The evolution of antibiotic resistance presents an escalating problem in the treatment of various infectious diseases worldwide. Although the origin of antibiotic resistance genes is not generally clearly documented, it has been thought that they evolved from specific genetic elements which eventually managed to spread to other microorganism of different strains and species through mobile genetics elements, transposons and plasmids. Extensively studying all aspects of these genes and their impact on the development of new treatments and drugs is of extreme importance. This study focuses on evolving and understanding how novel antibiotic resistance develops. Error prone PCR (EP-PCR) was used to introduce random mutation in an arr gene which confers high level resistance to rifampicin in E. coli. The clones obtained from EP-PCR were screened on different antibiotics with varying concentration in an attempt to isolate a clone with an increased minimum inhibitory concentration (MIC) as compared to the wild type parent strain (pBstN49). Several clones showed decreased levels of resistance against rifampicin but however none showed any significant increase in any of the other antibiotic MICs tested.

Degree of Master of Science in Medicine by Research Only Dissertation submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Master of Science in Medicine by Research. Johannesburg, 2012 / Genetic heterogeneity of Mycobacterium tuberculosis demonstrating mixed infections or affecting single strains has been previously described. A single sputum culture from five patients with drug-resistant tuberculosis treated at Sizwe Hospital was analysed in-depth for genotypic and phenotypic heterogeneity. IS6110-based restriction fragment length polymorphism (RFLP) was performed on 20 colonies from each sputum for detection of mixed infections and clonal heterogeneity. No mixed infections were found, but IS6110-RFLP-linked clonal heterogeneity was observed in one patient. Drug susceptibility testing (DST) and sequencing of nine drug-resistance-associated genes performed on a total of 99 colonies from the five patients failed to show genotypic hetero-resistance. On DST, however, discordant rifampicin resistance findings were encountered in one patient. Minimal inhibitory concentrations performed on these colonies were close to the rifampicin critical concentration used for resistance determination, suggesting failure of the BACTEC MGIT 960 assay to reliably determine rifampicin susceptibility in strains with borderline resistance.

Thesis advisor: Catherine Y. Read / Background and Significance: The metabolic syndrome is a heterogeneous disorder leading to increased morbidity and mortality. Components of the metabolic syndrome are known to be inherited, however efforts to identify genomic markers in humans have been unsuccessful and a candidate-gene/intermediate phenotype approach may be useful. Evidence supports a relationship between altered metabolic function and three candidate genes, caveolin-1 (CAV1), peroxisome proliferator receptor-activated gamma, and angiotensinogen (AGT). These genes may serve as markers for the co-aggregation of insulin resistance and hypertension. Research Question: To examine whether single nucleotide polymorphisms (SNPs) in the CAV1, PPARg and AGT genes are associated with the co-aggregation of insulin resistance and hypertension. Methods: Three gene association studies were conducted in a Caucasian hypertensive cohort (HyperPATH). The homeostasis assessment model (HOMA-IR), hyperinsulinemic euglycemic clamp, and salt sensitive blood pressure were determined in each subject. Statistical analyses were conducted using a general linear model accounting for relatedness and adjusting for the following covariates: age, gender, body mass index, study site. Replication was assessed in a hypertensive Mexican-American cohort (HTN-IR) for the CAV1 gene and a hypertensive African American cohort (HyperPATH) for the PPARg gene. Results: SNPs of the CAV1 gene were significantly associated with insulin resistance in Caucasians from HyperPATH. These results were replicated in the HTN-IR cohort. A SNP of the PPARg gene was associated with salt sensitive blood pressure and increased plasma renin levels in Caucasians and African Americans from HyperPATH. SNPs of the AGT gene were associated with insulin sensitivity in Caucasians from HyperPATH. Conclusion: CAV1 and AGT are genomic markers for the co-aggregation of insulin resistance and hypertension. The PPARg gene is a potential genomic marker for vascular dysfunction in hypertension. Clinical Perspective: Genomic markers for insulin resistance exist in human populations with hypertension. These markers explain the inter-individual variability of insulin resistance and hypertension and help identify potential underlying mechanisms. Use of these bio-markers in clinical practice may improve individualized prevention and treatment strategies, decreasing the incidence of and improving outcomes for this chronic disease. Promoting health through individualized care makes the incorporation of genomic markers into nursing practice essential. / Thesis (PhD) — Boston College, 2010. / Submitted to: Boston College. Connell School of Nursing. / Discipline: Nursing.

by Lai-chu Wu. / Bibliography: leaves 212-241 / Thesis (M.Ph.)--Chinese University of Hong Kong, 1984

by Ling Kin Wah, Thomas. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1991. / Includes bibliographical references (leaves 103-117). / ABSTRACT --- p.i / ACKNOWLEDGMENTS --- p.v / LIST OF ABBREVIATIONS --- p.vi / TABLE OF CONTENTS --- p.viii / LIST OF TABLES --- p.xv / LIST OF FIGURES --- p.xix / INTRODUCTION --- p.1 / LITERATURE REVIEW --- p.2 / Chapter 1. --- Structure of the bacterial cell envelope --- p.2 / Chapter 2 . --- The β-lactam antibiotics --- p.4 / Chapter 3. --- Mode of action of β-lactam antibiotics --- p.5 / Chapter 4. --- Penicillin-binding proteins (PBPs) --- p.6 / Chapter 5. --- Mechanisms of bacterial resistance to β-lactam antibiotics --- p.7 / Chapter 5.1 --- Non-enzymatic resistance --- p.7 / Chapter 5.1.1 --- Alteration in cell permeability --- p.8 / Chapter 5.1.2 --- Alteration of the target site --- p.9 / Chapter 5.1.3 --- Tolerance and persistence --- p.9 / Chapter 5.2 --- Enzyme-mediated resistance --- p.12 / Chapter 6. --- Transfer of resistance --- p.13 / Chapter 7. --- β-lactamases --- p.16 / Chapter 7.1 --- History --- p.16 / Chapter 7.2 --- Classification of β-lactamases --- p.17 / Chapter 7.2.1 --- Richmond and Sykes scheme --- p.17 / Chapter 7.2.2 --- Matthew scheme --- p.18 / Chapter 7.2.3 --- Bush scheme --- p.19 / Chapter 7.3 --- β-lactamases of Gram-negative bacteria --- p.19 / Chapter 7.3.1 --- Chromosomally-mediated β-lactamases --- p.19 / Chapter 7.3.2 --- Plasmid-mediated β-lactamases --- p.20 / Chapter 7.4 --- β-lactamase inhibitors --- p.25 / Chapter 7.5 --- Regulation of β-lactamase production --- p.28 / Chapter 7.5.1 --- β-lactamase induction --- p.28 / Chapter 7.5.2 --- Mutation to constitutive enzyme production --- p.29 / Chapter 7.5.3 --- β-lactam induced β-lactamase production --- p.30 / Chapter 8. --- Emergence of resistance due to production of β-lactamases --- p.31 / Chapter 8.1 --- Resistance in staphylococci --- p.32 / Chapter 8.2 --- Resistance in haemophili and gonococci --- p.33 / Chapter 8.3 --- Resistance in Enterobacteriaceae (non E. coli) --- p.34 / Chapter 8.4 --- Distribution of β-lactamases in E. coli --- p.35 / MATERIALS AND METHODS / Chapter 1. --- Bacterial strains --- p.38 / Chapter 1.1 --- Standard organisms --- p.38 / Chapter 1.2 --- Clinical isolates --- p.38 / Chapter 2. --- Antibiotics --- p.39 / Chapter 3. --- "Media, chemicals and culture conditions" --- p.39 / Chapter 4. --- Bacterial identification and viable bacterial count --- p.39 / Chapter 5. --- Antibiotic sensitivity testing --- p.40 / Chapter 5.1 --- Disk diffusion --- p.40 / Chapter 5.2 --- Determination of minimal inhibitory concentration (MIC) --- p.40 / Chapter 6. --- Plasmid analysis --- p.41 / Chapter 6.1 --- Transfer of drug resistance plasmids --- p.41 / Chapter 6. 2 --- Molecular studies of plasmids --- p.42 / Chapter 6.2.1 --- Extraction of plasmid DNA --- p.43 / Chapter 6.2.2 --- Agarose gel electrophoresis --- p.43 / Chapter 6.2.3 --- Molecular size determination --- p.44 / Chapter 7 . --- DNA hybridization --- p.44 / Chapter 7.1 --- DNA blotting --- p.44 / Chapter 7.1.1 --- Colony blotting --- p.45 / Chapter 7.1.2 --- Southern blotting --- p.45 / Chapter 7.2 --- Labeling of oligonucleotide probe --- p.46 / Chapter 7.3 --- Hybridization --- p.47 / Chapter 7.4 --- Autoradiography --- p.47 / Chapter 7.5 --- Re-use of blots --- p.48 / Chapter 8. --- Detection and screening for classification of β-lactamases --- p.48 / Chapter 8.1 --- Detection of β-lactamases --- p.48 / Chapter 8.1.1 --- Acidimetric --- p.48 / Chapter 8.1.2 --- Chromogenic substrate --- p.49 / Chapter 8.1.2.1 --- Whole cell --- p.49 / Chapter 8.1.2.2 --- Cell extract and filtrate --- p.49 / Chapter 8.2 --- Screening for classification of β-lactamases --- p.49 / Chapter 9. --- "Preparation, purification, qualitative and quantitative analyses of the β-lactamase from transconjugants TU117, TB117 and the recipient K12" --- p.51 / Chapter 9.1 --- Large scale preparation of enzyme --- p.51 / Chapter 9.2 --- Gel filtration --- p.52 / Chapter 9.3 --- Preparative isoelectric focusing (PIEF) --- p.53 / Chapter 9.4 --- Protein determination --- p.55 / Chapter 9.5 --- Qualitative analyses and characterization of β-lactamases --- p.56 / Chapter 9.5.1 --- Analytical isoelectric focusing --- p.56 / Chapter 9.5.1.1 --- Semi-quantitative determination of β-lactamases --- p.56 / Chapter 9.5.1.2 --- Polyacrylamide gel preparation --- p.57 / Chapter 9.5.1.3 --- Isoelectric focusing --- p.58 / Chapter 9.5.1.4 --- pH measurement --- p.58 / Chapter 9.5.1.5 --- Gel development and recording --- p.59 / Chapter 9.5.1.5.1 --- Nitrocefin staining --- p.59 / Chapter 9.5.1.5.2 --- Silver staining --- p.59 / Chapter 9.5.1.6 --- Isoelectric point (pI) determination --- p.60 / Chapter 9.5.2 --- Spectrophotometric assay of β-lactam substrates --- p.60 / Chapter 9.5.2.1 --- Absorption spectra of β-lactam antibiotics --- p.60 / Chapter 9.5.2.2 --- The molar extinction coefficient of β-lactam substrates --- p.60 / Chapter 9.5.2.3 --- Measurement of β-lactamase hydrolytic activities --- p.61 / Chapter 9.5.2.4 --- Determination of enzyme kinetics --- p.61 / Chapter 9.5.3 --- Molecular weight determination of proteins --- p.62 / Chapter 9.5.3.1 --- SDS-polyacrylamide gel preparation --- p.62 / Chapter 9.5.3.1.1 --- Resolving gel --- p.62 / Chapter 9.5.3.1.2 --- Stacking gel --- p.63 / Chapter 9.5.3.2 --- Electrophoresis --- p.63 / Chapter 9.5.3.3 --- Staining and recording --- p.64 / Chapter 9.5.3.4 --- Molecular weight determination --- p.64 / RESULTS / Chapter 1. --- Collection of organisms --- p.65 / Chapter 2 . --- Identification of organisms --- p.65 / Chapter 3. --- Antibiotic sensitivity testing --- p.66 / Chapter 4. --- Genetic and molecular studies of ampicillin- resistant plasmids --- p.68 / Chapter 4.1 --- Transfer of ampicillin-resistant factor --- p.68 / Chapter 4.1.1 --- E. coli K12 14R525 as recipient --- p.68 / Chapter 4.1.2 --- other Enterobacteriaceae --- p.68 / Chapter 4.2 --- Plasmid studies of E. coli --- p.69 / Chapter 5. --- Detection and identification of β-lactamases --- p.69 / Chapter 5.1 --- Analytical IEF --- p.70 / Chapter 5.2 --- DNA hybridization --- p.70 / Chapter 5.2.1 --- Colony blot hybridization --- p.70 / Chapter 5.2.2 --- Southern blot hybridization --- p.71 / Chapter 6. --- Characterization of TEM-1 producing E. coli --- p.71 / Chapter 6.1 --- Susceptibility testing --- p.71 / Chapter 6.2 --- Enzyme kinetic study --- p.72 / Chapter 6.2.1 --- Absorption spectra and molar extinction coefficient of β-lactam antibiotics --- p.72 / Chapter 6.2.2 --- Comparison of the substrate profiles --- p.73 / Chapter 6.3 --- Correlation of MICs to β-lactamase activities --- p.73 / Chapter 7. --- "Isolation, quantitation and characterization of β-lactamases isolated from three E. coli strains" --- p.74 / Chapter 7.1 --- Preparation of β-lactamases --- p.75 / Chapter 7. 2 --- Purification of β-lactamases --- p.76 / Chapter 7.2.1 --- Gel-filtration chromatography --- p.76 / Chapter 7.2.2 --- Preparative isoelectric focusing --- p.77 / Chapter 7.3 --- Characterization of the purified β-lactamases --- p.78 / Chapter 7.3.1 --- Isoelectric point --- p.78 / Chapter 7.3.2 --- Molecular weight assessment --- p.79 / Chapter 7.3.3 --- Enzyme kinetic study --- p.79 / DISCUSSION / Chapter 1. --- Epidemiology of ampici11in (or amoxycillin)- resistant E. coli --- p.81 / Chapter 2. --- Distribution of β-lactamases in ampicillin- resistant E. coli --- p.84 / Chapter 3. --- Correlation between level of resistance and β-lactamase activity --- p.86 / Chapter 4. --- Plasmid-mediated TEM-1 β-lactamase --- p.89 / Chapter 4.1 --- Transfer of resistance --- p.89 / Chapter 4 .2 --- Identification of β-lactamases by DNA hybridization --- p.91 / Chapter 5. --- Mechanism of high-level resistance --- p.93 / Chapter 5.1 --- Selection of resistant strains --- p.93 / Chapter 5.2 --- β-lactamases preparation and purification --- p.95 / Chapter 5.3 --- Hyperproduction of β-lactamase --- p.97 / SUMMARY AND CONCLUSIONS --- p.102 / REFERENCES --- p.103 / APPENDICES / Chapter 1. --- TABLES --- p.118 / Chapter 2. --- FIGURES --- p.153

Thesis (Ph.D.)--Boston University / PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you. / Antibiotic-resistant bacterial strains continually arise and their increasing prevalence poses significant clinical and societal challenges. Functional analyses of resistant mutants and the study of general stress responses perturbed by antibiotic treatment have yielded valuable insights into how resistance arises through mutations. However, less is known about the population dynamics and communal interactions that underlie the development of resistance through mutations. In this work, we utilize systems approaches to study the functional dynamics of bacterial populations evolving antibiotic resistance. We follow a continuous culture of Escherichia coli facing increasing levels of antibiotic and show that the vast majority of isolates are less resistant than the population as a whole. We find that the few highly resistant mutants improve the survival of the populations less resistant constituents, in part, by producing indole, a signaling molecule generated by actively growing and unstressed cells. We show, through transcriptional profiling, that indole serves to turn on drug efflux pumps and oxidative stress protective mechanisms. The indole production comes at a fitness cost to the highly resistant isolates, and wholegenome sequencing reveals that this bacterial altruism is enabled by drug-resistance mutations unrelated to indole production. This work establishes a population-based resistance mechanism constituting a form of kin selection whereby a small number of resistant mutants can, at some cost to themselves, provide protection to other more vulnerable cells, enhancing the survival capacity of the overall population in stressful environments. Deeper studies into cooperative strategies bacteria use to evade antibiotics may prove critical for the rational design of more effective antimicrobial interventions. / 2031-01-01

Hospital-acquired infections are the most frequent adverse event in healthcare delivery worldwide. Seven in ten hospital-acquired infections exhibit resistance to at least one antibiotic, and three in five doctors have encountered an infection unresponsive to any treatment at all. This prolongs hospitalisation, increases suffering, and causes long-term disability and unnecessary death. At present the antibiotic pipeline is unable to meet the demand for novel antibiotics needed to treat these infections. Herein I discuss how the lack of new scaffolds, limitations of target-based screening, and poor target validation each contribute to the current bottleneck in the antibiotic pipeline. In turn, I argue that the development and application of chemical probes is a more fruitful way to make progress towards new antibiotics with novel mechanisms of action. This dissertation describes a combined chemical-biology study in the search for novel inhibitors of the human pathogens Pseudomonas aeruginosa and Staphylococcus aureus. First we extend organocatalysis to the field of diversity-oriented synthesis as a powerful means to generate molecular diversity and complexity in small molecule screening collections. We then study a family of potential biofilm inhibitors identified from a diverse screening collection, and for which we suggest a possible mode of action upon the quorum sensing receptors LasR and RhlR. Thereafter we provide evidence that a novel macrocycle, based upon the cylindrocyclophane family of natural products, inhibits methicillin-resistant S. aureus through action upon the respiratory chain. Finally, we also report insights into the structure and small molecule inhibition of a key P. aeruginosa drug target, malate synthase G. Together the findings in this dissertation encourage the development and application of divergent synthesis and unbiased screening methods in antibacterial discovery.

by Li Yanchun. / "August 2000." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (p. 193-213). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web.

archives@tulane.edu / 1 / Brittany J Dodson

Thesis (PhD (Microbiological Pathology) --University of Limpopo (Medunsa Campus), 2010 / Background: Azoles and polyenes are antifungal agents used for treatment and/or prophylaxis of C. albicans infections, and a high increase in antifungal resistance in clinical isolates of C. albicans in HIV/AIDS patients has been reported. Five genetic clades were described among C. albicans isolates using DNA fingerprinting methods (clades I, II, III, SA and NG). Although these clades have been described, little is known about their phenotypic characteristics, and not much is known about antifungal resistance with regard to each of these clades. The widespread use of fluconazole has led to its increased resistance reported world-wide. Resistance to fluconazole can be caused by point mutations in the ERG11 gene or overexpression of this gene, however, not much is known about the contribution of these mutations and over-expression to fluconazole resistance among different clades of C. albicans, and whether mutations or over-expression are clade-related. There is evidence to suggest that phenotypic switching may play a significant role in the ability of Candida strains to survive under adverse conditions and perhaps cause more severe forms of disease in the immunocompromised host (Vargas et al., 2004). Only limited studies on the relationship between phenotypic switching and fluconazole resistance of C. albicans have been done, and not much is known about this relationship among different clades of C. albicans. Objectives: This study undertook to investigate: (1) the induction of antifungal resistance among South African C. albicans isolates belonging to different clades, (2) the contributions of mutations in the ERG11 gene to fluconazole resistance among C. albicans isolates belonging to different clades, (3) the contributions of over-expression of ERG11 gene to fluconazole resistance among C. albicans isolates belonging to different clades, (4) and the relationship between fluconazole resistance and phenotypic switching among C. albicans isolates belonging to different clades. Study populations and Methods: To investigate the induction of antifungal resistance among South African C. albicans isolates belonging to different clades, a total of 100 C. albicans isolates (20 from each of clades I, II, III, SA and NG) were used. These yeast isolates were obtained from surveillance cultures on patients attending HIV/AIDS clinics in the Pretoria region. Resistance to fluconazole, miconazole, amphotericin B and nystatin was induced in all 100 isolates according to the modified National Committee of Clinical vi Laboratory Standards (NCCLS) broth microdilution method. Survival and retention of resistance among fluconazole resistant (n=100), miconazole resistant (n=100), amphotericin B resistant (n=100) and nystatin resistant (n==100) isolates after two years of storage at -80oC was determined in the presence of highest concentrations of each antifungal. To investigate the contributions of mutations in the ERG11 gene to fluconazole resistance among C. albicans isolates belonging to different clades, 30 isolates were used. These consisted of 3 isolates with induced fluconazole resistance and their 3 matching fluconazole susceptible isolates from each of clades I, II, III, SA, and NG. DNA was extracted, PCR performed with a high-fidelity Pwo DNA polymerase), and PCR products sequenced using BigDye® Terminator v3.1 Cycle Sequencing Kit on the GeneAmp® PCR System 9700. Obtained sequences were compared with the published ERG11 sequence from a wild-type, fluconazole-susceptible C. albicans strain (Lai and Kirsch, 1989). To investigate the contributions of over-expression of the ERG11 gene to fluconazole resistance among C. albicans isolates belonging to different clades, 30 isolates were used. These consisted of 3 isolates with induced fluconazole resistance and their 3 matching fluconazole susceptible isolates from each of clades I, II, III, SA, and NG. RNA was extracted, cDNA synthesized and Real time PCR performed on a Rotor-Gene 6000 instrument. Relative gene expression of ERG11 gene among resistant isolates, relative to susceptible isolates was quantified after normalization with the 18SrRNA house-keeping gene. To investigate the relationship between fluconazole resistance and phenotypic switching among C. albicans isolates belonging to different clades, 30 isolates were used. These consisted of 3 isolates with induced fluconazole resistance and their 3 matching fluconazole susceptible isolates from each of clades I, II, III, SA, and NG. Primary and secondary cultures were prepared on Lee’s medium agar supplemented with arginine and zinc, and containing phloxine B. The switched colonies were counted and colony morphologies viewed and photographed. Phenotypic switching behavior and different colony morphologies obtained between the resistant and susceptible isolates from different clades were compared. Switch phenotypes among fluconazole resistant isolates in different clades were compared. Switch phenotypes and MIC levels among fluconazole resistant isolates from different clades were compared. Results: Resistance to nystatin, AmB, fluconazole and miconazole was successfully induced in all of 20 (100%) C. albicans isolates from each of clades I, II, III, SA and NG. When survival and retention of resistance were determined, all 20 (100%) fluconazole resistant vii isolates from clades I, II, SA, NG, and 19 (95%) from clade III survived and retained their resistance. Of miconazole resistant isolates, all 20 (100%) isolates from clade I, II, and SA, and 19 (95%) from clade III and NG survived and retained their resistance. Of AmB resistant isolates, 12 (60%) from Clade NG survived and retained their resistance; 9 (45%) from Clade I; 8 (40%) from Clade III; 7 (35%) from Clade II and 6 (30%) from Clade SA survived and retained their resistance. Of the isolates resistant to nystatin, 12 (60%) from clade I survived and retained their resistance, 8 (40%) from clade II, 10 (50%) from clade III, 11 (55%) from clade SA, and 15 (75%) from clade NG survived and retained their resistance. No mutations associated with fluconazole resistance were observed in all isolates from clades I and II. Mutations associated with fluconazole resistance were observed in 33.3% of isolates from each of clades III, SA and NG , and some of the mutations observed in resistant isolates from clades III and NG were novel. A total of 50 novel mutations that have not been described previously were observed in both fluconazole resistant and susceptible isolates from this study. Previously described mutations, which were associated with fluconazole resistance, namely, D116E, K128T, V437I and E266D were also observed in this study. When relative ERG11 gene expression was quantified among fluconazole resistant and susceptible isolates from various clades, over-expression of ERG11 gene was observed in 66.6% of isolates from each of clades I, II and SA, and in 33.3% of isolates from each of clades III and NG. When the relationship between fluconazole resistance and phenotypic switching was investigated, phenotypic switching was related to resistance in 66.6% of the resistant isolates tested from each of clades I, II and III, and in 33.3% of the resistant isolates tested from each of clades SA and NG. When the switch phenotypes and MIC levels of resistant isolates from different clades were compared, stipple was the most common switch phenotype observed in all clades, and it was associated with the highest fluconazole MIC levels among isolates from all clades. Conclusions: The results of this study showed that resistance to polyenes and azoles could readily be induced in C. albicans isolates from all clades, and that induction was not claderelated. The ease with which azole and polyene resistance could be induced in this study can hold serious implications, especially in HIV/AIDS patients who are already immunocompromised, and in whom azoles/polyenes are mostly used for C. albicans infections. viii The study also showed that mutations contributed to fluconazole resistance in isolates from clades III, SA and NG, but not clades I and II, showing clade-relatedness. Novel mutations were observed, and their contribution to fluconazole resistance is at this stage not known. Genetic analysis of these mutations needs to be studied further to determine their significance to azole resistance, especially in C. albicans isolates from HIV/AIDS patients in South Africa. The results of the study showed that over-expression of ERG11 gene contributed to fluconazole resistance in isolates from all clades. However, over-expression was observed in more isolates from clades I, II and SA, and in less isolates from clades III and NG, showing clade-relatedness of ERG11 over-expression. The occurrence of over-expression of ERG11 gene in these clades is a cause for concern, especially in HIV/AIDS patients with OPC, as the increased expression of ERG11 allows for the cells to persist within the host, which in turn leads to the subsequent development of other more stable resistant isolates. In this study, phenotypic switching was found to be related to fluconazole resistance in isolates from all clades, with a high number of switch phenotypes occurring more in isolates from clade II as compared to others. This suggests that isolates belonging to this clade may survive better under adverse conditions than isolates from other clades. These results suggest that further study of differences between different C. albicans clades may be warranted, and that isolates from this clade need to be studied further. The stipple phenotype was found to be the most dominant in isolates from all clades, and was found to be associated with the highest fluconazole MICs levels. These findings suggest that the evaluation of colony phenotypes and their antifungal susceptibilities in C. albicans isolates may be useful in therapy. Recommendations: A continued analysis of clade-specific phenotypic characteristics of C. albicans isolates is recommended. Pathogens that can potentially infect HIV-infected individuals need to be studied to subspecies level in order to improve treatment of these patients. Continued antifungal surveillance is needed to predict the evolution of resistance in a particular population and to take timely measures. Evaluation of colony phenotypes and their antifungal susceptibilities in C. albicans isolates is recommended as this may be useful in therapy. Genetic analysis of the novel mutations observed is recommended to determine their significance to azole resistance, especially in C. albicans isolates from HIV/AIDS patients in South Africa.