 1 to
20 Pa and assessed the detachment of the assemblages in
real-time using confocal live-cell imaging (Fig. 2A). We found
that surface-colonized S. mutans or C. albicans alone were
dissembled and readily detached from the sHA surface under
increased fluid shear stress. In contrast, the interkingdom
assemblage remained attached to the surface, maintaining its
structural stability even under high shear stress (Fig. 2B).
Quantitative analysis revealed distinctive detachment patterns
(Fig. 2C). Most of the aggregated S. mutans cells detached from
the sHA surface at low to intermediate shear stress (>90%
removal at 10 Pa). Notably, aggregated C. albicans could withstand intermediate shear stress levels (80% removal at 20 Pa). In contrast, most of the assemblages
remained on the surface under even the highest shear stresses
(90% removal at 20 Pa) (SI Appendix, Fig. S9), suggesting that EPS degradation weakened its attachment strength.
We then examined whether EPS-degradation could affect the
antimicrobial tolerance against both chlorhexidine (SI Appendix,
Fig. S10) and nystatin (SI Appendix, Fig. S8). We found that the
microbes were more effectively and homogeneously killed across
the entire interkingdom assemblage after pretreatment with glucanohydrolase, indicating a protective role provided by the locally
produced α-glucans within the interkingdom assemblage.
We also assessed whether the emergent properties are speciesspecific. We used Streptococcus gordonii, an early-colonizer oral
commensal species (13), to form interkingdom assemblages with
C. albicans. We found that the surface-attached C. albicans–
S. gordonii assemblages were readily removed by increased fluid
shear (>85% removal at 10 Pa) (SI Appendix, Fig. S9), suggesting much weaker attachment strength vs. the C. albicans–
S. mutans assemblage. We also observed that C. albicans and
S. gordonii within the assemblage were rapidly killed by chlorhexidine contrasting with the enhanced antimicrobial tolerance of
C. albicans–S. mutans assemblage (SI Appendix, Fig. S10). The
data suggest bacterial species-specificity associated with emergent
properties displayed by the interkingdom assemblage.
Taking these data together, we find that by cocolonizing as
an interkingdom assemblage, C. albicans and S. mutans display
enhanced tolerance against mechanical clearance and antimicrobial compounds, which promote the persistence and survival
of these biostructures that are acting as initial colonizers of
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surfaces. Importantly, these benefits were not observed in
monospecies aggregates, indicating a highly interdependent
partnership between bacterial and fungal cells.
Surface-Bound Interkingdom Assemblages Grow Faster and
Initiate Biofilms. Given that C. albicans and S. mutans can
coassemble in saliva, followed by attachment to surfaces as a
structured group, we investigated how the surface-attached cells
residing in this biostructure grow spatiotemporally into biofilms. To investigate the growth dynamics of the sHA surfaceattached assemblages, we employed a flow-cell microfluidic
biofilm culture system that mimics saliva flow coupled with
time-lapse confocal imaging and computational analyses (22)
(Fig. 3A). We tracked each surface colonizer individually
and calculated the dynamic change of its biovolume across the
surface. These experiments showed that the interkingdom
assemblage developed into larger biofilms compared to monospecies aggregates, eventually growing to cover the surface (Fig.
3B). Interestingly, when analyzing the biovolume of each species within the interkingdom assemblage, we found that the
biovolume of S. mutans increased more rapidly than the biovolume of C. albicans (Fig. 3 C, Inset). We then compared the
S. mutans biovolume growth dynamics within interkingdom
assemblages to that of aggregated S. mutans alone or in assemblages treated with fungicide (using a high concentration of nystatin, selectively killing C. albicans). These experiments showed that
the biovolume of S. mutans in the assemblages increased faster
than S. mutans alone (Fig. 3C). Similarly, C. albicans inactivation
with nystatin reduced the bacterial growth benefit from intact
assemblages (Fig. 3B; quantification in Fig. 3C). Measurements
of the surface coverage also show that the interkingdom assemblages spread much faster than S. mutans alone, or assemblages
treated with the fungicide nystatin (Fig. 3D).
We also monitored the spatiotemporal growth dynamics of
surface-attached aggregated S. mutans and interkingdom assemblages (Fig. 3E). When tracking two spatially distant assemblages,
we found that they grew and expanded toward each other and
eventually merged to create a new superstructure (Fig. 3 E,
Lower). In contrast, individual S. mutans aggregates grow separately without merging events across the same time span (Fig. 3 E,
Upper). As a result, the surface coverage occurred more rapidly by
the interkingdom assemblage, compared to aggregated S. mutans
or fungicide-treated assemblage (Fig. 3D).
Assemblage Mobility, Bacterial Hitchhiking on Migrating Fungi,
and Enhanced Surface Spreading. Next, we tracked the surface-
attached interkingdom assemblages on surfaces and found a
unique migratory behavior. In this migration process, the interkingdom assemblage deformed, and the “leading edge” (Fig.
4A, solid lines) moved significantly as time elapsed. In addition
to the movement of the leading edge, the centroid of the
S. mutans biovolume also moved across the surface (Fig. 4A,
hollow dots indicate the centroid at the initial time point t0
and filled dots indicates the centroid at selected time points),
suggesting a surface mobility of the assemblage. During the
growth process, which coincides with the migration process,
the interkingdom assemblage developed a changing directionality along the moving direction (Fig. 4A, purple arrow).
Since S. mutans cells are attached to C. albicans cells within
the assemblage, and the killing of fungal cells by nystatin disrupted the surface-spreading of the bacteria within the assemblage (Fig. 3D), we hypothesized that the bacterial mobility
across the surface by the interkingdom assemblages was driven
by fungal growth. Using high-resolution time-lapse confocal
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Fig. 3. Biofilm growth dynamics of interkingdom
assemblages on tooth-mimetic surface. (A) Schematic diagram of the flow-cell microfluidic culture
system coupled with time-lapse confocal microscopy for visualizing biofilm growth. (B) Confocal
images of the initial colonizers on the surface at
0 h, and the biofilm structure after 10 h, for interkingdom assemblage (Assembl), for aggregated
S. mutans (Agg S.m.), and for fungicide-treated
assemblage (250 μg/mL nystatin for 30 min).
Green, S. mutans; cyan, C. albicans. (Scale bar,
100 μm.) (C) Time-resolved biofilm biovolume of
S. mutans during the biofilm development. (Inset)
Time-resolved biovolume of each S. mutans and
C. albicans within the assemblage. Lines correspond to mean, shaded region to SD of n = 4
independent replicates. (D) Quantification of the
dynamics of biofilm surface spreading. Lines correspond to mean, shaded region to SD of n = 4
independent replicates. *P < 0.05 by one-way
analysis of variance with Tukey’s multiplecomparison test (t = 6.5 h). (E) Confocal image
time series showing merging behavior (yellow
arrowhead) of multiple individually developing
interkingdom assemblages on the tooth-mimetic
surface. Green, S. mutans; cyan, C. albicans. (Scale
bar, 100 μm.)
microscopy and computational quantification of the mobility,
we tested this hypothesis, and more generally explored how
S. mutans and C. albicans codeveloped from the initial surfacebound assemblages into large biofilms. The fungal and bacterial
behaviors within the surface-attached assemblages were analyzed
individually from the four-dimensional (x, y, z, time) confocal
datasets (orthogonal time-frames are shown in Fig. 4B and
Movies S1–S3).
Surprisingly, the images showed that bacterial clusters were
lifted away from the surface and transported laterally (purple
arrows in Fig. 4 B2 and Movie S2) while continuously growing
along with fungi, thus hitchhiking on the elongating hyphae. In
contrast, aggregated S. mutans alone remained in their initial
positions on the surface during the growth (Fig. 4 B1 and Movie
S1). Three-dimensional tracks of the centroid of the S. mutans
biovolume within the assemblage show that the bacteria displayed lateral and vertical movement (Fig. 4C), whereas the
monospecies S. mutans aggregates have very short tracks (Fig.
4D). Given that growing hyphae can generate mechanical forces
at the point of contact (23), we investigated whether the mobility
was resulting from the fungal hyphal growth. We specifically
deactivated fungal growth in the assemblage using nystatin and
examined the bacterial mobility behavior (Fig. 4 B3 and Movie
S3). Spatial tracking analyses revealed a lack of filamentation and
complete loss of lateral mobility within the interkingdom
assemblages exposed to the fungicide nystatin (Fig. 4 B, 3
and E), similar to that of monospecies bacterial growth (Fig. 4
B1 and D), suggesting that hyphal formation was required for
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bacterial hitchhiking mobility. We calculated the accumulated
path lengths of the biostructures relative to their initial positions
(t0). The resulting displacement curves showed that the interkingdom assemblage moved at remarkably high velocity (up to
40 μm/h) during the first 3 h, then slowed down, and reached a
total path length of ∼100 μm after 6 h (Fig. 4F). In contrast,
centroids of aggregated S. mutans alone (or assemblages treated
with nystatin to kill C. albicans) remained mostly stationary
(