Antimicrobial peptides (AMPs) are innate host defense peptides that protect against pathogenic microbes by neutralizing toxins or via a direct killing mechanism. AMPs are classified based on their physical properties such as charge, structure, and binding motifs. Here we investigated the antimicrobial and immune-modulating effects of the Regenerating Islet-Derived Protein 3 (REG3) family and LL-37 REG3 peptides are C-type lectins and have been demonstrated to have antimicrobial activity against Gram-positive bacteria by binding to sugars on the peptidoglycan membrane of these bacteria. A similar strategy is also employed by the lectin Surfactant Protein-D which has been shown to bind and neutralize Influenza A Virus (IAV). REG3 peptides were shown to be expressed in the lungs of mice infected with IAV. We observed reduction of IAV infected cells when IAV was pre-incubated with an Escherichia coliexpressed recombinant version of human REG3A peptide. This peptide also modified interaction of IAV with primary human neutrophils. However, these effects were lost when using a mammalian cell expressed recombinant REG3A. A second member of the REG3 family, REG3G, showed minimal inhibition of IAV infection. While the mechanism remains unclear, LL-37 has demonstrated killing activity against a spectrum of microbes including IAV. Previous work from our group identified the core domain of LL-37 responsible for IAV neutralization. In addition, our group showed that LL-37 modulates interaction of IAV with neutrophils. Here we tested three modified versions of LL-37 that retain the overall size and charge of LL-37, but with modifications in the core domain reducing hydrophobicity. We observed that these mutants retain IAV killing activity across multiple strains. In addition, these mutants retain the modulation of IAV induced neutrophil responses. We also found that the compounds sodium butyrate and Entinostat, which can upregulate endogenous expression of LL-37, have variable effects in IAV infection. We believe these findings will aid in the development of LL-37 derivatives to expand the repertoire of antimicrobials.

Introduction: Carbapenems and colistin are the last-line antibiotics for treating Gram-negative bacterial infections. However, increasing reports of resistance to these antibiotics is being reported in clinical settings, the environment and in animals. In this paper, we describe the molecular epidemiology and resistance mechanisms of colistin and carbapenem resistance in clinical, veterinary, and environmental Enterobacterales isolates in Pretoria, South Africa. Method: One hundred VITEK®-2-confirmed colistin and carbapenem-resistant clinical isolates were collected from the departmental isolate bank at the National Health Laboratory Service. A total of 88 porcine (stool) and 11 environmental (effluents) samples were collected in November 2018 and again in March 2019 from a farm in Pretoria. Both the porcine and environmental samples were screened using Eosin methylene blue agar with colistin and ertapenem disks. All isolates were identified and a minimum inhibitory concentration of colistin and carbapenems was determined using the MicroScan® WalkAway system. Isolates resistant to colistin were confirmed by the broth microdilution method. Isolates phenotypically resistant to colistin and carbapenems were selected for whole genome sequencing to determine the resistome and phylogenetic trees were drawn to determine the relatedness of isolates. Results: A total of 275 Gram-negative isolates were identified from the clinical (100), environmental (57) and veterinary (118) samples using the MicroScan® WalkAway system. The MicroScan® WalkAway system’s minimum inhibitory concentration results for clinical isolates revealed 88% and 93% resistance to colistin and carbapenems, respectively. BMD was found to be more reliable in all isolates, and it recorded higher MICs (increased resistance) than the MicroScan® WalkAway system. Overall, colistin susceptibility was higher among animal isolates compared to the clinical and environmental samples. Genomic analysis identified several resistance genes associated with resistance among the isolates and the CTX-M family were the dominant resistance genes. Phylogenomic analysis demonstrated closer evolutionary relationship between EB008 (environment), SW10B (animals), and C080 and C084 (both humans) strains as well as with strains from the United States of America, Canada, China, Russia and Durban (South Africa). Conclusion: The study established multiple resistance genes from different antibiotics to mediate resistance in Enterobacterales isolates from humans, animals and the environment. The presence of carbapenemases in animals is alarming and poses a public health concern. Strains EB008 (environment), SW10B (animals) and C080 and C084 (both human) were phylogenetically related with strains from the United States of America, China and Durban (South Africa) more commonly. Therefore, One Health approach studies are significant to ascertain colistin and carbapenem transmission from human to animals/the environment and vice versa to combat increasing resistance in Enterobacterales. / Dissertation (MSc)--University of Pretoria, 2020. / National Research Foundation (NRF) / National Health Laboratory Service research grant / Medical Microbiology / MSc / Unrestricted

archives@tulane.edu / Gram-negative bacteria secrete outer membrane vesicles (OMVs) that play critical roles in intraspecies, interspecies, and bacteria-environment interactions. Some OMVs, such as those produced by Pseudomonas aeruginosa, have previously been shown to possess antimicrobial activity against competitor species. In the current work, we demonstrate that OMVs from Burkholderia thailandensis inhibit the growth of drug-sensitive and drug-resistant bacteria and fungi and exhibit antibiofilm activity against methicillin-resistant S. aureus (MRSA) and Streptococcus mutans. We show that a number of compounds, including peptidoglycan hydrolases, 4-hydroxy-3-methyl-2-(2-non-enyl)-quinoline (HMNQ) and long-chain rhamnolipid present in B. thailandensis OMVs exert antimicrobial activity. Furthermore, we demonstrate that HMNQ and rhamnolipid possess antimicrobial and antibiofilm properties against various microbes. Rhamnolipid is superior at reducing the integrity of biofilms while HMNQ displays greater bactericidal activity. We attempted to use HMNQ and rhamnolipid to combat MRSA and promote wound healing in a murine full-thickness wound model. However, further optimization of the model and characterization of the molecules in antimicrobial efficacy, wound healing, and host immune responses are required. Overall, this work indicates that B. thailandensis secretes antimicrobial OMVs that may impart a survival advantage by eliminating competition. In addition, bacterial OMVs may represent an untapped resource of novel therapeutics effective against biofilm-forming and multidrug-resistant organisms. / 1 / Yihui Wang

In the early phases of drug design and development, scientists must overcome many challenges involved in identifying potential drug-like or lead-like compounds. This has led to the need of creating large sets of chemical data which will aid in improving the identification of pharmacophores and active compounds. Various scientific fields especially pharmacology, medicinal chemistry and biochemistry have begun to employ the use of computer sciences to aid in the screening for potential leads with more specificity with regards to drug-like compounds' or substances' bioactivity. The emphasis of this project was to create a database containing a collection of pyrazine compounds synthesized overtime in the Faculty of Pharmacy (Charles University, Hradec Kralove) with the aim of having anti-mycobacterium (and possible antibacterial and antifungal) activity, and further utilize this database to predict certain pharmacokinetic and bioavailability properties. This project seeks to demonstrate how certain molecular descriptors can be used as reliable chemoinformation to determine the likeliness or possibility of developing a lead-like or drug-like compound by utilizing computer software. An in-house database of 623 compounds saved in SMILES format was created and used in demonstrating quantitative...

Purpose: Use of ampicillin in outpatient parenteral antimicrobial therapy (OPAT) has historically been complicated by frequent dosing and short beyond use dates. However historic stability data relied on inaccurate testing methods. The purpose of this study is to evaluate the stability of ampicillin using high-pressure liquid chromatography (HPLC), the gold standard, in a real-world OPAT dosing model using continuous infusion at room temperature over 24 hours immediately following preparation compared to batches stored under refrigeration for 24 hours, 72 hours, and 7 days. Methods: An HPLC method was developed and validated as stability – indicating according to guidance in USP general Chapter . Method development included linearity, precision, accuracy, repeatability and forced degradation. Four batches were prepared using 4 different lots from 2 different manufacturers for each storage condition (immediate, 24 hours, 72 hours, and 7 days). Three 2-gram vials were each reconstituted with 10 mL of sterile water for injection (SWFI) and added to 250 mL of normal saline by a licensed pharmacist and stored in a laboratory refrigerator (2 – 8oC). A pump system was used to continuously circulate the solutions through medical grade tubing at room temperature. One milliliter aliquots were removed from each batch at time 0, 4 hours, 8 hours, 12 hours and 24 hours and analyzed for ampicillin concentration using the aforementioned HPLC method. The samples were filtered prior to analysis using a 0.22-micron syringe filter and analyzed in triplicates along with freshly prepared calibration samples (24 – 12 mg/mL). Peak area was used to determine percent recovery for each sample. Results: Each batch was assayed for initial concentration (20.34 – 21.50 mg/mL) upon preparation, and percent recovery was compared to that initial concentration thereafter. Acceptable recovery was defined as 90 – 110% of initial concentration. On the day of product preparation (immediate use), the average percent recovery over 24 hours was 96.4%. The other average percent recoveries were as follows: 95.8% (24-hour storage), 94.6% (72-hour storage) and 90.3% (7-day storage). These data represent the average percent recovery for all time points during the 24 hours sampling (n = 60 for each experiment). When evaluating individual time points, the percent recovery remained above 90% for all batches and time points except for the 7-day storage experiment. Under 7-day storage conditions, the percent recovery fell below 90% after 4 hours of circulation through the medical grade tubing. Furthermore, 95% confidence interval for percent recovery for ampicillin in the samples stayed within 90 – 110% of the initial concentration for the duration of the experiment for all test groups except 7-day storage. Conclusions and Relevance: Ampicillin can be prepared and stored in a refrigerator for up to 72-hours prior to continuously infusing at room temperature over 24 hours with less than a 10% loss of potency over the dosing period. This model supports twice weekly OPAT delivery of ampicillin.

Despite many medical advances, antibiotic resistant bacteria increasingly plague the modern world, necessitating discovery of new antibiotics. One area of nature that can provide inspiration for antibiotics is antimicrobial peptides. Many of these peptides exist in nature, with some classes that have not been studied or characterized well. One such class is the defensin-like peptides generated by the plant Medicago truncatula as part of their symbiotic relationship with Sinorhizobium meliloti. Nodule-specific Cysteine Rich (NCR) peptides are defined by the presence of multiple cysteines, and regulate the growth of S. meliloti within plant cells. While some of these NCR peptides have been shown to have antimicrobial properties, hundreds of peptides remain uncharacterized. We have developed an assay for further characterization of these peptides in E. coli. Of the seven peptides that have been tested using this assay, three have exhibited definitive antimicrobial properties against both E. coli and S. meliloti. Additionally, we have developed a system for discovering novel antimicrobial peptides. This platform, called PepSeq, uses the expression of random peptides in E. coli combined with deep sequencing to detect antimicrobial activity. This technology is capable of screening through millions of peptide molecules simultaneously. Using this platform, we have discovered and confirmed six novel antimicrobial peptides, with hundreds of additional predicted antimicrobial peptides. In addition to the peptides we have analyzed using PepSeq, additional peptide scaffolds could be used to discover more potent antimicrobial peptides.

A dissertation submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in fulfillment of the requirements for the degree of Master of Science in Medicine. Johannesburg, 2017. / Antimicrobial stewardship (AMS) programmes along with infection and prevention control measures have been shown to reduce the burden of antimicrobial resistance (AMR) in hospitals. There is a global campaign by infectious diseases physicians and other stakeholders for hospitals to implement AMS programmes. In Africa, there have been a limited number of AMS studies conducted although South African private hospitals have published some outcomes on initiation of these programmes in the continent, with the aim of improving patients’ clinical outcomes and reducing the development of resistance to prescribed antibiotics. A formal AMS programme is yet to be implemented in the surgery departments of the Charlotte Maxeke Johannesburg Academic Hospital. This study was conducted in two surgical wards of the Charlotte Maxeke Johannesburg Academic Hospital (CMJAH). It was a quantitative study combining a prevalence cross-sectional observational stage, and an intervention study. It involved a retrospective review of patient records in the baseline stage followed by an intervention which took the form of a weekly antibiotic round led by an infectious diseases specialist. The appropriateness of antibiotic prescriptions was assessed using the criteria developed by Gyssens and colleagues, while the appropriateness of surgical prophylaxis was determined based on the recommendations of the South African Antibiotic Stewardship Programme (SAASP) and current Standard Treatment Guidelines and Essential Medicines Lists for South Africa. The prices of the antibiotics used were obtained from the central pharmacy of the CMJAH and Masters Price Catalogue list of the National Department of Health, while the prices of laboratory tests were obtained from the Tariff database. The volume of antibiotics consumed was determined by Defined Daily Doses (DDDs)/1000 patient days. In both stages of the study amoxicillin/clavulanic acid was the most frequently used agent. The intravenous route was the most commonly used route of drug administration in both stages of the study. There was a reduction in the proportion of patients who were treated with antibiotics for more than seven days in the intervention stage, from 6.19% in the baseline stage to 2.07% in the intervention stage. A significant reduction in the duration of antibiotic therapy for two days and more was observed from 4.74 ± 4.58 days in the baseline stage compared to 3.96 ± 2.04 days in the intervention stage (p = 0.01). A shift from empiric to culture directed therapy was also observed in the intervention stage compared to the baseline stage. There was a significant reduction in the volume of antibiotic consumption from a total of 739.30 DDDs/1000 patient days in the baseline stage to 564.93 DDDs/1000 patient days in the intervention stage (p = 0.038). Overall, there was a significant reduction of inappropriate antibiotic utilisation from 35% in the baseline stage to 26% in the intervention stage (p = 0.006). A high percentage of inappropriate surgical prophylaxis was found which was mostly due to the incorrect choice of agent with 64.75% and 61.54% in the baseline and intervention stages, respectively. The average antibiotic cost per patient was reduced from R 268.23 ± 389.32 to R 228.03 ± 326.88 in the Vascular Surgery Ward compared to the General Surgery Ward where there was an increase in average cost per patient from R 219.80 ± 400.75 in the baseline stage to R 284.06 ± 461.28 in the intervention stage. Gram-negative bacteria were the most prevalent pathogens in both stages of the study at 53% in the baseline and 54% during the intervention stage. The findings of this study show an improvement in the appropriateness of antibiotic utilisation, reduction in antibiotic consumption and cost reduction in one of the study wards, following implementation of an AMS programme. Also, there was an improvement in culture directed therapy, requests for an appropriate biological specimen for culture, with a consequent increase in the cost of laboratory investigations per patient during the intervention stage, which was due to increases in culture request. Rational antimicrobial prescribing habits, strong AMS interventions along with infection and prevention control measures, sound government policies and surveillance of resistant organisms in Africa will go a long way in preserving our antibiotics and preventing the spread of multidrug-resistant pathogens. / LG2018

To improve the success of vegetative propagation of Sciadopitys verticillata, stem cuttings were subjected to three treatments designed to minimize the accumulation of a latex-like sap at the cut ends of stems. A 24-hour soak in water before a hormone dip significantly enhanced rooting success and root mass. The water soak pretreatment was more beneficial to hardwood cuttings compared with softwood cuttings. Cuttings from shade-grown source trees showed the highest rooting success, but source tree age, height, and place of origin were not important factors. The water-insoluble latex-like sap had strong antibacterial activity against 3 of 11 bacterial species tested, but activity was not related to bacterial Gram reaction or the bacterial natural environment. In contrast, pine resins and latexes from selected angiosperms showed no antibacterial activity. The antibacterial component of the Sciadopitys latex-like sap was heat stable and therefore probably not protein based.

Candida albicans is a fungus found in the normal biota of humans, but in immuno-compromised individuals, C. albicans forms complex biofilms on the surface of medical prosthetics, skin, oral cavities, the urinary tract, and other epithelial cell layers. Biofilms and the development of drug resistance has limited treatment options. Antimicrobial peptides (AMPs) are increasingly becoming attractive therapeutic agents for the treatment of these infections due to their multifunctional properties, multiple cellular targets, and the lower incidence of resistance development. Previous studies have shown that Os, an AMP derived from the tick defensin OsDef2, has antifungal activity against C. albicans. Preliminary antifungal mode of action studies indicated that Os induces the formation of reactive oxygen species although not a primary mode of killing. Os causes membrane permeabilization, which is inhibited by an excess of free laminarin and mannan. Furthermore, Os was shown to bind plasmid DNA but was inactive in high salt conditions. The aim of this study was to further explore the mode of action of Os in planktonic C. albicans (ATCC 90028) cells. A modified microbroth dilution assay was developed to allow rapid screening of salt sensitive AMPs such as Os. With this method the IC50 of the positive control, amphotericin B (AmpB), and Os were determined as 0.547 ± 0.056 μM and 1.163 ± 0.116 μM, respectively. The effects of AmpB and Os on cellular morphology were evaluated using scanning electron microscopy and transmission electron microscopy at their respective IC25, IC50 and IC75 values. When comparing the effects of Os with AmpB on the cell wall and membrane, Os had more severe and nonspecific effects. Os induced the formation of pits on the cell surface and pores in the cell membrane, as well as increased budding scars. Using isothermal titration calorimetry, no interaction between Os and the fungal cell wall components, mannan and laminarin, could be detected. Factors such as the lack of tryptophan and aspartate residues as well as β-sheet secondary structures may account for the lack of interaction. However, with the modified microbroth dilution assay in the presence of excess of mannan or laminarin (20 mg/mL), reduced activity from Os was observed. The formation of soluble macro-complexes between Os and the cell wall components at high concentrations may account for reduced activity. The ability of Os to cause membrane depolarization was evaluated with bis-(1,3-dibutylbarbituric acid) trimethine oxonol. The control, melittin, caused a linear increase in depolarization with a significant increase at 0.63 μM, while Os caused a sigmoidal increase in depolarization with a significant increase at 2.5 μM. Therefore, membrane depolarization occurs following membrane permeabilization which occurs at 2 μM. Finally, the localisation of 0.5 μM and 6.4 μM (IC25, IC75) 5-FAM-Os, and concurrently the effect on vacuoles loaded with CellTracker Blue-CMAC, was determined with flow cytometry and confocal laser scanning microscopy (CLSM). Findings were that Os, at a concentration below its IC50, binds to the cell membrane, then translocates and binds DNA. At a concentration above its IC50, Os accumulates in the cytoplasm and causes destruction of membranes, including that of vacuoles, leading to cell death. In conclusion, this study shows that Os is a membrane acting AMP that can be further developed for clinical application as an antifungal drug. / Dissertation (MSc (Biochemistry))--University of Pretoria, 2020 / NRF / Biochemistry / MSc (Biochemistry) / Unrestricted

The goal of antibiotic stewardship is to improve antibiotic use, often by reducing unnecessary treatment. Bedside dysphagia screening tools help identify patients at high risk of aspiration following stroke. Presence of dysphagia does not indicate a need for antibiotic treatment. Therefore, this retrospective, cohort study was developed to evaluate the association of dysphagia and antibiotic prescribing following stroke. There were 117 patients included. Patients were placed into 2 cohorts based on the results of the dysphagia screening, with 55 patients positive for dysphagia and 62 patients negative for dysphagia. Patients with dysphagia tended to be older, had higher National Institutes of Health stroke scores, and lower renal function. Patients with dysphagia were prescribed more empiric antibiotics than those without dysphagia (18.2% vs. 3.2%, p = 0.01). This resulted in 53 antibiotic days of therapy in the dysphagia cohort compared to 19 antibiotic days of therapy in the no dysphagia cohort (p = 0.1). No patients later developed pneumonia and only one patient was started antibiotics after 48 h. Two cases of Clostridioides difficile were reported. Both patients were in the dysphagia cohort and received antibiotics. Multivariable logistic regression demonstrated that positive chest x-ray findings and failed dysphagia screen were independent conditions associated with initiating antibiotics. These findings indicate that antibiotic use was higher in patients following stroke with a positive dysphagia screen. Close monitoring of stroke patients, particularly when positive for dysphagia, might be an under-recognized antibiotic stewardship opportunity.