Introduction: Antibiotics are among the most commonly prescribed drugs in the hospital setting. Because of an overall rise in health care costs, lack of uniformity in drug prescribing and the emergence of antibiotic resistance, monitoring and control of antibiotic use are of growing concern and strict antibiotic policies and prescribing guidelines are warranted. The aim of the study was to monitor the antibiotic prescriptions in a general outpatient setting in a rural regional hospital in Mpumalanga and to determine the impact of an education intervention on the prescribing patterns and adherences to the EDL/STD Method: Randomly selected adult outpatients files, at Themba Hospital, Mpumalanga, were selected to review the antibiotic prescribing patterns. The antibiotic prescriptions, diagnosis or symptom complex, and category of prescriber were recorded. The antibiotic prescriptions were cost according to the 2007 Hospital price list. Prescriptions were recorded in two phases; Phase 1; September, October and November 2006 and again January, February and March 2007 for Phase 2. Lectures on antibiotic prescribing, the use of the EDL/STG and the risk of antibiotic resistance were provided to all medical doctors in the first two weeks of January 2007. The prescription patterns, compliance with EDL/STG and the cost of the pre and post lectures were compared. Results: During the six-month study, a total of 1021 (Phase 1 = 505; Phase 2 = 520) prescriptions were reviewed and of those, 368 [Phase 1 = 230(46%); Phase 2 = 138 (27%)] prescriptions contained one or more antibiotics. Although the total number of antibiotics prescribed decreased from 338 in Phase 1 to 172 in Phase 2 the non-adherent to the EDL/STD for each antibiotic for the different diagnoses increased from 90% to 98% in Phases 1 and 2 respectively. There was no improvement in non-adherent prescriptions from the Community Medical Officers (CMO) and the Medical Interns after the lecture. The total antibiotic cost for the study period was R4 235.29 the calculated cost as per STG for the different diagnoses was R1 485.02 a decrease of 285% Conclusion: The number of prescriptions with antibiotics and the number of antibiotics decreased after the lecture. The antibiotics were not prescribed according to the EDL/STD. The lectures ix had no impact on the EDL/STG adherence. The EDL/STG should be included in the teaching and learning of medical students as the junior staff prescribed the most nonadherent antibiotic prescriptions and a once off in-service lecture had little impact. Prescribing according to the STG could have a significant impact in decreasing the cost of antibiotics.

archives@tulane.edu / The rise in prevalence of antimicrobial resistance is one the greatest public health concerns the world is facing today. As the incidence of antimicrobial resistance climbs, persistent wound and surgical site infections are becoming more common, with multidrug resistant Staphylococcus aureus and Pseudomonas aeruginosa being two of the most frequently identified causative agents. To combat these drug resistant infections, there is an urgent need to develop novel therapeutics. In this work, we developed and validated a splinted full thickness murine wound model used in tandem with bioluminescent strains of bacteria to study bacterial infection, wound healing, and novel therapeutics. Upon the validation of the model, we use it to evaluate the topical antimicrobial efficacy of three novel antimicrobials: bacterial derived outer membrane vesicles (OMVs), the antiseptic ‘ASP’, and the peptide D-CONGA. Though OMVs ultimately exacerbate wound infection and healing, we demonstrate that ‘ASP’ and D-CONGA possess broad spectrum antimicrobial and antibiofilm activity with minimal impact on wound healing. With success in evaluating antimicrobials we next use the wound infection model to evaluate the role of the immune cytokine IL-22 in bacterial clearance and healing in the context of MRSA and P. aeruginosa infections in addition to the potential use of IL-22 as a topical therapeutic. We demonstrate that enhanced IL-22 signaling positively influences both wound healing and gross wound pathology in P. aeruginosa wound infections and that topical treatment of IL-22 promotes wound healing in MRSA infected mice. Further we show that infection with P. aeruginosa enhances the expression of IL-22, while wounding enhances the expression of IL-22 receptor (IL-22R) regardless of infection status. Ultimately the completion of this work demonstrates the utility and reproducibility of the full thickness splinted wound model used in conjunction with bioluminescent bacteria for the study of infection progression, healing, antimicrobial therapeutics, and innate immunity, while also providing insight into IL-22’s role in skin infections. / 1 / Hoffmann, Joseph

Thesis advisor: Jianmin Gao / As the threat of microbial resistance to antibiotics grows, we must turn in new directions to find new drugs effective against resistant infections. Antimicrobial peptides (AMPs) and host-defense peptides (HDPs) are a class of natural products that have been well-studied towards this goal, though very few have found success clinically. However, as there is much known about the behavior of these peptides, work has been done to manipulate their sequences and structures in the search for more drug-like properties. Additionally, novel sequences and structures mimicking those seen in nature have been discovered and characterized. Herein, we demonstrate our ability to finely tune the antimicrobial activity of various peptides, such that they can be provided with more clinically desirable characteristics. Our results show that gramicidin A (gA) can be made to be less toxic via incorporation of unnatural cationic amino acids. This is achieved by synthesizing lysine analogues with diverse hydrophobic groups alkylated to the side-chain amine. Through exploring different groups, we achieved peptide structures with improved selectivity for bacterial over mammalian membranes. Additionally, we were able to achieve novel broad-spectrum gram-negative activity for gA peptides. In efforts to combat bacterial resistance to cationic antimicrobial peptides (CAMPs), we have directed our reported amine-targeting iminoboronate chemistry towards neutralizing Lys-PG in bacterial membranes. Originally incorporating 2-APBA into gA, we found this to hinder the peptide’s activity. However, we were successful in increasing the potency of gA3R, a cationic mutant of gA, towards S. aureus by using a co-treatment of this peptide with a Lys-PG binding structure. Currently, we are exploring this strategy further. Finally, we describe our work towards establishing a novel cyclic peptide library incorporating a 2-APBA warhead for iminoboronate formation with a given target. In this, we have achieved intermolecular reduction of iminoboronates, strengthening the stringency of library screening. Although we were unsuccessful in finding a potent hit for binding of the lipid II stem peptide, screening against human transferrin yielded selective hits. Currently we are investigating these hits to understand their activity and therapeutic potential. / Thesis (PhD) — Boston College, 2017. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

Aim of the study: Despite the extensive traditional use of Croton gratissimus Burch. var. gratissimus for medicinal purposes, scientific studies validating the therapeutic properties of this indigenous plant are lacking. As the bark, roots and leaves of C. gratissimus are used separately as well as in combination, this study focused on determining antimicrobial efficacies of the plant parts independently and in combination to assess possible pharmacological interactions (e.g. synergy, antagonism). Material and Methods: The hydro-distilled leaf essential oil and extracts of bark, root and leafwere comparatively assessed for antimicrobial activity by means of microdilution minimum inhibitory concentration (MIC). The fractional inhibitory concentrations (FIC) were determined for the leaf and root (1:1), bark and root (1:1), leaf and bark (1:1) combination. Isobolograms were plotted to demonstrate interactions between various ratios of the roots and leaves. Results: The MIC and FIC results indicated variable efficacies for the various plant part combinations, the greatest of which was noted for Cryptococcus neoformans in the root and leaf combination (MIC 0.4 mg/ml and FIC of 0.4). Isobolograms indicated the greatest synergy for Bacillus cereus, Candida albicans and Cryptococcus neoformans. Conclusion: The observed synergistic interactions clearly indicate that the reductionist approach may often be short-sighted and that biological activity may be improved through combination therapy, where different complex metabolic pools collectively contribute to the enhanced effect.

The antimicrobial, anti-oxidant and cytotoxic activities of the extracts obtained from 17 indigenous Agathosma species (19 samples) were investigated in order to validate the historic and continued use of Agathosma species in traditional healing. The antimicrobial activity was evaluated using the minimum inhibitory concentration (MIC) method on four pathogens, i.e. Staphylococcus aureus (ATCC 12600), Bacillus cereus (ATCC 11778), Klebsiella pneumoniae (NCTC 9633) and Candida albicans (ATCC 10231). The anti-oxidant activity was evaluated using the 2,2- diphenyl-1-picrylhydrazyl radical (DPPH) and 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assays, while the cytotoxic properties was determined using the MTT (3-[4,5-dimethyl-2-thiazol-yl]-2,5-diphenyl-2H-tetrazolium bromide) cellular viability assay. Agathosma ovata (round-leaf) displayed the best activity against S. aureus and B. cereus with MIC values of 0.16 mg/ml and 0.13 mg/ml, respectively. Most of the extracts had moderate to poor activity in the DPPH assay with the exception of A. capensis (Gamka) and A. pubigera which were the two most active species in the assay (IC50 values of 24.08±4.42 μg/ml and 35.61±0.86 μg/ml). The results obtained from the ABTS assay differed from that of the DPPH assay. All extracts showed greater activity in the ABTS assay with A. namaquensis and A. capensis (Besemfontein) being the most active species (IC50 values of 15.66±4.57 μg/ml and 19.84±0.09 μg/ml). Agathosma lanata (IC50 value of 26.17±9.58 μg/ml) and A. ovata (round-leaf) (IC50 value of 25.20±6.30 μg/ml) proved to be the most toxic in the MTT assay. Agathosma arida, A. collina, A. hirsuta, A. pubigera, A. roodebergensis, A. stipitata and A. zwartbergense also displayed some degree of toxicity.

The objective of this study was to determine the impact of an antimicrobial stewardship intervention in six non-intensive care services. A review of all patients on their 3rd and 10th day of broad-spectrum antibiotic therapy was conducted at Sunnybrook Health Sciences Centre using a stepped–wedge randomized design. The primary outcome was broad-spectrum antimicrobial utilization in the intervention period compared to the control period. Despite high volumes and uptake of stewardship recommendations, a non-significant reduction in targeted antimicrobial utilization was achieved; however, median length of stay of the patients included was ~4 days, limiting the ability of the intervention to make a significant impact on inpatient antimicrobial utilization. Careful consideration of the targeted patient population is, therefore, warranted when planning and implementing antimicrobial stewardship interventions. Future work may focus on intervening earlier in the course of therapy.

The objective of this study was to determine the impact of an antimicrobial stewardship intervention in six non-intensive care services. A review of all patients on their 3rd and 10th day of broad-spectrum antibiotic therapy was conducted at Sunnybrook Health Sciences Centre using a stepped–wedge randomized design. The primary outcome was broad-spectrum antimicrobial utilization in the intervention period compared to the control period. Despite high volumes and uptake of stewardship recommendations, a non-significant reduction in targeted antimicrobial utilization was achieved; however, median length of stay of the patients included was ~4 days, limiting the ability of the intervention to make a significant impact on inpatient antimicrobial utilization. Careful consideration of the targeted patient population is, therefore, warranted when planning and implementing antimicrobial stewardship interventions. Future work may focus on intervening earlier in the course of therapy.

The emergence of multidrug resistant strains of bacteria has resulted in the need for novel therapeutic agents. The ApoEdp peptide, derived from the receptor-binding region of the human apolipoprotein E, had previously been shown to have activity against herpes simplex viruses, human immunodeficiency virus and certain bacterial species. However, its antibacterial mode of action was not elucidated, therefore the present study aimed to determine this mechanism. The susceptibility of several different strains, including Pseudomonas aeruginosa, Staphylococcus aureus, Mycobacterium smegmatis, Staphylococcus epidermidis and Escherichia coli, to ApoEdp was investigated. No significant difference was observed between the minimal inhibitory concentrations (MICs) of ApoEdp against a range of Gram positive and Gram negative bacteria. The presence of E. coli K5 capsular polysaccharide in the growth medium led to a decrease in ApoEdp susceptibility of the non-capsulated E. coli MS101 DeltakfiC strain. Bacteria with non-functioning multidrug efflux pumps showed no difference in susceptibility. A mutation in the phoP gene of Salmonella enterica Serovar Typhimurium LT2, which regulates cell surface modifications led to an increase in ApoEdp susceptibility. Transmission electron microscopy (TEM) images showed changes in the membrane and internal structures of strains incubated with a minimal bactericidal concentration (MBC) of ApoEdp for 5 min. ApoEdp was able to depolarise the cytoplasmic membrane. The ability of ApoEdp to induce cell lysis was assessed by the release of β-galactosidase into the supernatant. There was no significant difference in the supernatant β-galactosidase levels of ApoEdp treated and unlysed cells. ApoEdp, however was able to form pores in artificial lipid bilayers and decrease intracellular ATP levels. The effect of ApoEdp on transcription and translation was determined using an in vitro transcription/translation system. Results showed that ApoEdp did not affect protein synthesis. ApoEdp also worked in synergy with rifampicin, chloramphenicol, ampicillin and ciprofloxacin against bacteria. Overall, the results showed that ApoEdp acts by targeting the cytoplasmic membrane, although it may also have intracellular targets. Its ability to work in combination with conventional antibiotics and antibacterial activity against a range of different bacteria species demonstrates its therapeutic potential.

During the course of evolution, the avian egg has developed multiple physical and chemical barriers in order to resist microbial challenges. These barriers are essential for the successful reproduction of avian species as well as to maintain safe and nutritious food for human consumption of the table egg. The calcified eggshell is a biomineralized barrier with an integrated organic matrix containing antimicrobial proteins, a hallmark of sophisticated biological structures. Calcium carbonate is deposited onto the outer shell membranes to form the calcified mammillary, palisade and vertical crystal layers; the final layer to be deposited is the outer eggshell cuticle. In this thesis, mass spectrometry-based technology was used to investigate the proteome of the outer cuticle, the mammillary cones and the shell membranes in order to gain insight into biomineralization and antimicrobial functions of the avian eggshell. Proteomics analysis of the eggshell cuticle revealed multiple antimicrobial proteins, supporting the hypothesis that the outermost cuticle layer is the first barrier against invading pathogens. The two most abundant cuticle proteins identified are similar to Kunitz-like protease inhibitor (ovocalyxin-25) and ovocalyxin-32. Multiple antimicrobial proteins were also revealed to be associated with the shell membrane fibres. Among the most abundant proteins were lysozyme C, avian β-defensin-11, ovotransferrin, ovocalyxin-36 and gallin. The biomineralized shell is also an important physical barrier against invading pathogens. Proteomics analysis of the mammillary cones, the initiation sites for shell calcification, revealed several candidate proteins involved in calcitic biomineralization. Promising candidates include nucleobindin-2 and SPARC, two calcium binding proteins previously shown to modulate mineralization. In-depth analysis of the comprehensive proteomes generated by this study revealed the presence of histones in the shell membranes, shell and cuticle compartments. Histones are cationic antimicrobial peptides, which are key molecules of the innate immune defense system of many species. This thesis reports the minimal inhibitory concentrations and minimal bactericidal concentrations of histones extracted from avian erythrocytes against Gram-positive, Gram-negative and antibiotic-resistant bacteria. Results suggest that the underlying antimicrobial mechanism is based on the interaction between histones and lipopolysaccharides / lipoteichoic acids, which are negatively charged components of bacterial cell membranes. Histones also inhibit the growth of Gram-positive biofilms; the minimal biofilm eradication concentrations were determined for S. aureus and methicillin-resistant S. aureus (MRSA). Sensitive proteomics analyses have provided great insight into the protein constituents of the eggshell matrix, with two primary roles in the innate immune defense of the egg: regulation of calcitic biomineralization and antimicrobial protection. Further research on these proteins and their functions can provide a new focus for selective breeding programs looking to enhance the egg’s natural defenses, or provide inspiration for alternatives to conventional antibiotics, such as the histones.

The rise of β-lactam-resistant bacteria from agricultural settings, including food-producing animals and their related food products has become a significant public health concern. Consumption of food contaminated by such bacteria may cause infection as well as the transmission of resistance genes. Here we used a mouse model to assess the impact of different antibiotic treatments on the composition of the gut microbiota and any impact on the transfer of β-lactam resistance genes between donor and recipient bacteria. Mice were inoculated with β-lactam resistant Escherichia coli and an antibiotic-susceptible Salmonella Heidelberg strain. The mice were treated with either streptomycin, ampicillin or both antibiotics. Mouse feces were collected at regular intervals and processed using selective culture techniques to capture potential transfer of resistance genes. Gene transfer was confirmed by whole genome sequencing. DNA extracted from the feces was used for monitoring changes in microbial profiles by 16S rDNA sequencing. In the absence of antibiotic treatment, the inoculated bacteria were only transiently detected and no transconjugants were recovered from the mouse feces. In comparison, antibiotic treatment changed microbial profiles in the mouse gut, enhanced colonization of the bacterial isolates, and facilitated the transfer of the resistance genes into both S. Heidelberg and commensal E. coli recipient strains. The results of this study indicated that the use of multiple antibiotics may enhance infection of opportunistic β-lactam resistant bacterial pathogens relative to single antibiotics and pose a greater risk in terms of antibiotic resistance gene transfer. Such process might occur in clinical settings where patients are under prolonged antibiotic treatments. Information gained through this study together with future work will inform the development of new policies guiding the prudent use of antibiotics.