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Chapter 1 [119] and flow cell reactors have been adapted and fitted with a glass chambers into the devices to facilitate real-time imaging [102]. This method involves staining the bacterial cells with a fluorescent dye before inoculating the reactor and assessing the accumulation of cells over time using microscopy. The Center for Disease Control and Prevention Biofilm Reactor (CBR) has been extensively used in recent studies [117, 120-124]. This continuous flow method was chosen for this research as it allows biofilm formation on multiple surfaces simultaneously. The CBR also has the benefits of incorporating sheer stress and examination of spatial growth (through microscopy) and evaluation of viable cells (though plate counts). The CDC reactor was the first model approved by the American Society for Testing Materials (ASTM) for the development of biofilm on surfaces [117]. As of 2012, both the CDC biofilm reactor (ASTM standard number E2871- 12)[125] and the MBEC Assay (ASTM standard number E2799-12)[126] have been approved by ASTM. The CBR is discussed in greater detail in chapter two. 1.16. Biocides and Disinfectants The term biocide is inclusive of a broad range of chemical agents such as disinfectants, antiseptics, herbicides, preservatives, insecticides, fungicides, enzymes and probiotic substances such as Lactobacillus species [127-128]. The term biocide does not generally include antibiotics [128-129] which usually target a specific organism by use of a compound at a low concentrations while biocides generally target all organisms through the use of high concentration of the compound. A disinfectant can be classified as a product that reduces the number of viable organisms on a surface [129]. Because of extensive variability in tests it is difficult to develop a Page 27
Chapter 1 standardised approach to disinfectant and biocide testing. The European Committee for Standardisation have developed protocols for documenting efficacy of products sold within the European Union [130]. According to the European suspension test (EN1040) a 5 log 10 reduction in the number of viable cells (99.999%) is necessary to prove the efficacy of a disinfectant [131-132]. However according to the EN 13697 bactericidal activity is defined as >4 log 10 reduction of organisms attached to a surface under examination [133-134]. 1.16.1. Tolerance to disinfectants When cells in a mature biofilm come into contact with disinfectants, it is hypothesised that the cells deep within the biofilm matrix are protected by the outer layers of cells and extracellular polymeric matrix. As a result of this protection these cells only come into contact with sub-lethal doses of the chemicals therefore this may be responsible for both phenotypic and genotypic adaptation [105]. The stress adaptation by biofilm cells may also give rise to an increase in population of resistant or tolerant cells [76]. This may result in limited antibiotic options in the case of salmonellosis infection [135]. Braoudaki and Hilton highlighted this concern when they demonstrated that the use of disinfectants (benzalkonium chloride, chlorhexidine and triclosan) against Salmonella enterica may encourage cross resistance to antibiotics [136]. This research demonstrated that if Salmonella Virchow was exposed to sub-inhibitory concentrations of benzalkonium chloride it became relatively more resistant to 8/18 antibiotics tested in comparison to the parent strain [136]. Similarly Karatzas et al. found that when S. Typhimurium was exposed to sub-lethal doses of three disinfectants (oxidising compound blend, quaternary ammonium blend and a tar-acid Page 28
Page 1: Salmonella enterica - biofilm forma
Page 4 and 5: Table of Contents Page Number 1.16.
Page 6 and 7: Table of Contents Page Number 4.1.2
Page 8 and 9: Table of Contents Page Number 6.8.4
Page 10 and 11: List of Abbreviations Table Page Nu
Page 12 and 13: List of Abbreviations List of Abbre
Page 14 and 15: Summary of Content Summary of Conte
Page 16 and 17: “To show your true ability is alw
Page 18 and 19: Acknowledgements ever met, Fiona th
Page 20 and 21: Acknowledgements This Ph.D. thesis
Page 22 and 23: Chapter 1 1.1. Salmonella Salmonell
Page 24 and 25: Chapter 1 may impose enormous costs
Page 26 and 27: Chapter 1 cracks or penetration of
Page 28 and 29: Chapter 1 detected by agglutination
Page 30 and 31: Chapter 1 become colonized with >10
Page 32 and 33: Chapter 1 antigens [z27],[z45] [1].
Page 34 and 35: Chapter 1 S. Agona has also been im
Page 36 and 37: Chapter 1 isolated from undercooked
Page 38 and 39: Chapter 1 The S. Typhimurium strain
Page 40 and 41: Chapter 1 ecosystem with the use of
Page 42 and 43: Chapter 1 [82]. Previous research h
Page 44 and 45: Chapter 1 to genes involved in flag
Page 46 and 47: Chapter 1 observation has also been
Page 50 and 51: Chapter 1 based disinfectant) the s
Page 52 and 53: Chapter 1 formation of two strains
Page 54 and 55: Chapter 1 shield the S. Agona cells
Page 56 and 57: Chapter 1 Key Objectives of this st
Page 58 and 59: 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
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Chapter 2 Figure 2.7: Graph of the
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Chapter 2 also used “high” soni
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Chapter 2 2.8. Summary All 13 strai
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Chapter 3 3. Abstract It has been e
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Chapter 3 classified as persistent
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Chapter 3 was plated onto TSA for e
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Chapter 3 Figure 3.1: The mean log
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Chapter 3 Figure 3.3: SEM image of
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Chapter 3 Table 3.2: Difference bet
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Chapter 3 3.4.3. Intra-serovar vari
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Chapter 3 3.4.4. Strain variation i
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Chapter 3 3.4.5. The density of bio
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Chapter 3 3.5. Discussion As illust
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Chapter 3 As discussed in section 3
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Chapter 3 increased biofilm appeare
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Chapter 4 Examining the efficacy of
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Chapter 4 through laboratory based
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Chapter 4 acid and quaternary ammon
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Chapter 4 the surface the temperatu
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Chapter 4 availability of multiple
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Chapter 4 peracetic acid (100, 200,
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Chapter 4 As discussed previously,
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Chapter 4 Dey/Engley (Difco) neutra
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Chapter 4 sterilisation was achieve
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Chapter 4 IX. Aliquots of 100µl of
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Chapter 4 XV. XVI. XVII. XVIII. XIX
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Chapter 4 4.3. Results 4.3.1. Suspe
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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
Page 242 and 243:
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.
Page 264 and 265:
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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Page 280 and 281:
Page 282 and 283:
Page 284 and 285:
Page 286 and 287:
Appendix 3 Appendix 3-Figure 2: PFG
Page 288 and 289:
Appendix 4 Poster Presentation Envi
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