Siyun Wang Kazun

Non-typhoidal Salmonella is a leading cause of foodborne diarrheal disease. The prevalence of antimicrobial resistant (AMR) isolates is rising, and the overexpression of the AcrAB–TolC efflux pump is one factor often linked to multidrug resistance. We propose a targeted strategy to elicit structural changes in the TolC protein via bacteriophage (phage) infection. We hypothesize that these alterations in TolC structure will restore antimicrobial sensitivity of the host Salmonella. The purpose of this thesis was to assess changes in AMR of bacteriophage-insensitive mutants (BIMs) of Salmonella Typhimurium resistant to chloramphenicol and tetracycline, following infection with a TolC-binding phage.Using a collection of newly-isolated phages (n=72), TolC dependence was determined via relative efficiency of plating using a ΔtolC mutant of S. Typhimurium ATCC 14028. BIMs were generated by co-inoculating S. Typhimurium with TolC binding phage in tryptic soy broth (TSB) in triplicate. Every day for three days, 5 µL of co-culture and an additional 5 µL of phage lysate (~10⁹ PFU/mL) was transferred to fresh TSB. Cultures were isolated on streak plates and ten colonies selected at random; which were confirmed as phage resistant over five rounds of subculture via spot test. Minimum inhibitory concentrations (MICs) of BIMs to chloramphenicol and tetracycline were determined via broth microdilution. Additionally, the ability of this phage to alter the frequency of AMR within populations of S. Typhimurium was investigated by a serial co-culture method.One phage, SeKF 80, showed reliance on TolC. Host resistance to this phage arose after three days. Of the ten randomly selected BIMs, #1, #5, and #9 displayed twofold increases in tetracycline resistance compared to the parental strain. In contrast, nine of the ten BIMs had unchanged MICs of chloramphenicol, with only BIM 5 showing a twofold reduction in chloramphenicol resistance. Phage SeKF 80 had minimal effect on the frequency of AMR when used to infect populations of S. Typhimurium.This study demonstrates that forced evolution of the TolC receptor through phage infection can lead to evolutionary trade-offs related to antimicrobial resistance; however, the phenotypes of bacteriophage-insensitive Salmonella mutants are complex and potentially specific to the host-phage pair.

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The full abstract for this thesis is available in the body of the thesis, and will be available when the embargo expires.

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Shiga-toxin-producing Escherichia coli (STEC) have been implicated with numerous outbreaks associated with the contamination of food products. Fresh produce, a minimally processed commodity, may become contaminated during production, processing, and distribution. Bacteriophages are viruses that specifically target and utilize bacterial hosts for replication. The availability of phage-based antibacterial strategies has provided the food industry with a sustainable, natural, and cost-effective method to control contamination. Although phage products for biocontrol are currently available, the risk of potential bacterial resistance mandates continuing the characterization of new strains for future formulations.Previously isolated phages (n = 13) were sequenced and assembled de-novo. Sequences were then probed for their phylogenetic origin and annotated for the identification of potential integrase genes, and virulence or pathogenic genes. Phages (n = 3) were selected based on their ease of propagation and ability to target STEC O157. Select phages were tested for their efficacy against four strains of STEC O157 both in broth culture and on fresh Romaine lettuce at a temperature of 10°C. Whole-genome nucleotide alignment based on the large subunit of the terminase gene identified that phages were closely related, clustering into three distinct clusters and two singletons. The lack of integrase, virulence, and pathogenic genes demonstrated their suitability for the food industry. Of the three phages tested for efficacy against STEC serogroup O157 strains, all were equally effective in reducing STEC populations significantly compared to the control in liquid culture (P0.05). Likewise, phages VE04, VE05 and VE07 were effective against the tested STEC O157 strains on Romaine lettuce. Compared to control groups, log-reduction ranged from 2.6 log colony forming units (CFU) /cm2 to approximately 6 log CFU/cm2 after 3 days of storage at a temperature of 10°C (P0.05). Phages remained stable and persisted despite the absence of a STEC host in broth culture and on Romaine lettuce, confirming their stability as biocontrol agents in the food industry during storage, transport, and retail.The added understanding of phage treatments will support optimized novel strategies for the control of STEC O157 contamination in fresh produce and therefore reduce the economic and health burden associated with outbreaks.

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Listeria monocytogenes, a highly virulent foodborne pathogen, remains a threat to the food system and has led to numerous foodborne outbreaks worldwide. L. monocytogenes can establish itself in food production facilities by adhering to surfaces, resulting in increased resistance to environmental stressors. The aim of this study was to evaluate the adhesion ability of a collection of L. monocytogenes at 8°C and to analyse associations between the observed phenotypes and genetic factors such as internalin A (inlA), stress survival islet 1 (SSI-1), and clonal complex (CC). L. monocytogenes isolates (n=184) were grown at 8°C and 100% relative humidity. Growth was determined by measurement of optical density at 600 nm. Adherent cells were stained using crystal violet and quantified spectrophotometrically at 595 nm. Genotyping of inlA and SSI-1, multi-locus sequence typing, and a genome wide association were performed to elucidate the phenotype-genotype relationships in L. monocytogenes cold adhesion. Among all inlA genotypes, truncated inlA isolates had the highest mean adhered cells (ANOVA; P0.05), while isolates with the three-codon deletion inlA had the least mean adhered cells (ANOVA; P0.05). When SSI-1 was present, more cells were adhered; and less cells were adhered when SSI-1 was absent (Welch’s t-test; P0.05). Increased adhesion was associated with clonal complexes with low clinical frequency while lack of adhesion was associated with clonal complexes with high clinical frequency. A genome wide association study revealed genes which were not previously known to be associated with 8°C adhesion. The associations of genotype and 8°C adhesion suggests that there may be an evolutionary trade off between virulence and adhesion in L. monocytogenes.

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Multiple outbreaks caused by Salmonella have been linked to fresh produce. Washing in sanitizing solutions has been shown to reduce microbial populations by 90%. Bacteriophage cocktails have been suggested as an alternative to chemical sanitizers due to their effective and specific antimicrobial activity, safety and lack of effects on organoleptic properties. Lettuce and tomato plants were separately inoculated with 43 Salmonella strains, 26 most commonly associated and 17 not commonly associated with fresh produce outbreaks. Salmonella populations were measured immediately after inoculation and after 5 days. Laser Scanning Confocal Microscopy (LSCM) was performed after staining with a fluorescein isothiocyanate (FITC) labelled anti-Salmonella antibody. Romaine lettuce was inoculated with a phage cocktail + 1.0mM calcium chloride or only 1.0mM calcium chloride one day before inoculation with 3 separate Salmonella strains. Salmonella populations were measured immediately after inoculation and after 1 and 2 days. Populations of 26 strains (60.5%) increased on all plant species and cultivars, although there were significant differences (p0.05) in the extent of population increase by different strains on the same plant species/cultivar. The remaining strains displayed differential ability to colonize lettuce and tomato plants depending on plant species or cultivar. Most strains not commonly associated with fresh produce outbreaks were able to colonize the plants. LSCM showed that cells or cellular aggregates were located within stomates, in surface depressions adjacent to stomata or in random microsites not associated with specific anatomical features. Application of a bacteriophage cocktail to Romaine lettuce leaf sections 24 hours before inoculation significantly reduced (P0.05) populations of Salmonella Saintpaul S204, Saintpaul S205 and Typhimurium S441 by 2-4 log CFU/cm². The results of this study showed that the interaction between plant host and colonizing Salmonella is complex and subject to several interacting factors. Moreover, the colonization potential of Salmonella is highly variable and should be carefully considered in the selection of experimental strains for future research on the ecology of this bacterial species on growing food plants.

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Verocytotoxigenic Escherichia coli (VTEC) are bacteria that have been implicated in foodborne outbreaks, and may be transmitted to food by contaminated irrigation water. Antibiotics are not generally used in VTEC infection treatment, however the presence of antimicrobial resistant (AMR) bacteria and genes in the environment pose a threat to human health due to the potential for horizontal gene transfer.The broth microdilution technique was used to determine if VTEC strains (n = 15) isolated in 2014-2016 from irrigation water in British Columbia (BC) were resistant or susceptible to the following antibiotics: ampicillin, ceftriaxone, chloramphenicol, gentamicin, nalidixic acid, streptomycin, trimethoprim-sulfamethoxazole, and tetracycline. Compared to VTEC isolated from 2012-2013, strains from 2014-2016 had overall lower rates of antibiotic resistance. Whole genome sequences of VTEC strains (n = 67) isolated from 2012-2016 were queried against the ResFinder database and acquired AMR genes such as aph(3”)-Ib, aph(6)-Id, blaTEM-1b. dfrA8, sul2, tetA and tetB were identified in antibiotic resistant VTEC. Bacteriophages (phages) are viruses that use bacteria as a host cell, and their use as an antimicrobial against pathogens has been examined. Phages (n = 15) that target VTEC were isolated from sewage and their spotting host range was determined. Further characterization of four VTEC phages was done to determine if they had characteristics that are suitable for use in the food industry. The four phages had similar morphology to the Myoviridae family from transmission electron microscope images, had latent periods ranging from 35-55 minutes, burst sizes ranging from 15 to 57 phages per cell, and were stable over 31 days at pH 8, 10 and at 4°C, varied stability at pH 6 or at -20°C or 22°C, and not at pH 2, 4 or at 37°C. The potential for Salmonella phages (n = 34) to infect VTEC was examined by the spotting and plaquing host range assays. Nine Salmonella phages were capable of lysing VTEC, however only six were able to form plaques on VTEC. This research provides insights into AMR patterns in VTEC isolated from irrigation water in BC, and about novel phages for potential use for VTEC biocontrol in the food industry.

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Consumption of alfalfa sprouts has increased worldwide due to the nutritional value and health benefits of sprouts. However, alfalfa sprouts contaminated with Salmonella enterica (S. enterica) have been the source of many foodborne outbreaks in Europe and North America. Antimicrobial treatments for sprouted seeds are recommended by the Canadian Food Inspection Agency but the influence of different antimicrobial sanitation seed treatments on the behaviour of S. enterica during seed germination remains unclear. The goals of this study were (1) to investigate the ability of S. enterica to grow on sprouting alfalfa seeds after three different sanitation seed treatments to reduce microbial load, and (2) to understand how colonization by S. enterica and different antimicrobial treatments affect metabolites released by sprouting alfalfa seed.Alfalfa seeds inoculated with five strains of S. enterica were subject to three different seed treatments: (1) sodium hypochlorite (NaClO), (2) hydrogen peroxide (H₂O₂), and (3) an organic treatment involving a hot water dip, treatment with H₂O₂ and acetic acid. The disinfected seeds were sprouted to identify the growth characteristics of S. enterica after sanitation stress (n = 3). The populations of all five S. enterica strains which were present at 10 CFU/g immediately after sanitation treatment increased to 4 - 8 log CFU/g after 6 days of germination. After 6 days of germination, most S. enterica strains recovered from sprouts germinated from seeds treated with H₂O₂ or the organic treatment were lower than those recovered from sprouts germinated from seeds treated with NaClO. Additionally, metabolites were identified by rinsing seeds after 24 hours of germination (n = 4). Almost all of the 535 identified compounds were affected by the application of antimicrobial treatments. Specifically, the NaClO treatment diminished the levels of metabolites on uninoculated, S. enterica Agona and Typhimurium colonized sprouting seed by almost half, possibly via oxidative destruction. The organic treatment increased and decreased similar numbers of metabolites, around 30% of all detected metabolites.This study provided new insight on the ecology of S. enterica on germinating seeds, an important consideration in the development of better strategies to lessen the risk associated with sprouted vegetables.

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Cucumbers have been associated with recent Salmonella enterica (S.enterica) outbreaks. The ability of S. enterica to attach or internalize into produce may be a factor that make these produce items more likely to be sources of S. enterica contamination. The purpose of this study was to evaluate the survival capability of S. enterica on mini cucumbers and explore the factors contributing to the survival of this foodborne pathogen on the surface of cucumbers.Five strains of S. enterica representing different serotypes were individually inoculated onto mini cucumbers and subsequently incubated at 22 ± 2 °C for 8 days or at 4 ± 2 °C for 19 days respectively. Crystal violet assay was performed to quantify the biofilm formation and attachment capability based on the value of optical density at 595 nm of the destaining crystal violet at the specific interval time (0, 3, 6, 9, 12, 24, 48, 72, 96 hours). The phenotypic evaluation of red dry and rough (rdar) morphotype formation of S. enterica were conducted on Luria-Bertani (LB) agar complemented with Congo red (40 μg/mL) and Coomassie brilliant blue (20 μg/mL). The results suggested different S. enterica strains showed differential survival rates at both temperatures. S. Poona exhibited the strongest survival ability at 22 ± 2 °C with the highest Δlog CFU and maximum achieved density (Nmax) of 0.84 ± 0.01 and 6.72 ± 0.05, respectively. However, at 4 ± 2 °C, S. Enteritidis survived better compared with S. Poona due to the least cell density decrease of -0.91 ± 0.01 Δlog CFU and maximum achieved density of 6.04 ± 0.09. Besides, survival behaviors of S. enterica were found to be associated with biofilm formation ability and the biofilm ability differed among different strains. This means that biofilm formation contributes to the survival ability of S. enterica on mini cucumbers. Lastly, different strains exhibited specific morphotypes on Congo red agar, indicating that both curli and cellulose contribute to biofilm formation of S. enterica. Unique survival characteristics among S. enterica reveal that corresponding interventions need to be applied to eliminate contamination of produce with specific S. enterica strains.

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Foodborne pathogens such as verotoxigenic Escherichia coli (VTEC), Salmonella, and Listeria monocytogenes may be present in surface waters, thus having the potential to contaminate fresh produce during crop irrigation. The objectives of this study were to determine the occurrence of VTEC, Salmonella, and L. monocytogenes in surface waters used for produce irrigation in the Lower Mainland of British Columbia, and to investigate potential predictors of their presence.Water samples (n = 223) were collected from three and four irrigation ditches in both the Serpentine and Sumas watersheds, respectively, between February 2015 and August 2016. VTEC colonies on water filters were detected using a verotoxin colony immunoblot developed for the detection of all VTEC serotypes, and isolates were confirmed via multiplex PCR for virulence genes vt1 and vt2. Detection of Salmonella and L. monocytogenes was completed using Health Canada Methods MFHPB-20 and MFHPB-30, respectively. Generic E. coli (EC) and fecal coliforms (FC) were enumerated using 3M™ Petrifilm™ Count Plates, and by membrane filtration with growth on m-FC agar with 0.01% rosalic acid; this was followed by transfer to nutrient agar containing 4-methylumbelliferyl-β-D-glucuronide. Meteorological data were collected from Environment Canada records, and agricultural data were collected from the British Columbia Agricultural Land Use Inventories.The most commonly occurring pathogen was L. monocytogenes (11.2%), followed by VTEC (4.93%), and Salmonella (2.68%). Pathogen recovery was more common in the Serpentine watershed (p 0.05), especially during the winter and fall seasons (p 0.05). Pathogen occurrence in these locations was correlated with FC (r = 0.431) and EC (r = 0.408), but only by using the membrane filtration method. Pathogens were still recovered when indicator concentrations were low. Pathogen occurrence was also correlated with the proximity to upstream livestock (rs = -0.886) and the level of precipitation the day before sample collection (r = 0.203).In conclusion, VTEC, Salmonella, and L. monocytogenes are present in surface waters used for irrigation in the Lower Mainland of British Columbia. There is potential to predict their presence, but further research is required to confirm factors affecting pathogen occurrence, which entail longer sampling times and increased sampling locations.

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In North America, outbreaks of Salmonella in recent years have been linked to low-water activity (aw) foods, such as tree nuts, peanut butter and chia seed powder. The unusual emergence in microenvironments that should otherwise limit bacterial survival highlights the need for the elucidation of mechanisms that enhance Salmonella survival in low aw foods, which are currently poorly understood. The purpose of this study was to evaluate the response of Salmonella enterica to two stressors commonly encountered in low-aw food processing, desiccation and heat treatment. Five strains representing different serotypes of S. enterica were inoculated onto food matrices with varying aw: peanut oil (aw 0.521 ± 0.003), peanuts (aw 0.321 ± 0.20) and chia seeds (aw 0.585 ± 0.003) to identify survival characteristics in low-aw environments. To assess the effect of stress pre-adaptation on survival, peanut oil-desiccated cells and/or cells shocked at 45°C were subsequently subjected to 70°C. Lastly, the relative expression levels of five stress response or virulence genes (i.e. invA, fadA, otsB, rpoE and dnaK) were assessed following heat treatment or desiccation using quantitative polymerase chain reaction (qPCR). S. enterica exhibited long-term survival in the low-aw foods (up to 105 days) and showed a strain-specific response. S. Hartford and S. Thompson were identified as persistent in these low-aw foods, while Typhimurium was identified as the least persistent serotype. Furthermore, cells pre-exposed to six days of desiccation in peanut oil and/or 45°C heat for three minutes exhibited significantly (p0.05) higher resistance to 70°C heat treatment. qPCR revealed various degrees of up- and down-regulation amongst the characterized genes and across different strains under the desiccation and heat treatments. Serotypes Hartford and Thompson displayed the highest up-regulation in otsB and fadA, genes vital in desiccation response, consistent with their persistence in the survival assays. Moreover, differential expression of dnaK, a gene important for heat-tolerance was also observed across all Salmonella strains. The current research emphasizes the adaptable nature of S. enterica to stresses encountered in low-aw food processing. Additionally, unique stress response characteristics among Salmonella strains highlight the need for tailored mitigation strategies regarding high-risk Salmonella strains in the food industry.

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