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Subject: search.filters.subject.Antimicrobials

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    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.
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    The molecular and cellular characterisation of the first glycocin, plantaricin KW30 : a thesis presented in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biochemistry at Massey University, Palmerston North, New Zealand
    (Massey University, 2010) Stepper, Judith
    Bacteriocins, typically secreted by Gram-positive and -negative bacteria, are ribosomally synthesised antimicrobial peptides which inhibit the growth of competing bacteria. We have purified a 43 amino acid bacteriocin, plantaricin KW30 (PlnKW30) produced by Lactobacillus plantarum KW30, that has little amino acid sequence similarity to any other characterised bacteriocin. The gene encoding plnKW30 is in a cluster with the genes required for maturation and export of, and immunity to, the bacteriocin. This arrangement of genes is similar to the genomic context of bacteriocin genes in other lactic acid bacteria. The plnKW30 gene cluster comprises six genes encoding a glycosyltransferase, a proteolytic ABC-transporter, two putative thioredoxins, a response regulator and PlnKW30 itself. PlnKW30 was found to possess two unusual post-translational modifications: an O-glycosylated serine and an unprecedented S-glycosylation of the C-terminal cysteine. The modified serine is located on an eight residue loop that is tethered by a disulfide bridge. Bothmodifications have been identified as N-acetylglucosamines (GlcNAc), making PlnKW30 the first described class IV bacteriocin. A post-translational modification with S-linked GlcNAc is unprecedented in bacteriocins as well as in all genera. The antimicrobial activity of PlnKW30 on L. plantarum ATCC 8014 was analysed using enzymatic dissection coupled with bioassays. It was found to be concentration dependent and both the N-and C-terminalfragments are necessary for activity. Furthermore, reduction of the disulfide bonds results in abolishment of antimicrobial activity and it appears that deglycosylation of the serine 18 decreases the antimicrobial activity by about two thirds. These results show that all posttranslational modifications contribute to the antimicrobial activity of PlnKW30. The addition of N-acetylglucosamine to cultures of the indicator strain L. plantarum ATCC 8014 protects it from the antimicrobial effect of the added PlnKW30. PlnKW30 probably targets an N-acetylglucosamine transporter in the target cell membrane, similar to the mannose phosphotransferase system targeted by lactococcin A.
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    Synthetic targets as mechanistic probes for the key biosynthetic enzyme, dehydroquinate synthase : a dissertation submitted to Massey University in partial fulfilment of the requirements for the degree of Doctor of Philosophy, Institute of Fundamental Sciences, Palmerston North
    (Massey University, 2009) Negron, Leonardo
    Dehydroquinate synthase (DHQS) catalyses the five-step transformation of the seven carbon sugar 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAH7P) to the carbacycle dehydroquinate (DHQ). Multiple studies have described in detail the mechanism of most of the steps carried out by DHQS with the exception of the final cyclisation step. In this study, (3S)-3-fluoro-DAH7P and (3R)-3-fluoro-DAH7P (fluorinated analogues of DAH7P) were produced and assayed across three phylogenetically distinct sources of DHQS in order to determine the role of the enzyme during the cyclisation step of the reaction. Incubation of (3S)-3-fluoro-DAH7P with DHQS from Escherichia coli, Pyrococcus furiosus, and Kiwifruit resulted in the production of different ratios of (6S)-6-fluoro-DHQ and 1-epi-(6S)-6-fluoro-DHQ for each enzyme. In addition, enzyme catalysis showed a slowing of reaction rates when (3S)-3-fluoro-DAH7P was used, suggesting that the fluorine at C-3 is stabilising the enol pyranose. An increase in the stabilisation of the fluoro-enol pyranose would allow release of this substrate intermediate from the enzyme to compete with the on-going on-enzyme reaction. The differences in the ratio of products formed suggest that the cyclisation occurs in part on the enzyme and that the epimeric product arises only by an abortive reaction pathway where the (3S)-3-fluoro-enol pyranose is prematurely released and allowed to cyclise free in solution. Once in solution, the (3S)-3-fluoro-enol pyranose could undergo a conformational change in the ring leading to the formation of the epimeric product. Furthermore, it is suspected that the position of fluorine influences the likely transition-state in carbacycle formation leading to the production of the epimeric product. This research has illuminated the role of the enzyme in guiding the correct stereochemistry of the product and illustrates the important molecular interplay between the enzyme and substrate.