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Item Optimising pre-incubation time and comparing sterility methods for the detection of thermophilic bacteria in UHT dairy products : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science (Biological Sciences) at Massey University, Manawatū, New Zealand(Massey University, 2025) Warren, JulieGlobally, UHT milk and UHT dairy products are an important source of nutrition and therefore have the requirement to be safe for consumption. Commercial sterility testing of these products ensures their safety and reduces reputational/financial losses for the dairy companies. However, temperature abuse during storage and transport has been highlighted as a concern due to potential spoilage effects from possible thermophilic contamination. Traditional commercial sterility testing methods, established by governing bodies, are time-consuming and labour-intensive, and they are not within the boundary of rapid food testing. This study compared two rapid methods: the commercially available Charm Epic ATP bioluminescence method and a flow cytometry method (newly developed as part of this study), with the traditional plate method, for assessing the commercial sterility of UHT dairy products when contaminated with low numbers of obligate thermophiles. Four different UHT product types were investigated (milk, in-house cream, whipping cream and a medical beverage). In addition, the effect of shortening the UHT pack pre-incubation time on method performance was investigated. For instance, pre-incubation times of 6-24 hours were compared with 48 hours. It was found that a pre-incubation time of 48 hours had only 50% agreement when comparing the ATP bioluminescence method with the plate reference method when measuring UHT milk. Whereas pre-incubation times of 6-24 hours had >95% agreement, thus greatly improving the performance of the ATP bioluminescence method. With UHT milk, the flow cytometry method had >95% agreement with the plate method at all pre-incubation times tested, however overall, the readings tended to be highest at 24 hours, indicating that a 24 hour pre-incubation time would have the lowest chance of a false negative result. Neither the ATP bioluminescence nor the flow cytometry method had acceptable agreement (i.e. ≥95%) with plate counting when measuring the in-house cream or medical beverage products, regardless of the pre-incubation time. Thus, it was concluded that the plate count method would be most appropriate for these matrices. Given these findings, an adjusted approach was applied to improve the performance of the flow cytometry method when analysing the whipping cream product. Firstly, additional sample preparation was used which involved mixing samples with a cation chelator, followed by centrifugation to obtain a bacterial pellet, assisting the extraction of the bacteria from the matrix. Secondly, the gate to capture the live bacterial cells on the flow cytometry plots was positioned further away from the non-bacterial background particles. These two approaches prevented the spillover of non-bacterial particles into the live gate, thereby preventing false positive results, and improving method sensitivity. This resulted in >95% agreement between the flow cytometry method and the plate method, at all pre-incubation times tested with the whipping cream. Furthermore, this contrasted results with the ATP bioluminescence method, where the highest agreement achieved was 44%, with the whipping cream. As UHT milk is the most common type of UHT dairy product, the shorter pre-incubation time in commercial sterility testing identified in this study would be highly beneficial to dairy companies, particularly when using methods alternative to the plate method. Not only would a shorter pre incubation time have potential to improve the performance of the method, but it would also allow for a faster turnaround time. There needs to be recognition of the rapid transition into the death phase of the thermophilic bacterial growth cycle, as this has potential to impact rapid testing methods, such as those that measure ATP. These findings may be adopted into industry standards and regulations in the future.Item The influence of cations on biofilm formation of Listeria monocytogenes persistence strains : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Food Technology, Institute of Food Science and Technology, Massey University, Manawatū, New Zealand(Massey University, 2024) Chalke, SailiListeria monocytogenes is a Gram-positive pathogen, that possess a considerable risk to the human health with a high mortality rate. The persistence of pathogens through severe environmental conditions could be associated with their biofilm forming abilities. In this study, four different L.monocytogenes isolates from the seafood industry, were examined for their biofilm formation ability in the presence of three the cations: magnesium, calcium and sodium that are readily available in the seafood industry. Out of four the two isolates 15G01 and 33H04, were the persistent isolates from different seafood industry in New Zealand. Isolate 15A04 was a low biofilm former and the last isolate 16A01 was associated with a mussel contamination outbreak. The divalent cations, magnesium and calcium had a significantly greater effect on biofilm formation compared to the monovalent cation, sodium, especially at a concentration of 50mM. To further understand the effect, comparative transcriptomics was used on L.monocytogenes isolate 15G01 (a persistent and high biofilm forming isolate) and 15A04 (a low biofilm former). Both the isolates were exposed to 50mM concentrations of magnesium and sodium. In the presence of magnesium, various genes related to the phosphotransferase system, flagellar assembly, chemotaxis and various signal transduction receptors were upregulated. In case of sodium, the results indicated limited effect on gene expression for both the isolates. As biofilm is a community of bacteria enclosed in a self-induced matrix called EPS (extracellular polymeric substances), understanding the influence of cations on the composition of the EPS and the structural stability of biofilm is important. Magnesium enhanced the polysaccharide content, thus enhancing biofilm formation particularly in 15G01. eDNA concentration increased in the presence of cations however there were no significant differences among the cations. A unique hexagonal structure with voids were observed for the first time in the presence of magnesium and calcium for isolate 15A04. These findings contribute insights into the role of cations in biofilm formation, their involvement in regulating the complex network in biofilms and maintaining their structural integrity.Item Unravelling the molecular and evolutionary mechanisms of copper resistance in plant-associated Pseudomonas spp. : Master Thesis, School of Natural Science, Massey University, Auckland, New Zealand(Massey University, 2024) Leung, Kon PingCopper (Cu) is a vital trace element for all living organisms but can be extremely harmful when in excess. Its antimicrobial properties have made copper compounds a popular choice in agriculture for managing plant diseases. However, the widespread use of copper-based bactericides has resulted in heavy metal pollution and the emergence of copper-resistant strains, which could potentially undermine disease control efforts. The mechanism by which copper inhibits pathogenic infection, the capacity of pseudomonads to evolve copper resistance, and the potential for co-evolution of resistance to copper and antibiotics are yet to be evaluated. Furthermore, the impact of the evolution of copper resistance on bacterial virulence remains a mystery. The central objective of my research project was to unravel the molecular and evolutionary underpinnings of copper resistance in pseudomonads, a group of bacteria that are closely associated with plants. This includes the study of Pseudomonas fluorescens SBW25, which is a model bacterium known for promoting plant growth. Additionally, my project also focused on Pseudomonas syringae pv. actinidiae (Psa) NZ13 and NZ47, the causative agent of bacterial canker in kiwifruit. By understanding the mechanisms of copper resistance in these bacteria, we can gain insights into how they adapt to environmental stressors, which could have significant implications for agricultural practices and disease management. In this work, I successfully devised a strategy to quantify bacterial infection using lux labelled bioreporter strains. This innovative technique has shed light on the inhibitory action of copper on kiwifruit infection by Psa. This newfound understanding of copper’s inhibitoryeffect can contribute to the sustainable use of copper bactericides in agriculture. Furthermore, my research has confirmed that prolonged exposure to copper can lead to the emergence of copper resistant pseudomonad strains. Interestingly, I found that resistance to aminoglycosides appears to co-evolve with copper resistance, particularly in strains derived from P. fluorescens SBW25. My work also provides insights into the potential roles of two-component regulatory systems (CopRS and EnvZ-OmpR) and transcriptional regulators (HutC and KefA). They may act as global regulators in copper resistance, antibiotic resistance, and Psa virulence. This understanding could pave the way for new strategies in managing bacterial diseases and promoting sustainable agriculture.Item Spoilage bacteria in ewe milk and the sheep dairy farm environment : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Veterinary Science/Microbiology at Massey University, Manawatū, New Zealand(Massey University, 2023-03) Risson, AlexisMilk spoilage bacteria are capable of impacting the stability, processability, and overall quality of dairy products. These contaminants are highly diverse and can be found at various stages of the dairy production, but the majority originate from the dairy farm environment where they contaminate raw milk. On bovine dairy farms, research has identified several routes of transmission of spoilage microorganisms into raw milk, as well as some of the factors that modulate this process. This knowledge has aided the design, development and implementation of interventions aiming to limit the on-farm contamination of raw milk and thereby improve the microbiological quality of cow dairy products. In contrast, the microbial communities associated with the ovine dairy production have been largely overlooked. This lack of understanding of the ecology and transmission of spoilage bacteria on sheep dairy farms and in dairy products hinders attempts at reducing the farm-borne contamination of ewe milk by spoilage organisms. In this thesis, I set out to identify and characterise the microbial communities associated with the ovine dairy production, with a focus on bacteria capable of impacting ewe milk quality. In the first part, I compiled, developed, and subsequently used an extensive set of culture methods targeting the main types of bacteria implicated in dairy spoilage, namely spore-forming bacteria, and Pseudomonas species. These methods were applied to survey the microbial communities present in the farm environment of dairy ewes that were grazed or housed. For the first time, this approach provides a detailed overview of the ecology of spoilage bacteria found on sheep dairy farms and enables the identification of numerous bacterial species with spoilage potential. The diversity and principal reservoirs of spoilage bacteria were found to vary between the two farms, possibly as a result of their farming practices. In the second part, I performed source-tracking of the contaminants isolated from milking cups using phylogenetic tools such as DNA fingerprinting and whole-genome sequencing. According to this approach, sources of milking cup, and thus possibly raw milk contamination were found to vary between the two farms. In the barn environment, the silage-faeces axis was identified as the main source of spoilage bacteria in the wider farm environment and in milking cups. There was also some contribution, albeit minimal, from bedding materials and animal drinking water as potential intermediaries. In the pasture environment, the transmission of bacteria in the environment was low, but the origin of the contaminants found in milking cups was multiple, dependent on the bacteria considered, and included soil, pasture, animal faeces and drinking water. In addition, the phylogeny of Thermoactinomyces spp., which were found to be particularly abundant in both housing and grazing environments, was studied in more depth using whole-genome sequencing. Finally, I surveyed the microbiological quality of ewe raw milk samples and sheep dairy products produced in New Zealand using the culture methodology developed for the farm studies. This work provided a glimpse of the diversity and abundance of the spoilage microbiota of sheep milk and dairy products produced in New Zealand. It was found that the microbiological quality of sheep dairy products was high, however, the presence of a highly diverse range of spoilage bacteria highlights their potential to impact the quality of a variety of dairy products.Item The maintenance and evolution of antibiotic resistance genes in the absence of antibiotic selection : a thesis submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Microbiology & Genetics at Massey University, Albany Campus, New Zealand(Massey University, 2023) Lai, Huei-YiThe rise of new antibiotic resistance in pathogenic bacteria combined with the stagnation of drug development has led to a crisis in treating bacterial infection. An understanding of factors that can influence the development and prevalence of antibiotic resistance in bacteria can help combat resistance. Bacteria can acquire antibiotic resistance via two types of genetic changes— antibiotic resistance mutations (ARMs) and acquisition of antibiotic resistance genes (ARGs). In the presence of antibiotics, these genetic changes are beneficial to bacteria but in the absence of antibiotic any fitness cost of the resistance genotype imposed on the bacteria is uncovered. The fitness cost of a resistance genotype creates a fitness difference between resistant and susceptible bacteria leading to purifying selection against resistant bacteria. As a result, the fitness cost of a resistance genotype can play an important role in the maintenance of resistant bacteria in a population, especially when the antibiotic selection is absent or weak. Previous studies have focused on the degree and mechanistic basis of the fitness cost of ARMs and of ARGs embedded in mobile genetic elements (MGEs), such as plasmids. Little is known about the fitness cost of individual ARGs, let alone its mechanistic basis. Moreover, ARGs are often associated with MGEs, which subject ARGs to frequent gene flow between bacteria. Because of this movement between host strains, any variation in the fitness cost of an ARG between different strains can influence its prevalence at the population level. Despite the potential importance of this effect in determining the success of ARGs, direct measurements of host specific fitness costs have been made for only a few distinct ARGs. Finally, compensatory evolution can alleviate the fitness cost of resistance genotypes so that both immediate and longterm costs of ARGs must be considered. In this thesis, I aim to investigate the fitness cost of individual ARGs and test its evolutionary significance. In Chapter 2, I quantify the fitness costs of six ARGs prevalent in published Escherichia coli genomes and determine the variation in costs across twelve Escherichia strains. While on average the fitness cost of the six ARGs is small, consistent with their high prevalence, the costs of most ARGs vary between hosts. I show that this variation can be consequential, resulting in host-dependent evolutionary dynamics of an ARG plasmid. In Chapter 3, I use whole genome sequencing and reverse genetics to dissect the genetic basis of the compensatory evolution observed in Chapter 2. I identify a mutation on a phage gene that can alleviate the fitness cost of a b-lactamase, and moreover, I demonstrate that the host-dependent cost of the b-lactamase is due to the negative interaction between the b-lactamase and the phage gene. Chapter 4 extends work on measuring ARG costs and determining their effect on ARG maintenance to investigate the influence of costs on the molecular evolution of an ARG. In Chapter 4, I examine if the host dependent fitness cost of the b-lactamase can influence the accumulation of genetic variation in that gene. Together, these chapters characterize the influence of the fitness cost of ARGs on their maintenance and evolution and demonstrate that, even without antibiotic selection, other selective forces continue to influence the persistence of antibiotic resistance genes in bacterial populations.Item Characterization of the effects of 'old' nitrofuran antibiotics on Gram-negative bacteria : Master of Science (Microbiology), Massey University, Manawatu, New Zealand(Massey University, 2022) Joseph, Robyn Sandy DoriannNitrofurans are “old” antibacterials that are regaining popularity over the past decade due to the low prevalence of resistance amongst formidable Gram-negative bacteria. According to reports by the WHO, Escherichia coli and Pseudomonas aeruginosa are on the list of critical pathogens for which antibiotics are urgently needed because of the emergence and widespread dissemination of antibiotic resistance. E. coli and P. aeruginosa are Gram-negative bacteria that cause various diseases, such as uncomplicated urinary tract infections and chronic infections. Furazolidone, nitrofurantoin, and nitrofurazone are three nitrofuran antibiotics currently being investigated for their utility in treating infections caused by these pathogens. Nitrofurans are prodrugs that require activation through reduction by bacterial enzymes. In E. coli, three oxidoreductases, NfsA, NfsB, and AhpF, were shown to activate nitrofurans. Nevertheless, in the absence of these three enzymes, nitrofurans can still kill E. coli, albeit at an increased concentration, suggesting that additional oxidoreductases were reducing furazolidone from a prodrug to its active form. Due to the failure to identify additional nitrofuran-activating enzymes using mutagenic screens, it was hypothesised that the putative oxidoreductases must be essential for E. coli growth. Using a bioinformatic approach, I identified 18 essential oxidoreductases as candidates for nitrofuran-activating enzymes. These were investigated via overexpression from a high-copy-number plasmid in the E. coli triple mutant ΔnfsA ΔnfsB ΔahpF. Among those enzymes, five oxidoreductases, FolD, CydA, HemA, HemG, and MurB, decreased nitrofurantoin MIC when overexpressed and are, therefore, candidates for the nitrofuran-activating factors in E. coli. Furazolidone and two other nitrofurans were also investigated for antivirulence activity in P. aeruginosa PAO1 to determine whether its potency was more pronounced than the previously studied nitrofurazone. Effect on swimming and swarming motilities, biofilm formation, and pyocyanin production were assessed. Despite being non-inhibitory to the P. aeruginosa PAO1 growth at concentrations that are lethal to E. coli, furazolidone was found to be more potent than nitrofurazone and was capable of limiting biofilm formation and pyocyanin production. Overall, the results show the value of reviving “old” antibiotics as they can be used to treat E. coli infections even in the “resistant” mutants lacking three known activation enzymes and prove effective as antivirulence compounds in P. aeruginosa.Item Identification and characterisation of rumen bacteria with prominent roles in the ruminal metabolism of forages : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy (Microbiology and Genetics) at Massey University, Palmerston North, New Zealand(Massey University, 2022) Mahoney-Kurpe, SamThis thesis documents the characterisation of two groups of rumen bacteria that are both prominent in forage-fed ruminants, with the aim to better understand their roles in ruminal metabolism. The first group, referred to as the R-7 group, has in recent years been shown to be one of the most abundant rumen bacterial groups, though the few isolated representative strains available were uncharacterised. Two strains of the group included in the Hungate1000 culture collection, R-7 and WTE2008, were selected for characterisation. To facilitate phylogenetic analyses of this group, the complete genomes of an additional three previously isolated R-7 group strains were sequenced. Genomic, phylogenetic and phenotypic characterisation of R-7 and WTE2008 demonstrated that despite their 16S rRNA gene sequences sharing 98.6-99.0% nucleotide identity, their genome-wide average nucleotide identity of 84% assigned them as separate species of a novel genus and family of the proposed order ‘Christensenellales’ using the Genome Taxonomy Database. Phenotypic characterisation showed that the strains were identical in morphology, and both possessed the ability to degrade plant cell wall polysaccharides xylan and pectin, but not cellulose. Acetate, ethanol, hydrogen and lactate were produced by both strains, though R-7 produced greater amounts of hydrogen than WTE2008, which instead produced more lactate. Based on these analyses, it is proposed that R-7 and WTE2008 belong to separate species (Aristaeella gen. nov. hokkaidonensis sp. nov. and Aristaeella lactis sp. nov., respectively) of a newly proposed family (Aristaeellaceae fam. nov.). The second bacterial group of interest, due to their dominant role in ruminal propionate production, was the Prevotella 1 group, following analyses of metatranscriptome datasets of rumen microbial communities of lucerne-fed sheep for dominant community members that express propionate pathway genes from succinate. Screening of 14 strains spanning the diversity of Prevotella 1 found that all except one P. brevis strain produced propionate in a cobalamin (vitamin B12)-dependent manner. To better understand the pathway and regulation of propionate production from succinate, a comparative multi-omics approach was used to test the hypothesis that propionate production is regulated by a cobalamin-binding riboswitch. Scanning of a completed genome assembly of Prevotella ruminicola KHP1 identified four ‘cobalamin’ family riboswitches. However, the riboswitches were not in close proximity to genes putatively involved in converting succinate to propionate, nor were these genes arranged in a single operon. Comparative genomics of the 14 screened strains found that all strains possessed all homologues of candidate propionate pathway genes identified in the KHP1 genome. However, the 13 propionate-producing strains possessed a putative transporter and three subunits encoding a putative methylmalonyl-CoA decarboxylase upstream but antisense to two genes encoding methylmalonyl-CoA mutase subunits, whereas the non-producing strain did not. Comparative transcriptomics and proteomics of KHP1 cultures in the presence and absence of cobalamin demonstrated that some gene candidates were upregulated by cobalamin at the transcriptome level, including co-located genes annotated as phosphate butyryltransferase and butyrate kinase, despite the strain not producing butyrate, suggesting that propionate production may occur via propionyl phosphate. However, only both subunits of methylmalonyl-CoA mutase showed greater transcript and protein abundances in the presence of cobalamin. These results show that while some propionate pathway candidate genes were differentially expressed between cobalamin treatments, they did not appear to be under direct control of a cobalamin-binding riboswitch. This study has contributed to our understanding of the roles of both Aristaeellaceae fam. nov. and Prevotella 1 in ruminal metabolism.Item Isolation and characterisation of Faecalibacterium prausnitzii in New Zealand calves : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Microbiology at Massey University, Manawatū, New Zealand(Massey University, 2022) Lim, Yun WenFaecalibacterium is an oxygen-sensitive bacterium found in the intestines of many animals and is of considerable interest due to the benefits it contributes to gastrointestinal tract (GIT) health and homeostasis. Faecalibacterium produces the short-chain fatty acid, butyrate, which is an important energy source for gut epithelial cells and acts as an anti-inflammatory agent in the GIT. In New Zealand, artificially reared calves that are raised in the absence of their dams are highly susceptible to gastrointestinal diseases which can result in morbidity and mortality. Understanding the roles of beneficial GIT microorganisms, and how these may enhance GIT health in calves, is highly desirable to improve calf health, growth and development. This project aimed to isolate and characterise Faecalibacterium strains from whole milk-fed New Zealand calves. Cultivation trials from calf faecal samples were conducted on Faecalibacterium enriching agar media in Petri plates or roll tubes and more than 300 colonies were screened to identify Faecalibacterium. A polymerase chain reaction (PCR) screen selective for Faecalibacterium was developed using a combination of universal and Faecalibacterium-specific 16S rRNA gene primers, and only one isolate was identified as Faecalibacterium via 16S rRNA gene sequencing. However, other closely related members of the Ruminococcaceae have also been isolated: two Fournierella massiliensis isolates and one member of Gemmiger. Genome sequencing and analyses showed that in both the Faecalibacterium and Gemmiger isolates, genes in the pathway from pyruvate to butyrate were present, consistent with their ability to produce butyrate. The Fournierella isolates were not observed to produce butyrate, and instead, ethanol was a major fermentation end-product. The genome of the Faecalibacterium isolate had an average nucleotide identity (ANI) of 96.05% compared to that of the human-derived F. prausnitzii strain A2-165, and an ANI of 85.01% compared to the F. prausnitzii type strain 27768T, suggesting genomic differences between calf and human-derived isolates. This study will provide a better understanding of microbial diversity and function within New Zealand calves, and the bacteria isolated in this study may serve as beneficial microbes to help reduce scours or other gastrointestinal diseases.Item Rapid phenotypic switching in a natural isolate of Escherichia coli : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Genetics at Massey University, Albany, New Zealand(Massey University, 2022) Pearless, Stella MargaretThe survivability of any given bacterial population is dependent on its genetic and phenotypic makeup. When cells replicate they usually produce genetically identical daughter cells through a process called binary fission. Although these daughter cells may remain genetically identical and form isogenic populations, bacteria also possess the ability to alter their phenotype independently of other cells in their population. This can result in subpopulations of phenotypically variable cells forming within a larger population, rendering them phenotypically heterogeneous. Phenotypic heterogeneity can arise from multiple molecular mechanisms that cause either genetic or epigenetic changes. Genetic mechanisms include site-specific DNA inversion, slipped-strand mispairing and homologous recombination. Epigenetic changes involve modifications to DNA at the structural level, and in bacteria most commonly refer to methylation. Heterogeneity can provide evolutionary advantages through processes like bet-hedging or the division of labour. A well documented example of evolutionarily advantageous phenotypic heterogeneity is the formation of persister cells within bacterial strains, a leading cause of antibiotic treatment failure. In this study, we have identified an Escherichia coli natural isolate strain (SC375) that is able to rapidly switch between two phenotypes. The phenotypic heterogeneity demonstrated in this strain results in varying colony morphologies influenced by their cellular composition. We initially proposed a DNA inversion mechanism for this switching but subsequently confirmed that all cells remain isogenic regardless of cell phenotype. Through RNA-sequencing we identified three virulence genes that were differentially regulated in both phenotypes, suggestive of an epigenetic regulatory mechanism. We then show, using reporter assays, that two of these genes are expressed in variable levels across subpopulations. We suggest that the rapid phenotypic reversibility of this strain is a possible indicator of epigenetic memory.Item The spore formation and toxin production in biofilms of Bacillus cereus : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Food Technology at Massey University, Palmerston North, New Zealand(Massey University, 2021) Huang, YiyingBacillus cereus (B. cereus) is a foodborne pathogen causing diarrhoea and emesis which are the consequences of enterotoxin and emetic toxin production, respectively. Sporulation and biofilm formation are used as survival strategies by B. cereus protecting cells from harsh environments. However, these survival strategies also make B. cereus more difficult to control in the food industry. The aim of this study is to investigate the spore formation and toxin production in the biofilm of B. cereus. In this study, higher sporulation and higher spore heat resistance were demonstrated in biofilms grown on stainless-steel (SS) compared to planktonic populations. The structure of coat in spores isolated from biofilms, the upregulated germination genes in planktonic cells and upregulated sigma factor B in biofilm cells are possible explanations for these observations. The levels of dipicolinic acid (DPA) did not affect the heat resistance of spores harvested from biofilms in this study. Haemolytic toxin (Hbl) was mainly secreted by cells into surrounding media while emetic toxin (cereulide) was associated with cells. Higher Hbl toxin was observed in the presence of biofilms grown on SS compared to either planktonic culture or biofilm grown on glass wool (GW) using the Bacillus cereus Enterotoxin Reverses Passive Latex Agglutination test (BCET-RPLA). This was supported by the significant (P < 0.05) increase in HblACD expression in biofilm cells on SS, using both real-time quantitative PCR (RT-qPCR) and RNA sequencing. The transcriptomic analysis also revealed that biofilms grown on SS had an upregulated secretion pathway, suggesting biofilms of B. cereus grown on SS are more pathogenic than planktonic cells. Unlike the Hbl toxin, cereulide was associated with biofilm cells/structures and attached to the biofilm-forming substrates including SS and GW used in this study. The expression of cerA and cerB was similar between biofilms and planktonic cells using RT-qPCR. This project highlights the importance of biofilms by B. cereus in food safety through the enhanced heat resistance of spores, the higher Hbl toxin production and attached cereulide toxin.