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Chapter 3 classified as persistent (4 S. Agona, 3 S. Montevideo) and 14 presumed non persistent (2 S. Agona, 3 S. Montevideo, 3 S. Senftenberg, 6 S. Typhimurium). Biofilm formation was assessed using the microtiter plate method measuring optical density (OD) and biofilm (pellicle formation) at the air-liquid interface. The S. Agona and S. Montevideo serovars resulted in a more dense biofilm and the biofilm formed more rapidly than the biofilm formed by strains of S. Senftenberg and S. Typhimurium [56]. Although there were significant limitations in the data in terms of limited assessment of reproducibility of observations which are further discussed in this thesis in chapter 6 where the reproducibility and repeatability of this work is compared the results achieved in this thesis. 3.1.2. Repeated S. Agona outbreaks – persistence in the environment As discussed in chapter 1, the S. Agona outbreak strain SAGOXB.0066 was isolated from various food products during the course of the outbreak and traced back to a processing line over the course of the investigation [58]. After a pharmaceutical grade cleaning of the food plant the outbreak strain was no longer identified within the facility. However the SAGOXB.0066 variant (distinguishable by 2 bands) was subsequently isolated from the drains of the campus for an extended period of time. The persistence of the strain prompted this research to assess the relative density of biofilm formed by this strain over an extended period. For purposes of comparison, representatives of the serovars S. Typhimurium (including a monophasic variant) and S. Enteritidis were also studied. 3.1.3. Biofilm formation over an extended period of time Most reported work on biofilm is based on studies of biofilm formed over 24 or 48 hours [56, 71, 96, 98, 111, 159, 165, 178, 191]. There is comparatively little work examining biofilm developed over a longer period of time [102, 148-149] and very little or no work comparing biofilm Page 91
Chapter 3 formation on multiple surfaces by a number of strains over short (≤48 hours) and long periods of time (≥72 hours). In previously published work in this area authors have examined biofilm formation of a single strain over an extended period of time [102, 106, 148-149, 188] or examined the use of two strains [71] or four strains of the same serovar (S. Enteritidis) [67] after biofilm formation after a specific duration of time. This is a significant gap in knowledge as it may be that there are important differences between early biofilm and late biofilm. It is likely that late biofilm is more relevant to the situation that can arise in food processing environments when a specific strain is repeatedly isolated over a period of months or years. 3.2. Method – 168 hour biofilm This section describes investigation of biofilm density when biofilm was established over an extended period of time (168 hours / 7days) and a comparison of density at 48 and 168 hours. Essentially, the method for the 168 hour biofilm was similar to the standardised method (method described in chapter 2) except the period of continuous flow was extended from 24 hours to 144 hours. The strains studied were an S. Agona from the outbreak (S09-0494), S. Agona reference strain (SL483), S. Typhimurium (S09-0419), S. Typhimurium reference strains SL1344 and a S. Enteritidis (S09-0717). The characteristics of these strains were previously described in Table 2.1 in chapter 2. 3.3. Validation of techniques Following SEM analysis (see Figure 3.1 and 3.2) it became clear that the entire biofilm was not removed from the surface using the conditions (20 kHz for 7 minutes) previously optimized in chapter 2 (see section 2.3 in chapter 2). In order to improve removal alternative sonication conditions were examined. Following the conditions described in chapter 2, sonication was performed at 20 kHz for 7, 10 and 14 minutes. The suspended biofilm Page 92
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Salmonella enterica - biofilm forma
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Table of Contents Page Number 1.16.
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Table of Contents Page Number 4.1.2
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Table of Contents Page Number 6.8.4
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List of Abbreviations Table Page Nu
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List of Abbreviations List of Abbre
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Summary of Content Summary of Conte
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“To show your true ability is alw
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Acknowledgements ever met, Fiona th
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Acknowledgements This Ph.D. thesis
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Chapter 1 1.1. Salmonella Salmonell
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Chapter 1 may impose enormous costs
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Chapter 1 cracks or penetration of
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Chapter 1 detected by agglutination
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Chapter 1 become colonized with >10
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Chapter 1 antigens [z27],[z45] [1].
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Chapter 1 S. Agona has also been im
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Chapter 1 isolated from undercooked
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Chapter 1 The S. Typhimurium strain
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Chapter 1 ecosystem with the use of
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Chapter 1 [82]. Previous research h
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Chapter 1 to genes involved in flag
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Chapter 1 observation has also been
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Chapter 1 [119] and flow cell react
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Chapter 1 based disinfectant) the s
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Chapter 1 formation of two strains
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Chapter 1 shield the S. Agona cells
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Chapter 1 Key Objectives of this st
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Chapter 2 2. Abstract Food-borne pa
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Chapter 2 indicated that there may
Page 62 and 63: Chapter 2 taken from industrial set
Page 64 and 65: Chapter 2 2.1.4. CDC Biofilm Reacto
Page 66 and 67: Chapter 2 can be performed without
Page 68 and 69: Chapter 2 To summarise, previous au
Page 70 and 71: Chapter 2 2.3. Methods for examinin
Page 72 and 73: Chapter 2 and the biofilm formed th
Page 74 and 75: Chapter 2 2.4. Statistical Analysis
Page 76 and 77: Chapter 2 Table 2.1: Strain charact
Page 78 and 79: Chapter 2 XII. The gas port and med
Page 80 and 81: Chapter 2 II. Primary fixative cons
Page 82 and 83: Chapter 2 IX. Once the conditions w
Page 84 and 85: Chapter 2 Figure 2.1: Image of CBR
Page 86 and 87: Chapter 2 2.6. Results 2.6.1. Asses
Page 88 and 89: Chapter 2 2.6.3. SEM Analysis of su
Page 90 and 91: Chapter 2 Figure 2.6: Complete remo
Page 92 and 93: Chapter 2 2.6.5. Mean log 10 densit
Page 94 and 95: Chapter 2 2.6.6. Mean log 10 densit
Page 96 and 97: Chapter 2 Table 2.5: Mean log 10 de
Page 98 and 99: Chapter 2 The results displayed in
Page 100 and 101: Chapter 2 Table 2.7: Difference bet
Page 102 and 103: Chapter 2 Table 2.8: The mean log 1
Page 104 and 105: Chapter 2 Figure 2.7: Graph of the
Page 106 and 107: Chapter 2 also used “high” soni
Page 108 and 109: Chapter 2 2.8. Summary All 13 strai
Page 110 and 111: Chapter 3 3. Abstract It has been e
Page 114 and 115: Chapter 3 was plated onto TSA for e
Page 116 and 117: Chapter 3 Figure 3.1: The mean log
Page 118 and 119: Chapter 3 Figure 3.3: SEM image of
Page 120 and 121: Chapter 3 Table 3.2: Difference bet
Page 122 and 123: Chapter 3 3.4.3. Intra-serovar vari
Page 124 and 125: Chapter 3 3.4.4. Strain variation i
Page 126 and 127: Chapter 3 3.4.5. The density of bio
Page 128 and 129: Chapter 3 3.5. Discussion As illust
Page 130 and 131: Chapter 3 As discussed in section 3
Page 132 and 133: Chapter 3 increased biofilm appeare
Page 134 and 135: Chapter 4 Examining the efficacy of
Page 136 and 137: Chapter 4 through laboratory based
Page 138 and 139: Chapter 4 acid and quaternary ammon
Page 140 and 141: Chapter 4 the surface the temperatu
Page 142 and 143: Chapter 4 availability of multiple
Page 144 and 145: Chapter 4 peracetic acid (100, 200,
Page 146 and 147: Chapter 4 As discussed previously,
Page 148 and 149: Chapter 4 Dey/Engley (Difco) neutra
Page 150 and 151: Chapter 4 sterilisation was achieve
Page 152 and 153: Chapter 4 IX. Aliquots of 100µl of
Page 154 and 155: Chapter 4 XV. XVI. XVII. XVIII. XIX
Page 156 and 157: Chapter 4 4.3. Results 4.3.1. Suspe
Page 158 and 159: Chapter 4 Table 4.3: Mean log 10 de
Page 160 and 161: Chapter 4 Benzalkonium chloride was
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Chapter 4 4.4. Discussion Suspensio
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Chapter 4 effective at eliminating
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Chapter 4 Surprisingly, Nguyen et a
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Chapter 4 though a contact time of
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Chapter 4 provide a more standardis
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Chapter 4 hours instead of the stan
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Chapter 5 5. Abstract Examining bio
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Chapter 5 dry and rough (bdar) morp
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Chapter 5 absence of curli and cell
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Chapter 5 studies such as the micro
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Chapter 5 discussed in chapter 4. T
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Chapter 5 5.2. Methods 5.2.1. Colon
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Chapter 5 XIII. XIV. XV. XVI. XVII.
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Chapter 5 XVI. XVII. XVIII. XIX. XX
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Chapter 5 5.3.2. Repeatability of m
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Chapter 5 Figure 5.2: Stained biofi
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Chapter 5 was formed at room temper
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Chapter 5 Table 5.3: The density of
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Chapter 5 5.3.4. The density of bio
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Chapter 5 Table 5.6: The difference
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Chapter 5 Table 5.7: The difference
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Chapter 5 Table 5.8: Summary of ass
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Chapter 5 interpretation therefore
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Chapter 5 measures of S. enterica b
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Chapter 5 cells peaks [165, 191]. D
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Chapter 5 5.5. Limitations of the s
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Chapter 6 Discussion of S. enterica
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Chapter 6 However, a number of auth
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Chapter 6 It is of interest to exam
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Chapter 6 were similar at 48 hours.
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Chapter 6 attributable to a small n
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Chapter 6 chosen as an appropriate
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Chapter 6 undetermined. However, th
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Chapter 6 reason for this may be du
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Chapter 6 Moreover, based on the re
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Chapter 6 responsible for increased
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Chapter 6 not fully capture the ran
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Chapter 6 However, the extent of bi
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Chapter 6 6.10. Conclusions and rec
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Chapter 6 studies may solve some of
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Bibliography Bibliography Page 221
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Bibliography 12. T. Takata, J.L., H
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Bibliography 29. Barker, J., M. Nae
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Bibliography 47. Threlfall, E.J., H
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Bibliography 65. CDC. Investigation
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Bibliography 83. Ledeboer, N., Frye
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Bibliography 100. Chia, T., Goulter
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Bibliography 117. Buckingham-Meyer,
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Bibliography 133. European Committe
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Bibliography Supernatant of a Hafni
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Bibliography 165. Díez-García, M.
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Bibliography cultivation of Staphyl
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Bibliography Compounds. 2012. Appli
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Bibliography 215. Buck, et al., A q
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Bibliography Canadian Journal of Ps
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Appendix 1 Appendix 1— List of Re
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Appendix 1 Appendix 1 - List of Equ
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Appendix 2 Colour index High outlie
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Appendix 3 Appendix 3-Figure 2: PFG
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Appendix 4 Poster Presentation Envi
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