Student Number : 9906314R - MSc dissertation - School of Pathology - Faculty of Science / Inappropriate use of antimicrobial agents has been shown to select for organisms with resistance mechanisms (eg. efflux pumps), which could lead to the development of antibiotic resistance. The objective of this study was to investigate a possible link between antibiotic resistant gastrointestinal pathogens and reduced susceptibility to anti microbial agents found in commonly used household disinfectants. Tetracycline and ofloxacin resistant and sensitive Salmonella and E.coli species were isolated from fresh poultry and clinical samples. Ethidium bromide accumulation assays were performed to assess the presence of active efflux pumps. Using spectrophotometric accumulation assays, the extrusion of the active components of commercial household agents by the efflux pumps was tested. To determine changes in the efficacy of these products, in-use disinfectant testing was performed. Active efflux pumps and extrusion of the active ingredients was observed in the resistant but not in the sensitive organisms. When the household products were used at the recommended concentrations, a significant reduction of both resistant and sensitive bacteria was observed after the in-use disinfectant testing procedure. However, if the household products were used at concentrations below the recommended concentration, the resistant bacteria were not eliminated as efficiently as the sensitive bacteria.

Bacterial infection and colonisation of polymeric biomaterials represents a major problem that is on the rise within the health care industry. Bacterial attachment and biofilm formation on medical polymers is often the cause of addition discomfort, pain and in the worse case scenario sepsis and even mortality. This, in combination to the overuse of antibiotics and evolution of resistant bacterial stains, means there is a need for a more intelligent approach in the prevention of biofilm formation and bacterial infection. In this work, the antimicrobial properties of metals (in particular silver and zinc) was utilized and incorporated into polymeric biomaterial to render them antimicrobial. Several methods of functionalising polymers with antimicrobial metals were assessed. The materials developed throughout this work were designed to respond to changes in environmental as a result of infection. These changes include differences in pH and temperature all of which are altered in response to infection. This smart design allows for the reduction of unnecessary release of antimicrobial, and will reduce the likelihood of toxicity and resistance. Polymer modifications in this research include modifications made during polymer synthesis, i.e. reaction with additional antimicrobial monomer. In this case, pH responsive zinc containing crosslinker molecule was designed to crosslink into any polymeric material. Post synthesis modifications were also investigated, and include the ‘grafting to’ and ‘grafting from’ of polymers which could then be functionalised with antimicrobial metals. This work demonstrated methods to modify non-woven polypropylene. A system, for the ‘grafting from’ approach to give a pH responsive release of antimicrobial metals from a polymer brush was investigated. Secondly a ‘grafting to’ approach to give a temperature responsive release of metals was investigated. Finally, several zinc compounds were synthesised and assessed for there ability to graft via plasma assisted grafting. The results presented in this work demonstrate novel ways of incorporating antimicrobial metal functionality into polymeric biomaterials and their antimicrobial efficacy.

Antimicrobial peptides are key actors in organisms€™ immune systems. They play an important role in phagocytosis, breaking bacteria membranes. They destroy the microbes, keeping them from repairing themselves, and therefore do not promote antimicrobial resistance. LL37 is a peptide produced by the human body. It is a short amino acid chain that is particularly active on the skin and mucous membranes. It has antimicrobial and fungal activity as well as wound healing properties, which makes it a very interesting active substance in wound treatment. However, its fragile and sensitive structure is a challenge to its use. Nowadays, encapsulation in a biocompatible polymer system is a promising technique in drug delivery, and presents a solution to LL37 administration and delivery. LL37 is a hydrophilic active substance, it will be trapped in PLGA (poly (lactic-co-glycolic acid)) by double emulsion and the microspheres will be shaped and stabilized by solvent evaporation. The capsules will be characterized by Dynamic Light Scattering (DLS) and Scanning Electron Microscopy. Their main features, drug loading, encapsulation efficiency and release profile, are determined using the Bradford assay. Since the peptide is expensive and delicate, it is important to optimize its encapsulation. For that reason, we will adapt the process to have the best drug loading as possible using water in oil in oil emulsions. For an external use, the capsules would be used over a few days, so having a fast release is very relevant. The larger the specific surface area, the faster the diffusion. For that reason, we will also study the impact of porosity on the release profile. As a result, different types of capsules will be synthesized, with higher porosity and by two processes: aqueous double emulsion and oil double emulsion. Their characteristic features and impact on bacterial pathogens will be determined and compared in order to determine their optimal synthesis process and formulation in given conditions of use.

Thesis (Msc.(Med.)(Pharmacy))--University of Limpopo, 2009. / Background: The study was conducted in primary healthcare facilities in Vhembe district in Limpopo province, South Africa. Seeing that the National Drug policy was implemented in 1996 and followed by the Standard Treatment Guidelines, it was necessary to investigate if PHC facilities are prescribing according to the guidelines. Although the data collected was from five clinics, iinformation on antimicrobial prescribing patterns is necessary for a constructive approach to the challenges that arise from the vast number of antibiotics that are available in the market and the occurrence of resistance. Objectives: The aim of the study was to investigate whether prescribers at Primary Health Care (PHC) facilities in Vhembe district adhered to the Standard Treatment Guidelines/Essential Drug List (STG/EDL) when prescribing antimicrobials. Method: Five hundred antimicrobial prescribing patterns and patient demographics were recorded from five PHC facilities (100 prescriptions in each) in the Vhembe District, Limpopo Province over a two weeks period. In all PHC facilities, data collection of prescriptions focused on the period from 01 August 2007 until the required number of 100 prescriptions was reached. x Results: Conditions where prescriptions complied to STG/EDL were skin infections and wound 91.67% (22/24), upper respiratory tract infection 86.49% (32/37), bronchitis 80% (4/5), ear/eye infections 75% (3/4), otitis media 57.14% (4/7), sore throat/tonsillitis 56.57% (56/99), urinary tract infection 37.5% (3/8), sexual transmitted infections 37.25% (19/51), bites 33.33% (1/3), and abscesses 25% (1/4). All other remaining 18 conditions that were listed as diagnosis did not comply at all. On overall only 29.2% (146/500) of prescriptions that were evaluated complied. Conclusion: Antimicrobial prescribing patterns adhering to the EDL/STD were low. Attention needs to be given to the prescribing of antimicrobials.

Thesis (PhD Med.(Microbiology))--University of Limpopo, 2010. / Studies were performed on local isolates of Neisseria gonorrhoeae to assess their aetiological role in different clinical populations, to determine the evolution of antimicrobial resistance and characterisation of quinolone resistance. In the study performed on women presenting for termination of pregnancy (TOP) the prevalence of common sexually transmitted pathogens (Neisseria gonorrhoeae, Chlamydia trachomatis and Trichomonas vaginalis) was much higher than reported from other studies in TOP populations. The commonest organism isolated was C. trachomatis. There were no significant differences in infection rates in women with or without signs and symptoms of vaginal discharge. Therefore all women presenting for TOP need to be screened and treated for sexually transmitted pathogens. In the study to determine the co-infection rates in men presenting with urethritis (discharge and/or burning on micturition (BOM)), the overall infection rate was 65% with co-infections (more than one pathogen detected) in 8%. N. gonorrhoeae was found in 45%, C. trachomatis in 15% and T. vaginalis in 6% of the men. T. vaginalis was found in higher percentages in men with BOM only, in the absence of visible discharge. There may be a need to add an anti-trichomonicidal agent in men presenting with BOM only. When comparing sexual partners and the pathogens isolated, significantly fewer pathogens were detected in males that had their wives as sole contact when comparing them to men who had sex with casual contacts, reflecting high sexual risk behaviour. M DE JONGH Page xi PhD Med Microbiology N. gonorrhoeae isolates were obtained from men presenting to a general practitioner in Pretoria with urethral discharge. The antimicrobial susceptibility profile was determined to currently used, previously used and potential antimicrobial agents by the disk diffusion, Etest and agar dilution methods. High-level ciprofloxacin resistance emerged in the Pretoria region at 7%. No isolate showed a MIC value of intermediate resistance, suggesting importation of resistant strains, rather than a step-wise incremental increase. Penicillin-resistant gonococcal isolates are entrenched in the community; overall there was 32% resistance (MIC≥2μg/mℓ), with 16% due to penicillinase-producing (PPNG) isolates. Tetracycline-resistant isolates has increased dramatically at 54% and with 36% showing high-level (plasmid-mediated) resistance. All isolates remained susceptible to ceftriaxone, cefoxitin and cefpodoxime. Local gonococcal isolates were sequenced using Neisseria gonorrhoeae Multi-Antigen Sequence Typing (NG-MAST). In this study a total of 18 isolates resolved into 13 different sequence types (STs). A cluster of four isolates of known sequence type, ST217, was found. Two other known sequence types (ST189 & ST523) have previously been seen in Durban. The ten quinolone-resistant isolates resolved into six STs, five of which were new STs and one cluster of four isolates of a known sequence type. This demonstrates the heterogeneity of ciprofloxacin-resistant strains suggesting introduction of strains from multiple sources rather than clonal spread of a single strain.

acase@tulane.edu / Development of antimicrobial peptides (AMPs) as next generation clinical antibiotics has been a pursuit of the scientific community for several decades. AMPs are attractive drug candidates because of their potent antibacterial activity and a low propensity for eliciting antibiotic resistant bacterial phenotypes. However, despite substantial efforts and myriad development approaches, AMPs have yet to make inroads in the clinic due to toxicity concerns and activity loss in vivo. We hypothesized that eukaryotic cytotoxicity and antibacterial activity loss are intricately related in that peptide-induced tissue or host cell damage corresponds to depletion of free peptide available to target bacterial cells. Using human red blood cells (RBCs) as a model eukaryotic cell, we demonstrate that a cross-section of AMPs lose appreciable antibacterial activity when preincubated with concentrated eukaryotic cells (1x109 red blood cells/mL) and that this behavior can be explained by plasma membrane binding. To approach this problem in a unique manner, we synthesized a combinatorial peptide library based on the potent AMP, ARVA, and screened the library for activity in the presence of concentrated RBCs. We isolated nine unique, but similar sequences from the screen. During the screening program, we discovered that RBC-peptide interactions lead to peptide degradation through the release of cytosolic proteases. We used this knowledge to design a consensus sequence based on the nine peptides isolated from the library screen and synthesized it using only D-isomer amino acids. The novel peptide displays excellent antimicrobial activity against several human pathogens in the presence and absence of concentrated RBCs, has reduced toxicity towards eukaryotic cells, and is not susceptible to cleavage by cellular proteases. We attempted to use this peptide, D-NOGCON, to combat P. aeruginosa in a mouse model of acute pneumonia, but were unable to ameliorate the negative outcomes associated with infection. We ultimately suggest alternative models of bacterial infection in which the peptide may be more effective and future approaches to further refining the sequence of D-NOGCON. / 1 / Charles Starr

Background: Antimicrobial resistance (AMR) is a widely recognized threat to global health that has been accelerated by the overuse of antimicrobials. Policy and regulatory interventions offer a useful approach to bring about widespread change in antimicrobial use. Objectives: This dissertation aims to investigate and characterize the role of policy interventions on antimicrobial use, to understand which policy levers have been used, which interventions have proven to be effective, and to identify analytic strategies that assist researchers and policymakers in developing evidence-informed approaches to address human AMR. Methods: To investigate effectiveness of policy interventions, I developed a systematic review and evidence map and appraised the quality of research on government policies to reduce antimicrobial use. To evaluate the extent of policy action on AMR in the Canadian context, I conducted an environmental scan of AMR action between 2008-2018. Finally, to facilitate advances in the scientific evidence base on antimicrobial resistance policy, I developed a framework for planning, conducting, and disseminating AMR intervention research. Findings: Governments have a variety of policy options at their disposal to respond to the growing threat of AMR, however, most of these policy options have not been rigorously evaluated and their effectiveness is unknown. Some regulatory, communication, guidelines, and fiscal interventions have been effective in particular settings, however, the interventions were too disparate to generalize about the effectiveness of any particular policy approach and reporting gaps in pose challenges for interpreting and replicating study results. In Canada, policy interventions have been too few, too small and too uncoordinated to achieve our AMR goals. Building on these findings, I developed a framework for planning, conducting, and disseminating AMR intervention research that identifies challenges, areas for increased coordination with policymakers, and best-practices for overcoming common limitations in AMR research. Discussion: Governments around the world have committed to respond to AMR, however, rigorous evidence to inform their policy and regulatory strategies is lacking. As the threat posed by AMR grows, it is increasingly important to strengthen the scientific evidence base on AMR policy interventions, to learn from existing policies and programs, and integrate scientific evidence into the global AMR response.

Cationic antimicrobial peptides (CAPs) offer a viable alternative to conventional antibiotics as they physically disrupt the bacterial membranes, leading to cell lysis and death. However, colonized bacteria often form “biofilms” – characterized by the overproduction of exopolysaccharides - that restrict the penetration of antibiotics; successful antimicrobial agents must evade this exopolysaccharide ‘matrix’ to reach the bacterial membrane. Since the Pseudomonas aeruginosa biofilm alginate traps CAPs by forming peptide-alginate complexes, the aim of this thesis is to better understand the mechanisms of interaction of CAPs with bacterial membranes and biofilm alginate. Using a series of CAPs designed in our lab derived from the sequence KKKKKK-AAFAAWAAFAA-NH2, we found that hydrophobicity, charge distribution, and amino acid composition of CAPs play important roles in their membrane disruptive power, bioactivities, alginate-binding and alginate-diffusion abilities. These findings suggest routes to an optimal balance of factors in CAP design to allow both biofilm penetration and bacterial membrane destruction.

Cationic antimicrobial peptides (CAPs) offer a viable alternative to conventional antibiotics as they physically disrupt the bacterial membranes, leading to cell lysis and death. However, colonized bacteria often form “biofilms” – characterized by the overproduction of exopolysaccharides - that restrict the penetration of antibiotics; successful antimicrobial agents must evade this exopolysaccharide ‘matrix’ to reach the bacterial membrane. Since the Pseudomonas aeruginosa biofilm alginate traps CAPs by forming peptide-alginate complexes, the aim of this thesis is to better understand the mechanisms of interaction of CAPs with bacterial membranes and biofilm alginate. Using a series of CAPs designed in our lab derived from the sequence KKKKKK-AAFAAWAAFAA-NH2, we found that hydrophobicity, charge distribution, and amino acid composition of CAPs play important roles in their membrane disruptive power, bioactivities, alginate-binding and alginate-diffusion abilities. These findings suggest routes to an optimal balance of factors in CAP design to allow both biofilm penetration and bacterial membrane destruction.

Limited information exists regarding the efficacy of consumer-available antimicrobials for the use on produce surfaces. There is a strong focus on eliminating pathogens from produce at a commercial level, but consumers can achieve pathogen reduction in a domestic setting. The objectives were to determine the ability of consumer-available antimicrobials to disinfect waxed green bell peppers, determine the efficacy of knife cleaning methods, and assess the transfer of contamination. Peppers were inoculated via immersion in a cocktail of rifampicin-resistant Salmonella serovars and Escherichia coli O157:H7 to a final concentration of 5.6 ± 0.5 log CFU/cm2. In study 1, samples of 3 10-cm2 pieces of inoculated pepper were excised from smooth tissue and immersed in 3% (v/v) hydrogen peroxide (H2O2), 2.5% (v/v) acetic acid, 70% (v/v) ethyl alcohol (EtOH), or sterile distilled water (SDW) for various lengths of time. Following treatment, samples were immersed for 30 s in a neutralizer solution. For study 2, inoculated peppers were chopped into 1-cm2 pieces. Knives were treated with one cleaning method: no treatment (control), towel wipe (TW), running hot water for 5 s (5SW), running hot water for 10 s (10SW) or 1% (v/v) detergent solution followed by hot running water for 10 s (ST). After treatments, knives were used to chop cucumbers. Surviving Salmonella and E. coli O157:H7 for both studies were selectively enumerated on lactose-sulfite-phenol red-rifampicin agar following aerobic incubation of plates for 24 h at 35 °C. Hydrogen peroxide exposure for 5 min resulted in reductions of 1.3 ± 0.3 log CFU/cm2 for both pathogens. Following 1 min exposure to EtOH, pathogens were reduced by 1.3 ± 0.1 3 log CFU/cm2; exposure for >1 min did not result in additional reduction. Acetic acid exposure after 5 min resulted in a Salmonella reduction of 1.0 ± 0.7 log CFU/cm2, but for E. coli O157:H7, exposure resulted in no significant reduction (p<0.05) of pathogens compared to SDW at the various points. For study 2, 5SW, 10SW, and ST were equally effective for knife decontamination. No significant difference (p<0.05) was found between log CFU/cm2 on knife blade and log CFU/cm2 transferred to surface of cucumber; therefore, viable organisms remaining on the knife blade were transferred onto the surface of the cucumber. Findings suggest EtOH and H2O2 may be effective consumer-deployable antimicrobials for surface decontamination of smooth produce, and contaminated produce can contaminate other produce. Further research of antimicrobial exposure on produce sensorial characteristics is also advised in order to determine how various antimicrobial exposure times will affect the quality and sensorial characteristics of the produce commodity.