Thesis (M.Sc.(Medical Sciences)) -- University of Limpopo, 2022 / Background: Wound infections continue to be problematic in clinical practice where empiric treatment of infections is a routine, with non-healing wounds being a burden to the health care system. A gap has been noted between antimicrobial resistance and demographic factors as an existing relationship. This necessitates an investigation of patterns of isolates and susceptibility profiles of microorganisms in wounds to modify the preventative and therapeutic strategies against the resistant strains leading to the stall of wound healing, which could aid in empiric treatment. Objective: The aim of this study was to determine the antimicrobial patterns and their associated demographic determinants in bacteria isolated from patients with non healing wounds at Pietersburg and Mankweng Hospitals, Limpopo Province. Methods: The study was conducted using antimicrobial susceptibility data collected from National Health Laboratory Service through Academic Affairs and Research Management System for the period 2016-2020. A total of 797 Antimicrobial Susceptibility Test results were analysed using Statistical Package for Social Sciences version 27.0. The susceptibility rates for the bacterial isolates by age and gender were calculated. The mean percentages for sensitivity and resistance were also calculated. Pearson’s Chi-square test was used to compare age and gender with drug susceptibility. A p-value of ≤ 0.05 was considered significant. Results: Of the 797 patient Antimicrobial Susceptibility Test results, 372 (46.7%) were males and 425 (53.3%) females, with mean age of 31.42 ± 21.75 years. The most common isolates were, Klebsiella pneumoniae (23%), Pseudomonas aeruginosa (21.7%), Escherichia coli (16%) and Proteus mirabilis (13.5%). Highest percentage of resistance to any antibiotic was amoxicillin, ampicillin (85.15%) then trimethoprim sulfamethoxazole (60.85%), amoxicillin ampicillin (49.1%), tigecycline (46.35%), cefepime (32.7%), gentamycin (25.4%), ciprofloxacin (22.5%), colistin (17.6%), and meropenem (12.3%). Furthermore, the general view of the study is no statistically clinical significance on the effect of age and gender on bacterial resistance although statistical significance was noted on age the resistance Acinetobacter baumannii vi (p=0.018), and gender on K. pneumoniae (p=0.015), P. mirabilis (p=0.024). Major resistance to A. baumannii, K. pneumoniae and P. mirabilis were from female patients. Conclusions: The most effective antibiotics were meropenem, colistin, and ciprofloxacin. The highest number of isolates were K. pneumoniae, E. coli, P. aeruginosa, P. mirabilis and A. baumannii with the most effective antibiotics gentamycin, meropenem, ciprofloxacin, and cefepime. Although the general view of the study is that no statistically clinical significance was noted on the effect of age and gender on bacterial resistance, it is important to note the significant observation that there was an observed relation of age to amoxicillin-clavulanic acid and Ciprofloxacin and gender to amoxicillin ampicillin. As such, there is insufficient evidence that supports the effect of age and gender on antimicrobial susceptibility. The study suggests caution against the use of amoxicillin ampicillin in the treatment of wound infections as it confers low levels of efficacy and high resistance and ultimately the call to revise minimum inhibitory concentrations and critical concentrations of all less effective drugs to increase their efficacy. / National Research Foundation (NRF)

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Engineering therapeutic approaches to wound healing can be divided into two major categories of fibrous and non-fibrous approaches. There has been significant progress in designing artificial skin products to replace autografting. For patients with non-healing/hard-to-heal wounds, there is an unmet clinical need for inexpensive skin substitutes to be transplanted. In skin regeneration area of research, electrospinning is a very commonly used method of production of grafts for wound healing applications, owing its popularity to the fibrous nature of the resultant product, which mimics the extracellular matrix of the native skin. Despite the high degree of porosity in conventional electrospun scaffolds, the small pore size effectively limits the penetration of cells into the scaffold. Transplantation of such scaffolds with poor cell infiltration abilities may lead to a range of negative consequences, from prolongation of the first/destructive phase of inflammation to rejection of the scaffolds. Several experimental approaches have been developed to generate interfibrillar space in the electrospun scaffolds, including but not limited to modifications of the electrospinning set-up and inclusion of sacrificial components. It has been reported that scaffolds with larger pore diameters in the range of ~ 40-100 μm can modulate, moderate and reduce acute inflammatory responses of the body, by influencing macrophages biological behavior, and direct the course of the wound healing process to the tissue remodeling phase. Macrophages are the major cell component of innate immune system and play critical roles in clearance of pathogens, resolution of inflammation and wound healing following an injury. Macrophages are characterized by their diversity and plasticity. In response to environmental stimuli, they acquire different functional phenotypes of pro-inflammatory (M1) or anti-inflammatory (M2). In this thesis, we developed a novel unique gradient porous structure from a plant-based “green” soy protein isolate (SPI) with improved pore size for macrophages to infiltrate. We further showed the ability of the scaffold to modulate phenotype switch in macrophages in vitro and in vivo. The proposed scaffold, moreover, appeared to support transition of the inflammation process from the destructive to the constructive phase in vivo. Based on the promising results of this thesis, we propose our newly developed scaffold has the ability to be used as a new therapeutic modality for treatment of non-healing chronic wounds. / Bioengineering

This study investigated the use of "naturalistic stressors" such as physical restraint and animal pheromones on the etiology of Posttraumatic Stress Disorder in a murine model. Pilot data suggest that stress effects may lead to an increase in the amount of time needed for cutaneous wounds to heal. Pilot data to support the creation of this model are presented suggesting that a delayed stress response may inhibit healing rates. In the present study an animal model of PTSD was used to investigate the effect of stress on the immune system. Yehuda and Antelman's (1993) nonhuman animal model of Posttraumatic Stress Disorder was tested with respect to the animals' immune response to cutaneous wounding. Additionally, effects of stress on exploratory behavior and activity were examined. The findings support the hypothesis that restraint and pheremonal stress and housing arrangements influence the ability of mice to heal a 1.5 mm punch biopsy, and exploratory behavior. The findings also support a profile for the Post-Traumatic Mouse. / Ph. D.

Whether innate immune cells may be adapted into potential memory states has becoming an important question in the field of immunity. Although previous conceptual paradigm failed to acknowledge this important question, emerging clinical and basic observations have started to shed intriguing clues to shake the previous dogma regarding innate immunity of being "simple", "raw", "first-line defense with no memory". We have aimed to further address this fundamental issue in this dissertation work, under the close guidance of Dr. Liwu Li. We have chosen to use the model system of Toll-Like-Receptor (TLR) signaling networks within primary monocytes. TLRs play fundamental roles in sensing pathogen-associated molecular patterns (PAMPs) and modulation of innate immunity. Lipopolysaccharide (LPS), an endotoxin found on the cell membrane of gram-negative bacteria, is the ligand of TLR4 and induces a range of inflammatory as well as anti-inflammatory responses. Higher dosages of LPS were known to cause robust yet transient expression of pro-inflammatory mediators. On the other hand, the effects of super-low dose LPS, commonly manifested in humans with adverse health conditions, have been largely ignored in the basic research field. Super-low dose LPS may skew host immune environment into a mild non-resolving pro-inflammatory state, which is a risk factor for inflammatory diseases such as atherosclerosis, compromised wound healing, and elevated risks for sepsis. Our central hypothesize is that monocytes may be adapted by super-low dose LPS into a non-resolving low-grade inflammatory state conducive for the pathogenesis of inflammatory diseases. We have employed both in vitro cell culture system as well as in vivo disease models to test this hypothesis. For the in vitro system, we have cultured primary murine monocytes with increasing signal strength of LPS. Monocyte phenotypes such as the expression of key inflammatory mediators including cytokines, chemokines, and cellular surface markers were studied. Potential molecular and cellular mechanisms were examined. We revealed a novel low-grade inflammatory monocyte phenotype termed ML adapted by super-low dose LPS, mediated through IRF5. For the in vivo system, we have employed both acute and chronic models of inflammation. For the chronic model, we have tested the effects of super-low dose LPS on monocyte polarization in vivo, as well as its contribution to the pathogenesis of atherosclerosis. Furthermore, we have tested the effects of programmed monocytes on wound healing. For the acute model, we have tested the effects of pre-conditioning with super-low dose LPS on the subsequence risks of sepsis elicited by cecal ligation and puncture. We have demonstrated aggravated atherosclerosis, compromised wound healing, and increased sepsis mortality in mice pre-conditioned with super-low dose LPS. Taken together, our findings reveal that monocytes can be differentially programmed into distinct states, depending on the signal strength of LPS. The differential programming and adaptation of monocytes can occur both in vitro and in vivo, and may bear profound pathological consequences. / Ph. D.

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