Influence of External pH and Organic Acids on Internal pH and Acid Anion Accumulation in Listeria monocytogenes and Escherichia coli

iii PUBLIC ABSTRACT . vi LIST OF TABLES . xi LIST OF FIGURES . xii LIST OF ABBREVIATIONS . xiii CHAPTER I. 2 Listeria monocytogenes . 2 Listeriosis infection . 3 Escherichia coli . 4 E. coli O157:H7 infection . 5 Organic acids . 6 Organic acids as antimicrobials . 6 Anion Accumulation . 8 Intracellular pH . 8 Measuring intracellular pH by Fluorescence Staining Technique . 10 Preliminary Studies . 11 CHAPTER II. 15 Abstract . 15 Introduction . 17 Objectives . 20 Materials and Methods . 21 Bacterial strains and growth conditions . 21 Experimental overview and statistical analysis . 22. 15 Introduction . 17 Objectives . 20 Materials and Methods . 21 Bacterial strains and growth conditions . 21 Experimental overview and statistical analysis . 22 x Accumulation of organic acid . 26 Measurement of pHi . 27 Results and Discussion . 31 Acid anion accumulation . 31 pHi values . 33 Conclusions & Further Research . 35 REFERENCES . 38 APPENDICES . 53 Appendix A: Anova DataMeasurement of pHi. 53 Appendix B: Additional Data from Measurement of pHi . 56 Appendix C: T-Tests from Accumulation of Organic Acid & Inulin . 61 xi LIST OF TABLES Table Page 2-1 Strains selected for this study.21 2-2 Objective 1 habituation treatments . 23 2-3 Objective 1A habituation treatments . 24 2-4 Objective 2 habituation treatments.25 A-1 ANOVA for measurement of pHi in L. monocytogenes R2-499 . 54 A-2 ANOVA for measurement of pHi in L. monocytogenes N1-227.54 A-3 ANOVA for measurement of pHi in E. coli O157:H7.55 C-1 T-test for accumulation of organic acid in L. monocytogenes R2-499 . 61 C-2 T-test for accumulation of organic acid in L. monocytogenes N1-227.62 C-3 T-test for accumulation of organic acid in L. monocytogenes N1-227.63 C-4 T-test from inulin control comparing N1-227 supernatant & pellet.64 C-5 T-test from inulin control comparing R2-499 supernatant & pellet . 64 C-6 T-test from inulin control comparing E. coli supernatant & pellet.65 xii LIST OF FIGURES Figure Page 2-1 pHi calibration curves for L. monocytogenes and E. coli at mid log phase.29 2-2 Acid anion accumulation values . 29 2-3 pHi values after one hour.34 B-1 Polynomial fitting for strain L. monocytogenes N1-227 . 56 B-2 Polynomial fitting for strain L. monocytogenes R2-499 . 57 B-3 Polynomial fitting for strain E. coli O157:H7 . 58 B-4 Preliminary results for L. monocytogenes N1-227 from tracking pHi.59 B-5 Preliminary results for L. monocytogenes R2-499 from tracking pHi.60 xiii LIST OF ABBREVIATIONS ACacetic acid LAlactic acid HClhydrochloric acid BCbaseline control NaOHsodium hydroxide KH2PO4 -potassium phosphate STECshiga toxin producing Escherichia coli EHECenterohemorrhagic Escherichia coli TSBtryptic soy broth TSAtryptic soy agar CFSE5(6)-carboxyfluorescein diacetate N-succinimidyl ester, a membrane permeant fluorescein-based dye LT50the lethal time until 50% population mortality DPMdisintegrations per minute CHAPTER I. LITERATURE REVIEW Listeria monocytogenes Listeria monocytogenes is a gram positive, non-spore forming, facultative anaerobic bacteria (20, 72). L. monocytogenes is environmentally ubiquitous. It lives saprophytically in the soil and on plants, and it can persist in the intestines of animals. Its survival can be influenced by temperature, the humidity of the soil, and the motility of the bacterium (54). Listeria is capable of transitioning into a pathogen when it encounters stressors, such as when it is ingested (23). These stressors include reduced oxygen, bile, endogenous microbiota and low pH (23, 48, 63). Because of its ubiquitous presence, L. monocytogenes has the potential to contaminate food processing facilities and food products (40). L. monocytogenes also has growth and survival properties that make it difficult for the food industry to control its growth. It is psychotropic and can grow at temperatures as low as 0°C and as high as 50°C. It grows within a wide pH range, from 4.6-9.5. It can grow at water activity less than 0.93 and in a concentration of salt up to 10% (46, 44). It is capable of transport and cross contamination through water, air, animals, and humans. It can also survive and persist in biofilms (20, 25, 64). Listeria can survive in hostile environments within a food processing facility including equipment, drains, and wet floors in refrigerated rooms of processing facilities (3). 3 Listeriosis infection Listeriosis is the infection in humans caused by the consumption of food contaminated with L. monocytogenes. In the United States, the incidence of listeriosis is relatively low-only 2.4 cases per million in the population (33). The CDC reported 126 cases of listeriosis in 2018, 121 of which resulted in hospitalization (96%) and 26 resulted in death (21%) (66). Thirteen different serotypes of L. monocytogenes have been described, and serotypes 1/2a, 1/2b, and 4b have been associate