AEM Accepts, published online ahead of print on 17 October 2014 Appl. Environ. Microbiol. doi:10.1128/AEM.02311-14 Copyright © 2014, American Society for Microbiology. All Rights Reserved.
Embedded biofilm: a new biofilm model based on the embedded
growth of bacteria
Yong-Gyun Jung1$, Jungil Choi2,3,4$, Soo-Kyoung Kim5, Joon-Hee Lee5*, and Sunghoon
Quantamatrix Inc, 104-213, Seoul National University, Seoul 151-742, Republic of Korea.
Center for Nanoparticle Research, Institute for Basic Science (IBS), Seoul 151-742, Republic of Korea. 3
School of Electrical Engineering and Computer Science, Seoul National University, Seoul 151-742, Republic of Korea. 4
Institutes of Entrepreneurial BioConvergence, Seoul National University, Seoul 151-742, Republic of Korea. 5
Department of Pharmacy, College of Pharmacy, Pusan National University, Busan 609-735, Republic of Korea.
Authors’ E-mail addresses: Yong-Gyun Jung ([email protected]
), Jungil Choi
), Soo-Kyoung Kim ([email protected]
), Joon-Hee Lee
), and Sunghoon Kwon ([email protected]
These authors contributed equally to this work
*Correspondence should be addressed to: Sunghoon Kwon ([email protected]
) or Joon-Hee Lee ([email protected]
Running Title; Embedded biofilm
A variety of systems have been developed to study biofilm formation. However, most of the
systems are based on the surface-attached growth of microbes under shear stress. In this study,
we designed a microfluidic channel device, called an MAC (microfluidic agarose channel),
and found that microbial cells in the MAC system formed an embedded cell aggregative
structure (ECAS). The ECASs were generated from the embedded growth of bacterial cells in
agarose matrix and better mimicked the clinical environment of biofilms formed within
mucus or host tissue under shear-free conditions. ECASs were developed with the production
of extracellular polymeric substances (EPS), the most important feature of biofilms, and
eventually burst to release planktonic cells, which resembles the full developmental cycle of
biofilms. Chemical and genetic effects have also confirmed that ECASs are a type of biofilm.
Unlike the conventional biofilms formed in the flow cell model system, this embedded-type
biofilm completes the developmental cycle in only 9~12 h and can easily be observed with
ordinary microscopes. We suggest that ECASs are a type of biofilm and that the MAC is a
system for observing biofilm formation.
Key words; embedded-type biofilm, biofilm model, embedded growth, extracellular polymeric
substance, microfluidic agarose channel (MAC), embedded cell aggregative structure
In natural habitats, bacterial cells often form biofilms to structurally organize and protect
their communities. A biofilm is an aggregate of microbial cells that adhere to a live/non-
living surface, and the adherent cells in biofilms are enclosed in a self-produced matrix of
extracellular polymeric substances (EPS), including nucleic acids, proteins, polysaccharides
and lipids (1). Bacterial biofilm formation is a complex developmental process involving
several stages, including migration and initial attachment, EPS production and irreversible
attachment, maturation, destruction and cell dispersion (2). The significance of biofilms is
witnessed in the environment, industry, and human health (3). Importantly, pathogenic
bacteria build biofilms on various medical implants and human tissues, and they have a
strong resistance to antimicrobial agents as well as persistently colonize in chronic diseases.
In the USA, the fouling of microorganisms on industrial equipment and architecture costs
billions of dollars annually (4).
To study biofilms, many researchers have developed a variety of in vitro model systems
that mimic the environment of biofilm habitats (5), and several models are commonly used
for biofilm assays, such as the agar plate system (6), multi-well plate system (7), biofilm
reactor system (8, 9) and flow cell system (10). Flow and biofilm reactor systems are based
on the surface-attached growth of microbes under shear stress. While these conventional
models may mimic some aspects of natural environments, biofilms sometimes form in very
different conditions that the conventional systems cannot mimic entirely. The clinically
relevant biofilms in host tissues are formed without shear stress within the mucosal layer on
epithelial cells or inside of host cells. Pseudomonas aeruginosa, which infects cystic fibrosis 3
(CF) patients, does not attach to the pulmonary epithelial surfaces of CF patients, but it forms
persistent biofilms within the thick mucosal layer on the epithelial cells of CF patients (11).
In urinary tract infections, intracellular bacteria mature into biofilms, forming pod-like bulges
in the mouse bladder lumen, showing a different structure from biofilms formed on surfaces
with shear stress (12). Additionally, bacteria reside on soil matrices or plant surfaces as
aggregates of cells in terrestrial habitats (non-shear stress conditions), and they are embedded
in exopolymeric substances that they generate and that can be a biofilm. These biofilms are
commonly unsaturated, and the size of the biofilms vary depending on the environmental
conditions (13-15). Therefore, it is necessary to develop a new biofilm-modeling system that
more closely resembles the clinical and terrestrial relevance of biofilms.
To address this need, we have developed a new biofilm-forming system based on the
embedded growth of bacteria using a microfluidic channel device, called an MAC
(microfluidic agarose channel, Fig. 1A and 1B), instead of based on conventional biofilm
generation using shear stress, such as the multi-well plate system (7), biofilm reactor system
(8, 9) and flow cell system (10). The developed device generated a novel type biofilm-like
structure in shear stress-free conditions. In the MAC system, bacterial cells multiplied while
embedded in an agarose matrix, and they generated embedded cell aggregative structures
(ECASs) that could represent the clinical biofilms formed in mucosal layers or intracellular
space (11, 12), while the conventional systems can only model attached biofilms using shear
stress conditions. The ECASs formed in our system demonstrated a sufficient number of
biofilm characteristics to be considered a type of biofilm. We suggest that the MAC can be a
good in vitro system that re-enacts biofilm formation in the mucosal or viscous environment 4
and that ECAS-derived biofilms provide a valuable model for studying the clinical relevance
of biofilms that are generated from the embedded growth of bacteria in host tissues.
Materials and Methods
Design and manufacture of the MAC For the microfluidic channel-based biofilm assay,
MACs were designed and manufactured by QuantaMatrix Inc. at Seoul National University,
as described in Fig. 1 and Fig. S1, where a straight channel for filling agarose-embedded cells
merges into a V-shape channel for flowing medium. The junction region of two channels has
an open-barrier to allow for the diffusion of nutrients, and it was manufactured as optically
transparent for direct microscopic observation. The embedded biofilms form and grow in this
area (indicated by dotted line in Fig. 1A and 1B). The MAC chip was fabricated with
polydimethylsiloxane (PDMS, (Sylgard 184, Dow Corning) and then assembled with a
PDMS-coated glass slide. The size of the chip was 50 mm by 25 mm (Fig. 1).
Bacterial strains and biofilm formation Four standard bacteria from the Clinical and
Laboratory Standard Institute (Pseudomonas aeruginosa ATCC 27853, Escherichia coli
ATCC 25922, Enterococcus faecalis ATCC 29212, and Staphylococcus aureus ATCC 29213)
and laboratory bacteria (Pseudomonas aeruginosa PA14 and its mutant strains as well as
Bacillus subtilis ATCC 6633) were studied for biofilm formation using the MAC system.
Bacterial strains were grown overnight and sub-cultured in 5 ml of fresh medium to an
optical density at 600 nm (OD600) ≈ 1.0. The cells were mixed with agarose liquid gel at a 1:3
ratio (final concentration of agarose: 0.5%, 1%, 1.5%, and 2%) and injected into the straight
channel with a syringe pump (model: 781230, KD Scientific. U.S.). After the solidification of 5
the agarose at room temperature, the nutrient was continuously supplied with flowing Luria-
Bertani (LB) broth (BD science Ltd) or pseudominimal medium 7 (PMM7) (Kisan Biotech
Ltd. South Korea) through the V-shape channel at a flow rate of 10 μl/h for 5 h with a syringe
pump. The entire MAC system was incubated at 37°C. As the ECASs were formed in the
microfluidic channel, as illustrated in Fig. 1, they were monitored by bright field microscopy.
EPS staining The ECASs were formed for 5 h in the MAC system. For SYTO 9 and
propidium iodine (PI) staining (LIVE/DEAD® BacLight™ Bacterial Viability Kit L7012,
Molecular Probes, Inc.) and for FilmTracer™ calcein (Invitrogen) staining, the staining
solutions were supplied with LB broth through a V-shaped channel for 30 min; then, the
ECASs were observed by fluorescent microscopy. The concentrations of the staining dyes
were prepared as recommended in the supplier’s manuals. For ConA (Concanavalin A
Conjugates, Invitrogen, Alexa Fluor® 350) staining, the tiny agarose block containing ECASs
was carefully removed from the MAC chip and placed on a glass slide, which was stained
overnight in ConA solution (200 μg/ml) and then rinsed with PBS (phosphate buffered
saline); the stained ECASs were then observed using fluorescent microscopy. For Congo red
staining, the agarose block was carefully removed and stained on a glass slide for 20 min in
the solution containing Congo red (40 mg/ml) and 10% tween 80 (3:1). After rinsing the
slides with water, the samples were observed using bright field microscopy.
Microscopic analysis. ECASs were monitored using an S Plan Fluor ELWD 40X lens (N.A.
0.6; Nikon Instruments, Tokyo, Japan) of an inverted optical microscope (Eclipse Ti - Nikon,
IX71, Olympus), and micrographs were obtained by employing a electron multiplying charge
coupled device camera (QuantEM:512SC – Photometrics for Eclipse Ti) and a true-color
CCD (charge-coupled device) camera (DP71 for IX71). Con A staining was examined by
epifluorescence with a Nikon inverted microscope equipped with a filter set for UV radiation
(UV-1A: DM400; Ex 365/10; BA 400); SYTO 9, FilmTracerTM, and GFP stainings were
observed under a filter set for blue light (FITC: DM 505; Ex 465-495; BA 515-555). For PI
staining, a filter set for green light (TRITC: DM 565; Ex 540/25; BA 605/55) was used.
Image analysis. For comparing the size of ECASs, ImageJ (V. 1.48) was used (16). After
adjusting the threshold values, the ECASs were detected by binarization of the raw images.
The area of the ECASs was calculated and plotted accordingly.
Scanning electron microscopy (SEM). For SEM observation of the biofilm, the tiny agarose
block containing the ECASs was carefully removed from the MAC chip and placed on a
glass slide. For ECAS fixation, the agarose block was exposed to osmium tetroxide vapor (2%
in H2O, Electron Microscopy Sciences) for 9 h in a Petri dish. The fixed agarose block was
observed under an SEM (Hitachi S-48000, acceleration voltage, 0.5 kV; current, 10 μA).
Embedded cell aggregative structure (ECAS) formation in a microfluidic agarose
When injected on the MAC system and incubated at 37°C, all bacteria tested in this study,
such as E. coli, E. faecalis, S. aureus, and P. aeruginosa, formed spherical ECASs within a
few hours (Fig. 2). When the growth of the ECASs was further monitored under time-lapse 7
microscopy, the ECASs grew larger and matured into a large spherical shape, and they
eventually burst to release individual cells (Fig. S2, Mov. S1 and S2). This growth pattern
resembles the developmental cycle of canonical biofilms. Although this embedded-type
biofilm structure derived from the embedded growth of bacteria in a gelling agent has not
been previously suggested as a biofilm, the appearance was similar to clinical biofilms found
in mucosal tissues or inside of host cells (11, 12). Additionally, while the mature ECASs
were derived from the aggregates of cells, they were apparently distinct from the simple
aggregation of individual cells or premature ECASs in microscopic images. While the
individual cells retained their cellular shapes and Brownian movement even in the
aggregation, the ECASs lacked cell shape or movement (Fig. 2, 3A, 3B, 4, 5 and Mov. S3).
Both individual cells and ECASs were often detected together in the MAC system, providing
clear contrast (Fig. 3A, 3B and Mov. S3). The ECASs generated by a GFP-expressing P.
aeruginosa strain showed a much stronger GFP signal than that of the simple aggregation of
individual cells in the MAC system (Fig. S3), demonstrating that the bacterial cells in the
biofilms of the MAC system were highly compact and also alive. We also confirmed ECAS
formation with other bacterial strains, including the well-known lab strain E. coli DH5α (data
not shown) and Bacillus subtilis ATCC 6633 (Fig. S4A, S4B, and S4C).
The maturation and integrity of the ECASs were affected by the hardness of the agarose
matrix. In the 0.5% agarose, ECAS formation in the MAC system seems to be delayed and
less rigid than ECAS formation in the 1% and 2% agarose, implying that the fixing stress
may influence ECAS formation (Fig. S5). ECAS formation was also affected by nutrients;
formation was delayed in minimum medium and was faster in complex medium (Fig. S6). 8
The results imply that ECAS formation may be influenced by environmental conditions, such
as fixing stress, viscosity, and nutrition.
ECASs are a type of biofilm containing EPS
Biofilms have some peculiar features. The most important feature of a biofilm is the
production of an extracellular polymeric substance (EPS) enclosing the bacterial cells. To
verify that the ECAS is a biofilm, the production of EPS was investigated by staining.
Several different EPS staining agents were used, such as ConA (to detect extracellular
polysaccharides), Congo red (to detect cellulose), SYTO 9 (to detect nucleic acids),
propidium iodide (to detect nucleic acids in dead cells), and FilmTracer™ (to detect both the
cellular and matrix esterase activity within biofilms). While ConA, which binds to
α-mannopyranosyl and α-glucopyranosyl residues of polysaccharides, stained the ECASs
well, simple aggregations of individual cells or premature ECASs were poorly stained,
demonstrating that mature ECASs contain a significant level of EPS (Fig. 3A, S4A).
FilmTracer™ also specifically stained ECASs (Fig. 3B, S4B). As for ConA, FilmTracer™
poorly stained individual cells even when densely aggregated (Fig. 3B, S4B). The staining
dyes for nucleic acids, SYTO 9 and propidium iodine (PI), stained the ECASs at the same
locations (Fig. 3C, S4C). PI is known to stain dead cells because the dye cannot pass through
intact cell membranes. However, because extracellular DNA is also an important EPS
component, PI can stain biofilms. Many bacteria, including P. aeruginosa, S. aureus, and E.
faecalis, generate extracellular DNA by the lysis of subpopulations (17, 18). Congo red, a dye
that is often used for biofilm staining due to its strong affinity to polysaccharides, such as
cellulose (19), also stained the ECASs well (Fig. 3D). All of these data indicate that the 9
ECASs contained abundant EPS and that cells were enclosed in this self-producing EPS.
ECASs reflect the previously reported features of biofilms
Several agents have been studied for their effect on bacterial biofilm formation. Indole has
been reported to repress the biofilm formation of E. coli but to enhance the biofilm formation
of P. aeruginosa (20). When we applied indole to our MAC system, the ECAS formation of
P. aeruginosa was significantly enhanced, whereas the ECAS formation of E. coli was
reduced (Fig. 4A and 4B, Fig. S7A and S7B). With E. coli, cells were incubated longer until most
ECASs were ruptured without indole treatment. However, the indole slowed the development of
ECASs, leaving most ECASs intact (Fig. 4B). Another biofilm-modulating agent, sodium
nitroprusside (SNP), which is a nitric oxide generator, significantly reduced ECAS formation
(Fig. 4C, Fig. S7C). Nitric oxide has been reported to repress biofilm formation by inducing
biofilm dispersion (21, 22). A previously reported anti-biofilm agent, 5f (23), also inhibited
ECAS formation in the MAC system (Fig. 4D, Fig. S7D). These results demonstrate that the
ECAS is a type of biofilm.
In P. aeruginosa, the wspF mutant is known to overproduce biofilm, and the pel mutant is
known to form less biofilm when generated using conventional systems (24, 25). When these
mutants and their parental wild-type strain, PA14, were applied into the MAC system and
their growth was monitored, the wspF mutant formed ECASs more vigorously, but the pel
mutant did not (Fig. 5, Fig. S8). The effect of the wspF mutation was not observed in the
presence of the pel mutation (Fig. 5). These results using the MAC system are consistent with
the previous results using conventional systems.
We investigated the direct image of the ECASs by scanning electron microscopy (SEM).
The SEM images show that the ECASs exhibit a biofilm-like structure, where cells are
densely aggregated, showing a slimy appearance that is distinct from individual cells (Fig. 6).
These images also demonstrate that the ECASs have morphological characteristics consistent
with a biofilm. Considering the combined results, because the ECASs formed in the MAC
system show most of the previously reported features of a biofilm, we define the ECAS as an
embedded-type biofilm and suggest that this embedded-type biofilm is a type of biofilm that
is derived from the embedded growth of bacteria within a matrix.
The MAC system was originally designed for the rapid antibiotic susceptibility test (26). In
the system, bacteria were immobilized using agarose in a microfluidic culture chamber in
which different concentrations of antibiotics were applied and single cell growth was tracked
by microscopy. This system reduced the time for the antimicrobial susceptibility test (AST)
assay to 3~4 h. Interestingly, there were two types of bacterial growth in the MAC system, a
dividing type and an aggregative type (26). We characterized the aggregative type as a new
type of biofilm.
Biofilms are often considered as “an aggregate of microbial cells adherent to a surface,
enclosed in EPS they produce”. However, it does not seem necessary to include “adherence
to a surface” in the biofilm definition. Flemming and Wingender (2010) include the other cell
aggregate types for biofilm, such as floating biofilm and sludge, which are not attached to an
surface but that show the characteristics of a biofilm (27). The aggregated forms of the 11
floating S. aureus and P. aeruginosa cells exhibit higher tolerances to antibiotics (28, 29). In
the MAC system, bacterial cells are not attached to a surface, but they are fixed within an
agarose matrix and form aggregates, which exhibit most biofilm characteristics. Bacterial
cells in the MAC system initially grew and were embedded in the matrix, produced EPS that
forms ECASs, and dispersed.
The ECASs showed previously reported features of biofilms, such as indole-mediated
biofilm enhancement in P. aeruginosa, biofilm repression by anti-biofilm agents, and some
mutational effects. Therefore, we suggest that ECASs in an agarose matrix are a new type
biofilm and have been defined as an embedded-type biofilm. The advantage of this
embedded-type biofilm as a new biofilm model is that it mimics infection-related clinical
biofilms formed in host mucosal tissues and inside of host cells. This system is clearly
distinct from the conventional flow cell systems based on the surface-attached biofilm
because clinical biofilms do not attach to hard surfaces in many cases, but they are embedded
in a matrix of viscous mucus or in the cytoplasm of host cells, such as the ECASs in the
MAC system. The flow-cell system is a more appropriate model system for environmental or
industrial biofilms formed on hard surfaces with shear stress under liquid flow, such as water-
supplying pipes, membranes of water purifying systems, industrial reactors, and medical
implants. In the chronic lung infection associated with cystic fibrosis (CF), the aggregates of
P. aeruginosa are not attached to epithelial surfaces; instead, they are within the thickened
viscous mucosal layer associated with airways (30, 31). Bjarnsholt et al. (2009) observed a
compact biofilm that formed microcolonies in chronic P. aeruginosa-infected CF patient
sputum. In urinary tract infections, E. coli forms biofilms, shown as pod-like bulges in the 12
bladder lumen, and bacteria in pods are embedded in a matrix in the host cell cytoplasm (12).
Anderson et al. (2003) discovered that the pods contained bacterial cells encased in a
polysaccharide-rich matrix surrounded by a protective shell of uroplakin. The pod-shaped
biofilm resembles the gel microcolony biofilm from the MAC system. There are other
biofilms on soil matrices or plant surfaces where bacterial cells aggregate by producing EPS
to form a biofilm (13-15). However, the type of biofilm that forms on a surface might have
rare shear stress, and its morphology is similar to the embedded type of biofilm in the MAC
system. Therefore, the MAC system, using embedded growth in a gelling agent such as
agarose, can be a more suitable model system for studying clinical biofilms, providing
researchers with an alternative choice of biofilm.
The MAC system provides a much faster, easier, and cheaper way to study biofilms. The
conventional flow cell systems take a few days to 1 week to observe the full developmental
cycle of biofilms. In the MAC system, biofilms form a few hours (3~4 h) after the inoculation
of a bacterial cell with agarose (Fig. 2), and the biofilms mature in 5~7 h. The full
developmental cycle, including dispersion, mostly terminates in 9~12 h (Fig. S2). Moreover,
conventional biofilm model systems require large and expensive equipment, including
complex tube and pump connections, bulky media for a long incubation time, and pricy
fluorescence microscopy. The MAC system requires small microfluidic channels (slide glass-
sized chip that includes multiple channels), simple tiny tubing, and several milliliters of
medium. Biofilm formation can be observed under a normal microscope without additional
accessories, and the MAC system makes it easy to observe single cells because they are
immobilized in a gel. 13
Notably, biofilm formation in the MAC system depended on the concentration of agarose.
Cell fixing stress in the agarose matrix might be a significant factor for biofilm formation and
therefore also influence clinical biofilm formation. In the flow cell systems, the flow rate of
fluid generating a shear force influences biofilm formation, EPS production, and the
induction of biofilm deformation and detachment (32, 33). These are other important features
discriminating the MAC system from conventional systems.
The MAC system can also provide some clues about single cell behavior during biofilm
formation. In the 1~1.5 % agarose matrix of the MAC system, there were simultaneously two
cell types, individual and ECAS-included cells. This observation reveals that the responses to
the same fixing stress are different among the bacterial population, although the cells
originated from the same inoculum and were cultured under the same conditions. It will be
interesting to identify the factors that predestine each cell to form a biofilm or remain as an
individual cell. The MAC system can help researchers perform single-cell level studies of
biofilm formation and inhibition.
This research was supported by a grant from the Korean Health Technology R&D Project,
Ministry of Health & Welfare, Republic of Korea (grant numbers: HI13C1468 and
HI13C0866), the Pioneer Research Center Program through the NRF of Korea funded by the
Ministry of Science, ICT & Future Planning (NRF-2012-0009555), National Research
(2012M3A7A9671610) and Basic Science Research Programs through the National Research
Foundation of Korea (NRF) funded by the Ministry of Education (formerly, the Ministry of
Education, Science and Technology) (2010-0006622 and 2013R1A1A2012220)
313 314 315
316 317 318
319 320 321
324 325 326
327 328 329
332 333 334 335
O'Toole G, Kaplan HB, Kolter R. 2000. Biofilm formation as microbial development. Annual Reviews in Microbiology 54:49-79. Monroe D. 2007. Looking for chinks in the armor of bacterial biofilms. PLoS Biol 5:e307. Hall-Stoodley L, Costerton JW, Stoodley P. 2004. Bacterial biofilms: from the natural environment to infectious diseases. Nature reviews. Microbiology 2:95-108. Mah TF, O'Toole GA. 2001. Mechanisms of biofilm resistance to antimicrobial agents. Trends Microbiol 9:34-39. McBain AJ. 2009. Chapter 4: In vitro biofilm models: an overview. Adv Appl Microbiol 69:99-132. Kearns DB, Chu F, Branda SS, Kolter R, Losick R. 2005. A master regulator for biofilm formation by Bacillus subtilis. Mol Microbiol 55:739-749. Ali L, Khambaty F, Diachenko G. 2006. Investigating the suitability of the Calgary Biofilm Device for assessing the antimicrobial efficacy of new agents. Bioresour Technol 97:1887-1893. Goeres DM, Loetterle LR, Hamilton MA, Murga R, Kirby DW, Donlan RM. 2005. Statistical assessment of a laboratory method for growing biofilms. Microbiology 151:757-762. Crabbe A, De Boever P, Van Houdt R, Moors H, Mergeay M, Cornelis P. 2008. Use of the rotating wall vessel technology to study the effect of shear stress on growth behaviour of Pseudomonas aeruginosa PA01. Environ Microbiol 10:2098-2110. Palmer RJ, Jr. 1999. Microscopy flowcells: perfusion chambers for real-time study of biofilms. Methods Enzymol 310:160-166. Hall-Stoodley L, Stoodley P, Kathju S, Høiby N, Moser C, William Costerton J, Moter A, Bjarnsholt T. 2012. Towards diagnostic guidelines for biofilm-associated infections. FEMS Immunology & Medical Microbiology 65:127-145. Anderson GG, Palermo JJ, Schilling JD, Roth R, Heuser J, Hultgren SJ. 2003. Intracellular Bacterial Biofilm-Like Pods in Urinary Tract Infections. Science 301:105-107. Monier J-M, Lindow S. 2005. Aggregates of resident bacteria facilitate survival of immigrant bacteria on leaf surfaces. Microbial Ecology 49:343-352. Chang W-S, Halverson LJ. 2003. Reduced Water Availability Influences the Dynamics, Development, and Ultrastructural Properties of Pseudomonas putida Biofilms. Journal of Bacteriology 185:6199-6204. Danhorn T, Fuqua C. 2007. Biofilm formation by plant-associated bacteria. Annu. Rev. Microbiol. 61:401-422. 15
339 340 341
348 349 350
351 352 353 354
355 356 357
358 359 360
361 362 363
364 365 366
369 370 371
372 373 374 375 376 377 378 379
Rasband W. 1997. ImageJ. US National Institutes of Health, Bethesda, Maryland, USA. Montanaro L, Poggi A, Visai L, Ravaioli S, Campoccia D, Speziale P, Arciola CR. 2011. Extracellular DNA in biofilms. The International journal of artificial organs 34:824-831. Whitchurch CB, Tolker-Nielsen T, Ragas PC, Mattick JS. 2002. Extracellular DNA required for bacterial biofilm formation. Science 295:1487. McKinney RE. 1953. Staining bacterial polysaccharides. Journal of bacteriology 66:453-454. Lee J, Jayaraman A, Wood T. 2007. Indole is an inter-species biofilm signal mediated by SdiA. BMC Microbiology 7:42. Barraud N, Hassett DJ, Hwang S-H, Rice SA, Kjelleberg S, Webb JS. 2006. Involvement of Nitric Oxide in Biofilm Dispersal of Pseudomonas aeruginosa. Journal of bacteriology 188:7344-7353. Barraud N, Schleheck D, Klebensberger J, Webb JS, Hassett DJ, Rice SA, Kjelleberg S. 2009. Nitric Oxide Signaling in Pseudomonas aeruginosa Biofilms Mediates Phosphodiesterase Activity, Decreased Cyclic Di-GMP Levels, and Enhanced Dispersal. Journal of bacteriology 191:7333-7342. Kim C, Kim J, Park H-Y, Park H-J, Lee J, Kim C, Yoon J. 2008. Furanone derivatives as quorum-sensing antagonists of Pseudomonas aeruginosa. Applied Microbiology and Biotechnology 80:37-47. Hickman JW, Tifrea DF, Harwood CS. 2005. A chemosensory system that regulates biofilm formation through modulation of cyclic diguanylate levels. Proc Natl Acad Sci U S A 102:14422-14427. Chung IY, Choi KB, Heo YJ, Cho YH. 2008. Effect of PEL exopolysaccharide on the wspF mutant phenotypes in Pseudomonas aeruginosa PA14. J Microbiol Biotechnol 18:1227-1234. Choi J, Jung YG, Kim J, Kim S, Jung Y, Na H, Kwon S. 2013. Rapid antibiotic susceptibility testing by tracking single cell growth in a microfluidic agarose channel system. Lab Chip 13:280-287. Flemming HC, Wingender J. 2010. The biofilm matrix. Nat Rev Microbiol 8:623633. Fux CA, Wilson S, Stoodley P. 2004. Detachment characteristics and oxacillin resistance of Staphyloccocus aureus biofilm emboli in an in vitro catheter infection model. J Bacteriol 186:4486-4491. Alhede M, Kragh KN, Qvortrup K, Allesen-Holm M, van Gennip M, Christensen LD, Jensen PO, Nielsen AK, Parsek M, Wozniak D, Molin S, Tolker-Nielsen T, Hoiby N, Givskov M, Bjarnsholt T. 2011. Phenotypes of nonattached Pseudomonas aeruginosa aggregates resemble surface attached biofilm. PLoS One 6:e27943. Worlitzsch D, Tarran R, Ulrich M, Schwab U, Cekici A, Meyer KC, Birrer P, Bellon G, Berger J, Weiss T, Botzenhart K, Yankaskas JR, Randell S, Boucher RC, Doring G. 2002. Effects of reduced mucus oxygen concentration in airway Pseudomonas infections of cystic fibrosis patients. J Clin Invest 109:317-325. 16
381 382 383
384 385 386 387 388
Bjarnsholt T, Jensen PO, Fiandaca MJ, Pedersen J, Hansen CR, Andersen CB, Pressler T, Givskov M, Hoiby N. 2009. Pseudomonas aeruginosa biofilms in the respiratory tract of cystic fibrosis patients. Pediatr Pulmonol 44:547-558. Stoodley P, Cargo R, Rupp CJ, Wilson S, Klapper I. 2002. Biofilm material properties as related to shear-induced deformation and detachment phenomena. J Ind Microbiol Biotechnol 29:361-367. Menniti A, Kang S, Elimelech M, Morgenroth E. 2009. Influence of shear on the production of extracellular polymeric substances in membrane bioreactors. Water Res 43:4305-4315.
Fig. 1 Schematic diagram of the microfluidic agarose channel (MAC) for developing an
embedded cell aggregative structure (ECAS). (A) The MAC chip was fabricated with
PDMS and assembled with a glass slide. One chip contained six parallel channel sets. The
tube connected to the PDMS chip is for supplying the culture medium. (B) The expanded
figure for A. The bacteria were mixed with agarose and then injected into the left hole of the
straight channel, and liquid medium was applied from a V-shaped channel. The interface of
two channels was connected by open capillary valves. Bacterial cells embedded in the
agarose grow in two forms, individually dividing cells and ECASs. (C) The full
developmental cycle of ECASs in the MAC system is illustrated. From left to right: single
cell, cell division and EPS production, ECAS formation, ECAS maturation, and ECAS
rupture and cell release. Scale bar, 100 μm.
Fig. 2. ECAS formation by various bacteria in a microfluidic channel. Four bacterial
strains, P. aeruginosa (ATCC 27853), E. coli (ATCC 25922), E. faecalis (ATCC 29212), and
S. aureus (ATCC 29213), were applied to the MAC system for biofilm formation. The sub-
cultured bacterial cells were mixed with agarose liquid gel (final concentration, 1.5%) and
injected into a straight channel by a syringe injector. After the solidification of the agarose at
room temperature, nutrients were supplied by flowing LB broth through the V-shape channel
at a flow rate of 10 μl/h for 5 h. The entire system was incubated at 37°C. ECAS formation in
the microfluidic channel was monitored by bright field microscopy for 5 h. The large white
pebble-like objects are ECASs. Some images of the biofilms were magnified for clearer
visualization. Scale bars, 50 μm.
Fig. 3. EPS production in ECASs. (A) ConA staining. The agarose patches containing the
ECASs were carefully separated from the MAC chip and directly observed on a bright field
(BF) microscope (top row) or stained overnight in ConA solution. After rinsing in PBS, the
samples were observed using a fluorescent microscope (bottom row). The bright field image
of P. aeruginosa is also recorded in a movie file, which is provided in the supplementary data
(Mov. S3). (B) FilmTracer™ calcein and (C) SYTO 9/propidium iodine (PI) staining. The
dyes were supplied through a V-shape channel for 30 min, and ECASs and individual cells
were observed with bright field and fluorescent microscopes. The premature ECASs or
simple aggregation of cells, which are poorly stained, are indicated by dotted red circles for
comparison with mature ECASs. (D) Congo red staining. The agarose block was removed
from the MAC system, stained with a Congo red solution, and observed with a microscope.
Microscopes with 40× objective lenses and bright field (BF), green (FilmTracer™ and SYTO
9), red (PI), and blue (ConA) filters were used in this study. The exposure time was 400 ms
for ConA, 300 ms for SYTO 9/PI, and 3 s for FilmTracer™. Scale bars, 50 μm.
Fig. 4. Chemical effects on ECAS formation; indole, SNP, and quorum sensing inhibitor
(5f). Bacterial cells were incubated for ~5 h in the MAC system, and during ECAS formation,
the chemicals were continually provided with LB medium through the side V-channel. ECAS
formation was monitored by bright field microscopy. (A) P. aeruginosa ATCC 27853 was
treated with 0.4 mM indole and compared with the untreated strain. (B) With E. coli ATCC
25922, the cells were incubated longer until ECAS rupture. Without indole treatment, most
ECASs ruptured. (C), (D) P. aeruginosa ATCC 27853 was treated with 10 µM SNP and 1
mM 5f, respectively, and compared with the untreated strain. The images were processed to
measure the size of the biofilms using ImageJ software (V. 1.48). The aggregative structures
in each strain were changed to a binary code, and the pixel number was measured to represent
the size of biofilms (Fig. S7). The averages (AVG) and standard deviations (STV) of the
biofilm size were calculated for comparison among different conditions. Scale bars, 50 μm.
Fig. 5. ECAS formation of biofilm mutants. The strains were incubated for ~5 h, and
ECAS growth was observed under a bright field microscope. The images were processed to
measure the size of the biofilms using ImageJ software (V. 1.48). The aggregative structures
in each strain were changed to a binary code, and the pixel number was measured to represent
the size of the biofilms (Fig S8). The average (AVG) and standard deviation (STV) values of
the biofilms from the biofilm images of each strain (P14, wspF-, pel-, and wspF-/pel-) were
compared with each other. Scale bars, 50 μm.
Fig. 6. Scanning electron microscopic (SEM) images of ECASs. Agarose patches
containing ECASs were segregated from the channel. The agarose block was fixed in osmium
tetroxide vapor for 9 h before SEM observation. Scale bars, 10 μm.