Class of 2012 / Specific Aims: The primary aim of this study was to determine the time to appropriate therapy for all patients with candidemia and/or bacteriemia (due to either Enterococcus or Streptococcus species) during a one year period in relation to time of blood culture, time of Gram-stain result, time of PNA FISH species result, and time of final species determination result. The secondary and third aims were to compare the time to appropriate therapy based on clinician group that was notified of Gram-stain result and PNA FISH result and compare the time to appropriate therapy based on PNA FISH assay results reported during the day and night microbiology laboratory shifts. Methods: This Institutional Review Board approved project is a retrospective, chart review evaluation of the 24 hour/ 7 days a week use of PNA FISH assays with therapeutic interventions by infectious diseases pharmacists and physicians on patient outcome measures and time to appropriate therapy. All patients admitted to an academic medical center during a one year period (April 2010-March 2011) with either Enterococcus, Streptococcus, or Candida species isolated from blood were included. Main Results: A total of 168 subjects were identified with Candida species isolated from 31 subjects and Enterococcus/Streptococcus species isolated from blood in 137 subjects. Conclusions: While reporting conditions can affect interpretation and intervention rates, rapid species identification assays such as PNA FISH can be used by pharmacists to provide antimicrobial therapy recommendations based on the species identification and to decrease the time to appropriate antimicrobial therapy.

Master of Science / Department of Diagnostic Medicine/Pathobiology / Sanjeev Narayanan / The use of antimicrobials in animal feed has come under increasing scrutiny from the public and regulatory agencies. Direct-fed microbials (DFM) are considered valuable alternatives to antimicrobials in food animal nutrition. DFM are products containing live (viable microorganisms). Studies in Europe have reported antimicrobial resistance (AMR) in organisms used in DFM. This is of serious concern because of the potential for transferring resistance to pathogenic bacteria in the gut. The aim of the present study is to characterize phenotypic and genotypic AMR profiles for 20 different antimicrobials in bacterial strains isolated from 10 commercially available DFM used in. Two antimicrobial susceptibility testing methods, disc diffusion and broth micro-dilution based assay were performed. Enterococcus faecium isolates showed resistance towards metronidazole (n=9/9) with a MIC of > 32 μg/mL, erythromycin (n=5/9) with a MIC of ≥ 8 μg/mL, ciprofloxacin (n=2/9) with a MIC ≥ 4 μg/mL, ceftriaxone (n=6/9) with a MIC ≥ 0.25 μg/mL, rifampin (n=8/9) with a MIC of > 4 μg/mL, trimethoprim/sulfamethoxazole (n=4/9) with a MIC ≥ 1 μg/mL and clindamycin (n=5/9) with a MIC of > 0.5 μg/mL. A Propionibacterium freudenreichii isolate showed resistance towards kanamycin with a MIC of > 64 μg/mL. The same strain also had a MIC of 16 μg/mL for levofloxacin. Two Lactobacillus acidophilus were resistant to vancomycin (n=2/6) with a MIC ≥ 32 μg/mL. All the Lactobacillus species including L. acidophilus (n=6), L. casei (n=4) and L. plantarum (n=2) were resistant to metronidazole, MIC > 32 μg/mL. Two strains of Bacillus subtilis showed resistance to clindamycin, with an MIC of 4 μg/mL and erythromycin with an MIC of > 8 μg/mL, and one strain had no zone of inhibition for metronidazole (MIC > 32 μg/mL). Microarray analysis revealed resistance genes in E. faecium strains of 3 different DFM, including aminoglycoside resistance genes, ant(4’)-Ia, erythromycin resistance genes, ere(A2) and ermB, tetracycline resistance genes, tet39, tet31, tetK and tetC, and beta-lactam resistance gene, pbp5. Conjugation with filter mating showed erythromycin resistance gene transfer, msrC gene, from donor strains to a recipient strain (E. faecium 45-24). These studies show that AMR is prevalent among bacterial strains used as DFM in the cattle industry in the U.S., justifying further characterization, detection and observation of transferable antibiotic resistance between the same genus. .

ABSTRACT The burning of selected indigenous plants and the inhalation of the smoke liberated from them has been a widely accepted and practised form of administration in traditional healing therapy dating back to as far as the Koi and San, and is a method still widely practised in South Africa today. Inhalation has various advantages as a method of administration in both allopathic and traditional practices. Not only is inhalation a highly effective mode of administration because of its direct and local effect on the lungs for the treatment of respiratory ailments, but also because of its ability to deliver drugs effectively systemically. This study elucidated the rationale behind this widely practised treatment by examining chromatographic and antimicrobial data. Five plants that are commonly administered traditionally through inhalation were chosen: Heteropyxis natalensis, Myrothamnus flabellifolius, Artemisia afra, Pellaea calomelanos, and Tarchonanthus camphoratus. An apparatus was designed and constructed and the burning process that occurs in the traditional setting was simulated with the selected plants. The induced volatile fraction (smoke) was captured for analysis. Control solvent extracts were made for each plant using conventional extraction solvents, methanol, acetone, water, and the essential oil of the aromatic plants was also investigated. Antimicrobial assays revealed that the extracts (smoke) obtained after burning had lower minimum inhibitory concentration (MIC) values than the corresponding solvent extracts in most cases. For Klebsiella pneumoniae all five inhalation samples were far more active than the conventional extracts. When tested against the pathogen B. cereus, M. flabellifolius and P. calomelanos inhalation samples proved to exhibit superior antimicrobial activity compared to the respective solvent extracts. Pellaea calomelanos inhalation extract had the lowest MIC values compared to the solvent extracts for all pathogens (P. calomelanos inhalation extract MIC values: 0.53; 1.00; 0.53; 0.53 mg/ml for S. aureus, B. cereus, K. pneumoniae and C. neoformans respectively). Inhalation extracts exhibited different chemical profiles from the solvent extracts of the same plant. For example, A. afra inhalation extract had an abundance of peaks at various retention times from 3.2 to 5.4 minutes, which were not present in the chromatograms of the acetone and methanol extracts of the same plant. These results, albeit preliminary, suggest that the chemistry and antimicrobial activity of plants are influenced by the combustion process which is often used in traditional healing rites.

A research report submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in partial fulfilment of the requirements for the degree of Master of Science in Medicine ( Pharmacotherapy) / Species of the genus Plectranthus, a member of the mint family ( Lamiaceae ), have been used in alternative medicines in third world countries dating back to the early Chinese empire. Plectranthus species have been used in the past for coughs and colds (P. ambiguus) and as a mouth-wash for loose and bleeding teeth (P. laxiflorus). The crushed leaves of P. madagascariensis are used by Xhosa as an ointment for scabies P. hadiensis is used orally as a cough mixture. Eight species from the genus were chosen to study the essential oil composition and antimicrobial activity. / IT2018

Faculty of Health SCiences School of Therapantic Sciences(pharmacy and Pharmaciology) 9604111j vanessa8@absamail.co.za / The genus Salvia, commonly known as the sages is a member of the mint family (Lamiaceae). In Latin, ‘sage’ means "to save" and the Romans called it "sacred herb". Throughout history it has been used for depression, fever, respiratory infections, women's complaints, sleep inducer, diuretic, gargles and sick room use. The essential oil is reported to have anti-inflammatory, antimicrobial, antioxidant, antiseptic, antispasmodic, astringent, diuretic, antihypertensive and insecticidal properties. Of the 900 species recorded worldwide, 30 are indigenous to South Africa where they are used extensively in traditional healing. The aerial parts of twelve samples were hydrodistilled and the essential oil analysed by GC-MS. The essential oil composition varied quantitatively and qualitatively within the different Salvia species analysed. Linalool was the only compound that was present in all the essential oils. β-caryophyllene and caryophyllene oxide were present in all essential oil with the exception of S. stenophylla. The essential oil as well as methanol and acetone extracts were tested for antimicrobial activity on a number of bacteria and fungi. No species showed activity against Cryptococcus neoformans, Candida albicans nor Alternaria alternata. All test samples studied demonstrated variable degrees of antibacterial activity with the exception of four test samples; S. disermas (methanol and acetone) from the Walter Sisulu Botanical Garden; S. disermas (methanol) from Mossel Bay and S. lanceolata (methanol). Gram-positive organisms were more sensitive to the test samples than the Gram-negative organisms. In general, the extracts were far more active than the essential oils. Thin layer chromatography indicated that all methanol extracts possess antioxidant activity. All methanol extracts contain the antioxidant compound, rosmarinic acid. It is evident that, in addition to rosmarinic acid, other polar and non-polar compounds are present in all Salvia species that also act as antioxidants.

Thesis advisor: Mary F. Roberts / There is substantial interest in exploring antibiotic alternatives with a new mode of action due to the increasing rates of bacterial resistance against current antibiotics. Antimicrobial peptides (AMPs) may take up the battle against bacteria in the future because as a result of their membrane-lysis mechanism, it is more difficult for bacteria to develop resistance against AMPs. Although AMPs could preferentially bind to and disrupt negatively charged bacterial membranes through electrostatic and hydrophobic interactions, there is still a great need to further increase the potency and selective toxicity towards bacteria for clinical applications. Herein, we present two strategies to improve the selectivity: light activation and environment-responsive moiety incorporation. Along the way, we also explored the effect of structure stabilization on AMPs action. A well-characterized antimicrobial peptide magainin 2 (mag2) was used as a prototype. Chemical manipulations of mag2 sequence were achieved by incorporation of unnatural amino acids. The selectivity was then tested on liposomes as a membrane model, as well as on bacterial cells and human red blood cells (hRBCs). Different extents of selectivity enhancement were observed from the modified peptides, and within the attempts to illustrate these results, we have gained useful information revealing the membrane-lysis mechanism, which may help us to rationally design and engineer AMPs as therapeutic drugs in the future. / Thesis (MS) — Boston College, 2011. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

The Copper Development Association (CDA) has identified over 450 copper alloys registered with the U.S. Environmental Protection Agency (EPA) as antimicrobial. With growing antibiotic resistance, there is a need for copper coatings with increased antimicrobial capability. Cold spray is a high velocity, high deposition rate process that forms dense coatings with little to no oxides or inclusions. It is possible that this process contributes to the increased antimicrobial capability of copper cold spray coatings as compared to other additive processes. The focus of this effort is to understand the effects of powder production and cold spray process parameters on copper cold spray coatings in order to optimize antimicrobial properties. Specifically, this work looks at the differences in conventional and nanomaterial copper cold spray coatings. Materials characterization and test methods show differences in adhesion, microstructure, corrosion, mechanical properties, and surface topography. Materials data is compared against Abaqus FEA software model outputs, and antimicrobial efficacy test data, based on the EPA approved procedure, is used to support materials observations and modelling outputs.

Abstract The genus Pteronia consists of approximately 80 species which are widely distributed in southern Africa. For hundreds of years the indigenous people of southern Africa have turned to the earth in order to provide healing for their people. The genus Pteronia has been amongst the first species to be used by the San and Khoi-San people for treating infections and stomach ailments. Ten species were selected for the purpose of this report. The essential oils were isolated by using a Clevenger-type apparatus while the non-volatiles were extracted with acetone and methanol. The essential oils and extracts were assessed for antimicrobial activity. The disc diffusion assays included three Gram-negative bacteria; Escherichia coli, Yersinia enterocolitica and Klebsiella pneumoniae, three Gram-positive bacteria; Staphylococcus aureus, Bacillus subtilis and Bacillus cereus as well as one yeast; Candida albicans. Results indicated that the species were primarily active against Gram-positive organisms. The minimum inhibitory concentration of the ten most active species (essential oils and extracts) were determined using the microdilution method. The most promising activity was noted for P. fasiculata which had a MIC of 0.22 mg/ml against S. aureus, 0.39 mg/ml against B. cereus and 2.08 mg/ml against B. subtilis. The essential oils analysis by GC/MS revealed two chemotypes. In Pteronia pallens, P. empetrifolia and P. flexicaulis rare compounds, such as presilphiperfolol-7-ene, 7-α-(H)-silphiperfol-5-ene, 7-β-(H)-silphiperfol-5-ene, α-campholene aldehyde, silphiperfol-5-ene, camaroonan-7-α-ol, silphiperfol-7- β -ol, presilphiperfolan-9- α -ol and presilphiperfolan-8-ol (a major compound in Pteronia pallens) were recorded. A cluster analysis of the essential oil data indicated that individual collections of P. camphorata within a population were tightly clustered. Similarly, P. pallens sampled from three different localities were also united in the cluster analysis. These results suggest minimal within and between population variations for some of the species studied.

Antimicrobial peptides (AMPs) are relatively-short chain molecules that living organisms use to defend themselves against a wide range of invading microorganisms such as bacteria and viruses. They selectively bind to and kill microbes over host cells by permeabilizing cell membranes or by inhibiting the biological functions of intra-cellular components. Despite its significance in determining their cell selectivity, however, the cell-concentration dependence of AMP's membrane-perturbing activity has not been criticality examined. In this thesis, we present a physical model for cell selectivity of AMPs, especially its cell-concentration dependence. To this end, we use a coarse-grained model that captures essential molecular details such as lipid composition (e.g., fraction of anionic lipids) and peptide amphiphilicity and charge. In particular, we calculate the surface coverage of peptides in the membrane-perturbing mode as a function of peptide and cell densities: those that bind to the interface between lipid headgroups and tails. This allows us to extract the minimum inhibitory concentration (MIC) and the minimum hemolytic concentration (MHC) of the peptides. Our results show that both MIC and MHC increase as the cell density increases so that the peptide selectivity (given by MHC/MIC) decreases with increasing cell density. Our results will help resolve conflicting interpretations of peptide-selectivity experiments.

This cross-sectional study described antimicrobial resistance (AMR) in healthy pigs in 20 Alberta and Saskatchewan herds. All herds used antimicrobials; the daily probability of antimicrobial exposure was 0.8 for nursery pigs and 0.3 for grow-finish pigs. Salmonella spp. (n = 468) were isolated from nursery, grow-finish pigs and sows while <i>Escherichia coli</i> (n = 1439) and <i>Campylobacter</i> spp. (n = 405) were isolated from grow-finish pigs. <p>Fifty-nine percent of the Salmonella were pansusceptible. Isolates from sows were more likely to be pansusceptible than those from other production phases, while Salmonella from nursery pigs were more likely to be multiresistant. All Salmonella and E. coli were susceptible to ceftriaxone and ciprofloxacin, drugs critically important to human medicine, while one E. coli was resistant to ceftiofur. Resistance was most common to tetracycline and sulfamethoxazole (Salmonella, 35% and 27%; E. coli, 68% and 46%). Although often considered an indicator organism, <i>E. coli</i> AMR was a poor sentinel for Salmonella AMR at the herd-level. <p>Antimicrobial resistance genes, described in 151 <i>E. coli</i>, were associated in two sets: aadA1 / sul1 / tetA and blaTEM / strA strB / sul2 / tetB. Associations between genes consistently matched associations between phenotypes suggesting phenotype data may be useful for predicting co-selection. Demonstrating dose-response relationships between various antimicrobial exposures and resistance phenotypes in E. coli reiterated the importance of co-selection. Significant predictors included exposures in other production phases and to unrelated drugs. Four <i>E. coli </i> resistance-phenotypes were associated with macrolide exposure; the most commonly used antimicrobial class in study herds. Additionally, 70% of the Campylobacter were resistant to a macrolide and this resistance was associated with macrolide exposure in nursery pigs. Study herds did not use quinolones. Despite this, 15% of Campylobacter were resistant to a quinolone. Both Campylobacter and <i>E. coli</i> AMR clustered within herds, indicating on-farm interventions could mitigate AMR in pigs.<p>This study described AMR in enteric bacteria from healthy pigs. Identifying dose-response relationships between antimicrobial resistances and exposures to unrelated drugs, and exposures of pigs in different production phases, emphasize the importance of judicious antimicrobial use in pig production.