Background There has been an increase in antibiotic consumption in the last decades in lower income countries, still, it is insufficient to meet everyone’s needs. Ensuring access to them is essential to lower the burden of disease and mortality, as well as to accomplish the Sustainable Developmental Goals. However, this consumption needs to be responsible to avoid faster spreading of antibiotic resistances. The recent COVID-19 pandemic has threatened the healthcare systems everywhere, both directly and indirectly. Nevertheless, there is a lack of research on how it has impacted antibiotics’ stock.  Methods This study used secondary data of a cross-sectional cohort design to follow the impact of the pandemic on the stock of three commonly used antibiotics in healthcare facilities of Uganda. The data was compared between three time periods: before the pandemic started (January 2019-February 2020), during the first months of the pandemic and coinciding with the harshest restrictions (March 2020-May 2020), and the second period of the pandemic (June 2020-December 2020). Results An increase in stock of the antibiotics was observed as the restrictions hardened, followed by a decrease when they were lifted. Likewise, this rise happened both in the consumption and arrival of antibiotics. Stratified analysis was preformed grouping by healthcare level and by region, seeing that the strongest change happened in facilities at level II and III in the Eastern region of Uganda. Conclusions  This suggests that the pandemic influenced the antibiotics stocks and consumption. Further research needs to be done to better understand this effect.

Infections caused by antibiotic resistant pathogenic bacteria is an increasing issue inhealthcare, and development of new antimicrobial substances could contribute to combat the continued spread. Plants have historically been used in traditional medicine, have intrinsic defense mechanisms against microbes, and could therefore be a source for new antimicrobial agents. At the Department of Chemistry and Biomedicine at Linnaeus University, Kalmar, a total of 18 extracts made from 9 different combinations of Swedish-growing plants were available.The purpose of the current thesis was to investigate possible antimicrobial effects of the plant extracts in vitro, on a selection of Gram-positive (n=3) and Gram-negative (n=4) bacterial strains. Initial screening of all 18 extracts was performed with agar-based methods including agar well diffusion and direct application on inoculated agar. Detection of concentration-dependent antimicrobial effects was performed with four extracts on Staphylococcus aureus and Enterococcus faecalis. At inhibitory concentrations, viability was estimated as colony forming units/ml (CFU/ml).Screening showed that 11 of 18 extracts affected the growth of at least one of the strains tested. Gram-positive species were affected to a greater extent than Gram-negatives. Estimation of concentration dependency showed inhibitory effects at 50 mg/l in the most potent extracts. Viability estimation revealed an average reduction for both S. aureus and E. faecalis, as compared to the positive control. In conclusion, the study showed possible antimicrobial effects of several extract-bacteria combinations, disclosing potential substances for further investigations.

Antibiotic resistance is a global threat that can severely imperil public health. To curb the spread of antibiotic resistance, it is imperative that efforts commensurate with a “One Health” approach are undertaken. Given that interconnectivities among ecosystems can serve as conduits for the proliferation and dissemination of antibiotic resistance, it is increasingly being recognized that a robust global environmental surveillance framework is required to promote One Health. The ideal aim would be to develop approaches that inform global distribution of antibiotic resistance, help prioritize monitoring targets, present robust data analysis frameworks to profile resistance, and ultimately help build strategies to curb the dissemination of antibiotic resistance. The work described in this dissertation was aimed at evaluating and developing different data analysis paradigms and their applications in investigating and characterizing antibiotic resistance across different resistomes. The applications presented in Chapter 2 illustrate challenges associated with various environmental data types (especially metagenomics data) and present a path to advance incorporation of data analytics approaches in Environmental Science and Engineering research and applications. Chapter 3 presents a novel approach, ExtrARG, that identifies discriminatory ARGs among resistomes based on factors of interest. The results in Chapter 4 provide insight into the global distribution of ARGs across human fecal and sewage resistomes across different socioeconomics. Chapter 5 demonstrates a data analysis paradigm using machine learning algorithms that helps bridge the gap between information obtained via culturing and metagenomic sequencing. Lastly, the results of Chapter 6 illustrates the contribution of phages to antibiotic resistance. Overall, the findings provide guidance and approaches for profiling antibiotic resistance using metagenomics and machine learning. The results reported further expand the knowledge on the distribution of antibiotic resistance across different resistomes. / Antibiotic resistance is a global threat that can severely imperil public health. To curb the spread of antibiotic resistance, it is imperative that efforts commensurate with a "One Health" approach are undertaken. Given that interconnectivities among ecosystems can serve as conduits for the proliferation and dissemination of antibiotic resistance, it is increasingly being recognized that a robust global environmental surveillance framework is required to promote One Health. The ideal aim would be to develop approaches that inform global distribution of antibiotic resistance, help prioritize monitoring targets, present robust data analysis frameworks to profile resistance, and ultimately help build strategies to curb the dissemination of antibiotic resistance. The work described in this dissertation was aimed at evaluating and developing different data analysis paradigms and their applications in investigating and characterizing antibiotic resistance across different resistomes. The applications presented in Chapter 2 illustrate challenges associated with various environmental data types (especially metagenomics data) and present a path to advance incorporation of data analytics approaches in Environmental Science and Engineering research and applications. Chapter 3 presents a novel approach, ExtrARG, that identifies discriminatory ARGs among resistomes based on factors of interest. The results in Chapter 4 provide insight into the global distribution of ARGs across human fecal and sewage resistomes across different socioeconomics. Chapter 5 demonstrates a data analysis paradigm using machine learning algorithms that helps bridge the gap between information obtained via culturing and metagenomic sequencing. Lastly, the results of Chapter 6 illustrates the contribution of phages to antibiotic resistance. Overall, the findings provide guidance and approaches for profiling antibiotic resistance using metagenomics and machine learning. The results reported further expand the knowledge on the distribution of antibiotic resistance across different resistomes. / Doctor of Philosophy / Antibiotic resistance is ability of bacteria to withstand an antibiotic to which they were once sensitive. Antibiotic resistance is a global threat that can pose a serious threat to public health. In order to curb the spread of antibiotic resistance, it is imperative that efforts commensurate with the "One Health" approach. Since ecosystem networks can act as channels for the spread and spread of antibiotic resistance, there is growing recognition that a robust global environmental monitoring framework is required to promote a true one-health approach. The ideal goal would be to develop approaches that can inform the global spread of antibiotic resistance, help prioritize monitoring objectives and present robust data analysis frameworks for resistance profiling, and ultimately help develop strategies to contain the spread of antibiotic resistance. The objective of the work described in this thesis was to evaluate and develop different data analysis paradigms and their applications in the study and characterization of antibiotic resistance in different resistomes. The applications presented in Chapter 2 illustrate challenges associated with various environmental data types (especially metagenomics data) and present a path to advance incorporation of data analytics approaches in Environmental Science and Engineering research and applications. The Chapter 3 presents a novel approach, ExtrARG, that identifies discriminatory ARGs among resistomes based on factors of interest. The chapter 5 demonstrates a data analysis paradigm using machine learning algorithms that helps bridge the gap between information obtained via culturing and metagenomic sequencing. The results of Chapters 4 provide insight into the global distribution of ARGs across human fecal and sewage resistomes across different socioeconomics. Lastly, the results of Chapter 6 illustrates the contribution of phages to antibiotic resistance. Overall, the findings provide guidance and approaches for profiling antibiotic resistance using metagenomics and machine learning. The results reported further expand the knowledge on the distribution of antibiotic resistance across different resistomes.

The main aim of the study was to analyse the impact of physico-chemical parameters on drinking water quality, biofilm formation and antibiotic resistant bacteria in the drinking water distribution system in Mafikeng, North West Province, South Africa. Another objective was to isolate and characterise Pseudomonas and Aeromonas species from drinking water distribution system and detect the virulence gene determinants in the isolates by PCR analysis. The physico-chemical data obtained were subjected to statistical analysis using Excel 2007 (Microsoft) and SPSS (version 14.0) programmes. Pearson’s correlation product of the moment was used to determine the correlation between EC, TDS, pH and temperature. The two tailed test of significance (p<0.05) was used in order to determine the significance of the result. Antibiotic susceptibility tests were performed using Kirby-Bauer disk diffusion method. Cluster analysis based on the antibiotic inhibition zone diameter data of different organisms isolated from different sites was determined and was expressed as dendograms using Wards algorithm and Euclidean distance of Statistica version 7. Specific PCR was used to determine the identities of presumptive Pseudomonas and Aeromonas species through amplification of the gyrB, toxA and the ecfX gene fragments. Virulence gene determinants for the confirmed Pseudomonas and Aeromonas species were detected by amplifying the exoA, exoS and exoT genes and the aerA and hylH gene fragments, respectively. A Gene Genius Bio imaging system (Syngene, Synoptics; UK) was used to capture the image using GeneSnap (version 3.07.01) software (Syngene, Synoptics; UK) to determine the relative size of amplicons. Physico-chemical parameters were monitored from three drinking water sources three times a week and bacteriological quality was monitored weekly for four months from raw and treated drinking water. Water samples were analysed for pH, temperature, total dissolved solids (TDS) and electric conductivity (EC). Bacterial consortia from drinking water samples were isolated using selective media and enumerated. The results revealed a good chemical quality of water. However, the microbial quality of the water is not acceptable for human consumption due to the presence of Pseudomonas, Aeromonas, faecal coliforms (FC), total coliforms (TC) and Heterotrophic bacteria. The results showed that the drinking water is slightly alkaline with pH value ranging between7.7 to 8.32. What is of concern was the microbial quality of the water. Pseudomonas sp., faecal coliforms (FC), total coliforms (TC) and heterotrophic bacteria were present in some of the treated water samples. The most significant finding of this study is that all drinking water samples were positive for Pseudomonas sp.(>100/100ml), but also that when one considers the TDS it demonstrates that water from the Modimola Dam has an impact on the quality of the mixed water. The prevalence and antibiotic resistance profiles of planktonic and biofilm bacteria isolated from drinking water were determined. The susceptibility of these isolates was tested against 11 antibiotics of clinical interest and the multiple antibiotic resistance (MAR) patterns were compiled. The most prevalent antibiotic resistance phenotype observed was KF-AP-C-E-OT-K-TM-A. All isolates from all samples were susceptible to ciprofloxacin. However, all faecal coliforms and Pseudomonas spp. were susceptible to neomycin and streptomycin. On the contrary all organisms tested were resistant to erythromycin (100%) trimethoprim and amoxycillin. Cluster analysis based on inhibition zone diameter data could not differentiate the various isolated into sample types. The highest prevalence of antibiotic resistant isolates was observed in Modimola Dam and Molopo eye. Biofilms were investigated in both raw water and treated drinking water sources for the presence of faecal coliforms, total coliforms, Pseudomonas spp., Aeromonas spp. and heterotrophic bacteria based on conventional microbiology and molecular methods. Drinking water biofilms were grown twice and the biofilm developing device containing copper and galvanized steel coupons were utilized. The Mini Tap filter, a home water treatment device which can be used at a single faucet, under constant flow was used during the second collection of treated water samples from cold water taps. Scanning electron micrograph revealed the existence of biofilms in all the sites investigated and the highest density was obtained on galvanized steel coupons. Isolates were tested against the antibiotics ampicillin (10μg), cephalothin (5μg), streptomycin (10μg), erythromycin (15μg), chloramphenicol (30μg), neomycin (30 μg), amoxycillin (10 μg), ciprofloxacin (5 μg), trimethoprim (25μg), kanamycin (30μg), and oxytetracycline (30μg). The multiple antibiotic resistance profiles and the presence of virulence related genes were determined. Various types of drug resistance and presence of virulence genes were observed. The most prevalent resistance phenotype observed was KF-AP-C-E-OT-TM-A. In conclusion, the results indicated the occurrence of faecal indicator bacteria in the drinking water destined for human consumption. Faecal indicator bacteria are the major contributors of poor drinking water quality and may harbour opportunistic pathogens. This highlighted survival of organisms to treatment procedures and the possible regrowth as biofilms in plumbing materials. The detection of large proportion of MAR Aeromonas and Pseudomonas species which possessed virulent genes was a cause of concern as these could pose health risks to humans. The data obtained herein may be useful in assessing the health risks associated with the consumption of contaminated water. / PhD (Microbiology), North-West University, Potchefstroom Campus, 2014

Water is considered the most vital resource on earth and its quality is deteriorating. Not all residents living in South Africa‘s rural areas have access to treated drinking water, and use water from rivers, dams, and wells. The quality of these resources is unknown, as well as the effects of the bacteria in the water on human health. The heterotrophic plate count (HPC) method is a globally used test to evaluate microbial water quality. According to South African water quality guidelines, water of good quality may not contain more than a 1 000 coliforming units (CFU)/mℓ. There is mounting evidence that HPC bacteria may be hazardous to humans with compromised, underdeveloped, and weakened immune systems. In this study the pathogenic potential of HPC bacteria was investigated. Samples were collected from boreholes in the North West Province and HPCs were enumerated with a culture-based method. Standard physico-chemical parameters were measured for the water. Different HPC bacteria were isolated and purified and tested for α- or β-haemolysis, as well as the production of extracellular enzymes such as DNase, proteinase, lecithinase, chondroitinase, hyaluronidase and lipase, as these are pathogenic characteristics. The isolates were identified with 16S rRNA gene sequencing. The model for the human intestine, Hutu-80 cells, were exposed to the potentially pathogenic HPC isolates to determine their effects on the viability of the human cells. The isolates were also exposed to different dilutions of simulated gastric fluid (SGF) to evaluate its effect on the viability of bacteria. Antibiotic resistant potential of each isolate was determined by the Kirby-Bauer disk diffusion method. Three borehole samples did not comply with the physico-chemical guidelines. Half of the samples exceeded the microbial water quality guideline and the greatest CFU was 292 350 CFU/mℓ. 27% of the isolate HPC bacteria were α- or β- haemolytic. Subsequent analysis revealed the production of: DNase in 72%, proteinase in 40%, lipase and lecithinase in 29%, hyaluronidase in 25% and least produced was chondroitinase in 25%. The HPC isolates identified included: Alcaligenes faecalis, Aeromonas hydrophila and A. taiwanesis, Bacillus sp., Bacillus thuringiensis, Bacillus subtilis, Bacillus pumilus, Brevibacillus sp., Bacillus cereus and Pseudomonas sp. All the isolates, except Alcaligenes faecalis, were toxic to the human intestinal cells to varying degrees. Seven isolates survived exposure to the most diluted SGF and of these, four isolates also survived the intermediate dilution but, only one survived the highest SGF concentration. Some isolates were resistant to selected antibiotics, but none to neomycin and vancomycin. Amoxillin and oxytetracycline were the least effective of the antibiotics tested. A pathogen score was calculated for each isolate based on the results of this study. Bacillus cereus had the highest pathogen index with declining pathogenicity as follows: Alcaligenes faecalis > B. thuringiensis > Bacillus pumilus > Pseudomonas sp. > Brevibacillus > Aeromonas taiwanesis > Aeromonas hydrophila > Bacillus subtilis > Bacillus sp. The results of this study prove that standard water quality tests such as the physico-chemical and the HPC methods are insufficient to provide protection against the effects of certain pathogenic HPC bacteria. / MSc (Environmental Sciences), North-West University, Potchefstroom Campus, 2014

The main aim of the study was to analyse the impact of physico-chemical parameters on drinking water quality, biofilm formation and antibiotic resistant bacteria in the drinking water distribution system in Mafikeng, North West Province, South Africa. Another objective was to isolate and characterise Pseudomonas and Aeromonas species from drinking water distribution system and detect the virulence gene determinants in the isolates by PCR analysis. The physico-chemical data obtained were subjected to statistical analysis using Excel 2007 (Microsoft) and SPSS (version 14.0) programmes. Pearson’s correlation product of the moment was used to determine the correlation between EC, TDS, pH and temperature. The two tailed test of significance (p<0.05) was used in order to determine the significance of the result. Antibiotic susceptibility tests were performed using Kirby-Bauer disk diffusion method. Cluster analysis based on the antibiotic inhibition zone diameter data of different organisms isolated from different sites was determined and was expressed as dendograms using Wards algorithm and Euclidean distance of Statistica version 7. Specific PCR was used to determine the identities of presumptive Pseudomonas and Aeromonas species through amplification of the gyrB, toxA and the ecfX gene fragments. Virulence gene determinants for the confirmed Pseudomonas and Aeromonas species were detected by amplifying the exoA, exoS and exoT genes and the aerA and hylH gene fragments, respectively. A Gene Genius Bio imaging system (Syngene, Synoptics; UK) was used to capture the image using GeneSnap (version 3.07.01) software (Syngene, Synoptics; UK) to determine the relative size of amplicons. Physico-chemical parameters were monitored from three drinking water sources three times a week and bacteriological quality was monitored weekly for four months from raw and treated drinking water. Water samples were analysed for pH, temperature, total dissolved solids (TDS) and electric conductivity (EC). Bacterial consortia from drinking water samples were isolated using selective media and enumerated. The results revealed a good chemical quality of water. However, the microbial quality of the water is not acceptable for human consumption due to the presence of Pseudomonas, Aeromonas, faecal coliforms (FC), total coliforms (TC) and Heterotrophic bacteria. The results showed that the drinking water is slightly alkaline with pH value ranging between7.7 to 8.32. What is of concern was the microbial quality of the water. Pseudomonas sp., faecal coliforms (FC), total coliforms (TC) and heterotrophic bacteria were present in some of the treated water samples. The most significant finding of this study is that all drinking water samples were positive for Pseudomonas sp.(>100/100ml), but also that when one considers the TDS it demonstrates that water from the Modimola Dam has an impact on the quality of the mixed water. The prevalence and antibiotic resistance profiles of planktonic and biofilm bacteria isolated from drinking water were determined. The susceptibility of these isolates was tested against 11 antibiotics of clinical interest and the multiple antibiotic resistance (MAR) patterns were compiled. The most prevalent antibiotic resistance phenotype observed was KF-AP-C-E-OT-K-TM-A. All isolates from all samples were susceptible to ciprofloxacin. However, all faecal coliforms and Pseudomonas spp. were susceptible to neomycin and streptomycin. On the contrary all organisms tested were resistant to erythromycin (100%) trimethoprim and amoxycillin. Cluster analysis based on inhibition zone diameter data could not differentiate the various isolated into sample types. The highest prevalence of antibiotic resistant isolates was observed in Modimola Dam and Molopo eye. Biofilms were investigated in both raw water and treated drinking water sources for the presence of faecal coliforms, total coliforms, Pseudomonas spp., Aeromonas spp. and heterotrophic bacteria based on conventional microbiology and molecular methods. Drinking water biofilms were grown twice and the biofilm developing device containing copper and galvanized steel coupons were utilized. The Mini Tap filter, a home water treatment device which can be used at a single faucet, under constant flow was used during the second collection of treated water samples from cold water taps. Scanning electron micrograph revealed the existence of biofilms in all the sites investigated and the highest density was obtained on galvanized steel coupons. Isolates were tested against the antibiotics ampicillin (10μg), cephalothin (5μg), streptomycin (10μg), erythromycin (15μg), chloramphenicol (30μg), neomycin (30 μg), amoxycillin (10 μg), ciprofloxacin (5 μg), trimethoprim (25μg), kanamycin (30μg), and oxytetracycline (30μg). The multiple antibiotic resistance profiles and the presence of virulence related genes were determined. Various types of drug resistance and presence of virulence genes were observed. The most prevalent resistance phenotype observed was KF-AP-C-E-OT-TM-A. In conclusion, the results indicated the occurrence of faecal indicator bacteria in the drinking water destined for human consumption. Faecal indicator bacteria are the major contributors of poor drinking water quality and may harbour opportunistic pathogens. This highlighted survival of organisms to treatment procedures and the possible regrowth as biofilms in plumbing materials. The detection of large proportion of MAR Aeromonas and Pseudomonas species which possessed virulent genes was a cause of concern as these could pose health risks to humans. The data obtained herein may be useful in assessing the health risks associated with the consumption of contaminated water. / PhD (Microbiology), North-West University, Potchefstroom Campus, 2014

Master of Science / Department of Diagnostic Medicine/Pathobiology / Tiruvoor G. Nagaraja / Copper, as copper sulfate, is often supplemented at elevated concentrations in swine diets, particularly in piglets, to promote growth. Growth promotional effects of copper are believed to be similar to that of antibiotics in that gut microbial flora is altered to reduce loss of nutrients and suppress pathogens. Bacteria exposed to copper may acquire resistance, and in Enterococcus faecium and E. faecalis, resistance is conferred by a plasmid-borne transferable copper resistance (tcrB) gene. The plasmid also carries macrolide [erm(B)] and glycopeptide (vanA) antibiotics resistance genes. The objectives of the research were to 1) determine the prevalence of tcrB gene in fecal enterococci of piglets in relation to normal (16.5 ppm) and elevated level (125 ppm) of copper supplementation, 2) determine the relationship of tcrB gene and susceptibilities to copper, erythromycin, and vancomycin, and 3) determine the transferability of tcrB gene in enterococci by conjugation. Weaned piglets, housed in pens, fed normal (16.5 ppm; control) or elevated level of copper (125 ppm) were used. Fecal samples were collected weekly for isolation of enterococci. Isolates were speciated by multiplex PCR and sodA gene sequence analysis. The prevalence of tcrB-positive enterococcal isolates was higher (P < 0.05) in the copper supplemented group than the control group. The prevalence of tcrB was affected by sampling days (P < 0.05) with a significant treatment and sampling time interaction (P < 0.05). The tcrB positive isolates were either E. faecium or E. faecalis, and majority of isolates was E. faecium. The mean MIC of copper for tcrB-positive isolates (21.1 mM) was higher (P < 0.001) compared to tcrB-negative isolates (6.1 mM). All isolates were resistant to erythromycin, tetracyclines and susceptible to vancomycin. The transferability of the tcrB gene from tcrB-positive strains to tcrB-negative strains was demonstrated by conjugation. The potential link between tcrB and antibiotic resistance genes and the propensity of enterococci to transfer tcrB to other strains suggests the possibility that copper supplementation may exert selection pressure for antibiotic resistance. The positive association between copper supplementation and prevalence of tcrB gene has important implications for antimicrobial resistance and food safety, which warrants further investigation.

Water is considered the most vital resource on earth and its quality is deteriorating. Not all residents living in South Africa‘s rural areas have access to treated drinking water, and use water from rivers, dams, and wells. The quality of these resources is unknown, as well as the effects of the bacteria in the water on human health. The heterotrophic plate count (HPC) method is a globally used test to evaluate microbial water quality. According to South African water quality guidelines, water of good quality may not contain more than a 1 000 coliforming units (CFU)/mℓ. There is mounting evidence that HPC bacteria may be hazardous to humans with compromised, underdeveloped, and weakened immune systems. In this study the pathogenic potential of HPC bacteria was investigated. Samples were collected from boreholes in the North West Province and HPCs were enumerated with a culture-based method. Standard physico-chemical parameters were measured for the water. Different HPC bacteria were isolated and purified and tested for α- or β-haemolysis, as well as the production of extracellular enzymes such as DNase, proteinase, lecithinase, chondroitinase, hyaluronidase and lipase, as these are pathogenic characteristics. The isolates were identified with 16S rRNA gene sequencing. The model for the human intestine, Hutu-80 cells, were exposed to the potentially pathogenic HPC isolates to determine their effects on the viability of the human cells. The isolates were also exposed to different dilutions of simulated gastric fluid (SGF) to evaluate its effect on the viability of bacteria. Antibiotic resistant potential of each isolate was determined by the Kirby-Bauer disk diffusion method. Three borehole samples did not comply with the physico-chemical guidelines. Half of the samples exceeded the microbial water quality guideline and the greatest CFU was 292 350 CFU/mℓ. 27% of the isolate HPC bacteria were α- or β- haemolytic. Subsequent analysis revealed the production of: DNase in 72%, proteinase in 40%, lipase and lecithinase in 29%, hyaluronidase in 25% and least produced was chondroitinase in 25%. The HPC isolates identified included: Alcaligenes faecalis, Aeromonas hydrophila and A. taiwanesis, Bacillus sp., Bacillus thuringiensis, Bacillus subtilis, Bacillus pumilus, Brevibacillus sp., Bacillus cereus and Pseudomonas sp. All the isolates, except Alcaligenes faecalis, were toxic to the human intestinal cells to varying degrees. Seven isolates survived exposure to the most diluted SGF and of these, four isolates also survived the intermediate dilution but, only one survived the highest SGF concentration. Some isolates were resistant to selected antibiotics, but none to neomycin and vancomycin. Amoxillin and oxytetracycline were the least effective of the antibiotics tested. A pathogen score was calculated for each isolate based on the results of this study. Bacillus cereus had the highest pathogen index with declining pathogenicity as follows: Alcaligenes faecalis > B. thuringiensis > Bacillus pumilus > Pseudomonas sp. > Brevibacillus > Aeromonas taiwanesis > Aeromonas hydrophila > Bacillus subtilis > Bacillus sp. The results of this study prove that standard water quality tests such as the physico-chemical and the HPC methods are insufficient to provide protection against the effects of certain pathogenic HPC bacteria. / MSc (Environmental Sciences), North-West University, Potchefstroom Campus, 2014

Dihydrodipicolinate Synthase (EC 4.3.3.7; DHDPS), the product of the dapA gene, is an enzyme that catalyzes the condensation of pyruvate and S-aspartate-β-semialdehyde (ASA) into dihydrodipicolinate via an unstable heterocyclic intermediate, (4S)-hydroxy-2,3,4,5-tetrahydro-(2S)-dipicolinic acid. DHDPS catalyzes the first committed step in the biosynthesis of ʟ-lysine and meso-diaminopimelate; each of which is a necessary cross-linking component between peptidoglycan heteropolysacharide chains of bacterial cell walls. Therefore, strong inhibition of DHDPS would result in disruption of meso-diaminopimelate and ʟ-lysine biosynthesis in bacteria leading to decreased bacterial growth and cell lysis. Much attention has been given to targeting the active site for inhibition; however DHDPS is subject to natural feedback inhibition by ʟ-lysine at an allosteric site. In DHDPS from Campylobacter jejuni ʟ-lysine is known to act as a partial uncompetitive inhibitor with respect to pyruvate and a partial mixed inhibitor with respect to ASA. Little is known about how the protein structure facilitates the natural inhibition mechanism and mode of allosteric signal transduction. This work presents ten high resolution crystal structures of Cj-DHDPS and the mutant Y110F-DHDPS with various substrates and inhibitors, including the first reported structure of DHDPS with ASA bound to the active site. As a body of work these structures reveal residues and conformational changes which contribute to the inhibition of the enzyme. Understanding these structure function relationships will be valuable for the design of future antibiotic lead compounds. When an inhibitor binds to the allosteric site there is meaningful shrinkage in the solvent accessible volume between 33% and 49% proportional to the strength of inhibition. Meanwhile at the active site the solvent accessible volume increases between 5% and 35% proportional to the strength of inhibition. Furthermore, inhibitor binding at the allosteric site consistently alters the distance between hydroxyls of the catalytic triad (Y137-T47-Y111') which is likely to affect local pKa's. Changes in active site volume and modification of the catalytic triad would inhibit the enzyme during the binding and condensation of ASA. The residues H56, E88, R60 form a network of hydrogen bonds to close the allosteric site around the inhibitor and act as a lid. Comparison of ʟ-lysine and bislysine bound to wt-DHDPS and Y110F-DHDPS indicates that enhanced inhibition of bislysine is most likely due to increased binding strength rather than altering the mechanism of inhibition. When ASA binds to the active site the network of hydrogen bonds among H56, E88 and R60 is disrupted and the solvent accessible volume of the allosteric site expands by 46%. This observation provides some explanation for the reduced affinity of ʟ-lysine in high ASA concentrations. ʟ-Lysine, but not other inhibitors, is found to induce dynamic domain movements in the wt-DHDPS. These domain movements do not appear to be essential to the inhibition of the enzyme but may play a role in cooperativity between monomers or governing protein dynamics. The moving domain connects the allosteric site to the dimer-dimer interface. Several residues at the weak dimer interface have been identified as potentially involved in dimer-dimer communication including: I172, D173, V176, I194, Y196, S200, N201, K234, D238, Y241, N242 and K245. These residues are not among any previously identified as important for formation of the quaternary structure.

Experimental studies into the use of cold atmospheric plasmas for inactivating foodborne pathogens are presented in this thesis. Eliminating the possibility that treatment delivered by a plasma to a population or assemblage of micro-organisms is unevenly distributed is an essential pre-requisite to attempting to interpret inactivation kinetics with a view to elucidating mechanisms of inactivation. A filtration method of depositing cells evenly on the surface of a membrane without cell stacking was developed and used throughout the work described here. Two atmospheric plasma systems were evaluated and each brought about microbial inactivation in a distinct way. A pulsed radio frequency plasma jet operated at 3.47 MHz caused gross morphological changes to L. innocua whereas a low frequency air mesh plasma system operated at a frequency of 24 kHz led to the inactivation of these bacteria without inducing observable structural changes. Changing the operating parameters of the plasma jet system had a significant effect on the composition of the reactive plasma species generated as revealed by changes to the mode of inactivation of bacteria. In addition to inactivating bacteria, the pulsed plasma jet was shown to be highly effective in degrading and removing amyloid aggregates from the surface of mica coupons. Amyloids have widely been used as a non-infectious model for prions, and the results obtained here show potential for the application of gas plasma technology for removing prions from abiotic surfaces in medical and other applications. It has widely been assumed that bacterial envelopes are the principal sites at which reactive plasma species bring about damage to cells. However, changing the composition of the bacterial membranes of E. coli and Listeria innocua by cultivating them at widely different temperatures to induce changes proved not to result in enhanced inactivation. Flow cytometry was also used to provide additional insights into possible mechanisms of inactivation. The following fluorescent dyes were used either singly or in combination; SYTO 13, DiBAC4(3), cFDA and PI. The results obtained with the dyes DiBAC4(3) and PI showed that Gram positive bacteria became depolarised prior to the bacterial membrane becoming compromised, possibly suggesting that the inactivating plasma species are affecting membrane proteins responsible for maintaining the bacterial charge. Differences between the fluorescent dye staining of Gram negative and Gram positive species were obtained using SYTO13 and PI demonstrating that the different membrane structures affect their interaction with the plasma. In additional studies, the air mesh plasma was used to treat multi-drug resistant strains of Methicillin resistant Staphylococcus aureus (MRSA) in an attempt to reverse antibiotic resistance. MRSA PM 64 was shown to reverse its antibiotic resistance to Oxacillin, Kanamycin and Trimethoprim. Culturing the bacteria in a nutrient limited media led to increased resistance towards plasma treatment and maintenance of their high levels of antibiotic resistance.