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.
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Embedded biofilm: a new biofilm model based on the embedded
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growth of bacteria
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Yong-Gyun Jung1$, Jungil Choi2,3,4$, Soo-Kyoung Kim5, Joon-Hee Lee5*, and Sunghoon
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Kwon1,2,3,4*
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6 7
8 9
10 11
1
Quantamatrix Inc, 104-213, Seoul National University, Seoul 151-742, Republic of Korea.
2
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
13
Department of Pharmacy, College of Pharmacy, Pusan National University, Busan 609-735, Republic of Korea.
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Authors’ E-mail addresses: Yong-Gyun Jung (
[email protected]), Jungil Choi
15
(
[email protected]), Soo-Kyoung Kim (
[email protected]), Joon-Hee Lee
16
(
[email protected]), and Sunghoon Kwon (
[email protected])
12
17
18
$
These authors contributed equally to this work
*Correspondence should be addressed to: Sunghoon Kwon (
[email protected]) or Joon-Hee Lee (
[email protected])
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Running Title; Embedded biofilm
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1
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Abstract
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A variety of systems have been developed to study biofilm formation. However, most of the
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systems are based on the surface-attached growth of microbes under shear stress. In this study,
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we designed a microfluidic channel device, called an MAC (microfluidic agarose channel),
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and found that microbial cells in the MAC system formed an embedded cell aggregative
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structure (ECAS). The ECASs were generated from the embedded growth of bacterial cells in
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agarose matrix and better mimicked the clinical environment of biofilms formed within
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mucus or host tissue under shear-free conditions. ECASs were developed with the production
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of extracellular polymeric substances (EPS), the most important feature of biofilms, and
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eventually burst to release planktonic cells, which resembles the full developmental cycle of
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biofilms. Chemical and genetic effects have also confirmed that ECASs are a type of biofilm.
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Unlike the conventional biofilms formed in the flow cell model system, this embedded-type
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biofilm completes the developmental cycle in only 9~12 h and can easily be observed with
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ordinary microscopes. We suggest that ECASs are a type of biofilm and that the MAC is a
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system for observing biofilm formation.
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Key words; embedded-type biofilm, biofilm model, embedded growth, extracellular polymeric
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substance, microfluidic agarose channel (MAC), embedded cell aggregative structure
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(ECAS)
2
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Introduction
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In natural habitats, bacterial cells often form biofilms to structurally organize and protect
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their communities. A biofilm is an aggregate of microbial cells that adhere to a live/non-
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living surface, and the adherent cells in biofilms are enclosed in a self-produced matrix of
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extracellular polymeric substances (EPS), including nucleic acids, proteins, polysaccharides
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and lipids (1). Bacterial biofilm formation is a complex developmental process involving
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several stages, including migration and initial attachment, EPS production and irreversible
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attachment, maturation, destruction and cell dispersion (2). The significance of biofilms is
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witnessed in the environment, industry, and human health (3). Importantly, pathogenic
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bacteria build biofilms on various medical implants and human tissues, and they have a
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strong resistance to antimicrobial agents as well as persistently colonize in chronic diseases.
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In the USA, the fouling of microorganisms on industrial equipment and architecture costs
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billions of dollars annually (4).
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To study biofilms, many researchers have developed a variety of in vitro model systems
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that mimic the environment of biofilm habitats (5), and several models are commonly used
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for biofilm assays, such as the agar plate system (6), multi-well plate system (7), biofilm
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reactor system (8, 9) and flow cell system (10). Flow and biofilm reactor systems are based
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on the surface-attached growth of microbes under shear stress. While these conventional
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models may mimic some aspects of natural environments, biofilms sometimes form in very
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different conditions that the conventional systems cannot mimic entirely. The clinically
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relevant biofilms in host tissues are formed without shear stress within the mucosal layer on
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epithelial cells or inside of host cells. Pseudomonas aeruginosa, which infects cystic fibrosis 3
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(CF) patients, does not attach to the pulmonary epithelial surfaces of CF patients, but it forms
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persistent biofilms within the thick mucosal layer on the epithelial cells of CF patients (11).
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In urinary tract infections, intracellular bacteria mature into biofilms, forming pod-like bulges
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in the mouse bladder lumen, showing a different structure from biofilms formed on surfaces
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with shear stress (12). Additionally, bacteria reside on soil matrices or plant surfaces as
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aggregates of cells in terrestrial habitats (non-shear stress conditions), and they are embedded
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in exopolymeric substances that they generate and that can be a biofilm. These biofilms are
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commonly unsaturated, and the size of the biofilms vary depending on the environmental
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conditions (13-15). Therefore, it is necessary to develop a new biofilm-modeling system that
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more closely resembles the clinical and terrestrial relevance of biofilms.
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To address this need, we have developed a new biofilm-forming system based on the
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embedded growth of bacteria using a microfluidic channel device, called an MAC
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(microfluidic agarose channel, Fig. 1A and 1B), instead of based on conventional biofilm
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generation using shear stress, such as the multi-well plate system (7), biofilm reactor system
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(8, 9) and flow cell system (10). The developed device generated a novel type biofilm-like
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structure in shear stress-free conditions. In the MAC system, bacterial cells multiplied while
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embedded in an agarose matrix, and they generated embedded cell aggregative structures
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(ECASs) that could represent the clinical biofilms formed in mucosal layers or intracellular
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space (11, 12), while the conventional systems can only model attached biofilms using shear
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stress conditions. The ECASs formed in our system demonstrated a sufficient number of
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biofilm characteristics to be considered a type of biofilm. We suggest that the MAC can be a
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good in vitro system that re-enacts biofilm formation in the mucosal or viscous environment 4
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and that ECAS-derived biofilms provide a valuable model for studying the clinical relevance
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of biofilms that are generated from the embedded growth of bacteria in host tissues.
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Materials and Methods
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Design and manufacture of the MAC For the microfluidic channel-based biofilm assay,
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MACs were designed and manufactured by QuantaMatrix Inc. at Seoul National University,
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as described in Fig. 1 and Fig. S1, where a straight channel for filling agarose-embedded cells
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merges into a V-shape channel for flowing medium. The junction region of two channels has
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an open-barrier to allow for the diffusion of nutrients, and it was manufactured as optically
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transparent for direct microscopic observation. The embedded biofilms form and grow in this
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area (indicated by dotted line in Fig. 1A and 1B). The MAC chip was fabricated with
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polydimethylsiloxane (PDMS, (Sylgard 184, Dow Corning) and then assembled with a
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PDMS-coated glass slide. The size of the chip was 50 mm by 25 mm (Fig. 1).
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Bacterial strains and biofilm formation Four standard bacteria from the Clinical and
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Laboratory Standard Institute (Pseudomonas aeruginosa ATCC 27853, Escherichia coli
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ATCC 25922, Enterococcus faecalis ATCC 29212, and Staphylococcus aureus ATCC 29213)
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and laboratory bacteria (Pseudomonas aeruginosa PA14 and its mutant strains as well as
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Bacillus subtilis ATCC 6633) were studied for biofilm formation using the MAC system.
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Bacterial strains were grown overnight and sub-cultured in 5 ml of fresh medium to an
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optical density at 600 nm (OD600) ≈ 1.0. The cells were mixed with agarose liquid gel at a 1:3
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ratio (final concentration of agarose: 0.5%, 1%, 1.5%, and 2%) and injected into the straight
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channel with a syringe pump (model: 781230, KD Scientific. U.S.). After the solidification of 5
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the agarose at room temperature, the nutrient was continuously supplied with flowing Luria-
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Bertani (LB) broth (BD science Ltd) or pseudominimal medium 7 (PMM7) (Kisan Biotech
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Ltd. South Korea) through the V-shape channel at a flow rate of 10 μl/h for 5 h with a syringe
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pump. The entire MAC system was incubated at 37°C. As the ECASs were formed in the
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microfluidic channel, as illustrated in Fig. 1, they were monitored by bright field microscopy.
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EPS staining The ECASs were formed for 5 h in the MAC system. For SYTO 9 and
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propidium iodine (PI) staining (LIVE/DEAD® BacLight™ Bacterial Viability Kit L7012,
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Molecular Probes, Inc.) and for FilmTracer™ calcein (Invitrogen) staining, the staining
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solutions were supplied with LB broth through a V-shaped channel for 30 min; then, the
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ECASs were observed by fluorescent microscopy. The concentrations of the staining dyes
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were prepared as recommended in the supplier’s manuals. For ConA (Concanavalin A
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Conjugates, Invitrogen, Alexa Fluor® 350) staining, the tiny agarose block containing ECASs
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was carefully removed from the MAC chip and placed on a glass slide, which was stained
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overnight in ConA solution (200 μg/ml) and then rinsed with PBS (phosphate buffered
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saline); the stained ECASs were then observed using fluorescent microscopy. For Congo red
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staining, the agarose block was carefully removed and stained on a glass slide for 20 min in
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the solution containing Congo red (40 mg/ml) and 10% tween 80 (3:1). After rinsing the
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slides with water, the samples were observed using bright field microscopy.
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Microscopic analysis. ECASs were monitored using an S Plan Fluor ELWD 40X lens (N.A.
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0.6; Nikon Instruments, Tokyo, Japan) of an inverted optical microscope (Eclipse Ti - Nikon,
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IX71, Olympus), and micrographs were obtained by employing a electron multiplying charge
6
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coupled device camera (QuantEM:512SC – Photometrics for Eclipse Ti) and a true-color
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CCD (charge-coupled device) camera (DP71 for IX71). Con A staining was examined by
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epifluorescence with a Nikon inverted microscope equipped with a filter set for UV radiation
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(UV-1A: DM400; Ex 365/10; BA 400); SYTO 9, FilmTracerTM, and GFP stainings were
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observed under a filter set for blue light (FITC: DM 505; Ex 465-495; BA 515-555). For PI
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staining, a filter set for green light (TRITC: DM 565; Ex 540/25; BA 605/55) was used.
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Image analysis. For comparing the size of ECASs, ImageJ (V. 1.48) was used (16). After
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adjusting the threshold values, the ECASs were detected by binarization of the raw images.
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The area of the ECASs was calculated and plotted accordingly.
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Scanning electron microscopy (SEM). For SEM observation of the biofilm, the tiny agarose
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block containing the ECASs was carefully removed from the MAC chip and placed on a
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glass slide. For ECAS fixation, the agarose block was exposed to osmium tetroxide vapor (2%
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in H2O, Electron Microscopy Sciences) for 9 h in a Petri dish. The fixed agarose block was
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observed under an SEM (Hitachi S-48000, acceleration voltage, 0.5 kV; current, 10 μA).
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Results
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Embedded cell aggregative structure (ECAS) formation in a microfluidic agarose
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channel (MAC)
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When injected on the MAC system and incubated at 37°C, all bacteria tested in this study,
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such as E. coli, E. faecalis, S. aureus, and P. aeruginosa, formed spherical ECASs within a
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few hours (Fig. 2). When the growth of the ECASs was further monitored under time-lapse 7
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microscopy, the ECASs grew larger and matured into a large spherical shape, and they
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eventually burst to release individual cells (Fig. S2, Mov. S1 and S2). This growth pattern
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resembles the developmental cycle of canonical biofilms. Although this embedded-type
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biofilm structure derived from the embedded growth of bacteria in a gelling agent has not
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been previously suggested as a biofilm, the appearance was similar to clinical biofilms found
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in mucosal tissues or inside of host cells (11, 12). Additionally, while the mature ECASs
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were derived from the aggregates of cells, they were apparently distinct from the simple
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aggregation of individual cells or premature ECASs in microscopic images. While the
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individual cells retained their cellular shapes and Brownian movement even in the
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aggregation, the ECASs lacked cell shape or movement (Fig. 2, 3A, 3B, 4, 5 and Mov. S3).
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Both individual cells and ECASs were often detected together in the MAC system, providing
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clear contrast (Fig. 3A, 3B and Mov. S3). The ECASs generated by a GFP-expressing P.
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aeruginosa strain showed a much stronger GFP signal than that of the simple aggregation of
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individual cells in the MAC system (Fig. S3), demonstrating that the bacterial cells in the
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biofilms of the MAC system were highly compact and also alive. We also confirmed ECAS
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formation with other bacterial strains, including the well-known lab strain E. coli DH5α (data
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not shown) and Bacillus subtilis ATCC 6633 (Fig. S4A, S4B, and S4C).
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The maturation and integrity of the ECASs were affected by the hardness of the agarose
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matrix. In the 0.5% agarose, ECAS formation in the MAC system seems to be delayed and
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less rigid than ECAS formation in the 1% and 2% agarose, implying that the fixing stress
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may influence ECAS formation (Fig. S5). ECAS formation was also affected by nutrients;
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formation was delayed in minimum medium and was faster in complex medium (Fig. S6). 8
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The results imply that ECAS formation may be influenced by environmental conditions, such
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as fixing stress, viscosity, and nutrition.
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ECASs are a type of biofilm containing EPS
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Biofilms have some peculiar features. The most important feature of a biofilm is the
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production of an extracellular polymeric substance (EPS) enclosing the bacterial cells. To
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verify that the ECAS is a biofilm, the production of EPS was investigated by staining.
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Several different EPS staining agents were used, such as ConA (to detect extracellular
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polysaccharides), Congo red (to detect cellulose), SYTO 9 (to detect nucleic acids),
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propidium iodide (to detect nucleic acids in dead cells), and FilmTracer™ (to detect both the
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cellular and matrix esterase activity within biofilms). While ConA, which binds to
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α-mannopyranosyl and α-glucopyranosyl residues of polysaccharides, stained the ECASs
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well, simple aggregations of individual cells or premature ECASs were poorly stained,
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demonstrating that mature ECASs contain a significant level of EPS (Fig. 3A, S4A).
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FilmTracer™ also specifically stained ECASs (Fig. 3B, S4B). As for ConA, FilmTracer™
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poorly stained individual cells even when densely aggregated (Fig. 3B, S4B). The staining
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dyes for nucleic acids, SYTO 9 and propidium iodine (PI), stained the ECASs at the same
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locations (Fig. 3C, S4C). PI is known to stain dead cells because the dye cannot pass through
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intact cell membranes. However, because extracellular DNA is also an important EPS
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component, PI can stain biofilms. Many bacteria, including P. aeruginosa, S. aureus, and E.
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faecalis, generate extracellular DNA by the lysis of subpopulations (17, 18). Congo red, a dye
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that is often used for biofilm staining due to its strong affinity to polysaccharides, such as
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cellulose (19), also stained the ECASs well (Fig. 3D). All of these data indicate that the 9
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ECASs contained abundant EPS and that cells were enclosed in this self-producing EPS.
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ECASs reflect the previously reported features of biofilms
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Several agents have been studied for their effect on bacterial biofilm formation. Indole has
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been reported to repress the biofilm formation of E. coli but to enhance the biofilm formation
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of P. aeruginosa (20). When we applied indole to our MAC system, the ECAS formation of
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P. aeruginosa was significantly enhanced, whereas the ECAS formation of E. coli was
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reduced (Fig. 4A and 4B, Fig. S7A and S7B). With E. coli, cells were incubated longer until most
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ECASs were ruptured without indole treatment. However, the indole slowed the development of
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ECASs, leaving most ECASs intact (Fig. 4B). Another biofilm-modulating agent, sodium
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nitroprusside (SNP), which is a nitric oxide generator, significantly reduced ECAS formation
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(Fig. 4C, Fig. S7C). Nitric oxide has been reported to repress biofilm formation by inducing
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biofilm dispersion (21, 22). A previously reported anti-biofilm agent, 5f (23), also inhibited
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ECAS formation in the MAC system (Fig. 4D, Fig. S7D). These results demonstrate that the
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ECAS is a type of biofilm.
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In P. aeruginosa, the wspF mutant is known to overproduce biofilm, and the pel mutant is
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known to form less biofilm when generated using conventional systems (24, 25). When these
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mutants and their parental wild-type strain, PA14, were applied into the MAC system and
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their growth was monitored, the wspF mutant formed ECASs more vigorously, but the pel
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mutant did not (Fig. 5, Fig. S8). The effect of the wspF mutation was not observed in the
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presence of the pel mutation (Fig. 5). These results using the MAC system are consistent with
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the previous results using conventional systems.
10
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We investigated the direct image of the ECASs by scanning electron microscopy (SEM).
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The SEM images show that the ECASs exhibit a biofilm-like structure, where cells are
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densely aggregated, showing a slimy appearance that is distinct from individual cells (Fig. 6).
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These images also demonstrate that the ECASs have morphological characteristics consistent
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with a biofilm. Considering the combined results, because the ECASs formed in the MAC
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system show most of the previously reported features of a biofilm, we define the ECAS as an
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embedded-type biofilm and suggest that this embedded-type biofilm is a type of biofilm that
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is derived from the embedded growth of bacteria within a matrix.
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Discussion
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The MAC system was originally designed for the rapid antibiotic susceptibility test (26). In
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the system, bacteria were immobilized using agarose in a microfluidic culture chamber in
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which different concentrations of antibiotics were applied and single cell growth was tracked
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by microscopy. This system reduced the time for the antimicrobial susceptibility test (AST)
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assay to 3~4 h. Interestingly, there were two types of bacterial growth in the MAC system, a
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dividing type and an aggregative type (26). We characterized the aggregative type as a new
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type of biofilm.
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Biofilms are often considered as “an aggregate of microbial cells adherent to a surface,
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enclosed in EPS they produce”. However, it does not seem necessary to include “adherence
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to a surface” in the biofilm definition. Flemming and Wingender (2010) include the other cell
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aggregate types for biofilm, such as floating biofilm and sludge, which are not attached to an
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surface but that show the characteristics of a biofilm (27). The aggregated forms of the 11
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floating S. aureus and P. aeruginosa cells exhibit higher tolerances to antibiotics (28, 29). In
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the MAC system, bacterial cells are not attached to a surface, but they are fixed within an
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agarose matrix and form aggregates, which exhibit most biofilm characteristics. Bacterial
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cells in the MAC system initially grew and were embedded in the matrix, produced EPS that
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forms ECASs, and dispersed.
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The ECASs showed previously reported features of biofilms, such as indole-mediated
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biofilm enhancement in P. aeruginosa, biofilm repression by anti-biofilm agents, and some
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mutational effects. Therefore, we suggest that ECASs in an agarose matrix are a new type
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biofilm and have been defined as an embedded-type biofilm. The advantage of this
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embedded-type biofilm as a new biofilm model is that it mimics infection-related clinical
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biofilms formed in host mucosal tissues and inside of host cells. This system is clearly
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distinct from the conventional flow cell systems based on the surface-attached biofilm
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because clinical biofilms do not attach to hard surfaces in many cases, but they are embedded
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in a matrix of viscous mucus or in the cytoplasm of host cells, such as the ECASs in the
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MAC system. The flow-cell system is a more appropriate model system for environmental or
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industrial biofilms formed on hard surfaces with shear stress under liquid flow, such as water-
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supplying pipes, membranes of water purifying systems, industrial reactors, and medical
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implants. In the chronic lung infection associated with cystic fibrosis (CF), the aggregates of
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P. aeruginosa are not attached to epithelial surfaces; instead, they are within the thickened
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viscous mucosal layer associated with airways (30, 31). Bjarnsholt et al. (2009) observed a
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compact biofilm that formed microcolonies in chronic P. aeruginosa-infected CF patient
253
sputum. In urinary tract infections, E. coli forms biofilms, shown as pod-like bulges in the 12
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bladder lumen, and bacteria in pods are embedded in a matrix in the host cell cytoplasm (12).
255
Anderson et al. (2003) discovered that the pods contained bacterial cells encased in a
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polysaccharide-rich matrix surrounded by a protective shell of uroplakin. The pod-shaped
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biofilm resembles the gel microcolony biofilm from the MAC system. There are other
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biofilms on soil matrices or plant surfaces where bacterial cells aggregate by producing EPS
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to form a biofilm (13-15). However, the type of biofilm that forms on a surface might have
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rare shear stress, and its morphology is similar to the embedded type of biofilm in the MAC
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system. Therefore, the MAC system, using embedded growth in a gelling agent such as
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agarose, can be a more suitable model system for studying clinical biofilms, providing
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researchers with an alternative choice of biofilm.
264
The MAC system provides a much faster, easier, and cheaper way to study biofilms. The
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conventional flow cell systems take a few days to 1 week to observe the full developmental
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cycle of biofilms. In the MAC system, biofilms form a few hours (3~4 h) after the inoculation
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of a bacterial cell with agarose (Fig. 2), and the biofilms mature in 5~7 h. The full
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developmental cycle, including dispersion, mostly terminates in 9~12 h (Fig. S2). Moreover,
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conventional biofilm model systems require large and expensive equipment, including
270
complex tube and pump connections, bulky media for a long incubation time, and pricy
271
fluorescence microscopy. The MAC system requires small microfluidic channels (slide glass-
272
sized chip that includes multiple channels), simple tiny tubing, and several milliliters of
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medium. Biofilm formation can be observed under a normal microscope without additional
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accessories, and the MAC system makes it easy to observe single cells because they are
275
immobilized in a gel. 13
276
Notably, biofilm formation in the MAC system depended on the concentration of agarose.
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Cell fixing stress in the agarose matrix might be a significant factor for biofilm formation and
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therefore also influence clinical biofilm formation. In the flow cell systems, the flow rate of
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fluid generating a shear force influences biofilm formation, EPS production, and the
280
induction of biofilm deformation and detachment (32, 33). These are other important features
281
discriminating the MAC system from conventional systems.
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The MAC system can also provide some clues about single cell behavior during biofilm
283
formation. In the 1~1.5 % agarose matrix of the MAC system, there were simultaneously two
284
cell types, individual and ECAS-included cells. This observation reveals that the responses to
285
the same fixing stress are different among the bacterial population, although the cells
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originated from the same inoculum and were cultured under the same conditions. It will be
287
interesting to identify the factors that predestine each cell to form a biofilm or remain as an
288
individual cell. The MAC system can help researchers perform single-cell level studies of
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biofilm formation and inhibition.
290
Acknowledgements
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This research was supported by a grant from the Korean Health Technology R&D Project,
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Ministry of Health & Welfare, Republic of Korea (grant numbers: HI13C1468 and
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HI13C0866), the Pioneer Research Center Program through the NRF of Korea funded by the
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Ministry of Science, ICT & Future Planning (NRF-2012-0009555), National Research
295
Foundation
296
(2012M3A7A9671610) and Basic Science Research Programs through the National Research
of
Korea
(NRF)
Grant
funded
14
by
the
Korean
Government
297
Foundation of Korea (NRF) funded by the Ministry of Education (formerly, the Ministry of
298
Education, Science and Technology) (2010-0006622 and 2013R1A1A2012220)
299
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Figure legends
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Fig. 1 Schematic diagram of the microfluidic agarose channel (MAC) for developing an
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embedded cell aggregative structure (ECAS). (A) The MAC chip was fabricated with
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PDMS and assembled with a glass slide. One chip contained six parallel channel sets. The
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tube connected to the PDMS chip is for supplying the culture medium. (B) The expanded
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figure for A. The bacteria were mixed with agarose and then injected into the left hole of the
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straight channel, and liquid medium was applied from a V-shaped channel. The interface of
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two channels was connected by open capillary valves. Bacterial cells embedded in the
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agarose grow in two forms, individually dividing cells and ECASs. (C) The full
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developmental cycle of ECASs in the MAC system is illustrated. From left to right: single
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cell, cell division and EPS production, ECAS formation, ECAS maturation, and ECAS
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rupture and cell release. Scale bar, 100 μm.
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Fig. 2. ECAS formation by various bacteria in a microfluidic channel. Four bacterial
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strains, P. aeruginosa (ATCC 27853), E. coli (ATCC 25922), E. faecalis (ATCC 29212), and
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S. aureus (ATCC 29213), were applied to the MAC system for biofilm formation. The sub-
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cultured bacterial cells were mixed with agarose liquid gel (final concentration, 1.5%) and
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injected into a straight channel by a syringe injector. After the solidification of the agarose at
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room temperature, nutrients were supplied by flowing LB broth through the V-shape channel
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at a flow rate of 10 μl/h for 5 h. The entire system was incubated at 37°C. ECAS formation in
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the microfluidic channel was monitored by bright field microscopy for 5 h. The large white
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pebble-like objects are ECASs. Some images of the biofilms were magnified for clearer
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visualization. Scale bars, 50 μm.
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Fig. 3. EPS production in ECASs. (A) ConA staining. The agarose patches containing the
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ECASs were carefully separated from the MAC chip and directly observed on a bright field
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(BF) microscope (top row) or stained overnight in ConA solution. After rinsing in PBS, the
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samples were observed using a fluorescent microscope (bottom row). The bright field image
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of P. aeruginosa is also recorded in a movie file, which is provided in the supplementary data
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(Mov. S3). (B) FilmTracer™ calcein and (C) SYTO 9/propidium iodine (PI) staining. The
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dyes were supplied through a V-shape channel for 30 min, and ECASs and individual cells
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were observed with bright field and fluorescent microscopes. The premature ECASs or
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simple aggregation of cells, which are poorly stained, are indicated by dotted red circles for
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comparison with mature ECASs. (D) Congo red staining. The agarose block was removed
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from the MAC system, stained with a Congo red solution, and observed with a microscope.
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Microscopes with 40× objective lenses and bright field (BF), green (FilmTracer™ and SYTO
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9), red (PI), and blue (ConA) filters were used in this study. The exposure time was 400 ms
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for ConA, 300 ms for SYTO 9/PI, and 3 s for FilmTracer™. Scale bars, 50 μm.
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Fig. 4. Chemical effects on ECAS formation; indole, SNP, and quorum sensing inhibitor
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(5f). Bacterial cells were incubated for ~5 h in the MAC system, and during ECAS formation,
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the chemicals were continually provided with LB medium through the side V-channel. ECAS
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formation was monitored by bright field microscopy. (A) P. aeruginosa ATCC 27853 was
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treated with 0.4 mM indole and compared with the untreated strain. (B) With E. coli ATCC
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25922, the cells were incubated longer until ECAS rupture. Without indole treatment, most
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ECASs ruptured. (C), (D) P. aeruginosa ATCC 27853 was treated with 10 µM SNP and 1
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mM 5f, respectively, and compared with the untreated strain. The images were processed to
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measure the size of the biofilms using ImageJ software (V. 1.48). The aggregative structures
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in each strain were changed to a binary code, and the pixel number was measured to represent
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the size of biofilms (Fig. S7). The averages (AVG) and standard deviations (STV) of the
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biofilm size were calculated for comparison among different conditions. Scale bars, 50 μm.
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Fig. 5. ECAS formation of biofilm mutants. The strains were incubated for ~5 h, and
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ECAS growth was observed under a bright field microscope. The images were processed to
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measure the size of the biofilms using ImageJ software (V. 1.48). The aggregative structures
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in each strain were changed to a binary code, and the pixel number was measured to represent
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the size of the biofilms (Fig S8). The average (AVG) and standard deviation (STV) values of
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the biofilms from the biofilm images of each strain (P14, wspF-, pel-, and wspF-/pel-) were
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compared with each other. Scale bars, 50 μm.
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Fig. 6. Scanning electron microscopic (SEM) images of ECASs. Agarose patches
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containing ECASs were segregated from the channel. The agarose block was fixed in osmium
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tetroxide vapor for 9 h before SEM observation. Scale bars, 10 μm.
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