Rhodococcus is a soil bacterium, member of the Actinobacteria, and a close relative of the prolific small molecule producer Streptomyces. Recent interest in Rhodococcus as an under investigated source of possible bioactive secondary metabolites is sparked by the discovery of many polyketide synthase and non-ribosomal peptide synthetase genes of unknown function from sequenced Rhodococcus genomes. Rhodococcus species strain MTM3W5.2 was recently shown to produce a strong inhibitory compound with activity against most strains of Rhodococcus and closely related genera. A goal of this investigation is to discover the gene(s) required to synthesize this inhibitory molecule. The engineered Rhodococcus transposon, pTNR, was used to generate random insertional mutations in the genome of MTM3W5.2. The transposon insertion sites for 8 non-producing mutants were cloned and sequenced. Genes that encode polyketide synthases usually form parts of large biosynthetic gene clusters responsible for the production of small polyketide molecules.

Thesis (M.Pharm. (Pharmaceutics) -- University of Limpopo, 2018 / Griseofulvin is an antibiotic fungistatic drug used in the treatment of dermatophyte and ringworm infections. About 50% of a dose of griseofulvin passes the gastro- intestinal tract unabsorbed and is excreted in faeces. Since griseofulvin is highly soluble in acidic pH, a gastro-retentive floating matrix system was developed to control dissolution rate and thereby enhance solubility in an effort to develop an improved and convenient dosage form. Preformulation studies included selection of excipients and evaluation of their compatibility with griseofulvin. Using the chosen excipients, floating tablets of griseofulvin were formulated. Floating tablets containing 100 mg of griseofulvin were prepared by wet granulation technique with varying ratios of Methocel™, Accurel MP and Polyvinylpyrrolidone as determined by Design Expert software. Pre and post-compression studies, buoyancy capability and dissolution studies were carried out to assess the influence of the tablet components. Results obtained revealed that a density of less than 0.00091 g/cm3 was necessary for tablet floatation. Tablets that float immediately upon contact with dissolution medium and continue floating for over 12 hours were achieved with at least 28% Accurel MP by mass of the tablet. Dissolution studies revealed that an increase in tablet hardness reduced the rate of griseofulvin release only up to 120 minutes. From 120 minutes onwards, tablet hardness had no significant influence on griseofulvin release from tablets. Methocel™ had the most significant influence on griseofulvin release. The amount of Methocel™ included in the formulation was indirectly proportional to the rate of griseofulvin release. Using Design Expert software, optimized formulation was achieved with 1% Polyvinylpyrrolidone, 30% Methocel™, 60% Accurel MP and hardness ranging between 8 – 9 N. Pre and post-compression parameters of the optimized tablets were found to be within pharmacopoeial limits and thus compressed tablets were of acceptable quality. Tablets produced floated immediately upon contact with the medium and remained floating for at least 12 hours. Griseofulvin was released from the optimized tablets in a near zero order fashion, with a total of 80.8% griseofulvin released at the end of the 12 hour dissolution test period. Results of accelerated stability studies indicated potential stability of the manufactured tablets months.

Legionella bacteria, the causative agent of Legionaries’ disease and Pontiac fever, are ubiquitous in fresh-water environments including man-made water systems. Incidence of legionellosis is increasing in the United States resulting in thousands of cases every year. Infection via aerosols generated by showers, faucets, cooling towers, spas, fountains, and other water fixtures has been identified as the primary source of transmission. Legionella bacteria pose a significant public health threat, particularly in health care and long term care settings as Legionella can readily colonize the plumbing systems and infect the vulnerable patient population. One species, Legionella pneumophila (Lp), is responsible for over 90% of the known cases of Legionnaires’ disease. The importance of genetic diversity of Lp and non-pneumophila strains in human disease remains an area of ongoing research. Little is known in regard to the phylogenetic diversity of environmental strains, particularly strains that colonize facilities with high risk populations such as hospitals. Whole-genome sequencing (WGS) analysis, is an emerging tool used to support epidemiological investigation of cases of legionellosis and can be used to describe and establish phylogenetic relationships between environmental strains and clinical cases. The advantage of this method is the ability to differentiate bacteria down to the level of single nucleotide polymorphisms (SNPs). However, it was unknown whether current WGS methods accurately represent the potential SNP diversity among Lp isolates from the same environmental sample. It is unclear as to why certain strains tend be associated with clinical cases more than others, but certain genes referred to as virulence factors may be related to the relative pathogenicity of Legionella strains. Further investigation into virulence factors and antibiotic resistance factors could be used in future risk assessment of environmental Legionella. Additionally, Legionella have the potential for high genetic diversity due to recombination events, and gene transfer can occur between distinct Legionella species and strains. There is a lack of research on the potential sharing of virulence factor genes between Legionella strains typically associated with disease and those considered to be non-virulent. The goal of the work presented in this thesis is to describe the diversity of phylogenetic relationships between Lp isolates found in hospital premise plumbing systems, to estimate the genetic diversity among Lp found in the same environmental sample, and to identify virulence and antibiotic resistance genes shared between Legionella strains. A better understanding of the genetic diversity of environmental Lp could inform future surveillance and outbreak investigations by demonstrating the need to collect samples from multiple sites within a facility, and identifying shared virulence and antibiotic resistance genes between Legionella species and strains could apprise future risk assessment. WGS was utilized to describe the phylogenetic relationships of 81 Lp isolates from five hospitals. Individual hospitals were found to have distinct strains of Lp. For some strains, highly conserved subpopulations were collected from the same room over time, whereas other strains did not cluster by room. Using prospectively collected isolates from two hospitals, the mean number of SNP differences among isolates from the same environmental sample was found to differ between hospitals (0.4 versus 7.5). The presence of virulence factors and antibiotic resistance genes in Legionella species and strains was described. An analysis of 10 virulence factor genes revealed that Lp likely did not share these genes with Legionella anisa, a species generally considered to be non-virulent. Within Lp strains there was no clear difference between the Lp strains considered to be more virulent and those considered to be less virulent. A few antibiotic resistance genes were also identified. Following an in vitro assay, only the identified genes associated with macrolide resistance, LpeA and LpeB, were found to impact a quantifiable measure of antimicrobial resistance. The results of these studies emphasize the importance of understanding the context of an individual facility in Legionella related studies. Importantly, the observations or trends of one facility should not necessarily be applied to another. Legionella genetic diversity was highly conserved in some facilities, whereas in others there was greater diversity as measured by SNP differences. Within sample SNP differences was also variable between hospitals. The virulence findings gave a clear indication of the limited virulence capacity of L. anisa. These findings could explain the limited potential of L. anisa to cause disease in humans. However, a lack of difference among Lp strains may be cause to reassess the potential risk of these other strains especially in diagnostic practices. Finally, some strains of Lp have genes that may contribute to resistance to the leading antibiotic treatments for Legionnaires’ disease. Overall, this research further demonstrates the power of WGS as multiple questions can be addressed using this methodology.

In October 2017, the United States government declared a state of public health emergency in response to the burgeoning prescription opioid epidemic. Opioid analgesics are the gold standard of therapy for moderate to severe pain, but their clinical utility is greatly limited by analgesic tolerance – a primary driver of diminished pain control and opioid dose escalations. Integral in this process are primary afferent sensory neurons in dorsal root ganglia (DRG), the first-order components of nociceptive sensation. With surmounting evidence that morphine and other narcotics can alter gut microbial composition and promote bacterial translocation to other tissues, a question arises of whether the secondary release of bacterial products and pro-inflammatory cytokines can modulate antinociceptive tolerance development. This dissertation examines how gut bacteria depletion with antibiotics modulates the pharmacodynamic properties of chronic morphine in mice. Utilizing a “top-down” experimental approach, this is characterized at the whole-animal, single-cell, and molecular level via behavioral assays of antinociception, whole-cell patch-clamp recordings in DRG neurons, and analysis of tetrodotoxin-resistant (TTX-R) Na+ channel kinetics, respectively. Our findings collectively indicate that the gastrointestinal microbiome is an important modulator of antinociceptive tolerance development with chronic morphine administration.

Multi-drug resistance in bacterial pathogens has become a global health crisis. Each year, millions of people worldwide are infected with bacterial strains that are resistant to currently available antibiotics. Diseases such as tuberculosis, pneumonia, and gonorrhea have become increasingly more difficult to treat. It is essential that novel drugs and cellular targets be identified in order to treat this resistance. Bacterial topoisomerase IA is a novel drug target that is essential for cellular growth. As it has never been targeted by existing antibiotics, it is an attractive target. Topoisomerase IA is responsible for relieving torsional strain on DNA by relaxing supercoiled DNA following processes such as replication and transcription. The aim of this study is to find novel compounds that can be developed as leads for antibiotics targeting bacterial type IA topoisomerase. Various approaches were used in order to screen thousands of compounds against bacterial type IA topoisomerases, including mixture-based screening and virtual screening. In the mixture-based screen, scaffold mixtures were tested against the M. tuberculosis topoisomerase I enzyme and subsequently optimized for maximum potency and selectivity. The optimized compounds were effective at inhibiting the enzyme at low micromolar concentrations, as well as killing the tuberculosis bacteria. In a virtual screen, libraries with hundreds of thousands of compounds were screened against the E. coli and M. tuberculosis topoisomerase I crystal structures in order to find new classes of drugs. The top hits were effective at inhibiting the enzymes, as well as preventing the growth of M. smegmatis cells in the presence of efflux pump inhibitors. Organometallic complexes containing Cu(II) or Co(III) were tested as well against various topoisomerases in order to determine their selectivity. We discovered a poison for human type II topoisomerase that has utility as an anticancer agent, as it killed even very resistant cell lines of breast and colon cancer. The Co(III) complexes were found to inhibit the bacterial topoisomerase I very selectively over other topoisomerases. The various methods of drug discovery utilized here have been successful at identifying new classes of compounds that may be further developed into antibiotic drugs that specifically target bacterial type IA topoisomerases.

The genus Salmonella, comprised of 2400 serotypes, is one of the leading causes of foodborne illnesses in the US and has been used for the deliberate contamination of food. A rapid system for detection, isolation, typing and antibiotic susceptibility profiling is essential for diagnosis and source tracking in natural outbreaks or a bioterrorism event. Pure culture is essential for molecular typing and antibiotic resistance testing. The virulence and the resistance mechanisms of Salmonella are rapidly evolving and many are still unexplained. The first aim of the study was to rapidly detect and isolate Salmonella from intentionally contaminated food. The second aim was to build a DNA fingerprinting database for accurate identification of the subtype. The third objective was to study the antibiotic susceptibility patterns and the underlying mechanisms of resistance. A correlation between the DNA subtypes and antibiograms was hypothesized. An association between the resistance determinants and pathogenicity genes was expected. A total of 114 isolates including environmental and clinical sources were tested. General and selective enrichments and immunomagnetic separation (IMS) were tested for rapid detection and isolation of Salmonella from eight food groups. Isolates were subtyped by pulsed field gel electrophoresis (PFGE) and automated RiboPrinter®. Resistance to 31 drugs was tested by the Sensititre® system and integrons were identified by PCR. The association between virulence and resistance was verified by Southern hybridization. Of the three genes tested, ompF was found to be the most reliable target for identifying Salmonella subspecies I, III and IV. Detection by real time PCR after enrichment in buffered peptone water and isolation by IMS provided the fastest results. Sixty two ribotypes and 74 pulsotypes were observed for the 100 isolates subtyped. Sixty isolates were resistant to one or more antimicrobials and 12 had class-1 integrons. In conclusion, pure culture was achieved in 25 hours by IMS. Ribotyping, a comparatively rapid technique was found to be ideal for initial identification. PFGE, which was more discriminatory, was appropriate for source tracking. Contrary to the original hypothesis, no correlation between subtyping and antibiograms was observed and no association of integrons with the virulence genes tested was demonstrated

N-Thiolated β-lactams (1) represent a promising new group of compounds with potent inhibition effects on bacteria, like Bacillus anthracis and methicillin resistant Staphylococcus aureus, and onco-systems, like breast cancer and leukemia. Originally developed as part of a synthetic pathway to bicyclic lactams, N-thiolated β-lactams have been shown in this laboratory to possess intriguing biological activities. The antibacterial activities of this new class of agents rely on novel structural features unlike those of any existing family of β-lactam drugs. The lactams seem to exert their effects intracellularly, requiring passage of the bioactive species through the cellular membrane, rather than acting extracellularly on cell wall components in the manner of penicillin and related antibiotics. The lipophilic nature of these molecules, which lack the polar side chain functionality of all other microbially-active β-lactams, suggests the compounds do not target the penicillin binding proteins within bacterial membranes but instead pass through these membranes. The biological target of these compounds has been investigated. The most active members of this β-lactam class appear to be those bearing a small branched alkyl chain on the sulfur atom. The effects of stereochemistry, branching and chain length of the sulfur group on bioactivities were studied. This dissertation is divided into six chapters. A review of organosulfur anti-infectives is discussed in Chapter 1. The types of existing antibiotics and their modes of action will be discussed in Chapter 2. The synthesis of these novel agents is discussed in Chapter 3. A structure-activity relationship of these lactam analogues is discussed in Chapter 4. And Chapters 5 and 6 demonstrate a novel mode of action and biological target for these drugs using techniques which include target identification, metabolic effects, and reactivity kinetics.

Philosophiae Doctor - PhD / Polyamic acid (PAA) nanostructured materials were successfully produced by electrochemical deposition and electrospinning using polyvinlypyrrolidone (PVP) as supporting polymer. Polyamic acid thin film and nanofibers were deposited directly at the surface of a screen-printed carbon electrode (SPCE) as electro-catalysts for reliable analytical reporting of sulphonamide as contaminants of emerging concern by electrochemical techniques. Fourier transform infrared (FTIR) spectroscopy was used to confirm the structural integrity of the PAA electrospun nanofibers compared to the chemical synthesized PAA. Brunauer-Emmett-Teller (BET) was used to determine the surface area of the nanofibers. The surface morphology and surface thickness of the polyamic acid (PAA) nanofibers on the screen-printed electrodes was studied using scanning electron microscopy (SEM) and atomic force microscopy (AFM). Cyclic voltammetry (CV) was used to study redox behavior of the nanostructured PAA modified screen-printed carbon electrodes. Electrochemical parameters surface concentration, diffusion coefficient, formal potential and peak separation were determined. Three sulphonamides were selected based on the United States of protection agency (US EPA) and World Health Organization (WHO) list of emerging contaminants and detected sulphonamides in environmental waters in South Africa and other African regions. The selected sulfonamides were evaluated at the unmodified and modified screen-printed carbon electrodes. The sulphonamides were evaluated in three different supporting electrolytes at pH < 7 and >7 to enhance electrochemical signal reporting. Sulfadiazine (SDZ), sulfamethoxazole (SMX) and sulfamethazine (SMZ) displayed peaks at 0.80 V vs Ag/AgCl in 0.1 M tris-HCl using square wave voltammetry at the unmodified transducer. At the PAA thin film transducer, SDZ, SMX and SMZ displayed well-defined analytical oxidative peaks at 0.77 V 0.82 V and 0.83 V vs Ag/AgCl respectively. The LOD (n=3) for SDZ was found to be 12.14 ųM with a correlation coefficient of 0.9950. The LOD (n=3) for SMX and SMZ was found to 14.59 ųM (R2 =0.9928) and 10.41 ųM (R2 =0.9963). These sulphonamides were also electro-analytical evaluated at the screen-printed carbon PAA nanofiber modified transducer. SDZ, SMX and SMZ produced well-defined analytical signals at 0.79 V, 0.81 V and 0.78 V vs Ag/AgCl respectively. The determined LOD (n=3) for the individual sulphonamides was 8.26 ųM, 16.59 ųM and 8.81 ųM SDZ, SMX and SMZ respectively. The linearity correlation coefficient (R2) was determined to be 0.9977, 0.9956 and 0.9974 respectively. The efficacy of the proposed nanostructured PAA thin film modified screen-printed carbon sensor was evaluated by performing recovery studies for the selected sulphonamides using square wave voltammetry. Tap water was used to simulate environmental matrix. The recoveries of SDZ with respect to each concentration were 98.84% (RSD 4.98%) to 40.58% (RSD 6.74%). For SMX the recoveries were 154.17% (RSD 11.00%) to 111.03% (RSD 16.80%). The recoveries for SMZ with respect to each concentration were 184% (RSD 8.19%) to 90.26 (RSD 18.26%) indicating the reliability of the analytical results. / 2021-09-01

Objectives: To evaluate the influence of pharmacist interventions on drug and dosage prescribing in pediatric settings. Method: Demographic, clinical, and prescribing data and parents’ measurement data were evaluated by pre- and post studies including time series studies and control groups. The data was evaluated against Australian Therapeutic Guidelines. Educational intervention strategies were designed and administered and a post-intervention evaluation was conducted. Group comparisons were made using x2 and Student’s t-test statistics. Time series analysis involved multiple linear regression analysis. Results: The major study involved antibiotics and analgesic drugs and dosages in appendectomy in children. Significant improvements occurred in the selection and dosages of prophylactic antibiotics @<0.001) and in subsequent ward antibiotic treatments @<0.001) also showed marked conformity with the guidelines Other pediatric studies involved liquid medication dosing and prescribing accuracy for paracetamol in a developing country where a simple intervention produced very marked improvements @<0.001). An intervention in severe community-acquired pneumonia showed an improvement in the prescription of appropriate drugs @<0.001) and appropriate dosages of paracetamol (p<O.OOl) according to the guidelines. In drug utilisation evaluation of cefiriaxone, flucloxacillin and Liquigesic COB, there was a significant improvement in the dosage prescribing of ceftriaxone and flucloxacillin and no change in Liquigesic COB following the intervention. O f the total, 38/218 (17%) o f the patients received appropriate post-operative antibiotic dosages. 286/368 (78%) of the analgesic prescriptions and 31/218 (14%) of the patients on postoperative antibiotic choice and dosage that were identified as appropriate in tonsillectomy. / Conclusion: This study has identified deficiencies related to the prescribing of antibiotics and analgesics in children. There was a varied level of improvement in the drug dosage prescribing of pediatricians following the pharmacist educational intervention. Locally developed guidelines are more likely to be accepted and followed than those developed nationally without local input.

<p>The essay investigates the economic burden of skin, soft tissue, bone and lung infections in Sweden 2003. The cost-of-illness method, based on the human capital theory, is used in the estimation. A prevalence approach and a top-down method were chosen for direct as well as indirect costs. Also there is a discussion concerning health economic aspects of antibiotic resistance and evidence of the increasing costs because of it. The lack of data leads to a result within a large interval of uncertainty; the direct costs are estimated to 1 072 million SEK and indirect costs are estimated to 4 655 million SEK.</p>