Accepted Manuscript Corneal Biofilms: From planktonic to microcolony formation in an experimental keratitis infection with Pseudomonas aeruginosa Padmanabhan Saraswathi, PhD, Roger W. Beuerman, PhD PII:
S1542-0124(15)00109-3
DOI:
10.1016/j.jtos.2015.07.001
Reference:
JTOS 139
To appear in:
Ocular Surface
Received Date: 24 March 2014 Revised Date:
1 July 2015
Accepted Date: 9 July 2015
Please cite this article as: Saraswathi P, Beuerman RW, Corneal Biofilms: From planktonic to microcolony formation in an experimental keratitis infection with Pseudomonas aeruginosa, Ocular Surface (2015), doi: 10.1016/j.jtos.2015.07.001. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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ACCEPTED MANUSCRIPT SECTION: Original Research, Ali Djalilian, MD, Editor TITLE: Corneal Biofilms: From planktonic to microcolony formation in an experimental keratitis infection with Pseudomonas aeruginosa Padmanabhan Saraswathi, PhD,1 and Roger W. Beuerman, PhD 1- 3
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SHORT TITLE: CORNEAL BIOFILMS AND PSEUDOMONAS AERUGINOSA /Sawaswathi and Beuerman FOOTNOTES
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Accepted for publication July 2015.
From 1Singapore Eye Research Institute (SERI), 2Duke-NUS SRP Neuroscience and
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Behavioural Disorders and Emerging Infectious Diseases, and 3Ophthalmology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore. Funding and support: This study was supported by SERI-PILOT grant R959/68/2012, NMRC/TCR/002-SERI/2012/R1018 and ETPL/10-S10FSH-008.
in this article.
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The authors have no commercial or proprietary interest in any concept of product discussed
Single-copy reprint requests to Roger W Beuerman, PhD (address below). Corresponding author: Roger W Beuerman, PhD, Singapore Eye Research Institute (SERI),
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The Academia, Level 6, 20 College Road, Singapore-169856. Tel: 65 6576 7215 Fax: 65
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6225 2568. E-mail address:
[email protected]
ABSTRACT
Purpose: Microbial biofilms commonly comprise part of the infectious scenario, complicating the therapeutic approach. The purpose of this study was to determine in a mouse model of corneal infection if mature biofilms formed and to visualize the stages of biofilm formation. Methods: A bacterial keratitis model was established using Pseudomonas aeruginosa ATCC 9027 (1x108 CFU/ml) to infect the cornea of C57BL/6 black mouse. Eyes were examined post-infection (PI) on days 1, 2, 3, 5, and 7, and imaged by slit lamp microscopy, and light, confocal, and electron microscopy to identify the stages of biofilm
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ACCEPTED MANUSCRIPT formation and the time of appearance. Results: On PI day 1, Gram staining showed rodshaped bacteria adherent on the corneal surface. On PI days 2 and 3, bacteria were seen within webs of extracellular polymeric substance (EPS) and glycocalyx secretion, imaged by confocal microscopy. Scanning electron microscopy demonstrated microcolonies of active infectious cells bound with thick fibrous material. Transmission electron microscopy
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substantiated the formation of classical biofilm architecture with P. aeruginosa densely packed within the extracellular polymeric substances on PI days 5 and 7. Conclusion: Direct visual evidence showed that biofilms routinely developed on the biotic surface of the mouse cornea. The mouse model can be used to develop new approaches to deal therapeutically with
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biofilms in corneal infections.
KEY WORDS biofilm, imaging, microscopy, keratitis, polymeric extracellular substance,
Outline I.
Introduction
II.
Materials and Methods
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Pseudomonas aeruginosa
Animal Use
B.
Bacteria
C.
Experimental Corneal Infections
D.
Slit Lamp Microscopy
E.
Light Microscopy
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A.
F.
Confocal Laser Scanning Microscopy
G.
Scanning Electron Microscopy
H.
Transmission Electron Microscopy
III.
Results
IV.
Discussion
V.
Conclusion
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ACCEPTED MANUSCRIPT I.
Introduction Bacterial and fungal infections of the ocular surface are common worldwide,
particularly in Southeast Asia, and can damage the structure of the cornea, leading to visual disability or blindness.1-3 Recent microbiological studies have emphasized that most pathogens prefer to live in specialized communities called biofilms rather than as isolated
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organisms, the so-called planktonic state.4-6 Consequently, the role of biofilms in infection management has begun to receive more attention. Bacteria as individual organisms are directly susceptible to environmental conditions, including the presence of antibiotics; however, biofilms are associated with antibiotic resistance.7-9
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Costerton and co-workers10,11 defined a biofilm as “a structural community of bacterial cells enclosed in a self-promoted matrix and adherent to an inert or living surface.”
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The dynamic process of biofilm formation is initiated by adherence on a surface (abiotic or biotic) followed by proliferation and attachment within a secreted matrix, which enhances the modified phenotypes to form resistant infections.12-15 The secreted matrix referred to as extracellular polymeric substances (EPS) protects the microbial community, and antibiotic resistance of up to 1000 times has been reported.4,5,16 Thus, it is currently thought that while most infections will have a biofilm phase, chronic infections may be more prone to
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developing mature biofilms, which may account for a lack of response to antibiotics.11,17 Development of biofilms on inert surfaces under laboratory conditions have been well studied. Biofilms on mucosal surfaces have been documented in tonsillitis,18 rhinosinusitis,19
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cystic fibrosis,20 and otitis media.21 Although biofilms in the eye have been discussed,22 there is no evidence that corneal infections develop mature biofilms, which are the most likely to contribute to treatment resistance.
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Corneal infections are common complications of both daily and extended wear
contact lens wear, accounting for 12-66% of all microbial keratitis in Europe and the US.22-30 P. aeruginosa, an ubiquitous biofilm producer, is often associated with contact lens wear, with a somewhat greater incidence of infection in tropical climates.31-33 Bacterial adhesion, an initial step in biofilm formation may be influenced by surface hydrophobicity,34 and subsequently formation of a sessile biofilm anchors the bacteria to the surface of the lenses. A worldwide outbreak of fungal keratitis due to Fusarium solani infection was linked with a contaminated multipurpose contact lens disinfecting solution.35 Although biofilms were not investigated at that time as a possible cause, shortly thereafter Imamura et al showed that Fusarium readily forms biofilms on contact lenses.36 Biofilms have also been associated with
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ACCEPTED MANUSCRIPT contact lens cases, and it was found that 82/101 cases were contaminated; in 78 of those 82, the primary contaminant was bacteria.37 Gram-positive cocci are also common corneal pathogens reported in non-contact lensrelated keratitis38-41 and conjunctivitis.42 Infective crystalline keratopathy, an indolent corneal infection may be caused by the accumulation of bacterial EPS and is often due to
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Streptococcus viridans, as well as Staphylococcus, Enterococcus or Candida.43-46 Medical treatments of infective crystalline keratopathy can be difficult due to the presence of a
biofilm, and laser therapy in addition to antibiotics has been used to disrupt the biofilm.47 Endopthalmitis, a potentially blinding intraocular infection, is a rare complication of cataract surgery48 in which Staphyloccocus aureus and Staphyloccocus epidermidis biofilms have
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been identified.49-51 Additionally, materials like sutures, scleral buckles, glaucoma tubes, stents, corneal transplants, and other ocular prostheses are solid, abiotic substrates that may
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contribute to the risk of an infection complicated by biofilm formation transferring into a biotic biofilm.52-53
Antimicrobial treatments for biofilm infections could be problematic if based on the Minimum Inhibitory Concentration (MIC) breakpoints.54 The MIC of an antibiotic is based on the Clinical Laboratory Standards Institute (CLSI) microbiology testing method using
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planktonic bacteria in the microbiology laboratory, which may be inadequate for killing organisms embedded in biofilms. This has prompted the use of a new term called the Minimum Biofilm Eradication Concentration (MBEC), as higher concentrations of antibiotics could be necessary to penetrate the biofilm EPS matrix and kill the genetically
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diverse population existing in the biofilm community.55 Biofilm treatment should involve 1) exploration of high potency specialized drugs, 2) examination of drug delivery mechanisms,
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and 3) determination of biofilm vulnerabilities. Developing a corneal model of biofilms as presented here may be particularly relevant, as there is opportunity to correlate stage of biofilm formation and antibiotic efficacy optimized for therapeutic outcomes and host immune responses.
Structurally, a biofilm consists of bacteria compacted by a matrix of polymeric
substances, built up over a substrate, resulting in a three dimensional biomass.56 Imaging methods, including electron microscopy, are needed to visualize the biofilm architecture.57 Real time imaging of live biofilm structures with horizontal and vertical sectioning of hydrated biofilms by optical methods is feasible using Confocal Laser Scanning Microscopy (CLSM).58 The purpose of the present study was to utilize different imaging methods to visualize the development of mature bacterial biofilms in an experimental infection of the
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ACCEPTED MANUSCRIPT mouse cornea with an ATCC strain of P. aeruginosa. It was found that biofilms routinely develop in a standard model of infection in the mouse, and the imaging results showed that all stages of biofilm development existed in experimental corneal infections with Pseudomonas. The corneal biofilm model is particularly useful, as the infection can be easily
II.
Materials and Methods
A.
Animal Use
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followed and observed.
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Wild type C57BL/6 mice (7-8 weeks old) purchased from the National University of Singapore (NUS) were used in this study. Handling and care of all animals were performed according to the guidelines adopted by SingHealth Institutional Animal Care and Use
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Committee (IACUC), and the study was carried out in accordance with the Association for Research in Vision and Ophthalmology (ARVO) guidelines for animal experimentation.
B.
Bacteria
P. aeruginosa ATCC 9027 strain was grown overnight in Tryptic Soy Agar (TSA)
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Plates at 350 C. Colonies were picked up and suspended in sterile saline at a concentration of 1×108 CFU/mL for use in this study.
Experimental Corneal Infections
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C.
Initially, all eyes of the mice were examined by slit lamp to ensure that there were no corneal abnormalities, such as vascularization or other defects. Mice were anesthetized by an
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intraperitoneal injection of xylazine (10mg/kg, Troy Laboratories, Smithfield, Australia) and ketamine (80mg/kg, Ketamine, Parnell Laboratories, Australia). With the aid of a dissecting microscope (Zeiss, Stemi-2000 C), four superficial abrasions of 1-2 mm in length were made on the corneal epithelium, using a sterile miniblade (BD-Beaver no-376400), and immediately a 5-µl aliquot of P. aeruginosa suspension was applied topically on the cornea. The eyes were examined daily by slit lamp and sacrificed at PI days 1, 2, 3, 5, and 7 for imaging. Separate groups of mice were used for slit lamp microscopy, light microscopy, CLSM, SEM and TEM analysis (n=6 mice/group).
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ACCEPTED MANUSCRIPT D.
Slit Lamp Microscopy The corneal infection was followed by slit lamp microscopy over the course of the
study. The corneal surface was photographed under normal light and after fluorescein instillation (2%) by slit lamp (NS-2D, Righton, Tokyo, Japan).
Light Microscopy
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E.
At each time point, globes were enucleated and fixed in Hartmann’s fixative (Sigma) and stored at 40C. Eyes were washed and processed in a Sakura Tissue-Tek R VIPTM 5 and embedded (Tissue-Tek R TEC TM Paraffin Embedding Centre). Sections were cut at 5 µm using a Leica RM2255 microtome, then dried, deparaffinized and hydrated using a graded
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series of alcohols, and stained with Brown-Hopps Gram stain.59 Selected areas of the stained
F.
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sections were imaged (Axioplan 2; Carl Zeissmeditec GmbH, Oberkochen, Germany). Confocal Laser Scanning Microscopy
CLSM was used with dual staining to assess the presence of bacterial biofilms on the corneal surface. Confocal imaging using fluorescent material is a useful tool to study biofilm on its cellular biochemical identification of the biofilm glycocalyx, and it does not require fixation.60 At each time point, corneas were dissected from the globe, washed with phosphate
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buffered saline (PBS) and stained with propidium iodide (Live/Dead BacLight TM bacterial viability kit –Invitrogen, Molecular Probes, Eugene, OR) for 15 minutes at room temperature. After being washed in PBS, the corneas were stained with 50 µg/mL of fluorescein
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isothiocyanate-conjugated concanavalin A (FITC-con A, Sigma-Aldrich Corp, St. Louis, MO, USA) for 15 minutes at room temperature to stain the extracellular secretions. After staining, corneas were washed three times with PBS followed by sterile deionized water, and
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mounted on slides for imaging. Propidium Iodide was imaged at 520nm, the emission was monitored at 620 nm, and FITC-con A was excited and monitored at 495 nm and 525 nm, respectively. Images were collected using a Carl Zeiss LSM710 confocal microscope and Zen software was used for image processing.
G.
Scanning Electron Microscopy Bacteria on the corneal surface were imaged with SEM at 1, 2, 3, 5, and 7 days PI.
The globes were enucleated, washed in PBS and prefixed in 1 ml of a mixed aldehyde fixative (2% glutaraldehyde and 2% paraformaldehyde in 0.1 M sodium cacodylate buffer,
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ACCEPTED MANUSCRIPT pH 7.2) for 24 hours. Then the samples were washed in sodium cacodylate buffer (Electron Microscopy Sciences, Washington, USA) and subsequently post-fixed in 1% osmium tetroxide (Electron Microscopy Sciences, Washington, USA). After post-fixation, samples were washed in sodium cacodylate buffer and dehydrated, using an ascending series of ethanol solutions. Following dehydration, the samples were critical-point-dried, attached to
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carbon stubs, and sputter-coated with 10 nm of gold. All samples were seen and photographed on a FESEM at an accelerating voltage of 3 kV at the Carl Zeiss Microscopic facility at the National University of Singapore. Transmission Electron Microscopy
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H.
TEM was utilized to view the adherence, invasion, and biofilm formation on the cornea. On PI days 1, 2, 3, 5, and 7, corneas were dissected from the globe, washed in PBS,
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and prefixed in 1 ml of a mixed aldehyde fixative (2% glutaraldehyde and 2% paraformaldehyde in 0.1 M sodium cacodylate buffer, pH 7.2) for 24 hours. Samples were subsequently washed in sodium cacodylate buffer (Electron Microscopy Sciences, Washington, USA) and subsequently post-fixed in 1% osmium tetroxide (Electron Microscopy Sciences, Washington, USA). For post-fixation, samples were again washed in
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sodium cacodylate buffer and dehydrated, using an ascending series of ethanol solutions and embedded in araldite (Electron Microscopy Sciences, Washington, USA). Ultrathin sections (90 nm) were cut from blocks with a Reichert-Jung Ultracut E Ultramicrotome (C.Reichert Optiche Werks AG, Vienna Austria). All ultrathin sections were collected on copper grids
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and doubled-stained with uranyl acetate and lead citrate for 20 minutes, dried, and viewed under a TEM (JEOL- JEM1010, NUS Electron Microscopy Facility, National University of Singapore, Singapore) at 100 kV and the areas of interest were photographed. Other than
III.
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brightness and contrast, no other image processing adjustments were made to the images.
Results
The normal structure of the mouse cornea was, of course, dramatically affected by the progress of the experimental bacterial infection. In this study, we were primarily interested in the appearance of immune cells, especially neutrophils, and also in determining if the bacteria remained as in a planktonic stage or if the classic signs of biofilm formation were discernible. Although the steps leading to biofilm formation are described below in detail, it may be helpful to refer to an overall chart of the events below in Table 1.
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ACCEPTED MANUSCRIPT The mouse cornea prior to infection was structurally intact, and fluorescent images did not reveal staining (Figures 1-A1 and A2). CLSM images were negative for FITC-con A and propidium iodide staining revealed corneal epithelial cells (Figure 1-B). Imaging the glycocalyx provided a means of showing the EPS as a biofilm marker. Histology with Gram staining displayed well-defined corneal layers with normally arranged epithelial cells and
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compact stroma with keratocytes, but without neutrophils (Figure 1-C). SEM showed a smooth layer of hexagonal epithelial cells with well-defined lateral borders (Figure 1-D1) and microvilli, visible at high magnification (Figure 1-D2). TEM revealed the normal corneal epithelial organization (Figure 1-E1), and in Figure 1-E2 the epithelial cells and microvilli were seen undamaged in higher magnification. The overall appearance was similar to what
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has been previously described for the human cornea.61 At PI day 1, corneal structural changes reflected the rapid pace of the infection, the epithelium was damaged, and an opacity forming
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(Figures 2-A1 and A2) with very little FITC-con A staining as in Figure 2-B. Gram staining showed P. aeruginosa, as typical rod-shaped appearance, pink in color (Figure 2-C). Histologically, the corneal surface was occupied with remnants of epithelium and numerous neutrophils migrated into the stroma (Fig 2-C). SEM images showed adherent bacteria at sites over the surface of the cornea (Figure 2-D1). A fine network extended over the bacteria was
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likely the release of Neutrophil Extracellular Traps (NETs), which were visible at high magnification (Figure 2-D2). TEM corroborated epithelial damage and P. aeruginosa had already invaded into the stroma, with some bacteria engulfed by neutrophils (Fig 2-E). Thus, on PI day 1, P. aeruginosa infection was accompanied by corneal damage with
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the rod-shaped bacteria seen scattered discretely in small groups, along with neutrophil activity. On PI day 2, increasing opacity and epithelial damage were seen along with positive
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glycocalyx staining. Figure 3-B shows the corneal epithelium with faint red staining of propidium iodide, while neutrophils were distinguished by a dark red color with a few positioned above the green-stained areas (Figure 3-B). Light microscopy revealed clusters of darkly staining bacteria adhering to the stroma (Figure 3-C). By SEM, small colonies of bacteria were seen (Figure 3-D1) at higher magnification; the bacteria were trapped within a more dense fibrous material (Figure 3-D2). These microcolonies did not cover large areas, but were localized as patches with active cells (Figure 3-D2). At PI day 2, bacteria became arranged in a unique layered architecture, confirming the formation of a structured biofilm (Figure 3-E1). Bacteria in the basal layer were loosely fixed in the secreted matrix, but in the middle layer, the bacteria appeared tightly packed with an electron-dense fibrous substance
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ACCEPTED MANUSCRIPT bound closely to the organisms. However, bacteria in the top layer appeared more mobile, as in Figure 3-E2. Increased corneal opacity, hypopyon, and vascularization were characterized on PI day 3 (Figure 4-A1). An epithelial defect (Figure 4-A2) with an uneven distribution of fluorescein staining was seen in some mice (Table 1). FITC-con A staining revealed an
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amorphous material within the ulcer, occupying much of the corneal surface (Figure 4-B). Distinct bacteria were not clear, but vaguely visible embedded within the fibrous matter.
Light microscopy imaging revealed bacterial clusters and numerous neutrophils in the stroma (Figure 4-C). SEM showed a mature biofilm on the severely damaged corneal surface,
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including the diagnostic mushroom-shaped bodies and tower-like structures in which the bacteria are firmly attached within the matrix (Figure 4-D1). Free bacteria were seen in less involved areas of the cornea and below the biofilm clusters (Figures 4-D2 and D3).
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Neutrophils were noted on the surface of the biofilm, while bacteria were deep-packed within the EPS. At this stage, the ultrastructure of the biofilm diverged from the previous stages. Bacterial cells were arranged compactly as a rigid layer with some neutrophils (Figure 4-E1). Thus, the established biofilm with the secreted EPS incorporating the bacteria were seen, as in images in Figure 4-E2. On PI days 5-7, the corneal opacity with hypopyon and
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vascularization were largely unchanged (Figures 5-A1 and 6-A1) with a marked fluorescein uptake (Figures 5-A2 and 6-A2). FITC-con A staining was the same as the earlier stages of the biofilm (Figures 5-B and 6-B). During these later stages of infection, neutrophils were observed in the anterior stroma. Individual bacteria were not clearly visible and were
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probably masked by the EPS, which could be seen by light microscopy (Figures 5-C and 6C). SEM images showed P. aeruginosa and neutrophils on the surface of the biofilm, as in
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the earlier biofilm stages without release of NETs (Figures 5-D1 and 5-D2). Some bacterial constellations were seen occupying large areas of the cornea, and the biofilm assembly appeared slightly compressed on PI day 7 (Figures 6-D1 and D2); however, the buildup of the biofilm body was alike from PI days 5 to 7. TEM also indicated a strong attachment of the P. aeruginosa biofilm onto the mouse cornea (Figures 5-E1 and 6-E1), and the bacterial cells appeared immobile inside the secreted material (Figures 5-E2 and 6-E2). Overall, the areal extent of the biofilm enlarged with bacteria more deeply embedded within the secreted matrix. Thus, the architectural shift of planktonic bacteria to microcolonies proceeded fairly rapidly in all corneas and then evolved into a mature biofilm covering larger areas of the cornea.
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ACCEPTED MANUSCRIPT IV.
Discussion Research on corneal infections has detailed many aspects of an infection: microbial
adhesion, corneal pathogenicity, role of immune cells, and mechanisms of innate and adaptive immunity on ocular microbiology.66-69 Biofilm infections of the eye have been discussed in a recent review.22 The development of corneal biofilm has not been observed;
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this is important, as antibiotic resistance is found more when the EPS compacts the bacteria. The present study has provided structural evidence for the classical stages of biofilm
formation in an experimental infection in the mouse cornea. This model can be used to determine how biofilms affect therapeutic outcomes.
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The biofilm required more than 24 hours to form in the mouse cornea and was
accompanied by a slight opacity at PI day 1 without FITC-con A staining, indicating that the bacteria had not yet begun to form an EPS. Bacteria were planktonic in the early stages of the
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infection, as evidenced by the presence of rod-shaped P. aeruginosa as discrete single organisms. Additionally, SEM images confirmed that P. aeruginosa were not embedded within electron-dense substances, but were shown to be trapped by NETs. Furthermore, phagocytosis by neutrophils, as observed in TEM images, indicated an early immune defense. The strain of Pseudomonas ATCC 9027 has been found to be a moderate biofilm producer, and as used in our laboratory for several years, shows consistent sensitivity to most
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antimicrobials at low MIC levels.70-72 ATCC 9027 does have some ability to invade epithelial cells, which would be in counter-distinction to their ability to form biofilms.73 The obvious corneal opacity on PI day 2 and FITC-con A staining were evident and
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detected by CLSM imaging, corroborating other studies.74 Positive staining for FITC-con A revealed the secretion of a bacterial glycocalyx, the EPS, which promotes adherence to
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nearby bacilli as well as to the corneal surface, producing the sessile biofilm.75 Gram staining showed distinct clusters of attached bacteria on PI day 1. Similarly, the presence of densely packed masses of bacterial biofilms in nasal polyps of patients was confirmed using Gram staining.76
Small microcolonies of P. aeruginosa seen by SEM were organized as a biofilm with
bacterial cells embedded inside the fibrous material. The arrangement of microcolonies as patches could be clearly distinguished from the ridges and folds of the damaged corneal surface. Bacterial cells appeared healthy and the dividing cells within the microcolony indicated viability. TEM further defined biofilm architecture of P. aeruginosa with the bacteria spatially arranged as basal, middle, and top layer. Thus, by PI day 2, considerable evidence for the initiation of biofilm formation on the mouse cornea was obtained.
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ACCEPTED MANUSCRIPT At PI day 3, FITC-con A staining of fibrous structures and masses of bacteria denoted the EPS, glycocalyx, and maturation of the biofilm. Imaging bacterial cells as well as the matrix on mucosal biofilms was achieved by using a dual staining technique in combination with CLSM.75,78 Confocal imaging was also used to demonstrate the presence of biofilms in patients
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with tonsillitis.18 FITC-con A has a high affinity for sugar residues, which specifically bind to α-mannose (2-epimer of D-glucose) residues of the polysaccharides and is considered as a marker for detecting the biofilm glycocalyx.74 Neutrophils seen by light and electron
microscopy around masses of bacteria were notable. Patches of bacteria within network-like organizations consisting of active cells were imaged on the surface of the tonsillar epithelial
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surface by SEM.18 In another study, Robert et al used SEM to detect group A Streptococcus biofilms on the epithelial crypts of tonsillar tissues of pediatric patients.79 Planktonic bacteria
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scattered underneath the mushroom-shaped bodies, indicated possible detachment from the mature biofilm. The ultrastructure of the biofilm differs from the preceding stage as it was comprised of packed bacteria as a dense rigid stratum.
On PI day 5, CLSM as well as light and electron microscopy observations, revealed the continuous presence of the mature biofilm, which was apparently active even with the
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host immune response. SEM images of pneumococcal biofilms on nasopharyngeal tissues and thick bacterial biofilms of nontypable Haemophilus influenzae encrusted on chinchilla middle-ear mucosa were similarly described.80 Another study of imaging with SEM and CLSM provided evidence for biofilm formation in an experimental model of otitis media.81 In
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the later stages of infection, the bacteria were not clearly visible and were probably masked by the increased secretion of EPS. In this study, biofilm formation persisted until PI day 7
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with corneal perforation in a few cases (2 mice were immediately sacrificed at that point). Our results using Gram staining were useful in differentiating planktonic and biofilm
stages of P. aeruginosa in the infected eye. Gram staining, a simple but older technique, is considered as a “gold standard’’ in clinical microbiology. Normal hematoxylin and eosin staining have also been useful to detect and confirm the existence of biofilms in patient samples.82 Recently, Fluorescence in Situ Hybridization (FISH)83 has also been used; however, Gram staining seems to be a simpler technique, particularly when corroborated by other methods. The imaging techniques applied here are particularly advantageous in microbiology studies, especially the combination of CLSM and SEM. CLSM also enables the quantification of 3-dimensional biofilm structures in mucosal tissues.84
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ACCEPTED MANUSCRIPT The biofilm-forming ability of Staphylococcus aureus, a major pathogen for bovine mastitis was also detected by glycocalyx production.85 In damaged skin, CLSM was used to detect glycocalyx of S. aureus biofilms 4 hours after bacterial inoculation.86 CLSM was used to detect biofilms in skin lesions of bullous impetigo, atopic dermatitis, and Pemphigus foliaceus,87 and upper airways in otolaryngology diseases.88 Thus, CLSM imaging has an
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important role in biofilm research, as it does not require dehydration of tissues and provides better preservation of biofilm structure.
Our SEM study substantiated biofilm development from P. aeruginosa on the infected eye with morphologically well-distinguished stages of mature biofilm formation. TEM
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images revealed that P. aeruginosa maintained its rod-shaped morphology irrespective of planktonic stage or matrix embedded biofilm microcolonies. Chole et al observed more complex biofilm colonies with densely packed bacteria of varying morphological appearance
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by TEM and the cells were seen in a homogeneous amorphous matrix.89
In an ultrastructural study of endodontic biofilm communities, lytic bacteria were seen anchored in a collagenous matrix with a variety of morphologies with significant differences in precise spatial arrangements.90 However, the corneal infections, as reported here, and subsequent biofilm formation are a site where all stages of biofilm formation have been easily
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imaged.
Limitations of the present study may be in the control group; the cornea was imaged prior to infection, and separate groups of mice were used for various imaging procedures. The aim of the current study was to determine the organizational details of P. aerguinosa in
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planktonic and biofilm modes in vivo. An experimental infection of the mouse cornea is straight-forward and particularly useful for biofilm studies, as the corneal surface changes
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can be correlated with in vivo observations.The current experiments have used real-time imaging, documenting the developing biofilms on the biotic surfaces of the cornea in the absence of abiotic material.91 The results explained the existence of biofilm formation in the eye in a standard infection and may be useful to determine the impact of biofilms on therapeutic efforts and as one of the targeted domains of the Human Microbiome Project launched by the National Institutes of Health.92
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Conclusion This study provides strong direct evidence for the occurrence of bacterial biofilm
formation on the corneal surface of the mice in an experimentally induced P. aeruginosa keratitis infection. The results suggest that mature biofilms are a common component of
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ACCEPTED MANUSCRIPT keratitis and need to be considered as a source of therapeutic difficulty when resistance to treatment occurs.
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Acknowledgements We extend our apprecaition to the Carl Zeiss facility at National University of Singapore for use of the FE-SEM and special appreciation to Chris Park for assistance. We thank
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Guillaume Mercy Thierry for assistance with CLSM imaging at DUKE- NUS, Singapore
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Figure Legends:
Figure 1: Structural status of the cornea before infection. The corneas of the mice used in
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these studies were initially examined by slit lamp and appeared normal. A1. Image taken in normal light and (A2) after instillation of fluorescein. B. CLSM imaging, propidium iodide produced little staining of the normal corneal epithelial cells (X63) and FITC-con A was not detected. C. Cellular organization of the normal cornea following Gram staining (X63); welldefined epithelial cell organization (Epi) and compact stroma (St) were present. D1. SEM image of corneal surface cells showed the hexagonal outer epithelial cell (Epi) layer with
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tightly adherent lateral cell borders. D2. Higher magnification SEM image showing microvilli (Mv). E1. TEM image of corneal epithelium; microvilli (Mv) and cell features such as mitochondria (Mc) and desmosomes were seen at high magnification (E2).
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Figure 2: Post infection day 1. A1. Slit lamp images of mice cornea on PI day 1 with corneal opacity and (A2) epithelial defect and fluorescein staining. B. CLSM image (X40) showed limited staining by FITC-con A, and corneal epithelium faint red in color. C. Gram staining
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revealed P. aeruginosa on the surface of the cornea with remnants of the epithelium (Epi). Stroma (St) and Neutrophils (N) (Black arrow indicates bacteria). D1. SEM image of infected mice cornea; bacteria are scattered on the corneal surface and organized in small groups. (Black arrow). D2.Magnified view of a cluster of P. aeruginosa on the corneal stroma (St). Network fibrils were seen over the surface of bacterial clusters (White arrow), which were probably the extracellular traps from the neutrophils. E. TEM image of infected mice cornea. P. aeruginosa were seen within the damaged epithelium (Epi) and stroma (St). Some bacteria were observed to be engulfed by neutrophils (black arrow).
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ACCEPTED MANUSCRIPT Figure 3: Post-infection day 2. A1. Corneal opacity. A2. Intense fluorescein staining across the cornea. B. Appearance of strong FITC-con A (green) staining by CLSM (X40) revealed the presence of glycocalyx. Propidium iodide left the corneal epithelium with a faint red color while neutrophils were stained a darker red and were distributed around and above the glycocalyx. C. Small clusters of P. aeruginosa (colonies) attached to the stroma were seen by
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Gram staining. D1. Black arrows indicate attached colonies of P. aeruginosa with a few detached cells. D2. SEM shows bacteria in microcolonies (black arrow) attached on the
corneal surface. Magnified view of a microcolony; bacteria were seen in the matrix (black arrow) and dividing cells were present inside the colony (white arrow). E1 and E2. TEM
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images showing an organized biofilms. Densely packed P. aeruginosa accumulated on the corneal surface. E1. At high magnification the bacteria appeared in layers. E2. Bacteria within the electron-dense matrix formed the basal (b) and middle layers (m). Bacteria in the
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top layer were seen to be less attached and free to move to a planktonic mode (t). Figure 4: Post-infection day 3. A1. On PI day 4, hypopyon and vascularization were observed. A2.Pan corneal fluorescein staining. B. CLSM showed more intense staining, indicating an increase in the biofilm glycocalyx (X40). C. Clusters of bacteria appeared on the corneal surface, and neutrophils were seen in the anterior stroma. D1. SEM revealed a
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mature biofilm with mushroom-shaped structures (thick black arrow) along with extensive epithelial loss and damage. D2 and D3. The area marked by an
are seen in higher
magnification. In D2, a tower-like structure consisted of a dense matrix and embedded bacteria. The portion of the biofilm in D3 showed bacteria emerging from the biofilm. E1.
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TEM microphotograph of a compact layer of bacteria with neutrophils, about 60 microns thick. E2. As on the previous day, E2 shows that bacteria can emerge from the compact
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Figure 5: Post-infection day 5. On PI day 5, slit lamp images A1 and A2 reveal the continued severity of the infection as a continuation of PI Day 3, characterized by hypopyon, fluorescein staining, and vascularization. B. FITC-con A staining using CLSM (X40). C. Gram staining revealed disorganized layers of epithelial cells immersed in a matrix with background staining along with neutrophils. SEM image in D1 demonstrated dense mats of P. aeruginosa biofilm attached to the corneal surface.
- Area magnified further illustrates
masses of bacteria embedded in a dense matrix (D2). TEM images (E1 and E2) confirm P. aeruginosa biofilm with similar morphology and organized as in PI day 3.
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Figure 6: Post-infection day 7. On PI day 7, slit lamp iamges A1 and A2 with fluorescein show little change from PI day 5. An increase in secreted matrix as well as release of material from the cornea and neutrophils changed the microscopic views, as observed in Figure 5. B. CLSM image continued to reveal large glycocalyx covered biofilm, but was somewhat less
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intense than at PI Day 5 (X40). C. Gram staining appears as an exudate overlying the corneal surface containing epithelial cells and neutrophils. SEM (D1) and TEM (E1) photographs demonstrate P. aruginosa biofilm outsized the corneal surface,
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in D2, presence of ghosts of bacteria in the matrix (E2).
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ACCEPTED MANUSCRIPT Table 1 Slit-lamp Imaging
Electron Microscopy Imaging
Before Infectio n
No
PI-Day 1
Yes 6/6
Yes 6/6
Negative 6/6
Pink stained rod shaped bacteria
Typical rod shaped bacteria with clear boundaries and smooth morphology
PI-Day 2
Yes 6/6
Yes 6/6
Positive 6/6
Bacteria organised into clusters
Small micro colonies with fibrous, hard extracellular substances
PI-Day 3
Yes 6/6
Yes 6/6
Highly positive 6/6
Bacteria formed a filmy sheet of hazy cover
PI-Day 5
Yes 6/6
Yes Even 4/6 Uneven 2/6
Bacteria intermingled with dense fibrous substances and organised as mushroom shaped bodies and tower like structures Consolidated biofilm bacteria evident with increased secretion
PI-Day 7
Yes 6/6
Glycocaly x staining
Light Microscop y Imaging Gram staining
Epitheli al Defect No
No bacteria
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Highly positive 6/6
Bacteria formed a thick layer on the surface
Highly Positive 6/6
Bacteria formed a thick layer on the surface
Biofilm bacteria occupied large area but with a slightly compressed form
Bacterial Architectu re
TEM No bacteria
No bacteria
Planktonic bacteria
Planktonic bacteria
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Yes Even 4/6 Uneven 2/6
SEM
Negative
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Corneal Opacity
CLSM Imaging
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Time of Infectio n
Bacteria settled into basal, middle and top layered organisation Thick rigid layer of biofilm in which bacteria were gotten embedded in extracellular substances
Attachment and colony formation
Biofilm bacteria
Compact mass of biofilm in which bacteria were entrenched inside the extracellular substances
Biofilm bacteria
Strong attachment of biofilm and the bacteria were static inside the secreted substances
Biofilm bacteria
A1 C
B
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