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Class of 2010 Abstract / OBJECTIVES:The main purpose of this study was to review recent and good quality studies of the antimicrobial therapy of for moderate to severe (“limb threatening”) DFI. The analysis of these studies was to conclude with one or two “standard” approach to the routine management of this clinical entity. METHODS: This literature review study consisted of an evaluation of clinical trials that compare two or more active systemic antimicrobial regimens for the treatment of moderate to severe (i.e., “limb-threatening”) diabetic foot infections in human patients. Literature sources were identified primarily from OVID MEDLINE, but also included additional tertiary sources. The primary criteria for the clinical studies were: prospective, controlled, randomized and investigator blinded. Studies had to be published after the year 2003, and be available in full-text in English. RESULTS: Ultimately, only four studies were found that met the criteria for consideration. Trials differed in numerous features. All four studies were sponsored by the manufacturer of one of the comparator drugs. Three of the four were non-inferiority design. Evidence is lacking that any of the suggested regimens are superior. CONCLUSIONS: Instead of meeting our original goal of concluding that one or two regimens could be the “standard” management of DFI, we were limited to commentary on the quality and applicability of the current literature on this clinical entity. Numerous suggestions for improvement in the clinical information provided by DFI studies were offered. We eagerly anticipate the publication of the updated IDSA guideline document on DFI.

Master of Science / Food Science Institute / Randall Phebus / Shiga toxin-producing E. coli (STEC) are frequently associated with foodborne illness outbreaks, especially attributable to beef. Intervention methods, such as water washes and organic acid application, are widely implemented across the beef industry to combat this risk. This research evaluates the efficacy of intervention methods applied to chilled beef subprimal pieces and pre-rigor beef carcasses to reduce STEC contamination. Beef strip loins were inoculated (ca. 5 log CFU/cm²) with a 7-serotype STEC cocktail and sprayed with increasing concentrations of peracetic acid (200-1800 ppm; ambient temperature), lactic acid (3-10%; 55°C), or a water control before being vacuum-packaged and stored for 24 h at 4°C. Meat surface excision samples and color readings (L*, a*, and b*) were obtained from each subprimal at three sampling points: post-inoculation, 5 min post chemical spray, and post-24 h vacuum packaged chilling. Peracetic acid spray and lactic acid spray reduced STEC populations by 0.5 -1.3 log CFU/cm² and 0.2 – 0.7 log CFU/cm², respectively, across the incremental concentration increases. All concentrations of peracetic acid and lactic acid concentrations ≥3.5% reduced (P ≤ 0.05) STEC populations compared to their respective control. Application of higher concentrations of lactic acid (7-10%) decreased (P ≤ 0.05) L* and b* values compared to the control, indicating that quality attributes of the subprimals were negatively effected. Carcass intervention methods were evaluated using a three-stage commercial carcass washing cabinet (Chad Equipment). Four pre-rigor carcass sides were inoculated by electrostatically spraying with a 7-serogroup STEC cocktail (ca. 6.5 log CFU/100 cm²). Three treatments were applied, in order, to each side: ambient water wash, hot water wash (82-92°C at the nozzle head), and antimicrobial mist. Meat surface excision samples were taken from the bottom, middle, and top section of each carcass side at five sampling points: 30 min post-inoculation, post-ambient water wash, post-hot water wash, post-antimicrobial spray, and after 18 h spray chilling. The combination of the high-volume ambient water wash stage and subsequent hot water wash stage reduced STEC populations on sides by 3.5, 4.7, and 4.8 log CFU/100 cm² at the bottom, middle, and top of the carcass, respectively. Due to STEC populations declining to very low or undetectable levels after the hot water stage, minimal additional STEC reductions were observed after chemical spray application and chilling. Sequential antimicrobial treatments applied using a three-stage Chad carcass wash cabinet and a subsequent chill step reduced STEC populations on pre-rigor beef carcasses by 4.5 – 5.3 log CFU/100 cm².

Background: Cassia angustifolia Vahl. (commonly known as senna makkai or cassia senna), native to Saudi Arabia, Egypt, Yemen and also extensively cultivated in Pakistan, is a medicinal herb used traditionally to cure number of diseases like liver diseases, constipation, typhoid, cholera etc. This study was conducted to evaluate the in-vitro antimicrobial, antioxidant and anticancer assays and phytochemical constituents of aqueous and organic extracts of C. angustifolia leaves. Methods: The antimicrobial activities of C. angustifolia aqueous and organic (methanol, ethanol, acetone, ethyl acetate) extracts were investigated by the disk diffusion method. These extracts were further evaluated for antioxidant potential by the DPPH radical scavenging assay. Anticancer activities of the extracts were determined by the MTT colorimetric assay. The total phenolic and flavonoid contents of C. angustifolia extracts were evaluated by the Folin-Ciocalteu method and aluminum chloride colorimetric assay, respectively. The structures of the bioactive compounds were elucidated by NMR and ESI-MS spectrometry. Results: Bioactivity-guided screening of C. angustifolia extracts, led to the isolation and identification of three flavonoids quercimeritrin (1), scutellarein (2), and rutin (3) reported for the first time from this plant, showed significant anticancer activity against MCF-7 (IC50, 4.0 mu g/mu L), HeLa (IC50, 5.45 mu g/mu L), Hep2 (IC50, 7.28 mu g/mu L) and low cytotoxicity against HCEC (IC50, 21.09 mu g/mu L). Significant antioxidant activity was observed with IC50 2.41 mu g/mL against DPPH radical. Moreover, C. angustifolia extracts have the potential to inhibit microbial growth of E. cloacae, P. aeruginosa, S. mercescens and S. typhi. Conclusion: C. angustifolia extracts revealed the presence of quercimeritrin (1), scutellarein (2), and rutin (3), all known to have useful bioactivities including antimicrobial, antioxidant and anticancer activities.

Contact antimicrobials for use in the medical device industry are being studied extensively to minimize the risk of hospital acquired infections, which are among the top ten leading causes of death in the US. Surfaces modified with quaternary ammonium containing side chains have been known to demonstrate excellent antimicrobial properties. Prior work has indicated that polyurethane surfaces with copolyoxetane soft blocks consisting of fluorinated and quaternary ammonium side chains can act as good antimicrobials. However, stabilizing the positive charge on the surface has been a challenge. The dissertation is aimed at creating a surface modifier that would confer a stable contact kill antimicrobial surface at very low modifier content, that is, less than 2 wt%. To achieve this objective, the study explored the introduction of a different fluorous group in the soft block to enhance stability. In particular, prior studies by other groups and early work by Kurt have shown that replacement of one of the terminal “chaperone” C-F bonds by C-H decreased surface tension. This led to the hypothesis that a –CF2H terminated “chaperone” group would be “amphiphilic” resulting in surface stability under both dry and wet conditions. Keeping this hypothesis in mind, a –CF2-CF2H (4F) terminal “chaperone” group was created in a modifier having two different 4F to quaternerary C12 ratios. It was found that polyurethanes prepared with a 66:34 ratio of 4F:C12 as the diol, performed as a very good surface modifier with high zeta potentials over a long period of time compared to the –CF3 based modifier. Antimicrobial tests performed within one week and four weeks after coating preparation have provided promising results that demonstrate improved biocidal stability. Guided by improved antimicrobial properties obtained with surface modifier polyurethanes made from P[(4F)(C12)-66:34-Mn], a new concept was explored by end-capping the same diol with isocyanatopropyltriethoxysilane and blending the end-capped diol with base polyurethane along with a 10 wt % cross linker. These modifiers show excellent antimicrobial properties (100% kill of bacteria) over one month with no observable changes in the zeta potential or surface morphologies. XPS analysis confirms the presence of quaternary ammonium on the surface. Preliminary kinetic studies show excellent antimicrobial properties for a 2 wt% modifier and 100% kill within 1 hr.

While genetic improvement of susceptible crop species may enhance resistance to microbial pathogens and facilitate reduced pesticide load, the possibility for transmission of novel genes to wild relatives has hampered acceptance of GM crops in some markets. Chloroplast transformation presents an attractive alternative to nuclear transformation and offers the potential to ameliorate these environmental concerns. Most agronomically important species exhibit maternal inheritance of organellar genomes which eliminates the threat of transgene escape through pollen. Gene silencing is absent due to site directed, single copy insertion by homologous recombination. Foreign proteins can accumulate to high levels (up to 50% of total soluble protein) and are retained within the chloroplast envelope protecting them from degradation by host cytoplasmic proteases. A bacterial chloroperoxidase gene (cpo-p) was transformed into the tobacco chloroplast genome to test its efficacy against plant pathogens and the mycotoxin producing saprophyte Aspergillus flavus.

Masters Research - Fuculty of Health Sciences / Tuberculosis (TB) is one of the leading causes of mortality in the developing world. Mycobacterium tuberculosis, the causative organism, infects approximately a third of the world’s population. With high rates of HIV infection, particularly in sub-Saharan Africa, TB rates are inevitably soaring. Treatment regimens are based on multi-drug therapy taken over a prolo nged period, leading to poor patient compliance, in turn resulting in the development of multi-drug resistant TB (MDR-TB) which is difficult to treat. The need for new anti-TB drugs that can decrease the period of treatment or the number of doses and that will be effective against latent and MDR-TB is desperate. An added advantage would be the ability of a new class of anti-TB drugs to have a novel target to avoid potential crossresistance to existing drugs. Various approaches have been taken to antituberculosis drug development, including the high-throughput screening of plants, which represent an enormous, largely untapped resource of novel compounds. A further parameter to increase the chances of finding promising lead compounds is to focus research on plants that have traditionally been used to treat symptoms associated with TB. Traditional herbalists prescribing plant-based treatments have long played an important role in the provision of primary healthcare, especially in rural areas where most of the population is poor and unable to afford modern drugs. The aim of this study is to research literature resources pertaining to medicinal plants in southern Africa used to treat symptoms associated with TB, collect these plants, prepare methanol and acetone extracts for the antimicrobial and antimycobacterial testing, identify a plant with promising activity, and to isolate the active principle/s. These compounds in turn would be identified structurally and tested for activity against a range of micro-organisms, including mycobacteria, as well as for cytotoxicity. Twenty-three plant parts from nineteen different species were collected and 46 extracts prepared. These extracts were tested against a range of Gram-positive and –negative bacteria and fungi using the disc diffusion and broth micro-dilution methods. The effects of these extracts were also tested against non-pathogenic mycobacteria using the broth microdilution method, and against M. tuberculosis using the radiometric BACTEC 460 ABSTRACT V method. Te n of the nineteen species exhibited activity against two or more of the eleven organisms tested, namely Xerophyta retinervis bark, Helichrysum odoratissimum leaves, Ozoroa paniculosa bark, Eriocephalus africanus leaves, Siphonochilus aethiopicus roots, Conyza scabrida leaves, Syzigium cordatum bark, Tetradenia riparia leaves, Datura stramonium leaves and Dioscorea sylvatica tubers. The acetone extract of O. paniculosa was further pursued for the isolation of its active principles as it exhibited potent activity against Enterococcus faecalis, Pseudomonas aeruginosa, M. tuberculosis and Mycobacterium aurum with MIC’s of less than 1mg/ml. Previous work on related species has indicated anti-cancer, anti-helminthic, anti- malarial and anti-schistosomiasis activities, but no antimicrobial or antimycobacterial properties have been researched. Moronic, anacardic, and ginkgolic acids have previously been isolated from related species. Bio-assay guided fractionation led to the isolation of a known C15:1 anacardic acid (compound 1) and two HPLC fractions (HPLC2 and HPLC3) of which the predominant components of HPLC3 is the saturated analogue of anacardic acid. NMR data of HPLC2 suggest it is also a C15:1 anacardic acid, although the location of its double bond is as yet unkown. These compounds were cytotoxic to CHO cells at 44.9 – 64μg/ml. Compound 1 and HPLC2 were moderately active against M. tuberculosis with MIC’s of 125μg/ml, while HPLC3 had increased activity with an MIC of 31.3μg/ml, work not previously reported. Similarly, HPLC3 had the greatest activity a gainst M. smegmatis with an IC50 value of 22.1μg/ml. M. aurum had higher IC 50 values ranging from 98.4 to 112.5μg/ml for all three compounds. Compound 1 had potent activity against a range of Grampositive bacteria with IC50 values of 1.3, 2.1 and 6.5μg/ml against Bacillus cereus, E. faecalis and Staphylococcus aureus respectively. Furthermore, this compound had good activity against one of two drug-resistant strains of S. aureus tested with IC50’s of 6.9 and 43.2μg/ml. The saturated anacardic acid (HPLC3) in general had poorer activity against Gram-positives than its unsaturated analogues, consistent with reported literature. The anacardic acids had decreased activity against yeast and Gram-negative organisms tested, with IC50 values of around 80μg/ml against Candida albicans, 19 ->250μg/ml for Serratia odorifera, 26 - >250μg/ml for Klebsiella pneumoniae and 17 – 68μg/ml for ABSTRACT VI Pseudomonas aeruginosa, results comparable to those previously reported. The saturated anacardic acid appeared to have better activity than its unsaturated analogues against Gram- negatives and yeast. The current literature suggests that lipophilic compounds have greater activity against M. tuberculosis. Results obtained in this project are in support of these findings, as the saturated anacardic acid, believed to be the major constituent of HPLC3 based on NMR data available, had increased activity against this organism compared to the unsaturated and less hydrophobic compound 1 and HPLC2. It is possible that these findings implicate anacardic acid in the disruption of the mycobacterial cell wall.

Hydroxyapatite (HA) is a biologically active ceramic used in surgery to replace bone. While HA promotes bone growth, it suffers from weak mechanical properties and does not possess any antibacterial property. Multi walled carbon nanotubes (MWCNTs), as one of the strongest and stiffest materials, have the potential to strengthen and toughen HA, thus expanding the range of clinical uses for the material. Furthermore, Silver nanoparticles (Ag NPs) can be decorated to sidewalls of the MWCNTs which could be released over a period of time to prevent infection following surgery. This work sought to develop and characterise Ag NPs- MWCNTs – HA composites in four main areas: 1) production and characterisation of the composite, 2) evaluation of mechanical properties, 3) investigation of antimicrobial property and 4) assessment of biological response to in vitro cell culture. Pristine (p-MWCNTs) and acid treated MWCNTs (f-MWCNTs) were decorated with Ag NPs. In the presence of 0.5 wt % Ag NPs-MWCNTs, HA was precipitated by the wet precipitation method in the presence of either poly vinyl alcohol (PVA) or Hexadecyl trimethyl ammonium bromide (HTAB) as the surfactant. Composites were characterised using various techniques and the diameteral tensile strength and compressive strength of the composites were measured. The antibacterial effect of these composites was investigated against clinically relevant microbe, Staphylococcus aureus. To determine the ability of the HOB cells to differentiate and mineralize in the presence of the composite, HOB cells were cultured on the composites for 21 days. Gene expression studies was performed along with the biochemical assays and scanning electron microscopy was used for qualitative analysis. Pure HA was used as control in all the studies. The study revealed that both the MWCNTs and surfactants play a crucial role in the nucleation and growth of the HA. XRD and FTIR characterisation revealed that HA was the primary phase in all the synthesised powders. Composites made with f-MWCNTs were found to have better dispersion and better interaction with the HA compared to composites with p-MWCNTs. Although mechanical strength was improved in all the composites, p-MWCNTs composites exhibiting maximum strength. Antibacterial studies showed 80% bacterial reduction in the treatment composites compared to pure HA. The biocompatibility study showed reduced activity of the HOB cells, however, no significant difference was observed between the control and the treatments. This systematic study of the synthesis and properties of the Ag NPs- MWCNTs-HA composites has resulted in improved understanding of the production and processing of these materials and the effect of MWCNTs and silver nanoparticles on primary human osteoblast cells. Additionally, it has yielded interesting biocompatibility result favouring the use of MWCNTs in the development of implants. There is potential to translate Ag NPs-MWCNTs-HA composites into clinically approved product.

Enteric fever continues to affect people living in endemic settings substantially causing at least 20 million cases of febrile illnesses every year with 1% mortality. Over the last decade there has been considerable debate surrounding the burden and disease profile of enteric fever in the paediatric population. This is partially due to the similarity of the clinical features of paediatric enteric fever to most other febrile illness seen in endemic settings. The treatment of enteric fever is proving to be a challenge with the emergence of antimicrobial resistant strains, particularly the 4.3.1 genotype (H58 haplotype), which is spreading rapidly. Multi-drug resistant (MDR) enteric fever, defined as infection with typhoidal Salmonellae that exhibit a combined resistance to ampicillin, cotrimoxazole and chloramphenicol emerged in the 1990s and was mediated primarily via the 4.3.1 genotype population through the horizontal acquisition of antimicrobial resistance determinants. Subsequently, fluoroquinolones became the drug of choice and the treatment of enteric fever following which fluoroquinolone resistance emerged, again through the 4.3.1 genotype. However, these antimicrobial trends may not be uniform across endemic regions and an understanding of these differing patterns as well the temporal changes in these trends are important in planning treatment strategies. In the short and medium term work needs to be focused on achieving the greatest benefits from the prudent use of the recently WHO pre-qualified Vi-TT conjugate vaccine candidate. Whilst the long term vision towards eradicating enteric fever needs to focus on better understanding the underlying the biology of this disease through the use of contemporary technologies while simultaneously improving infrastructure for the provision of clean water, adequate sanitation and hygiene. This thesis aims to age-characterise the disease burden of typhoid fever in endemic regions of South and South-East Asia as well as the African continent. Following this, the molecular epidemiology of enteric fever in two endemic settings in the Indian subcontinent is delineated with a keen focus on the 4.3.1 genotype (H58) population as well the phenotypic patterns and molecular determinants of antimicrobial resistance. This thesis finally systematically reviews the global trends of antimicrobial resistance of S. Typhi isolates over time both from a phenotypic and molecular perspective. The key results from this thesis include; the age stratification of disease occurrence in endemic regions which showed a substantial proportion occurs in the youngest age group in both Africa and Asia, the uniform dominance of 4.3.1 genotypes conferring a high degree of fluoroquinolone resistance contrary to earlier suggestions of younger children being more susceptible to a broader range of infecting genotypes, the dissimilarities between the antimicrobial resistance carrying capabilities of lineage I and lineage II strains of the 4.3.1 genotype as well as novel AMR gene arrangements and finally the temporal trends of AMR in S. Typhi which were different between Asia an Africa. The high prevalence of lineage I strains in Africa and South-East Asia in contrast to the high prevalence of lineage II strains in the Indian subcontinent reflect the antimicrobial selection pressures as well the evolutionary characteristics of circulating pathogen populations in these regions. The implications of the data reported in this thesis have implications for treatment and prevention strategies. For the first time in history an opportunity has risen to effectively vaccinate the youngest age group (0-4 years) from typhoid through the Vi-TT conjugate vaccine. As highlighted in this thesis the youngest age group (0-4 years) have a high disease occurrence in endemic areas as seen in a meta-analysis as well as through data from two endemic sites collated and reported in this thesis. The older age groups also suffer greatly from this disease calling for a broad based vaccine strategy. The implications for treatment of enteric fever are however more relevant in the immediate term which suggest that in endemic regions in Asia, fluoroquinolones have little role to play in treatment protocols while fluoroquinolones are still relevant in the African setting. In Asia, reverting back to former first-line antimicrobials might be an option but the possibility of re-emergence of widespread resistance to these currently sensitive antimicrobials is very high exemplifying the ability of S. Typhi to adapt to changing antimicrobial pressures.

Bacterial infection is a growing concern in hospital and community settings, where the issue of biofilms is a major problem. Most current methods of preventing microbial attachment and biofilm formation are limited due to application, process or inherent flaws. It was proposed that thin films containing an organometallic element could be deposited using plasma, a quick, clean surface modification technique; to create antimicrobial films which could then be applied to a range of substrates. <br /> Several novel antimicrobial monomer systems were synthesised and characterised based on silver, copper and zinc as the active constituent with phosphines, phosphites, maleimide and a novel Schiff base among the ligand systems. All monomers were found to greatly inhibit the growth of P. aeruginosa and S. aureus in solution and on solid media. Successful monomers were deposited onto suitable substrates (glass, gold, plastics, non-woven polypropylene) using continuous wave and pulse plasma, with the films characterised and low levels of active metal found in analysis using XPS and SIMS. Films were tested against solutions of pathogenic bacteria using a number of traditional and modern microbiological techniques and found to inhibit growth under a range of conditions, potentially due to the synergistic action of metal and ligand on bacterial cells. Effective control of bacteria was exhibited at times varying from 1h to 24h+. Highly volatile compounds were produced which allowed quick deposition of plasma films, which showed excellent activity against bacteria (99.9%+ growth reduction), indicating viability for potential application. All films tested showed no inhibition or toxicity to eukaryotic cells.