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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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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(p0.05), 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). 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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235 Attachment of ΔfliC strains

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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).

249 E. coli O157:H12 ΔfliC::cat attached in significantly lower numbers to Teflon than its

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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
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

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fashion (Table 2). These results show that the strains included in this study with the

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ability to express fliCH12 have an increased ability to attach to a variety of abiotic

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surfaces such as glass, SS and Teflon when compared with strains lacking fliCH12 or

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strains expressing fliCH7 in the majority of cases.

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374 While flagella have been shown to influence attachment in other studies [9, 16, 17],

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numerous factors may be involved in the attachment process that are likely to differ

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between strains. These include surface charge [14], the production of other surface

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structures such as fimbriae [31], outer membrane proteins such as Ag43 [32], among

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others. Growth media also influences these factors and others; future studies

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including various other growth media and the relationship between expression of

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bacterial surface structures and attachment is warranted. In order to fully understand

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the attachment mechanism for individual strains many factors must therefore be

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considered.

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The emergence of Shigatoxigenic E. coli (STEC) strains of various O and H antigenic

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types other than E. coli O157:H7 causing severe outbreaks and disease [33] shows the

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study and previously by our laboratory [5]. If non-pathogenic E. coli strains were to

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acquire virulence genes and had the added capacity to adhere strongly to surfaces

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commonly used in food processing environments, controlling such a potential

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pathogen would be difficult. For this reason, further studies into the control of

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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

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pathogenic and non-pathogenic E. coli strains with the ability to attach readily to a

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number of surfaces are required. This study has identified the importance of H12

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antigens in attachment as indicated by the strains included in this study, and as such

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has identified a potential target for the control of this organism.

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400 ACKNOWLEDGEMENTS

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R. M. Goulter acknowledges the financial support of the Department of Employment,

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Economic Development and Innovation of the Queensland Government, Australia

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through the Smart State PhD Scholarships Program and the financial support of the

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Australian Government through the Australian Postgraduate Award. This work was

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also supported through funding from CSIRO Food and Nutritional Sciences.

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Figure 1: Hydrophobicity of strains following growth in Luria Bertani Broth as

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determined using the Bacterial Adherence to Hydrocarbons method. Data shown are

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means of three independent experiments + standard deviation. *Results also shown in

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Table 1.

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Figure 2: Hydrophobicity of strains following growth in Luria Bertani Broth as

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determined using the Contact Angle Measurements Method. Data shown are means of

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three independent experiments + standard deviation. *Results also shown in Table 1.

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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 (p0.05), 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

Escherichia coli strains expressing H12 antigens demonstrate an increased ability to attach to abiotic surfaces as compared with E. coli strains expressing H7 antigens.

The role of Escherichia coli H antigens in hydrophobicity and attachment to glass, Teflon and stainless steel (SS) surfaces was investigated through c...
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