title: Survival and recovery of Escherichia coli O157:H7 during starvation at sub-optimal temperatures

authors: Xavier, Ron Neo

subjects: escherichia coli

abstract: Escherichia coli O157:H7 is an important foodborne pathogen with a dual lifestyle of growth within a host and persistence in non-host environments. Environmental survival of E. coli O157:H7 is particularly important in New Zealand due to the increasing trend in notified clinical cases, with most being associated with rural environments rather than contaminated regulated foods. Starvation and a decrease in temperature are characteristic of the conditions encountered by bacteria during transition from a reservoir host, normally a ruminant, to the environment; cf. feast to famine. These conditions are known to induce bacterial entrance into the viable but non-culturable (VBNC) state where cell viability can only be detected using culture-independent methods with the number of colonies recovered decreasing over the duration of the starvation period. Findings from this study showed that formation of a VBNC sub-population can be induced by starving E. coli O157:H7 at sub-optimal temperatures. 

Five E. coli O157:H7 strains were used in this study; NCTC12900 as a reference strain, three New Zealand clinical isolates and a New Zealand bovine isolate. Cells were starved in phosphate buffered saline for 84 days at sub-optimal temperatures. Cell viability was determined using the SYTO 9-PI staining system coupled with a haemocytometer counting method developed in this study. Cell culturability was assessed using the nutrient-rich tryptic soy agar (TSA), nutrient-limited plate count agar (PCA), and PCA with additional supplements. The distribution of colony size provided information on the population dynamics of starved cells and their ability to recover on agar. The role of quorum sensing (LuxS/autoinducer-2) on starved cell recovery was investigated by supplementing PCA with quorum sensing molecules and using a mutant strain deficient in luxS, a gene encoding the synthase of autoinducer-2. The ability of starved and non-starved cells to metabolise single carbon sources were compared using the Biolog MicroPlatesTM.
	
When held at 4°C, a three- to six-log reduction in culturability on PCA was observed after 84 days of starvation. The reduction in culturability observed for cells starved at 15°C was between one to two logs on PCA. Despite the decline in culturability on PCA, the number of viable cells as determine by LIVE/DEAD® BacLightTM staining remained within one log of the initial viable count. Supplementation of PCA with 0.2% sodium pyruvate resulted in a significant increase in the number of colonies recovered from 4°C-starved cultures for all E. coli O157:H7 strains tested. Recovery on TSA was consistently at least two-logs higher than recovery on PCA for E. coli O157:H7 cells after 84 days of starvation at 4°C. The distribution of colony diameter was markedly wider for 4°C-starved cells compared to non-starved cells when recovered under aerobic conditions. In addition to the strain variation observed, culturability of starved E. coli O157:H7 was found to be dependent on the starvation temperature, the nutrient level of recovery media, and the presence of specific resuscitation factors such as pyruvate. While the culturability phenotype was similar between reference, clinical and bovine E. coli O157:H7 strains, metabolic profiles determined using carbon source MicroPlatesTM differed. Analysis of the carbon utilisation kinetics allowed comparison of carbon metabolism between exponential phase, stationary phase and starved E. coli O157:H7. The duration of lag in substrate utilisation was significantly different between starved and non-starved cells. Supplementing PCA with quorum sensing molecules and using a mutant strain deficient in luxS, a gene encoding the synthase of autoinducer-2, did not result in a significant improvement in the recovery of starved VBNC cells.

Based on current knowledge, models for the change in E. coli O157:H7 population dynamics during its lifecycle, the role of pyruvate resuscitation of VBNC cells, and factors involved in E. coli O157:H7 infection of bovine and human hosts were proposed. The ability of New Zealand clinical and bovine isolates to enter the VBNC state, which was demonstrated in this study, highlighted the potential for culture-based assessment methods to underestimate the number of viable E. coli O157:H7. While reports of the use of pyruvate for resuscitating VBNC cells are in the literature, this study is the first to propose a mechanism of pyruvate resuscitation. By understanding the physiology of VBNC cells and mechanism of their resuscitation, it may be possible to eliminate the potential for underestimation of viable E. coli O157:H7 in the environment and in food by tailoring culture methods to promote their growth.

text: Survival and recovery of Escherichia coli O157:H7 during starvation at sub-optimal temperatures Escherichia coli O157:H7 is an important foodborne pathogen with a dual lifestyle of growth within a host and persistence in non-host environments. Environmental survival of E. coli O157:H7 is particularly important in New Zealand due to the increasing trend in notified clinical cases, with most being associated with rural environments rather than contaminated regulated foods. Starvation and a decrease in temperature are characteristic of the conditions encountered by bacteria during transition from a reservoir host, normally a ruminant, to the environment; cf. feast to famine. These conditions are known to induce bacterial entrance into the viable but non-culturable (VBNC) state where cell viability can only be detected using culture-independent methods with the number of colonies recovered decreasing over the duration of the starvation period. Findings from this study showed that formation of a VBNC sub-population can be induced by starving E. coli O157:H7 at sub-optimal temperatures. 

Five E. coli O157:H7 strains were used in this study; NCTC12900 as a reference strain, three New Zealand clinical isolates and a New Zealand bovine isolate. Cells were starved in phosphate buffered saline for 84 days at sub-optimal temperatures. Cell viability was determined using the SYTO 9-PI staining system coupled with a haemocytometer counting method developed in this study. Cell culturability was assessed using the nutrient-rich tryptic soy agar (TSA), nutrient-limited plate count agar (PCA), and PCA with additional supplements. The distribution of colony size provided information on the population dynamics of starved cells and their ability to recover on agar. The role of quorum sensing (LuxS/autoinducer-2) on starved cell recovery was investigated by supplementing PCA with quorum sensing molecules and using a mutant strain deficient in luxS, a gene encoding the synthase of autoinducer-2. The ability of starved and non-starved cells to metabolise single carbon sources were compared using the Biolog MicroPlatesTM.
	
When held at 4°C, a three- to six-log reduction in culturability on PCA was observed after 84 days of starvation. The reduction in culturability observed for cells starved at 15°C was between one to two logs on PCA. Despite the decline in culturability on PCA, the number of viable cells as determine by LIVE/DEAD® BacLightTM staining remained within one log of the initial viable count. Supplementation of PCA with 0.2% sodium pyruvate resulted in a significant increase in the number of colonies recovered from 4°C-starved cultures for all E. coli O157:H7 strains tested. Recovery on TSA was consistently at least two-logs higher than recovery on PCA for E. coli O157:H7 cells after 84 days of starvation at 4°C. The distribution of colony diameter was markedly wider for 4°C-starved cells compared to non-starved cells when recovered under aerobic conditions. In addition to the strain variation observed, culturability of starved E. coli O157:H7 was found to be dependent on the starvation temperature, the nutrient level of recovery media, and the presence of specific resuscitation factors such as pyruvate. While the culturability phenotype was similar between reference, clinical and bovine E. coli O157:H7 strains, metabolic profiles determined using carbon source MicroPlatesTM differed. Analysis of the carbon utilisation kinetics allowed comparison of carbon metabolism between exponential phase, stationary phase and starved E. coli O157:H7. The duration of lag in substrate utilisation was significantly different between starved and non-starved cells. Supplementing PCA with quorum sensing molecules and using a mutant strain deficient in luxS, a gene encoding the synthase of autoinducer-2, did not result in a significant improvement in the recovery of starved VBNC cells.

Based on current knowledge, models for the change in E. coli O157:H7 population dynamics during its lifecycle, the role of pyruvate resuscitation of VBNC cells, and factors involved in E. coli O157:H7 infection of bovine and human hosts were proposed. The ability of New Zealand clinical and bovine isolates to enter the VBNC state, which was demonstrated in this study, highlighted the potential for culture-based assessment methods to underestimate the number of viable E. coli O157:H7. While reports of the use of pyruvate for resuscitating VBNC cells are in the literature, this study is the first to propose a mechanism of pyruvate resuscitation. By understanding the physiology of VBNC cells and mechanism of their resuscitation, it may be possible to eliminate the potential for underestimation of viable E. coli O157:H7 in the environment and in food by tailoring culture methods to promote their growth.

year: 2014

title: Synthesis of Poly(galactaramides) from Alkylene- and Substituted Alkylenediammonium Galactarates

authors: Kiely, Donald E.

subjects: polyamides

abstract: Polyhydroxypolyamides (PHPAs) represent a class of synthetic polyamides derived from aldaric acid and diamine monomer units. This paper describes the synthesis of some poly(galactaramides), a class of polyhydroxypolyamides, that employs alkylene and substituted alkylenediammonium galactarate salts, with 1:1 molar equivalency of the diacid and diamine monomer components, as precursors for the polyamides. The salts were treated with acid/alcohol and then base in order to initiate the polymerization in methanol. The polyamides, labeled as prepolyamides, precipitated from solution and were then subjected to a second polymerization (postpolymerization) in a different solvent to produce a generally larger polyamide, labeled as a postpolyamide.

text: Synthesis of Poly(galactaramides) from Alkylene- and Substituted Alkylenediammonium Galactarates Polyhydroxypolyamides (PHPAs) represent a class of synthetic polyamides derived from aldaric acid and diamine monomer units. This paper describes the synthesis of some poly(galactaramides), a class of polyhydroxypolyamides, that employs alkylene and substituted alkylenediammonium galactarate salts, with 1:1 molar equivalency of the diacid and diamine monomer components, as precursors for the polyamides. The salts were treated with acid/alcohol and then base in order to initiate the polymerization in methanol. The polyamides, labeled as prepolyamides, precipitated from solution and were then subjected to a second polymerization (postpolymerization) in a different solvent to produce a generally larger polyamide, labeled as a postpolyamide.

year: 2009

title: Toi Tuku Iho: An exploration of oranga and mana motuhake through toi

authors: Waititi, Kahurangi Rora

subjects: Toi tuku iho

abstract: This research examines how mahi toi as process, theory, and output can contribute to oranga and mana motuhake for whānau, hapū, and iwi which is explored through the tribal storytelling practices of Te Whānau a Apanui. In this thesis, I argue that kōrero tuku iho is the umbilical link to our ancestors that sustains us with teachings; cultivating a richness and depth of self and connection through story. I demonstrate that the vehicles of sharing are as varied as the stories themselves and are known as “toi” or our creative expressions of kōrero tuku iho. 
The key focus of this thesis is to reveal how toi can be used to decolonise and re-indigenise research methodologies and facilitate transformation through praxis. Iwi storytellers were interviewed, sharing that within our toi practices lie a myriad of ways to challenge colonial structures and ideologies, ground us in tīpuna knowledge systems, and provide a space to innovate solutions to the challenges of te ao hurihuri (a changing world). The kaikōrero provided a collective and relational lens that fostered the development of a Toi Tuku Iho methodology. This was processed through a creative output that I created of a digital story that was filmed to reveal how knowledge through stories can become taonga tuku iho. A Toi Tuku Iho methodology is proffered to understand the complex processes of iwi storytelling and reveal the relationality between innovation and what this thesis refers to as the tipuna continuum.
Themes explored in the thesis are an ecosystem of learning through people, place, story, whakapapa and time; how iwi oranga is viewed and contributed to by toi; and how mana motuhake is embraced and enacted through kōrero tuku iho. Furthermore, the application of kaupapa Māori creative practices to this research expands the academic confines of knowledge production to include Indigenous stories and toi. The thesis demonstrates that the research process, potential products, and outcomes, are transformed by using these practices.  Storyscapes, timescapes, landscapes and generations past and future merge together, in this research, to facilitate dreams and aspirations for future generations. The collapsing of timescapes is explored through audio-visual digital storytelling, a medium known to manoeuvre time to enhance a story. I seek to amplify the power of tribal storytelling through toi to transform the spaces we occupy both within and outside of our iwi. Mahi toi ignites our creative selves to dream and can activate a belief to explore beyond what we know. I argue that Toi Tuku Iho has the potential to engage communities, on their own terms, in their own ways and for their own means.

text: Toi Tuku Iho: An exploration of oranga and mana motuhake through toi This research examines how mahi toi as process, theory, and output can contribute to oranga and mana motuhake for whānau, hapū, and iwi which is explored through the tribal storytelling practices of Te Whānau a Apanui. In this thesis, I argue that kōrero tuku iho is the umbilical link to our ancestors that sustains us with teachings; cultivating a richness and depth of self and connection through story. I demonstrate that the vehicles of sharing are as varied as the stories themselves and are known as “toi” or our creative expressions of kōrero tuku iho. 
The key focus of this thesis is to reveal how toi can be used to decolonise and re-indigenise research methodologies and facilitate transformation through praxis. Iwi storytellers were interviewed, sharing that within our toi practices lie a myriad of ways to challenge colonial structures and ideologies, ground us in tīpuna knowledge systems, and provide a space to innovate solutions to the challenges of te ao hurihuri (a changing world). The kaikōrero provided a collective and relational lens that fostered the development of a Toi Tuku Iho methodology. This was processed through a creative output that I created of a digital story that was filmed to reveal how knowledge through stories can become taonga tuku iho. A Toi Tuku Iho methodology is proffered to understand the complex processes of iwi storytelling and reveal the relationality between innovation and what this thesis refers to as the tipuna continuum.
Themes explored in the thesis are an ecosystem of learning through people, place, story, whakapapa and time; how iwi oranga is viewed and contributed to by toi; and how mana motuhake is embraced and enacted through kōrero tuku iho. Furthermore, the application of kaupapa Māori creative practices to this research expands the academic confines of knowledge production to include Indigenous stories and toi. The thesis demonstrates that the research process, potential products, and outcomes, are transformed by using these practices.  Storyscapes, timescapes, landscapes and generations past and future merge together, in this research, to facilitate dreams and aspirations for future generations. The collapsing of timescapes is explored through audio-visual digital storytelling, a medium known to manoeuvre time to enhance a story. I seek to amplify the power of tribal storytelling through toi to transform the spaces we occupy both within and outside of our iwi. Mahi toi ignites our creative selves to dream and can activate a belief to explore beyond what we know. I argue that Toi Tuku Iho has the potential to engage communities, on their own terms, in their own ways and for their own means.

year: 2023