The Correlation between Tolerance to Low Water Activity and High Hydrostatic Pressure in Shiga Toxin Producing Escherichia coli
BY
Fahad Bazaid
A Thesis Presented to The University of Guelph
In partial fulfillment of requirements for the degree of Doctor of Philosophy in Food Science
Guelph, Ontario, Canada © Fahad Bazaid, March 2019
ABSTRACT
THE CORRELATION BETWEEN TOLERANCE TO LOW WATER ACTIVITY AND HIGH HYDROSTATIC PRESSURE IN SHIGA TOXIN PRODUCING ESCHERICHIA COLI
Fahad Bazaid University of Guelph, 2019 Advisors: Dr. Keith Warriner Dr. Andrew Kropinski Dr. Don Mercer Dr. Tatiana Koutchma
High hydrostatic pressure processing (HHP) is extensively applied for the non-thermal pasteurization of foods. One notable feature of HHP mediated inactivation of microbes is the large variation in lethality between strains and the influence of the food matrix. In the following it was hypothesized that the barotolerance of Shiga Toxin producing
Escherichia coli could be correlated to tolerance to low water activity environments. The study evaluated the barotolerance of members of the Top 6 STEC serotypes and found variation with O26: H11 and O45: H2 exhibiting higher tolerance to pressure compared to the other bacterial types tested. A negative correlation was found between resistance to high pressure and the NaCl concentration under which E. coli could grow under. For example, E. coli O26: H11 exhibited the highest tolerance to pressure, within 300 MPa, but could only grow under NaCl up to 2.4% w/v. In contrast, E. coli O121: H19 was sensitive to pressure, but could grow in the presence of 6% NaCl. The results suggested
ii that the induction of the stress-response was the likely reason for high barotolerance in salt sensitive strains vs resistant. It was also found that supplementing the suspension medium with amino acids, sugar alcohols and starch could increase the barotolerance of
E. coli. Although it is possible that the solutes acted directly as osmolytes it is also proposed that metabolic products and other indirect protective mechanisms were also operating. The study suggests that early induction of the osmotic stress response can be used to predict the resistance to high hydrostatic pressure.
iii DEDICATION
I would like to dedicate this thesis to the greatest and precious person in my life my mother, who passed away twelve years ago. The person who I haven’t got to spent much time with, and the person who I always missed. Even though she passed away twelve years ago, she is always present in my heart and soul. I would like to dedicate this thesis to my wonderful father, who was always there for me when I needed support and encouragement. Also, would like to dedicate this thesis to my little angels Joud and
Wesaam, who will be proud of their father when they grow up. Finally would like to dedicate this thesis to my sisters, brothers, friends, and professors.
iv ACKNOWLEDGMENT
I would like to express my thanks to Allah who guided me and gave me blessing to complete my PhD. I would also like to express my deep appreciation and thanks to my advisor Dr. Keith Warriner for giving me the opportunity to peruse my PhD. Dr.
Warriner provided me with his guidance, advice, patience and encouragement throughout my research. The door for Dr. Warriner office was open to me whenever I needed guidance and advice. I would also like to express my thanks to my advisory committee
Dr. Andrew Kropinski, Dr. Don Mercer, and Dr. Tatiana Koutchma for their advice, experience and help during my research. Special thanks to the government of Saudi
Arabia for giving me this scholarship and funding me to peruse my graduate studies.
My thanks also go to the Department of Food Science, and all my friends in Dr.
Warriener’s lab. Also, special thanks to my father, my siblings, and my kids for their unconditional support and encouragement through my entire study. Finally, thanks to all my friends who supported me during my study.
v Table of Content ABSTRACT ...... ii DEDICATION ...... iv ACKNOWLEDGMENT ...... v Table of Content ...... vi List of Tables ...... viii List of Figures ...... x General Introduction ...... 1 Chapter 1 ...... 3 1.Literature review: ...... 3 1.1. Shiga Toxin Producing E. coli STEC O157 and non-O157 ...... 3 1.1.2. STEC Outbreaks: ...... 6 1.1.3. STEC Outbreaks in Low Water Activity Food: ...... 8 1.2 High Hydrostatic Pressure ...... 11 1.2.1.Components of the High Hydrostatic Pressure Equipment: ...... 12 1.2.2.High Pressure Process: ...... 13 1.2.3. The Principle of High Hydrostatic Pressure: ...... 14 1.2.4. Effect of High Pressure on Proteins: ...... 15 1.2.5. Microbial Inactivation by HHP ...... 15 1.3. Water Activity and Osmotic Stress ...... 18 1.3.1. Effect of Water Activity on Barotolerance and Other Stresses: ...... 21 1.3.2. Accumulation of Osmolytes: ...... 23 1.4 Strain Variation in Resisting High Hydrostatic Pressure: ...... 24 1.5 Glycolysis (Embden-Meyerhof-Parnas pathway) and Krebs cycle Tricarboxylic acid (TCA) cycle: ...... 25 1.6. Dormancy State in Bacteria: ...... 27 1.6.1.Resuscitation of VBNC: ...... 29 1.6.2.Detection of Dormant Cells (VBNC): ...... 30 Research Hypothesis and Objectives ...... 32 Chapter 2 ...... 33 The Correlation between Tolerance to Low Water Activity and High Pressure Processing in Shiga Toxin Escherichia coli ...... 33 2.1 Abstract ...... 33 2.2 Introduction: ...... 34 2.3 Materials and Methods ...... 36 2.3.1 Bacteria and Cultivation ...... 36 2.3.2 Preparation of Suspensions ...... 36 2.3.3 Persistence of the STEC Strains under osmotic stress (Colony counts) ...... 37 2.3.3.1 Live counts under fluorescent microscope (Viability staining) ...... 38 2.3.3.2 Recovery of the STEC dormant cells (Resuscitation) ...... 38 2.3.3.3 Growth limits of STEC (The gradient Plate Technique) ...... 39 2.3.4 High Pressure Treatment ...... 39 2.3.4.1 High Pressure inactivation (barotolerance) ...... 40 2.3.4.2 Microscopic counts ...... 40 2.3.4.3 High-pressure tolerance and STEC osmotic growth limits ...... 41 2.3.4.4 Effect of peptone (the accumulation of osmolytes) on the barotolerance of STEC ...... 41
vi 2.3.4.5 Effect of amino acids (osmolytes) and other supplements (non- osmolytes) on the barotolerance of STEC ...... 42 2.3.4.6The effect of Different water activity levels on the barotolerance of STEC in the presence of 1% Glycine ...... 42 2.3.5 Dry weight of STEC (Accumulation of osmolytes) ...... 43 2.3.6 The substrate utilization profile test ...... 44 2.3.7 Enumeration of Bacteria ...... 44 2.3.7.1 Persistence of STEC Strains under osmotic stress (Colony counts) ...... 44 2.3.7.2 Live counts under fluorescent microscope ...... 45 2.3.7.3 High Pressure-Treatment ...... 45 2.3.8 Experimental Design and Statistics ...... 45 2.4 Results: ...... 46 2.4.1 Growth limits of STEC under osmotic stress (Gradient Plate Technique): ...... 46 2.4.2 Persistence of the STEC Strains under osmotic stress (Plate counts and viability staining): ...... 46 2.4.3 High-Pressure inactivation (barotolerance): ...... 48 2.4.4 High-pressure tolerance and STEC osmotic growth limits in NaCl: ...... 49 2.4.5 Underlined mechanisms ...... 51 2.4.5.1 Accumulation of osmolytes ...... 51 2.4.5.1.1 The dry weight of STEC ...... 51 2.4.5.1.2 The effect of amino acids (osmolytes) on the barotolerance of STEC ...... 51 2.4.5.2 Metabolic precursors ...... 52 2.4.5.2.1 The effect non-osmolytes (supplements) on the barotolerance of STEC ...... 52 2.4.5.2.2 Effect of peptone (non-osmolyte) on the barotolerance of STEC strains: ...... 53 2.4.5.2.3 The Effect of Fermented and non-fermented sugars on the barotolerance of STEC ... 54 2.4.6 The effect of Different water activity levels on the barotolerance of STEC in the presence of 1% glycine ...... 55 2.5 Discussion ...... 75 Chapter 3 ...... 83 The effect of low water activity (glycerol, NaCl) on barotolerance of non pathogenic Escherichia coli P36 ...... 83 3.1 Abstract ...... 83 3.2 Introduction ...... 84 3.3 Materials and Methods ...... 85 3.3.1 Bacteria and Cultivation ...... 85 3.3.2 Preparation of Suspensions ...... 85 3.3.3 Effect of low water activity (glycerol, NaCl) on HHP inactivation of E.coli ...... 86 3.3.4 Enumeration of Bacteria ...... 87 3.3.5 Experimental Design and Statistics ...... 87 3.4 Results and Discussion ...... 87 Chapter 4 ...... 93 General Conclusion and Future Work ...... 93 4.1 General Conclusion ...... 93 4.2 Future Work ...... 95 References ...... 97
vii List of Tables
Table 1.1: Outbreaks of STEC in water, food and fresh produce worldwide…….………8
Table 1.2: STEC outbreaks in low water activity foods in North America and Europe……………………………………………………………………………………10
Table 2.1 A: Persistence of STEC O157 and O121 under osmotic stress with the 3 different inoculation levels and with incubation of enumeration plates at 37°C and 22°C…………………………………………………………………………………..….59
Table 2.2 B: Persistence of STEC O1O3 and O111 under osmotic stress with the 3 different inoculation levels and with incubation of enumeration plates at 37°C and 22°C………………..…………………………………………………………………….60
Table 2.3 C: Persistence of STEC O45 and O26 under osmotic stress with the 3 different inoculation levels and with incubation of enumeration plates at 37°C and 22°C……………..……………………………………………………………………….61
Table 2.4. STEC culturable counts and microscopic counts (live counts) at pressure 300 MPa (1- min) after 24 h and 72 h of the pressure treatment...……………………...……66
Table 2.5 STEC counts after pressure treatment 400 MPa (1-3min) after 24 h and 72 h of the pressure treatment………………………………...…………………………...……..66
Table 2.6 The effect of pressure 300 MPa (Osmotic growth limit) (1-3 min) after 24 h and 72 h of pressure treatment…………………………………………………………...67
Table 2.7 The dry weight of STEC strains after incubating in 10% NaCl solution with 1% peptone and with no peptone added. …………...………………………………………..68
Table 2.8 The effect of 1% Peptone on barotolerance of STEC at pressure 300 MPa (1-3 min)………………………………………………………………………………….69
Table 2.9 The effect of amino acids (osmolytes) on the barotolerance of STEC after 24 h and 72 h of pressure treatment 300 MPa/ 1-3 min………………………………….71
Table 2.10 The effect of non osmolytes (supplements) on the barotolerance of STEC after 24 h and 72 h of the pressure treatment 300 MPa (1-3 min)………………………..72
Table 2.11 The effect of fermented and non- fermented sugars (non osmolytes) on the barotolerance of STEC at pressure 300 MPa/ 1-3 min……………………………....73
Table 2.12 STEC O157:H7 counts after 72 h of the pressure treatment 400 MPa (1- 3 min) in the different water activity solutions with 1% glycine....………………...... 75
viii Table 2.13 STEC counts after 72 h of the pressure treatment 400 MPa (1- 3 min) in the different water activity solutions with 1% glycine ……………………………….....75
Table 3.1.The effect of low aw by using NaCl on the barotolerance of E. coli…………..89
Table 3.2 The effect of low aw by using glycerol on the barotolerance of E. coli………..90
Table 3.3 The effect of HHP on non-stressed E. coli……………………………………91
ix List of Figures
Figure 1.1 A scheme illustrating the six pathogenic groups of E.coli and demonstrating the unique features for each group in interacting with eukaryotic cells………………………………...... 5
Figure 1.2: Schematic diagram on the mode of infection of STEC…………………..…...6
Figure 1.3: HHP commercial unit…………………………………………………….….13
Figure 1.4: The effect of temperature on the efficacy of pressure on inactivation of Model microbes to reach 5 log reduction………………………………...……………...16
Figure 1.5: Illustrating the bacterial response to the increase and decrease in osmotic pressure…………………………………………………………………………………..20
Figure 1.6: Glycolysis (EMP) pathway for glucose metabolism in Eukaryotic and prokaryotic cells………………………………………………………………………….26
Figure 1.7: TCA (Tricarboxylic acid Cycle), Krebs cycle pathway in E.coli…………...27
Figure 2.1 The culturable counts (log CFU/mL) compared to the viable counts by viability staining (microscopic, Log cell/ mL) in peptone water with 10% NaCl for STEC O1O3:H2 ………………………………………………………………………………...62
Figure 2.2 The culturable counts log CFU/mL compared to the viable counts by viability staining (microscopic) Log cell/ mL in peptone water with 10% NaCl for STEC O121:H19 ……………………………………………………………………...... 62
Figure 2.3 The culturable counts log CFU/mL compared to the viable counts by viability staining (microscopic) Log cell/ mL in peptone water with 10% NaCl for STEC O157:H7 ………………………………………………………...………………………………….63
Figure 2.4 The culturable counts log CFU/mL compared to the viable counts by viability staining (microscopic) Log cell/ mL in peptone water with 10% NaCl for STEC O111:NM …………………………………………………………………………….….63
Figure 2.5 The culturable counts log CFU/mL compared to the viable counts by viability staining (microscopic) Log cell/ mL in peptone water with 10% NaCl for STEC O45:H2 …………………………………………………………………………...……………….64
Figure 2.6 The culturable counts log CFU/mL compared to the viable counts by viability staining (microscopic) Log cell/ mL in peptone water with 10% NaCl for STEC O26:H11 ……………………………………………………………………………………..…...... 64
x Figure 2.7 The effect of pressure 600 MPa on STEC counts after 24 h of pressure the treatment…………………………………………………………………………………65
Figure 2.8 The log reduction in STEC counts after pressure treatment 300 MPa/ 3 min. The log reductions are from lowest to highest log reduction…………………………….65
Figure 2.9 The relation between barotolerance of STEC at 300 MPa/ 3 min and their growth limits on 10% NaCl gradient plate………………………………………………76
Figure 2.10 The effect of peptone on barotolerance of STEC after 24 h of pressure treatment pressure 600 MPa/ 3min………………………………………………………69
Figure 2.11 The effect of peptone on the barotolerance of STEC after 72 h of the pressure treatment 600 MPa/ 3 min………………………………………………………………..70
Figure 2.12 The effect of HHP 600 MPa (1-3 min) on the barotolerance of STEC O157:H7 in the different water activity solutions (0.98, 0.94, 0.85)…………………….74
Figure 2.13 The effect of HHP 400 MPa (1 -3 min) on the barotolerance of STEC O157:H7 in the different water activity solutions (0.98, 0.94, 0.85)…………………….74
Figure 2.14. HHP profile for samples processed at 400 MPa (1-3min).………………...67
xi General Introduction
The Public Health Agency of Canada estimates that 4 million cases of foodborne diseases occur in Canada every year (Public Health Agency, 2016). Shiga toxin producing
Escherichia coli (STEC) causes serious human foodborne disease; Hemolytic Uremic
Syndrome (HUS) (i.e., kidney failure) with STEC O157 accounting for more than 73,000 illness cases yearly in the USA; and non O157 STEC strains accounting for over 100,000 illness cases (Public Health Agency, 2016; CDC, 2012). Foods from animal sources such as beef and dairy products are linked to STEC. Therefore, there is a need to implement processing methods to inactivate the pathogen while retaining the raw sensory of food characteristics (Hussein & Sakuma, 2005; Erickson & Doyle, 2007). High Hydrostatic
Pressure (HHP) is a food preservation method that has been reported to extend the shelf life of fresh products by the inactivation of microbial cells (Zhou, 2013; Martin et al.,
2002). Non-thermal HHP has been used since 2000 in North America as a pathogen reduction step to increase the safety of products such as juices, deli meats, and other products. However, the HHP process is unpredictable due to gaps in knowledge relating to barotolerance (i.e., pressure resistance) of different microbes and factors affecting the barotolerance (Alpas et al., 1999; Martin et al., 2002; Warriner & Namvar, 2014). Lianou et al. (2013) reported a significant variation in log reduction among nine strains of
Listeria monocytogenes when investigating their pressure resistance at 345 MPa with a range of 0.9-3.5 logs reduction. In addition, a variation of 0.6-3.4 log reduction has been reported in 17 strains of E. coli O157: H7 that were treated with HHP at 500 MPa, 23°C and for 1 min (Malone et al., 2007). Moreover, Robey et al. (2001) reported broad
1 differences in resisting pressure among six strains of E. coli O157: H7 when applying
HHP. Also, Koseki and Yamamoto (2007) reported differences in resisting pressure for six strains of Listeria monocytogenes at the same water activity when applying pressures of 400 and 600 MPa at 25°C. In addition, other stresses can influence the efficacy of
HHP inactivation. Therefore, cells exposed to stresses such as heat or low water activity will have more tolerance response to pressure inactivation (Hormann et al., 2006). For example, Aertsen et al. (2004) reported an increase in barotolerance of E. coli that were exposed to heat shock than regular non-stressed E. coli. In addition, an increase in barotolerance of E. coli K12 was reported when decreasing water activity from 0.99 to
0.90 at pressures 400 and 600 MPa (Setikaite et al., 2009). Moreover, Sagarzazu et al.
(2009) reported an increase in resistance of Campylobacter jejuni to HHP after exposing to cold temperature (i.e., 0°C) compared to non-stressed C. jejuni that were incubated at
37°C.
Previously there have been attempts to correlate barotolerance to thermal resistance, pH, cold shock, or genetic determinants (Cutter et al., 2012; Martin et al., 2002). However, none of the parameters have proven successful in predicting barotolerance of microbes. It is known that high pressure is less effective against pathogens with low water activity matrices, and it is known that osmolytes, solutes that regulate osmosis by accumulating inside the cell, such as amino acids, have a protective effect on bacteria. Therefore, it follows that a correlation may exist between tolerance to low water activity and high pressure. It is this relationship that was studied in this research with the focus being placed on STEC given the relevance of the pathogenic group.
2 Chapter 1 1.Literature review: 1.1. Shiga Toxin Producing E. coli STEC O157 and non-O157
Escherichia coli is a Gram-negative facultative anaerobic bacillus that belongs to the family Enterobacteriaceae. It is commonly found in the intestine of warm-blooded animals as intestinal microflora, and it has an optimum growth temperature of 37°C
(Ljubovic et al., 2009). Escherichia coli includes a large group of bacteria that are considered mostly non-pathogenic to humans. However, some strains of E. coli cause serious human illness by producing different toxins. These pathogenic strains are divided into groups and include enteropathogenic E. coli EPEC, enterotoxigenic E. coli ETEC, enteroinvasive E. coli EIEC, enteroaggregative E. coli EAEC, and enterohemorrhagic E. coli EHEC (Grant et al., 2011). Members of these groups produce attachment-effacement lesions that facilitate attachment to the epithelial cells in the intestine (Jay, 2000), Figure
1.1. EHEC is considered as a sub-group of STEC, which is classified as one of the pathogenic groups of E. coli producing shiga toxin (Stx) (Kalchayanand et al., 2011;
Warriner, 2011; Grant et al., 2011). Cattle and ruminants, such as sheep, are often considered as main carriers of STEC O157:H7 and non-O157 serotypes. In addition, healthy adult cattle are known to shed VTEC including STEC normally, and carriage is different from season to season (Pradel et al., 2004; Sanchez et al., 2010; Kalchayanand et al., 2011; Ferens & Hovde, 2011; Gill & Gill, 2010).
STEC causes hemolytic uremic syndrome HUS, a severe kidney failure that mostly affects young children and older people (Mayer et al., 2012). STEC O157: H7 is known to be one of the concerning serotypes. In 1975, STEC O157: H7 was classified as a human pathogen after it was found in a patient with bloody diarrhea in California (Doyle
3 et al., 2006). In 1982, STEC O157: H7 was first associated with a foodborne outbreak of ground beef (Doyle et al., 2006). In 1993, STEC O157: H7 caused a huge historical multistate outbreak that was related to contaminated beef burgers from a well-known fast food restaurant Jack in the Box (Rangel et al., 2005). However, illness cases and outbreaks associated with non-O157 serotypes have increased significantly in Canada, the United States, and Europe (Taylor, 2008; Maccallum et al., 2013; Chui et al., 2011).
In North America, studies found that non-O157 is responsible for 30-50% of VTEC illness including STEC infections (Gill& Gill, 2010). In addition, in the USA, by examination of samples from patients, it has been found that the number of non-O157 associated with VTEC illness is increasing in several states. Moreover, in Canada and
USA, it has been reported that non-O157 are possibly linked to 50% of VTEC illness cases (Gill & Gill, 2010). There are six main serotypes that are called top STEC including O26, O45, O103, O111, O121 and O145 that were responsible for 71% of the
HUS illness cases reported in the U.S.A in 2000 and hence called top six (Grant et al.,
2011). In the U.S.A, during 2010, 66% of the outbreaks associated with non-O157 were caused by the serotypes O26 and O111 (Luna-Gierke et al., 2014).
The virulence feature associated with the STEC group is due to the production of Stx1,
Stx2 and intimin (eae) protein, which is an attaching and effacing protein. It is reported that Stx2 is more virulent than Stx1 and it increases the risk of HUS (Mccallum et al.,
2013). In addition, the STEC group produces other virulence factors such as Tir receptors that are effector proteins introduced by type three secretion system TTSS. The TTSS is a transport system that transfers Tir receptors and other bacterial proteins into the host cell
(Mohawk and O’Brien, 2011), Figures 1.1.and 1.2. In addition, Stx is an AB5 toxin
4 consisting of AB subunits. The 5-B subunits have the ability to attach to the host cell by the recognition receptors, while the active A subunit enters the cell causing catalytic activities (Tozzoli et al., 2010). Complement of the virulence factors determines the ability of the STEC to cause disease as in O157.
(Figure 1.1: A scheme illustrating the pathogenic groups of E. coli and demonstrating the unique features for each group in interacting with host cells) Adapted from (Nataro& Kaper, 1998). ETEC attach and colonize on intestinal cells and secret enterotoxins LT and ST. EHEC have intimate attachment to the intestinal cells and produce Stx. EAEC aggregate on the surface of intestinal cells and produce cytotoxins. EPEC have initial attachment to the intestinal cells from distance and then intimate attachment. EIEC penetrate intestinal cells and can extent to adjacent cells.
5
(Figure 1.2: Schematic diagram on the mode of infection of STEC) Adapted from (Mohawk and O’Brien, 2011)
1.1.2. STEC Outbreaks:
STEC O157 and non-O157 serotypes have been implicated in outbreaks and illness cases around the globe (Mathusa, 2010). Infections associated with STEC non-O157 are increasing in the United States, Canada and all around the world (Gill & Gill, 2010;
Mathusa, 2010). For example, in USA, during the period 2000-2005, the number of illness cases associated with non O157 STEC increased from 171 cases to 501 cases
(Mathusa et al., 2010). In 2000, STEC O157: H7 caused one of the largest outbreaks that occurred through the history of Canada, which is the contamination of water in
Walkerton, Ontario (Salvadori et al., 2009). The outbreak was caused by STEC O157: H7 and resulted in 2,300 illness cases and 7 death cases (Salvadori et al., 2009). In 2017, the
Public Health Agency of Canada reported a recent outbreak of 42 illness cases in five
6 provinces (Ontario, Quebec, New Brunswick, Nova Scotia, and Newfoundland) that is caused by STEC O157:H7 and associated with consumption of romaine lettuce (Public
Health Agency of Canada, 2018). Moreover, STEC O157: H7 caused a recent multistate outbreak of 25 illness cases that is associated with leafy greens in USA, 2017 (CDC,
2018). In 2016, STEC O26 caused an outbreak of 55 illness cases associated with consumption of food at Chipotle Mexican Grill restaurant in 11 states (CDC, 2016). In
2014, an outbreak of 119 illness cases was caused by STEC O157: H7 in Alberta, Canada and was associated with the consumption of contaminated pork at Asian Style restaurants
(Honish et al., 2017). In addition, in 2014, STEC O157: H7 caused an outbreak of 12 illness cases associated with contaminated ground beef from Wolverine Packing
Company in USA (CDC, 2018). Also, in 2014, CDC reported an outbreak in six states caused by STEC O121 and causing 18 illness cases associated with consumption of raw clover sprouts (CDC, 2014). Furthermore, STEC O121 caused a multistate outbreak of 35 illness cases that was associated with frozen food from Farm Rich brand in 2013 (CDC,
2013). In 2011, STEC O1O4: H4 caused one of the largest outbreaks in Europe, specifically, Germany. The outbreak caused over 4,000 illness cases, most of which have been reported in Germany, and was associated with contaminated sprouts (CDC, 2011).
In 2008, a multistate outbreak of 49 illness cases that was caused by STEC O157: H7 and associated with Kroger’s ground beef was reported (CDC, 2008). Moreover, a multistate outbreak of 40 illness cases was reported in 2007 and was associated with consumption of frozen ground beef and caused by STEC O157: H7 (CDC, 2007). The previous cases caused by STEC show the increase in the implication of STEC O157 and non-O157 in outbreaks and illness cases around the world.
7
Table 1.1: Outbreaks of STEC in water, food and fresh produce worldwide.
STEC Food Place Outbreak cases Year Reference serotype O157:H7 Romaine lettuce Canada 42 2017 Public Health Agency of Canada, 2018 O157:H7 Leafy greens USA 25 2017 CDC, 2018
O26 Contaminated food USA 55 2016 CDC, 2016
O157:H7 Contaminated pork Canada 119 2014 Honish et al., 2017 O157:H7 Ground beef USA 12 2014 CDC, 2014
O121 Clover sprouts USA 18 2014 CDC, 2014
O121 Frozen food USA 35 2013 CDC, 2013
O1O4: H4 Sprouts Germany > 4,000 2011 CDC, 2011
O157:H7 Ground beef USA 49 2008 CDC, 2008
O157:H7 Frozen ground beef USA 40 2007 CDC, 2007
O157:H7 Contaminated water Canada 2,300 2000 Salvadori et al., 2009
1.1.3. STEC Outbreaks in Low Water Activity Food:
STEC have been involved in outbreaks associated with low water activity and dry foods such as dry fermented sausage, cookie dough, and flour. (Böhnlein et al., 2017; Shah et al., 2016; Hiramatsu et al., 2005) In 2009, the FDA reported a multistate outbreak of 72 illness cases caused by STEC O157:H7 and associated with consumption of raw cookie dough (CDC, 2009; Crowe et al., 2017). Flour is a low water activity ingredient that does not permit the growth of pathogens. However, in 2017, an outbreak of 29 illness cases that is caused by STEC O121 was reported in Canada in six provinces (Ontario, Alberta,
Saskatchewan, British Columbia, Quebec, and Newfoundland) and associated with
8 consumption of flour (Morton et al., 2017). In addition, in 2016, a multistate outbreak with 63 illness cases caused by STEC O121and O26 and associated with consumption of flour was reported in the USA (CDC, 2016; Crowe et al., 2017). Moreover, STEC have been involved in outbreaks associated with dry fermented sausage and salami (Rode et al., 2017). For example, STEC O1O3: H25 was associated with an outbreak of 17 illness cases and one death related to traditional cured sausage consumption in Norway in 2006
(Schimmer et al., 2008). In addition, in 2007, an outbreak of 20 illness cases caused by
STEC O26:H11 and associated with beef sausage was reported in Denmark (Ethelberg, et al., 2009). STEC O157: H7 has been involved in an outbreak in western Canada associated with consumption of raw fermented Hungarian style sausage in 1999 (Health
Canada, 2000). In addition, an outbreak of 143 illness causes caused by STEC O157: H7 and associated with salami was reported in Canada, British Columbia, in 1999
(MacDonald et al., 2003). Furthermore, STEC have been involved in outbreaks associated with low moisture foods. In 2017, the public health agency in the United
States reported an outbreak caused by E.coli O157: H7 and associated with consumption of SoyNut butter in several states (CDC, 2017; Burgess et al., 2016; Shah et al., 2016). In addition, CDC reported a multi-state outbreak of 13 illness cases caused by STEC O157:
H7 and associated with consumption of Lebanon bologna in 2011 (CDC, 2011). Table
1.3. All the previous cases mentioned demonstrate the involvement of STEC in outbreaks associated with dry and low water activity foods. The previous cases exhibit the ability of
STEC to tolerate and survive under low water activity conditions. The current study will explain if the phenotype is related to the barotolerance.
9
Table 1.2: STEC outbreaks in low water activity foods in North America and Europe
STEC serotype Food Place Outbreak Year Reference cases O121 Flour Canada 29 2017 Morton et al., 2017 O157:H7 SoyNut USA 2017 CDC, 2017; Burgess et al., 2016; Shah et al., 2016 O26, O121 Flour USA 63 2016 CDC, 2016 O157:H7 Lebanon USA 13 2011 CDC, 2011 bologna O157:H7 Raw cookie USA 72 cases 2009 CDC, 2009 dough O26:H11 Beef sausage Denmark 20 2007 Ethelberg, et al., 2009 O1O3: H25 Cured sausage Norway 17 2006 Schimmer et al., 2008 O157:H7 Fermented Canada 1999 Health Canada, Hungarian 2000 sausage O157:H7 Salami Canada 143 1999 MacDonald et al, 2003
10 1.2 High Hydrostatic Pressure
High Hydrostatic Pressure (HHP) is a non-thermal processing method used to process food products under elevated pressure between 100-800 MPa by using a liquid transmitter that is usually water to achieve microbial inactivation (Zhou, 2013; FDA,
2014; Martin et al., 2002). During the last decade, HHP has been considered as one of the emerging technologies in food manufacturing that provides uniform transmission of pressure through the food sample (Koutchma, 2014). HHP can be used to process both liquid and solid food products (Yordanov & Angelova, 2010; USFDA, 2014). In contrast to thermal processing technologies, HHP can maintain the quality of food due to its minimal effect on the texture, taste and nutritional values of the food being processed
(Yordanov & Angelova, 2010). Also, compared to thermal processing technologies, the color change in the food product being processed by HHP is significantly lower
(Considine et al., 2008).
HHP is considered as one of the widespread technologies in food manufacturing due to the microbial inactivation effect and the shelf life extension in ready to eat meats
(Koutchma, 2014). Hite (1899) was the first to demonstrate the process of high-pressure processing of foods in the late 19th century. He reported that high-pressure processing of foods has the ability to extend the shelf-life for milk and fresh produce. However, during that period, the HHP was considered hazardous and unreliable due to the limitation in technology and hence was not used for food processing (Hogan et al., 2005). In the
1990s, the progress and revolution in technology introduced the first commercial application of the pressure treatment of food in Japan including the extension of low risk foods such as jams (Heinz and Buckow, 2009; Martin et al., 2002; Yamamoto, 2017).
11 Starting from 2000, the use of HHP as a step to reduce pathogens to enhance the safety of food such as juices and deli meats has increased in North America (Demazeau &
Rivalain, 2011; Martin et al., 2002; Deliza et al., 2002). HHP can be used as a thermal food processing technology or a non-thermal food processing technology by using a range of temperature that goes below 0°C and over 100°C (FDA, 2014). In addition, at high pressures, there is an increase in the temperature of food, which is related to the adiabatic heating caused by the compression of water and other food components. This increase in temperature is about 3°C/ 100 MPa and depends on the food components
(fellows 2009).
1.2.1.Components of the High Hydrostatic Pressure Equipment:
Food can be processed under HHP by using either a batch system, which is most commonly used especially with packaged food or a semi-continuous system
(Balasubramaniam et al., 2015; Koutchma, 2014).
The primary components for HHP batch system include:
o Pressure Vessel.
o Two closures to seal the pressure vessel.
o Yoke, which is a structure used to restrain the end closures while under pressure.
o High-pressure pump.
o Process control for controlling and monitoring pressure
o Handling system for loading and removing the product (Balasubramaniam et al.,
2015).
The pressure vessel should be made of stainless steel to prevent corrosion, and it can be in a horizontal, vertical, or tailing mode. The pressure vessel should be able to stand the
12 maximum pressure designed. The two end closures close and seal both ends of the pressure vessel while under pressure with the support of the yoke. The pressure pump compresses the transmitting liquid, usually water, to the target pressure. The control system is needed to control and monitor pressure, temperature and holding time
(Balasubramaniam et al, 2015).
Figure 1.3: HHP commercial unit (source: http://www.hiperbaric.com/en/ hiperbaric55)
1.2.2.High Pressure Process:
When using HHP in processing of foods, food is usually packaged in flexible pouches before applying to HHP to protect food and prevent contamination from the pressure medium (Balasubramaniam et al., 2015; Zhang et al., 2011). The packaging should allow a 15% decrease in volume and should be able to return back to the original volume
(USFDA, 2000). In addition, the package should be able to stand the high pressure
13 without leaking or being damaged (Fellows, 2009). Solid materials such as metal containers will not be able to stand the pressure treatment and will break (Fellows, 2009).
During HHP processing, the packaged food is placed in a basket and then transferred to a high-pressure vessel. The vessel is sealed, filled with the pressure transmitting liquid and compressed by the use of a high-pressure pump that pumps more liquid into the vessel.
The packaged food is compressed under the same pressure as in the pressure vessel. Once the desired pressure has been reached and the food has been held for the desired time, decompression of the vessel, which is releasing the pressure transmitting liquid, takes place (Zhou, 2013; Martin et al., 2002; Balasubramaniam et al., 2015).
1.2.3. The Principle of High Hydrostatic Pressure:
HHP technology is based on some basic principles including the Isotactic principle, Le
Chatelier’s principal, and the Microscopic ordering principle. The Isotactic principle
(Pascal’s principle) states that pressure is transformed uniformly in all directions through the food sample without being affected by the size and shape of the food
(Balasubramaniam et al., 2015). This uniform pressure compresses the food sample resulting in a decrease in the volume of the sample, yet it will be able to return to its original shape when pressure is released (Balasubramaniam et al., 2015; Barbosa et al.,
2005). Le Chatelier’s principle states that HHP enhances some chemical reactions in food systems that cause a reduction in the volume (Rupasinghe & Yu, 2012; Chawla et al.,
2011; Balasubramaniam et al., 2015). The Microscopic ordering principle states that increasing pressure at a constant temperature increases the degree of ordering for molecules of a given substance (Balasubramaniam et al., 2015; Koutchma, 2014).
14 1.2.4. Effect of High Pressure on Proteins:
High pressure can have an inactivation effect on enzymes including food quality enzymes such as lipoxygenase that causes the off flavor and color change in food (Fellow, 2009).
The high-pressure treatment does not affect the primary structure of proteins. However, when using very high pressures, the hydrogen bonds break in the secondary structure of proteins leading to irreversible denaturation (Kilimann, 2005).
1.2.5. Microbial Inactivation by HHP
The effect of High-pressure treatment on microorganisms can be observed as inhibition of enzymes and proteins, cell membrane and cell morphology changes, and inhibition in the transcription and translation of genetics (Considine et al., 2008). Protein denaturation is observed at low pressure of 50 MPa with ribosomal function inhibition at 100 MPa and membrane damage at 200 MPa (Abe 2007). The membrane and protein damage that is caused by HHP can be partially reversible up to 300 MPa, which means that the cells have the ability to recover. As the pressure goes over 300 MPa, the damage becomes irreversible leading to cell death (Abe, 2007). Loss of nutrient transport and energy generating enzymes and proteins, such as ATPase, from the cells that have been exposed to high pressure are evidence that support the effect of pressure on membranes. In addition, there are different effects of membrane damage on microbial cells including leakage of the inner components of the cell through the membrane (Considine et al.,
2008). It is important to note that HHP does not cause degradation of nucleic acids such as DNA, but it causes nucleic acids to become more compact, which disables the replication of the cell (Simpson & Gilmour, 1997). Replication of the cells is disabled as
15 HHP breaks the non-covalent bonds in proteins including proteins involved in replication
(Fellows, 2009).
HHP is used in combination with heat to increase the rate of microbial and enzyme inactivation (FDA, 2014). In addition, temperature is considered as one of the main parameters that affects the inactivation kinetics in HHP processing with an increase in the efficacy of HHP lower or higher than the range of 20-40°C (Heinz and Buckow, 2009),
Figure1.5. For example, Alpas et al. (1999), reported increased inactivation of E. coli
O157: H7 and other pathogenic microorganisms when using a pressure of 345 MPa at
50°C for 5 min. In addition, Liu et al. (2011), reported an increase in the inactivation of
Campylobacter jejuni, Escherichia coli, and spoilage microbiota when using a pressure of 400 MPa at 40°C compared to when using pressure of 300 MPa at 30°C. The log reduction observed was more than 6 logs for all microorganisms except E. coli that had a
4.5-log reduction. However, chemical changes in food are influenced by temperature and time (FDA, 2014).
Figure 1.4: The effect of temperature on the efficacy of pressure on inactivation of model microbes to reach 5 log reduction. (Heinz and Buckow, 2009)
16 There has been a lack of the complete understanding of the factors other than temperature that affect the efficacy of HHP process (Martin et al., 2002; Warriner & Namvar, 2014).
There are critical parameters for high pressure including time, pH, and water activity. The pH of the food is one of the parameters affecting high pressure. Acidic pH values enhance pressure inactivation of microbes and inhibit the recovery of injured cells
(Hogan et al., 2005). For example, Ganzle et al., 2015, reported that the barotolerance of
STEC O157 and non- O157 strains decreased significantly by 6 log CFU/mL in low acidic fruit juice when treating with pressure 400 and 600 MPa. In addition, the inactivation of microorganisms by HHP is affected by treatment time, pressure, and processing temperature (Setikaite et al., 2009). For example, it has been reported that under the same pressure treatment in PBS (Phosphate Buffer Saline), strains of E. coli
O157: H7 and Listeria monocytogenes were more resistant to high pressure at 20°C-25°C compared to when at 35°C (Alpas et al., 1999). In addition, differences in pressure resistance at two temperatures between strains of Listeria monocytogenes,
Staphylococcus aureus, E. coli O157: H7, and Salmonella were investigated in a study
(Alpas et al., 1999). The study reported that under a pressure of 345 MPa for 5 min at
25°C, some strains within the same species of bacteria were more barotolerant than others. The log reduction achieved at these conditions (345 MPa, 5 min, 25°C) ranged from 0.9 to 3.5 for 9 strains of L. monocytogenes; 0.7 to 7.8 for 7 strains of S. aureus; 2.8 to 5.6 for 6 strains of E. coli O157:H7; and 5.5 to 8.3 for 6 strains of Salmonella.
However, when pressurized at 345 MPa for 5 min at 50°C, the log reduction achieved for all strains except for one resistant strain of S. aureus was more than 8 logs. For the resistant strain of S. aureus, only 6.3 log reduction was achieved after 15 min (Alpas et
17 at., 1999). These studies show that there are variations in pressure resistance between strains of the same species.
1.3. Water Activity and Osmotic Stress
Some of the food preservative methods used in industries include the addition of high concentrations of salt and sugars. This, in turn, lowers the water activity in food creating osmotic pressure (Burgess et al., 2016; Ueno et al., 2011; Stewart et al., 2005). Osmosis is the movement of water molecules from solutions of lower solute concentration to higher solute concentration through a semi-permeable membrane (Lodish et al., 2000).
The semi-permeability of cell membranes makes osmosis to be one of the important physiological challenges for cells (Cheung et al., 2009). Increasing the external osmotic pressure causes outflow of water resulting in shrinkage and dehydration of cells, while decreasing the external osmotic pressure causes inflow of water and swelling of cells
(Woods, 2015). In addition, inflow and outflow of water cause changes in the cytoplasmic features, changes in the concentration of metabolites and affects membrane transportation (Woods, 2011). Bacteria including E. coli can maintain balance by using different mechanisms including increasing the uptake of K+ ions and biosynthesis or transport of osmolytes, solutes that regulate osmosis (Cheung et al., 2009; Ueno et al.,
2011). It has been reported that the main response of bacteria to high osmotic pressure is the increase in the uptake of K+ followed by the accumulation or synthesis of osmolytes as a secondary response (Sleator &Hill, 2001) Figure 1.5. When osmolytes are not available, they can be biosynthesized from internal substrates (Woods, 2011). Osmolytes can be accumulated inside the cell in high concentrations without affecting the function of the cell, and they are accumulated by active transporters. When osmotic pressure
18 drops, cytoplasmic solutes can be discharged by mechanosensitive channels to prevent lysis (Woods, 2015), Figure 1.5. Bacteria survive high osmotic pressure by accumulation of osmolytes that significantly enhances tolerance to low aw conditions, while releasing osmolytes promotes survival under low osmotic pressure (Woods, 2015). Osmolytes are accumulated by regulation of specific osmotic transporters including ProP and ProU
(Wood, 2015; Murdock et al., 2014). It has been reported that the accumulation of osmolytes is one of the main changes found in microbes when exposed to high osmotic pressure (Teixido et al., 2005). In E. coli, the osmotic stress is controlled by accumulation of K+ and other osmolytes including proline and glycine betaine (Burgess et al., 2016;
Burg & Ferraris, 2008). When osmolytes are not available, E. coli can control osmotic stress by synthesizing trehalose (Styrvold & StrØm, 1991). In addition, accumulation of
K+ glutamate in E. coli provides osmotic tolerance in the absence of osmolytes (Murdock et al., 2014). Moreover, compatible solutes increase stability of proteins and enzymes and maintain integrity of biological membranes (Hoppner et al., 2003; Sleator &Hill, 2001).
Increasing the stability of enzymes by osmolytes provides protection not only against osmotic pressure but also against increased temperature, freezing, and drying (Sleator &
Hill, 2001). Studies reported that the type of solute accumulated affects bacterial growth and rehydration (Woods, 2015). For example, accumulation of K+ glutamate causes incomplete rehydration of cells and allows bacterial growth, yet not within the same rate of growth as in pre-stressed conditions (Woods, 2015). On the other hand, accumulation of organic osmolytes rehydrates cells and allows bacteria to grow within their preferable growth rate that is usually in the range of 0.90-0.99aw. In some pathogenic bacteria, the aw limit for growth is as low as 0.86 aw (Woods, 2015). Also, it has been reported that
19 some molecules such as glycerol diffuse freely and easily through the membrane to maintain balance due to the concentration gradient under low water activity conditions
(Sperber, 2012; Hoppner et al., 2003; Chang et al, 2014). This shows that the different solutes accumulated inside the cells during low water activity conditions (osmotic stress) would affect bacterial growth differently.
Figure 1.5: Illustrating the bacterial response to the increase and decrease in osmotic pressure. When osmotic pressure increases outside the cell, the K+ starts to accumulate in the cell as well as the osmolytes that are accumulated by specific transporters. The osmolytes can be synthesized inside the bacterial cells if the osmolytes are not available in the outside soundings of the cell. When osmotic pressure decreases out the cell, osmolytes are released to the outside by specific transporter channels (Wood, 2011).
20 1.3.1. Effect of Water Activity on Barotolerance and Other Stresses:
Water activity aw has been known as one of the main factors affecting the inactivation and growth of pathogens in extended shelf life foods (Setikaite et al, 2007). Exposure to a single stress such as osmotic stress has been reported to increase tolerance to other stresses such as thermal stress (Huang et al., 2009). In addition, low water activity provides protection to microbes against HHP (Considine et al., 2007; Setikaite et al,
2007). It has been reported that resistance of microorganism to HHP increases when increasing osmotic stress (Hoppner et al., 2003). Furthermore, survival of bacterial cells under high pressure is affected significantly by the type of solute used (i.e. sugar or salt)
(Considine et al., 2007). For example, sucrose and salt content were reported to provide significant protection for pathogens against HHP inactivation (Considine et al., 2007). In addition, Palou et al, 1997, reported that using sucrose to lower the water activity increased barotolerance of Zygosaccharomyces bailii when exposing to high pressures
(Setikaite et al, 2007). Furthermore, the usage of NaCl and sugars had a protective effect for E.coli, saccharomyces cerevisiae, and Lactococcus lactis against HHP (Setikaite et al,
2007). Hayman et al. (2008) reported that decreasing the water activity caused an increase in the barotolerance of Listeria innocua. In addition, Goodridge et al. (2005) reported less than one log reduction, 0.83 log, in Salmonella enteritidis inoculated into raw almonds was achieved 413 MPa at 50°C for 5 min. Oxen and Knorr (1993) reported an increase in the barotolerance of Rhodotorula rubra when lowering the water activity from 0.98 to below 0.91, while a 7 log reduction was reported when water activity was more than 0.96 (Setikaite et al., 2009). In addition, it has been reported that decreasing water activity can protect microorganisms from other stresses such as heat. Scott (1955)
21 reported that proteins and cells of microorganisms were protected against heat when decreasing water activity (Setikaite et al., 2009). However, information regarding the effect of water activity on bacteria that cause foodborne illness was insufficient (Setikaite et al, 2007).
The type of substrate has been reported to be one of the underlying reasons for the effect of HHP on barotolerance of microbes (Considine et al., 2008). It has been reported that magnesium is important for stabilizing the structure of ribosomes, which gives more protection to the cells against HHP. In addition, when the concentration of calcium is increased, it provides more stabilization to the outer membrane of the cell, which in turn decreases the effect of HHP in the inactivation of cells (Considine et al., 2008).
Moreover, sucrose has been reported to stabilize the function of the membrane proteins, which gives the cells more protection against high pressure treatment. Furthermore, osmolytes were reported to protect cells against high pressure by stabilizing the function of enzymes (Considine et al., 2008). Another reason for the effect of HHP on the barotolerance of microbes is the increase in the levels of unsaturated fatty acids in the cell membrane of the bacterial cells exposed to HHP. This increase in the unsaturated fatty acids in the membrane increases survival of the cells under high pressure (Tamber, 2017).
In addition, studies reported that mutant E.coli that lacked the gene responsible for fatty acid synthesis were more sensitive to high pressure (Tamber, 2017). Some other reasons that were reported to affect the ability of HHP to inactivate microbes include the effect of other stresses such as heat shock and cold shock (Tamber, 2017). Aertsen et al., 2004, reported that heat shock proteins and cold shock proteins were observed in E.coli exposed to high-pressure treatment. They reported an increase in barotolerance of E. coli that were
22 applied to heat shock than regular non-stressed E. coli. In addition, Sagarzazu et al.
(2009) reported an increase in resistance of Campylobacter jejuni to HHP after exposing to cold temperature (i.e., 0°C) compared to non-stressed C. jejuni that were incubated at
37°C. However, none of the stresses were able to successfully predict the barotolerance of microbes.
1.3.2. Accumulation of Osmolytes:
Solutes such as salts and sugars are used to control and lower the water activity, which causes accumulation of osmolytes such as amino acids in the osmotic stressed microbial cells (Sperber, 2012; Csonka, 1989; Teixido et al., 2005). Accumulation of osmolytes inside bacterial cells allows the survival of bacteria at high osmotic pressure (Wood,
2015). In addition, the accumulation of osmolytes provides protection for cells against osmotic stress and other stresses such as temperature and drying. Moreover, resistance of microorganisms to HHP has been found to increase when lowering water activity, yet it was different when using NaCl than when using sucrose (Hayman et al., 2008). Also, solutes such as sucrose and NaCl have been reported to provide protection to microorganism against HHP by the accumulation of amino acids, and the physiological features of HHP treated cells did not have the same effect when using sucrose or NaCl, ionic and non-ionic solutes (Hoppner et al., 2003). In addition, Molina-Höppner et al.,
2004, reported that accumulation of solutes such as NaCl and sucrose occurs as a result of high osmotic stress in microbes such as E.coli and Lactococcus lactis. This, in turn, retains the membrane integrity of the cells leading to high resistance during high-pressure treatment (Sevenich et al., 2015).
23 1.4 Strain Variation in Resisting High Hydrostatic Pressure:
There is strain variation with regards to pressure resistance of bacterial species even between strains of the same species. It has been reported that there is variation in pressure resistance within E.coli strains (Liu et al., 2015; Tamber, 2017). In addition, studies reported that when exposing different species of bacteria to HHP treatment within the same conditions, there is difference in the inactivation within the strains of the species
(Vanlint et al., 2012; Liu et al., 2011). For example, Tamber, 2017, reported a variation of 0.9- 6 log CFU/mL in pressure resistance within 99 strains of Salmonella enterica when treating with pressure 600 MPa/ 3 min. Furthermore, Liu et al., 2011, reported that when treating 19 strains of campylobacter jejuni with pressure 300 MPa/ 3 min at 30°C, there was a variation of 0.5-5 log CFU/mL in the barotolerance of the C. jejuni strains. In addition, Liu et al., 2015, reported a variation of 1.1 – 5.5 log CFU/mL in pressure resistance of 100 strain of E.coli including VTEC strains when exposing to pressure 600
MPa/ 5 min at 25°C in PBS. Moreover, under pressure treatment of 345 MPa/ 3 min at
25°C, there was variation in pressure resistance between strains of the same species of
STEC O157: H7, Listeria monocytogenes, Staphylococcus aureus, and Salmonella when studying the variation in the pressure resistance of these pathogens. The variation in the pressure resistance ranged from 0.9-3.5 within L. moncytogenes, 0.7-7.8 S. aureus, 2.8-
5.6 STEC O157: H7, and 5.5-8.3 Salmonella (Alpas et al., 1999). Whitney et al., 2007, reported variation of 0.26 to 3.95 log CFU/mL in pressure resistance of 5 strains
Salmonella enterica when examining pressure resistance at pressure 300 MPa/ 2 min in
TSB. They also reported variation of 0.28 to 4.39 log CFU/mL in pressure resistance of 6 strains STEC O157:H7 when examining pressure resistance at 550 MPa/ 2min. The
24 previous examples illustrate the strain variation in pressure resistance of the different pathogens and even within strains of the same species.
1.5 Glycolysis (Embden-Meyerhof-Parnas pathway) and Krebs cycle Tricarboxylic acid (TCA) cycle:
Glycolysis is a metabolic pathway for metabolizing carbohydrates to pyruvate thereby producing energy by substrate-level phosphorylation (Kim & Gadd, 2008; Chaudhry &
Bhimji, 2018). Glucose is metabolized by the EMP pathway in E. coli (Kim & Gadd,
2008). Sorbitol, which is a polyol, is converted into fructose. Fructose then is phosphorylated to fructose-6-phosphate to enter the glycolysis pathway (Jeffery &
Jörnvall, 1983). Starch is a large polysaccharide compound that cannot pass through the bacterial cell membrane. It is hydrolyzed out of the cell by amylase that eventually breaks it down to glucose units, and the glucose units can be then metabolized by glycolysis
(Kim & Gadd, 2008; Petersen and McLaughlin, 2016; Figure 1.6).
There are different points of entry in which amino acids can enter Krebs cycle. Some of the amino acids are degraded into intermediates that can participate in the Krebs cycle.
Alanine, glycine, and tryptophan can enter the cycle by converting to pyruvate, and then pyruvate is converted into acetyl CoA. The acetyl CoA is then converted to citrate, which is the first intermediate in the Krebs cycle. Asparagine can enter the cycle by breaking down into oxaloacetate, which is the final intermediate in the Krebs cycle. Pyruvate can be converted into acetyl CoA, and then converted into the first intermediate in the krebs cycle citrate (Schowen 1993). Figure 1.7.
25
Figure 1.6: Glycolysis (EMP) pathway for glucose metabolism in Eukaryotic and prokaryotic cells, and the participation of sorbitol and starch in EMP pathway. Glucose is converted into pyruvate as an end product through a sequence of reactions. Retrieved from (Wolfe, 2015).
26
Figure 1.7: TCA (Tricarboxylic acid Cycle), Krebs cycle pathway in E.coli, and the participation of amino acids in the krebs cycle. 1- Citrate. 2- Iso citrate. 3- α- ketoglutartae. 4- Succinyl CoA. 5- Succinate. 6- Fumarate. 7- Malate. 8- Oxaloacetate.
1.6. Dormancy State in Bacteria:
Under stressful environmental conditions, microbes have the ability to develop survival strategies that lead to down-regulation of metabolic activities, which is known as dormancy state (Lebre et al., 2017; Dworkin & Shah, 2010). The dormancy state is a survival strategy that allows bacteria to survive stresses for a long period of time (Lebre
27 et al., 2017; Ayrapetyan et al., 2015). In addition, it has been reported that when pathogens are in the dormant state, they have the ability to persist till the environment becomes best for their growth (Kearns et al., 2016; Aanderud et al., 2016). Under specific conditions, many of which remain unknown, dormant cells can be resuscitated
(Ayrapetyan et al., 2014). Dormant state has been reported to include two well-defined forms that are antibiotic persisters and Viable but non-culturable cells (VBNC) (Lebre et al., 2017; Ayrapetyan et al., 2015). Persisters are defined as a part of the bacterial population (1%) that are in a metabolic inactive state (non-growing) and can resist antibiotics, yet are able to remain sensitive to the same antibiotic after re-growing
(Zhang, 2014; Wood et al., 2013). Viable but non-culturable state VBNC is a state of dormancy known as a special survival strategy that is developed by bacteria when facing stressful environmental conditions (Lebre et al., 2017; Ramamurthy et al., 2014). In 1982,
VBNC state was first detected in E.coli and Vibrio choleare (Li et al., 2014). VBNC are viable cells that have lost their ability to form colonies on regular agar, and they are known to have low metabolic activity and intact cell membrane with undamaged DNA
(Ayrapetyan et al., 2015; Pienaar et al, 2015; Li et al., 2014). Studies stated that different environmental and experimental conditions such as salinity, osmotic stress, starvation, addition of food preservatives, and exposure to decontaminants cause bacteria including
E.coli to enter VBNC state (Ayrapetyan et al., 2015; Morishige et al., 2015; Pienaar et al,
2015; Fakruddin et al., 2013). In addition, pathogenic strains of E.coli including STEC
O157: H7 have been reported to enter VBNC state when exposed to sub lethal conditions
(Pienaar et al, 2015). Food characteristics, food processing environment and storage are important factors for pathogens to enter a VBNC state in food (Fakruddin et al., 2013).
28 VBNC are dormant, yet they can resuscitate and regain their pathogenicity in suitable hosts (Zhao et al., 2017). They are undetectable by culturable methods due to their down- regulation, and they are able to resuscitate under suitable conditions. (Ayrapetyan et al.,
2014; Pienaar et al, 2015; Li et al., 2014).
1.6.1.Resuscitation of VBNC:
VBNC state bacteria can regain their ability to form colonies under certain suitable conditions, which is called resuscitation (Oliver, 2005; Gupte et al., 2003; Liu et al.,
2017). The food-processing environment could provide these appropriate conditions promoting resuscitation of VBNC bacteria, which pauses a significant food risk (Ding et al., 2017). The presence of VBNC or injured bacteria may cause underestimation in the number of pathogens. Therefore, when plate counts are negative for food samples, environmental samples, and clinical samples, they cannot be considered as pathogen free
(Fakruddin et al, 2013). It has been stated in a study that E.coli responsible for urinary tract infection in the VBNC state was the reason behind the frequent urinary tract infections in a large number of people (Fakruddin et al, 2013). Resuscitation of VBNC
E.coli has been mostly studied and reported by shifting to a suitable temperature, using media supplemented with high nutrients, and by the addition of chemicals and substances such as sodium pyruvate and amino acids (Ding et al., 2017; Pinto et al., 2011).
Reissbrodt et al., 2002, reported resuscitation of EHEC strains by adding Oxyrase to TSB after they became non-culturable in water microcosms. Moreover, Ohtomo and Saito,
2000, reported resuscitation of VBNC E.coli to culturable cells when removing the high salinity conditions that affected their culturability.
29 1.6.2.Detection of Dormant Cells (VBNC):
One of the most important needs is to apply dependable methods for detection of dormant cells including VBNC to determine the accurate number of viable cells (Li et al., 2014).
The presence of metabolic activity and the inability to be culturable by routine methods raise concerns when it comes to testing of food and water. Therefore, different methods have been proposed to detect dormant state cells in bacterial cultures (Morishige et al.,
2015; Riedel et al., 2013). The existence of dormant cells can be indicated by comparing the number of culturable cells to that of viable cells in the sample (Li et al., 2014). The number of culturable cells can be determined by using the plate count technique, while the number of viable cells can be determined by using different staining methods along with microscopy and flow cytometer (Li et al., 2014). Viable cells have intact cell membranes that do not allow stains to pass through, while injured and dead cells have a collapsed cell membrane (Stiefel et al., 2015). To distinguish between these cells that have different membranes integrities, staining methods such as LIVE/DEAD® BacLight assay can be used (Li et al., 2014). This assay includes the fluorescent dyes SYTO 9 and propidium iodide (PI) that have the ability to detect intact cell membranes (Morishige et al., 2015; Stiefel et al., 2015; Ramamurthy et al., 2014; Li et al., 2014). PI is a red fluorescent stain that is used to detect dead and injured cells as it has the ability to penetrate cells with damaged membranes only (Stiefel et al., 2015; Fakruddin et al.,
2013). On the other hand, SYTO 9 is a green fluorescent stain that has the ability to enter live and dead cells, yet it shows a strong fluorescent signal when it bounds to nucleic acids, and it gets reduced when combined with PI in dead cells (Stiefel et al., 2015;
Fakruddin et al., 2013). Therefore, live cells including VBNC cells and culturable cells
30 will be stained with green, while dead and injured cells will be stained with red (Li et al.,
2014; Fakruddin et al., 2013). This LIVE/DEAD® assay can be used with instruments such as flow cytometer and fluorescent microscope to detect and enumerate live and dead cells (Stiefel et al., 2015; Ramamurthy et al., 2014; Li et al., 2014; Khan et al., 2010).
31
Research Hypothesis and Objectives
Hypothesis:
“The barotolerance of STEC can be correlated to the osmotic stress resistance.
Accumulation of osmolytes provide cross-protection between osmotic stress and high pressure”
Objectives:
1- To determine the variation in osmotic stress resistance of Shiga Toxin
producing Escherichia coli and barotolerance.
2- To correlate the sodium chloride growth limit of Shiga Toxin producing
Escherichia coli to barotolerance.
3- To elucidate the mechanisms by which low water activity can enhance
barotolerance in Shiga Toxin producing Escherichia coli.
32 Chapter 2
The Correlation between Tolerance to Low Water Activity and High Pressure Processing in Shiga Toxin Escherichia coli
2.1 Abstract
High hydrostatic pressure processing (HHP) is extensively applied for the non-thermal pasteurization of foods. One notable feature of HHP mediated inactivation of microbes is the large variation in lethality between strains and the influence of the food matrix. In the following, it was hypothesized that the barotolerance of Shiga Toxin producing
Escherichia coli could be correlated to resistance to low water activity environments. The study evaluated the barotolerance of members of the Top 6 STEC serotypes and found variation with O26:H11 and O45:H2 exhibiting higher tolerance to pressure compared to the other bacterial types tested. A negative correlation was found between resistance to high pressure and sodium chloride concentration under which the test E. coli strains could grow. For example, E. coli O26:H11 exhibited the highest tolerance to pressure but could only grow up to a NaCl concentration of 2%. In contrast, E. coli O121:H19 was sensitive to pressure, but could grow in the presence of 6% NaCl. The results suggested that the induction of the stress-response was the likely reason for high barotolerance in salt sensitive strains vs resistant. It was also found that supplementing the suspension medium with amino acids, sugar alcohols and starch could increase the barotolerance of
E. coli. Although it is possible that the solutes acted directly as osmolytes it is also proposed that metabolic products and other indirect protective mechanisms were also operating. The study illustrated that under high pressure (600 MPa) the barotolerance is
33 related to the food matrix, while under lower pressures the food matrix along with physiological state of the cell contribute.
2.2 Introduction:
Shiga toxin producing Escherichia coli (STEC) causes serious human foodborne disease;
Hemolytic Uremic Syndrome (HUS) (i.e., kidney failure) with STEC O157 accounting for more than 73,000 illness cases yearly in the USA; and non-O157 STEC strains accounting for over 100,000 illness cases (Public Health Agency, 2016; CDC, 2012).
Foods from animal sources such as beef and dairy products, fresh produce, and sprouts are linked to STEC. To decrease the risk and inactivate the pathogen while keeping the raw sensory of food characteristics, different food processing technologies have been introduced (Hus et al., 2015; Erickson & Doyle, 2007; Hussein & Sakuma, 2005; Kumar et al., 2004). High hydrostatic pressure is an emerging non-thermal food processing technology that increases the safety of foods. It has minimal effect on the texture, taste, and nutritional values of the food being processed (Yordanov & Angelova, 2010;
Considine et al., 2008). Since 2000, HHP has been used in North America to increase the safety of food products such as juices, deli meats, seafood, fruits and vegetables
(Demazeau & Rivalain, 2011; Martin et al., 2002; Deliza et al., 2002).
One notable feature of HHP mediated inactivation of microbes is the large variation in lethality between strains and the influence of the food matrix (Gänzle and Liu, 2015;
Whitney et al., 2007; Malone et al., 2007). For example, Malone et al., 2007, reported a variation of 0.6-3.4 log reduction in 17 strains of E. coli O157:H7 that were treated with
HHP at 500 MPa, 23°C for 1 min (Malone et al., 2007). In addition, Liu et al. (2015), reported a variation of 1.1 – 5.5 log CFU/mL in pressure resistance of 100 strain of E.
34 coli when exposing to pressure 600 MPa/ 5 min at 25°C in phosphate buffered saline.
Moreover, Tamber, 2017, reported a variation of 0.9- 6 log CFU/mL in pressure resistance within 99 strains of Salmonella enterica when treating with pressure 600 MPa/
3 min.
Efforts have been made to link barotolerance of microbes to thermal resistance, pH, and cold shock. However, the parameters did not successfully predict the barotolerance of microbes (Liu et al., 20015; Cutter et al., 2012; Martin et al., 2002; Metrick et al., 1989;
Huban et al., 1997). There are critical parameters for high pressure including time, pH, and water activity. Studies reported that high pressure is less effective on microbes in low aw matrices (Huang et al., 2009; Considine et al., 2007; Setikaite et al., 2007; Mussa et al., 2006) Hayman et al., (2008), reported increase in the barotolerance of listeria monocytogenes (2.5 log reduction) when aw was decreased to 0.83 compared to when aw was 0.99-0.87 (6.5-7 log reductions). In addition, Setikaite, et al., (2007), reported that decreasing aw from 0.99 to 0.90 in glycerol solutions increased barotolerance of E.coli k12 as no log reduction was observed. Moreover, Moussa et al., (2006), reported that decreasing aw to 0.85 in glycerol solutions increased barotolerance of E. coli k12 when exposed to pressure 450 MPa/ 10 min. However, information regarding the effect of water activity on bacteria that cause foodborne illness was insufficient (Setikaite et al,
2007). The objective of the current study was to determine if the tolerance to low water activity could be correlated with the high pressure resistance in STEC.
35 2.3 Materials and Methods
2.3.1 Bacteria and Cultivation
The STEC isolates used O157:H7, O121:H19, O1O3:H2, O111:NM, O45:H2, and
O26:H11 were obtained from Dr. Roger Johnson, Public Health Agency of Canada,
Guelph, Ontario. The pure isolates were cultured on TSA, while suspensions were stored in 50% TSB and 50% glycerol solution at -80°C.
2.3.2 Preparation of Suspensions
The STEC isolates were grown overnight on Tryptone Soy Agar (TSA; OXOID Ltd,
Basingstoke, Hampshire, England) at 37°C. A loop-full of isolated colonies was suspended and dispersed in 0.9% w/v saline to achieve a final O.D of 0.2 (108 log
CFU/mL), and the suspension was stored at 4°C until required.
For the osmotic stress experiment, the STEC isolates were grown overnight on Tryptone
Soy Agar (TSA; OXOID Ltd, Basingstoke, Hampshire, England) at 37°C. A loop-full of isolated colonies was suspended and dispersed in 0.9% w/v saline to achieve a final O.D of 0.2 (108 logs CFU/mL), and serial dilutions in 0.9% w/v saline were made to achieve
106 and 105 CFU/mL suspensions with the suspensions stored at 4°C until required.
For the gradient plates, the isolates were grown overnight on Tryptone Soy Agar (TSA;
OXOID Ltd, Basingstoke, Hampshire, England) at 37°C. A loop-full of isolated colonies was suspended and dispersed in 0.9% w/v saline to achieve a heavy suspension of (109
CFU/mL) 2 O.D, and the suspension was stored at 4°C until required.
For the dry weight experiment, the STEC strains used were grown overnight on Tryptone
Soy Agar (TSA; OXOID Ltd, Basingstoke, Hampshire, England) at 37°C. A loop-full of isolated colonies was enriched in Tryptone Soy Broth (TSB; OXOID Ltd, Basingstoke,
36 Hampshire, United Kingdom) at 37°C/ 24 h. The cells were centrifuged at 5000 rpm/ 10 min and then the pellet was used in the experiment.
For the substrate utilization profile test, the STEC strains were grown overnight on
Tryptone Soy Agar (TSA; OXOID Ltd, Basingstoke, Hampshire, England) at 37°C. Then a single isolated colony was taken to prepare a 5mL bacterial suspension for each strain and used to inoculate the API strip.
2.3.3 Persistence of the STEC Strains under osmotic stress (Colony counts)
Sample preparation
To determine the persistence of the STEC strains under osmotic stress by plate count, the
STEC strains were suspended in 0.9% w/v saline to prepare three different suspensions including 108, 106, and 105 CFU/mL as described in subdivision 2.1. Then 1 mL of the suspension was inoculated into tubes that contain 9 mL peptone water with 10 % NaCl
(50g). After that, tubes were incubated at room temp (22°C) for 40 days. Samples were prepared by adding 0.1 mL of the sample into 0.9 mL saline (0.9% w/v) and serial dilutions were carried out. Then 0.1 mL of the dilutions was plated on TSA with plates incubated at 37°C/24 h. Samples were taken for plating every 3 days for the higher inoculum 107 CFU/mL and every 2 days for the lower inoculums 105 CFU/mL and 104
CFU/mL.
The incubation temperature also was tested to determine if it affects recovery of cells on enumeration plates (i.e., injured cells) by incubating the enumeration plates at room temperature (22°C)/48 h.
37 2.3.3.1 Live counts under fluorescent microscope (Viability staining)
The LIVE/DEAD® BacLight Bacterial Viability kit (L7007) for fluorescent microscopy
(Molecular Probes, USA) that includes PI and SYTO9 fluorescent dyes was used to determine the viability of cells as will be described below.
To determine the number of live cells under fluorescent microscope for the STEC cultures during osmotic stress, 1 mL of the bacterial suspension was inoculated in tubes containing 9 mL peptone water with 10% NaCl. Tubes were incubated at room temperature 22°C for 24 days. Samples were stained with LIVE/DEAD ® BacLight kit
(L7007) for microscope by adding 3µl of the mixed stains (SYTO 9 and PI in equal amounts) to 1mL of the sample in a tube according to the manufacturer. Five fields were examined under an Olympus optical fluorescent microscope (Olympus Optical, MA,
USA) by regular slide using a filter set with an excitation wavelength of 460-490 nm.
Samples were examined after 0, 3, 6, 10, 14, 18, 22, and 24 days to determine the number of live cells under microscope. Viable cells including VBNC will be stained with green
(SYTO9), while non-viable cells will be stained with red (P1).
2.3.3.2 Recovery of the STEC dormant cells (Resuscitation)
Recovery of cells was tested to evaluate the ability of the dormant non-culturable STEC cells to recover and gain their ability to form colonies.
Sample preparation
Tubes containing 9 mL peptone water with 10% NaCl were inoculated with 1 mL of the bacterial suspension. Tubes were incubated at room temperature 22°C and left under osmotic stress for 50 days. After 50 days, 0.1 mL of the samples was plated on TSA and
38 incubated at 37°C/ 24 h to determine if they decreased to the level of enumeration. Once the STEC cultures have reached the level of enumeration, 1 mL of the non-culturable
STEC cultures was inoculated in tubes containing TSB with 5% glycerol and tubes containing TSB alone. Tubes were incubated at room temperature (22°C) for 2 h, and then 0.1 mL of the samples was platted on TSA. Plates were incubated for 48-72 h at
22°C to allow the dormant or injured cells to grow.
2.3.3.3 Growth limits of STEC (The gradient Plate Technique)
The gradient plate consists of 2 layers of agar. The bottom layer is TSA agar, while the upper layer is TSA agar with NaCl (10%, 12%). The plates (9x9 cm) were placed in a slightly sloping position to allow the TSA agar to cover the bottom of the plate forming a slope. Once the TSA agar has solidified, the plate was placed in a horizontal position and the modified TSA agar that contains NaCl was added. Each of the NaCl concentrations was performed in a separate gradient plate. After that, 0.1 mL of the suspension was spread on each of the gradient plates, and the plates were incubated at 37°C for 24-48 h.
After incubation, the plates were checked and the growth limit for the STEC strains was measured in cm.
2.3.4 High Pressure Treatment
The HHP treatment was carried out in a commercial HHP unit, which is Hyperbaric 55 L unit (Hyperbaric, USA, Miami, FL), located in Stoney Creek, ON, Canada. The unit has a capacity of 55 L and uses a range of pressure up to 600 MPa.
39 2.3.4.1 High Pressure inactivation (barotolerance)
To show the barotolerance of the STEC strains and the variation in barotolerance of the strains, the STEC strains were exposed to HHP (400-600 MPa) in a saline solution with
(10% NaCl), and the plate counts and viable counts by viability staining were determined.
Sample preparation
Normal saline was supplemented with 10% NaCl (50g/ 500 mL) to prepare 10% NaCl and kept in the fridge at 4 °C till it was dispensed by a volume of 9 mL into HHP pouches. After that 1 mL of the suspension was inoculated into pouches that contain 9 mL of the 10% NaCl solution. The pouches were sealed and kept in a cooler at 4° C while transferring to the HHP facility. The pouches were placed in a high-pressure chamber and exposed to pressures 400 MPa, 500 MPa, and 600 MPa for 3 min. Then a lower pressure (300 MPa) was used to show the plate counts of the STEC strains and compare it to the viable counts under fluorescent microscope (barotolerance). Samples were prepared the same method as mentioned above, and the pouches were exposed to pressure 300 MPa and 400 MPa for 1-3 min. The samples were held at 4°C till enumeration after HHP treatment and post recovery.
2.3.4.2 Microscopic counts
After the samples were exposed to HHP at pressures 300-600 MPa for 3 min, the viable counts under fluorescent microscope were determined as described previously in section
(2.3.2).
40 2.3.4.3 High-pressure tolerance and STEC osmotic growth limits
To show the correlation between high pressure tolerance and STEC growth limits under lowering water activity, the STEC strains isolated from Gradient plates were exposed to
HHP in 10% NaCl at 300 MPa (1 - 3 min); 600 MPa (3 min).
Sample preparation
Samples were prepared by transferring 9 mL of the 10% NaCl solution (50g/ 500 mL) to
HHP pouches. After that 1 mL of the suspension was inoculated into the pouches that are sealed and kept in a cooler at 4° C while transferring to the HHP facility. The pouches were placed in a high-pressure chamber and exposed to pressures 600 MPa / 3 min and
300 MPa for 1-3 min. The samples were held at 4°C after HHP treatment for enumeration and post recovery.
2.3.4.4 Effect of peptone (the accumulation of osmolytes) on the barotolerance of STEC
STEC strains were exposed to HHP in 10% NaCl solution with 1% peptone water to see the effect of peptone on the barotolerance of STEC.
Sample preparation
NaCl (50g) was added to 500 mL normal saline to prepare 10% NaCl solution, and then
1% peptone (5g) was added to the solution. The solution was kept in the fridge at 4 °C till it was dispensed by a volume of 9 mL into HHP pouches that contain 10% NaCl solution with 1%peptone. After that 1 mL of the bacterial suspension was inoculated into pouches that contain 9 mL of the NaCl solution (10% NaCl +1% peptone). The pouches were sealed and kept in a cooler at 4 °C while transferring to the HHP facility. The pouches were placed in a high-pressure chamber and exposed to pressures 600 MPa / 3 min and
41 300 MPa for 1-3 min. The samples were held at 4°C till enumeration after HHP treatment and post recovery.
2.3.4.5 Effect of amino acids (osmolytes) and other supplements (non- osmolytes) on the barotolerance of STEC
To show the effect of amino acids (osmolytes), other supplements (non osmolytes) and sugars on the barotolerance of STEC, the amino acids, supplements, and sugars were added to the NaCl solution and exposed to high pressure.
Sample preparation
Normal saline was supplemented with 10% NaCl (50g/ 500 mL) to prepare 10% NaCl solutions, and then 1% of the following was added to the NaCl solution: amino acids
(glycine, betaine, or asparagine), other supplements (alanine, pyruvate, or tryptophan), or sugars (sorbitol, or starch). The solutions were kept in the fridge at 4°C till they were dispensed by a volume of 9 mL into HHP pouches. After that 1 mL of the bacterial suspension was inoculated into pouches that contain 9 mL of the NaCl solutions with 1% supplements. The pouches were sealed and kept in a cooler at 4°C while transferring to the HHP facility. The pouches were placed in a high-pressure chamber and exposed to pressure 300 MPa for 1-3 min. The samples were held at 4°C till enumeration after HHP treatment and post recovery.
2.3.4.6The effect of Different water activity levels on the barotolerance of STEC in the presence of 1% Glycine
To show if 1% glycine would provide a stable level of protection to STEC in the different aw levels, STEC O157:H7 was treated with high pressure 400 and 600 MPa for 1-3 min in different water activity solutions containing 1% glycine.
42 Sample preparation
Saline bottles (500 mL) supplemented with 1% glycine (5g) were used to prepare solutions with different water activity values by the addition of NaCl (0.98, 0.94, and
0.85 aw). Water activity values were measured using a water activity meter (Dew point water activity meter, AquaLab 4(TE), Pullman, WA). The solutions were autoclaved and kept in the fridge at 4°C till they were dispensed by a volume of 9 mL into HHP pouches.
After that 1 mL of the bacterial suspension was inoculated into pouches that contain 9 mL of the water activity solutions with 1% glycine. The pouches were sealed and kept in a cooler at 4°C while transferring to the HHP facility. The pouches were placed in a high- pressure chamber and exposed to pressure 400 MPa and 600 MPa for 1, 2, and 3 min.
The samples were held at 4°C till enumeration after HHP treatment and post recovery.
2.3.5 Dry weight of STEC (Accumulation of osmolytes)
To show the accumulation of osmolytes, the dry weight for the STEC strains was taken after incubation in 10% NaCl solution with 1% peptone compared to incubation in NaCl solution with no peptone.
Sample preparation and bacterial weight
NaCl (50g) was added to 500 mL normal saline to prepare 10% NaCl solution. Then 1% peptone (5g) was added to one of the NaCl solutions (10% NaCl + 1% peptone). The
STEC pellets, individually, were suspended in 300 mL of the NaCl solution with peptone and with no peptone for 24 h at room temperature (22°C). After 24 h, the culture was centrifuged at 5000 rpm/ 10 min, and the pellet was washed in distilled water in a weighing dish. The weighing dish was left to dry in the oven at 60°C for 3 days with the
43 weight taken every day. To get the dry weight, the average weight of the STEC strains after 3 days was subtracted from the weight of the control dish (empty).
2.3.6 The substrate utilization profile test
The substrate utilization profile test was used to confirm that STEC O157:H7 is sorbitol non-fermenter and the other non O157 STEC are sorbitol fermenters to see the effect of fermented and non-fermented sugars on high-pressure tolerance of STEC.
Sample preparation
Using a pipet the compartments on the API strip were filled with the bacterial suspensions. The compartments that are surrounded with a box were filled up completely with the bacterial suspension, while the other compartments were filled just to the top of the compartments. The compartments that are underlined were sealed by overlaying with mineral oil. The strips were incubated for overnight at 37°C, and the compartments were checked for the color change as shown in the manual table.
2.3.7 Enumeration of Bacteria 2.3.7.1 Persistence of STEC Strains under osmotic stress (Colony counts)
Enumeration was done by adding 0.1 mL of the sample to 0.9 mL saline (0.9% w/v) and a dilution series was carried out. Then 0.1 mL of the dilutions was plated on TSA with plates incubated at 37°C/24 h. Samples were taken for plating every 3 days for the higher inoculum 1x107 CFU/mL and every 2 days for the lower inoculums 1x106 CFU/mL and
1x105 CFU/mL.
44 2.3.7.2 Live counts under fluorescent microscope
Samples were stained with LIVE/DEAD® BacLight kit (L7007) for microscope by adding
3µl of the mixed stains (SYTO 9 and PI in equal amounts) to 1 mL of the sample in a tube according to the manufacturer. Five fields were examined under Olympus Optical fluorescent microscope (Olympus Optical, MA, USA) using a regular slide and by using a filter set with an excitation wavelength of 460-490 nm.
2.3.7.3 High Pressure-Treatment
For all the high-pressure treatments, enumeration was done by adding 0.1 mL of the sample to 0.9 mL sterile saline (0.9% w/v) and a dilution series was carried out. Then 0.1 mL of the dilutions was inoculated into TSA and incubated at 37°C/24 h. The same enumeration method was done after 72 h for the post recovery of cells.
2.3.8 Experimental Design and Statistics
Experiments were performed in triplicates, and the number of colonies was converted to log10. The data obtained were analyzed using two-way ANOVA and Tukey test of the
SPSS statistical software for Mac (IBM SPSS statistics 21).
45 2.4 Results:
2.4.1 Growth limits of STEC under osmotic stress (Gradient Plate Technique):
STEC strains varied in their growth limits on the gradient plate ranging from 2.4 - 6%
NaCl w/v. The limit of NaCl to inhibit the growth of O121: H19 was 6% and it was the highest limit. The limits of NaCl to inhibit the growth of O111: NM, O1O3: H2, O157:
H7, and O45: H2 were 4.8%, 4.7%, 3.2%, and 2.8% respectively. The limit of NaCl to inhibit the growth of O26: H11 was 2.4% and it was the lowest limit.
2.4.2 Persistence of the STEC Strains under osmotic stress (Plate counts and viability staining):
The plate count revealed that STEC O157 and non-O157 strains were significantly variable (p<0.05) in their persistence (die-off) in peptone water with 10% NaCl compared to each other (Tables 2.1 A, 2.2 B, 2. 3 C). The results show that the population for all
STEC strains decreased progressively and significantly with time (p<0.05) with the different inoculums used (Tables 2.1 A, 2.2 B, 2. 3 C). We used the Ct value, which is the time at a given salt level, required to achieve a 2-log reduction. STEC O157: H7 was the most tolerant with a Ct2 value of 18 days and then decreased to the level of detection
(<0.69 log CFU/mL) by day 36. In contrast, STEC O45: H2 was the most sensitive with a
Ct2 value of 9 days and then decreased to the level of detection (<0.69 log CFU/mL) by day 27. There was significant difference (p<0.05) between the STEC strains at the time when they decreased to the level of detection (0.69 log CFU/mL) with the lower inoculums 105 and 104 log CFU/ mL. For the higher inoculum 107 log CFU/ mL, there was a significant difference between STEC O157: H7 and O121: H19 (p<0.05) compared with the other STEC strains at the time when they decreased to the level of detection
(0.69 log CFU/mL). For example, it was found that STEC O157:H7 and STEC O121:
46 H19 were culturable after 33 days of incubation under osmotic stress, while the other strains O1O3: H2, O111: NM, O45:H2, and O26:H11 decreased to the level of detection within 27-30 days (Tables 2.1 A, 2.2 B, 2. 3 C).
It was found that incubation of plates at 22°C increased recovery of cells in some STEC strains significantly (p<0.05) compared to when plates incubated at 37°C. On the other hand, incubation of plates at 22°C decreased recovery in other STEC strains significantly
(P<0.05) compared to when plates incubated at 37°C. For example, the Ct value to get a
2-log reduction (Ct2) for STEC O121: H19 increased to 21 days when incubating plates at
22°C compared with 10 days when incubated at 37°C. On the other hand the Ct value to get a 2-log reduction (Ct2) for STEC O157: H7 decreased to 12 days when incubating plates at 22°C compared to 15 days when incubated at 37°C (Tables 2.1 A, 2.2 B, 2. 3 C).
The fluorescent microscope observation found that when the STEC strains were exposed to osmotic stress in peptone water with 10%NaCl, the population of viable cells for STEC O157: H7 and all non-O157 had significant decrease after 3 days (p<0.05) then stabilized for up to 24 days. When comparing the plate counts for the STEC strains with the number of viable cells counted under epifluorescent microscopy, the results show that the plate counts for all STEC had a progressive decrease till they reached the level of detection, while the microscopic counts (viable counts) for all STEC had significant decline after 3 days and stabilized with time thereafter (figures 2.1, 2.2, 2.3, 2.4, 2.5, 2.6).
In addition, there was a significant difference (P<0.05) between the viability staining compared to the plate counts at the end point (24 days) for all the STEC strains. The results indicate that during osmotic stress STEC lose their culturability, yet they are still viable.
47 It was found that after the extended exposure to osmotic stress (50 days), STEC O157 and all non-O157 decreased to the level of detection (<0.69 log CFU/mL), and none of the cells were able to recover after a 2-h recovery step in TSB. However, the cells were able to recover after a 2- h recovery step in TSB with 5% glycerol due to the presence of glycerol that enhanced recovery of the dormant non-culturable cells. This finding shows that the culturability of cells was dependent on the imposition of a recovery step in glycerol that in turn would have impacted on the perceived tolerance to NaCl.
2.4.3 High-Pressure inactivation (barotolerance):
When exposing STEC strains to pressure higher than 300 MPa (400-600 MPa), the pressure inactivation of STEC was significantly increased (p<0.05) compared to when pressure 300 MPa was used. In addition, variation was observed in pressure resistance between STEC strains. A significant difference in barotolerance (p<0.05) was observed between STEC strains at pressures 400 and 500 MPa. When the cell density was 107 log
CFU/mL at pressure 400 MPa (3 min), 1.4 log CFU/ mL survivals was detected by plate counts for STEC O1O3: H2, while the other strains decreased to the level of detection
(<0.69 log CFU/mL) (Figure 2.7). At pressure 500 MPa/ 3min, STEC O157: H7 and
O121: H19 had a surviving population of 1.9 and 1.5 log CFU respectively, while the other four strains decreased to the level of detection (<0.69 log CFU/mL) (Figure 2.7).
After exposure of STEC strains to pressure 600 MPa/ 3min, the population decreased to the level of detection (<0.69 CFU/mL) (Figure 2.7). This shows the increase in the sensitivity of STEC when exposed to pressures higher than 300 MPa (400-600). It also shows the variation in resisting pressure between STEC strains when exposed to pressure higher than 300 MPa. However, after 72 h of the pressure treatment 400- 600 MPa, all
48 STEC cells reached the level of detection (<0.69 log CFU/mL) due to the irreversible damage that led to cell death. In addition, no viable cells were observed under fluorescent microscope after pressure treatment 400-600 MPa. This would suggest that high pressure causes loss of cellular viability and not only culturability.
Exposure of STEC to 300 MPa (1-3 min) caused a lower reduction in the population of cells as the plate counts did not reach the level of detection (>0.69 log CFU/mL), and variation in pressure resistance between the strains was observed. There was significant log reduction (p<0.05) in the population of STEC compared to time (0 min) after exposure to pressure 300 MPa. STEC O45: H2, O1O3: H2, and O157: H7 had lower log reductions ranged from 1.8-2.1 log CFU. STEC O111: NM, O26: H11, and O121: H19 had higher log reductions ranged from 3.3- 3.6 log CFU, and there was significant difference compared to the strains with lower log reductions (p<0.05) (Figure 2.8.). It was found that the viable cell counts and the plate counts for STEC were not significantly different (P>0.05) at pressure 300 MPa suggesting that the barotolerance is not related to the non-culturable (dormant) cells. Table 2.4.
Exposure of STEC strains to pressure 400 MPa (1-3 min) resulted in significant log reduction (p<0.05) in the population compared to pressure 300 MPa and time 0 min as the population decreased to the level of detection (<0.69 log CFU/mL) (Table 2.5). In addition, no viable cells were observed by fluorescent microscope, which is due to the irreversible effect of the high pressure on the bacterial cells that caused cell death.
2.4.4 High-pressure tolerance and STEC osmotic growth limits in NaCl:
The results revealed a major finding in the research as an inverse relation was found between the barotolerance of STEC strains and their growth limits in the presence of
49 NaCl. The exception was with E. coli O157: H7 that was pressure and salt sensitive. The pressure treatment at 300 MPa resulted in a non-significant reduction (p>0.05) in bacterial counts of STEC O45: H2, and O26: H11 compared to the other strains with no significant difference between them (p>0.05) (figure 2.8). On the other hand, the pressure treatment at 300 MPa (1-3 min) resulted in significant reduction in bacterial counts of
STEC O121: H19, O1O3: H2, O111: NM, and O157: H7 (p<0.05) compared to STEC
O45: H2 and O26: H11. Based on the log reductions after the pressure treatment, STEC
O26: H11 and O45: H2 were found to be pressure tolerant having lower log reductions
(1.46±0.30 and 1.66±0.28) with no significant differences between them (p>0.05). Then comes STEC O111: NM, O1O3: H2, O121: H19, and O157: H7 respectively (figure2.8).
The log reduction for the pressure sensitive strains (O111: NM, O1O3: H2, O121: H19, and O157: H7) was ranged from 3.53±0.23 to 5.36±0.32 log. When comparing the growth limits for the STEC strains in the presence of NaCl, it shows that STEC O121:
H19 had the highest growth limit being able to grow up to 6% NaCl, while under high- pressure treatment O121: H19 was ranked as one of the lowest pressure tolerance having high log reduction (4.70±0.17 log CFU) (Figure 2.9). In addition, STEC O26: H11 had the lowest growth limit being able to grow only up to 2.4% NaCl, while under high- pressure treatment (300 MPa) it was ranked as the highest pressure tolerance having the lowest log reduction (1.46±0.30 log CFU) (Figure 2.9). Moreover, STEC O45: H2 was ranked as second highest-pressure tolerance having low log reduction (1.66±0.28 log
CFU), while under osmotic stress it had one of the lowest growth limits under osmotic stress. The same finding applies with STEC O111: NM, and O1O3: H2 having higher log
50 reductions after the pressure treatment (3.53±0.23, and 4.10±0.26), while being able to grow on higher salt concentrations (4.8, and 4.7%) (Figure 2.9).
2.4.5 Underlined mechanisms
2.4.5.1 Accumulation of osmolytes 2.4.5.1.1 The dry weight of STEC
The plate counts showed that there was no significant difference (p>0.05) in the dry weight of STEC cells when incubated in 10% NaCl with peptone compared to when incubated with no peptone. When comparing the dry weight values for the STEC strains incubated in the 10% NaCl solution with peptone to that incubated with no peptone, it shows that the dry weight was non-significantly higher in the absence of peptone Table
2.7.
2.4.5.1.2 The effect of amino acids (osmolytes) on the barotolerance of STEC
The addition of 1% w/v of the amino acids (glycine, betaine, or asparagine) in 10% NaCl resulted in non-significant log reduction (p>0.05) in the population of STEC cells
(0.56±0.30 -1.23±0.26 log CFU) (at 300 MPa) compared to the control containing no amino acids (Table 2.9). In addition, there was no significant difference (P>0.05) in the log reduction between the STEC strains, and there was no significant variation (p>0.05) in the log reduction with respect to the different amino acids used. In the absence of the amino acids, exposure to 300 MPa pressure resulted in significant log reduction (p<0.05) in the population of STEC in the range of 1.70±0.14 -3.00±0.20 log CFU (Table 2.9).
This shows that STEC becomes pressure sensitive in the absence of osmolytes. After 72 h post-pressure treatment, the plate counts decreased compared to the counts after 24 h of
51 the pressure treatment. However, the counts of survivors in the amino acid solutions were still significantly higher compared to the cells suspended with no amino acids (control)
(Table 2.9). The results show that the addition of osmolytes to the media increases barotolerance of STEC, which could be related to the accumulation of osmolytes.
2.4.5.2 Metabolic precursors 2.4.5.2.1 The effect non-osmolytes (supplements) on the barotolerance of STEC
The addition of precursors that could be fed into the main metabolic pathway via anaplerotic reactions to eventually form osmolytes. The addition of 1% w/v of the supplements (alanine, pyruvate, tryptophan, or glycine) in 10% NaCl resulted in no significant log reduction (p>0.05) in the population of STEC (0.36±0.24 -1.37±0.20 log
CFU) (at 300 MPa) (Table 2.10) compared to the population at time 0 min. In addition, there was no significant difference (p>0.05) in the log reduction between the STEC strains, and there was non-significant difference (p>0.05) in the log reductions with respect to the different supplements used. This shows that the presence of non-osmolytes in 10% NaCl also gives STEC resistance to the high-pressure treatment.
After 72 h post- pressure treatment, the plate counts decreased compared to the counts after 24 h suggesting an increased number of cells were losing viability with time (Table
2.10). In addition, after 72 h of the pressure treatment, there was a significant difference
(p<0.05) in the log reductions in the samples containing supplements compared to the control (Table 2.10). The results show that non-osmolytes can elicit a protective effect against moderate pressure treatments.
52 2.4.5.2.2 Effect of peptone (non-osmolyte) on the barotolerance of STEC strains:
The presence of 1% peptone in 10% NaCl provided protection to STEC against pressure treatment. When adding 1% w/v peptone in 10% NaCl (at 300 MPa), the number of
STEC surviving cells was significantly higher (p<0.05) compared to when no peptone was added (control). After exposing STEC strains to pressure 300 MPa in the presence of peptone, the log reductions for the strains ranged from 0.80±0.30 to 0.96±0.20 log CFU, while in the absence of peptone the log reductions for STEC strains ranged from
2.06±0.31 to 3.36±0.20 log CFU (Table 2.8). In addition, there was no significant difference (p>0.05) in the pressure assisted log reduction between the STEC strains in the presence of 1% peptone. In the absence of peptone, there was a significant difference
(p>0.05) in the log reduction between the STEC strains having lower log reductions
(O1O3: H2, O45: H2, O157: H7) compared to the STEC strains having higher log reductions (O121: H19, O111: NM, O26: H11).
At 600 MPa/ 3min, the addition of 1% w/v peptone also provided protection to the STEC strains compared to when no peptones was added. When adding 1% peptone in 10%
NaCl (at 600 MPa/ 3min), there was a significant decrease in the number of cells
(p<0.05), compared to the counts of non-treated controls. The number of cells decreased to the range 2.3±0.15 to 3.9±0.15 log CFU/mL when the inoculum was in the range of
6.93±0.11 to 7.10±0.10 log CFU/ mL (Figure 2.10). However, in the absence of peptone
(at 600 MPa/ 3min), the STEC population decreased below the level of detection (<0.69 log CFU/mL) (figure 2.10). After 72 h of the pressure treatment (at 600 MPa), the plate counts decreased compared to the counts after 24 h of the pressure treatment. This is due to the irreversible damage caused by the pressure 600 MPa (Figure 2.11).
53 2.4.5.2.3 The Effect of Fermented and non-fermented sugars on the barotolerance of STEC
The substrate utilization profile test showed that all STEC non O157 strains O121: H19,
O1O3: H2, O111: NM, O45: H2, and O26: H11 had the ability to ferment sorbitol
(sorbitol positive), while STEC O157: H7 did not have the ability to ferment sorbitol
(sorbitol negative). Regardless of the ability of the STEC strain to ferment the sugar or not, the addition of 1% w/v starch or sorbitol increased barotolerance of the STEC strains against high pressure compared to the control that did not have any sugars. The addition of sorbitol and starch increased the barotolerance of STEC O157: H7 and O121: H19 significantly (p<0.05) compared to the control (10% NaCl solution containing no supplements). The addition of 1% w/v sugars (ferment or non fermented) in 10% NaCl resulted in a non-significant (p>0.05) log reduction in the population of STEC O157: H7 and O121: H19 (0.65±0.19-0.75±0.20 log CFU) (at 300 MPa) (Table 2.11). Exposing
STEC O157: H7 and O121: H19 to pressure 300 MPa in the absence of sugars (control) resulted in a significant log reduction (p<0.05) in the population of STEC in the range
1.91±0.36-1.93±0.32 log CFU (Table 2.11). In addition, there was a non-significant difference (p>0.05) in the log reduction between the STEC strains and in the effect of starch or sorbitol on the STEC strains. Even though, both STEC O157: H7 and O121:
H19 did not have the ability to metabolize starch, it provided protection to the STEC cells against high-pressure treatment. In addition, there was non-significant difference in the protection provided by sorbitol to both STEC O157: H7 and O121: H19 although STEC
O157:H7 did not have the ability to metabolize sorbitol. This might suggest the barotolerance of STEC is not related to the ability of the STEC strain to metabolize the substrates found in the media.
54 2.4.6 The effect of Different water activity levels on the barotolerance of STEC in the presence of 1% glycine
Under 600 MPa HHP treatment, glycine did not contribute to the barotolerance of STEC
O157: H7. There was a significant variation (p<0.05) in the level of STEC inactivation between the aw solutions of 0.85 to 0.98 both with or without glycine. It was evident that the log reduction was dependent on the water activity of the matrix. The barotolerance of
STEC O157: H7 increased significantly (p<0.05) with lower water activity solutions.
Treatment of low aw solutions (0.85, 0.94, and 0.98) with pressure 600 MPa in the presence of glycine resulted in 1.62±0.15, 4.69±0.34 and 6.65±0 log reduction, respectively. In addition, treatment of the control samples (aw 0.95) having no glycine with pressure 600 MPa resulted in 6.31±0.57 log CFU reduction (Figure 2.12).
It was expected that STEC O157: H7 treated with pressure 400 MPa would follow the same relation as 600 MPa, in which the level of reduction would be dependent on the aw.
However, the presence of glycine in the different aw solutions (0.85-0.98 aw) resulted in a non-significant variation (p>0.05) in the log reduction of STEC O157: H7 between the aw solutions suggesting there is a protective effect of glycine. The log reductions for STEC
O157: H7 in the aw solution with glycine ranged from 0.77±0.09-1.61±0.20 log reductions after exposing to pressure 400 MPa (Figure 2.13). On the other hand, the absence of glycine in 0.95 aw (control) resulted in a significant log reduction (p<0.05) compared to the high aw solutions having glycine. The log reduction for STEC O157:H7 in the control sample having no glycine (0.95aw) was 2.88±0.30 logs after exposing to pressure 400 MPa (Figure 2.13).
55 After 72 h of the pressure treatments 400 MPa and 600 MPa, the plate counts decreased compared to the counts after 24 h of the pressure treatment due to the irreversible effect of high pressure 400 and 600 MPa (Tables 2.12 and 2.13).
56 Table 2.1 A: Persistence of STEC O157: H7 and O121: H19 under osmotic stress with the 3 different inoculation levels and with incubation of enumeration plates at 37°C and 22°C
STEC Time Average Log Temp STEC Time Average Log Temp (Day) CFU/mL (Day) CFU/mL O157:H7 0 7.06 ±0.15 37°C O121:H19 0 7.00± 00.01 37°C 3 6.52± 0.20aA 3 6.30± 0.20aB 6 6.23± 0.15bA 6 6.03± 0.15bB 9 5.93± 0.25cA 9 5.70± 0.10cB 12 5.63± 0.25dA 12 5.33± 0.15dB 15 5.30± 0.20eA 15 5.13± 0.32eB 18 5.17± 0.25fA 18 4.97± 0.31fB 21 4.63± 0.15gA 21 4.47± 0.26gB 24 3.43± 0.15hA 24 3.40± 0.10hB 27 2.53± 0.35iA 27 2.33± 0.15iB 30 2.30± 0.20jA 30 2.10± 0.20jB 33 1.27± 0.33kA 33 1.37± 0.31kB 36 0.69± 0.00a 36 0.69± 0.00a 38 0.69± 0.00a 38 0.69± 0.00a
O157:H7 0 5.07± 0.06a 37°C O121:H19 0 5.09 ± 0.12a 37°C 3 4.73± 0.15bA 3 3.70 ± 0.10bB 6 4.67± 0.21cA 6 3.43 ± 0.15bB 9 3.43± 0.11dA 9 2.67 ± 0.15dB 12 2.43± 0.15eA 12 1.47 ± 0.12eB 15 2.10± 0.10fA 15 0.69 ± 0.00aB 18 1.70± 0.20aA 18 0.69 ± 0.00aB
20 1.23± 0.25a 22 0.69± 0.00a 24 0.69 ±0.00a
O157:H7 0 4.06±0.06a 37°C O121:H19 0 4.14 ± 0.11a 37°C 2 3.84 ± 0.10aA 2 3.61 ± 0.22aB 4 3.63± 0.15bA 4 2.96 ± 0.15bB 6 3.33 ± 0.15cA 6 2.72 ± 0.11cB 8 2.47± 0.25dA 8 2.57 ± 0.15dB 10 2.03 ± 0.15eA 10 1.73 ± 0.25eB 12 1.03± 0.35fA 12 0.69 ± 0.00aB 14 0.69± 0.00fA 14 0.69 ± 0.00aA
O157:H7 0 5.11 ± 0.10a 22°C O121:H19 0 5.11 ± 0.05a 22°C 3 4.07 ± 0.15bA 3 4.47 ± 0.15bB 6 2.69 ± 0.34cA 6 3.70 ± 0.15cB 9 2.48 ± 0.23dA 9 3.52 ± 0.16dB 12 2.34 ± 0.32eA 12 3.20 ± 0.25eB 15 1.31 ± 0.27fA 15 3.01 ± 0.26fB 18 0.69 ± 0.00aA 18 2.44 ± 0.25gB 21 0.69 ± 0.00aA 21 2.13 ± 0.15hB
24 1.86 ± 0.17i 27 1.52 ± 0.16j 30 1.22 ± 0.15a 33 0.69 ± 0.00a
±Value means Standard Deviation for 3 samples, CFU= Colony Forming Unit. Means followed by same small letter in columns and same capital letter in rows are non significantly different (p>.05). Means followed by different small letters in columns and different capital letters in rows are significantly different (p<0.05).
57
Table 2.2 B: Persistence of STEC O1O3: H2 and O111: NM under osmotic stress with the 3 different inoculation levels and with incubation of enumeration plates at 37°C and 22°C.
STEC Time Average Log Temp STEC Time Average Log Temp (Day) CFU/mL (Day) CFU/mL
O1O3:H2 0 7.17± 0.15 37°C O111:NM aA 0 6.97 ± 0.12 37°C 3 6.03± 0.25 aB bA 3 6.13 ± 0.15 6 5.73±0.15 bB cA 6 5.67 ± 0.21 9 5.23±0.25 cB dA 9 5.27 ± 0.15 12 5.07±0.21 dB eA 12 5.03 ± 0.15 15 4.80±0.10 eB fA 15 4.88 ± 0.25 18 4.00±0.30 fB gA 18 4.07 ± 0.15 21 2.90± 0.26 gB hA 21 3.57 ± 0.15 24 1.91±0.20 hB iA 24 2.33 ± 0.25 27 1.06± 0.35 iB a 27 1.23 ± 0.32 30 0.69± 0.00 a a 30 0.69 ± 0.00 33 0.69± 0.00 a 33 0.69 ± 0.00
O1O3:H2 a 37°C O111:NM a 0 5.07 ± 0.06 0 4.93 ± 0.21 37°C 3 3.60± 0.10bA 3 3.81 ± 0.20bA 6 3.33± 0.15cA 6 3.40 ± 0.10cA 9 2.50± 0.20dA 9 2.55 ± 0.20dA 12 1.83 ± 0.15eA 12 1.37 ± 0.25eA 15 1.31 ± 0.12fA 15 0.69 ± 0.00aA 0.69 ± 0.00aA 18 0.69 ± 0.00aA 18 0.69 ± 0.00a 20
O1O3:H2 a 37°C O111:NM 0 4.09 ± 0.15 0 4.10 ±0.10a 37°C 2 3.56 ± 0.16bA 2 3.29 ± 0.19bB 4 3.23 ± 0.18cA 4 2.83 ± 0.25cB 6 2.91 ± 0.10dA 6 2.35 ± 0.15dB 8 2.47 ±0.14eA 8 1.29 ± 0.25eB 10 1.84 ± 0.15fA 10 0.69 ± 0.00aB 12 0.69 ± 0.00aA 12 aA 0.69 ± 0.00
14 0.69 ± 0.00a a a O1O3:H2 0 5.06 ± 0.13 22°C O111:NM 0 5.04 ± 0.11 22°C 3 4.36 ± 0.18bA 3 4.27 ± 0.15bA 6 3.62 ± 0.17cA 6 3.72 ± 0.12cA 9 3.34 ± 0.20dA 9 3.46 ± 0.18dA 12 3.17 ± 0.25eA 12 3.18 ± 0.20eA 15 2.74 ± 0.21fA 15 2.97 ± 0.20fA 18 2.39 ± 0.22gA 18 2.72± 0.19gA 21 2.14 ± 0.19hA 21 2.27 ± 0.18hA 24 1.79 ± 0.16iA 24 2.03 ± 0.19iA 27 1.47 ± 0.16jA 27 1.34 ± 0.21jA 30 1.22 ± 0.21kA 30 0.69 ± 0.00aA 33 0.69 ± 0.00lA 33 0.69 ± 0.00aA
±Value means Standard Deviation for 3 samples, CFU= Colony Forming Unit. Means followed by same small letter in columns and same capital letter in rows are non significantly different (p>.05). Means followed by different small letters in columns and different capital letters in rows are significantly different (p<0.05).
58 Table 2. 3 C: Persistence of STEC O45:H2 and O26:H11 under osmotic stress with the 3 different inoculation levels and with incubation of enumeration plates at 37°C and 22°C. STEC Time Average Log Temp STEC Time Average Log Temp (Day) CFU/mL (Day) CFU/mL
O45:H2 0 7.10± 0.17 37°C O26:H11 0 6.97±0.11 37°C 3 6.03± 0.25aA 3 6.13±0.25aB 6 5.63± 0.21bA 6 5.67±0.21bB 9 5.07± 0.25cA 9 5.47±0.25cB 12 4.63± 0.25dA 12 4.88±0.25dB 15 4.30± 0.30eA 15 3.67±0.15eB 18 3.23± 0.25fA 18 2.90±0.15fB 21 2.37± 0.15gA 21 2.22±0.25gB 24 1.70± 0.10hA 24 1.81±0.15hB 27 0.69± 0.00aA 27 1.20±0.32iB 30 0.69± 0.00a 30 0.69±0.00a 33 0.69± 0.00a 33 0.69±0.00a
a a O45:H2 0 5.10± 0.10 37°C O26:H11 0 5.03 ± 0.05 37°C 3 4.41 ± 0.10aA 3 4.01 ± 0.20aB 6 3.70 ± 0.12bA 6 3.40 ± 0.10bB 9 2.73 ± 0.15cA 9 2.43 ± 0.15cB 12 1.92 ± 0.20dA 12 1.62 ± 0.26dB 15 1.10 ± 0.10aA 15 0.69 ± 0.00aA 18 0.69 ± 0.00aA 18 0.69 ± 0.00aA
20 0.69 ± 0.00a a O45:H2 0 4.09± 0.10a O26:H11 0 4.13 ± 0.10 37°C bA 37°C 2 2.91 ± 0.15bA 2 3.24 ± 0.14 cA 4 2.54 ± 0.15cA 4 2.75 ± 0.25 dA 6 2.07± 0.15dA 6 2.27 ± 0.51 eA 8 1.68 ± 0.20eA 8 1.27 ± 0.25 aA 10 0.69 ± 0.00aA 10 0.69 ± 0.00 12 aA 12 0.69 ± 0.00aA 0.69 ± 0.00
O45:H2 0 5.10 ± 0.10 22°C O26:H11 0 5.10 ± 0.11 22°C 3 4.14 ± 0.24aA 3 4.00± 0.28aB 6 2.61 ± 0.20bA 6 3.55 ± 0.21bB 9 2.26 ± 0.26cA 9 3.19 ± 0.24cB 12 1.78 ± 0.16dA 12 2.98 ± 0.37dB 15 1.57 ± 0.20eA 15 2.75 ± 0.31eB 18 1.19 ± 0.22fA 18 2.34 ± 0.30fB 21 0.69 ± 0.00aA 21 2.09 ± 0.34gB 24 0.69 ± 0.00aA 24 1.46 ± 0.32hB
27 0.69 ± 0.00a 30 0.69 ± 0.00a
Value means Standard Deviation for 3 samples, CFU= Colony Forming Unit. Means followed by same small letter in columns and same capital letter in rows are non significantly different (p>.05). Means followed by different small letters in columns and different capital letters in rows are significantly different (p<0.05).
59 Log CFU/mL O1O3 Log Cell/mL O1O3
8
7
6
5
4
3
2 Log CFU/mL-Log cell/mL 1
0 0 5 10 15 20 25 30 35 Days
Figure 2.1 The culturable counts (log CFU/mL) compared to the viable counts by viability staining (microscopic, Log cell/ mL) in peptone water with 10% NaCl for STEC O1O3:H2 left for 24-36 days. Samples were taken and dilutions plated on TSA for log CFU/mL. samples were taken and stained with LIVE/DEAD® BacLight for viable counts.
Log CFU/mL 0121 Log Cell/mL O121
8 7 6 5 4 3 2 Log cfu/ml- Log cell/ml 1 0 0 5 10 15 20 25 30 35 40 Days
Figure 2.2 The culturable counts log CFU/mL compared to the viable counts by viability staining (microscopic) Log cell/ mL in peptone water with 10% NaCl for STEC O121:H19 left for 24-36 days. Samples were taken and dilutions plated on TSA for log CFU/mL, samples were taken and stained with LIVE/DEAD® BacLight for viable counts.
60 Series1 Series2
8 7 6 5 4 3 2 Log cfu/ml- Log cell/ml 1 0 0 5 10 15 20 25 30 35 40 Days
Figure 2.3 The culturable counts log CFU/mL compared to the viable counts by viability staining (microscopic) Log cell/ mL in peptone water with 10% NaCl for STEC O157:H7 left for 24-36 days. Samples were taken and dilutions plated on TSA for log CFU/mL, samples were taken and stained with LIVE/DEAD® BacLight for viable counts.
LogCFU/mL O111 Log Cell/mL O111
8 7 6 5 4 3 2 Log cfu/ml-Log cell/ml 1 0 0 5 10 15 20 25 30 35 Days
Figure 2.4 The culturable counts log CFU/mL compared to the viable counts by viability staining (microscopic) Log cell/ mL in peptone water with 10% NaCl for STEC O111:NM left for 24-36 days. Samples were taken and dilutions plated on TSA for log CFU/mL, samples were taken and stained with LIVE/DEAD® BacLight for viable counts.
61 Log CFU/mL O45 Log Cell/mL O45
8 7 6 5 4 3 2 Log cfu/ml- Log cell/ml 1 0 0 5 10 15 20 25 30 35 Days
Figure 2.5 The culturable counts log CFU/mL compared to the viable counts by viability staining (microscopic) Log cell/ mL in peptone water with 10% NaCl for STEC O45:H2 left for 24-36 days. Samples were taken and dilutions plated on TSA for log CFU/mL, samples were taken and stained with LIVE/DEAD® BacLight for viable counts.
Log CFU/mL O26 Log Cell/mL O26
8
7
6
5
4
3
2 Log cfu/ml- Log cell/ml 1
0 0 5 10 15 20 25 30 35 Days
Figure 2.6 The culturable counts log CFU/mL compared to the viable counts by viability staining (microscopic) Log cell/ mL in peptone water with 10% NaCl for STEC O26:H11 left for 24-36 days. Samples were taken and dilutions plated on TSA for log CFU/mL, samples were taken and stained with LIVE/DEAD® BacLight for viable counts.
62
400 Mpa 500 Mpa 600 Mpa
7 Inoculum 6 5
4
3 Log CFU/mL 2
1 Detection Limit 0 O157 O121 O1O3 O111 O45 O26 STEC
Figure 2.7 The effect of pressure 400-600 MPa on STEC counts after 24 h of pressure the treatment showing the variation in pressure resistance between STEC.
4
3.5
3
2.5
2
1.5 Log Reduction
1
0.5
0 O45 O1O3 O157 O111 O26 O121 STEC
Figure 2.8 The log reduction in STEC counts after pressure treatment 300 MPa/ 3 min. The log reductions are from lowest to highest log reduction.
63 Table 2.4 STEC culturable counts and microscopic counts (live counts) at pressure 300 MPa (1- 3 min) after 24 h and 72 h of the pressure treatment
STEC Log CFU/mL Log reduction Log reduction Log reduction (0 min) (3 min)/ 24 h (3 min)/ 72 h (3 min) Cell/ mL O157: H7 7.06±0.11 2.10±0.36A 5.56±0.30 B 1.67±0.36A O121: H19 7.10±0.10 3.20±0.25A 4.60±0.26 B 1.85±0.36A O1O3: H2 7.10±0.10 2.06±0.31A 3.73±0.35 B 1.80±0.36A O111: NM 7.00±0.10 2.10±0.20A 6.07±0.20 B 1.95±0.36A O45: H2 6.93±0.11 1.93±0.19A 4.46±0.15 B 1.96±0.36A O26: H11 7.06±0.11 2.80±0.20A 5.90±0.20 B 1.98±0.36A
± Value means standard Deviation for 3 samples, CFU= Colony Forming Unit. Means followed by same capital letter in rows are non significantly different (p>0.05). Means followed by different capital letters in rows are significantly different (p<0.05).
Table 2.5 STEC counts after pressure treatment 400 MPa (1-3min) after 24 h and 72 h of the pressure treatment
STEC Log CFU/mL Log reduction Log reduction (0 min) (3 min)/ 24 h (3 min)/ 72 h B O157:H7 7.06±0.11 6.37±0.00 aA 6.37±0.00 aA B O121:H19 7.10±0.10 6.41±0.00 aA 6.41±0.00 aA B O1O3:H2 7.10±0.14 6.41±0.00 aA 6.41±0.00 aA B O111:NM 7.00±0.10 6.31±0.00 aA 6.31±0.00 aA B O45:H2 6.93±0.05 6.37±0.00 aA 6.37±0.00 aA B O26:H11 7.06±0.14 6.37±0.00 aA 6.37±0.00 aA
±Value means Standard Deviation for 3 samples, CFU= Colony Forming Unit. Means followed by same small letter in columns and same capital letter in rows are non significantly different (p>0.05). Means followed by different capital letter in rows are significantly different (p< 0.05).
64 Table 2.6 The effect of pressure 300 MPa (Osmotic growth limit) (1-3 min) after 24 h and 72 h of pressure treatment
STEC Log CFU/mL Log reduction Log reduction (0 min) (3 min)/ 24 h (3 min)/ 72 h O157: H7 7.03±0.15 5.36±0.32 a A 5.80±0.25 a A O121: H19 7.06±0.11 4.70±0.17 a A 5.13±0.20 a A O1O3: H2 7.10±0.10 4.10±0.26 a A 4.30±0.28 a A O111: NM 6.96±0.10 3.53±0.23 c A 4.83±0.20 a A O45: H2 7.00±0.05 1.66±0.28 b A 2.46±0.20 b A O26: H11 6.96±0.11 1.46±0.30 b A 2.36±0.25 b A
±Value means Standard Deviation for 3 samples, CFU= Colony Forming Unit. Means followed by the same small letter in columns and same capital letter in rows are non significantly different (p>0.05). Means followed by different small letters in columns are significantly different (p<0.05).
HHP 300 MPa Growth limit
6 7
5 6
5 4 4 3
3 % NaCl
Log Reduction 2 2
1 1
0 0 O26 O45 O111 O1O3 O121 O157 STEC
Figure 2.9 The relation between barotolerance of STEC at 300 MPa/ 3 min and their growth limits on 10% NaCl gradient plate. Comparing the log reductions of STEC at pressure 300 MPa/ 3 min with their growth limits on the 10% NaCl gradient plate. The log reductions were rated from lower log reductions to higher log reductions.
65 Table 2.7 The dry weight of STEC strains after incubating in 10% NaCl solution with 1% peptone and with no peptone added. Strain W (g)/ Control Plate Average (Peptone) 72 h/60°C W (g) W (g) O157:H7 2.4405a 2.0860 0.3545±0.02a O121:H19 2.5052a 2.0881 0.4171±0.04 a O1O3:H2 2.4628a 2.0889 0.3739±0.01 a O111:NM 2.4755a 2.0866 0.3889±0.04 a O45:H2 2.4986a 2.0882 0.4104±0.06 a O26:H11 2.4508a 2.0888 0.3620±0.05 a (No peptone) O157:H7 2.4977a 2.0952 0.4025±0.07 a O121:H19 2.5179a 2.0872 0.4301±0.05 a O1O3:H2 2.5009a 2.0868 0.4141±0.02 a O111:NM 2.5104a 2.0919 0.4185±0.03 a O45:H2 2.5766a 2.0946 0.4820 ±0.05 a O26:H11 2.4464a 2.0900 0.3564 ±0.02 a
±Value means Standard Deviation for 3 samples. Means followed by same small letter in columns are non significantly different (p<0.05).
66 Table 2.8 The effect of 1% Peptone on barotolerance of STEC at pressure 300 MPa (1-3 min)
STEC Peptone Log CFU/mL Log Reduction Log Reduction (0 min) (3 min) /24 h (3 min) /72 h O157: H7 Peptone 7.03±0.10 0.96±0.20aA 1.32±0.13aA No peptone 7.06±0.11 2.10±0.36 bA 5.56±0.30bB O121: H19 Peptone 7.10±0.10 0.80±0.30 aA 1.13±0.20aA No peptone 7.10±0.10 3.20±0.25 bA 4.60±0.26bB O1O3: H2 Peptone 7.06±0.11 0.86±0.20 aA 1.21±0.18 aA No peptone 7.10±0.10 2.06±0.31 bA 3.73±0.35 bB O111: NM Peptone 7.03±0.15 0.83±0.26 aA 1.14±0.17 aA No peptone 7.00±0.10 3.30±0.20 bA 6.07±0.20 bB O45: H2 Peptone 7.00±0.10 0.83±0.15 aA 1.13±0.20 aA No peptone 6.93±0.11 1.93±0.19 bA 4.46±0.15bB O26: H11 Peptone 7.10±0.10 0.96±0.32 aA 1.17±0.17aA No peptone 7.06±0.11 3.36±0.20 bA 5.90±0.20bB ±Value means Standard Deviation for 3 samples, CFU= Colony Forming Unit. Means followed by same small letter in columns and same capital letter in rows is non significantly different (p>0.05). Means followed by different small letters in columns and different capital letter in rows are significantly different (p<0.05).
3 min 1% peptone 3 min no peptone (Original)
7 Inoculum
6
5
4
3 Log CF/mL
2
1 Detection Limit
0 O157 O121 O1O3 O111 O45 O26 STEC Strain
Figure 2.10 The effect of peptone on barotolerance of STEC after 24 h of the pressure treatment 600 MPa/ 3min compared to when no peptone was added.
67
1% PEPTONE No peptone
7 Inoculum
6
5
4
3 Log CFU/ mL 2
1 Detection Limit 0 O157 O121 O1O3 O111 O45 O26 STEC
Figure 2.11 The effect of peptone on the barotolerance of STEC after 72 h of the pressure treatment 600 MPa/ 3 min compared to when no peptone was added.
68 Table 2.9 The effect of amino acids (osmolytes) on the barotolerance of STEC after 24 h and 72 h of pressure treatment 300 MPa/ 1-3 min
STEC Amino Acid Log CFU/mL Log Reduction Log Reduction (mM) (0 min) (3 min)/ 24 h (3 min)/ 72 h O157: H7 (0.13) Glycine 7.10±0.14 0.93±0.20 aA 1.76±0.64aA (0.08) Betaine 0.93±0.15 aA 1.43±0.30 aA (0.07) Asparagine 1.10±0.35 aA 1.96±0.35 aA No Amino acid 2.20±0.25 bA 3.00±0.30 bB O121: H19 (0.13) Glycine 7.00±0.07 0.76±0.20 aA 1.46±0.30 aA (0.08) Betaine 0.60±0.20 aA 1.16±0.32 aA (0.07) Asparagine 0.70±0.20 aA 1.83±0.36 aA No Amino acid 2.90±0.60 bA 3.80±0.25 bB O1O3: H2 (0.13) Glycine 6.85±0.07 0.95±0.25 aA 2.63±0.36 aA (0.08) Betaine 0.75±0.30 aA 1.60±0.35 aA (0.07) Asparagine 1.23±0.26 aA 2.00±0.25 aA No Amino acid 2.22±0.20 bA 3.31±0.14 bB O111: NM (0.13) Glycine 7.00±0.14 0.80±0.25 aA 1.63±0.49 aA (0.08) Betaine 0.70±0.35 aA 1.43±0.30 aA (0.07) Asparagine 0.70±0.15 aA 1.63±0.30 aA No Amino acid 1.70±0.14 bA 3.23±0.35 bB O45: H2 (0.13) Glycine 6.90±0.14 0.56±0.30 aA 1.43±0.40 aA (0.08) Betaine 0.56±0.25 aA 1.60±0.35 aA (0.07) Asparagine 0.73±0.25 aA 1.83±0.50 aA No Amino acid 2.10±0.25 bA 3.16±0.35 bB O26: H11 (0.13) Glycine 7.00±0.07 0.90±0.26 aA 2.00±0.36 aA (0.08) Betaine 0.73±0.15 aA 1.46±0.35 aA (0.07) Asparagine 0.90±0.12 aA 1.90±0.36 aA No Amino acid 3.00±0.20 bA 3.50±0.50 bB
±Value means Standard Deviation for 3 samples, CFU= Colony Forming Unit. Means followed by same small letter in columns and same capital letter in rows are non significantly different (p<0.05). Means followed by different small letter in columns and different capital letter in rows is significantly different (p<0.05).
69 Table 2.10 The effect of non-osmolytes (supplements) on the barotolerance of STEC after 24 h and 72 h of the pressure treatment 300 MPa (1-3 min)
STEC Supplement Log CFU/mL Log Reduction Log Reduction (mM) (0 min)/ 24 h (3 min)/ 24 h (3 min)/ 72 h O157: H7 (0.11) Alanine 7.10±0.07 1.17±0.32 aA 1.88±0.29 aA (0.11) Pyruvate 1.37±0.20 aA 1.92±0.28 aA (0.04)Tryptophan 1.47±0.35 aA 2.32±0.23 aA (0.13)Glycine (control) 0.70±0.20 aA 1.33±0.23 bA O121:H19 (0.11) Alanine 7.10±0.15 1.00±0.20 aA 1.42±0.39 aA (0.11) Pyruvate 1.13±0.26 aA 1.55±0.22 aA (0.04)Tryptophan 1.20±0.25 aA 1.53±0.25 aA (0.13)Glycine (control) 0.60±0.35 aA 0.92±0.37 bA O1O3:H2 (0.11) Alanine 7.00±0.07 1.20±0.20 aA 1.83±0.25 aA (0.11) Pyruvate 1.16±0.32 aA 2.07±0.11 aA (0.04)Tryptophan 1.23±0.21 aA 1.72±0.25 aA (0.13)Glycine (control) 0.45±0.07 aA 1.03±0.25 bA O111:NM (0.11) Alanine 7.00±0.14 1.00±0.15 aA 1.36±0.34 aA (0.11) Pyruvate 0.90±0.15 aA 1.48±0.33 aA (0.04)Tryptophan 1.26±0.32 aA 1.75±0.32 aA (0.13)Glycine (control) 0.45±0.14 aA 0.97±0.28 bA O45:H2 (0.11) Alanine 7.10±0.14 0.83±0.15 aA 1.23±0.31 aA (0.11) Pyruvate 1.06±0.21 aA 1.55±0.33 aA (0.04)Tryptophan 1.26±0.26 aA 1.44±0.21 aA (0.13)Glycine (control) 0.36±0.24 aA 0.96±0.19 bA O26:H11 (0.11) Alanine 7.00±0.14 0.96±0.20 aA 1.55±0.21 aA (0.11) Pyruvate 0.93±0.30 aA 1.70±0.25 aA (0.04)Tryptophan 0.96±0.30 aA 1.55±0.27 aA (0.13)Glycine (control) 0.49±0.28 aA 0.97±0.21 bA
±Value means Standard Deviation for 3 samples, CFU= Colony Forming Unit. Means followed by same small letter in columns and same capital letter in rows are non significantly different (p>0.05). Means followed by different small letter in columns are significantly different (p<0.05).
70 Table 2.11 The effect of fermented and non- fermented sugars (non osmolytes) on the barotolerance of STEC at pressure 300 MPa/ 1-3 min
STEC Sugar Log CFU/mL Log Reduction Log Reduction (mM) (0 min) (3 min) /24 h (3 min) /72 h aB O157: H7 (0.05) Sorbitol 7.34±0.14 0.74±0.27aA 1.87±0.28 (0.01) Starch aA 1.29±027 aB 0.75±0.20 bB Control 1.91±0.36bA 3.03±0.38 aA O121: H19 (0.05) Sorbitol 7.26±0.23 0.73±0.39aA 0.98±0.35 (0.01) Starch aA 2.28±0.47 bB 0.65±0.19 cB Control 1.93±0.32bA 3.28±0.43
±Value means Standard Deviation for 3 samples, CFU= Colony Forming Unit. Means followed by same small letter in columns and same capital letter in rows are non significantly different (p>0.05). Means followed by different small letter in columns and different capital letter in rows is significantly different (p<0.05).
71 Log CFU/mL O157 0.98 (1%Glycine) Log CFU/mL O157 0.94 (1%Glycine) Lof CFU/mL O157 0.85 (1%Glycine) LogCFU/mL 10 NaCl (Control)
8 7 6 5 4 3 Log cfu/ml 2 1 0 0 1 2 3 4 Time (m)
Figure 2.12 The effect of HHP 400 MPa (1-3 min) on the barotolerance of STEC O157:H7 in the different water activity solutions 0.98 Δ, 0.94 x, and 0.85 ¢ with 1% glycine compared with the control (0.95, no glycine) n.
Log CFU/mL O157 0.98 (1%Glycine) Log CFU/mL O157 0.94 (1%Glycine) Lof CFU/mL O157 0.85 (1%Glycine) Log CFU/mL 10% NaCl (Control)
8
7
6
5
4
Log cfu/ml 3
2
1
0 0 1 2 3 4 Time (m)
Figure 2.13 The effect of HHP 600 MPa (1 -3 min) on the barotolerance of STEC O157:H7 in the different water activity solutions 0.98 Δ, 0.94 x, and 0.85 ¢ with 1% glycine compared with the control (0.95, no glycine) n.
72
Table 2.12 STEC O157: H7 counts after 72 h of the pressure treatment 400 MPa (1- 3 min) in the different water activity solutions with 1% glycine
STEC Log CFU/mL Log Reduction Water activity aw (3 min) /72 h O157:H7 7.34±0.09A 2.19±0.09aB 0.98a 7.34±0.09A 0.95±0.10bA 0.94b 7.34±0.09A 1.12±0.17bA 0.85b 7.34±0.09A 3.76±0.27cB Control (0.95)c ±Value means Standard Deviation for 3 samples, CFU= Colony Forming Unit. Means followed by same small letter in columns and same capital letter in rows are non significantly different (p>0.05). Means followed by different small letter in columns and different capital letter in rows is significantly different (p<0.05).
Table 2.13 STEC counts after 72 h of the pressure treatment 400 MPa (1- 3 min) in the different water activity solutions with 1% glycine STEC Log CFU/mL Log Reduction Water activity aw (3 min) /72 h O157:H7 7.34±0.09A 6.64±0.00aB 0.98a 7.34±0.09A 5.13±0.19bB 0.94b 7.34±0.09A 1.63±0.15cB 0.85c 7.34±0.09A 6.64±0.00dB Control (0.95)d ±Value means Standard Deviation for 3 samples, CFU= Colony Forming Unit. Means followed by different small letter in columns and different capital letters in rows are significantly different (p<0.05).
73
Figure 2.14 HHP profile for samples processed at 400 MPa (1-3 min).
74 2.5 Discussion
The results obtained in the study confirmed the pressure sensitivity of STEC when treatments > 300 MPa were applied. The log reduction of STEC was proportional to the applied pressure with treatments of 600 MPa decreasing populations below the limit of detection (<0.69 log CFU/mL). The results are in agreement with other published works that illustrated that >6 log CFU reductions can be achieved at pressure in the order of 600
MPa (Calvo et al., 2014; Hus et al., 2013; Hiremath and Ramaswamy, 2012). However, the differences in barotolerance between STEC was more evident at lower pressure treatments whereby the intrinsic resistance of strains became more apparent. The results are in agreement with others that have demonstrated inter-strain variation of STEC when relatively lower pressure treatments are applied (Liu et al., 2015; Tamber, 2017). For example, Liu et al., 2015, reported a variation of 1.1 – 5.5 log CFU/mL in pressure resistance of 100 strain of E. coli including VTEC strains when exposing to pressure 600
MPa/ 5 min at 25°C in phosphate buffered saline at pH 7. The workers found that E. coli
O111: NM serotype exhibited the highest barotolerance compared to the other strains tested. Whitney et al., 2007, reported that there was a variation of 0.6 - 4.39 log CFU/mL in resisting pressure between 6 strains of STEC O157: H7 after exposure to pressure 500
MPa (2 min) in tryptic soy broth TSB. The isolates used in the study were isolated from outbreaks associated with fresh produce, beef, and apple juice. The pressure sensitivity could not be attributed to isolation source nor did the researchers put forward any theories on the basis on barotolerance between the different strains.
75 Malone et al., 2007, found that after exposing 17 strains of E.coli O157: H7 to pressure
500 MPa (1min) in Phosphate Buffer Saline (PBS), there was a variation of 0.6- 3.4 log
CFU/mL.
The results from the study performed also highlighted the difference in barotolerance of the different STEC strains tested. The intra strain variation of barotolerance in E. coli has been previously attributed to the food matrix due to the interactions between low pH, low water activity, and food composition with pressure (Gänzle and Liu, 2015). In addition, the intra strain variation in resisting pressure has been attributed to the existence of different stress responses within the same species. For example, some bacterial strains such as E.coli accumulate osmolytes including proline, glycine, and betaine that protect
E.coli from high pressure. On the other hand, other strains induce stress response in response to high pressure (Gänzle and Liu, 2015; Liu et al., 2015). Also, the intra strain variation of barotolerance has been attributed to the change in the membrane integrity during high-pressure treatment that causes changes in the membrane composition and membrane repair capability (Whitney et al., 2007). Moreover, the intra strain variation in resisting pressure in E.coli has been attributed to the type of fatty acids in the membrane and the membrane fluidity (Gänzle and Liu, 2015). Furthermore, the inter strain variation in resisting pressure has been attributed to the variation in the genes expressed when exposing E.coli strains to pressure and the stress proteins that are expressed by the RpoS
(Gänzle and Liu, 2015; Malone at al., 2007; Whitney et al., 2007). Yet, from the results of the current study, it was found that there was an inverse relationship between salt tolerance and barotolerance. There are various means by which salt tolerance can be measured with each providing a different ranking of the STEC strains tested. Specifically,
76 salt tolerance can be determined by plate counts or viability staining after holding the bacterial cells in NaCl solutions. In the current study culturable STEC suspended in 10%
NaCl progressively declined to below the level of detection within 27-33 days. A similar finding has been reported by others, for example, Stasic et.al (2012), determined tolerance of E. coli O157: H7 and reported viable cells could be cultured from a 12%
NaCl for up to 8 days. In a further example, Hrenovic et al. (2009), determined tolerance of E. coli to NaCl (10-20%) by plate counts and reported a rapid decline in numbers after
72 h. In the current study, there was a significant difference in the Ct values of the different STEC strains as determined by plate counting. However, it was noted that numbers of STEC recovered depended on the culture conditions applied to recover the cells. Specifically, by incubating the cells in (5%) glycerol-TSB prior to plating the number of cells recovered were above the limit of detection (>0.69 log CFU/mL). It has been reported that the recovery of Salmonella from low moisture environments can be increased by increasing the incubation period in glycerol- BHI prior to plating (Kinsella et al., 2006; Mattick et al., 2001). The pre-incubation step reduces the osmotic shock of cells thereby enabling repair then subsequent growth (Kinsella et al., 2006). In addition,
Poirier et al., 1997, reported that when water activity was decreased gradually in glycerol solution, there was no loss in the viability of E.coli. The increase in recovery of STEC by a pre-incubation step in glycerol-TSB suggests that plate counts are an unsuitable approach to assess salt tolerance given that the result would depend on the method of analysis. This fact was further confirmed when viability staining was applied. Even though plate counts suggested a decline in numbers, it was evident that a high proportion of cells remain viable as determined using viability staining. The same lack of agreement
77 between enumerating cells using culture and non-culture based methods has been previously reported. For example, Lothigius et al., 2010, also found that the plate counts of six strains ETEC decreased to the level of detection after 2 weeks of incubation in seawater (3.5% NaCl), while the total number of viable cells determined by real-time
PCR remained constant. The researchers suggested that the E. coli cells underwent a transition to the VBNC state in the course of exposure to osmotic stress. A similar result was described by Darcan et al. (2009), who also reported a significant proportion of E. coli populations enter the VBNC state in high NaCl solutions. In the current study, it is also probable that the STEC entered a VBNC state. The VBNC state is a type of dormancy whereby the cells become inherently stress tolerant (Lebre et al., 2017;
Ayrapetyan et al., 2014). In the strict definition, VBNC cannot be cultured but as observed in the current study, this was not found to be the case given the recovery in glycerol-TSB. Yet, even by applying a recovery step in glycerol the viability counts did not match plate counts that would suggest that at least a proportion of STEC cells had entered a deeper state of dormancy. Regardless of this fact, the use of viability staining did not reveal significant differences in the NaCl tolerance between the strains tested.
However, it was noted that the limits of NaCl that E. coli could grow under both differed between strains and moreover, could be inversely correlated to barotolerance, which is a major finding in the study. Against intuition, it was found that those STEC where the growth was inhibited by relatively low NaCl (2% w/v) concentrations exhibited the highest barotolerance. Conversely, STEC that could continue to grow under relatively high NaCl concentrations (6% w/v) were contrastingly pressure sensitive. The growth limits of E. coli and other bacteria in NaCl (and low moisture environments in general) is
78 controlled by the ability of the cell to continue growing by maintaining homeostasis of the cytosol (Wood, 2011). However, in bacteria that are sensitive to low water activity there is an induction of the stress response in cells exposed to low moisture environments thereby resulting in the growth inhibition and down-regulation of cellular metabolism
(Battesti et al., 2015; Malone et al., 2007; Chang et al., 2002; Moat et al., 2002; Hengge-
Aronis et al., 1993; Sperber, 1982). The hypothesis is supported by studies that reported the induction of stress response in E.coli after exposure to stresses that block bacterial growth (Malone et al., 2007). For example, it has been reported that RpoS is involved in resistance to osmotic stress in E. coli (Culham et al., 2001; Chung et al., 2006). In addition, RpoS has been reported to control the transcription of genes involved in stress responses including osmotic stress (Mata et al., 2017). Mata et al., 2017, found that mutant EPEC strains that lacked the rpoS gene were sensitive to osmotic stress (20-25% survivals) when exposed to 2 M NaCl (11.6%) up to 6 h compared to the parent strain that was resistant to osmotic stress (80% survivals). In addition, Cheville et al., 1996, reported that mutant strain of STEC O157: H7 that lacked the rpoS was significantly more sensitive to salt (15% NaCl) and heat (55°C) compared to the parent strain that was more tolerant. The survival population of the mutant strain of E.coli O157: H7 in 15%
NaCl was 51.1% survivals, while the survival population for the parent STEC O157: H7 was 93.8% survivals.
It is well established that E. coli under low water activity have enhanced barotolerance.
Consequently, it is not unexpected to find that cells grown under reduced water activity also exhibit barotolerance (Scheyhing et al., 2003; Ueno et al., 2011). However, in the current, study the STEC strains were not cultivated under osmotic stress but yet the
79 growth limit for NaCl still correlated to barotolerance. The results suggest that in the course of applyi1ng pressure the same stress response was induced given the association with growth limits of NaCl and pressure sensitivity. However, it is possible the response to applied pressure induced a general stress response thereby increasing the stability of the cell. In contrast, with pressure sensitive strains the delay in the stress response ultimately cells susceptible to irreversible damage (Charoenwong et al., 2011). The theory is supported by the fact that rpoS mutants of E. coli exhibit significantly greater pressure sensitivity compared to wild type strains (Charoenwong et al., 2011). In addition, (Robey et al., 2001), found that after pressure treatment 500 MPa (15 min), the mutant strains of STEC O157: H7 lacking the RpoS had significant log reduction (2.2 log
CFU) compared to the parent strain that had < 0.5 log reduction. In this respect, the early induction of rpoS in both cells grown on NaCl gradient plates or exposed to high pressure is the underlying mechanism of the association of NaCl growth limits and barotolerance.
However, additional mechanisms likely exist given that the E. coli O157: H7 tested was relatively sensitive to both pressure and NaCl. In this respect, it was interesting to note that the barotolerance of the STEC strains could be enhanced through provision of supplements such as glycine into the suspending media. It was found that provision of glycine enhances barotolerance under lower pressure treatments as there was no significant difference in the level of protection of STEC O157: H7 against high pressure within the different aw solutions contacting glycine. On the other hand, under higher- pressure treatments such as 600 MPa, it was evident that the barotolerance was dependent on the aw level. Accumulation of osmolytes during osmotic stress provides protection to microbes against osmotic stress and other stresses such as starvation and dehydration.
80 Glycine and betaine are amino acids that have been considered as osmolytes and can be accumulated in E. coli (Styrvold & Strøm, 1991; Sleator & Hill, 2006). The type of solute and the compassion of the media affect the high-pressure inactivation of STEC. It has been reported that the addition of a high solute concentration in low water activity solutions increases barotolerance of microbes (Setikaite et al., 2009; Considine et al.,
2011; Van Opstal et al., 2003). Smiddy et al. (2004), found that when the osmolytes glycine betaine (5mM) and L-carnitine (5mM) were added in 3% NaCl and exposed to pressure 400 MP (5min), the surviving population of Listeria innocua increased to 3.9 log CFU/mL compared to 2.1 log CFU/mL when no osmolytes were added. In addition,
Considine et al., 2011, found that at high-pressure treatment 400 and 500 MPa, mutant strains of Listeria monocytogenes lacking the gene responsible for proline synthesis was
1 log lower than the wild strain of listeria monocytogenes that had the ability to overproduce proline. Pleitner et al. (2012), found a correlation between salt tolerance and heat resistance of E. coli AW1.7. The correlation was found to be that the accumulation of osmolytes at 60°C increases the stability of ribosomes. On the basis that the mode-of- inactivation of bacteria at moderate pressure of 300 MPa is linked to ribosomal dissociation, it can be theorized that ribosome stability through osmolyte accumulation could also be part of a protective mechanism. Yet, it was also found that other solutes that were not traditionally considered osmolytes could also exert a protective mechanism against high pressure in STEC. Specifically, pyruvate, tryptophan, alanine, and peptone can provide protection to STEC against high-pressure treatment. It is possible that such non-osmolytes were assimilated by STEC then transformed into osmolytes via branches of the metabolic pathway. Essential amino acids can be converted to different
81 intermediates that can participate in anaplerotic reactions (Schowen 1993). However, it was noted in E. coli O157: H7 that the provision of sorbitol or starch in the suspension medium also provided barotolerance despite neither being directly metabolized by the bacterium. It was noted by Li et al. (2016), that barotolerance in E. coli AW1.7 could be enhanced by the provision of calcium, magnesium or glutamate to the suspending media.
In addition, Hauben et al., 1998, reported that the pressure tolerance of E. coli could be increased by the presence of calcium. It was hypothesized that the presence of Ca, Mg and glutamate stabilized the cell membranes and consequently barotolerance although no direct evidence was provided. If starch and sorbitol, in addition to the other solutes, have a membrane protecting effect remains unclear as does the mechanism by which it would do so.
The results from the study underlined the challenge of generating predictive models for pathogen inactivation by high-pressure processing. The dependence of barotolerance on strain, stress-response, and presence of solutes would make for complex predictive models. Yet, it should be noted that at high pressure (600 MPa) there were no strain differences observed and moreover, the provision of osmolytes on barotolerance was less evident. Therefore, in standard high-pressure processing regimes, it is likely that the food matrix will have a major influence on the barotolerance of STEC. However, at lower pressures, a combination of food matrix effects along with the inherent strain characteristics need to be taken into account.
82 Chapter 3
The effect of low water activity (glycerol, NaCl) on barotolerance of non pathogenic Escherichia coli P36
3.1 Abstract
The usage of High hydrostatic pressure (HHP) in the processing of foods has been increased during the last decades due to the ability to achieve microbial inactivation by non-thermal treatment while maintaining the quality. High-pressure inactivation of pathogens has been challenging in low water activity matrices. The objective of the study was to investigate the effect of low water activity and the type of solute in the barotolerance of E.coli p36. The study evaluated the effect of different water activity
(0.95, 0.90, 0.88 aw) by using solutions of glycerol and NaCl. It was found that the low water activity solutions with glycerol and NaCl increased barotolerance of E. coli p36 significantly (p<0.05) compared to PBS (no solutes) when exposed to pressure 400-600
MPa. The study found that the low water activity solutions containing glycerol provided more protection to E.coli against high-pressure inactivation than NaCl solutions. The results suggested that the permeability of glycerol into the cell increased recovery of
E.coli when exposed to high pressure compared to NaCl that is non-permeable. The finding supports the idea that low water activity increases barotolerance of E. coli and the degree of protection is dependent on the solute used.
83 3.2 Introduction
High hydrostatic pressure is a non-thermal food preservation method that can extend the shelf life of food products by the inactivation of pathogens. During the last decade, HHP has been considered as one of the emerging technologies in food manufacturing that has the ability to maintain the quality of foods (Yordanov & Angelova, 2010; Hayman et al.,
2008). Different factors can affect high-pressure inactivation of microbes including water activity (Georget et al., 2015; Setikaite et al, 2007). It is known that low water activity has the ability to inhibit the growth of pathogens, and it is used to control microbial growth in food (Burgess et al., 2016; Ueno et al., 2011; Beuchat et al., 2013). However, It has been reported that low water activity provides protection to microbes against stresses such as heat (Hayman et al., 2008; Huang et al., 2009). Studies reported that low water activity protects microbes against high pressure (Considine et al., 2007; Setikaite et al,
2007). In addition, it has been reported that resistance of microorganism to HHP increases when increasing osmotic stress (Hoppner et al., 2003). Hayman et a., 2008, reported an increase in the barotolerance of Listeria monocytogenes (2.5 log reduction) when aw was decreased to 0.83 compared to when aw was 0.99-0.87 (6.5-7 log reductions). Goodridge et al. (2005) reported that less than one log reduction, 0.83 log, in
Salmonella enteritidis inoculated into raw almonds was achieved when exposed to 413
MPa at 50°C for 5 min. However, it was found that the type of solute used to lower the water activity affects the survival of bacterial cells under high pressure (Considine et al.,
2007). For example, sucrose and salt content were reported to provide significant protection for pathogens against HHP inactivation (Considine et al., 2007). In addition, studies reported that the usage of NaCl and sugars had a protective effect on E.coli,
84 saccharomyces cerevisiae, and Lactococcus lactis against HHP (Setikaite et al, 2007). In addition, Setikaite, et al., 2007, reported that decreasing aw from 0.99 to 0.90 in glycerol solutions increased barotolerance of E.coli k12 as no log reduction was observed.
Moreover, Moussa et al., 2006, reported that decreasing aw to 0.85 in glycerol solutions increased barotolerance of E.coli k12 when exposed to pressure 450 MPa/ 10 min. The objective of the current study was to investigate the effect of low water activity and the type of solutes in the inactivation of E.coli by using NaCl and glycerol.
3.3 Materials and Methods 3.3.1 Bacteria and Cultivation
The non-pathogenic E.coli strain used in this experiment was E.coli P36 isolated from spinach (Warriner et al., 2003). The pure isolate was cultured on TSA, while suspensions were stored in 50% TSB and 50% glycerol solution at -80°C.
3.3.2 Preparation of Suspensions
The E.coli p36 strain was grown overnight on Tryptone Soy Agar (TSA; OXOID Ltd,
Basingstoke, Hampshire, England) at 37°C. A loop-full of isolated colonies was suspended and dispersed in 0.9% w/v saline to achieve a final O.D of 0.2 (108 log
CFU/mL), and the suspension was stored at 4°C until required.
85
3.3.3 Effect of low water activity (glycerol, NaCl) on HHP inactivation of E.coli
Sample Preparation
E.coli P36 was exposed to HHP in solutions that have different water activates
(0.90,0.85.0.80) and different osmolytes under a range of pressure (400, 500, 600 MPa) for 1 -3 min. Different solutes including NaCl and glycerol were added to phosphate buffer saline (pH 7.1, 1X) to lower the water activity. The solutes were added to the buffer forming 3 different water activity values as follows. NaCl was added by a concentration of 31% (0.80), 25% (0.85) and 16% (0.90). Glycerol was added by a concentration of 85.5 mL/100 mL (0.80), 65.5 mL/100 mL (0.85) and 44.4 mL/100 mL
(0.90). Water activity values were measured using a water activity meter (Dew point water activity meter, AquaLab 4(TE), Pullman, WA) and the solutions were autoclaved and stored at 4°C till dispensed by a volume of 9 mL into HHP pouches Then 1mL of the bacterial suspension, 8 log, was inoculated into 9 mL of the solutions (phosphate buffer saline pH 7.1, 1X and solute) in sterile flexible pouches and the aw value measured. The pouches then were placed in a high-pressure chamber and a range of pressure (400, 500,
600 MPa) was applied for different holding times (1-3min). The samples were held at
4°C till enumeration after HHP treatment for post recovery, and the log reduction was determined.
86 3.3.4 Enumeration of Bacteria
The high-pressure treatments, enumeration was done by adding 0.1 mL of the sample to
0.9 mL sterile saline (0.9% w/v) and a dilution series was carried out. Then 0.1 mL of the dilutions was inoculated into TSA and incubated at 37°C/24 h. The same enumeration method was done after 72 h for the post recovery of cells.
3.3.5 Experimental Design and Statistics
Experiments were performed in triplicates, and the number of colonies was converted to log10. The data obtained were analyzed using two-way ANOVA and Tukey test of the
SPSS statistical software for Mac (IBM SPSS statistics 21).
3.4 Results and Discussion
Decreasing the aw of the solutions containing glycerol or NaCl increased barotolreance of
E. coli p36. In general, the log reduction of E. coli p36 (at 400 MPa) in glycerol or NaCl solutions was significantly low (P<0.05) compared to when no solutes were added in
PBS. The log reduction of E. coli in the low aw solutions containing glycerol was significantly (P<0.05) low compared to when containing NaCl. The log reduction of
E.coli in the different glycerol solutions (at 400 MPa) ranged from 0.82±0.10 to
2.14±0.11 log CFU, while the log reduction in the different NaCl solutions (at 400 MPa) ranged from 1.31±0.17 to 6.21±0.00 log CFU (Tables 3.7, 3.8, 3.9). On the other hand, in the absence of glycerol or NaCl, the treatment of E.coli with pressure 400-600 MPa decreased the population to the limit of detection (<0.69 log CFU/mL). The log reduction was proportional to the pressure and time when pressure >400 MPa was applied to the aw
87 solutions with treatment of 600 MPa decreasing the populations below the limit of detection (<0.69 log CFU/mL). It was found that treatment of E.coli with pressure >400
MPa for 2 min increased the log reduction in aw solutions containing glycerol, yet the plate counts did not reach the limit of detection. However, when the holding time exceeded 2 min, the counts reached the lowest detectable limits for all aw solutions containing glycerol (Table 3.2). On the other hand, the treatment of E.coli with pressure
>400 MPa for 1-3 min in NaCl solutions decreased the population to the level of detection (<0.69 log CFU/mL) (Table 3.1).
In the presence of glycerol and NaCl, protection against high pressure was observed.
E.coli can respond to stress by the accumulation of osmolytes such as proline, glycine, and betaine that provide protection to E.coli against high pressure (Gänzle and Liu,
2015). Glycerol is freely permeable into the cell, and hence permeability of glycerol provides more protection to the cell and stabilizes the membrane acting as osmolytes.
Another response of E.coli to high pressure can be by induction of stress response that provides initial protection against stresses including high pressure and osmotic stress (Liu et al., 2015; Gänzle and Liu, 2015; Malone et al., 2007). It is suggested that in the presence of NaCl, the stress response is induced providing less protection to E.coli against high pressure. On the other hand, in the absence of solutes in PBS, no protection is provided and hence the pressure applied causes damage to the E.coli cell leading to cell death. Setikaite et al., 2009, reported an increase in the barotolerance of E. coli K12 in the presence of osmotic regulators such as NaCl, glycerol, and sucrose when testing the effect of water activity on high-pressure inactivation of E. coli K12 by using different solutes. Moussa et al., 2006, found that decreased water activity (0.85aw) by the addition
88 of glycerol increased barotolerance of E.coli k12 significantly (3.5 log reduction) compared to the control 0.99 aw (5 log reduction) at pressure 450 MPa.
The results in the study underlined that low water activity increases pressure resistance of
E.coli depending on the type of solutes present, and the stress response of E.coli to high pressure differs depending on the solute present in the surrounding environment.
89 Table 3.1.The effect of low aw by using NaCl on the barotolerance of E. coli
Treatment Pressure Time Log Average Average log Average log MPa (min) CFU/mL Log (24 h) reduction reduction Time (0) (24 h) (72 h) NaCl 0.95 aw 400 MPa 1min 6.90±0.15 4.26 ± 0.13 2.64a 1.80a NaCl 0.95 aw 400 MPa 2min 6.90±0.15 4.10 ± 0.18 2.80b 5.21b NaCl 0.95 aw 400 MPa 3min 6.90±0.15 <0.69 ± 0.00 6.21b 2.21c NaCl 0.90 aw 400 MPa 1min 6.79±0.13 4.28 ± 0.17 2.51a 2.79a NaCl 0.90 aw 400 MPa 2min 6.79±0.13 4.11 ± 0.15 2.68b 6.10b NaCl 0.90 aw 400 MPa 3min 6.79±0.13 <0.69 ± 0.00 6.10b 6.10b NaCl 0.88 aw 400 MPa 1min 6.70±0.10 5.39 ± 0.17 1.31a 1.70a NaCl 0.88 aw 400 MPa 2min 6.70±0.10 5.30 ± 0.12 1.40b 6.01b NaCl 0.88 aw 400 MPa 3min 6.70±0.10 <0.69 ± 0.00 6.01b 6.01b NaCl 0.95 aw 500 MPa 1min 6.90±0.15 <0.69 ± 0.00 6.21a 6.21a NaCl 0.95 aw 500 MPa 2min 6.90±0.15 <0.69 ± 0.00 6.21a 6.21a NaCl 0.95 aw 500 MPa 3min 6.90±0.15 <0.69 ± 0.00 6.21a 6.21a NaCl 0.90 aw 500 MPa 1min 6.79±0.13 <0.69 ± 0.00 6.10a 6.10a NaCl 0.90 aw 500 MPa 2min 6.79±0.13 <0.69 ± 0.00 6.10a 6.10a NaCl 0.90 aw 500 MPa 3min 6.79±0.13 <0.69 ± 0.00 6.10a 6.10a NaCl 0.88 aw 500 MPa 1min 6.70±0.10 <0.69 ± 0.00 6.01a 6.01a NaCl 0.88 aw 500 MPa 2min 6.70±0.10 <0.69 ± 0.00 6.01a 6.01a NaCl 0.88 aw 500 MPa 3min 6.70±0.10 <0.69 ± 0.00 6.01a 6.01a NaCl 0.95 aw 600 MPa 1min 6.90±0.15 <0.69 ± 0.00 6.21a 6.21a NaCl 0.95 aw 600 MPa 2min 6.90±0.15 <0.69 ± 0.00 6.21a 6.21a NaCl 0.95 aw 600 MPa 3min 6.90±0.15 <0.69 ± 0.00 6.21a 6.21a NaCl 0.90 aw 600 MPa 1min 6.79±0.13 <0.69 ± 0.00 6.10a 6.10a NaCl 0.90 aw 600 MPa 2min 6.79±0.13 <0.69 ± 0.00 6.10a 6.10a NaCl 0.90 aw 600 MPa 3min 6.79±0.13 <0.69 ± 0.00 6.10a 6.10a NaCl 0.88 aw 600 MPa 1min 6.70±0.10 <0.69 ± 0.00 6.01a 6.01a NaCl 0.88 aw 600 MPa 2min 6.70±0.10 <0.69 ± 0.00 6.01a 6.01a NaCl 0.88 aw 600 MPa 3min 6.70±0.10 <0.69 ± 0.00 6.01a 6.01a
±Value means Standard Deviation for 3 samples, CFU= Colony Forming Unit. Means followed by same small letter in columns and same capital letter in rows are non significantly different (p<0.05). Means followed by different small letter in columns and different capital letter in rows is significantly different (p<0.05).
90
Table 3.2 The effect of low aw by using glycerol on the barotolerance of E. coli
Treatment Pressure Time Log Average Average log Average log MPa (min) CFU/mL Log (24 h) reduction reduction Time (0) (24 h) (72 h) Glycerol 0.95 aw 400 MPa 1min 7.00 ±0.16 5.54 ± 0.10 1.46a 1.91a Glycerol 0.95 aw 400 MPa 2min 7.00 ±0.16 4.99 ± 0.09 2.01b 4.21b Glycerol 0.95 aw 400 MPa 3min 7.00 ±0.16 4.85 ±0.11 2.15b 5.71c Glycerol 0.90 aw 400 MPa 1min 6.79±0.18 5.97 ± 0.12 0.82a 2.71a Glycerol 0.90 aw 400 MPa 2min 6.79±0.18 5.53± 0.06 1.26a 1.81a Glycerol 0.90 aw 400 MPa 3min 6.79±0.18 4.99± 0.09 1.81a 4.31b Glycerol 0.88 aw 400 MPa 1min 6.99±0.20 6.10 ± 0.10 0.89a 0.89a Glycerol 0.88 aw 400 MPa 2min 6.99±0.20 6.10 ± 0.12 0.89a 1.69a Glycerol 0.88 aw 400 MPa 3min 6.99±0.20 6.10 ± 0.11 0.89a 1.40a Glycerol 0.95 aw 500 MPa 1min 7.00 ±0.16 <0.69 ± 0.00 6.31a 6.31a Glycerol 0.95 aw 500 MPa 2min 7.00 ±0.16 <0.69 ± 0.00 6.31a 6.31a Glycerol 0.95 aw 500 MPa 3min 7.00 ±0.16 <0.69 ± 0.00 6.31a 6.31a Glycerol 0.90 aw 500 MPa 1min 6.79±0.18 5.86 ± 0.18 0.84a 4.01a Glycerol 0.90 aw 500 MPa 2min 6.79±0.18 4.45 ± 0.1 2.30b 4.01a Glycerol 0.90 aw 500 MPa 3min 6.79±0.18 <0.69 ± 0.00 6.11c 6.11b Glycerol 0.88 aw 500 MPa 1min 6.99±0.20 5.94 ± 0.11 0.96a 1.20a Glycerol 0.88 aw 500 MPa 2min 6.99±0.20 5.02 ± 0.20 1.97a 4.21b Glycerol 0.88 aw 500 MPa 3min 6.99±0.20 <0.69 ± 0.00 6.30b 6.30c Glycerol 0.95 aw 600 MPa 1min 7.00 ±0.16 <0.69 ± 0.00 6.31b 6.31b Glycerol 0.95 aw 600 MPa 2min 7.00 ±0.16 <0.69 ± 0.00 6.31b 6.31b Glycerol 0.95 aw 600 MPa 3min 7.00 ±0.16 <0.69 ± 0.00 6.31b 6.31b Glycerol 0.90 aw 600 MPa 1min 6.79±0.18 4.07 ± 0.10 2.72a 3.00a Glycerol 0.90 aw 600 MPa 2min 6.79±0.18 <0.69 ± 0.00 6.11b 6.11b Glycerol 0.90 aw 600 MPa 3min 6.79±0.18 <0.69 ± 0.00 6.11 b 6.11b Glycerol 0.88 aw 600 MPa 1min 6.99±0.20 4.39 ± 0.22 2.61a 4.21a Glycerol 0.88 aw 600 MPa 2min 6.99±0.20 <0.69 ± 0.00 6.30b 6.30b Glycerol 0.88 aw 600 MPa 3min 6.99±0.20 <0.69 ± 0.00 6.30b 6.30b ±Value means Standard Deviation for 3 samples, CFU= Colony Forming Unit. Means followed by same small letter in columns are non significantly different (p<0.05). Means followed by different small letter in columns is significantly different (p<0.05).
91
Table 3.3.The effect of HHP on non-stressed E. coli in (PBS)
Treatment Pressure Time Log Average Average Average MPa (min) CFU/mL log log log Time (0 min) (24 h) reduction reduction (24 h) (72 h) No stress 400 MPa 1min 7.10±0.14A <0.69 ± 0B 6.41a 6.41a No stress 400 MPa 2min 7.10±0.14A <0.69 ± 0B 6.41a 6.41a No stress 400 MPa 3min 7.10±0.14A <0.69 ± 0B 6.41a 6.41a No stress 500 MPa 1min 7.10±0.14A <0.69 ± 0B 6.41a 6.41a No stress 500 MPa 2min 7.10±0.14A <0.69 ± 0B 6.41a 6.41a No stress 500 MPa 3min 7.10±0.14A <0.69 ± 0B 6.41a 6.41a No stress 600 MPa 1min 7.10±0.14A <0.69 ± 0B 6.41a 6.41a No stress 600 MPa 2min 7.10±0.14A <0.69 ± 0B 6.41a 6.41a No stress 600 MPa 3min 7.10±0.14A <0.69 ± 0B 6.41a 6.41a ±Value means Standard Deviation for 3 samples, CFU= Colony Forming Unit. Means followed by same small letter in columns are non significantly different (p<0.05). Means followed by different small letter in columns and different capital letter in rows is significantly different (p<0.05).
92 Chapter 4
General Conclusion and Future Work
4.1 General Conclusion
The objective of the study was to determine if the tolerance to low water activity could be correlated with the high-pressure tolerance of the STEC strains. The underlying hypothesis was that mechanisms that protect STEC in low water activity environments would be the same as those under high pressure.
• STEC strains varied in their growth limits on the gradient plate in the presence of
NaCl. The limit of NaCl to inhibit the growth of STEC O121: H19 was the
highest limit 6% w/v NaCl, whereas the limit of NaCl to inhibit the growth of
STEC O26: H11 was the lowest limit 2.4% w/v NaCl.
• STEC strains varied in their persistence when placed in 10% NaCl solution and
left for an extended period (36 days). STEC O157: H7 and O121: H19 were able
to grow after 33 days incubation under osmotic stress. The pre-incubation (2 h) in
5% glycerol TSB increased recovery of STEC cells after they lost their
culturability when exposed to 10% NaCl for an extended period (50 days).
• Under osmotic stress conditions (10% NaCl), STEC cells are losing their
culturability, yet they stay viable, and there was no difference between serotypes
in viability staining.
• The tolerance to high pressure could be inversely correlated to the growth limits
for NaCl. STEC strains that were able to grow on high concentration of NaCl had
93 lower pressure tolerance, whereas STEC strains that were able to grow on low
NaCl concentration had higher pressure tolerance.
• The addition of osmolytes (amino acids) and non-osmolytes (supplements) and
sugar all increased the protection of STEC against high pressure. The potential
mechanisms proposed for the barotolerance associated with the addition of amino
acids is the accumulation of osmolytes, whereas the potential mechanism
proposed for the barotolerance associated with the addition of non-osmolytes
(supplements) is the assimilation of the supplements by STEC.
• At pressure 600 MPa, the barotolerance of STEC was dependent on the low aw
level and glycine did not contribute to the barotolerance of STEC.
• At pressure 400 MPa, glycine had a protective effect for STEC against high
pressure.
• Low aw in glycerol and NaCl solutions increased pressure resistance of E. coli p36
and glycerol provided higher protection to E.coli p36 than NaCl due to the
permeability of glycerol.
94 4.2 Future Work
The study illustrated that there is an inverse relation between STEC growth limits under low aw and their barotolerance. Also, the study illustrated that the addition of peptone, amino acids, sugars and other supplements in the media provided protection to the STEC strains against high pressure.
• Future work is needed to investigate a broad selection of pathogenic E. coli and
see if the same inverse relation exists between their growth limits under low aw
and their barotolerance. In addition, further analysis is needed to investigate if the
early stress response elicited under relatively low salt concentrations is
responsible for enhanced barotolerance. This could be established by
transcriptome analysis and assessing both the salt and barotolerance of mutant
STEC strains relative to parents.
• Further work is needed to verify the accumulation or synthesis of osmolytes
inside the STEC cells by using radiolabeled osmolytes that can be detectable
inside the cells after accumulation. This can help to modulate the barotolerance of
microbes by the inclusion of osmolytes. It can also help to understand the
underplaying mechanism behind the barotolerance of STEC when amino acids
and peptone are added.
95 • Further analysis is needed to verify the metabolism of the supplements added by
following the fate of a substrate and the byproducts to see if the metabolism is the
mechanism behind barotolerance when supplements are added.
• By using knowledge gain between the relationship between salt growth limits and
barotolerance, it will be possible to construct predictive models that can be
applied by industry in establishing high pressure processing protocols.
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