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Chapter 3 3. Abstract It has been established that Salmonella enterica serovar Agona, serovar Typhimurium and serovar Enteritidis strains can form a biofilm on food contact surfaces. However there is limited information on Salmonella biofilm development over longer periods of time. A dense biofilm formed over an extended period of time may prove particularly difficult to eradicate in food manufacturing environments. This may result in recurrent outbreaks of particular strains of food-borne pathogens. The purpose of this work is to assess change in density of Salmonella enterica biofilm after a longer period of time than what is currently used in many studies. For this element of the thesis the biofilm was allowed to develop over 168 hours (7 days) and culturable counts were performed to investigate change in biofilm density over time. In general, biofilm became more dense after the extended period of time. At 168 hours the S. Agona outbreak associated strain (S09-0494) produced a significantly more dense biofilm than the other S. Agona SL483 (p=0.004 on all 5 surfaces). The S. Agona outbreak strain (S09-0494) also resulted in a greater density of cells recovered from the surface than S. Typhimurium S09-0419 (p
Chapter 3 previously [163]. These environmental persisters may be related to the formation of a mature biofilm [56]. After a mature biofilm has developed it may be difficult to eradicate the pathogen from the environment due to the number of metabolic processes as outlined in earlier chapters. 3.1.1. Repeated S. enterica outbreaks As discussed in detail in chapter 1 the serovars studied in this research (S. Agona, S. Enteritidis and S. Typhimurium) have been involved in multiple food-borne outbreaks. Repeated isolation of PFGE indistinguishable isolates from a source suggests that the isolates represent a strain that is resident in that niche. Isolation of PFGE indistinguishable isolates from different sources is generally interpreted as indicative of a recent common origin and my help to link cases of infection with a specific niche [194]. Nesse and colleagues analysed Salmonella isolated from the environment and the raw ingredients used in four fish feed factories over an extended period of time [163]. All isolates collected over three years were assessed by PFGE and plasmid analysis. In addition, strains taken retrospectively from the factories spanning a ten year period were also examined. It was evident from the results that there were numerous distinguishable PFGE profiles found within the ingredients [163]. Specific clonal lineages of Salmonella (based on indistinguishable PFGE profiles) were recognized to persist within the environment over the 3 year period, with the same serovar (no PFGE data) reported in the environment for up to ten years [163].This indicates a particular strain may enter and persist in a premises and that contamination of products may come from this source rather than from raw materials [163]. Following the PFGE analysis of S. enterica collected in the fish feed factories the same research group sought to link persistence in the environment with dense biofilm formation [56]. Seven strains were Page 90
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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
Page 60 and 61: 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 112 and 113: Chapter 3 classified as persistent
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
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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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