The emergence of CTX-M class-A extended-spectrum β-lactamases, which confer resistance to second and third-generation cephalosporins, poses a serious health threat to the public. CTX-M β-lactamases use a catalytic serine to hydrolyze the β-lactam ring. Specifically, the hydrolysis reaction catalyzed by CTX-M β-lactamase proceeds through a pre-covalent complex, a high-energy tetrahedral acylation intermediate, a low-energy acyl-enzyme complex, a high-energy tetrahedral deacylation intermediate after attack via a catalytic water, and lastly, the hydrolyzed β-lactam ring product which is released from the enzyme complex. The crystallographic structure of CTX-M at sub-angstrom resolution has enabled us to study enzyme catalysis as well as perform computational molecular docking in our efforts to develop new inhibitors against CTX-M. The goal of this project was to determine the hydrogen bonding network and proton transfer process at different stages of the reaction pathway as well as develop novel inhibitors against CTX-M β-lactamases. The results I have obtained from the project have elucidated the catalytic mechanism of CTX-M β-lactamase in unprecedented detail and facilitated the development of novel inhibitors for antibiotic drug discovery. The first aim of the project focused on developing high affinity inhibitors against class A β-lactamase using a structure-based drug discovery approach, which ultimately led to the identification of CTX-M9 inhibitors with nanomolar affinity. Compound design was based on the initial use of computational molecular docking results along with x-ray crystal structures with known inhibitors bound in the active site. In addition, chemical synthesis was used to build and extend the existing inhibitor scaffold to improve affinity to CTX-M9 and related serine β-lactamases. Through a fragment-based screening approach, we recently identified a novel non-covalent tetrazole-containing inhibitor of CTX-M. Structure-based design was used to improve the potency of the original tetrazole lead compound more than 200-fold with the use of small, targeted structural modifications. A series of compounds were used to probe specific binding hotspots present in CTX-M. The designed compounds represent the first nM-affinity non-covalent inhibitors of a class A β-lactamase. The complex structures of these potent compounds have been solved using high resolution x-ray crystallography at ~ 1.2-1.4 Å, which provides valuable insight about ligand binding and future inhibitor design against class A β-lactamases. Specifically, the first aim of the project was to use ultra-high resolution x-ray crystallography to study β-lactamase catalysis. Through the use of ultra-high resolution x-ray crystallography with non-covalent and covalent inhibitors, I was able to structurally characterize the critical stages of the enzyme mechanism. Here we report a series of ultra-high resolution x-ray crystallographic structures that reveal the proton transfer process for the early stages of the class A β-lactamase catalytic mechanism. The structures obtained include an a 0.89 Å crystal structure of CTX-M β-lactamase in complex with a recently-developed 89 nM non-covalent inhibitor, and a 0.80 Å structure in complex with an acylation transition state boronic acid inhibitor. Nearly all the hydrogen atoms in the active site, including those on the ligand, polar protein side chains and catalytic water, can be identified in the unbiased difference electron density map. Most surprisingly, compared with a previously determined 0.88 Å apo structure determined under the same conditions, the hydrogen-bonding network has undergone a series of reshuffling upon the binding of the non-covalent ligand. Two key catalytic residues, Lys73 and Glu166, appear to have both changed from a charged state to being neutral. Interestingly, structural evidence suggests the presence of a low barrier hydrogen bond (LBHB) shared between Lys73 and Ser70. These unprecedented detailed snapshots offer direct evidence that ligand binding can alter the pKa's of polar protein side chains and their affinities for protons. Such effects can be a common mechanism utilized by enzymes to facilitate the proton transfer process of a reaction pathway. They also have important implications for computational modeling of protein-ligand interactions. Ultra-high resolution x-ray crystallography allowed us to determine the hydrogen atom positions for key active site residues involved in catalysis. As a result, the ability to characterize the hydrogen bonding network led to the determination of the specific proton transfer process that occurs during the reaction stages of the CTX-M β-lactamase mechanism. Overall, the results from this project demonstrate the effectiveness of using ultra high resolution x-ray crystallography as a useful tool to study enzyme catalysis as well as develop and discover novel inhibitors.

A recent study using human fecal samples in conjunction with a culture-independent approach revealed immense diversity of antibiotic resistance (AR) genes in the human gut microflora. We hypothesize that food animal gut microflora also contain diverse and novel AR genes which could contribute to the emergence and transmission of AR in pathogens important in animal and human health. To test this, we examined AR reservoir in chicken gut microflora using a metagenomic, functional cloning method. Total genomic DNA was extracted from individual cecal contents of two free range chickens and two conventionally raised chickens. The DNAs were physically sheered into 1 to 3 kb fragments, cloned into expression vector pZE21-MCS, and transformed into E. coli TOP10 host strain, resulting in four metagenomic libraries of a total size of 108 base pairs per library. The AR transformants from the libraries were selected on plates containing the specific antibiotic of interest; six antibiotics including ampicillin, tetracycline, chloramphenicol, spectinomycin, ciprofloxacin and norfloxacin were used for screening. Plasmids from selected transformants were extracted and subjected to sequence analysis of inserted fragments. Identified AR genes were annotated and aligned with homologs that have been deposited in GenBank. A total of 12 AR genes and 3 AR genes were identified from the microbiome in conventionally raised chickens and free-range chickens, respectively. Of the identified 15 AR genes, 8 genes that confer resistance to ampicillin, spectinomycin or chloramphenicol shared low sequence similarity (58% - 76% at amino acid level) with the corresponding AR genes previously identified using culture-dependent approaches. Notably, among the 8 novel AR genes identified in this study, 4 genes also shared low sequence similarities (59%-76% at amino acid level) with recently identified AR genes in human gut. An E. coli-Campylobacter shuttle vector bearing the flaA sigma 28 promoter was constructed. Two novel genes conferring resistance to ampicillin (FRAmp1.1) and spectinomycin (FRSpe1.1) were cloned into this new expression vector, respectively. The derived vectors have conferred increased AR in C. jejuni, a leading zoonotic bacterial pathogen causing human gastroenteritidis in many industrialized countries. Together, findings from this study showed the effectiveness of the metagenomic approach for examination of AR reservoir in food animals, revealed novel AR resistance genes in chicken gut microflora, and demonstrated the functionality of such AR genes in foodborne human pathogens.

Introduction: Nosocomial infections remain a major cause of mortality and morbidity. The problem is most apparent in intensive care units (ICUs). Most ICU patients are compromised and vulnerable as a result of disease or severe trauma. One in ten people admitted to hospital is given an antibiotic for infection. The risk of acquiring a nosocomial infection in a European ICU is approximately 20%. It is vitally important that ways are found to prevent transmission between patients and personnel, and that local hygiene routines and antibiotic policies are developed. This thesis is a holistic work focused particularly on antimicrobial antibiotic resistance, antibiotic consumption and to some extent on hygiene in Swedish ICUs. Aims: The general aim of this thesis was to investigate bacterial resistance and antibiotic consumption in Swedish ICUs and to try to correlate ICU demographic data with antibiotic consumption and antibiotic resistance. Additional aims were to investigate on which clinical indications antibacterial drugs are prescribed in the ICU, and to investigate the emergence of resistance and transmission of Pseudomonas aeruginosa in the ICU using cluster analysis based on antibiograms and genotype data obtained by AFLP. Material and methods: In paper 1-3, antibiotic consumption data together with bacterial antibiotic resistance data and specific ICU-demographic data were collected from an increasing number of ICUs over the years 1997-2001. Data from ICUs covering up to six million out of Sweden’s nine million inhabitants were included. In paper 4, the indications for antibiotic prescribing were studied during two weeks in 2000. Paper 5 investigated Pseudomonas aeruginosa isolates in order to detect cross-transmission with genotype obtained by AFLP, and antibiogram-based cluster analysis was also performed in order to see if this could be a quicker and easier substitute for AFLP. Results: This thesis has produced three important findings. Firstly, antibiotic consumption in participating ICUs was relatively high during the study period, and every patient received on average more than one antimicrobial drug per day (I-IV). Secondly, levels of antimicrobial drug resistance seen in S. aureus, E. coli and Klebsiella spp remained low when data were pooled from all ICUs throughout the study period, despite relatively high antibiotic consumption (I-V). Thirdly, the prevalence of antibiotic resistance in CoNS and E. faecium, cefotaxime resistance in Enterobacter, and ciprofloxacin and imipenem resistance in P. aeruginosa was high enough to cause concern. Conclusion: For the period studied, multidrug resistance in Swedish ICUs was not a major problem. Signs of cross-transmission with non-multiresistant bacteria were observed, indicating a hygiene problem and identifying simple improvements that could be made in patient care guidelines and barrier precautions. A need for better follow up of prescribed antibiotics was evident. With further surveillance studies and monitoring of antibiotics and bacterial resistance patterns in the local setting as well as on a national and international level, some of the strategic goals in the prevention and control of the emergence of antimicrobial-resistant microbes may be achievable.

Bacterial infections are again becoming difficult to treat because the microbes are growing increasingly resistant to the antibiotics in use today. The need for novel antimicrobial compounds is urgent and to achieve this new targets are crucial. In this thesis we present a strategy for identification of such targets via a bioinformatics approach. In our first study we compared proteins with unknown and hypothetical function of the spirochete Treponema pallidum to five other pathogens also causing chronic or persistent infections in humans (Yersinia pestis, Neisseria gonorrhoeae, Helicobacter pylori, Borrelia burgdorferi and Streptococcus pneumoniae). T. pallidum was used as a starting point for the comparisons since this organism has a condensed genome (1.1 Mb). As we aimed at identifying conserved proteins important for in vivo survival or virulence of the pathogens we reasoned that T. pallidum would have deleted genes not important in the human host. This comparison yielded 17 ORFs conserved in all six pathogens, these were deleted in our model organism, Yersinia pseudotuberculosis, and the virulence of these mutant strains was evaluated in a mouse model of infection. Five genes were found to be essential for virulence and thus constitute possible antimicrobial drug targets. We have studied one of these virulence associated genes (vags), vagH, in more detail. Functional and phenotypic analysis revealed that VagH is an S-adenosyl-methionine dependent methyltransferase targeting Release factor 1 and 2 (RF1 and RF2). The analysis also showed that very few genes and proteins were differentially expressed in the vagH mutant compared to wild-type Yersinia. One major finding was that expression of the Type III secretion system effectors, the Yops, were down regulated in a vagH mutant. We dissected this phenotype further and found that the down regulation was due to lowered amounts of the positive regulator LcrF. This can be suppressed either by a deletion of yopD or by over expression of the Ribosomal Recycling Factor (RRF). These results indicate that YopD in addition to its role in translational regulation of the Yops also plays a part in the regulation of LcrF translation. We suggest also that the translation of LcrF is particularly sensitive to the amount of translation competent ribosomes and that one effect of a vagH mutation in Y. pseudotuberculosis is that the number of free ribosomes is reduced; this in turn reduces the amount of LcrF produced thereby causing a down regulation of the T3SS. This down regulation is likely the cause of the attenuated virulence of the vagH mutant. Finally, we set up a high throughput screening assay to screen a library of small molecules for compounds with inhibiting the VagH methyltransferase activity. Five such compounds were identified and two were found to inhibit VagH also in bacterial culture. Furthermore, analogues to one of the compounds showed improved inhibitory properties and inhibited the T3SS-dependent cytotoxic response induced by Y. pseudotuberculosis on HeLa cells. We have successfully identified five novel targets for antimicrobial compounds and in addition we have discovered a new class of molecules with antimicrobial properties.

Antibiotic resistance is a growing problem on a global scale. Increasing numbers of bacteria resistant toward one or multiple antibiotics could return us to the high mortality rates for infectious diseases of the pre-antibiotic era. The need for development of new classes of antibiotics is great as is increased understanding of the mechanisms underlying the development of antibiotic resistance. We have investigated the emergence of resistance to peptide deformylase inhibitors, a new class of antibiotics that target bacterial protein synthesis. The fitness of resistant mutants as well as their propensity to acquire secondary compensatory mutations was assessed in order to gain some insight into the potential clinical risk of resistance development. Most of this work was done in the bacterium Salmonella typhimurium, due to the availability of excellent genetic tools to study these phenomena. In addition, we have studied the bacterium Staphylococcus aureus as peptide deformylase inhibitors have been shown to have the greatest effect on Gram-positive organisms. In the course of this work we also examined the mechanistic aspects of translation initiation. Using a cell-free in vitro translation system we studied the effects of various components on translation initiation. These results have been combined with results obtained from resistant and compensated bacterial strains in vivo to gain new insights into the mechanisms of translation initiation.

As one of the mechanisms of antibiotic resistance, bacteria use several families of membrane-embedded α-helical transporters to remove cytotoxic molecules from the cell. The small multidrug resistance protein family (SMR) is one such group of drug transporters that because of their relative small size [ca. 110 residues with four transmembrane (TM) helices] must form at the minimum dimers to efflux drugs. We have used the SMR homologue Hsmr from Halobacterium salinarum to investigate the oligomerization properties of the protein family at TM helix 4. We produced point mutations along the length of the TM4 helix in the full length Hsmr protein and assayed their dimerization and functional properties via SDS-PAGE and bacterial cell growth assays. We found that Hsmr forms functionally dependent dimers via an evolutionarily conserved 90GLxLIxxGV98 small residue heptad repeat. Upon investigation of the large hydrophobic residues in this motif by substituting each large residue to Ile, Leu, Met, Phe, and Val, we determined that Hsmr efflux function relies on an optimal level of dimerization. While some substitutions led to either decreased or increased dimer and substrate-binding strength, several Ile94 and Val98 mutants were equal to wild type dimerization levels but were nonfunctional, leading to the hypothesis of a mechanistic role at TM4 in addition to the locus of dimerization. The functionally sensitive TM4 dimer represents a potential target for SMR inhibition using a synthetic TM4 peptide mimetic. Using exponential decay measurements from a real-time cellular efflux assay, we observed the efflux decay constant was decreased by up to ~60% after treatment with the TM4 peptide inhibitor compared to control peptide treatments. Our results suggest that this approach could conceivably be used to design hydrophobic peptides for disruption of key TM-TM interactions of membrane proteins, and represent a valuable route to the discovery of new therapeutics.

Surface mucus of a freshwater fish, Alburnus alburnus (bleak), caught from Lake Mogan, situated in south of Ankara, was collected in different seasons. The total cultivable bacteria were enumerated by spread plate method on nine different media. Bacteria were isolated based on colony morphologies and pigmentation. A total of sixty bacterial isolates obtained. The mucus-dwelling bacteria were first tested for resistance against ampicillin and kanamycin / then streptomycin and chloramphenicol were added to the experimental set up. The resistance levels of isolates were determined in terms of four antibiotics by tube dilution method. About 90% of the isolates were resistant to chloramphenicol, about 84% to kanamycin, about 88% to streptomycin and about 98% to ampicillin. These high levels of antibiotic resistance are rather interesting from a standpoint that the lake has no record of antibiotics exposure of any sort. The plasmid isolations were carried out to determine if the multiple antibiotic resistance could be attributed to plasmids for starting assumption. But we found no direct relationship between the presence of plasmids and multiple antibiotic resistance. Our study indicated that multiple antibiotic resistance at high levels is among the current phenotypes of the fish mucus-dwelling bacterial populations in Lake Mogan.

To gain a better understanding of contaminating bacteria in bioethanol industry, we profiled the bacterial community structure in corn-based bioethanol fermentations and evaluated its correlation to environmental variables. Twenty-three batches of corn-mash sample were collected from six bioethanol facilities. The V4 region of the collective bacterial 16S rRNA genes was analyzed by Illumina Miseq sequencing to investigate the bacterial community structure. Non-metric multidimensional scaling (NMDS) ordination plots were constructed to visualize bacterial community structure groupings among different samples, as well as the effects of multiple environmental variables on community structure variation. Our results suggest that bacterial community structure is facility-specific, although there are two core bacterial phyla, Firmicutes and Proteobacteria. Feedstock, facility, and fermentation technology may explain the difference in community structure between different facilities. Lactic acid, the most important environmental variable that influences bacterial community structure grouping, could be utilized as an indicator of bacterial contamination. We also identified genes responsible for the multiple antibiotic-resistance phenotype of an Enterobacter cloacae strain isolated from a bioethanol fermentation facility. We performed PCR assays and revealed the presence of canonical genes encoding resistance to penicillin and erythromycin. However, a gene encoding resistance to virginiamycin was not detected.

Bakgrund: ESBL är ett enzym som kan produceras av bakterier i tarmens normalflora och gör bakterien motståndskraftig –resistent- mot många antibiotika. För den enskilde individen får det konsekvenser vid en infektion orsakad av ESBL-bildande tarmbakterier, och då behandling krävs. De som infekteras med dessa bakterier riskerar att svara dåligt på behandling med våra vanligaste antibiotika, och det kan krävas inläggning på sjukhus även vid banala infektioner. Syfte: Att fördjupa kunskapen om vad det innebär för den enskilda individen att drabbas av ESBL-bildande tarmbakterier. Metod. En modifierad variant av Grounded Theory användes som analysmetod av sju öppna intervjuer. Resultat: I analysen växte kärnkategorin Att bli utkastad i det skrämmande och okända utan karta och kompass fram. Samtliga informanter upplevde att de fått bristande information, eller ingen information alls om sin diagnos. Informationsvägen från läkaren var antingen via telefon eller genom ett brev via posten. Konsekvensen blev att det uppstod många tankar och funderingar efteråt, och det innefattade även frågan om hur man blivit smittad; genom sjukvården eller om de själva orsakat att bli smittade. I mötet med sjukvården upplevdes att okunskapen hos personalen i vissa fall orsakade stigmatisering. Här förekom såväl extrema hygienåtgärder i form av “skyddsmundering“ som alltför bristande hygienrutiner. Därutöver framkom också upplevda attitydproblem från personalens sida, nonchalans, bristande förståelse, ingen villighet eller tid att svara på frågor. Allt detta ledde till att informanterna i sin egen vardag fick ta saken i egna händer. Samtliga försökte skaffa sig information på annat sätt, exempelvis via internet. I oron för att smitta andra konstruerades egna åtgärder av kvinnorna i studien, exempelvis att instruera andra om handtvätt, att själv desinfektera föremål vid vårdbesök och i bostaden, att inte åka med tunnelbana eller buss, att inte umgås med andra och att själv uppleva informationsplikt om sin smitta. Männen i studien vidtog, trots bristande eller ingen information alls, inte några speciella åtgärder i sitt vardagsliv, de fortsatte att leva som tidigare. Ingen ville oroa sina anhöriga/närstående. Det förekom att barnen ej informerats alls om diagnosen. Konklusion: För att kunna hantera sin livssituation är det av stor betydelse att den som drabbas av en ESBL-bildande bakterie får en god information av patientansvarig läkare. / Background: Extended spectrum beta lactamase (ESBL), an enzyme produced by bacteria in normal intestinal flora, renders such bacteria resistant to many antibiotics. Some patients infected with ESBL respond poorly to antibiotic treatment, and even trivial infections may require hospitalization.Purpose: To increase understanding of the emotional impact of ESBL-producing intestinal bacteria.Method: This study used a modified version of grounded theory during seven open interviews to analyze coping mechanisms for ESBL infection.Results: Our analysis identified a core category (i.e., being thrown into scary and unknown territory without a map and compass). All respondents felt they received no or insufficient information about the diagnosis, and reported that any information they did receive arrived only by phone or letter. Consequently, respondents questioned whether they had been infected through medical care or through their own actions. They believed that lack of knowledge and attitude problems among healthcare providers (perceived as carelessness, lack of understanding, and unwillingness or lack of time to answer questions) stigmatizes patients. Such deficits led respondents to take matters into their own hands as they tried to obtain information by other means (e.g., the Internet). Respondents described extreme hygiene measures as a "protective suit" against inadequate hygiene. Female respondents constructed individual coping mechanisms (e.g., instructing others about hand washing technique, disinfecting objects during healthcare visits and at home, avoiding metro or bus travel, avoiding social interactions, and informing others of the infection). Conversely, male respondents took no special measures and lived as they did before infection. No one wanted to worry relatives/significant others, and no one told their children about the diagnosis.Conclusion: It is to important that attending doctors provide good information to individuals infected by ESBL-producing bacteria. Moreover, such individuals must develop good life management and coping skills. / <p>ISBN 978-91-86739-12-6</p>

Contamination of groundwater that is suitable for drinking is of growing concern as the water supply of South Africa is becomingincreasingly limited. This is especially the case in the North West province, with its semi – arid climate and variable rainfall patterns. The aim of the study was to evaluate the microbiological and physico – chemical qualities of groundwater obtained from selected DWA (Department of Water Affairs) monitoring boreholes in the Mooi River and Harts River catchment areas. Physico -chemical parameters included temperature, pH, electrical conductivity (EC), salinity, total dissolved solids (TDS), sulphate and nitrate concentrations. Physical parameters were measured using a calibrated submerge-able multimeter and chemical parameters using specialised kits and a spectrophotometer. Microbiological parameters included heterotrophic plate counts and total and faecal coliform enumeration. Membrane filtration and culture based methods were followed for enumeration of bacteria. During the identification procedures multiplex PCR for E. coli identification and 16S rRNA gene sequencing for identification of heterotrophic plate count bacteria and amoeba resistant bacteria were used. For antibiotic resistance, the Kirby- Bauer (1996) disk diffusion method was used. During the warm and wet season high electrical conductivity and salinity were observed in the Trimpark (65.3 mS/m; 325 ppm), School (125.1 mS/m; 644 ppm), Warrenton (166.9 mS/m; 867 ppm) and Ganspan (83.3 mS/m; 421 ppm) boreholes. Warrenton borehole had a high sulphate level (450 mg/l) as well. High chemical oxygen demand was observed in the Blaauwbank (62 mg/l) and Warrenton (98.5 mg/l) boreholes. In the dry and cold season similar observations were made for the various boreholes. Electrical conductivity and salinity levels remained high for the Trimpark (70.1 mS/m; 427.5 ppm), School (127 mS/m; 645 ppm), Warrenton (173.3 mS/m; 896.5 ppm) and Ganspan (88.1 mS/m; 444.5 ppm) boreholes. Nitrate levels for the Trimpark (14.1 mg/l) and School (137 mg/l), as well as sulphate levels for the Warrenton (325 mg/l) borehole were also high. Total coliforms, faecal streptococci and HPC bacteria were enumerated from water samples from all boreholes, except Blaauwbank where no faecal streptococci were enumerated. Faecal coliforms were enumerated from 5 of the possible 7 boreholes during a warm and wet season (Trimpark – 42 cfu/100ml; School – 2 cfu/100ml; Cemetery – 175 cfu/100ml; Warrenton – 3.84 x 10³ cfu/100ml; Ganspan – 1.9 x 10³ cfu/100ml). Indicator bacteria (FC, TC, HPC) exceeded target water quality ranges (TWQR) for drinking water in each case. During the cold and dry sampling season, faecal coliforms were enumerated mainly from the Trimpark (11 cfu/100ml) borehole. Total coliforms, faecal streptococci and HPC bacteria were enumerated from all the boreholes, except for Blaauwbank that contained no faecal streptococci or total coliforms. Enumerated indicator bacteria levels again exceeded TWQR for domestic use. Total coliform counts for the Pad dam borehole, however, complied with TWQR for domestic use. Identified E. coli were resistant to Erythromycin, Cephalothin and Amoxicillin and susceptible to Ciprofloxacin. Escherichia coli isolated from the Mooi River catchment shared the same antibiotic resistance phenotype. The most abundant HPC bacterial genus identified was Pseudomonas spp. (7 isolates). Opportunistic pathogens isolated included Pseudomonas aeruginosa, Acinetobacter, Aeromonas, Alcaligenes, Flavobacterium, Bacillus cereus and Mycobacterium spp. Varying degrees of antibiotic resistance were observed. Generally, the same pattern between the same genera were observed. All HPC isolates were resistant to Cephalothin and Amoxicillin and a lower degree Erythromycin and Streptomycin. The most abundant amoeba resistant bacteria was identified as Pseudomonas spp. Other isolates included Alcaligenes faecalis and Ochrobactrum sp. and Achromobacter sp. All of these are opportunistic pathogens, except for Achromobacter. Resistance to more antibiotics (Streptomycin, Chloramphenicol, Cephalothin, and Amoxicillin) was observed in ARBs compared to HPC (Cephalothin, Amoxicillin) from bulk water from the same borehole. The water of all the aquifers sampled is of very poor physico - chemical or microbiological quality or both. Water may be used for irrigation or livestock watering only in the case where these boreholes comply with TWQR for said purposes. Results obtained indicate that the groundwater is faecally contaminated. Amongst the bacteria, opportunistic pathogens displaying various degrees of antibiotic resistance were frequently isolated. These results indicate health risks if untreated groundwater is consumed. Therefore groundwater needs to be treated before distribution especially if the water is for human consumption. / Thesis (MSc (Environmental Sciences))--North-West University, Potchefstroom Campus, 2013.