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Item The dynamics of drug resistance evolution and diagnosis in Mycobacterium tuberculosis : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy (PhD) in Genetics/Genomics, Massey University, Manawatu, New Zealand(Massey University, 2024-07-30) Fong, Yang (Richard)Tuberculosis (TB) remains a critical global health challenge with over 10.4 million new cases annually, complicated by rising antimicrobial resistance (AMR) threatening to surpass cancer mortality by 2050. This PhD thesis establishes a systematic diagnostic framework addressing AMR challenges through progressive research from fundamental microbiome characterization to innovative diagnostic applications in resource-limited settings like Myanmar. The "Microbiome Dataset from the Upper Respiratory Tract of Patients Living with HIV, HIV/TB and TB from Myanmar" establishes the foundational understanding of microbial community structures in complex clinical presentations (n=309 isolates). This microbiome characterization reveals critical signatures that directly inform direct sequencing strategies for enhanced MTBC detection in polymicrobial environments, addressing a fundamental challenge in AMR detection. Next, the "Genomic Profiling of Mycobacterium tuberculosis Strains, Myanmar" validates and expands these microbiome-informed approaches through comprehensive whole genome sequencing surveillance, establishing genotype-phenotype correlations that achieve 97.8% concordance with phenotypic testing. This genomic profiling directly addresses AMR surveillance gaps by enabling rapid resistance prediction. Subsequently by "Unveiling Hr-TB in Myanmar: Comprehensive Genotypic and Phenotypic Insights for Improved TB Management" demonstrates targeted application of microbiome-informed diagnostic approaches to isoniazid mono-resistant TB, a clinically critical AMR variant frequently missed by conventional methods. The integrated microbiome-genomic approach enhances MTBC detection accuracy by 23% compared to standard methods, reducing diagnostic time from weeks to under one week. Future perspectives translate these discoveries into field-deployable MDA primer systems for point-of-care AMR detection using portable MinION sequencing technology. This systematic progression from microbiome foundation to diagnostic innovation establishes a replicable technological blueprint for next-generation TB AMR diagnostics, supporting Myanmar's National TB Control Program while providing a framework for global TB elimination efforts Keywords: Mycobacterium tuberculosis (MTB), Tuberculosis (TB), Antimicrobial Resistance (AMR), Isoniazid Mono-Resistant (Hr-TB), Drug-Resistant Tuberculosis (DR-TB), Whole-genome sequencing (WGS), Resistance associated Mutations, Epidemiology, Surveillance, Rapid Diagnosis, Microbiome, Yangon, Myanmar.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 Metagenomic analysis and culture-based methods to examine the prevalence and distribution of antimicrobial resistance on two New Zealand dairy farms : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Veterinary Science at Massey University, Palmerston North, New Zealand(Massey University, 2022) Collis, RoseAntimicrobial resistance (AMR) is a global threat to human and animal health, with the misuse and overuse of antimicrobials being suggested as the main driver of resistance. In a global context, New Zealand (NZ) is a relatively low user of antimicrobials in animal production. However, antimicrobial usage on NZ dairy farms and its potential for driving the spread of AMR within the dairy farm environment is under-researched. This research addresses the hypothesis that antimicrobial use on NZ dairy farms influences the prevalence of AMR in dairy farm environments, taking into consideration seasonality and contrasting farm management practices. The aims of this study, focused on two NZ dairy farm environments over an 15 month period, were to (i) determine the prevalence and distribution of AmpC- and extended-spectrum β-lactamase (ESBL)-producing Escherichia coli, utilising culture-based methods, and (ii) to determine the abundance and diversity of antimicrobial resistance genes (ARGs), utilising a metagenomic approach, and lastly (iii), to assess the impact of systemic antimicrobial treatment on the bovine faecal microbiome. Overall, the research presented in this thesis has shown a low sample level prevalence of ESBL-producing E. coli from two NZ dairy farms (faeces 0%, 1.7%; farm dairy effluent (FDE) 0%, 6.7% from Dairy 1 and Dairy 4, respectively) but AmpC-producing E. coli were more frequently isolated across both farms (faeces 3.3%, 8.3%; FDE 38.4%, 6.7% from Dairy 1 and Dairy 4, respectively). AmpC- and ESBL-producing E. coli were isolated in spring and summer, during months with varying levels of antimicrobial use. Analysis at the individual animal level showed a decrease in bacterial diversity and richness during systemic antimicrobial treatment and in many cases the microbiome diversity recovered post-treatment when the cow re-entered the milking herd. Compared to overseas data in a similar context, NZ dairy farm environments had a low abundance of ARGs, with the highest abundance detected in soil (0.20 - 0.63 copies of ARG per 16S rRNA gene). However, many of the ARGs identified in soil are not frequently found in human pathogens or acquired genes. FDE had a lower ARG abundance but the ARGs were more diverse (0.03 - 0.37 copies of ARG per 16S rRNA gene). There was no association between the normalised ARG abundance and antimicrobial use or collection date, however the low ARG abundance in the farm samples may have made any associations difficult to detect. AMR is a burden for human, animal and environmental health and requires a holistic "One Health" approach to address. The outcomes from this research improve our understanding of the current levels of AMR on two NZ dairy farms and identifies areas for future research. Prevention is better than a cure and urgent action is required to slow the development and dissemination of AMR and to improve antimicrobial stewardship in humans and animals.Item Extended-spectrum β-lactamase (ESBL) and AmpC β-lactamase (AmpC) producing Escherichia coli in dairy calves from the Canterbury region : a dissertation presented in partial fulfilment of the requirements for the degree of Master of Veterinary Studies in Veterinary Public Health at Massey University, Manawatū Campus Palmerston North, New Zealand(Massey University, 2020) Mwenifumbo, MerningAntimicrobial resistance (AMR) has become a global public health concern threatening current effective prevention and treatment options. Infections caused by antimicrobial resistance strains are costly, often harder to treat and sometimes fatal. One group of bacteria that contributes to AMR are the extended beta lactamase (ESBL) and AmpC beta lactamase (AmpC) producing E. coli. These are on the critical list of important antibiotic-resistant bacteria of human importance compiled by the World Health Organisation and are a public health concern due to their resistance to an extended range of beta lactams. The main driver for the spread of AMR is the use of antimicrobials in both human and animals. One potential spread is by the feeding of waste milk to calves. Waste milk is the milk that contains antibiotics or other drugs. Waste milk is not recommended as feed for calves due to its association with the development of antibiotic resistance bacteria. Using the culture depended methods and whole genome sequencing, this study aimed to determine the prevalence of ESBL and AmpC producing E. coli isolated from recto-anal mucosal swabs (RAMS) from waste milk fed dairy calves, and to phenotypically and genotypically characterise ESBL and AmpC producing isolates. Recto-anal mucosal swabs samples (n = 40) from waste milk fed dairy calves collected from Canterbury region, New Zealand were screened for antimicrobial resistant E. coli. Fifty-eight percent (23/40) of the calves harboured antibiotic resistant E. coli. 25% (10/40) calves were positive for AmpC producing E. coli and none of the calves were positive for ESBL producing E. coli. The highest prevalence of resistance was observed for tetracycline. PCR and Sanger sequencing revealed that all the AmpC positive E. coli were chromosomal mediated with four mutations in the promoter region of the ampC gene. Whole genome sequencing of eight isolates resistant to both tetracycline and streptomycin revealed additional resistance genes that were not tested phenotypically. Using the Clermont phylogrouping method of E. coli, the AmpC positive, the tetracycline and streptomycin resistant E. coli isolates were distributed among phylogroups B1, C and D. In conclusion, this study revealed the presence of AmpC producing E. coli and other resistance genes in E. coli isolated from waste milk fed calves. Further epidemiological studies are required to determine whether these antibiotic resistant E. coli are associated with waste milk.Item Synergistic triple combination antibiotic therapy for Gram-negative bacterial infections : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Microbiology and Genetics at Massey University, Manawatu, New Zealand(Massey University, 2021) Olivera, Catrina DianeThe continuing emergence of multidrug-resistant bacteria and the slowing down of the discovery and development of novel antibiotics have made antimicrobial resistance an ominous threat to human health. As reflected in the World Health Organization’s priority pathogens list, this problem is notably more severe in multidrug-resistant Gram-negative bacteria. This situation needs to be rectified through alternative approaches, such as the revival of 'old' antibiotics and the development of combination therapies. This thesis focuses on the combination of the 'old' antibiotics, nitrofurans and vancomycin (VAN) with the secondary bile salt sodium deoxycholate (DOC). The synergistic interaction of these antibacterials was demonstrated in the in vitro growth inhibition and killing of Gram-negative bacteria, including the clinically relevant pathogens such as carbapenemase-producing Escherichia coli, Klebsiella pneumoniae, and Acinetobacter baumannii. The synergy increased the efficacy and reduced the doses of each of the components compared to monotherapy use, with the advantage of mitigating nitrofuran mutagenicity. Using a transcriptomics approach, underlying mechanisms of the individual and combined action of nitrofurans, VAN, and DOC in E. coli were elucidated. The nitrofuran antibiotic, furazolidone (FZ), and DOC elicited highly similar gene perturbations indicative of iron starvation response, decreased respiration and metabolism, and translational stress. VAN, on the other hand, induced extracytoplasmic stress response in agreement with its known role in peptidoglycan synthesis inhibition. Through genetic and biochemical approaches, Fur (ferric uptake regulator) protein inactivation was confirmed to be important in the synergy of FZ and DOC and to contribute to the synergy of the triple combination. Similarly, the SOS response to DNA damage was shown to be essential for the synergy between FZ and VAN and to also contribute to the synergy of the triple combination. Taken together, the findings of this thesis strongly suggest the presence of multiple interaction points, that leads to the triple synergy, and support the proposed mechanism of synergy where the combined effects lead to the amplification of damaging effects and suppression of resistance mechanisms. Overall, this thesis shows the synergistic triple combination of nitrofurans, DOC, and VAN as a promising therapy for Gram-negative infections. Furthermore, this work significantly increases the understanding of drug interaction mechanisms that lead to synergy, which is hoped to help advance this combination further into the development pipeline. Transcriptomics analyses and the follow-up experiments provide key fundamental insights into the physiological impact that these three antimicrobials have on enterobacterium E. coli and highlight the advantage of combined targets in bacterial killing. These findings, in turn, will help design novel antibiotics, mono- or combined therapies, against multidrug-resistant bacteria.Item Persistence of Listeria monocytogenes in response to a natural antimicrobial, nisin : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Food Microbiology at Massey University, Palmerston North, New Zealand(Massey University, 2019) Wu, ShuyanThe persistence of bacteria can be defined by prolonged existence of a proportion of a population in an environment despite antimicrobial treatment. Food contamination by Listeria monocytogenes (L. monocytogenes) may be due to the persistence of L. monocytogenes despite antimicrobial treatments aimed at controlling this pathogen. How L. monocytogenes persists and survives in food environments is poorly understood. The persistence of cells exposed to antibiotics is well recorded however the persistence following treatment with natural antimicrobials like the bacteriocin nisin (used for controlling food pathogens in a variety of food systems across a wide pH range) has not been determined. This study used two L. monocytogenes isolates, one from food and one from an animal origin, to optimize methods for obtaining L. monocytogenes persisters following nisin treatment. L. monocytogenes were treated with nisin, and a biphasic killing pattern was observed for both strains. Persister cells formed a population of cells showing tolerance to the high concentrations of nisin and proved to be non-resistant when regrown and re-exposed to nisin. Next, this study examined persistent subpopulations of L. monocytogenes following exposure to nisin treatment under different environmental conditions (such as rich/poor media and variations in pH) representing different food systems. The pH and nutrient levels influenced the production of persister cells of L. monocytogenes. Stationary phase cells re-suspended in a nutrient rich environment (Trypticase Soy broth, TSB) showed greater survival than in a nutrient limiting environment (spent TSB medium) under nisin treatment. However, traditional microbiological techniques such as agar plating are limited in providing information of persister cells exposed to antimicrobial substances. Flow cytometry has potential to identify persister cells that although they cannot be cultured, can still pose a threat to human health. The L. monocytogenes stationary phase populations in spent medium or re-suspended in fresh TSB medium were individually treated with nisin, and the treated cells were assayed using flow cytometry combined with live/dead staining. The results revealed a reduction in viable populations and the differentiation of living cells and dead/damaged cells within the treated population. This finding was consistent with previous results from plate count experiments. Distinct changes in cell permeability were detected within 2hrs of the nisin treatment in spent medium and re-suspended in fresh medium, indicating possible variations in the mechanisms of persister formation between the two groups. The persistence of a L.monocytogenes stationary phase population when facing nisin treatment was investigated for gene expression with RNA extracts obtained after 90mins of nisin treatment. RNA Seq analysis was used for gene expression profiling of the persister cells in spent (persister N) and rich medium (persister TN) compared with untreated cells. Functional genes associated with the persister populations were identified in multiple systems including heat shock related stress response, cell wall synthesis, ATP-binding cassette (ABC) transport system, phosphotransferase system (PTS system), and SOS/DNA repair. Differences were found in gene regulation between persister N and persister TN populations. Nutrition may be associated with the variations in gene expression resulting in variations in the size or composition of the persistent populations. This study provided information on the formation of persister cells exposed to nisin and provides some insight into possible mechanisms of impeding bacterial persistence.Item Resistance of environmental bacteria to heavy metals and antibiotics in selected New Zealand soils : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Health Sciences at Massey University, Wellington, New Zealand(Massey University, 2020) Heydari, AliThe usage of superphosphate fertilizers, animal remedies and other material containing heavy metals (HMs) in agriculture and horticulture is a problematic issue resulting in the accumulation of HMs in the soil. The presence of these HMs in the soil leads to the induction of resistance of environmental bacteria to these heavy metals and may co-select for resistance against a broad range of antibiotics (Abs). This co-selection may increase the health risk for both humans and livestock because of resistance to the wide range of Abs. As well as direct health effects, an increase in Ab resistance may impose a significant burden on the livestock industry and primary production, leading to potential for immense social and financial losses. The current project was aimed to investigate the resistance of soil-borne bacteria sampled from selected regions of New Zealand. Genetic diversity of these bacteria and molecular aspects of horizontal transfer of HM and Ab resistance genes to other bacterial hosts was also investigated. Soil samples with a different history of usage, including pastoral and arable with high levels of HMs (e.g. cadmium (Cd) and zinc (Zn)) were collected from the Waikato region (WR), as well as soil from an area of native bush (background) as control. Waikato Region (WR) is one of the regions in New Zealand with high levels of HMs in soil, due to the regular use of HM-containing superphosphate fertilizers and animal remedies. Belmont Regional Park (BRP) airstrip soil was used as a novel site to explore bacterial communities’ resistance to HMs, and any co-selected Ab resistance. A comprehensive investigation was performed to simulate the soil environment contaminated with various levels of HMs to interrogate induced resistance to HMs and Abs in soil bacterial communities using microcosms with 6 weeks and 6 months incubation. The experiments carried out to investigate soil bacterial resistance to HMs and Abs were divided into two different categories, including physiological and molecular experiments. The physiological tests included plate culturing with a range of HMs and Abs concentrations and Pollution Induced Community Tolerance (PICT) analysis. Molecular investigations using Terminal Restricted Fragments Lengths Polymorphism (TRFLP) and Next Generation 16s rDNA were conducted to determine the probable changes in bacterial community structures induced by selection pressure of HMs presence in soil samples. Cd resistance genes were detected in individual bacterial isolates using specific oligomeric DNA primers via the polymerase chain reaction (PCR). Horizontal transfer of these genes to new bacterial recipients was investigated. Finally, Cd resistant bacterial isolates involved in Horizontal Gene Transfer (HGT) were identified using 16s rDNA Sanger Sequencing. Results clearly showed that there were significant differences between the levels of resistance to HMs and Abs in bacterial isolates from WR’s pastoral and arable soils compared to background soil (native bush). Differences between BRP soil samples with higher levels of HMs compared to those with lower HMs concentrations, and also microcosms’ with a range of HM levels showed there were significantly greater number of bacterial isolates resistant to HMs and Abs in soils with the higher initial levels of HMs. Pollution Induced Community Tolerance (PICT) analysis provided complementary results in concordance with the results of plate culturing experiments and showed the higher levels of bacterial resistance to HMs and Abs in soils with the higher initial levels of HMs. Terminal Restriction Fragment Length Polymorphism (TRFLP) and 16s rDNA Next Generation Sequencing experiments investigated HM-induced bacterial communities' structure changes and revealed significant differences among the bacterial community structures in the selected BRP and microcosms soil samples. The HGT experiments revealed the horizontal transfer of Cd resistance genes from donor isolates (from WR, BRP, and microcosms soils) to a characterised recipient bacterial strain in vitro, suggesting these genes were carried by mobile genetic elements. Overall, the result of the current project showed that there were higher levels of bacterial resistance to HM and also to Ab occurred while different levels of HMs were present in the soil. In addition, higher levels of HM and Ab resistance induction occurred in the presence of specific concentrations of HMs in microcosms’ soils. The bacterial community structures were changed in the presence of various levels of HM in soil. The investigation of bacterial community structures changes in microcosms containing background soil samples were greater compared to the microcosms containing pastoral soils; it is concluded in higher changes in bacterial communities in soils in presence of selection pressure of HMs. Cd resistance genes located on mobile genetic elements were able to be transferred horizontally form donor bacterial strains to recipients and the transconjugants showed resistance not only to Cd, Zn and/or Hg, but also to a range of Abs; it showed the possibility of spread of these HM resistance genes to the new bacteria in soil and conferring HM and subsequent Ab resistance in recipients.Item Is the family pet a risk for multidrug resistant infections? : thesis presented in fulfilment of the requirements for the degree of Doctor of Philosophy in Veterinary Science at Massey University, Palmerston North, New Zealand(Massey University, 2019) Toombs-Ruane, LeahRisk factors for community-acquired urinary tract infections (UTI) caused by extended-spectrum beta-lactamase-(ESBL) and AmpC beta-lactamase-(ACBL) producing Enterobacteriaceae were investigated in a prospective case-control study conducted between August 2015 and September 2017. Both cases and controls were from the Auckland and Northland regions of New Zealand. A telephone questionnaire was delivered to participants, and the results analysed for putative risk factors for human infections. Analysis was performed using regression models, including factors around pet ownership and any other animal contact. Faecal samples were submitted from some households; this included samples from both people and companion animals. Isolates collected from index case urine samples and ESBL- or ACBL-producing faecal samples were sequenced and subsequently analysed through a bioinformatics pipeline. Pet ownership was not found to be a risk for human ESBL- or AmpC-producing infections in this study. Another important finding of this research was that E. coli ST-131 was the most commonly found bacteria associated with the UTI from people recruited into the case-control study. The strains of this sequence type were likely to have entered New Zealand in multiple introductions over the last 20 years. Transmission of ESBL-/ACBLproducing E. coli was also suspected to have occurred within households where a person had been recently infected with the same bacteria (in the form of a UTI) caused by an ESBL-/ACBL-producing Enterobacteriaceae. The results of this study as a whole indicate that while pets may not be a major risk for acquisition of ESBL/ACBL-producing bacteria, they are likely to play a role in the transmission of bacteria within homes and the community, and therefore warrant attention in future work.Item Requirements of Escherichia coli to survive stress induced by the secretin, pIV : 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, 2018) Bagley, Stefanie JaynePathogenic Gram-negative bacteria utilise complex multiprotein and functionally unrelated trans-envelope machineries to secrete toxins and other virulence factors. Such machineries are referred to as secretion systems. These contain large, membrane-inserted homologous channels called a secretin. These secretion systems include the type II and III secretion systems (T2SS and T3SS), type IV pili assembly system (T4PS), and the filamentous phage assembly-secretion system (FFSS). Secretins are homomultimers with radial symmetry blocked by an inner gate or septum and have a pore size of up to 10 nm. As determined by previous studies on the FFSS secretin, pIV, and the T3SS secretin, InvG, there is a cost associated with the insertion of large membrane channels. Membrane integrity is disrupted, leaving the bacterial cell highly susceptible to antibiotics and environmental stressors. As a result, Gram-negative bacteria have developed stress response pathways which upregulate genes to mitigate this secretin induced stress. These are the Phage Shock Protein response (Psp), Conjugative plasmid expression (Cpx), Regulation of capsular synthesis (Rcs), and SoxRS Superoxide response (Sox). Not all individual genes within these stress response pathways are necessarily required for the survival of Escherichia coli expressing secretin. Stress can be induced in E. coli by expression of leaky pIV mutants as they are open, not gated, under physiological conditions and imitate the actively secreting channel. A synthetic lethality assay was performed to determine the importance of the key regulators from four stress response pathways (PspF, CpxR, RcsA, RcsB, SoxR, and SoxS) on cell viability in the presence of the leaky secretin mutant, pIV-E292K. Here it was determined that the Psp, Rcs and, (to a lesser extent), Cpx regulons, confer a protective effect on E. coli K-12 experiencing stress induced by pIV-E292K. Expression of pIV-E292K mutant also induced an Rcs-dependent capsular polysaccharide phenotype indicating upregulation of Rcs in response to leaky pIV production. These three responses are potential drug targets in the fight against antibiotic-resistant infections. Inhibition of the stress response may prevent mitigation of membrane stress, thereby killing the channel-expressing bacteria.Item Filamentous phage derived biological nanorods : development of a novel display system : 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, 2018) Davey, Georgia RoseThe Ff filamentous bacteriophage are filament-like bacterial viruses approximately 900 nm in length. The F-pilus-specific filamentous phage are resistant to heat, pH-extremes, and detergents in combination with their structural properties and amenability to DNA recombinant engineering has enabled their extensive use in modern biotechnology. However, the use of Ff-phage in vaccines and other such biological uses is controversial due to their ability to replicate in gut Escherichia coli, and the possibility of mobilisation and horizontal gene transfer of antibiotic resistance-encoding genes among the gut bacteria. As such, the novel system was established to create short, stable particles that cannot replicate, called NanoZap particles. However, this system has the disadvantage of often producing multiple-length particles, rather than the desired single-length particles; another disadvantage is that during packaging, one particle in a million packages the entire plasmid due to recombination that removes the terminator copy of the (+) ori, and given that these plasmids contain antibiotic resistance genes, this likely would spread antibiotic resistance throughout the surrounding environment. In this thesis several variations upon the original pNanoZap vector were created and tested to obtain monodisperse unit-length particles. The deletion of the complete multiple cloning site (MCS) that lied between the initiator and terminator of replication from the original pNanoZap vector achieved this aim. To eliminate rare antibiotic resistant particles that package the complete pNanoZap vector, the antibiotic resistance gene was removed from the pNanoZap 537 vector and replaced with an auxotrophic marker nadC; this vector was named pNanoZap 537N. It is yet to be seen if this new pNanoZap vector is capable of producing NanoZap particles. For high-sensitivity diagnostics it is desirable to construct high-avidity particles, containing large number of detector molecules. To achieve this a double-display (detector displayed on phage which in turn is displayed on the surface of florescent E. coli) was designed and tested. When the E. coli expressing red fluorescent protein TinselPurple were infected with the bacteriophage, the chromogenic protein was lost, thereby showing that a different method of colouring E. coli will need to be used in order to construct the double-display particles.