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    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, Saili
    Listeria 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.
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    Novel hyperthermoacidic archaeal enzymes for removal of thermophilic biofilms from stainless steel
    (Oxford University Press on behalf of Applied Microbiology International, 2023-06) Nam Y; Barnebey A; Kim HK; Yannone SM; Flint S
    AIMS: To test the efficacy of novel hot/acid hyperthermoacidic enzyme treatments on the removal of thermophilic spore-forming biofilms from stainless steel surfaces. METHODS AND RESULTS: The present study measured the efficacy of hyperthermoacidic enzymes (protease, amylase, and endoglucanase) that are optimally active at low pH (≈3.0) and high temperatures (≈80°C) at removing thermophilic bacilli biofilms from stainless steel (SS) surfaces. Plate counts, spore counts, impedance microbiology, as well as epifluorescence microscopy, and scanning electron microscopy (SEM) were used to evaluate the cleaning and sanitation of biofilms grown in a continuous flow biofilm reactor. Previously unavailable hyperthermoacidic amylase, protease, and the combination of amylase and protease were tested on Anoxybacillus flavithermus and Bacillus licheniformis, and endoglucanase was tested on Geobacillus stearothermophilus. In all cases, the heated acidic enzymatic treatments significantly reduced biofilm cells and their sheltering extracellular polymeric substances (EPS). CONCLUSIONS: Hyperthermoacidic enzymes and the associated heated acid conditions are effective at removing biofilms of thermophilic bacteria from SS surfaces that contaminate dairy plants.
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    Efficacy of commercial peroxyacetic acid on Vibrio parahaemolyticus planktonic cells and biofilms on stainless steel and Greenshell™ mussel (Perna canaliculus) surfaces.
    (Elsevier B.V., 2023-11-16) Wang D; Palmer JS; Fletcher GC; On SLW; Gagic D; Flint SH
    The potential of using commercial peroxyacetic acid (PAA) for Vibrio parahaemolyticus sanitization was evaluated. Commercial PAA of 0.005 % (v/v, PAA: 2.24 mg/L, hydrogen peroxide: 11.79 mg/L) resulted in a planktonic cell reduction of >7.00 log10 CFU/mL when initial V. parahaemolyticus cells averaged 7.64 log10 CFU/mL. For cells on stainless steel coupons, treatment of 0.02 % PAA (v/v, PAA: 8.96 mg/L, hydrogen peroxide: 47.16 mg/L) achieved >5.00 log10 CFU/cm2 reductions in biofilm cells for eight strains but not for the two strongest biofilm formers. PAA of 0.05 % (v/v, PAA: 22.39 mg/L, hydrogen peroxide: 117.91 mg/L) was required to inactivate >5.00 log10 CFU/cm2 biofilm cells from mussel shell surfaces. The detection of PAA residues after biofilm treatment demonstrated that higher biofilm production resulted in higher PAA residues (p < 0.05), suggesting biofilm is acting as a barrier interfering with PAA diffusing into the matrices. Based on the comparative analysis of genomes, robust biofilm formation and metabolic heterogeneity within niches might have contributed to the variations in PAA resistance of V. parahaemolyticus biofilms.
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    Comparative genome identification of accessory genes associated with strong biofilm formation in Vibrio parahaemolyticus.
    (Elsevier B.V., 2023-04-01) Wang D; Fletcher GC; Gagic D; On SLW; Palmer JS; Flint SH
    Vibrio parahaemolyticus biofilms on the seafood processing plant surfaces are a potential source of seafood contamination and subsequent food poisoning. Strains differ in their ability to form biofilm, but little is known about the genetic characteristics responsible for biofilm development. In this study, pangenome and comparative genome analysis of V. parahaemolyticus strains reveals genetic attributes and gene repertoire that contribute to robust biofilm formation. The study identified 136 accessory genes that were exclusively present in strong biofilm forming strains and these were functionally assigned to the Gene Ontology (GO) pathways of cellulose biosynthesis, rhamnose metabolic and catabolic processes, UDP-glucose processes and O antigen biosynthesis (p < 0.05). Strategies of CRISPR-Cas defence and MSHA pilus-led attachment were implicated via Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation. Higher levels of horizontal gene transfer (HGT) were inferred to confer more putatively novel properties on biofilm-forming V. parahaemolyticus. Furthermore, cellulose biosynthesis, a neglected potential virulence factor, was identified as being acquired from within the order Vibrionales. The cellulose synthase operons in V. parahaemolyticus were examined for their prevalence (22/138, 15.94 %) and were found to consist of the genes bcsG, bcsE, bcsQ, bcsA, bcsB, bcsZ, bcsC. This study provides insights into robust biofilm formation of V. parahaemolyticus at the genomic level and facilitates: identification of key attributes for robust biofilm formation, elucidation of biofilm formation mechanisms and development of potential targets for novel control strategies of persistent V. parahaemolyticus.
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    Biofilm formation of Vibrio parahaemolyticus : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Food Microbiology at Massey University, Campus Manawatū, New Zealand
    (Massey University, 2024-05-20) Wang, Dan
    Vibrio parahaemolyticus in seafood can cause food poisoning. There is increasing concern with the increase in reports of illness globally believed to be due to climate change affecting sea temperatures. Biofilm formation of V. parahaemolyticus is an additional concern as biofilms are more resistant to cleaning and sanitation than planktonic cells. However, little is known about the biofilm formation of V. parahaemolyticus. Strain variation and the factors determining biofilm formation were investigated in this study with the aim to provide information that can be used to design more effective control strategies. This study identified two robust biofilm forming strains (PFR30J09 and PFR34B02) from nine V. parahaemolyticus seafood isolates. Comparative genome analysis unveiled 136 unique accessory genes in robust biofilm formers. Protein-protein-interaction analysis showed interactions between UDP-glucose metabolism (Gene ontology (GO): 0006011), cellulose biosynthesis (GO: 0030244), rhamnose metabolism (GO: 0019299) and O antigen biosynthesis (GO: 0009243). Cellulose contributed to robust biofilm formation. Cellulose biosynthesis was identified as being acquired from within the order Vibrionales. The cellulose synthase operons consisting of genes bcsG, bcsE, bcsQ, bcsA, bcsB, bcsZ, bcsC were present in 15.94% (22/138) of V. parahaemolyticus. Strong biofilm-forming V. parahaemolyticus showed greater resistance to sanitizers of biofilm cells than the weaker biofilm forming cells. The effective concentrations of sodium hypochlorite for inactivating most V. parahaemolyticus biofilm cells were higher than the recommended concentration. Available chlorine of 1176 mg/L inactivated 1.74-2.28 log10 CFU/cm2 of biofilm on stainless steel surfaces and 4704 mg/L inactivated > 7.00 log10 CFU/cm2 of biofilm (to undetectable levels, < 10 CFU/cm2), except for biofilms formed by the strong biofilm formers. Peracetic acid (PAA) at 200 ppm (89.56 mg/L PAA, 471.64 mg/L hydrogen peroxide) inactivated > 5.00 log10 CFU/cm2 of biofilm from stainless steel surfaces (except for those the strong biofilm formers, see Figure 4.4). RNA sequencing (RNA-seq) identified 74 differentially expressed genes when comparing planktonic and biofilm cells of V. parahaemolyticus. These represented the rearrangement of nucleotide and energy metabolism in biofilm cells. Biosynthesis of secondary metabolites, purine and pyrimidine metabolism, propanoate metabolism, and valine, leucine and isoleucine degradation were deemed essential in the young V. parahaemolyticus biofilms. Genes of purH, purF, pdhA are potential genetic targets for biofilm prevention and control of V. parahaemolyticus. Understanding V. parahaemolyticus biofilm formation will help to design strategies to overcome the limitations of chemical sanitizers, improving product safety and quality in the seafood industry.
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    The Flagellar Transcriptional Regulator FtcR Controls Brucella melitensis 16M Biofilm Formation via a betI-Mediated Pathway in Response to Hyperosmotic Stress
    (MDPI (Basel, Switzerland), 2022-09) Guo J; Deng X; Zhang Y; Song S; Zhao T; Zhu D; Cao S; Baryshnikov PI; Cao G; Blair HT; Chen C; Gu X; Liu L; Zhang H
    The expression of flagellar proteins in Brucella species likely evolved through genetic transference from other microorganisms, and contributed to virulence, adaptability, and biofilm formation. Despite significant progress in defining the molecular mechanisms behind flagellar gene expression, the genetic program controlling biofilm formation remains unclear. The flagellar transcriptional factor (FtcR) is a master regulator of the flagellar system’s expression, and is critical for B. melitensis 16M’s flagellar biogenesis and virulence. Here, we demonstrate that FtcR mediates biofilm formation under hyperosmotic stress. Chromatin immunoprecipitation with next-generation sequencing for FtcR and RNA sequencing of ftcR-mutant and wild-type strains revealed a core set of FtcR target genes. We identified a novel FtcR-binding site in the promoter region of the osmotic-stress-response regulator gene betI, which is important for the survival of B. melitensis 16M under hyperosmotic stress. Strikingly, this site autoregulates its expression to benefit biofilm bacteria’s survival under hyperosmotic stress. Moreover, biofilm reduction in ftcR mutants is independent of the flagellar target gene fliF. Collectively, our study provides new insights into the extent and functionality of flagellar-related transcriptional networks in biofilm formation, and presents phenotypic and evolutionary adaptations that alter the regulation of B. melitensis 16M to confer increased tolerance to hyperosmotic stress.
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    Biofilm formation, sodium hypochlorite susceptibility and genetic diversity of Vibrio parahaemolyticus
    (Elsevier BV, 2023-01-16) Wang D; Fletcher GC; On SLW; Palmer JS; Gagic D; Flint SH
    Vibrio parahaemolyticus is a marine oriented pathogen; and biofilm formation enables its survival and persistence on seafood processing plant, complicating the hygienic practice. The objectives of this study are to assess the ability of V. parahaemolyticus isolated from seafood related environments to form biofilms, to determine the effective sodium hypochlorite concentrations required to inactivate planktonic and biofilm cells, and to evaluate the genetic diversity required for strong biofilm formation. Among nine isolates, PFR30J09 and PFR34B02 isolates were identified as strong biofilm forming strains, with biofilm cell counts of 7.20, 7.08 log10 CFU/cm2, respectively, on stainless steel coupons after incubation at 25 °C. Free available chlorine of 1176 mg/L and 4704 mg/L was required to eliminate biofilm cells of 1.74-2.28 log10 CFU/cm2 and > 7 log10 CFU/cm2, respectively, whereas 63 mg/L for planktonic cells, indicating the ineffectiveness of sodium hypochlorite in eliminating V. parahaemolyticus biofilm cells at recommended concentration in the food industry. These strong biofilm-forming isolates produced more polysaccharides and were less susceptible to sodium hypochlorite, implying a possible correlation between polysaccharide production and sodium hypochlorite susceptibility. Genetic diversity in mshA, mshC and mshD contributed to the observed variation in biofilm formation between isolates. This study identified strong biofilm-forming V. parahaemolyticus strains of new multilocus sequence typing (MLST) types, showed a relationship between polysaccharide production and sodium hypochlorite resistance.
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    Phenotypic properties and genotyping analysis of Bacillus cereus group isolates from dairy and potato products
    (Elsevier Ltd, 2021-04) Huang Y; Flint SH; Yu S; Ding Y; Palmer JS
    Bacillus cereus group (B. cereus sensu lato) are ubiquitously distributed in diverse environments. In this study, eight isolates including B. cereus, B. paranthracis and B. toyonensis species, from dairy and potato products, were assessed for biofilm formation, sporulation and genetic information including biofilm-related genes and toxin genes. The isolates varied in their ability to form biofilm (either at the stainless steel-liquid-air interface or floating pellicles). The amounts of biofilms of B. cereus s.l., were increased when incubated in agitation condition varied between isolates. Sporulation within the planktonic and biofilm modes of growth was compared, suggesting that biofilm is a favourable environment for B. cereus s.l. to form spores. Whole genome sequencing (WGS) was used to compare these B. cereus s.l. isolates. New sequence types (STs) of B. cereus were found in this study. Isolates that shared similar genomes had different biofilm-forming and sporulation abilities. Most of isolates tested, possessed biofilm-related genes. Different combinations of toxin-producing genes were identified in different isolates, with all isolates containing nhe while only some contained hbl and cytK. None of the food isolates contained the emetic ces gene. This study highlights the diversity of B. cereus s.l. in biofilm formation, sporulation and their genetic variables.
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    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, Yiying
    Bacillus 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.
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    The effect of calcium and milk formulations on biofilm formation of Geobacillus stearothermophilus : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Food Microbiology, the School of Food and Advanced Technology, Massey University, Palmerston North, New Zealand
    (Massey University, 2021) Wang, Tianyang
    G. stearothermophilus contaminates milk powder products from bacteria spores released from biofilms on product contact surfaces in dairy manufacturing plants. The dairy industry has observed that calcium-reduced milk protein powder is associated with reduced spore contamination of thermophilic bacteria during milk protein powder manufacture. Calcium, as a major component of milk minerals, was previously found to affect G. stearothermophilus biofilms grown on stainless steel exposed to milk formulations. Additionally, G. stearothermophilus cells cultured with additional calcium showed an increased attachment to stainless steel. The current study investigated the effect of calcium and milk formulations on cell attachment, biofilm formation, spore production and spore heat resistance of G. stearothermophilus to pinpoint the potential factors contributing to the reduced thermophile contamination in the calcium-reduced milk protein powder. The effect of calcium on biofilm formation of G. stearothermophilus dairy isolates A1, P3, and one reference strain 7953 in modified TSB media was studied to gain more insights into the role of calcium in biofilm formation of G. stearothermophilus. The presence of calcium increased biofilm cell numbers of strain A1, but reduced biofilm cell numbers of the reference strain and showed minimal effect on strain P3. Extracellular polymeric substances (EPS) quantity, in particular extracellular protein, varied between strains. Unlike the consistent biofilm promotive effect of calcium in milk formulations found in a previous study, the current findings suggest that a strain-to-strain difference exists in biofilm formation of G. stearothermophilus species in the presence of calcium. Calcium plays an important role in cell attachment by changing cell surface properties, cell physiology and metabolism, modifying cell surface structure and bridging between bacteria and substrata. In the dairy industry, milk formulations with different cation profiles may affect the cell attachment of a common contaminant G. stearothermophilus. The current study investigated the effect of calcium on cell attachment of G. stearothermophilus strains A1, P3 and 7953 to stainless steel and polystyrene substrata and characterized the cell surface charge, hydrophobicity and cell surface polymers. In addition, cell attachment in milk formulations on stainless steel was characterized. The presence of calcium increased the cell attachment of dairy isolates on polystyrene but not the reference strain, while calcium did not affect cell attachment of all strains on stainless steel. The presence of calcium changed the amount of cell surface polymers produced but not hydrophobicity. Although calcium affected the zeta potential, this did not correlate with the trend in cell attachment. It is assumed that the cell attachment of G. stearothermophilus is affected by the substrata, strain specific cell surface polymers, as well as calcium induced changes in cell surface polymers. Milk formulations (MF1 and MF2) with different cation profiles showed little effect on cell attachment of G. stearothermophilus on stainless steel, indicating a minimal effect of cell attachment to contamination levels of different cation-modified milk protein powder products in dairy manufacturing plant. The effect of total calcium concentration, total sodium concentration and bacteria growth history were investigated on biofilm formation of G. stearothermophilus in MF1 and MF2. The numbers of culturable biofilm cells of G. stearothermophilus strain A1, P3 and 7953 were compared in MF1 and MF2 over 18 h. MF1 and MF2 have similar protein, lactose and fat content except for cation profiles, where MF2 has relatively high sodium and low calcium concentrations. Biofilm formation of all three strains was lower in MF2 than in MF1, but the inhibition of MF2 was conditional and was highly dependent on the growth history of bacterial inocula. The inhibition of MF2 on dairy isolates A1 and P3 were further investigated by cation supplementation. Supplementation of MF1 with sodium decreased biofilm formation at 18 h for A1 and P3. Supplementation of MF2 with either 2 or 26 mM calcium increased biofilm formation of A1 at 18 h, and P3 at 10 h. High sodium and low calcium concentrations in the milk formulation seem to be required to inhibit biofilm formation of G. stearothermophilus. However, it is not known if the inhibitory effect of MF2 was due to the direct effect of cations on biofilms or the change of milk protein structure on biofilms due to calcium removal. Cations such as calcium and sodium were shown to affect sporulation and spore heat resistance of G. stearothermophilus. MF1 and 2 have distinctive cation profiles and therefore the sporulation and spore heat resistance of G. stearothermophilus A1, P3 and 7953 in milk formulations were investigated. MF2 effectively reduced the total biofilm spore numbers of A1 and P3 over the 18 h culture period compared to MF1, but the effect was not observed in 7953. The sporulation percentage of A1 was higher in MF1 than MF2 at 14 and 18 h, and a similar effect was observed for P3 at 10 and 14 h. Supplementation of calcium to MF2 increased the sporulation percentage of A1 at 14 and 18 h, as well as P3 at 14 h. Supplementation of sodium to MF1 decreased the sporulation percentage of A1 at 18 h. No significant difference in spore heat resistance of A1, P3 and 7953 was observed between spores produced from MF1 and MF2. Overall, the reduced biofilm formation and sporulation percentage of G. stearothermophilus in MF2 provided evidence to the reduced thermophile contamination in the calcium-reduced milk protein powder.