Attachment of Escherichia coli to Abiotic Surfaces

Rebecca Goulter (2011). Attachment of Escherichia coli to Abiotic Surfaces PhD Thesis, , The University of Queensland.

       
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s40781752_phd_finalthesis.pdf Rebecca Goulter 40781752 Final Thesis application/pdf 3.05MB 13
Author Rebecca Goulter
Thesis Title Attachment of Escherichia coli to Abiotic Surfaces
Institution The University of Queensland
Publication date 2011-03
Thesis type PhD Thesis
Supervisor Professor Ian Gentle
Professor Gary Dykes
Dr Kari Gobius
Total pages 220
Total colour pages 15
Total black and white pages 205
Subjects 11 Medical and Health Sciences
Abstract/Summary Escherichia coli O157:H7 are important foodborne pathogens with the ability to attach to materials commonly used in food processing environments such as stainless steel (SS). The aim of this work was to investigate the roles of a number of factors which may influence attachment of E. coli to glass, Teflon and SS surfaces. These factors were; 1) curli production, 2) surface roughness of SS, 3) DNA protection during starvation protein (Dps) expression and 4) different H antigens (encoded on the fliC gene). Six E. coli isolates were characterised for curli production, autoaggregation, hydrophobicity and attachment to glass and Teflon. Curli production and autoaggregation were determined by absorbance assays; hydrophobicity by bacterial adherence to hydrocarbons (BATH), hydrophobic interaction chromatography and contact angle measurements (CAM); and attachment by epifluorescence microscopy. Curli production varied and for some strains correlated with autoaggregation. Curli production correlated with decreased hydrophobicity for two strains. Results of attachment assays were variable between both strain and substrate. This study suggests that attachment of some E. coli to abiotic surfaces may be influenced by curli production, autoaggregation and hydrophobicity. Curli expression and attachment of E. coli to glass, Teflon and SS was further investigated through csgA knockout mutants in two isolates of E. coli O157:H7. Attachment assays and adhesion force measurements using atomic force microscopy (AFM) force mapping were used to determine differences between wildtype (wt) and csgA negative (ΔcsgA) strains. Hydrophobicity was determined using BATH and CAM. Attachment assay results indicated ΔcsgA strains attached in lower numbers to glass and Teflon surfaces compared with wt counterparts in 7/8 and 3/8 cases respectively (p<0.05), but in higher numbers to SS in 4/8 cases (p<0.05). No correlation was seen between BATH and CAM results (R2<0.70). Hydrophobicity differed between wt and ΔcsgA strains in some cases (p<0.05). AFM force mapping revealed no difference in adhesion force to glass and SS surfaces between wt and ΔcsgA strains (p>0.05), but a significantly greater adhesion force to Teflon for one of the two wt strains compared with its ΔcsgA counterpart (p<0.05). These results show for the first time through the use of csgA negative strains and AFM, that curli expression may influence attachment of E. coli to glass, Teflon and SS. The role of DNA protection during starvation protein (Dps) expression in E. coli attachment to a number of surfaces, including SS was investigated through the construction of dps knockout mutants. The Dps protein was not found to influence hydrophobicity, but did have a putative role in attachment in a strain and substrate dependent manner. The interactions of six E. coli strains with SS type 304, finishes #2B, #4 and #8 were investigated. Attachment assays, detachment assays and AFM were used to make this comparison. Attachment data suggested that E. coli attach in greater numbers to smoother SS#8, however, detachment assays and AFM data suggest cells are easily removed from this finish. Conversely, attachment to SS#2B was lower, and AFM data suggests E. coli may adhere more strongly to this finish. Attachment and detachment data to SS#4 was variable suggesting complex attachment mechanisms. The data from this study indicates bacterial interactions with SS#4 are complex and less easily predicted than SS of different finishes. This may be of concern to the food industry as SS#4 is the most common material used in food processing. The role of different H antigens attachment to glass, Teflon and SS surfaces was investigated through the construction of fliC knockout mutants in strains of E. coli O157:H7, O1:H7 and O157:H12. The loss of FliCH12 in O157:H12 decreased attachment to glass, Teflon and SS surfaces (p<0.05). Complementing O157:H12 ΔfliCH12 with fliCH12 restored the attachment to all surfaces to wt levels. The loss of FliCH7 in O157:H7 and O1:H7 did not always alter attachment (p>0.05), but complementation with fliCH12, as opposed to fliCH7, significantly increased attachment to all surfaces for both strains compared with wt counterparts (p<0.05). Hydrophobicity determined using BATH and CAM was influenced by fliC expression but was not correlated to attachment. Purified FliC was used to functionalise AFM probes which were used to measure adhesion forces between FliC and substrates. Although no significant difference in adhesion force was observed between FliCH12 and FliCH7 probes, differences in force curves suggest different mechanism of attachment for FliCH12 as compared with FliCH7. This study indicates that E. coli strains expressing flagellar H12 antigens have an increased ability to attach to certain abiotic surfaces as compared with E. coli strains expressing H7 antigens. The results of this work will be useful in assisting the food industry to modify practices; as many potential factors aiding the attachment of E. coli have been identified. Future work into the alteration of cleaning practices and procedures within the food industry targeting these attachment mechanisms may result in a decrease in the contamination of food products.
Keyword Escherichia coli (E. coli)
Attachment
Abiotic surfaces
Curli
Flagella
Outer membrane proteins
Cell surface hydrophobicity
Dps
H antigens
Additional Notes Colour Pages: 75-77, 102, 117, 164, 172-173, 180, 182-183, 192-195. Landscape Pages: 51, 73, 96-97, 99, 140, 154-159, 169-170, 177, 189-191.

 
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Created: Mon, 30 May 2011, 13:50:49 EST by Miss Rebecca Goulter on behalf of Library - Information Access Service