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    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-Yi
    The 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.
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    Diagnostic investigation into summer mortality events of farmed Chinook salmon (Oncorhynchus tshawytscha) in New Zealand : a thesis submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy at Massey University, Manawatū, New Zealand.
    (Massey University, 2020) Brosnahan, Cara
    Salmon farming is the second highest value aquaculture species in New Zealand and produces approximately 88% of the global market of farmed Chinook salmon, Oncorhynchus tshawytscha (Tucker, 2014). New Zealand salmon are free of many significant diseases affecting salmonids globally (Diggles, 2016). Therefore, disease is one of the greatest threats to this New Zealand aquaculture species. Biosecurity, early detection, and characterisation of new or emerging diseases is vital for management and sustainability of the aquaculture industry. Elevated mortalities termed ‘summer mortalities’ with no cause identified have occurred in certain farmed Chinook salmon populations in the Marlborough Sounds since 2012. This study identified two potential bacterial pathogens involved in summer mortalities; New Zealand rickettsia-like organism (NZ-RLO) and Tenacibaculum maritimum. Distribution of NZ-RLO and T. maritimum within farmed Chinook salmon populations, phylogenetic analysis of these pathogens and the pathogenicity of two strains of NZ-RLO were assessed to provide an understanding of the role of NZ-RLO and T. maritimum in summer mortalities. Additionally, new diagnostic tests were developed to efficiently detect these pathogens. Identification of NZ-RLO in the summer mortalities was the first detection in New Zealand. Tenacibaculum maritimum had been reported in New Zealand previously, however it had not been associated with mortalities. This study confirmed three strains of NZ-RLO with restricted geographical distribution. Two strains of NZ-RLO were found exclusively in areas where fish experienced summer mortalities and were associated with clinical signs of disease, indicating certain strains of NZ-RLO were likely primary pathogens. Widespread distribution of T. maritimum was detected within farmed salmon and no association was found with T. maritimum and clinical signs of disease in areas experiencing summer mortalities, indicating T. maritimum was unlikely to be a primary pathogen. This study proves that laboratory exposure of salmon to two strains of NZ-RLO caused disease and mortalities however, the differences between the two strains suggest NZ-RLO2 may be more pathogenic. This study suggests NZ-RLOs are likely to be involved in summer mortalities as primary pathogens however, the interaction between the pathogens and environment is likely to have amplified the levels of mortalities during these events.
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    Temperature- and host-dependent transcriptional responses in the entomopathogenic bacterium, Yersinia entomophaga MH96 : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Genetics at Massey University, Albany Campus, New Zealand
    (Massey University, 2020) Paulson, Amber Rose
    Yersinia entomophaga MH96 is a virulent pathogenic bacterium that is infective towards a broad range of insects and is under development as a biopesticide. MH96 produces insecticidal toxin complex called Yen-TC that is secreted at temperatures of 25 °C and below and has been shown to be the primary virulence factor (VF) during per os challenge against the New Zealand grass grub, Costelytra giveni and other agricultural pests (Hurst et al., 2011a, 2019). New insights into the pathobiology of MH96 during insect infection were gained from the in vivo transcriptome, including identification of a core secreted weaponry of co-expressed/co-secreted VFs, including Yen-TC and other exoenzymes; however, many other diverse types of VFs, including toxins, effectors, fimbriae, secretion systems, efflux pumps, iron acquisition, stress response and metabolic adaptation were also identified as highly expressed under in vivo conditions. A small DNA-binding protein, Yen6, was shown to be under thermoregulation at the transcriptional level and host-dependent-regulation at the post-transcriptional level and contributed to virulence during intrahemocoelic infection of Galleria mellonella at 37 °C. The in vivo transcriptome of Δyen6 and in vitro DNA-binding specificity analysis provided evidence that Yen6 is a novel LytTR-containing regulator that activates a ribose uptake/metabolism gene cluster, rbsD-xylG-rbsC-xylF-rbsK-ccpA, and represses a fructose uptake/metabolism gene cluster, IIA-fruK-IIB and a gene for RNA-binding protein yhbY during infection at 37 °C. Another small DNA-binding protein, Yen7, was also implicated as a potential temperature-dependent activator of Yen-TC component genes and over-expression of yen7 resulted in restored secretion by MH96 at 37 °C; however, deletion of yen7 did not abrogate Yen-TC production. Experimental investigations into potential regulatory linkages between Yen6 and yen7 were undertaken, and evidence to date does not support Yen6 as transcriptional repressor of yen7. A 17.5 Kb unstable element within the genome of MH96 with linkages to Yen-TC and toxin secretion, motility and cell shape was identified. Overall the findings presented in this thesis represent the most detailed investigation of MH96 pathogenesis to date, reinforcing MH96 as one of the most highly entomopathogenic bacteria known to humankind; yet suggesting MH96 has possibly maintained at least one core thermoregulatory mechanism more typical of an opportunistic pathogen.
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    Contact killing of bacterial pathogens on metallic copper : a thesis presented in partial fulfillment of the requirements for the degree of Master of Science in Microbiology at Massey University, Auckland, New Zealand
    (Massey University, 2016) Liu, Sha
    Hospital-acquired infections (HAIs) are a serious health concern worldwide. Currently in New Zealand, about one in ten patients admitted to hospitals will acquire an infection while receiving treatments for other medical or surgical conditions. An emerging strategy for HAIs prevention is to use self-sanitising copper surfaces on items commonly touched in hospitals, which can provide sustained protection against microbial contamination. This is due to the fact that a wide range of microorganisms can be rapidly killed on copper in a process termed “contact killing”. However, the mechanisms of copper-mediated contact killing are not fully understood; and moreover, the potential of bacterial pathogens to develop resistance to metallic copper has so far not been examined. Here we hypothesize that bacteria are predominantly killed by a burst release of toxic copper ions resulted from chemical reactions between surface components of bacterial cell and metallic copper. To test this copper ion burst release hypothesis, we isolated and phenotypically characterized small colony variants (SCVs) derived from the two most common nosocomial pathogens, Staphylococcus aureus and Pseudomonas aeruginosa. Consistent to our expectation, SCV mutants overproducing exopolysaccharides (EPS) are more rapidly killed than wild type on the surfaces of pure copper (99.9% Cu) and brass (63.5% Cu). Similar results were obtained with a panel of mutants with altered production of cell surface components (EPS, lipopolysaccharides, capsules, flagella and pili) in a non-pathogenic model organism of Pseudomonas fluorescens SBW25. Next, a unique approach of experimental evolution was used to assess the potential emergence of bacterial resistance to metallic copper. Specifically, P. fluorescens SBW25 was subjected to daily passage of sub-lethal conditions on the surfaces of brass. After 100 daily transfers, the evolved strains had a slight increase of survival rate on brass; but importantly, ~97% of cells can still be killed on brass within one hour. Taken together, our results clearly indicate that the rate of bacterial killing on copper is largely determined by surface components of a bacterial cell, providing support for the copper ion burst release hypothesis. Our primary data of experimental evolution showed that bacteria have limited ability to evolve resistance to metallic copper.
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    Extracytoplasmic stress responses induced by a model secretin : a dissertation presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biochemistry at Massey University, Manawatū, New Zealand
    (Massey University, 2015) Spagnuolo, Julian
    Pathogenic bacteria export large proteins and protein complexes, including virulence factors, using dedicated transenvelope multiprotein machinery, collectively called secretion systems. Four of these protein export machines found in Gram-negative bacteria, type 2/3 secretion systems, filamentous phage assembly-secretion system and the type 4 pilus assembly system contain large homologous gated channels, called secretins, in the outer membrane. Secretins are radially symmetrical homomultimers (luminal diameter 6-8 nm) interrupted by an internal septum or gate. Expression of these channels imposes a fitness cost to bacteria. While stress induced by model secretin pIV has been previously investigated using microarrays, this thesis is the first RNAseq characterisation of secretin stress responses. Furthermore, this is the first comparison of stress imposed by a closed-gate secretin (wildtype pIV), vs. an isogenic leaky-gate variant, the latter serving as a model of an open-gate substrate-secreting channel. The high sensitivity to changes in gene expression and low background noise of the RNA-seq approach have greatly expanded the known secretin stress responses to include the SoxS, CpxR and RcsB/RcsAB regulons, in addition to the known involvement of the Psp response. A synthetic lethality analysis of candidate genes in these pathways suggested that the leaky-gate secretins, besides rendering the Psp response essential for survival, also stimulate the SoxS and RcsB/RcsAB regulons for protection of the cells. Knowledge of the secretin stress expanded by this work helped identify potential targets for development of much-needed antibiotics against toxinsecreting Gram-negative bacteria.
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    Thermophilic Campylobacter in animals and man : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Veterinary Pathology and Public Health at Massey University
    (Massey University, 1984) Kakoyiannis, Charalambos Kleanthous
    During the course of the work reported in this thesis, more than 2,000 samples and isolates from animals and from humans, were examined for the presence of intestinal thermophilic Campylobacter. This figure does not include samples and isolates examined during the course of experimental studies. Culture of either rectal swabs from pigs or cloacal swabs from poultry usually provides an accurate assessment of the prevalence of distal gastro-intestinal (G.I.) tract infection by C. coli and C. jejuni, provided that the swabs are kept at 4°C and cultured within 6 hours. Cultures of isolates of C. jejuni can be preserved for at least 21 months if stored in FBP broth or PBS at -70°C. The major site of colonisation by C. coli in pigs, is the distal part of the G.I. tract and the proximal G.I tract in the case of C. jejuni. In poultry, the major sites of colonisation by both C. jejuni and C. coli are the distal ileum and the large intestine, especially the caeca. Surveys of pigs showed that 60 - 100% of post weaning pigs are infected with C. coli. In neonates the rates of infection increase with age, and infected sows are the major source of infection for piglets. C. jejuni is rarely isolated from pigs. Twenty eight percent (28%) of all poultry flocks examined were infected with intestinal thermophilic Campylobacter. In infected broiler flocks the rate of infection is almost 100%, while in older birds lower rates were found. Of all the isolates examined, 86% were C. jejuni and 14% C. coli. All poultry carcases and edible viscera, derived from infected broiler flocks, are contaminated with Campylobacter. The levels of contamination of these products by both C. jejuni and C. coli are approximately 106 and 104 cfu per whole carcase and packet of edible viscera respectively. Poultry chilled products for sale in supermarkets were also heavily contaminated by these two species Campylobacter which remained viable for the 'shelf life' of the products (ten days). The prevalence of infection by intestinal thermophilic Campylobacter in gulls was 59%, in ducks 29%, and in rats 60%. Infected sparrows were not detected. With the exception of an isolate from one pig, C. laridis is a species apparently host specific for sea birds, and isolates from gulls of what, on conventional taxonomic criteria, would be classified as C. coli are shown on more detailed examination to be C. laridis. The level of excretion in faeces of Campylobacter in most infected pigs is between 102 - 105 cfu/g, in poultry approximately 108 cfu/g, in rats 105 cfu/g, and in gulls 103 cfu/g. The infectivity of C. jejuni isolated from poultry for experimental chickens is in the region of 5 x 102 cfu and that of C. coli approximately 5 x 105. Both species of Campylobacter were a 100 to 1000-fold less infective for rats. Infection in both chickens and rats was self-limiting and attempts to reinfect chickens with the same species or organism, were usually unsuccessful. C. laridis was not infective for either chickens or rats at dose rates of up to 109 organisms. Campylobacter infections are the most common notified human enteric infection in New Zealand with an annual incidence rate of 31/100,000. Approximately 94% of the human cases of campylobacteriosis are due to C. jejuni and 6% to C. coli. People between 1 to 4, and 15 to 35 years of age are the most commonly affected, and patients who no longer have clinical signs of infection may shed up to 108 cfu of C. jejuni/g of faeces. By use of a bacterial restriction endonuclease DNA technique (BRENDA), up to 80 different types C. jejuni and C. coli were identified from 338 isolates from humans. One hundred and ninety four (61%) of the 316 isolates of C. jejuni from humans had similar BRENDA patterns to isolates of C. jejuni from animals. Poultry appear to be the major source of C. jejuni, and possibly C. coli, for humans, while pigs apparently are an insignificant source of C. coli for humans. Rats can be infected with strains of C. jejuni which can infect poultry, humans and other animals. BRENDA types recovered from gulls and ducks were not similar to any of the isolates from humans or other animals examined. Some strains of C. jejuni and C. coli developed an in-vitro resistance to nalidixic acid. This is an important finding in relation to conventional taxonomic criteria for differentiating C. laridis from other intestinal thermophilic Campylobacter. Isolates of 'C. coli' from gulls are phenotypic variants of C. laridis which may be either resistant, or non resistant, to nalidixic acid. Only by determining whether or not, anaerobic growth in the presence of TMAO occurs, can C. coli be differentiated from C. laridis.