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Escherichia coli strains expressing H12 antigens demonstrate an increased ability to attach to abiotic surfaces as compared with E. coli strains expressing H7 antigens
Accepted Manuscript Title: Escherichia coli strains expressing H12 antigens demonstrate an increased ability to attach to abiotic surfaces as compared with E. coli strains expressing H7 antigens Author: Rebecca M. Goulter Elena Taran Ian R. Gentle Kari S. Gobius Gary A. Dykes PII: DOI: Reference:
S0927-7765(14)00201-X http://dx.doi.org/doi:10.1016/j.colsurfb.2014.04.003 COLSUB 6380
To appear in:
Colloids and Surfaces B: Biointerfaces
Received date: Revised date: Accepted date:
25-11-2013 10-4-2014 13-4-2014
Please cite this article as: R.M. Goulter, E. Taran, I.R. Gentle, K.S. Gobius, G.A. Dykes, Escherichia coli strains expressing H12 antigens demonstrate an increased ability to attach to abiotic surfaces as compared with E. coli strains expressing H7 antigens, Colloids and Surfaces B: Biointerfaces (2014), http://dx.doi.org/10.1016/j.colsurfb.2014.04.003 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.
Escherichia coli strains expressing H12 antigens demonstrate an increased ability
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to attach to abiotic surfaces as compared with E. coli strains expressing H7
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antigens.
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Authors:
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Rebecca M. Goulter1, 2*, Elena Taran3, Ian R. Gentle2, Kari S. Gobius1 and Gary A.
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Dykes4
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Author’s affiliations:
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1
CSIRO Food and Nutritional Sciences, Coopers Plains, Queensland 4108, Australia
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School of Chemistry and Molecular Biosciences, The University of Queensland,
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Brisbane, Queensland 4072, Australia
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Nanotechnology, Australian National Fabrication Facility, Queensland Node,
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Brisbane, Queensland 4072 Australia.
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Running title: H antigens and attachment of E. coli
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*Corresponding author:
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The University of Queensland, Australian Institute for Bioengineering and
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School of Science, Monash University, Bandar Sunway, Selangor, Malaysia
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Key words: Escherichia coli, atomic force microscopy, hydrophobicity, attachment,
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food processing, H antigens, flagella.
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Tel: +1 919 515 3558;
Fax: +1 919 515 0014;
E-mail: [email protected]; Present Address: Food, Bioprocessing and Nutrition Sciences, Campus Box 7642, North Carolina State University, Raleigh, NC, 27695, USA.
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Abstract
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The role of Escherichia coli H antigens in hydrophobicity and attachment to glass,
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Teflon and stainless steel (SS) surfaces was investigated through construction of fliC
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knockout mutants in E. coli O157:H7, O1:H7 and O157:H12. Loss of FliCH12 in E.
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coli O157:H12 decreased attachment to glass, Teflon and stainless steel surfaces
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(p<0.05). Complementing E. coli O157:H12 ΔfliCH12 with cloned wildtype (wt)
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fliCH12 restored attachment to wt levels. The loss of FliCH7 in E. coli O157:H7 and
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O1:H7 did not always alter attachment (p>0.05), but complementation with cloned
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fliCH12, as opposed to cloned fliCH7, significantly increased attachment for both strains
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compared with wt counterparts (p<0.05). Hydrophobicity determined using bacterial
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adherence to hydrocarbons and contact angle measurements differed with fliC
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expression but was not correlated to the attachment to materials included in this study.
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Purified FliC was used to functionalise silicone nitride atomic force microscopy
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probes, which were used to measure adhesion forces between FliC and substrates.
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Although no significant difference in adhesion force was observed between FliCH12
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and FliCH7 probes, differences in force curves suggest different mechanism of
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attachment for FliCH12 compared with FliCH7. These results indicate that E. coli strains expressing flagellar H12 antigens have an increased ability to attach to certain abiotic surfaces compared with E. coli strains expressing H7 antigens.
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INTRODUCTION
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Escherichia coli O157:H7 is an important foodborne pathogen that can cause serious
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infections ranging from mild diarrhoea to potentially life threatening conditions such
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as haemolytic uraemic syndrome. Other H types of E. coli O157 such as E. coli
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O157:H12, are non pathogenic but demonstrate increased resistance to processing
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procedures such as high pressure [1]. Food products may become adulterated with E.
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coli O157:H7 through contact with a contaminated surface [2]. The process of
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contamination of a surface initially occurs though transfer of cells to a surface, the
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attachment of those cells, followed by growth and biofilm formation [3, 4].
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Many studies have demonstrated the ability of E. coli to attach to a variety of surfaces
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including stainless steel (SS), Teflon, glass, polystyrene and biotic surfaces such as
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fruits, beef muscle and cultured cell lines [2, 5-9]. Bacterial surface properties such
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as hydrophobicity, surface charge, and the expression of surface structures such as
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curli and flagella have all been shown to play possible roles in attachment of E. coli to
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many of these surfaces [4]. Cell surface hydrophobicity has been shown to play a
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positive role in attachment in a number of studies [10-12], whereas others have shown no such relationship [2, 13, 14]. In the study of Rad et al. [11] it was shown that hydrophobic bacteria are more likely to interact with hydrophobic rather than hydrophilic surfaces. The expression of surface structures has also been shown to influence cell surface hydrophobicity [15].
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The expression of flagella has been shown to play a positive role in attachment for E.
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coli in some studies [9, 16-18], however a number of studies have reported the
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contrary [2]. The flagellum consists of three main structural regions; the basal body,
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the hook and the filament [19]. The single protein subunit of the filament is flagellin
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(FliC), with thousands of subunits forming the main body of the flagella. The fliC
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gene encodes for the H antigen, and variability in H antigen types arises from
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differences in the middle region of the fliC gene, which encodes for surface exposed
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portions of the protein [19]. In the study of Rivas et al. [2] the attachment of a
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number of strains of E. coli, belonging to the serotypes O157:H7, O26:H11, and
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O111:H- (non motile; NM) to SS was investigated. Numbers of attached cells did not
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differ between motile and NM strains. In the study of Giron et al [16], flagella
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expression was shown to be critical in the adherence to HeLa cells for a number of E.
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coli strains, including serotypes O111:H6, but not for serotype O157:H7. In contrast,
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in the study of Mahajan et al [9], E. coli strains harbouring FliCH7 attached in higher
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numbers to bovine primary epithelial cells compared with E. coli strains harbouring
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FliCH6. To our knowledge, the role of different E. coli O157 H types (H12 and H7) in
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attachment to SS, Teflon and glass surfaces through the use of knock out mutants and
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atomic force microscopy (AFM) is yet to be investigated.
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of the growth conditions employed. The aim of this work was to further investigate
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the role of FliCH12 and FliCH7 in attachment to SS, Teflon and glass surfaces through
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the construction of ΔfliC knockouts. The influence of different FliC H types was
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We have previously investigated attachment behaviour of a number of wildtype E. coli O157 strains, including E. coli O157:H7 (n=4), O1:H7 (n=1) and O157:H12
(n=1) isolates to glass, SS and Teflon surfaces [5]. Although the level of attachment was dependent on growth conditions, E. coli O157:H12 was shown to attach in higher
numbers compared to all other strains to all of the abiotic surfaces studied under most
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confirmed through the complementation of all knockout strains with fliCH12 and
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fliCH7.
95 MATERIALS AND METHODS
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Bacterial strains and culture conditions
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Three E. coli strains were selected for this study based on previous attachment data
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[5] and different antigenic determinants. Specifically, E. coli EC614 (O157:H12)
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attaches to a variety of surfaces (SS, Teflon and glass) in high numbers and has the
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H12 antigen. Alternatively, E. coli EDL933 (O157:H7) attaches to the same surfaces
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in fewer numbers than E. coli EC614 and has the same O157 antigen but the different
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H7 antigen. E. coli ATCC11775 (O1:H7) attaches in similar numbers to EDL933 and
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has the same H7 antigen.
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Working cultures were prepared monthly from Protect beads (Technical Service
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Consultants, Lancashire, UK) stored at -80°C and maintained on Nutrient Agar (NA;
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Oxoid, Basingstoke, UK) plates at 4°C. Isolates were cultured in Nutrient Broth (NB;
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Bacterial cells were harvested by centrifuging 1 ml of broth culture at 13,000 x g or
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by suspending colonies from agar surfaces in 150 mM Phosphate Buffered Saline
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(PBS; 2.7 mmol l-1KCl, 10 mmol l-1 Na2HPO4, 17 mmol l-1 KH2PO4, 150 mmol l-1
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NaCl, pH 7.4). Cell suspensions were prepared by washing bacterial cells twice and
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Oxoid), Luria Bertani Broth (LB; Oxoid), NA or Luria Bertani Agar (LBA; Oxoid) consisting of LB with 1.5% bacteriological agar (Oxoid), at 37°C for 18 ± 2 h for all experiments unless stated otherwise. Growth media was selected for consistency with previous studies [5].
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resuspending in PBS for all experiments unless otherwise stated. All experiments
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were performed in triplicate with independently grown cultures.
120 Construction of ΔfliC strains
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Strains of EC614, EDL933 and ATCC11775 with the inability to express FliC were
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constructed using the λ Red System described by Datsenko and Wanner [20]. The
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fliC gene was targeted for mutagenesis with PCR products containing a
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chloramphenicol antibiotic resistance cassette (cat) amplified from pKD3. Briefly, 60
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bp primers were designed with 40 bp of 5’ homology to the fliC gene and 20 bp of 3’
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homology to the cat gene cassette. Primers used were fliC-mut-F
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(AATATAGGATAACGAATCATGGCACAAGTCATTAATACCAAGTGTAGGC
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TGGAGCTGCTTC) and fliC-mut-R
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(TTAATCAGGTTACAACGATTAACCCTGCAGCAGAGACAGAATGGGAATT
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AGCCATGGTCC). A commercial kit (Promega) was used to purify PCR products
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which were electroporated into electrocompetent cells of EC614, EDL933 or
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ATCC11775 carrying the pKD46 plasmid and induced to express the λ Red
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fliCH12 was amplified from EC614. fliCH7(O157) and fliCH7(O1) were amplified from
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EDL933 and ATCC11775 respectively. Primers used were fliC-comp2-F
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(AAAAAGAATTCCGGCATGATTATCCGTTTCT) and fliC-comp2-R
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(AAAAAGAATTCCCCAGCGATGAAATACTTGC). fliC PCR products were
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recombinase functions. Replacement of the fliC gene with cat was confirmed using
primers flanking the site of substitution. Loss of FliC was confirmed using motility assays described below.
Complementation of ΔfliC strains with pfliCH12, pfliCH7(O157) or pfliCH7(O1)
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cloned into pGem T-Easy as per manufacturer’s instructions (Promega) and
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transferred into ΔfliC strains (Table 1). Sequencing was used to confirm the presence
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of fliC with upstream promoter elements. The sequence of the fliC gene from EC614
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was submitted to Genbank (accession number JF308285). Functional expression of
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fliC was confirmed using motility assays described below. Experiments involving
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complemented strains were conducted using LB as growth media.
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149 Motility assays
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Motility agar plates, consisting of LB (Oxoid) with 0.3% agar (Agar technical #3;
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Oxoid) were inoculated with 10 µl overnight culture grown in LB broth and incubated
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at 37°C for 18 ± 2 h. Motility was confirmed by a halo of growth around the
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inoculation area.
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Determination of bacterial hydrophobicity
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Hydrophobicity was determined using Contact Angle Measurements (CAM) and
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Bacterial Adherence to Hydrocarbons (BATH) as described by Busscher et al. [21]
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s (Ab). A second tube containing 4 ml of untreated cell suspension was used as a
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control (Ac). All tubes were incubated in a waterbath at 37°C for 30 min. Following
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incubation, 1 ml of the lower aqueous layer was removed using a pipette and the OD
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and Li and McLandsborough et al. [14] respectively, with minor modifications as described by Rivas et al. [22]. Briefly, bacterial suspensions were prepared as described above and adjusted to an OD at 540nm of 1.0 ± 0.2 in PBS (approximate concentration of 8 x 108 cells/ml) for BATH assays. A 4 ml volume of the suspension
was added to 1 ml of xylene (Ajax Chemicals, Sydney, Australia) and vortexed for 20
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at 540 nm was measured. The percentage of bacterial cells which bound to the xylene
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hydrocarbon was calculated as (%) = (Ac – Ab)/Ac × 100.
169 Contact angle measurements (θ) were determined using a goniometer (KSV
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Instruments Ltd, Helsinki, Finland) and KSV CAM software (KSV Instruments Ltd)
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using the sessile drop method. Cell suspensions were prepared as described above
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using sterile distilled water (SDW) and the OD600nm adjusted to 1.2 ± 0.2
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(approximately 9 x 108 cells/ml). Bacterial lawns were prepared by filtering 25 ml of
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the suspension onto a HA filter (0.45 µm pore diameter, 25 mm filter diameter,
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Millipore, Bedford, MA, USA) by negative pressure filtration. Filters were fixed to
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glass slides using double sided adhesive tape and allowed to dry for 30 min in a
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desiccator. A drop of SDW was placed onto the lawn using a 1 ml syringe. An image
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of the drop was recorded using a digital video camera (30 frames per second FireWire
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IEEE 1394) and contact angles were determined using aforementioned KSV software.
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A minimum of five drops were recorded per filter, and reported angles (θ) were
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determined from the means of three independent filters per sample.
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described above and bacterial attachment was allowed to take place for 20 min at
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room temperature with gentle swirling for 5 s at 5 min intervals. Following
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attachment, slides were rinsed twice by dipping slides up and down three times in 50
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ml of fresh PBS, removing loosely adhered cells from the surfaces. Slides were
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Attachment assays
Assays enumerating cell attachment to SS surfaces were conducted as described by Rivas et al. [2] and to glass and Teflon surfaces as described by Goulter et al. [5]. Briefly, slides of each material were placed into bacterial suspensions prepared as
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stained with 0.01% acridine orange and the number of cells in 50 randomly selected
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fields was counted using an epifluorescence microscope (Leica DFC420 C, Leica
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Microsystems, Wetzlar, Germany). Results were presented as Log Direct
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Microscopic Count (DMC) / cm2.
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196 AFM probe modification and force mapping
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Outer membrane protein extractions were prepared and analysed using 2D gel
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electrophoresis as described by Rivas et al. [23]. In all cases two gels were run
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concurrently, with one of the two gels being stained with coomassie blue overnight.
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The location of the FliC protein on the stained gel was used to define the location of
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FliC in the unstained gel for excision. Gel pieces containing FliC were crushed and
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suspended in protein elution buffer (50 mMTris-HCl, 150 mMNaCl, and 0.1 mM
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EDTA; pH 7.5). Silicon nitride AFM probes with attached borosilicate glass particle
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(5µm diameter) were purchased precoated with 3-aminopropyltriethoxysilane by the
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manufacturer (Novascan Technologies Inc., IA, USA). Purified proteins were
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immobilised on AFM tips as described [24]. Spring constants of the protein coated
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cantilever. Immobilization efficiency of the different proteins were not significantly
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different.
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AFM tips were determined using software (Igor Pro, Asylum Research, Santa Barbara, CA). AFM tips were calibrated against glass slides prior to force mapping measurements, which were conducted at a speed of 1µm s-1. Measurements were
taken over a 5 µm x 5µm area, taking measurements at 32 x 32 (1024) total points. The immobilization of the protein to the probe was confirmed by comparison to a bare
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Statistical analysis
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One way analysis of variance and comparison of means (Tukey’s method) were
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performed using Minitab software (MINITAB 15; Minitab Inc., USA). Significant
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differences between individual strain fliC variations for a single growth media were
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determined for hydrophobicity and attachment studies. Significant differences
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between the three FliC proteins for each substrate were determined for the force
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measurement studies.
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RESULTS
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Hydrophobicity of ΔfliC strains
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In some cases, ΔfliC strains were more hydrophilic than the wt counterparts under
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some growth conditions studied; the statistical significance (p < 0.05) of these
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differences are shown in Table 1. The magnitude of differences in hydrophobicity
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was dependent on both growth media and method used to determine hydrophobicity.
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Greater differences were seen when the BATH method was used in comparison to
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CAM. In no combination studied was the ΔfliC strain more hydrophobic than its wt
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counterpart. When using the BATH method, all ΔfliC strains were more hydrophilic
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than wt counterparts (p < 0.05), with the exception of LBA (p > 0.05). When using
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the CAM method, there was again no difference when strains were cultured on LBA
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(p > 0.05), and variable results were seen for the remaining growth media.
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No significant difference in attachment to glass was seen between the E. coli
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O157:H7 and E. coli O1:H7 and their ΔfliC counterparts under any of the growth
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growth conditions (p < 0.05; Table 2). ΔfliC strains of E. coli O157:H12 and E. coli
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O157:H7 attached in significantly fewer numbers than wt counterparts following
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growth in LB and LBA (p < 0.05). E. coli O1:H7 ΔfliCH7::cat attached in
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conditions studied (p > 0.05; Table 2). In contrast, E. coli O157:H12 ΔfliCH12::cat was shown to attach in significantly fewer numbers to glass than E. coli O157:H12
under all growth conditions (p < 0.05; Table 2).
Differences in attachment to SS were seen between wt and ΔfliC pairs under some
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significantly fewer numbers than its wt counterpart following growth in LBA only (p
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< 0.05).
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wt counterpart following growth in all media (p < 0.05; Table 2), with the exception
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of LBA (p > 0.05). E. coli O157:H7 ΔfliCH7::cat attached in significantly fewer
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numbers to Teflon than the wt E. coli O157:H7 following growth in NB and NA alone
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(p < 0.05). No significant difference in attachment to Teflon between E. coli O1:H7
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and its ΔfliCH7 counterpart were seen under the majority of the growth conditions
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studied (p > 0.05), with the exception being growth in LBA (p < 0.05).
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Hydrophobicity of ΔfliC strains complemented with wtfliCH12 or wtfliCH7
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Complementing ΔfliC strains with cloned fliCH12 or cloned fliCH7 restored
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hydrophobicity to the same level (p < 0.05) as those of wt strains following growth in
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LB when hydrophobicity was determined by BATH (Figure 1). No significant
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difference (p > 0.05) was found for any of the isolate variations when hydrophobicity
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attachment to all materials for E. coli O157:H12 ΔfliCH12::cat was restored to wt
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levels when complemented with wtfliCH12 (Figure 3, upper panel).
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was determined using CAM following growth in LB (Figure 2).
Attachment of ΔfliC strains complemented with cloned fliCH12 or cloned fliCH7 Results of attachment of ΔfliC strains when complemented with cloned fliCH12 or
cloned fliCH7 to the selected abiotic surfaces is shown in Figure 3. Most notably,
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Attachment of E. coli O157:H7 ! fliCH7::cat/pfliCH12 to glass was significantly higher
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than wt E. coli O157:H7, E. coli O157:H7 ΔfliCH7::cat and E. coli O157:H7
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ΔfliCH7::cat/pfliCH7(O157) (p < 0.05; Figure 3, middle panel). There was no significant
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difference in attachment numbers to glass for wt E. coli O157:H7, E. coli O157:H7
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ΔfliCH7::cat and E. coli O157:H7 ΔfliCH7::cat/pfliCH7(O157) (p > 0.05; Figure 3, middle
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panel). No significant difference in attachment to Teflon was seen for any E. coli
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O157:H7 strain variation (p > 0.05; Figure 3, middle panel). Attachment of E. coli
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O157:H7 ! fliCH7::cat/pfliCH12 to SS was significantly higher than E. coli O157:H7
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ΔfliCH7::cat and E. coli O157:H7 ΔfliCH7::cat/pfliCH7(O157) (p < 0.05; Figure 3, middle
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panel), but not significantly different to wt E. coli O157:H7 (p > 0.05; Figure 3,
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middle panel). Attachment of E. coli O157:H7 ! fliCH7::cat/pfliCH12 was not
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significantly different from wt E. coli O157:H7 or E. coli O157:H7ΔfliC::cat (p <
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0.05; Figure 3, middle panel).
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Attachment of E. coli O1:H7 ! fliCH7::cat/pfliCH12 was significantly higher than wt E.
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coli O1:H7, E. coli O1:H7 ΔfliC::cat and E. coli O1:H7 ! fliCH7::cat/pfliCH7(O1) to all
materials studied (p < 0.05; Figure 3, lower panel). No significant difference was seen in attachment numbers of wt E. coli O1:H7, E. coli O1:H7 ΔfliCH7::cat and E. coli O1:H7 ΔfliCH7::cat/pfliCH7(O1) to any of the materials studied (p > 0.05; Figure 3,
lower panel).
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AFM force mapping
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Histograms generated from force mapping data are shown in Figure 4. The number of
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times (frequency; y axis) each unit of force was measured (x axis) can be seen in this
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figure. Probes coated with purified FliCH12 adhered significantly more strongly to 13
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glass and SS surfaces than probes coated with either FliCH7 protein (p < 0.05; Figure
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4, upper and middle panel). FliCH7(O1) had a significantly higher adhesion force to
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Teflon than FliCH12 (p < 0.05; Figure 4, lower panel). Due to high standard deviation,
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FliCH7(O157) was not significantly different (p > 0.05) to either protein in adhesion
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force to Teflon (Figure 4, lower panel). Example force distance curves to Teflon are
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shown in Figure 5. Figure 5 shows multiple pull off events for the FliCH12 protein,
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but only a single pull off event for both FliCH7 proteins. The shape of the force curves
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are representative of all force curves for these proteins to all materials (data not
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shown), albeit the force scale differed when measuring adhesion forces to glass and
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SS due to smaller measured forces.
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306 DISCUSSION
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A number of studies have shown that differences in the fliC gene of E. coli encoding
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different H antigens can influence attachment to biotic surfaces [9, 16]. Studies
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investigating the influence of flagella expression on the attachment of E. coli to
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abiotic surfaces often include a number of different strains, and have not used genetic
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eliminate flagella expression. To further investigate the role of different flagella H
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antigens, the deleted fliC gene of each strain was complemented with the cloned wt
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fliC gene. The influence of these modifications on surface hydrophobicity was also
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determined.
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modification tools in order to study the effects of loss of flagella expression on attachment for individual strains [2]. In these previous studies no correlation was found between attachment and flagellar expression. In the present study, the fliC genes from three E. coli strains belonging to serotypes O157:H12, O157:H7 and
O1:H7 were replaced by a chloramphenicol antibiotic resistance cassette (cat) to
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321 The loss of fliC and flagella expression resulted in a decrease in hydrophobicity for all
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strains in a growth media dependent fashion when determined using the BATH
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method (Table 1); demonstrating the hydrophobic nature of FliC proteins. This was
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also demonstrated by using AFM force mapping, where increased adhesion force was
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measured between FliC proteins and Teflon, the most hydrophobic surface included in
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this study, compared with glass and SS [25]. The lowest adhesion force was
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measured between FliC proteins and glass, followed by SS. These results support
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studies which suggest hydrophobic surfaces interact more readily with other surfaces
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which are hydrophobic in nature [11]. However, it should also be noted that BATH
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measurements can be influenced by the surface charge of bacterial cells [26]. Due to
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the lack of significant difference found when hydrophobicity was determined by
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CAM, this study highlights the complexity of determining bacterial hydrophobicity
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and highlights that other factors may also influence measurements and consequent
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correlations with attachment. In particular, the CAM method uses the Young
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equation to calculate hydrophobicity. This equation makes the assumption that the
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FliCH7 protein (Figure 4). Other studies utilising AFM to study bacterial flagella are
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often restricted to imaging alone [27, 28], and to our knowledge this is the first AFM
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force mapping study investigating the interactions between purified FliC proteins and
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glass, Teflon and SS surfaces. Force distance curves to glass and SS surfaces were
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surface is perfectly smooth, which is not the case for bacterial lawns. This may also explain the differences seen between BATH and CAM measurements in the current study.
FliCH12 was shown to have a higher average adhesion force to glass and SS than either
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similar, albeit, on a lower force scale (data not shown). As shown in Figure 5, single
347
pull off events were recorded for FliCH7 proteins, whereas the retraction of FliCH12
348
from the surface resulted in multiple, stepwise pull off events as indicated by the
349
multiple peaks seen in the retraction curve (Figure 5). This may indicate that FliCH12
350
adheres to these surfaces in a different fashion than FliCH7 proteins. Sequencing
351
results revealed that the fliC gene from the E. coli O157:H12 strain was slightly
352
longer than the fliC sequence in the E. coli O157:H7 or E. coli O1:H7 strains resulting
353
in an additional 10 predicted amino acids (595 as opposed to 585) in the FliCH12
354
protein. This suggests that additional amino acids resulting in longer proteins (or a
355
potential change in tertiary structure) may require multiple pull off events from a
356
surface than shorter proteins. In the study of Sethuraman et al. [29] adhesion forces
357
were measured between seven proteins of various sizes and solid surfaces. Proteins of
358
larger size were shown to have force distance curves extending over a few hundred
359
nanometres with a stepwise, multiple pull off event pattern similar to that seen in the
360
current study. Smaller proteins were shown to have a single pull off event [29]. A
361
more recent study investigating adhesion forces of single protein molecules has
363 364 365 366
cr
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M
d
te
Ac ce p
362
ip t
346
demonstrated that force curves with multiple pull off events are due to the breaking of individual bonds as the protein retracts from the surface [30]. These studies suggest that the larger FliCH12, as compared to the FliCH7 protein, results in force distance
curves with multiple, stepwise, pull off events, which may aid in stronger adhesion to the surfaces included in this study.
367 368
Our data indicated that FliCH12 plays an important role in attachment for the E. coli
369
O157:H12 strain included in this study to all surfaces in a growth media dependent
370
fashion (Table 2). These results show that the strains included in this study with the
16
Page 16 of 37
371
ability to express fliCH12 have an increased ability to attach to a variety of abiotic
372
surfaces such as glass, SS and Teflon when compared with strains lacking fliCH12 or
373
strains expressing fliCH7 in the majority of cases.
ip t
374 While flagella have been shown to influence attachment in other studies [9, 16, 17],
376
numerous factors may be involved in the attachment process that are likely to differ
377
between strains. These include surface charge [14], the production of other surface
378
structures such as fimbriae [31], outer membrane proteins such as Ag43 [32], among
379
others. Growth media also influences these factors and others; future studies
380
including various other growth media and the relationship between expression of
381
bacterial surface structures and attachment is warranted. In order to fully understand
382
the attachment mechanism for individual strains many factors must therefore be
383
considered.
us
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384
cr
375
The emergence of Shigatoxigenic E. coli (STEC) strains of various O and H antigenic
386
types other than E. coli O157:H7 causing severe outbreaks and disease [33] shows the
391
Ac ce p
385
392
study and previously by our laboratory [5]. If non-pathogenic E. coli strains were to
393
acquire virulence genes and had the added capacity to adhere strongly to surfaces
394
commonly used in food processing environments, controlling such a potential
395
pathogen would be difficult. For this reason, further studies into the control of
387 388 389 390
importance of studying the properties and attachment behaviour of non O157:H7 STEC strains. The transfer of virulence genes from one strain of E. coli to another is
also well documented [34-36]. E. coli O157:H12 strains have been shown to be resistant to processes such as high pressure treatment [1], and have been shown to attach more readily than other strains to a variety of surfaces as demonstrated in this
17
Page 17 of 37
pathogenic and non-pathogenic E. coli strains with the ability to attach readily to a
397
number of surfaces are required. This study has identified the importance of H12
398
antigens in attachment as indicated by the strains included in this study, and as such
399
has identified a potential target for the control of this organism.
ip t
396
400 ACKNOWLEDGEMENTS
402
R. M. Goulter acknowledges the financial support of the Department of Employment,
403
Economic Development and Innovation of the Queensland Government, Australia
404
through the Smart State PhD Scholarships Program and the financial support of the
405
Australian Government through the Australian Postgraduate Award. This work was
406
also supported through funding from CSIRO Food and Nutritional Sciences.
M
an
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Page 18 of 37
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[35] B.G. Kelly, A. Vespermann and D.J. Bolton, Horizontal gene transfer of
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Toxicology, 47 (2009) 969-977.
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[36] M. Muniesa, J. Jofre, C. Garcia-Aljaro and A.R. Blanch, Occurrence of
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cr
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518
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526
Ac ce p
528
te
d
M
an
527
529 530
25
Page 25 of 37
Figure 1: Hydrophobicity of strains following growth in Luria Bertani Broth as
532
determined using the Bacterial Adherence to Hydrocarbons method. Data shown are
533
means of three independent experiments + standard deviation. *Results also shown in
534
Table 1.
ip t
531
535
Ac ce p
te
d
M
an
us
cr
536
26
Page 26 of 37
us
cr
ip t
536
Ac ce p
538
te
d
M
an
537
539
27
Page 27 of 37
540
Figure 2: Hydrophobicity of strains following growth in Luria Bertani Broth as
541
determined using the Contact Angle Measurements Method. Data shown are means of
542
three independent experiments + standard deviation. *Results also shown in Table 1.
ip t
543 544
Ac ce p
te
d
M
an
us
cr
545
28
Page 28 of 37
ip t cr Ac ce p
2
te
d
M
an
us
1
3 4 5
30
Page 29 of 37
1
Figure 3: Attachment (Log Direct Microscopic Count (DMC) / cm2) of strains
2
following growth in LB;
3
Data shown are means of three independent experiments + standard deviation.
Ac ce p
te
d
M
an
us
cr
ip t
wt; ΔfliC::cat; pfliCH12; pfliCH7(O157); pfliCH7(O1).
31
Page 30 of 37
100
Glass
Frequency
80 60 40
0 0.0
0.2
0.4
0.6
0.8
nN
us
100
Stainless Steel
80 60
an
Frequency
1.0
cr
FliCH12 0.32 ± 0.09 nN FliCH7(O157) 0.09 ± 0.02 nN FliCH7(O1) 0.10 ± 0.06 nN
ip t
20
40
M
20 0 0.0
0.2
0.4
0.6
0.8
1.0
nN
50
Teflon
Frequency
Ac ce p
40
te
d
FliCH12 0.45 ± 0.16 nN FliCH7(O157) 0.07 ± 0.02 nN FliCH7(O1) 0.05 ± 0.09 nN
30 20 10
0
0
10
20
30
40
50
nN
FliCH12 3.2 ± 1.1 nN FliCH7(O157) 25.9 ± 15.4 nN FliCH7(O1) 15.1 ± 2.8 nN
1 2 3
Figure 4: Histograms of adhesion force to Glass, SS and Teflon measured using AFM
4
force mapping for purified FliCH12 and FliCH7 proteins. Measurements are average
5
adhesion force ! standard deviation. 32
Page 31 of 37
us
cr
ip t
1
FliC
an
H12
FliC
H7(O157)
FliC
H7(O1)
M
2 3
Figure 5: Example force distance curves for purified FliCH12 and FliCH7 proteins to
5
Teflon.
te
7
Ac ce p
6
d
4
33
Page 32 of 37
ip t cr
BATH (% Adherence) NB
LB*
O157:H12
54.1 ± 1.2a
18.6 ± 3.0a
O157:H12 ΔfliCH12::cat
27.8 ± 7.3b
2.02 ± 1.9b
O157:H7
57.7 ± 3.0a
5.7 ± 0.4a
O157:H7ΔfliCH7::cat
23.3 ± 8.0b
O1:H7
20.5 ± 1.0a
O1:H7 ΔfliCH7::cat
12.1 ± 1.9b
CAM (θ)
NA
LBA
NB
LB*
NA
LBA
19.2 ± 2.9a
13.2 ± 2.4a
15.4 ± 0.7a
15.8 ± 0.3a
14.4 ± 0.3a
14.5 ± 0.7a
9.7 ± 2.7b
15.1 ± 1.6a
12.7 ± 0.6b
13.6 ± 0.9a
11.0 ± 0.3b
13.2 ± 0.8a
32.7 ± 1.3a
14.3 ± 2.7a
14.7 ± 0.5a
15.4 ± 1.0a
15.3 ± 0.8a
13.6 ± 0.5a
2.0 ± 1.8b
5.6 ± 5.4b
11.4 ± 5.8a
12.5 ± 0.4b
12.1 ± 0.7b
11.5 ± 0.3b
12.0 ± 0.8a
9.1 ± 1.2a
9.8 ± 1.1a
0.4 ± 0.4a
20.8 ± 0.5a
17.2 ± 0.7a
16.5 ± 1.1a
17.7 ± 1.7a
3.5 ± 2.0b
3.2 ± 2.8b
3.3 ± 2.9a
15.0 ± 3.8a
12.6 ± 2.5b
17.8 ± 3.4a
16.1 ± 2.8a
M an
E. coli Strain
us
Table 1: Hydrophobicity of E. coli strains cultured in Nutrient Broth (NB), Luria Bertani Broth (LB), Nutrient Agar (NA) and LB Agar (LBA)
ce pt
ed
1
2
a, b
3
*Results also shown in Figures 1 and 2.
Ac
Different superscripts indicate significant differences for parent and ΔfliC::cat pairs for that growth media (p < 0.05).
34 Page 33 of 37
ip t cr
Table 2: Attachment (Log Direct Microscopic Count (DMC) / cm2 ± standard deviation of three independent experiments) of E. coli strains
2
cultured in Nutrient Broth (NB), Luria Bertani Broth (LB), Nutrient Agar (NA) and LB Agar (LBA) to glass, stainless steel (SS) and Teflon
O157:H7ΔfliCH7::cat
O1:H7
O1:H7 ΔfliC::cat
NA
LBA
NB
LB
NA
LBA
NB
LB
NA
LBA
4.2 ±
4.8 ±
3.9 ±
5.2 ±
3.9 ±
4.7 ±
2.9 ±
4.8 ±
3.5 ±
3.9 ±
3.9 ±
2.7 ±
0.1a
0.5a
0.1a
0.2a
0.0a
0.4a
0.2a
0.3a
0.2a
0.1a
0.1a
0.1a
2.8 ±
3.0 ±
2.9 ±
2.9 ±
3.5 ±
3.5 ±
2.7 ±
3.0 ±
2.6 ±
2.7 ±
2.6 ±
2.6 ±
0.2b
0.2b
0.2b
0.2b
0.3a
0.2b
0.5a
0.1b
0.2b
0.5b
0.2b
0.1a
4.0 ±
3.8 ±
3.7 ±
4.1 ±
2.2 ±
4.0 ±
2.8 ±
3.9 ±
3.6 ±
3.0 ±
4.2 ±
2.8 ±
0.1a
0.1a
0.2a
0.3a
0.3a
0.1a
0.1a
0.4a
0.2a
0.5a
0.1a
0.4a
3.7 ±
3.5 ±
3.7 ±
3.6 ±
2.7 ±
3.1 ±
2.8 ±
2.7 ±
2.7 ±
2.7 ±
2.2 ±
2.3 ±
0.0a
0.2a
0.2a
0.3a
0.2a
0.9b
0.2a
0.4b
0.1b
0.2a
0.3b
0.2a
Ac
O157:H7
LB
ce pt
O157:H12 ΔfliCH12::cat
Teflon (Log DMC / cm2)
NB
ed
O157:H12
SS (Log DMC / cm2)
M an
Glass (Log DMC / cm2)
us
1
3.7 ±
4.0 ±
3.6 ±
4.1 ±
2.8 ±
3.6 ±
2.7 ±
3.7 ±
3.2 ±
3.2 ±
3.4 ±
4.0 ±
0.1a
0.2a
0.0a
0.2a
0.1a
0.2a
0.2a
0.3a
0.2a
0.3a
0.2a
0.3a
3.8 ±
3.8 ±
3.8 ±
3.6 ±
2.9 ±
3.2 ±
2.6 ±
2.6 ±
3.4 ±
3.4 ±
3.4 ±
3.4 ±
0.2a
0.3a
0.2a
0.1a
0.2a
0.1a
0.4a
0.4b
0.2a
0.1a
0.2a
0.2b
35 Page 34 of 37
ip t cr
a, b
Different superscripts indicate significant differences for parent and ΔfliC::cat pairs for that growth media (p < 0.05)
us
1
Ac
ce pt
ed
M an
2
36 Page 35 of 37
ip t cr us
5
4
M an
200
µm
100
2
pN
150
3
ed
50 1
0
1
2
3
4
5
µm
Ac
0
1 2
ce pt
0
37 Page 36 of 37
Highlights •
Loss of FliCH12 in O157:H12 decreased attachment to glass, Teflon and stainless steel surfaces (p<0.05). Complementing O157:H12 ΔfliCH12 with cloned wildtype (wt) fliCH12 restored
ip t
•
•
cr
attachment to wt levels.
The loss of FliCH7 in O157:H7 and O1:H7 did not always alter attachment
us
(p>0.05), but complementation with cloned fliCH12, as opposed to cloned fliCH7, significantly increased attachment for both strains compared with wt counterparts
Although no significant difference in adhesion force was observed between
M
•
an
(p<0.05).
FliCH12 and FliCH7 AFM probes, differences in force curves suggest different
These results indicate that E. coli strains expressing flagellar H12 antigens have
te
•
d
mechanism of attachment for FliCH12 compared with FliCH7.
an increased ability to attach to certain abiotic surfaces compared with E. coli
Ac ce p
strains expressing H7 antigens.
Page 37 of 37
S0927-7765(14)00201-X http://dx.doi.org/doi:10.1016/j.colsurfb.2014.04.003 COLSUB 6380
To appear in:
Colloids and Surfaces B: Biointerfaces
Received date: Revised date: Accepted date:
25-11-2013 10-4-2014 13-4-2014
Please cite this article as: R.M. Goulter, E. Taran, I.R. Gentle, K.S. Gobius, G.A. Dykes, Escherichia coli strains expressing H12 antigens demonstrate an increased ability to attach to abiotic surfaces as compared with E. coli strains expressing H7 antigens, Colloids and Surfaces B: Biointerfaces (2014), http://dx.doi.org/10.1016/j.colsurfb.2014.04.003 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.
Escherichia coli strains expressing H12 antigens demonstrate an increased ability
2
to attach to abiotic surfaces as compared with E. coli strains expressing H7
3
antigens.
4
Authors:
5
Rebecca M. Goulter1, 2*, Elena Taran3, Ian R. Gentle2, Kari S. Gobius1 and Gary A.
6
Dykes4
7
Author’s affiliations:
8
1
CSIRO Food and Nutritional Sciences, Coopers Plains, Queensland 4108, Australia
9
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School of Chemistry and Molecular Biosciences, The University of Queensland,
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Brisbane, Queensland 4072, Australia
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Nanotechnology, Australian National Fabrication Facility, Queensland Node,
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Brisbane, Queensland 4072 Australia.
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Running title: H antigens and attachment of E. coli
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*Corresponding author:
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The University of Queensland, Australian Institute for Bioengineering and
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School of Science, Monash University, Bandar Sunway, Selangor, Malaysia
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Key words: Escherichia coli, atomic force microscopy, hydrophobicity, attachment,
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food processing, H antigens, flagella.
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Tel: +1 919 515 3558;
Fax: +1 919 515 0014;
E-mail: [email protected]; Present Address: Food, Bioprocessing and Nutrition Sciences, Campus Box 7642, North Carolina State University, Raleigh, NC, 27695, USA.
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Abstract
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The role of Escherichia coli H antigens in hydrophobicity and attachment to glass,
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Teflon and stainless steel (SS) surfaces was investigated through construction of fliC
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knockout mutants in E. coli O157:H7, O1:H7 and O157:H12. Loss of FliCH12 in E.
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coli O157:H12 decreased attachment to glass, Teflon and stainless steel surfaces
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(p<0.05). Complementing E. coli O157:H12 ΔfliCH12 with cloned wildtype (wt)
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fliCH12 restored attachment to wt levels. The loss of FliCH7 in E. coli O157:H7 and
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O1:H7 did not always alter attachment (p>0.05), but complementation with cloned
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fliCH12, as opposed to cloned fliCH7, significantly increased attachment for both strains
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compared with wt counterparts (p<0.05). Hydrophobicity determined using bacterial
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adherence to hydrocarbons and contact angle measurements differed with fliC
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expression but was not correlated to the attachment to materials included in this study.
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Purified FliC was used to functionalise silicone nitride atomic force microscopy
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probes, which were used to measure adhesion forces between FliC and substrates.
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Although no significant difference in adhesion force was observed between FliCH12
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and FliCH7 probes, differences in force curves suggest different mechanism of
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attachment for FliCH12 compared with FliCH7. These results indicate that E. coli strains expressing flagellar H12 antigens have an increased ability to attach to certain abiotic surfaces compared with E. coli strains expressing H7 antigens.
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INTRODUCTION
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Escherichia coli O157:H7 is an important foodborne pathogen that can cause serious
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infections ranging from mild diarrhoea to potentially life threatening conditions such
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as haemolytic uraemic syndrome. Other H types of E. coli O157 such as E. coli
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O157:H12, are non pathogenic but demonstrate increased resistance to processing
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procedures such as high pressure [1]. Food products may become adulterated with E.
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coli O157:H7 through contact with a contaminated surface [2]. The process of
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contamination of a surface initially occurs though transfer of cells to a surface, the
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attachment of those cells, followed by growth and biofilm formation [3, 4].
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Many studies have demonstrated the ability of E. coli to attach to a variety of surfaces
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including stainless steel (SS), Teflon, glass, polystyrene and biotic surfaces such as
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fruits, beef muscle and cultured cell lines [2, 5-9]. Bacterial surface properties such
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as hydrophobicity, surface charge, and the expression of surface structures such as
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curli and flagella have all been shown to play possible roles in attachment of E. coli to
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many of these surfaces [4]. Cell surface hydrophobicity has been shown to play a
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positive role in attachment in a number of studies [10-12], whereas others have shown no such relationship [2, 13, 14]. In the study of Rad et al. [11] it was shown that hydrophobic bacteria are more likely to interact with hydrophobic rather than hydrophilic surfaces. The expression of surface structures has also been shown to influence cell surface hydrophobicity [15].
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The expression of flagella has been shown to play a positive role in attachment for E.
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coli in some studies [9, 16-18], however a number of studies have reported the
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contrary [2]. The flagellum consists of three main structural regions; the basal body,
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the hook and the filament [19]. The single protein subunit of the filament is flagellin
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(FliC), with thousands of subunits forming the main body of the flagella. The fliC
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gene encodes for the H antigen, and variability in H antigen types arises from
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differences in the middle region of the fliC gene, which encodes for surface exposed
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portions of the protein [19]. In the study of Rivas et al. [2] the attachment of a
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number of strains of E. coli, belonging to the serotypes O157:H7, O26:H11, and
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O111:H- (non motile; NM) to SS was investigated. Numbers of attached cells did not
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differ between motile and NM strains. In the study of Giron et al [16], flagella
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expression was shown to be critical in the adherence to HeLa cells for a number of E.
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coli strains, including serotypes O111:H6, but not for serotype O157:H7. In contrast,
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in the study of Mahajan et al [9], E. coli strains harbouring FliCH7 attached in higher
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numbers to bovine primary epithelial cells compared with E. coli strains harbouring
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FliCH6. To our knowledge, the role of different E. coli O157 H types (H12 and H7) in
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attachment to SS, Teflon and glass surfaces through the use of knock out mutants and
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atomic force microscopy (AFM) is yet to be investigated.
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of the growth conditions employed. The aim of this work was to further investigate
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the role of FliCH12 and FliCH7 in attachment to SS, Teflon and glass surfaces through
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the construction of ΔfliC knockouts. The influence of different FliC H types was
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We have previously investigated attachment behaviour of a number of wildtype E. coli O157 strains, including E. coli O157:H7 (n=4), O1:H7 (n=1) and O157:H12
(n=1) isolates to glass, SS and Teflon surfaces [5]. Although the level of attachment was dependent on growth conditions, E. coli O157:H12 was shown to attach in higher
numbers compared to all other strains to all of the abiotic surfaces studied under most
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confirmed through the complementation of all knockout strains with fliCH12 and
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fliCH7.
95 MATERIALS AND METHODS
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Bacterial strains and culture conditions
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Three E. coli strains were selected for this study based on previous attachment data
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[5] and different antigenic determinants. Specifically, E. coli EC614 (O157:H12)
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attaches to a variety of surfaces (SS, Teflon and glass) in high numbers and has the
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H12 antigen. Alternatively, E. coli EDL933 (O157:H7) attaches to the same surfaces
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in fewer numbers than E. coli EC614 and has the same O157 antigen but the different
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H7 antigen. E. coli ATCC11775 (O1:H7) attaches in similar numbers to EDL933 and
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has the same H7 antigen.
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Working cultures were prepared monthly from Protect beads (Technical Service
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Consultants, Lancashire, UK) stored at -80°C and maintained on Nutrient Agar (NA;
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Oxoid, Basingstoke, UK) plates at 4°C. Isolates were cultured in Nutrient Broth (NB;
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Bacterial cells were harvested by centrifuging 1 ml of broth culture at 13,000 x g or
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by suspending colonies from agar surfaces in 150 mM Phosphate Buffered Saline
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(PBS; 2.7 mmol l-1KCl, 10 mmol l-1 Na2HPO4, 17 mmol l-1 KH2PO4, 150 mmol l-1
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NaCl, pH 7.4). Cell suspensions were prepared by washing bacterial cells twice and
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Oxoid), Luria Bertani Broth (LB; Oxoid), NA or Luria Bertani Agar (LBA; Oxoid) consisting of LB with 1.5% bacteriological agar (Oxoid), at 37°C for 18 ± 2 h for all experiments unless stated otherwise. Growth media was selected for consistency with previous studies [5].
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resuspending in PBS for all experiments unless otherwise stated. All experiments
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were performed in triplicate with independently grown cultures.
120 Construction of ΔfliC strains
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Strains of EC614, EDL933 and ATCC11775 with the inability to express FliC were
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constructed using the λ Red System described by Datsenko and Wanner [20]. The
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fliC gene was targeted for mutagenesis with PCR products containing a
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chloramphenicol antibiotic resistance cassette (cat) amplified from pKD3. Briefly, 60
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bp primers were designed with 40 bp of 5’ homology to the fliC gene and 20 bp of 3’
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homology to the cat gene cassette. Primers used were fliC-mut-F
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(AATATAGGATAACGAATCATGGCACAAGTCATTAATACCAAGTGTAGGC
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TGGAGCTGCTTC) and fliC-mut-R
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(TTAATCAGGTTACAACGATTAACCCTGCAGCAGAGACAGAATGGGAATT
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AGCCATGGTCC). A commercial kit (Promega) was used to purify PCR products
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which were electroporated into electrocompetent cells of EC614, EDL933 or
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ATCC11775 carrying the pKD46 plasmid and induced to express the λ Red
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fliCH12 was amplified from EC614. fliCH7(O157) and fliCH7(O1) were amplified from
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EDL933 and ATCC11775 respectively. Primers used were fliC-comp2-F
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(AAAAAGAATTCCGGCATGATTATCCGTTTCT) and fliC-comp2-R
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(AAAAAGAATTCCCCAGCGATGAAATACTTGC). fliC PCR products were
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recombinase functions. Replacement of the fliC gene with cat was confirmed using
primers flanking the site of substitution. Loss of FliC was confirmed using motility assays described below.
Complementation of ΔfliC strains with pfliCH12, pfliCH7(O157) or pfliCH7(O1)
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cloned into pGem T-Easy as per manufacturer’s instructions (Promega) and
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transferred into ΔfliC strains (Table 1). Sequencing was used to confirm the presence
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of fliC with upstream promoter elements. The sequence of the fliC gene from EC614
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was submitted to Genbank (accession number JF308285). Functional expression of
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fliC was confirmed using motility assays described below. Experiments involving
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complemented strains were conducted using LB as growth media.
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149 Motility assays
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Motility agar plates, consisting of LB (Oxoid) with 0.3% agar (Agar technical #3;
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Oxoid) were inoculated with 10 µl overnight culture grown in LB broth and incubated
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at 37°C for 18 ± 2 h. Motility was confirmed by a halo of growth around the
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inoculation area.
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Determination of bacterial hydrophobicity
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Hydrophobicity was determined using Contact Angle Measurements (CAM) and
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Bacterial Adherence to Hydrocarbons (BATH) as described by Busscher et al. [21]
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s (Ab). A second tube containing 4 ml of untreated cell suspension was used as a
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control (Ac). All tubes were incubated in a waterbath at 37°C for 30 min. Following
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incubation, 1 ml of the lower aqueous layer was removed using a pipette and the OD
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and Li and McLandsborough et al. [14] respectively, with minor modifications as described by Rivas et al. [22]. Briefly, bacterial suspensions were prepared as described above and adjusted to an OD at 540nm of 1.0 ± 0.2 in PBS (approximate concentration of 8 x 108 cells/ml) for BATH assays. A 4 ml volume of the suspension
was added to 1 ml of xylene (Ajax Chemicals, Sydney, Australia) and vortexed for 20
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at 540 nm was measured. The percentage of bacterial cells which bound to the xylene
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hydrocarbon was calculated as (%) = (Ac – Ab)/Ac × 100.
169 Contact angle measurements (θ) were determined using a goniometer (KSV
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Instruments Ltd, Helsinki, Finland) and KSV CAM software (KSV Instruments Ltd)
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using the sessile drop method. Cell suspensions were prepared as described above
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using sterile distilled water (SDW) and the OD600nm adjusted to 1.2 ± 0.2
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(approximately 9 x 108 cells/ml). Bacterial lawns were prepared by filtering 25 ml of
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the suspension onto a HA filter (0.45 µm pore diameter, 25 mm filter diameter,
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Millipore, Bedford, MA, USA) by negative pressure filtration. Filters were fixed to
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glass slides using double sided adhesive tape and allowed to dry for 30 min in a
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desiccator. A drop of SDW was placed onto the lawn using a 1 ml syringe. An image
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of the drop was recorded using a digital video camera (30 frames per second FireWire
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IEEE 1394) and contact angles were determined using aforementioned KSV software.
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A minimum of five drops were recorded per filter, and reported angles (θ) were
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determined from the means of three independent filters per sample.
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described above and bacterial attachment was allowed to take place for 20 min at
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room temperature with gentle swirling for 5 s at 5 min intervals. Following
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attachment, slides were rinsed twice by dipping slides up and down three times in 50
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ml of fresh PBS, removing loosely adhered cells from the surfaces. Slides were
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Attachment assays
Assays enumerating cell attachment to SS surfaces were conducted as described by Rivas et al. [2] and to glass and Teflon surfaces as described by Goulter et al. [5]. Briefly, slides of each material were placed into bacterial suspensions prepared as
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stained with 0.01% acridine orange and the number of cells in 50 randomly selected
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fields was counted using an epifluorescence microscope (Leica DFC420 C, Leica
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Microsystems, Wetzlar, Germany). Results were presented as Log Direct
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Microscopic Count (DMC) / cm2.
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196 AFM probe modification and force mapping
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Outer membrane protein extractions were prepared and analysed using 2D gel
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electrophoresis as described by Rivas et al. [23]. In all cases two gels were run
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concurrently, with one of the two gels being stained with coomassie blue overnight.
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The location of the FliC protein on the stained gel was used to define the location of
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FliC in the unstained gel for excision. Gel pieces containing FliC were crushed and
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suspended in protein elution buffer (50 mMTris-HCl, 150 mMNaCl, and 0.1 mM
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EDTA; pH 7.5). Silicon nitride AFM probes with attached borosilicate glass particle
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(5µm diameter) were purchased precoated with 3-aminopropyltriethoxysilane by the
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manufacturer (Novascan Technologies Inc., IA, USA). Purified proteins were
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immobilised on AFM tips as described [24]. Spring constants of the protein coated
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cantilever. Immobilization efficiency of the different proteins were not significantly
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different.
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AFM tips were determined using software (Igor Pro, Asylum Research, Santa Barbara, CA). AFM tips were calibrated against glass slides prior to force mapping measurements, which were conducted at a speed of 1µm s-1. Measurements were
taken over a 5 µm x 5µm area, taking measurements at 32 x 32 (1024) total points. The immobilization of the protein to the probe was confirmed by comparison to a bare
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Statistical analysis
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One way analysis of variance and comparison of means (Tukey’s method) were
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performed using Minitab software (MINITAB 15; Minitab Inc., USA). Significant
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differences between individual strain fliC variations for a single growth media were
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determined for hydrophobicity and attachment studies. Significant differences
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between the three FliC proteins for each substrate were determined for the force
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measurement studies.
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RESULTS
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Hydrophobicity of ΔfliC strains
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In some cases, ΔfliC strains were more hydrophilic than the wt counterparts under
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some growth conditions studied; the statistical significance (p < 0.05) of these
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differences are shown in Table 1. The magnitude of differences in hydrophobicity
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was dependent on both growth media and method used to determine hydrophobicity.
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Greater differences were seen when the BATH method was used in comparison to
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CAM. In no combination studied was the ΔfliC strain more hydrophobic than its wt
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counterpart. When using the BATH method, all ΔfliC strains were more hydrophilic
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than wt counterparts (p < 0.05), with the exception of LBA (p > 0.05). When using
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the CAM method, there was again no difference when strains were cultured on LBA
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(p > 0.05), and variable results were seen for the remaining growth media.
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No significant difference in attachment to glass was seen between the E. coli
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O157:H7 and E. coli O1:H7 and their ΔfliC counterparts under any of the growth
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growth conditions (p < 0.05; Table 2). ΔfliC strains of E. coli O157:H12 and E. coli
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O157:H7 attached in significantly fewer numbers than wt counterparts following
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growth in LB and LBA (p < 0.05). E. coli O1:H7 ΔfliCH7::cat attached in
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conditions studied (p > 0.05; Table 2). In contrast, E. coli O157:H12 ΔfliCH12::cat was shown to attach in significantly fewer numbers to glass than E. coli O157:H12
under all growth conditions (p < 0.05; Table 2).
Differences in attachment to SS were seen between wt and ΔfliC pairs under some
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significantly fewer numbers than its wt counterpart following growth in LBA only (p
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< 0.05).
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wt counterpart following growth in all media (p < 0.05; Table 2), with the exception
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of LBA (p > 0.05). E. coli O157:H7 ΔfliCH7::cat attached in significantly fewer
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numbers to Teflon than the wt E. coli O157:H7 following growth in NB and NA alone
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(p < 0.05). No significant difference in attachment to Teflon between E. coli O1:H7
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and its ΔfliCH7 counterpart were seen under the majority of the growth conditions
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studied (p > 0.05), with the exception being growth in LBA (p < 0.05).
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Hydrophobicity of ΔfliC strains complemented with wtfliCH12 or wtfliCH7
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Complementing ΔfliC strains with cloned fliCH12 or cloned fliCH7 restored
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hydrophobicity to the same level (p < 0.05) as those of wt strains following growth in
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LB when hydrophobicity was determined by BATH (Figure 1). No significant
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difference (p > 0.05) was found for any of the isolate variations when hydrophobicity
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attachment to all materials for E. coli O157:H12 ΔfliCH12::cat was restored to wt
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levels when complemented with wtfliCH12 (Figure 3, upper panel).
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was determined using CAM following growth in LB (Figure 2).
Attachment of ΔfliC strains complemented with cloned fliCH12 or cloned fliCH7 Results of attachment of ΔfliC strains when complemented with cloned fliCH12 or
cloned fliCH7 to the selected abiotic surfaces is shown in Figure 3. Most notably,
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Attachment of E. coli O157:H7 ! fliCH7::cat/pfliCH12 to glass was significantly higher
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than wt E. coli O157:H7, E. coli O157:H7 ΔfliCH7::cat and E. coli O157:H7
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ΔfliCH7::cat/pfliCH7(O157) (p < 0.05; Figure 3, middle panel). There was no significant
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difference in attachment numbers to glass for wt E. coli O157:H7, E. coli O157:H7
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ΔfliCH7::cat and E. coli O157:H7 ΔfliCH7::cat/pfliCH7(O157) (p > 0.05; Figure 3, middle
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panel). No significant difference in attachment to Teflon was seen for any E. coli
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O157:H7 strain variation (p > 0.05; Figure 3, middle panel). Attachment of E. coli
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O157:H7 ! fliCH7::cat/pfliCH12 to SS was significantly higher than E. coli O157:H7
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ΔfliCH7::cat and E. coli O157:H7 ΔfliCH7::cat/pfliCH7(O157) (p < 0.05; Figure 3, middle
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panel), but not significantly different to wt E. coli O157:H7 (p > 0.05; Figure 3,
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middle panel). Attachment of E. coli O157:H7 ! fliCH7::cat/pfliCH12 was not
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significantly different from wt E. coli O157:H7 or E. coli O157:H7ΔfliC::cat (p <
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0.05; Figure 3, middle panel).
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Attachment of E. coli O1:H7 ! fliCH7::cat/pfliCH12 was significantly higher than wt E.
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coli O1:H7, E. coli O1:H7 ΔfliC::cat and E. coli O1:H7 ! fliCH7::cat/pfliCH7(O1) to all
materials studied (p < 0.05; Figure 3, lower panel). No significant difference was seen in attachment numbers of wt E. coli O1:H7, E. coli O1:H7 ΔfliCH7::cat and E. coli O1:H7 ΔfliCH7::cat/pfliCH7(O1) to any of the materials studied (p > 0.05; Figure 3,
lower panel).
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AFM force mapping
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Histograms generated from force mapping data are shown in Figure 4. The number of
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times (frequency; y axis) each unit of force was measured (x axis) can be seen in this
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figure. Probes coated with purified FliCH12 adhered significantly more strongly to 13
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glass and SS surfaces than probes coated with either FliCH7 protein (p < 0.05; Figure
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4, upper and middle panel). FliCH7(O1) had a significantly higher adhesion force to
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Teflon than FliCH12 (p < 0.05; Figure 4, lower panel). Due to high standard deviation,
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FliCH7(O157) was not significantly different (p > 0.05) to either protein in adhesion
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force to Teflon (Figure 4, lower panel). Example force distance curves to Teflon are
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shown in Figure 5. Figure 5 shows multiple pull off events for the FliCH12 protein,
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but only a single pull off event for both FliCH7 proteins. The shape of the force curves
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are representative of all force curves for these proteins to all materials (data not
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shown), albeit the force scale differed when measuring adhesion forces to glass and
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SS due to smaller measured forces.
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306 DISCUSSION
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A number of studies have shown that differences in the fliC gene of E. coli encoding
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different H antigens can influence attachment to biotic surfaces [9, 16]. Studies
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investigating the influence of flagella expression on the attachment of E. coli to
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abiotic surfaces often include a number of different strains, and have not used genetic
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eliminate flagella expression. To further investigate the role of different flagella H
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antigens, the deleted fliC gene of each strain was complemented with the cloned wt
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fliC gene. The influence of these modifications on surface hydrophobicity was also
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determined.
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modification tools in order to study the effects of loss of flagella expression on attachment for individual strains [2]. In these previous studies no correlation was found between attachment and flagellar expression. In the present study, the fliC genes from three E. coli strains belonging to serotypes O157:H12, O157:H7 and
O1:H7 were replaced by a chloramphenicol antibiotic resistance cassette (cat) to
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321 The loss of fliC and flagella expression resulted in a decrease in hydrophobicity for all
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strains in a growth media dependent fashion when determined using the BATH
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method (Table 1); demonstrating the hydrophobic nature of FliC proteins. This was
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also demonstrated by using AFM force mapping, where increased adhesion force was
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measured between FliC proteins and Teflon, the most hydrophobic surface included in
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this study, compared with glass and SS [25]. The lowest adhesion force was
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measured between FliC proteins and glass, followed by SS. These results support
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studies which suggest hydrophobic surfaces interact more readily with other surfaces
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which are hydrophobic in nature [11]. However, it should also be noted that BATH
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measurements can be influenced by the surface charge of bacterial cells [26]. Due to
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the lack of significant difference found when hydrophobicity was determined by
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CAM, this study highlights the complexity of determining bacterial hydrophobicity
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and highlights that other factors may also influence measurements and consequent
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correlations with attachment. In particular, the CAM method uses the Young
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equation to calculate hydrophobicity. This equation makes the assumption that the
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FliCH7 protein (Figure 4). Other studies utilising AFM to study bacterial flagella are
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often restricted to imaging alone [27, 28], and to our knowledge this is the first AFM
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force mapping study investigating the interactions between purified FliC proteins and
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glass, Teflon and SS surfaces. Force distance curves to glass and SS surfaces were
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surface is perfectly smooth, which is not the case for bacterial lawns. This may also explain the differences seen between BATH and CAM measurements in the current study.
FliCH12 was shown to have a higher average adhesion force to glass and SS than either
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similar, albeit, on a lower force scale (data not shown). As shown in Figure 5, single
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pull off events were recorded for FliCH7 proteins, whereas the retraction of FliCH12
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from the surface resulted in multiple, stepwise pull off events as indicated by the
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multiple peaks seen in the retraction curve (Figure 5). This may indicate that FliCH12
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adheres to these surfaces in a different fashion than FliCH7 proteins. Sequencing
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results revealed that the fliC gene from the E. coli O157:H12 strain was slightly
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longer than the fliC sequence in the E. coli O157:H7 or E. coli O1:H7 strains resulting
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in an additional 10 predicted amino acids (595 as opposed to 585) in the FliCH12
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protein. This suggests that additional amino acids resulting in longer proteins (or a
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potential change in tertiary structure) may require multiple pull off events from a
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surface than shorter proteins. In the study of Sethuraman et al. [29] adhesion forces
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were measured between seven proteins of various sizes and solid surfaces. Proteins of
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larger size were shown to have force distance curves extending over a few hundred
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nanometres with a stepwise, multiple pull off event pattern similar to that seen in the
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current study. Smaller proteins were shown to have a single pull off event [29]. A
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more recent study investigating adhesion forces of single protein molecules has
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demonstrated that force curves with multiple pull off events are due to the breaking of individual bonds as the protein retracts from the surface [30]. These studies suggest that the larger FliCH12, as compared to the FliCH7 protein, results in force distance
curves with multiple, stepwise, pull off events, which may aid in stronger adhesion to the surfaces included in this study.
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Our data indicated that FliCH12 plays an important role in attachment for the E. coli
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O157:H12 strain included in this study to all surfaces in a growth media dependent
370
fashion (Table 2). These results show that the strains included in this study with the
16
Page 16 of 37
371
ability to express fliCH12 have an increased ability to attach to a variety of abiotic
372
surfaces such as glass, SS and Teflon when compared with strains lacking fliCH12 or
373
strains expressing fliCH7 in the majority of cases.
ip t
374 While flagella have been shown to influence attachment in other studies [9, 16, 17],
376
numerous factors may be involved in the attachment process that are likely to differ
377
between strains. These include surface charge [14], the production of other surface
378
structures such as fimbriae [31], outer membrane proteins such as Ag43 [32], among
379
others. Growth media also influences these factors and others; future studies
380
including various other growth media and the relationship between expression of
381
bacterial surface structures and attachment is warranted. In order to fully understand
382
the attachment mechanism for individual strains many factors must therefore be
383
considered.
us
an
M
d te
384
cr
375
The emergence of Shigatoxigenic E. coli (STEC) strains of various O and H antigenic
386
types other than E. coli O157:H7 causing severe outbreaks and disease [33] shows the
391
Ac ce p
385
392
study and previously by our laboratory [5]. If non-pathogenic E. coli strains were to
393
acquire virulence genes and had the added capacity to adhere strongly to surfaces
394
commonly used in food processing environments, controlling such a potential
395
pathogen would be difficult. For this reason, further studies into the control of
387 388 389 390
importance of studying the properties and attachment behaviour of non O157:H7 STEC strains. The transfer of virulence genes from one strain of E. coli to another is
also well documented [34-36]. E. coli O157:H12 strains have been shown to be resistant to processes such as high pressure treatment [1], and have been shown to attach more readily than other strains to a variety of surfaces as demonstrated in this
17
Page 17 of 37
pathogenic and non-pathogenic E. coli strains with the ability to attach readily to a
397
number of surfaces are required. This study has identified the importance of H12
398
antigens in attachment as indicated by the strains included in this study, and as such
399
has identified a potential target for the control of this organism.
ip t
396
400 ACKNOWLEDGEMENTS
402
R. M. Goulter acknowledges the financial support of the Department of Employment,
403
Economic Development and Innovation of the Queensland Government, Australia
404
through the Smart State PhD Scholarships Program and the financial support of the
405
Australian Government through the Australian Postgraduate Award. This work was
406
also supported through funding from CSIRO Food and Nutritional Sciences.
M
an
us
cr
401
407
Ac ce p
te
d
408
18
Page 18 of 37
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[35] B.G. Kelly, A. Vespermann and D.J. Bolton, Horizontal gene transfer of
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Toxicology, 47 (2009) 969-977.
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[36] M. Muniesa, J. Jofre, C. Garcia-Aljaro and A.R. Blanch, Occurrence of
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environment, Environmental Science & Technology, 40 (2006) 7141-7149.
cr
ip t
518
us
524 525
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te
d
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an
526
24
Page 24 of 37
us
cr
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526
Ac ce p
528
te
d
M
an
527
529 530
25
Page 25 of 37
Figure 1: Hydrophobicity of strains following growth in Luria Bertani Broth as
532
determined using the Bacterial Adherence to Hydrocarbons method. Data shown are
533
means of three independent experiments + standard deviation. *Results also shown in
534
Table 1.
ip t
531
535
Ac ce p
te
d
M
an
us
cr
536
26
Page 26 of 37
us
cr
ip t
536
Ac ce p
538
te
d
M
an
537
539
27
Page 27 of 37
540
Figure 2: Hydrophobicity of strains following growth in Luria Bertani Broth as
541
determined using the Contact Angle Measurements Method. Data shown are means of
542
three independent experiments + standard deviation. *Results also shown in Table 1.
ip t
543 544
Ac ce p
te
d
M
an
us
cr
545
28
Page 28 of 37
ip t cr Ac ce p
2
te
d
M
an
us
1
3 4 5
30
Page 29 of 37
1
Figure 3: Attachment (Log Direct Microscopic Count (DMC) / cm2) of strains
2
following growth in LB;
3
Data shown are means of three independent experiments + standard deviation.
Ac ce p
te
d
M
an
us
cr
ip t
wt; ΔfliC::cat; pfliCH12; pfliCH7(O157); pfliCH7(O1).
31
Page 30 of 37
100
Glass
Frequency
80 60 40
0 0.0
0.2
0.4
0.6
0.8
nN
us
100
Stainless Steel
80 60
an
Frequency
1.0
cr
FliCH12 0.32 ± 0.09 nN FliCH7(O157) 0.09 ± 0.02 nN FliCH7(O1) 0.10 ± 0.06 nN
ip t
20
40
M
20 0 0.0
0.2
0.4
0.6
0.8
1.0
nN
50
Teflon
Frequency
Ac ce p
40
te
d
FliCH12 0.45 ± 0.16 nN FliCH7(O157) 0.07 ± 0.02 nN FliCH7(O1) 0.05 ± 0.09 nN
30 20 10
0
0
10
20
30
40
50
nN
FliCH12 3.2 ± 1.1 nN FliCH7(O157) 25.9 ± 15.4 nN FliCH7(O1) 15.1 ± 2.8 nN
1 2 3
Figure 4: Histograms of adhesion force to Glass, SS and Teflon measured using AFM
4
force mapping for purified FliCH12 and FliCH7 proteins. Measurements are average
5
adhesion force ! standard deviation. 32
Page 31 of 37
us
cr
ip t
1
FliC
an
H12
FliC
H7(O157)
FliC
H7(O1)
M
2 3
Figure 5: Example force distance curves for purified FliCH12 and FliCH7 proteins to
5
Teflon.
te
7
Ac ce p
6
d
4
33
Page 32 of 37
ip t cr
BATH (% Adherence) NB
LB*
O157:H12
54.1 ± 1.2a
18.6 ± 3.0a
O157:H12 ΔfliCH12::cat
27.8 ± 7.3b
2.02 ± 1.9b
O157:H7
57.7 ± 3.0a
5.7 ± 0.4a
O157:H7ΔfliCH7::cat
23.3 ± 8.0b
O1:H7
20.5 ± 1.0a
O1:H7 ΔfliCH7::cat
12.1 ± 1.9b
CAM (θ)
NA
LBA
NB
LB*
NA
LBA
19.2 ± 2.9a
13.2 ± 2.4a
15.4 ± 0.7a
15.8 ± 0.3a
14.4 ± 0.3a
14.5 ± 0.7a
9.7 ± 2.7b
15.1 ± 1.6a
12.7 ± 0.6b
13.6 ± 0.9a
11.0 ± 0.3b
13.2 ± 0.8a
32.7 ± 1.3a
14.3 ± 2.7a
14.7 ± 0.5a
15.4 ± 1.0a
15.3 ± 0.8a
13.6 ± 0.5a
2.0 ± 1.8b
5.6 ± 5.4b
11.4 ± 5.8a
12.5 ± 0.4b
12.1 ± 0.7b
11.5 ± 0.3b
12.0 ± 0.8a
9.1 ± 1.2a
9.8 ± 1.1a
0.4 ± 0.4a
20.8 ± 0.5a
17.2 ± 0.7a
16.5 ± 1.1a
17.7 ± 1.7a
3.5 ± 2.0b
3.2 ± 2.8b
3.3 ± 2.9a
15.0 ± 3.8a
12.6 ± 2.5b
17.8 ± 3.4a
16.1 ± 2.8a
M an
E. coli Strain
us
Table 1: Hydrophobicity of E. coli strains cultured in Nutrient Broth (NB), Luria Bertani Broth (LB), Nutrient Agar (NA) and LB Agar (LBA)
ce pt
ed
1
2
a, b
3
*Results also shown in Figures 1 and 2.
Ac
Different superscripts indicate significant differences for parent and ΔfliC::cat pairs for that growth media (p < 0.05).
34 Page 33 of 37
ip t cr
Table 2: Attachment (Log Direct Microscopic Count (DMC) / cm2 ± standard deviation of three independent experiments) of E. coli strains
2
cultured in Nutrient Broth (NB), Luria Bertani Broth (LB), Nutrient Agar (NA) and LB Agar (LBA) to glass, stainless steel (SS) and Teflon
O157:H7ΔfliCH7::cat
O1:H7
O1:H7 ΔfliC::cat
NA
LBA
NB
LB
NA
LBA
NB
LB
NA
LBA
4.2 ±
4.8 ±
3.9 ±
5.2 ±
3.9 ±
4.7 ±
2.9 ±
4.8 ±
3.5 ±
3.9 ±
3.9 ±
2.7 ±
0.1a
0.5a
0.1a
0.2a
0.0a
0.4a
0.2a
0.3a
0.2a
0.1a
0.1a
0.1a
2.8 ±
3.0 ±
2.9 ±
2.9 ±
3.5 ±
3.5 ±
2.7 ±
3.0 ±
2.6 ±
2.7 ±
2.6 ±
2.6 ±
0.2b
0.2b
0.2b
0.2b
0.3a
0.2b
0.5a
0.1b
0.2b
0.5b
0.2b
0.1a
4.0 ±
3.8 ±
3.7 ±
4.1 ±
2.2 ±
4.0 ±
2.8 ±
3.9 ±
3.6 ±
3.0 ±
4.2 ±
2.8 ±
0.1a
0.1a
0.2a
0.3a
0.3a
0.1a
0.1a
0.4a
0.2a
0.5a
0.1a
0.4a
3.7 ±
3.5 ±
3.7 ±
3.6 ±
2.7 ±
3.1 ±
2.8 ±
2.7 ±
2.7 ±
2.7 ±
2.2 ±
2.3 ±
0.0a
0.2a
0.2a
0.3a
0.2a
0.9b
0.2a
0.4b
0.1b
0.2a
0.3b
0.2a
Ac
O157:H7
LB
ce pt
O157:H12 ΔfliCH12::cat
Teflon (Log DMC / cm2)
NB
ed
O157:H12
SS (Log DMC / cm2)
M an
Glass (Log DMC / cm2)
us
1
3.7 ±
4.0 ±
3.6 ±
4.1 ±
2.8 ±
3.6 ±
2.7 ±
3.7 ±
3.2 ±
3.2 ±
3.4 ±
4.0 ±
0.1a
0.2a
0.0a
0.2a
0.1a
0.2a
0.2a
0.3a
0.2a
0.3a
0.2a
0.3a
3.8 ±
3.8 ±
3.8 ±
3.6 ±
2.9 ±
3.2 ±
2.6 ±
2.6 ±
3.4 ±
3.4 ±
3.4 ±
3.4 ±
0.2a
0.3a
0.2a
0.1a
0.2a
0.1a
0.4a
0.4b
0.2a
0.1a
0.2a
0.2b
35 Page 34 of 37
ip t cr
a, b
Different superscripts indicate significant differences for parent and ΔfliC::cat pairs for that growth media (p < 0.05)
us
1
Ac
ce pt
ed
M an
2
36 Page 35 of 37
ip t cr us
5
4
M an
200
µm
100
2
pN
150
3
ed
50 1
0
1
2
3
4
5
µm
Ac
0
1 2
ce pt
0
37 Page 36 of 37
Highlights •
Loss of FliCH12 in O157:H12 decreased attachment to glass, Teflon and stainless steel surfaces (p<0.05). Complementing O157:H12 ΔfliCH12 with cloned wildtype (wt) fliCH12 restored
ip t
•
•
cr
attachment to wt levels.
The loss of FliCH7 in O157:H7 and O1:H7 did not always alter attachment
us
(p>0.05), but complementation with cloned fliCH12, as opposed to cloned fliCH7, significantly increased attachment for both strains compared with wt counterparts
Although no significant difference in adhesion force was observed between
M
•
an
(p<0.05).
FliCH12 and FliCH7 AFM probes, differences in force curves suggest different
These results indicate that E. coli strains expressing flagellar H12 antigens have
te
•
d
mechanism of attachment for FliCH12 compared with FliCH7.
an increased ability to attach to certain abiotic surfaces compared with E. coli
Ac ce p
strains expressing H7 antigens.
Page 37 of 37