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Item Is early life antibiotic-use a risk factor for the development of Type 1 diabetes? : a longitudinal data linkage study : a thesis with publications presented in partial fulfillment to the requirements for the degree of Doctor of Philosophy in Public Health (Epidemiology), Massey University, Wellington, New Zealand(Massey University, 2025-09-01) Ram, SharanIntroduction: Early-life antibiotic-use may disrupt the gut microbiota, potentially increasing the risk of Type 1 diabetes (T1D). This thesis assessed associations between prenatal and early childhood antibiotic exposure and T1D. Methods: A meta-analysis of 14 studies (2006-2020), encompassing 3,066,063 participants, assessed associations between early-life antibiotic-use and T1D. Subsequently, a longitudinal nation-wide linkage study examined this association in 315,789 New Zealand children born between 2005-2010 and followed until 2021, using Cox proportional hazards regression controlled for potential confounders. Patterns of antibiotic-use during pregnancy and early childhood were also analysed across demographic characteristics. Associations between both pre and post natal antibiotic exposure were assessed, including analyses by antibiotic class and spectrum, and stratification by delivery mode. T1D was identified using insulin dispensing and hospitalisation records. Results: The meta-analysis, showed a pooled risk estimates for prenatal exposure of 1.10 (95% CI: 1.00–1.21); for postnatal antibiotic-use a pooled risk estimate of 1.11 (95%CI 1.04–1.18) was found, with ≥5 courses resulting in a pooled estimate of 1.36 (95%CI 1.15–1.61). Broad-spectrum antibiotics were associated with higher risk (HR: 1.13, 95% CI: 1.03–1.23). In New Zealand, 30% of pregnant women received antibiotics, predominantly penicillin (73.7%). Higher usage was observed among Pacific (38.7%) and Māori (35.7%) women, those most deprived (i.e. those from the lowest socio-economic group) (39.5%). Those who had caesarean deliveries had higher rates of antibiotic use, with incidence rate ratios (IRRs) of 1.27 for elective and 1.09 for emergency procedures. By age five, 96% of children had received antibiotics, with similar subgroup patterns as observed for pregnant women. Prenatal antibiotic-use was associated with an increased T1D risk in a dose-dependent fashion (≥3 courses, HR 1.86; 95%CI:1.44–2.39), with the highest risk for broad-spectrum antibiotics (HR: 1.30; 95%CI: 1.12–1.57). Postnatal antibiotic-use was associated, also in a dose-dependent way, with T1D (≥13 courses, HR 1.93; 95%CI 1.18–3.17; broad-spectrum antibiotics, HR 1.74; 95%CI 1.10-2.78). Stratified analyses by delivery mode resulted in mixed results across different analyses. Conclusion: These findings show high antibiotic-use in New Zealand and among specific ethnic and socio-economic subgroups. It also showed clear associations with the development of childhood T1D, which is consistent with international studies as shown in the meta-analysis, underscoring the need for judicious antibiotic stewardshipItem 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 The role of emerging organic contaminants in the development of antimicrobial resistance(KeAi Communications Co. Ltd. Publishing services by Elsevier BV on behalf of KeAi Communications Co Ltd, 2021-08-05) Alderton I; Palmer BR; Heinemann JA; Pattis I; Weaver L; Gutiérrez-Ginés MJ; Horswell J; Tremblay LAAntimicrobial resistance (AMR) threatens human and ecological health worldwide. Unless major changes occur across the human, animal and environmental sectors, the problem will continue to expand. An important component of AMR that deserves greater attention is the influence of emerging organic contaminants (EOCs) – ubiquitous compounds found, amongst others, in pharmaceuticals, personal care products, food, industrial and agricultural products, plastics and building materials. EOCs are widely used and can accumulate in the environment from varied sources, predominantly via waste streams. EOCs can interact with microbial communities potentially leading to the emergence and spread of AMR. Biocides and pharmaceuticals have been demonstrated to promote AMR development. Antimicrobial resistance is a multi-faceted problem that requires input from all sectors, with robust strategies and policies needed to make headway with solving the issues of this important threat.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 Characterisation of the synergistic vancomycin-furazolidone action against Escherichia coli : a thesis presented in partial fulfilment of the requirements for the degree of Masters in Biochemistry at Massey University, Manawatū, New Zealand(Massey University, 2017) Weerasinghe, Raveen MarlonThe use of antibiotic combinations is garnering increased interest in the recent years due to the spread of antibiotic-resistant bacteria. The shortage of antibacterial therapy options is particularly severe for infections caused by Gram-negative bacteria, due to the formidable barrier to molecules > 600 Da imposed by the outer membrane. Vancomycin is a large glycopeptide antibiotic to which the outer membrane is poorly permeable, hence the minimal inhibitory concentration of this antibiotic for Escherichia coli is very high (~500 mg/L). Due to the resistance of E. coli and other Gram-negative pathogens to an increasing number of < 600 Da antibiotics including beta lactams, aminoglycosides and quinolones, enabling vancomycin use on Gram-negative bacteria would be valuable. Furazolidone was reported to increase sensitivity of E. coli to vancomycin, and this interaction has been investigated in this thesis in order to explore the potential of the vancomycin-furazolidone combination for clinical applications. The initial analysis of the vancomycin-furazolidone synergy demonstrated that their interaction is synergistic rather than merely additive. Furthermore, effectiveness of this combination for growth inhibition and eradication of E. coli biofilm was investigated. However, despite the synergy between vancomycin and furazolidone, the concentration of vancomycin in combinations required for growth inhibition and killing of E. coli in a planktonic mode and as a biofilm was above the nephrotoxicity (toxicity in the kidneys) threshold and therefore too high to treat infections with this organism systemically. However, by adding deoxycholic acid to the combination, the bactericidal vancomycin concentration was decreased below the nephrotoxicity threshold. The mechanism of synergy in the planktonic mode of growth was investigated through the analysis of E. coli gene-knock-out mutants and it was observed that TolC, the outer membrane channel common to a number of efflux systems (exporting enterobactin, xenobiotics and metabolites) is likely to be involved in vancomycin-furazolidone synergy. However, it was not possible to reliably pinpoint any particular efflux pump or enterobactin accumulation as factors in synergy. Using the genetic approach, it was found that DNA excision repair endonuclease UvrABC was ruled out as a factor involved in synergy. Overall this study characterised the synergy between vancomycin and furazolidone, initiated the enquiry into the mechanisms of interaction between these two antibiotics and examined its effectiveness against biofilms.Item Characterizing the removal of antibiotics in algal wastewater treatment ponds : a case study on tetracycline in HRAPs: a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Environmental Engineering at Massey University, Turitea Campus, Palmerston, New Zealand(Massey University, 2016) Norvill, ZaneAntibiotics are ubiquitous pollutants in wastewater, owing to their usefulness in both animal and human treatment. Antibiotic pollution is a growing concern because of the risk of encouraging antibiotic resistance in wastewater treatment (WWT) systems and downstream of effluent discharge. The aim of this thesis was to investigate the fate of antibiotics in algal WWT ponds, which have unique ecological and environmental characteristics (e.g. presence of algae; diurnal variation in pH, dissolved oxygen, and temperature) compared with conventional biological WWT. The research in this thesis focused on a case study of the fate of tetracycline (TET, an antibiotic) in high rate algal ponds (HRAP). Indoor lab scale HRAP studies were used to investigate the fate of TET under several operating conditions. Outdoor pilot scale studies (900 L and 180 L HRAPs) under Oceanic and Mediterranean climates were used to validate the lab scale findings. Results showed that high removal (85% to >98%) of TET was possible in the lab and pilot scale HRAPs with HRTs of 4 and 7 days. Sorption was consistently a low contributor (3-10% removal by sorption) during continuous HRAP studies, based on the amount of TET extracted from biomass. Batch experimentation was used to further distinguish mechanisms of TET removal. The majority of TET removal was caused by photodegradation. Indirect photodegradation of TET was dominant over direct photolysis, with 3-7 times higher photodegradation observed in wastewater effluent than for photodegradation in purified water during batch tests incubated in sunlight. Under dark conditions sorption was the dominant removal mechanism, and biodegradation was negligible in batch tests since aqueous TET removed was recovered (± 10%) by extraction of sorbed TET from the biomass. Irreversible abiotic hydrolysis was not observed during TET removal batch tests in purified (MQ) water. A kinetic model was developed and used to predict TET removal in the pilot HRAPs, based on parameters derived from batch experiments. The model predictions for aqueous TET concentrations were successfully validated against initial TET pulse tests in the 180 L pilot scale HRAP. However TET removal decreased in subsequent pulse tests in the pilot HRAP, resulting in over-prediction of TET removal by the kinetic model. This decrease in TET removal was associated with decrease in pH, dissolved oxygen concentrations, and biomass settleability, but causal relationships between TET removal and these variables could not be quantified. Until the predictive kinetic model is developed further, this model may serve as a preliminary estimate of TET fate in algal WWT ponds of different design and operation. Future research should also investigate the potential formation and toxicity (including antibiotic efficiency) of TET degradation products, but this was outside the scope of this thesis. Predictions from the model were sensitive to the daily light intensity, suggesting that TET removal would be reduced in the winter months.Item An experimental challenge model in lactating dairy cows using Streptococcus uberis for antibiotic efficacy testing : a thesis presented in partial fulfilment of the requirements for the degree of Master of Philosophy in Veterinary Science at Massey University, Palmerston North, New Zealand(Massey University, 2013) Notcovich, ShirliThe aim of this project was to develop a challenge model to test the efficacy of novel intramammary antimicrobial treatments for clinical mastitis. The use of the model, can reduces the costs of testing efficacy and accelerate the process of registration of new products. It provides controlled conditions which safeguard animal welfare. The experimental challenge model using Streptococcus uberis developed in this thesis can provide the pharmaceutical industry and animal health research groups with a cost-effective method to test the efficacy of new antimicrobial products for treatment of mastitis in a safe and controlled environment. Two Cloxacillin-based antimicrobials with different formulations and treatment frequency were tested for their efficacy to cure S. uberis infections after infections were induced using the challenge model developed as described in the third chapter of this thesis. The objective of the first study presented in this thesis was to choose one suitable strain from four strains of S. uberis, to be used in future challenge studies. Four strains were tested for their virulence and susceptibility to antibiotic therapy. A further study objective was to determine the dose (number of pathogens infused, expressed as colony forming units (CFU)) required for the tested strains to produce an acceptable proportion of clinical mastitis cases to enable future studies. The strain which accomplished the desired characteristics was then chosen and was utilised for experimental challenge in further studies (Chapters 4 and 5). The overall incidence of clinical mastitis obtained in this study at a quarter level was 54% (26/48). This study showed significant differences in the ability of different strains of S. uberis to cause clinical mastitis when inoculated via the intramammary route. However, only one of the four strains tested demonstrated favourable characteristics as a strain to be used in experimentally induced clinical mastitis studies. Chapters 4 and 5 describe two challenge studies conducted using the experimental challenge model (Chapter 3) to test the efficacy of different antimicrobial drug formulations. In Chapter 4, the cure rate of one cloxacillin based product applied every 24 hr. was compared with the cure rate of a penicillin-based product applied every 12 hr. During the observation period of this investigation all challenged cows developed clinicalThe aim of this project was to develop a challenge model to test the efficacy of novel intramammary antimicrobial treatments for clinical mastitis. The use of the model, can reduces the costs of testing efficacy and accelerate the process of registration of new products. It provides controlled conditions which safeguard animal welfare. The experimental challenge model using Streptococcus uberis developed in this thesis can provide the pharmaceutical industry and animal health research groups with a cost-effective method to test the efficacy of new antimicrobial products for treatment of mastitis in a safe and controlled environment. Two Cloxacillin-based antimicrobials with different formulations and treatment frequency were tested for their efficacy to cure S. uberis infections after infections were induced using the challenge model developed as described in the third chapter of this thesis. The objective of the first study presented in this thesis was to choose one suitable strain from four strains of S. uberis, to be used in future challenge studies. Four strains were tested for their virulence and susceptibility to antibiotic therapy. A further study objective was to determine the dose (number of pathogens infused, expressed as colony forming units (CFU)) required for the tested strains to produce an acceptable proportion of clinical mastitis cases to enable future studies. The strain which accomplished the desired characteristics was then chosen and was utilised for experimental challenge in further studies (Chapters 4 and 5). The overall incidence of clinical mastitis obtained in this study at a quarter level was 54% (26/48). This study showed significant differences in the ability of different strains of S. uberis to cause clinical mastitis when inoculated via the intramammary route. However, only one of the four strains tested demonstrated favourable characteristics as a strain to be used in experimentally induced clinical mastitis studies. Chapters 4 and 5 describe two challenge studies conducted using the experimental challenge model (Chapter 3) to test the efficacy of different antimicrobial drug formulations. In Chapter 4, the cure rate of one cloxacillin based product applied every 24 hr. was compared with the cure rate of a penicillin-based product applied every 12 hr. During the observation period of this investigation all challenged cows developed clinical mastitis in at least one quarter. The incidence of clinical mastitis at the quarter level was high, with 91.25% (73/80) of challenged quarters being affected. After diagnosis of infections, the cows were randomly allocated to two treatment groups and treated accordingly. Clinical cases in which the quarter did not respond to three applications of the allocated antimicrobial product received an extended treatment of the same product. As the allocation to the extended treatment was not random, clinical and bacteriological cures were statistically evaluated for the short treatment only. Clinical cure rates for the short treatment (3 syringes) were 52.63% and 43.75% for the cloxacillin- and penicillin-based products, respectively. There was no significant difference between the treatments (P = 0.8) in their efficacy for the treatment of experimentally induced S. uberis clinical mastitis. In Chapter 5, two long-acting cloxacillin containing products were compared in their efficacy to cure experimentally induced S. uberis infections. One commercially available product was compared with a novel long acting product (applied every 48 hr.). Out of 80 challenged quarters, 41 quarters developed clinical mastitis after inoculation (51.25%). Treatment with the novel product resulted in a total treatment success rate of 93.1% based on clinical examination, and 96.0% based on the bacteriological cure rate. Treatment with the control product resulted in total treatment success rate of 100% based on clinical and bacteriological cure rate. There was no significant difference between the products (P=0.19) in their efficacy for the treatment of experimentally induced S. uberis clinical mastitis. Results in this thesis showed that experimental challenge models can be a useful tool in animal research to test the efficacy of new products in a safe and cost effective manner.Item The bisspiroketal moiety of epi-17-Deoxy-(0-8)-salinomycin : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy at Massey University(Massey University, 1991) Williams, Geoffrey MartynThe synthesis of 2-(3,4-epoxy-3-methylbutan-1-yl)-1 ,7 -dioxaspiro[S.S]undec-4-ene 188 is described, the key step in it's formation being an addition of the lithium acetylide derivative of S-tert-butyldiphenylsilyloxy-2-methyl-2-trimethylsilyloxy-7 -octyn-1-ptoluenesulphonate 182 to o-valerolactone. The epoxide 188 was then converted to the hydroxy spiroketal 4-(1,7-dioxaspiro[S.S]undec-4-en-2-yl)-2-methyl-2-butanol 149 which underwent a Barton-type oxidative cyclisation to afford both the cis- and trans-2,2-dimethyl- 1,6,8-trioxadispiro[ 4.1.S.3]pentadec-13-enes 192 and 152. The ring system of this latter compound is analogous to the unsaturated bisspiroketal present in the polyether antibiotic epi-17-deoxy-(0-8)-salinomycin 8. Subsequently the route was modified to afford the trans- and cis-(2-methyl-1,6,8- trioxadispiro[4.1.S.3]pentadec-13-en-2-yl)methanols 211-214, since it was expected this terminal hydroxyl group would provide a 'handle' by which these molecules could be further elaborated. This required conversion of the epoxide 1 8 8 t o 4-(1,7- dioxaspiro[S.S]undec-4-en-2-yl)-1-iodo-2-methyl-2-butanol 200, which was followed by a Barton-type oxidative cyclisation, to give the cis- and trans-2-iodomethyl-2-methyl-1,6,8- trioxadispiro[4.1.S.3]pentadec-13-enes 201-204, which were then converted to the alcohols 211-214. The techniques used to construct these relatively simple bisspiroketal analogues were then applied to an enantioselective synthesis of the bisspiroketal portion of epi-17 -deoxy-(0- 8)-salinomycin. The two key intermediates required for this were (l'S, 3R, SS, 6S)-(+)-6- [1'-(tert-butyldiphenylsilyloxymethyl)propyl]-3,S-dimethyl-tetrahydropyran-2-one 84 and (SR, 2S)- and (SS, 2S)-2-methyl-2,S-bis(trimethylsilyloxy)-7-octyn-1-p-toluenesulphonate 231. The lactone 84 was prepared, using Evans' directed aldol methodology, from (4R, SS)-(+ )-4-methyl-3-(1'-oxobutyl)-S-phenyloxazolidin-2-one 219 and (S)-( + )-2,4-dimethyl- 4-pentenal 218. The acetylene 231 was prepared from levulinic acid 174, and the procedure incorporated a resolution step which enabled the 2S configuration of 231 to be introduced. The lactone 84 and the the lithium acetylide derivative of acetylene 231 were combined and subsequently converted to the (1"S, 2S, 2'S, 6'R, 8'S, 9'S, ll'R)-(-)- and (l"S, 2S, 2'R, 6'R, 8'S, 9'S, ll'R)-(+)-4-{8-[1-(tert-butyldiphenylsilyloxymethyl)propyl] -9,11-dimethyl-1,7 -dioxaspiro[S.S]undec-4-en-2-yl}-1-iodo-2-methyl-2-butanols 245 and 246. These hydroxy spiroketals were transformed, again using the Barton-type oxidative cyclisation methodology, to the cis-(l'S, 2S, SR, 7S, 9S, lOS, 12R)-(-)- and the trans( l'S, 2S, SS, 7S, 9S, lOS, 12R)-(-)-9-[l-(tert-butyldiphenylsilyloxymethyl)propyl]-2- iodomethyl-2,10,12-trimethyl-1 ,6,8-trioxadispiro[ 4.1.S.3]pentadec-13-ene 248 and 247, the latter of which resembles precisely the corresponding portion of epi-17-deoxy-(0-8)salinomycin. In addition, the termini of the bisspiroketal 247 are selectively functionalised, which will allow further elaboration to the entire natural product 8. The synthesis of the cis- and trans- 2, 2 - dimethyl-1S-hydroxy-1, 6, 8 - trioxadispiro[4.1.S.3]pentadec-13-enes 156 and 159, and of cis-2,2-dimethyl-13-hydroxy- 1,6,8-trioxadispiro[4.1.S.3]pentadec-14-ene 268 is described. These were formed firstly by allylic bromination of the cis- and trans-2,2-dimethyl-1,6,8-trioxadispiro[4.1.S.3]pentadec- 13-enes 192 and 152 to give the cis- and trans-1S-bromo - 2 , 2- dimethyl - 1 , 6 , 8- trioxadispiro[ 4.1.S. 3 ]pentadec-13-enes 262 and 265, and cis-13-bromo-2,2-dimeth y l- 1,6,8-trioxadispiro[4.1.S.3]pentadec-14-ene 261. These bromides were then displaced by an oxygen nucleophile to afford the alcohols 268, 156, 159, a procedure which involved both SN2 and anti-SN2' processes.Item Safety studies on probiotic strains Lactobacillus rhamnosus HN001, Lactobacillus acidophilus HN017, and Bifidobacterium lactis HN019 : a thesis submitted for the degree of Doctor of Philosophy at Massey University, Palmerston North, New Zealand(Massey University, 2001) Zhou, Joseph ShengliLactic acid bacteria (LAB) have been consumed in foods by human beings for several centuries without any obvious adverse effects. But the safety of consumption of these organisms, especially novel strains, which are added to foods as probiotics, has been questioned recently due to occasionally reported infections implicated with some particular LAB strains. Evaluation of the safety or potential toxicity of probiotic candidate strains, especially novel strains for which no prior safety data exist, is highly recommended. The LAB strains Lactobacillus rhamnosus HN001 (DR20TM), Lb, acidophilus HN017 and Bifidobacterium lactis HN019 (DR10TM) are three newly identified probiotic organisms with immune-enhancing properties. Their safety/potential toxicity was investigated in this study through a series of both in vitro and in vivo experiments. The mucus layer coating the surface of the gastrointestinal tract plays an important role in the gut mucosal defence system. Platelet activation and /or aggregation is a critical factor in the pathogenesis of infective endocarditis (IE). In the first part of this study, the potential pathogenicity of LAB strains was examined by in vitro mucin degradation (HN001, HN017, and HN019) and platelet aggregation (HN001 and HN019) assays. Following incubation with hog gastric mucin (HGM) in a minimal medium, the mucin degradation activity of test strains was determined via changes in the carbohydrate and protein concentration of the culture media and molecular weight changes of mucin glycoproteins (SDS-polyacrylamide gel electrophoresis, SDS-PAGE). The mucinolytic activity of test strains was also measured in an agarose petri dish assay. The results from these experiments suggested that HN001, HN017 and HN019 had no ability to degrade HGM in vitro. Flow cytometry analysis using platelet specific monoclonal antibodies demonstrated an inability of the test strains HN001 and HN019 to induce or enhance human platelet aggregation. These experiments indicated that the test strains are unlikely to degrade the mucin layer of the gastrointestinal mucosal surface or participate in the pathogenesis of endocarditis. Resistance of LAB strains to commonly used antibiotics has caused safety concerns regarding the genetic stability of these resistance properties. The antibiotic susceptibility and plasmid profiles of test organisms were investigated in another series of experiments. The susceptibility of the test strains to 18 antibiotics in common clinical use was examined by disk diffusion method. No extraordinary antimicrobial resistance was detected among the test strains (HN001, HN017, HN019, and HN067), and there were several antibiotics that efficiently suppressed the growth of test bacterial cells. A plasmid screening experiment demonstrated that all LAB strains examined were plasmid-free, this was verified by Southern blotting and DNA hybridisation techniques. These results indicate that the probiotic organisms tested here do not express or carry plasmid-associated antibiotic resistance, so their antibiotic resistance attributes are unlikely to disseminate to other clinically significant strains. To investigate the oral toxicity of test strains (HN001, HN017, and HN019), conventional BALB/c mice were inoculated with a high dose (10 11cfu/mouse/day) of the test probiotic LAB strains for 8 consecutive days. The feed and water intake, body weight gain, and general health status, of the mice were monitored. The potential translocation of inoculated LAB strains and gut mucosal histological changes following feeding were also investigated. Random amplified polymorphic DNA (RAPD) fingerprinting techniques were used for bacterial identification. Results showed that the test LAB strains had no adverse effects on the parameters observed; no viable bacteria were recovered from blood or tissue samples (mesenteric lymph nodes, liver, and spleen). These results suggest that the test strains had no acute toxicity and had no potential to result in infection in normal mice at the high dose applied in this study. To observe the consequences of longer-term consumption of test LAB strains, groups of BALB/c mice were orally administered with test LAB strains (HN001, HN017 and HN019) at doses of 5 x 10 7, 10 9 or 5 x 10 10 cfu/mouse/day for 4 weeks. In addition to the indicators observed in the acute toxicity study, the animals' haematological parameters; total and differential leucocyte counts; and blood biochemistry (plasma total protein, albumin, cholesterol, and glucose) were also investigated. Similar results to those of the acute toxicity study were obtained, i.e. 4 weeks consumption of HN001, HN017, and HN019 had no significant effects on the animals' general health status, haematology, blood biochemistry, or gut mucosal histological parameters. No dose-related effects were detected for any of the observed indicators. Translocation of test LAB strains was not observed. These results suggest that longer-term consumption of test strains is unlikely to cause any obvious health problems in host animals. In the final stage of this study, the potentially detrimental effects of HN001 and HN019 on hosts with sub-optimal immune functions were tested. To characterise the potential infectivity of test strains in immune deficient hosts, a group of adult male BALB/c mice pre-treated with dexamethasone (200µg/mouse/48 hrs) were fed with freshly cultured living HN001 or HN019 at doses of 1.5 ~ 2.5 x 10 7 cfu/mouse/day for 7 days; similar safety indicators to those outlined above were monitored. Results showed that no significant changes were noted in any of the safety parameters measured. No translocation of dietary LAB or systemic infection was detected. These findings suggest that HN001 and HN019 are well tolerated in immunocompromised mice without any significant safety concerns. To investigate the effects of consumption of test LAB strains in hosts with a preexisting immunological dysfuction, a group of female CBA/CaH mice (6 to 8 weeks) with experimentally induced autoimmune thyroiditis (EAT) were fed with freshly prepared probiotic preparations (HN001 4.2 x 10 8 cfu/mouse/day; HN019 2.16 x 10 8 cfu/mouse/day) for 5 to 8 weeks. Probiotic feeding was commenced one week prior to the immunization with auto antigens (MTg, mouse thyroglobulin). Antibody titres and spleen cell proliferative responses to the autoimmune inducing antigens (MTg) were determined via in vitro immunoassays. Lymphocyte (or mononuclear leucocyte) infiltration into thyroid tissue was also examined. Results showed that HN001 or HN019 feeding did not exacerbate spleen cell proliferative responses to MTg or lymphocyte infiltrations in thyroid tissues. These results indicate that feeding of HN001 or HN019 had no adverse effect on the induction or progress of autoimmune responses in CBA/CaH mice. Overall, the combined results from these studies suggest that the probiotic LAB strains HN001, HN017, and HN019 are non-pathogenic for experimental animals and are likely to be safe for human consumption.Item In vitro and in vivo studies on treatment and prevention of bovine mastitis : a thesis presented in partial fulfilment of the requirements for the degree of Philosophy Doctor in Veterinary Science at Massey University, Palmerston North, New Zealand(Massey University, 2011) Petrovski, Kiro R.Mastitis prevalence on dairy farms depends on the number of infected cows and the duration of each intramammary infection. Strategies aiming to influence these factors are the subject of research presented in this thesis. Decreasing the duration of infection can be achieved by successfully treating infected quarters. Treatment of mastitis can occur during lactation or in the dry period. Treatment success is influenced by the concentration of antimicrobial achieved at the site of infection and the length of time it is present. The concentration of antimicrobial should exceed the relevant minimal inhibitory concentration. The susceptibility of mastitis-causing organisms varies among geographical areas and over time. New Zealand’s susceptibility data demonstrated a high susceptibility to penicillin. A formulation containing this antimicrobial was administered to healthy lactating cows milked once or twice daily. The concentrations of penicillin in milk were above the minimal inhibitory concentrations for the entire inter-dosing interval. Doubling the number of treatments or milking once-a-day resulted in a significantly increased time above the minimal inhibitory concentrations. The number of new infections is greatest during the early dry period in mature cows and in the pre-calving period in both heifers and mature cows. Pre-partum administration of delayed release antimicrobial formulations in heifers decreased the incidence of clinical mastitis and resulted in better reproductive performance, but not in increased milk production, when compared to control heifers. More effective prevention of new infections within the dry period was achieved by administering a novel teat sealant to mature cows when compared to a commercial teat sealant and untreated controls. Strategies for shortening the duration of intramammary infections and decreasing the number of affected cows at the start of lactation investigated in this thesis should reduce the prevalence of mastitis on dairy farms in New Zealand.