Flavomycin is a phosphoglycolipid antibiotic, which is used exclusively as a growth- promoting feed additive. Existing data, from both in vitro and in vivo ruminal studies, give conflicting results regarding its mode of action, as well as no clear microbiological basis for the observed responses. Studies however do indicate that the principal site of action of the antibiotic is the rumen. From the available data, flavomycin appears to promote growth in a manner distinct from that of other feed antibiotics, for which the growth-promoting mechanisms have been elucidated. This study aimed to characterise the effect that flavomycin has on the microflora of the rumen, allowing its growth promoting mechanism in ruminants to be determined. In vitro analysis demonstrated that flavomycin has only antibacterial activity, as ruminal species of protozoa, fungi and archaea were unaffected by the antibiotic. Of the ruminal bacterial species tested, Fusobacterium necrophorum, Fibrobacter spp. and certain hyper-ammonia producing (HAP) bacteria (Atopobium oviles, Desulfomonas sp. and Peptostreptococcus anaerobius) were highly sensitive to the antibiotic. The sensitivity of the Fibrobacter spp. to flavomycin suggested that flavomycin is likely to select for a cellulolytic bacterial flora comprised predominantly of Ruminococcus spp., as has been previously proposed on the basis of in vitro fermentation studies. In vivo, suppression of ruminal numbers of F. necrophorum and flavomycin sensitive HAP bacteria occurred as a result of flavomycin supplementation. It was demonstrated that these bacterial populations were highly variable, between individual sheep and days respectively, suggesting why previous studies produced conflicting results. Assessment of ruminal fermentation parameters demonstrated that flavomycin caused a significant decrease in the production of ruminal ammonia, which could be directly attributed to decreased numbers of ruminal HAP bacteria. A small increase in the ruminal concentration of lactate also occurred, which con-elated with the suppression of ruminal numbers of the lactate utilising F. necrophorum. No change in the balance of individual volatile fatty acids (VFA) occurred, however total VFA production was significantly decreased. This was likely to be due to the total viable anaerobic bacterial counts being lower during flavomycin supplementation, although this result was not statistically significant. Uncultured rumen bacteria were also implicated in the growth promoting mechanism of flavomycin. Molecular investigation of the rumen bacterial population by denaturing gradient gel electrophoresis (DGGE) demonstrated that several changes occurred, which correlated with flavomycin supplementation. Analysis of the sequence data obtained from excised DGGE bands highlighted that the majority of the operational taxonomic units (OTU) detected were represented by presently uncultured species of bacteria, of which almost half had not been previously identified. Identification of the flavomycin sensitive bacterial populations was not possible, however, due to the recovery of multiple sequences from individual DGGE bands. Existing bacterial 16S rDNA sequence data, from published ruminal clone libraries, also demonstrated the poor cultural representation of rumen bacterial diversity, with only 10% of the OTU detected being represented by cultured bacterial species. Based on these results, flavomycin has the ability to increase the efficiency of dietary protein utilisation, although the role of uncultured bacteria in the growth promoting mechanism of the antibiotic is not clear. Protein retention in the rumen is increased as a consequence of decreased deamination by ruminal HAP bacteria. F. necrophorum has the ability to attach to and damage rumen epithelium, as well as being the principal aetiological agent in the development of liver abscesses. Suppression of F. necrophorum is likely to decrease metabolic and immune burdens within the animal, as well as potentially reducing the rate of rumen wall tissue turnover. The use of flavomycin as a feed additive is to be banned in Europe in 2005. However, it is not known if presently available feed additives or treatments will be able to act as an effective replacement for this antibiotic. Characterisation of an adaptive resistance mechanism against flavomycin, in the ruminal bacterium Prevotella bryantii, demonstrated that cross-resistance to therapeutic antibiotics can occur. As a result of this finding, and the interest in development of phosphoglycolipid antibiotics for therapeutic application, it can be concluded that the withdrawal of the use of a flavomycin as a feed additive is a wise precautionary measure to ensure the long-term efficacy of this class of antibiotics.

Thesis (Ph.D.)--Boston University / PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you. / Antibiotic-resistant bacterial strains continually arise and their increasing prevalence poses significant clinical and societal challenges. Functional analyses of resistant mutants and the study of general stress responses perturbed by antibiotic treatment have yielded valuable insights into how resistance arises through mutations. However, less is known about the population dynamics and communal interactions that underlie the development of resistance through mutations. In this work, we utilize systems approaches to study the functional dynamics of bacterial populations evolving antibiotic resistance. We follow a continuous culture of Escherichia coli facing increasing levels of antibiotic and show that the vast majority of isolates are less resistant than the population as a whole. We find that the few highly resistant mutants improve the survival of the populations less resistant constituents, in part, by producing indole, a signaling molecule generated by actively growing and unstressed cells. We show, through transcriptional profiling, that indole serves to turn on drug efflux pumps and oxidative stress protective mechanisms. The indole production comes at a fitness cost to the highly resistant isolates, and wholegenome sequencing reveals that this bacterial altruism is enabled by drug-resistance mutations unrelated to indole production. This work establishes a population-based resistance mechanism constituting a form of kin selection whereby a small number of resistant mutants can, at some cost to themselves, provide protection to other more vulnerable cells, enhancing the survival capacity of the overall population in stressful environments. Deeper studies into cooperative strategies bacteria use to evade antibiotics may prove critical for the rational design of more effective antimicrobial interventions. / 2031-01-01

Thesis (M.D.)--Boston University

Class of 2007 Abstract / Objectives: The purpose of this study is to determine if an antibiotic formulary is beneficial in an inpatient ICU setting. The main goal, of course, is to ensure patients receive the most appropriate antimicrobial therapy resulting in the least amount of resistance, by using an antibiotic formulary and ICU antibiotic intervention. Methods: This project will use a retrospective design in which one-year post-intervention antibiotic resistant trends will be compared with pre-intervention trends at Yuma Regional Medical Center (YRMC). As is common at YRMC, patients started on antibiotic therapy had susceptibility testing performed to determine the best treatment for the patient. This susceptibility data will be the data used for comparison. Comparison of patient charges and hospital costs associated with these patients will also be performed. YRMC employed an ICU antibiotic intervention documentation form that was used to monitor and extrapolate intervention data. Hospital lab percent susceptibility data will be looked at to determine isolate susceptibility data to determine if any trends are present in antibiotic resistance between the time period when the antibiotic formulary was implemented and the previous corresponding period of time before the formulary. This data will also be compared with the hospital trends in resistant isolates as a whole. The data is desensitized, as individual patient data is not being reviewed. In looking at patient charges and hospital costs, charts will be reviewed. These charts will be de-identified to the investigators of this study. Of further note, YRMC placed the intervention in action in February 2006 and began collecting post-intervention data at that time. This study will be using post intervention data collected from February 2006 thru February 2007. Results: Conclusions:

Graduation date: 1997

Daptomycin is a lipopeptide antibiotic that contains 13 amino acids and an N-terminally attached fatty acyl residue. The antibiotic kills Gram-positive bacteria by membrane depolarization. It has long been assumed that the mode of action of daptomycin involves the formation of oligomers on the bacterial cell membrane; however, at the outset of my studies, this had not been experimentally demonstrated. In the work described in this thesis, I have used fluorescence energy transfer (FRET) between native daptomycin and an NBD-labeled daptomycin derivative to demonstrate that the antibiotic indeed forms oligomers on bacterial cell membranes. In a liposome model, oligomer formation depends on calcium and on phosphatidylglycerol (PG). The oligomer forms rapidly and is stable for a length of time longer than required for the bactericidal effect. Through variation of the ratio of FRET donor (native daptomycin) and acceptor (NBD-daptomycin), I have determined that the oligomer consists of approximately 6–7 molecules, or, depending on the structure of the oligomer, possibly up to twice that number. Oligomer formation on liposomes and on bacterial membranes was confirmed using excimer fluorescence of a perylene-labeled daptomycin derivative. Excimer fluorescence was also used to demonstrate a stoichiometric interaction between daptomycin and PG. It has previously been shown that the bactericidal activity of daptomycin requires calcium and correlates with the concentration of PG in the bacterial cell membrane; these requirements mirror those observed here for oligomer formation. Furthermore, membrane permeabilization is selective, and electron microscopy of bacterial membranes exposed to daptomycin has revealed no discontinuities or accretions of electron density. Both of these findings suggest formation of a small membrane lesion, which is compatible with the small size of the oligomer that was determined here. In conjunction with these previous findings, the experiments contained in my thesis strongly suggest that the oligomer is the bactericidal form of daptomycin.

No description available.

Abstract Aims: Due to the emergence of multi-drug resistance, alternatives to conventional antimicrobial therapy are needed. This study aims to investigate the in vitro pharmacological interactions between essential oils (considered valuable as natural therapeutic treatments) and conventional antimicrobials (ciprofloxacin ⁄ amphotericin B) when used in combination. Methods and Results: Interactions of the essential oils (Melaleuca alternifolia, Thymus vulgaris, Mentha piperita and Rosmarinus officinalis) when combined with ciprofloxacin against Staphylococcus aureus indicate mainly antagonistic profiles. When tested against Klebsiella pneumoniae the isobolograms show antagonistic, synergistic and additive interactions depending on the combined ratio. The R. officinalis ⁄ ciprofloxacin combination against K. pneumoniae displayed the most favourable synergistic pattern. The interactions of M. alternifolia (tea tree), T. vulgaris (thyme), M. piperita (peppermint) and R. officinalis (rosemary) essential oils with amphotericin B indicate mainly antagonistic profiles when tested against Candida albicans. Conclusion: While a number of interactions show complete antagonism, others show varied (synergistic, additive and ⁄ or antagonistic) interactions, thus the efficacy is dependent on the ratio in which the two components co-exist. Significance and Impact of the Study: The predominant antagonistic interactions noted here, suggests that some natural therapies containing essential oils should be used with caution when combined with antibiotics.

Abstract Aims: Due to the emergence of multi-drug resistance, alternatives to conventional antimicrobial therapy are needed. This study aims to investigate the in vitro pharmacological interactions between essential oils (considered valuable as natural therapeutic treatments) and conventional antimicrobials (ciprofloxacin ⁄ amphotericin B) when used in combination. Methods and Results: Interactions of the essential oils (Melaleuca alternifolia, Thymus vulgaris, Mentha piperita and Rosmarinus officinalis) when combined with ciprofloxacin against Staphylococcus aureus indicate mainly antagonistic profiles. When tested against Klebsiella pneumoniae the isobolograms show antagonistic, synergistic and additive interactions depending on the combined ratio. The R. officinalis ⁄ ciprofloxacin combination against K. pneumoniae displayed the most favourable synergistic pattern. The interactions of M. alternifolia (tea tree), T. vulgaris (thyme), M. piperita (peppermint) and R. officinalis (rosemary) essential oils with amphotericin B indicate mainly antagonistic profiles when tested against Candida albicans. Conclusion: While a number of interactions show complete antagonism, others show varied (synergistic, additive and ⁄ or antagonistic) interactions, thus the efficacy is dependent on the ratio in which the two components co-exist. Significance and Impact of the Study: The predominant antagonistic interactions noted here, suggests that some natural therapies containing essential oils should be used with caution when combined with antibiotics.

Herein we describe the synthesis of (±) platensimycin, a potent antibiotic against gram-positive bacteria. The first chapter reviews (±) platensimycin’s isolation, biological profile and previously reported studies relevant to the area. The second chapter describes our initial efforts to synthesize (±) platensimycin. The third chapter accounts our second generation synthesis and its completion. The fourth chapter entails the experimental details of the important compounds in our synthesis. / text