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
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Quantamatrix Inc, 104-213, Seoul National University, Seoul 151-742, Republic of Korea.
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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
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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
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([email protected]), Soo-Kyoung Kim ([email protected]), Joon-Hee Lee
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([email protected]), and Sunghoon Kwon ([email protected])
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17
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$
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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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)
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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
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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).
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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.
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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
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complex tube and pump connections, bulky media for a long incubation time, and pricy
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fluorescence microscopy. The MAC system requires small microfluidic channels (slide glass-
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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
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immobilized in a gel. 13
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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
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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
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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
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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.
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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
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(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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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
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384 385 386 387 388
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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.
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17
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Figure legends
440
Fig. 1 Schematic diagram of the microfluidic agarose channel (MAC) for developing an
441
embedded cell aggregative structure (ECAS). (A) The MAC chip was fabricated with
442
PDMS and assembled with a glass slide. One chip contained six parallel channel sets. The
443
tube connected to the PDMS chip is for supplying the culture medium. (B) The expanded
444
figure for A. The bacteria were mixed with agarose and then injected into the left hole of the
445
straight channel, and liquid medium was applied from a V-shaped channel. The interface of
446
two channels was connected by open capillary valves. Bacterial cells embedded in the
447
agarose grow in two forms, individually dividing cells and ECASs. (C) The full
448
developmental cycle of ECASs in the MAC system is illustrated. From left to right: single
449
cell, cell division and EPS production, ECAS formation, ECAS maturation, and ECAS
450
rupture and cell release. Scale bar, 100 μm.
451
Fig. 2. ECAS formation by various bacteria in a microfluidic channel. Four bacterial
452
strains, P. aeruginosa (ATCC 27853), E. coli (ATCC 25922), E. faecalis (ATCC 29212), and
453
S. aureus (ATCC 29213), were applied to the MAC system for biofilm formation. The sub-
454
cultured bacterial cells were mixed with agarose liquid gel (final concentration, 1.5%) and
455
injected into a straight channel by a syringe injector. After the solidification of the agarose at
456
room temperature, nutrients were supplied by flowing LB broth through the V-shape channel
457
at a flow rate of 10 μl/h for 5 h. The entire system was incubated at 37°C. ECAS formation in
458
the microfluidic channel was monitored by bright field microscopy for 5 h. The large white
21
459
pebble-like objects are ECASs. Some images of the biofilms were magnified for clearer
460
visualization. Scale bars, 50 μm.
461
Fig. 3. EPS production in ECASs. (A) ConA staining. The agarose patches containing the
462
ECASs were carefully separated from the MAC chip and directly observed on a bright field
463
(BF) microscope (top row) or stained overnight in ConA solution. After rinsing in PBS, the
464
samples were observed using a fluorescent microscope (bottom row). The bright field image
465
of P. aeruginosa is also recorded in a movie file, which is provided in the supplementary data
466
(Mov. S3). (B) FilmTracer™ calcein and (C) SYTO 9/propidium iodine (PI) staining. The
467
dyes were supplied through a V-shape channel for 30 min, and ECASs and individual cells
468
were observed with bright field and fluorescent microscopes. The premature ECASs or
469
simple aggregation of cells, which are poorly stained, are indicated by dotted red circles for
470
comparison with mature ECASs. (D) Congo red staining. The agarose block was removed
471
from the MAC system, stained with a Congo red solution, and observed with a microscope.
472
Microscopes with 40× objective lenses and bright field (BF), green (FilmTracer™ and SYTO
473
9), red (PI), and blue (ConA) filters were used in this study. The exposure time was 400 ms
474
for ConA, 300 ms for SYTO 9/PI, and 3 s for FilmTracer™. Scale bars, 50 μm.
475
Fig. 4. Chemical effects on ECAS formation; indole, SNP, and quorum sensing inhibitor
476
(5f). Bacterial cells were incubated for ~5 h in the MAC system, and during ECAS formation,
477
the chemicals were continually provided with LB medium through the side V-channel. ECAS
478
formation was monitored by bright field microscopy. (A) P. aeruginosa ATCC 27853 was
479
treated with 0.4 mM indole and compared with the untreated strain. (B) With E. coli ATCC
22
480
25922, the cells were incubated longer until ECAS rupture. Without indole treatment, most
481
ECASs ruptured. (C), (D) P. aeruginosa ATCC 27853 was treated with 10 µM SNP and 1
482
mM 5f, respectively, and compared with the untreated strain. The images were processed to
483
measure the size of the biofilms using ImageJ software (V. 1.48). The aggregative structures
484
in each strain were changed to a binary code, and the pixel number was measured to represent
485
the size of biofilms (Fig. S7). The averages (AVG) and standard deviations (STV) of the
486
biofilm size were calculated for comparison among different conditions. Scale bars, 50 μm.
487
Fig. 5. ECAS formation of biofilm mutants. The strains were incubated for ~5 h, and
488
ECAS growth was observed under a bright field microscope. The images were processed to
489
measure the size of the biofilms using ImageJ software (V. 1.48). The aggregative structures
490
in each strain were changed to a binary code, and the pixel number was measured to represent
491
the size of the biofilms (Fig S8). The average (AVG) and standard deviation (STV) values of
492
the biofilms from the biofilm images of each strain (P14, wspF-, pel-, and wspF-/pel-) were
493
compared with each other. Scale bars, 50 μm.
494
Fig. 6. Scanning electron microscopic (SEM) images of ECASs. Agarose patches
495
containing ECASs were segregated from the channel. The agarose block was fixed in osmium
496
tetroxide vapor for 9 h before SEM observation. Scale bars, 10 μm.
23
1
Embedded biofilm: a new biofilm model based on the embedded
2
growth of bacteria
3
Yong-Gyun Jung1$, Jungil Choi2,3,4$, Soo-Kyoung Kim5, Joon-Hee Lee5*, and Sunghoon
4
Kwon1,2,3,4*
5
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.
14
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])
19
20
Running Title; Embedded biofilm
21
1
22
Abstract
23
A variety of systems have been developed to study biofilm formation. However, most of the
24
systems are based on the surface-attached growth of microbes under shear stress. In this study,
25
we designed a microfluidic channel device, called an MAC (microfluidic agarose channel),
26
and found that microbial cells in the MAC system formed an embedded cell aggregative
27
structure (ECAS). The ECASs were generated from the embedded growth of bacterial cells in
28
agarose matrix and better mimicked the clinical environment of biofilms formed within
29
mucus or host tissue under shear-free conditions. ECASs were developed with the production
30
of extracellular polymeric substances (EPS), the most important feature of biofilms, and
31
eventually burst to release planktonic cells, which resembles the full developmental cycle of
32
biofilms. Chemical and genetic effects have also confirmed that ECASs are a type of biofilm.
33
Unlike the conventional biofilms formed in the flow cell model system, this embedded-type
34
biofilm completes the developmental cycle in only 9~12 h and can easily be observed with
35
ordinary microscopes. We suggest that ECASs are a type of biofilm and that the MAC is a
36
system for observing biofilm formation.
37
Key words; embedded-type biofilm, biofilm model, embedded growth, extracellular polymeric
38
substance, microfluidic agarose channel (MAC), embedded cell aggregative structure
39
(ECAS)
2
40
Introduction
41
In natural habitats, bacterial cells often form biofilms to structurally organize and protect
42
their communities. A biofilm is an aggregate of microbial cells that adhere to a live/non-
43
living surface, and the adherent cells in biofilms are enclosed in a self-produced matrix of
44
extracellular polymeric substances (EPS), including nucleic acids, proteins, polysaccharides
45
and lipids (1). Bacterial biofilm formation is a complex developmental process involving
46
several stages, including migration and initial attachment, EPS production and irreversible
47
attachment, maturation, destruction and cell dispersion (2). The significance of biofilms is
48
witnessed in the environment, industry, and human health (3). Importantly, pathogenic
49
bacteria build biofilms on various medical implants and human tissues, and they have a
50
strong resistance to antimicrobial agents as well as persistently colonize in chronic diseases.
51
In the USA, the fouling of microorganisms on industrial equipment and architecture costs
52
billions of dollars annually (4).
53
To study biofilms, many researchers have developed a variety of in vitro model systems
54
that mimic the environment of biofilm habitats (5), and several models are commonly used
55
for biofilm assays, such as the agar plate system (6), multi-well plate system (7), biofilm
56
reactor system (8, 9) and flow cell system (10). Flow and biofilm reactor systems are based
57
on the surface-attached growth of microbes under shear stress. While these conventional
58
models may mimic some aspects of natural environments, biofilms sometimes form in very
59
different conditions that the conventional systems cannot mimic entirely. The clinically
60
relevant biofilms in host tissues are formed without shear stress within the mucosal layer on
61
epithelial cells or inside of host cells. Pseudomonas aeruginosa, which infects cystic fibrosis 3
62
(CF) patients, does not attach to the pulmonary epithelial surfaces of CF patients, but it forms
63
persistent biofilms within the thick mucosal layer on the epithelial cells of CF patients (11).
64
In urinary tract infections, intracellular bacteria mature into biofilms, forming pod-like bulges
65
in the mouse bladder lumen, showing a different structure from biofilms formed on surfaces
66
with shear stress (12). Additionally, bacteria reside on soil matrices or plant surfaces as
67
aggregates of cells in terrestrial habitats (non-shear stress conditions), and they are embedded
68
in exopolymeric substances that they generate and that can be a biofilm. These biofilms are
69
commonly unsaturated, and the size of the biofilms vary depending on the environmental
70
conditions (13-15). Therefore, it is necessary to develop a new biofilm-modeling system that
71
more closely resembles the clinical and terrestrial relevance of biofilms.
72
To address this need, we have developed a new biofilm-forming system based on the
73
embedded growth of bacteria using a microfluidic channel device, called an MAC
74
(microfluidic agarose channel, Fig. 1A and 1B), instead of based on conventional biofilm
75
generation using shear stress, such as the multi-well plate system (7), biofilm reactor system
76
(8, 9) and flow cell system (10). The developed device generated a novel type biofilm-like
77
structure in shear stress-free conditions. In the MAC system, bacterial cells multiplied while
78
embedded in an agarose matrix, and they generated embedded cell aggregative structures
79
(ECASs) that could represent the clinical biofilms formed in mucosal layers or intracellular
80
space (11, 12), while the conventional systems can only model attached biofilms using shear
81
stress conditions. The ECASs formed in our system demonstrated a sufficient number of
82
biofilm characteristics to be considered a type of biofilm. We suggest that the MAC can be a
83
good in vitro system that re-enacts biofilm formation in the mucosal or viscous environment 4
84
and that ECAS-derived biofilms provide a valuable model for studying the clinical relevance
85
of biofilms that are generated from the embedded growth of bacteria in host tissues.
86
Materials and Methods
87
Design and manufacture of the MAC For the microfluidic channel-based biofilm assay,
88
MACs were designed and manufactured by QuantaMatrix Inc. at Seoul National University,
89
as described in Fig. 1 and Fig. S1, where a straight channel for filling agarose-embedded cells
90
merges into a V-shape channel for flowing medium. The junction region of two channels has
91
an open-barrier to allow for the diffusion of nutrients, and it was manufactured as optically
92
transparent for direct microscopic observation. The embedded biofilms form and grow in this
93
area (indicated by dotted line in Fig. 1A and 1B). The MAC chip was fabricated with
94
polydimethylsiloxane (PDMS, (Sylgard 184, Dow Corning) and then assembled with a
95
PDMS-coated glass slide. The size of the chip was 50 mm by 25 mm (Fig. 1).
96
Bacterial strains and biofilm formation Four standard bacteria from the Clinical and
97
Laboratory Standard Institute (Pseudomonas aeruginosa ATCC 27853, Escherichia coli
98
ATCC 25922, Enterococcus faecalis ATCC 29212, and Staphylococcus aureus ATCC 29213)
99
and laboratory bacteria (Pseudomonas aeruginosa PA14 and its mutant strains as well as
100
Bacillus subtilis ATCC 6633) were studied for biofilm formation using the MAC system.
101
Bacterial strains were grown overnight and sub-cultured in 5 ml of fresh medium to an
102
optical density at 600 nm (OD600) ≈ 1.0. The cells were mixed with agarose liquid gel at a 1:3
103
ratio (final concentration of agarose: 0.5%, 1%, 1.5%, and 2%) and injected into the straight
104
channel with a syringe pump (model: 781230, KD Scientific. U.S.). After the solidification of 5
105
the agarose at room temperature, the nutrient was continuously supplied with flowing Luria-
106
Bertani (LB) broth (BD science Ltd) or pseudominimal medium 7 (PMM7) (Kisan Biotech
107
Ltd. South Korea) through the V-shape channel at a flow rate of 10 μl/h for 5 h with a syringe
108
pump. The entire MAC system was incubated at 37°C. As the ECASs were formed in the
109
microfluidic channel, as illustrated in Fig. 1, they were monitored by bright field microscopy.
110
EPS staining The ECASs were formed for 5 h in the MAC system. For SYTO 9 and
111
propidium iodine (PI) staining (LIVE/DEAD® BacLight™ Bacterial Viability Kit L7012,
112
Molecular Probes, Inc.) and for FilmTracer™ calcein (Invitrogen) staining, the staining
113
solutions were supplied with LB broth through a V-shaped channel for 30 min; then, the
114
ECASs were observed by fluorescent microscopy. The concentrations of the staining dyes
115
were prepared as recommended in the supplier’s manuals. For ConA (Concanavalin A
116
Conjugates, Invitrogen, Alexa Fluor® 350) staining, the tiny agarose block containing ECASs
117
was carefully removed from the MAC chip and placed on a glass slide, which was stained
118
overnight in ConA solution (200 μg/ml) and then rinsed with PBS (phosphate buffered
119
saline); the stained ECASs were then observed using fluorescent microscopy. For Congo red
120
staining, the agarose block was carefully removed and stained on a glass slide for 20 min in
121
the solution containing Congo red (40 mg/ml) and 10% tween 80 (3:1). After rinsing the
122
slides with water, the samples were observed using bright field microscopy.
123
Microscopic analysis. ECASs were monitored using an S Plan Fluor ELWD 40X lens (N.A.
124
0.6; Nikon Instruments, Tokyo, Japan) of an inverted optical microscope (Eclipse Ti - Nikon,
125
IX71, Olympus), and micrographs were obtained by employing a electron multiplying charge
6
126
coupled device camera (QuantEM:512SC – Photometrics for Eclipse Ti) and a true-color
127
CCD (charge-coupled device) camera (DP71 for IX71). Con A staining was examined by
128
epifluorescence with a Nikon inverted microscope equipped with a filter set for UV radiation
129
(UV-1A: DM400; Ex 365/10; BA 400); SYTO 9, FilmTracerTM, and GFP stainings were
130
observed under a filter set for blue light (FITC: DM 505; Ex 465-495; BA 515-555). For PI
131
staining, a filter set for green light (TRITC: DM 565; Ex 540/25; BA 605/55) was used.
132
Image analysis. For comparing the size of ECASs, ImageJ (V. 1.48) was used (16). After
133
adjusting the threshold values, the ECASs were detected by binarization of the raw images.
134
The area of the ECASs was calculated and plotted accordingly.
135
Scanning electron microscopy (SEM). For SEM observation of the biofilm, the tiny agarose
136
block containing the ECASs was carefully removed from the MAC chip and placed on a
137
glass slide. For ECAS fixation, the agarose block was exposed to osmium tetroxide vapor (2%
138
in H2O, Electron Microscopy Sciences) for 9 h in a Petri dish. The fixed agarose block was
139
observed under an SEM (Hitachi S-48000, acceleration voltage, 0.5 kV; current, 10 μA).
140
Results
141
Embedded cell aggregative structure (ECAS) formation in a microfluidic agarose
142
channel (MAC)
143
When injected on the MAC system and incubated at 37°C, all bacteria tested in this study,
144
such as E. coli, E. faecalis, S. aureus, and P. aeruginosa, formed spherical ECASs within a
145
few hours (Fig. 2). When the growth of the ECASs was further monitored under time-lapse 7
146
microscopy, the ECASs grew larger and matured into a large spherical shape, and they
147
eventually burst to release individual cells (Fig. S2, Mov. S1 and S2). This growth pattern
148
resembles the developmental cycle of canonical biofilms. Although this embedded-type
149
biofilm structure derived from the embedded growth of bacteria in a gelling agent has not
150
been previously suggested as a biofilm, the appearance was similar to clinical biofilms found
151
in mucosal tissues or inside of host cells (11, 12). Additionally, while the mature ECASs
152
were derived from the aggregates of cells, they were apparently distinct from the simple
153
aggregation of individual cells or premature ECASs in microscopic images. While the
154
individual cells retained their cellular shapes and Brownian movement even in the
155
aggregation, the ECASs lacked cell shape or movement (Fig. 2, 3A, 3B, 4, 5 and Mov. S3).
156
Both individual cells and ECASs were often detected together in the MAC system, providing
157
clear contrast (Fig. 3A, 3B and Mov. S3). The ECASs generated by a GFP-expressing P.
158
aeruginosa strain showed a much stronger GFP signal than that of the simple aggregation of
159
individual cells in the MAC system (Fig. S3), demonstrating that the bacterial cells in the
160
biofilms of the MAC system were highly compact and also alive. We also confirmed ECAS
161
formation with other bacterial strains, including the well-known lab strain E. coli DH5α (data
162
not shown) and Bacillus subtilis ATCC 6633 (Fig. S4A, S4B, and S4C).
163
The maturation and integrity of the ECASs were affected by the hardness of the agarose
164
matrix. In the 0.5% agarose, ECAS formation in the MAC system seems to be delayed and
165
less rigid than ECAS formation in the 1% and 2% agarose, implying that the fixing stress
166
may influence ECAS formation (Fig. S5). ECAS formation was also affected by nutrients;
167
formation was delayed in minimum medium and was faster in complex medium (Fig. S6). 8
168
The results imply that ECAS formation may be influenced by environmental conditions, such
169
as fixing stress, viscosity, and nutrition.
170
ECASs are a type of biofilm containing EPS
171
Biofilms have some peculiar features. The most important feature of a biofilm is the
172
production of an extracellular polymeric substance (EPS) enclosing the bacterial cells. To
173
verify that the ECAS is a biofilm, the production of EPS was investigated by staining.
174
Several different EPS staining agents were used, such as ConA (to detect extracellular
175
polysaccharides), Congo red (to detect cellulose), SYTO 9 (to detect nucleic acids),
176
propidium iodide (to detect nucleic acids in dead cells), and FilmTracer™ (to detect both the
177
cellular and matrix esterase activity within biofilms). While ConA, which binds to
178
α-mannopyranosyl and α-glucopyranosyl residues of polysaccharides, stained the ECASs
179
well, simple aggregations of individual cells or premature ECASs were poorly stained,
180
demonstrating that mature ECASs contain a significant level of EPS (Fig. 3A, S4A).
181
FilmTracer™ also specifically stained ECASs (Fig. 3B, S4B). As for ConA, FilmTracer™
182
poorly stained individual cells even when densely aggregated (Fig. 3B, S4B). The staining
183
dyes for nucleic acids, SYTO 9 and propidium iodine (PI), stained the ECASs at the same
184
locations (Fig. 3C, S4C). PI is known to stain dead cells because the dye cannot pass through
185
intact cell membranes. However, because extracellular DNA is also an important EPS
186
component, PI can stain biofilms. Many bacteria, including P. aeruginosa, S. aureus, and E.
187
faecalis, generate extracellular DNA by the lysis of subpopulations (17, 18). Congo red, a dye
188
that is often used for biofilm staining due to its strong affinity to polysaccharides, such as
189
cellulose (19), also stained the ECASs well (Fig. 3D). All of these data indicate that the 9
190
ECASs contained abundant EPS and that cells were enclosed in this self-producing EPS.
191
ECASs reflect the previously reported features of biofilms
192
Several agents have been studied for their effect on bacterial biofilm formation. Indole has
193
been reported to repress the biofilm formation of E. coli but to enhance the biofilm formation
194
of P. aeruginosa (20). When we applied indole to our MAC system, the ECAS formation of
195
P. aeruginosa was significantly enhanced, whereas the ECAS formation of E. coli was
196
reduced (Fig. 4A and 4B, Fig. S7A and S7B). With E. coli, cells were incubated longer until most
197
ECASs were ruptured without indole treatment. However, the indole slowed the development of
198
ECASs, leaving most ECASs intact (Fig. 4B). Another biofilm-modulating agent, sodium
199
nitroprusside (SNP), which is a nitric oxide generator, significantly reduced ECAS formation
200
(Fig. 4C, Fig. S7C). Nitric oxide has been reported to repress biofilm formation by inducing
201
biofilm dispersion (21, 22). A previously reported anti-biofilm agent, 5f (23), also inhibited
202
ECAS formation in the MAC system (Fig. 4D, Fig. S7D). These results demonstrate that the
203
ECAS is a type of biofilm.
204
In P. aeruginosa, the wspF mutant is known to overproduce biofilm, and the pel mutant is
205
known to form less biofilm when generated using conventional systems (24, 25). When these
206
mutants and their parental wild-type strain, PA14, were applied into the MAC system and
207
their growth was monitored, the wspF mutant formed ECASs more vigorously, but the pel
208
mutant did not (Fig. 5, Fig. S8). The effect of the wspF mutation was not observed in the
209
presence of the pel mutation (Fig. 5). These results using the MAC system are consistent with
210
the previous results using conventional systems.
10
211
We investigated the direct image of the ECASs by scanning electron microscopy (SEM).
212
The SEM images show that the ECASs exhibit a biofilm-like structure, where cells are
213
densely aggregated, showing a slimy appearance that is distinct from individual cells (Fig. 6).
214
These images also demonstrate that the ECASs have morphological characteristics consistent
215
with a biofilm. Considering the combined results, because the ECASs formed in the MAC
216
system show most of the previously reported features of a biofilm, we define the ECAS as an
217
embedded-type biofilm and suggest that this embedded-type biofilm is a type of biofilm that
218
is derived from the embedded growth of bacteria within a matrix.
219
Discussion
220
The MAC system was originally designed for the rapid antibiotic susceptibility test (26). In
221
the system, bacteria were immobilized using agarose in a microfluidic culture chamber in
222
which different concentrations of antibiotics were applied and single cell growth was tracked
223
by microscopy. This system reduced the time for the antimicrobial susceptibility test (AST)
224
assay to 3~4 h. Interestingly, there were two types of bacterial growth in the MAC system, a
225
dividing type and an aggregative type (26). We characterized the aggregative type as a new
226
type of biofilm.
227
Biofilms are often considered as “an aggregate of microbial cells adherent to a surface,
228
enclosed in EPS they produce”. However, it does not seem necessary to include “adherence
229
to a surface” in the biofilm definition. Flemming and Wingender (2010) include the other cell
230
aggregate types for biofilm, such as floating biofilm and sludge, which are not attached to an
231
surface but that show the characteristics of a biofilm (27). The aggregated forms of the 11
232
floating S. aureus and P. aeruginosa cells exhibit higher tolerances to antibiotics (28, 29). In
233
the MAC system, bacterial cells are not attached to a surface, but they are fixed within an
234
agarose matrix and form aggregates, which exhibit most biofilm characteristics. Bacterial
235
cells in the MAC system initially grew and were embedded in the matrix, produced EPS that
236
forms ECASs, and dispersed.
237
The ECASs showed previously reported features of biofilms, such as indole-mediated
238
biofilm enhancement in P. aeruginosa, biofilm repression by anti-biofilm agents, and some
239
mutational effects. Therefore, we suggest that ECASs in an agarose matrix are a new type
240
biofilm and have been defined as an embedded-type biofilm. The advantage of this
241
embedded-type biofilm as a new biofilm model is that it mimics infection-related clinical
242
biofilms formed in host mucosal tissues and inside of host cells. This system is clearly
243
distinct from the conventional flow cell systems based on the surface-attached biofilm
244
because clinical biofilms do not attach to hard surfaces in many cases, but they are embedded
245
in a matrix of viscous mucus or in the cytoplasm of host cells, such as the ECASs in the
246
MAC system. The flow-cell system is a more appropriate model system for environmental or
247
industrial biofilms formed on hard surfaces with shear stress under liquid flow, such as water-
248
supplying pipes, membranes of water purifying systems, industrial reactors, and medical
249
implants. In the chronic lung infection associated with cystic fibrosis (CF), the aggregates of
250
P. aeruginosa are not attached to epithelial surfaces; instead, they are within the thickened
251
viscous mucosal layer associated with airways (30, 31). Bjarnsholt et al. (2009) observed a
252
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
254
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
256
polysaccharide-rich matrix surrounded by a protective shell of uroplakin. The pod-shaped
257
biofilm resembles the gel microcolony biofilm from the MAC system. There are other
258
biofilms on soil matrices or plant surfaces where bacterial cells aggregate by producing EPS
259
to form a biofilm (13-15). However, the type of biofilm that forms on a surface might have
260
rare shear stress, and its morphology is similar to the embedded type of biofilm in the MAC
261
system. Therefore, the MAC system, using embedded growth in a gelling agent such as
262
agarose, can be a more suitable model system for studying clinical biofilms, providing
263
researchers with an alternative choice of biofilm.
264
The MAC system provides a much faster, easier, and cheaper way to study biofilms. The
265
conventional flow cell systems take a few days to 1 week to observe the full developmental
266
cycle of biofilms. In the MAC system, biofilms form a few hours (3~4 h) after the inoculation
267
of a bacterial cell with agarose (Fig. 2), and the biofilms mature in 5~7 h. The full
268
developmental cycle, including dispersion, mostly terminates in 9~12 h (Fig. S2). Moreover,
269
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
273
medium. Biofilm formation can be observed under a normal microscope without additional
274
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.
277
Cell fixing stress in the agarose matrix might be a significant factor for biofilm formation and
278
therefore also influence clinical biofilm formation. In the flow cell systems, the flow rate of
279
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.
282
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
286
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
289
biofilm formation and inhibition.
290
Acknowledgements
291
This research was supported by a grant from the Korean Health Technology R&D Project,
292
Ministry of Health & Welfare, Republic of Korea (grant numbers: HI13C1468 and
293
HI13C0866), the Pioneer Research Center Program through the NRF of Korea funded by the
294
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
440
Fig. 1 Schematic diagram of the microfluidic agarose channel (MAC) for developing an
441
embedded cell aggregative structure (ECAS). (A) The MAC chip was fabricated with
442
PDMS and assembled with a glass slide. One chip contained six parallel channel sets. The
443
tube connected to the PDMS chip is for supplying the culture medium. (B) The expanded
444
figure for A. The bacteria were mixed with agarose and then injected into the left hole of the
445
straight channel, and liquid medium was applied from a V-shaped channel. The interface of
446
two channels was connected by open capillary valves. Bacterial cells embedded in the
447
agarose grow in two forms, individually dividing cells and ECASs. (C) The full
448
developmental cycle of ECASs in the MAC system is illustrated. From left to right: single
449
cell, cell division and EPS production, ECAS formation, ECAS maturation, and ECAS
450
rupture and cell release. Scale bar, 100 μm.
451
Fig. 2. ECAS formation by various bacteria in a microfluidic channel. Four bacterial
452
strains, P. aeruginosa (ATCC 27853), E. coli (ATCC 25922), E. faecalis (ATCC 29212), and
453
S. aureus (ATCC 29213), were applied to the MAC system for biofilm formation. The sub-
454
cultured bacterial cells were mixed with agarose liquid gel (final concentration, 1.5%) and
455
injected into a straight channel by a syringe injector. After the solidification of the agarose at
456
room temperature, nutrients were supplied by flowing LB broth through the V-shape channel
457
at a flow rate of 10 μl/h for 5 h. The entire system was incubated at 37°C. ECAS formation in
458
the microfluidic channel was monitored by bright field microscopy for 5 h. The large white
21
459
pebble-like objects are ECASs. Some images of the biofilms were magnified for clearer
460
visualization. Scale bars, 50 μm.
461
Fig. 3. EPS production in ECASs. (A) ConA staining. The agarose patches containing the
462
ECASs were carefully separated from the MAC chip and directly observed on a bright field
463
(BF) microscope (top row) or stained overnight in ConA solution. After rinsing in PBS, the
464
samples were observed using a fluorescent microscope (bottom row). The bright field image
465
of P. aeruginosa is also recorded in a movie file, which is provided in the supplementary data
466
(Mov. S3). (B) FilmTracer™ calcein and (C) SYTO 9/propidium iodine (PI) staining. The
467
dyes were supplied through a V-shape channel for 30 min, and ECASs and individual cells
468
were observed with bright field and fluorescent microscopes. The premature ECASs or
469
simple aggregation of cells, which are poorly stained, are indicated by dotted red circles for
470
comparison with mature ECASs. (D) Congo red staining. The agarose block was removed
471
from the MAC system, stained with a Congo red solution, and observed with a microscope.
472
Microscopes with 40× objective lenses and bright field (BF), green (FilmTracer™ and SYTO
473
9), red (PI), and blue (ConA) filters were used in this study. The exposure time was 400 ms
474
for ConA, 300 ms for SYTO 9/PI, and 3 s for FilmTracer™. Scale bars, 50 μm.
475
Fig. 4. Chemical effects on ECAS formation; indole, SNP, and quorum sensing inhibitor
476
(5f). Bacterial cells were incubated for ~5 h in the MAC system, and during ECAS formation,
477
the chemicals were continually provided with LB medium through the side V-channel. ECAS
478
formation was monitored by bright field microscopy. (A) P. aeruginosa ATCC 27853 was
479
treated with 0.4 mM indole and compared with the untreated strain. (B) With E. coli ATCC
22
480
25922, the cells were incubated longer until ECAS rupture. Without indole treatment, most
481
ECASs ruptured. (C), (D) P. aeruginosa ATCC 27853 was treated with 10 µM SNP and 1
482
mM 5f, respectively, and compared with the untreated strain. The images were processed to
483
measure the size of the biofilms using ImageJ software (V. 1.48). The aggregative structures
484
in each strain were changed to a binary code, and the pixel number was measured to represent
485
the size of biofilms (Fig. S7). The averages (AVG) and standard deviations (STV) of the
486
biofilm size were calculated for comparison among different conditions. Scale bars, 50 μm.
487
Fig. 5. ECAS formation of biofilm mutants. The strains were incubated for ~5 h, and
488
ECAS growth was observed under a bright field microscope. The images were processed to
489
measure the size of the biofilms using ImageJ software (V. 1.48). The aggregative structures
490
in each strain were changed to a binary code, and the pixel number was measured to represent
491
the size of the biofilms (Fig S8). The average (AVG) and standard deviation (STV) values of
492
the biofilms from the biofilm images of each strain (P14, wspF-, pel-, and wspF-/pel-) were
493
compared with each other. Scale bars, 50 μm.
494
Fig. 6. Scanning electron microscopic (SEM) images of ECASs. Agarose patches
495
containing ECASs were segregated from the channel. The agarose block was fixed in osmium
496
tetroxide vapor for 9 h before SEM observation. Scale bars, 10 μm.
23
