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Item Global expansion of Vibrio parahaemolyticus threatens the seafood industry: Perspective on controlling its biofilm formation(Elsevier Ltd, 2022-03-15) Wang D; Flint SH; Palmer JS; Gagic D; Fletcher GC; On SLWAs global warming increases the geographical range and frequency of Vibrio parahaemolyticus infections, its formation of biofilms providing bacteria greater resistance to stress and contributing to the persistence of pathogens, is threatening the seafood industry. V. parahaemolyticus has a number of advantages leading to biofilm formation. This study reviews recent advances in understanding V. parahaemolyticus biofilm formation on biotic and abiotic surfaces, discusses research gaps in the mechanism of biofilm formation and examines promising biofilm control strategies to overcome current limitations of chemical disinfectant. This information will deepen our understanding of V. parahaemolyticus biofilm formation, as well as help design and optimize V. parahaemolyticus biofilm control strategies for the seafood industry.Item 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 SHVibrio 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.Item Enzymes produced by bacteria within biofilms of dairy origin and their effect on dairy products : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy, Massey University, Palmerston North, New Zealand(Massey University, 2013) Teh, Koon HoongPrior to the current study, there was no scientific evidence that enzymes produced by bacteria within biofilms in milk transport tanker could have a detrimental effect on the quality of dairy products. Bacteria attached to the internal surfaces of milk tankers were isolated, identified, and characterized in terms of their ability to produce heat-stable enzymes (protease and lipase) and to form biofilms. Twelve of the bacterial isolates were identified by 16s DNA sequencing as belonging to the genera Bacillus, Staphylococcus, Streptococcus, Pseudomonas, and Serratia. Six of the dairy bacterial isolates were evaluated for their ability to produce proteolysis in milk when growing within either single culture or co-culture biofilms in an in vitro model system that simulated the upper part of a milk tanker during a typical summer’s day of milk collection in New Zealand. Proteolysis per cfu decreased as the temperature of incubation increased (20-37 °C), and proteolysis per cfu was generally higher within biofilms compared with the corresponding planktonic cultures. Lipolysis by bacteria within biofilms in the in vitro model was investigated using single or co-culture biofilms or planktonic cultures of four dairy bacteria and a known lipase-producing bacterium. The hydrolysis of p-nitrophenol palmitate was at least 10 times higher by bacteria within biofilms (0.01 to 8.35 nU/CFU) than in planktonic cultures (0.01 to 0.07 nU/CFU). The effect of proteases on UHT skim milk was determined by exposing sterile skim milk to a multispecies biofilm formed on an in vitro model of a milk tanker. The amount of free peptides which indicated proteolysis in the UHT milk was monitored over five months of storage. Free peptides were higher in UHT milk that had been made from milk exposed to the multispecies biofilm, than in UHT milk that had been made from milk that had not been exposed to the biofilm. Enzymes that are secreted from biofilms into raw milk during transportation can potentially reduce the quality of dairy products. Improvements at this early stage of dairy manufacture may reduce economic loss in the dairy industry.Item The effect of silver ion-implantation of stainless steel on bacterial adhesion and biofilm formation : a thesis presented in partial fulfilment of the requirements for a Master of Science in Microbiology at Massey University, Palmerston North, New Zealand(Massey University, 2012) Dhillon, MehakBiofilms comprise an assembly of microbial communities attached to a surface and enclosed in a polysaccharide matrix. Biofilms are a problem in dairy manufacturing plants where they cause biofouling of the stainless steel surface, resulting in product contamination and the need to shorten manufacturing runs for frequent cleaning. The equipment has to be thoroughly cleaned at regular intervals to remove biofilms. Clean-In-Place (CIP) procedures used in dairy manufacturing plants are not effective enough to remove all the biofilm cells as the extracellular polysaccharide (EPS), an integral part of a biofilm, reduces the penetration of cleaning agents. One possible alternative method to control biofilm growth involves surface modification of the stainless steel by implanting silver ions to prevent the attachment of viable bacteria that would otherwise form biofilms. Stainless steel coupons, implanted with 1 × 1016 silver ions per cm2, and control stainless steel coupons were tested for the attachment of Streptococcus thermophilus and Pseudomonas fluorescens in various media for up to 30 minutes. Biofilm formation and EPS production for up to 24 hours was studied on the silver-implanted and control coupons in whole milk, skimmed milk and whey. It was found that there was higher attachment (0.49 and 0.18 log CFU per cm2 of S. thermophilus and P. fluorescens, respectively) on the stainless steel coupons than on the silver-implanted coupons in saline. In the presence of milk and whey, the difference in the attachment of bacteria on the two coupons reduced. Biofilm studies showed that the number of bacteria colonising both types of coupons was not statistically significantly different (P > 0.05). While the Live/Dead® BacLight™ Bacterial Viability stain showed that there were a few dead cells on the silver-implanted surfaces, scanning electron micrographs showed that the bacteria attached to a conditioning layer formed by the milk and whey proteins. Furthermore, both bacteria produced EPS, which, along with the conditioning film, might have masked the effect of silver ions from bacteria, resulting in similar numbers of bacteria present on the test and control coupons. Thus, due to the shielding effect of EPS and the conditioning film, the silver-implanted surfaces may be of limited practical value in the dairy industry.Item Mechanistic, neural network, and intelligent hybrid models for a three-phase fluidised-bed biofilm reactor : a thesis presented in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Environmental Engineering at Institute of Technology and Engineering, Massey University(Massey University, 1998) Hong, Yoon-SeokOver the past three decades, considerable amount of research efforts have been undertaken in order to develop a mathematical model for a three-phase fluidised-bed biofilm reactor (TPFBBR). Although biofilm properties such as biofilm thickness and its density are allowed to vary with biofilm growth in the model to simulate the real TPFBBR system, they are assumed to be constant in the majority of models developed for a TPFBBR. The main goal of this thesis is to develop mathematical models incorporating dynamic biofilm growth for a TPFBBR using three different modelling approaches such as a mechanistic model, a neural network model, and an intelligent hybrid model with a neurofuzzy model. This thesis consists of three parts. Firstly, a dynamic biofilm growth model, which reflects the variation of biofilm thickness and its density in time, is developed. This model is derived from a biomass balance equation and is solved by the method of characteristics. The biofilm detachment model is proposed and incorporated within the dynamic biofilm growth model. The dynamic biofilm growth model with detachment is then combined with a reaction-diffusion model and reactor model to form an integrated model of a TPFBBR. Simulation method of integrated model incorporating the dynamic biofilm growth model is developed. It is observed that results predicted are in good agreement with experimental data and the integrated model proposed provides a valuable tool to predict performance of a TPFBBR. Secondly, the sequential neural network model, which is composed of two parts, namely, the neural process estimator and the neural process predictor, is developed to describe the task of process estimation and prediction for a TPFBBR. In order to implement the sequential neural network model, multilayer feedforward neural network (MFNN) with cascaded-correlation (C-C) learning and extended Kalman filtering (EKF) learning, and generalized regression neural network (GRNN) are used. Results shows that the sequential neural network model has the feasibility as intelligent estimators and dynamic predictors and gives considerably good results in process estimation and prediction for a TPFBBR. Finally, this thesis shows how a combination of both mechanistic and empirical modelling approaches, called a hybrid model, can be implemented and utilised for modelling a TPFBBR. The neurofuzzy model as an empirical part of hybrid model is used to estimate the variation of the biofilm thickness and biofilm density, and is combined with mechanistic model-based reaction-diffusion and axial-dispersion models to predict the dynamic behavior and performance of a TPFBBR according to the variation of biofilm density and biofilm thickness. This hybrid modelling approach due to its flexibility shows a unified framework through incorporation of strong points of both mechanistic and empirical models, and provides a new modelling framework with a great potential to be applied to other types of biofilm reactors.Item Surface characteristics of an adhesive thermophilic spore-forming Bacillus, isolated from milk powder : 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, 2008) Palmer, Jon StuartThe growth of thermophiles during the manufacture of milk powder leads to a progressive increase in the number of thermophilic bacteria contaminating the final product. The limited residence time of the milk in the plant during milk powder manufacture and the concentration effect of converting milk into milk powder cannot explain the number of thermophiles found in the final product. This suggests that thermophiles are attaching to the large surface area of stainless steel found within a milk powder plant and then growing and developing into biofilms, with individual cells and/or biofilm fragments sloughing off into the product line and thus contaminating the final product. The aim of the present study was to investigate the attachment mechanisms that enable the thermophile Anoxybacillus flavithermus (B 1 2) to attach to stainless steel surfaces. Passing a B 1 2 culture through a column of stainless steel chips, collecting the first cells to pass through, re-culturing and repeating the process six times, resulted in the isolation of a mutant, labelled X7, with lO-fold reduced ability to attach to stainless steel as well as a reduced ability to attach to plastic and glass. A comparison of bacterial cell surface properties indicated that X7 was less hydrophobic than its parental strain B 1 2 . Cell surface charge measurements also suggest that X7 has less net negative surface charge. Disruption of extracellular polysaccharides and DNA appeared to have no effect on the attachment process. Removal of surface proteins caused a reduction in attachment of B 1 2 and X7 as well as a reduction in surface hydrophobicity suggesting surface protein involvement in both. Analysis by two-dimensional gel electrophoresis of lysozyme/mutanolysin extracted surface proteins revealed two proteins expressed at reduced levels in X 7 compared with B 1 2 . One protein was identified by mass spectrometry as the cytoplasmic enzyme Formate acetyltransferase. The role of Formate acetyltransferase and the second unidentified protein on the attachment process of Anoxybacillus flavithermus remains unclear.Item Biofilm formation of Enterobacter sakazakii on three different materials of infant feeding tube : a thesis presented in partial fulfillment of the requirements for the degree of Master of Technology in Food Microbiology at Massey University, Palmerston North, New Zealand(Massey University, 2009) Md Zain, Siti Norbaizura BintiThe aim of this study was to observe biofilm formation by Enterobacter sakazakii (E. Sakazakii) from different clinical, dairy and environmental origins on three infant feeding tubes made of different materials. Infant formula milk was selected as the medium for E. sakazakii growth. Seventeen isolates from different origins were retrieved and tested for purity, using a plating method and biochemical tests to eliminate the non E. sakazakii strains from this study. A method to rapidly and accurately detect viable cells of E. sakazakii on infant feeding tube surfaces using of the BacTrac® 4000 microbiological growth analyser was developed. The sources of errors such as from cleaning, operation and handling procedures were assessed prior to experimental runs. The strength of biofilm formation by different isolates of E. sakazakii on plastic surfaces was scrutinised using a microtiter plate assay. The results from the microtitre plate assay were based on the absorbance at 550 nm of crystal violet stained films and showed that all the clinical isolates were able to attach and form strong biofilms on the plate. Some environmental isolates formed strong or weak biofilms and some did not produce biofilm at all. However, dairy isolates formed both strong and weak biofilms in the microtitre plate when incubated in 10% reconstituted infant formula milk. The further studies were to quantify biofilm formation by three isolates of different origin on three different materials of infant feeding tubes using a batch system. Tubing pieces were incubated with infant formula milk inoculated with E. sakazakii cells at approximately 8 log CFU mL-1 and the biofilm formation was assessed at three time intervals: 4, 12 and 24 hours. Biofilm formation on the tubing by clinical isolates was also observed using epifluorescence microscopy and the scanning electron microscope. E. sakazakii from clinical, dairy and environmental isolates were able to form biofilm on three different materials of infant feeding tubes. The results showed that the initial attachment at 4 h on silicone tubing was low compared with the other two tubes. The scanning electron micrographs showed the surface characteristics of each tubing and the biofilm formation by E. sakazakii clinical isolates after 4, 12 and 24 hours. Silicone tubing appeared to be the best choice for premature babies that need feeding using feeding tubes, as it was slow to become colonised compared with the PVC and polyurethane tubing.Item Biofilm formation by Campylobacter jejuni in controlled mixed-microbial populations : a thesis presented in partial fulfillment of the requirements for the degree of Master of Technology in Food Technology at Massey University, Palmerston North, New Zealand(Massey University, 2008) Teh, Koon HoongPoultry meat consumption in New Zealand has been increasing since 1975 with the highest peak reported in 2006. The total poultry meat consumption was 36.5 kg per capita in the year ending September 2006. Consumption of contaminated food with raw poultry can lead to campylobacteriosis, which is a food-borne disease that causes gastroenteritis in humans and it is a major problem in New Zealand. There were 12,776 reported cases of campylobacteriosis in 2007, which accounts for 65.9% of the overall notified diseases. Campylobacteriosis can lead to Guillain-Barré syndrome in some patients, an autoimmune disorder of the peripheral nervous system. Campylobacteriosis is caused by consumption of either Campylobacter jejuni or Campylobacter coli. Campylobacter spp. have been found in commercially raised poultry being infected predominantly by C. jejuni. C. jejuni has been found associated with biofilms of other bacterial species in the watering supplies and plumbing systems of animal husbandry facilities and animalprocessing plants. A biofilm is an assemblage of microbial cells that is associated with a surface and the cells are enclosed in a matrix of polysaccharides, which provides a survival advantage to the bacteria in the film. In this study, the ability to form biofilm was measured in a laboratory assay using microtitre plates. C. jejuni strains in monoculture were shown to attach to the abiotic surface and form biofilms to various degrees, thus potentially enhancing their survivability in the poultry environment. C. jejuni was also shown to have the ability to attach and survive in mixed-microbial populations. Biofilm formation may play a role in the epidemiology of C. jejuni infections. Enterococcus faecalis and Staphylococcus simulans may play a role in the biofilm formation in the poultry environment as both of these microorganisms were able to form, and harbour C. jejuni in their biofilms. Pseudomonas aeruginosa seemed to inhibit biofilm formation and C. jejuni in the mixed-microbial population. Further studies are required to establish control measures against the formation of biofilms containing C. jejuni in poultry processing plants and farms in New Zealand to reduce the reservoir of contamination and thus reduce the incidence of campylobacteriosis.