Thesis (MScMedSc (Pathology. Medical Microbiology))--Stellenbosch University, 2008. / The genus Mycobacterium is a group of acid–fast, aerobic, slow- growing organisms which include more than 90 different species. A member of this genus, Mycobacterium tuberculosis, belonging to the Mycobacterium tuberculosis complex (MTB), is the causative agent of tuberculosis (TB). This disease is currently considered a global emergency, with more than 2 million deaths and over 8 million new cases annually. TB is the world’s second most common cause of death after HIV/AIDS. About one-third of the world’s population is estimated to be infected with TB. This catastrophic situation is further compounded by the emergence of Multi Drug Resistant tuberculosis (MDR-TB) and in more recent times, Extensive Drug Resistant tuberculosis (XDR-TB). Early diagnosis is critical to the successful management of patients as it allows informed use of chemotherapy. Also, early diagnosis is also of great importance if the menace of MDR-TB and XDR-TB is to be curbed and controlled. As MTB is highly infectious for humans, it is of paramount importance that TB be diagnosed as early as possible to stop the spread of the disease. Traditional conventional laboratory procedures involving microscopy, culture and sensitivity tests may require turnaround times of 3-4 weeks or longer. Tremendous technological advancement over the years such as the advent of automated liquid culture systems like the BACTEC® 960 and the MGITTM Tube system, and the development of a myriad of molecular techniques most of which involves nucleic acid amplification (NAA) for the rapid identification of mycobacterial isolates from cultures or even directly from clinical specimens have contributed immensely to the early diagnosis of tuberculosis. Most of these NAA tests are nevertheless fraught with various limitations, thus the search for a rapid, sensitive and specific way of diagnosing tuberculosis is still an active area of research. The search has expanded to areas that would otherwise not have been considered ‘conventional’ in diagnostic mycobacteriology. One of such areas is mass spectrometry. This study joins the relatively few studies of its kind encountered in available literature to establish the ground work for the application of mass spectrometry, specifically Matrix Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-ToF MS) in the field of diagnostic mycobacteriology. This is an area which is in need of the speed, sensitivity and specificity that MALDI-ToF technique promises to offer. Since this technology is still in its infancy, the use of utmost care in the preparation of reagents, and the handling and storage of the organisms used to generate reference mass spectra for the database cannot be overemphasized. Similarly, the optimization of certain crucial experimental factors such as inactivating method and choice of matrix is of paramount importance. The main aim of this thesis was to generate a database of reference mass spectra fingerprints of selected (repository) Mycobacterium species. This necessitated the standardization of an experimental protocol which ensured that experimental factors and the various instrument parameters were optimized for maximum spectra generation and reproducibility. A standard operating procedure (SOP) for generating the database of reference mass spectra finger print of selected Mycobacterium species was developed and used to investigate the ability of the database to differentiate between species belonging to the same clinical disease complex as well as the nontuberculosis complex. The findings of this study imply that if the defined protocol is followed, the database generated has the potential to routinely identify and differentiate (under experimental conditions) more species of Mycobacterium than is currently practical using PCR and its related techniques. It is therefore a realistic expectation that when the database is clinically validated and tested in the next phase of the study, it will contribute immensely to the diagnosis of tuberculosis and other mycobacterioses. It will also aid in the identification of emerging pathogens particularly amongst the non-tuberculous mycobacteria.

Thesis (MScMedSc (Pathology. Medical Microbiology))--University of Stellenbosch, 2006. / Acquired immunodeficiency syndrome (AIDS) was first reported in 5 homosexual men in Unite States of America in 1981 as a series of opportunistic infections which occasionally occurred in adults. Subsequently, it has been achieved that human immunodeficiency virus type 1 (HIV-1) is the cause of AIDS and this aetiological agent has spread all over the world. The virus primarily attacks CD4+ T cells and gradually leads to progressive depletion of CD4 T lymphocytes from peripheral blood and lymphoid organs. Since CD4+ T cells are vital immune cells in induction and regulation of both cell-mediated and humoral immune responses, depletion of these cells ultimately results in a profound immunodeficiency characterized by susceptibility to variety of opportunistic infection. Apoptosis have been commonly proposed as the mechanism of CD4 depletion because elevated levels of apoptosis were observed in HIV-1 infected individuals (Ameisen et al., 1991; Groux et al., 1992 & Oyaizu et al., 1993). Nevertheless, there was evidence showing that HIV-1 infected cells died not from apoptosis (Bolton et al., 2002) and another study reported that inhibition of apoptosis resulted in high viral production (Antoni et al., 1995). These controversial views indicated that the mechanism of CD4 depletion and the immuno-pathogenesis of apoptosis should be considered. As a pilot sub-study, eight HIV-1 infected subjects were enrolled to determine the methods in measuring apoptosis. Three different cell separations: (1) whole blood cells, (2) buffy coat cells and (3) isolated PBMCs were prepared to determine whether different cell preparations result in different measurements of apoptosis. In addition, FITC-labelled Annexin V, an early marker of apoptosis, and flow-cytometer based scatter methods based on characteristics of apoptotic cells were used to investigate the difference in analytical methods in determining the levels of apoptosis. Firstly, it was found that whole blood samples yielded more precise measurements in measuring apoptosis, followed by Buffy coat and then PBMC samples. Secondly, this sub-study also indicated that the scatter method as well as fluorenscent labelled Annexin V could be useful markers for apoptosis. Secondly, different surface markers of apoptosis were used to investigate apoptosis in HIV-1 infected adults. Fifty-eight HIV-1 infected adults were involved in this sub-study. They were classified into three categories based on CDC CD4 category classification (CDC, 1993). According to the data, the level of apoptotic CD4+ T cells measured by the scatter method was high in CD4 category 1, decreased in category 2 and finally increased again in category 3. This tendency was in parallel with CD95 (Fas) expression on CD4+ T cells. The curve formed a “V” shape according to the three CD4 categories. Together with the gradually increased plasma viral load, these data reflect an activated immune response at early stage of infection and under controlled viraemia. This possibly represents the immune response trying to eliminate infected cells as a means of survival. The high level of apoptosis in category 3 could indicate a disordered immune system accounting for the rapid loss of CD4+ T cells and progression to AIDS. A novel finding of this study was the presence of two CD4+ populations in 10 HIV-1 infected subjects, which were CD4dim and CD4bright. These 10 subjects had relatively high CD4 count and low viral replication. Statistical analysis showed they had significantly higher levels of apoptosis in CD4 and CD8 T lymphocytes, measured by the scatter method, than those subjects presenting single CD4 population. In addition, when comparing the two CD4 subpopulations, it was found that CD4dim cells had significant higher level of apoptosis and CD95 expression than the CD4bright cells. Finally, the virological and immunological effects of antiretroviral therapy (ART) were investigated in two cohorts of HIV-1 infected children. Fourteen HIV-1 infected children were involved in investigation of 12-month long-term effect, while another five children were involved in a short-term 1-month follow-up study. In addition, a different assay of detecting apoptosis: terminal deoxynucleotidyltransferase deoxyuridine triphosphates nick end labeling (TUNEL) was conducted to measure the level of apoptotic PBMCs. According to the findings from 12-month and 1-month sub-studies, it appeared that ART could be effective in suppression of viral replication at an early stage. However, the immunological effect, such as CD4 reconstitution, could only be seen as a long-term effect, since immune recovery would take a long time. In addition, different regimens containing protease inhibitors (PIs) might be more effective in inhibiting apoptosis than non-nucleoside reverse transcriptase inhibitors (NNRTIs).

Thesis (MScMedSc (Pathology. Medical Microbiology))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: S. aureus causes serious infections in the hospital and community settings. The rate of MRSA infections are rapidly increasing worldwide. Currently, at Tygerberg hospital, approximately a third of S. aureus isolates are MRSA. This was the first epidemiological study of S. aureus conducted at Tygerberg Hospital that included prospective clinical data on patients with S. aureus bacteraemia together with spa typing of strains and the detection of the mecA and pvl genes in a multiplex PCR. Clonal cluster groups of S. aureus isolates were obtained by BURP analysis and compared to international important clones. The molecular epidemiology of hospital acquired (HA), health-care associated (HCA) and community acquired (CA) S. aureus bacteraemic strains at this hospital was examined. Lastly, repeat isolates of patients were collected to analyse any possible organism-related factors associated with persistent and recurrent bacteraemia. We investigated a total number of 113 S. aureus strains from 104 patients (70% MSSA, 30% MRSA). Repeat strains consisted of nine isolates (from 5 patients). All isolates were obtained from blood cultures collected during the period March 2008 to May 2009. Phenotypic and genotypic detection of methicillin resistance correlated well. According to the literature, most CA-MRSA strains are distinguishable from HA-MRSA strains based upon the presence of the PVL toxin. However, no CA-MRSA was detected in our study, therefore the association between HA-MRSA versus CA-MRSA strains could not be analysed. In this study, CA-MSSA was identified in 22% of all MSSA isolates versus 0% CA-MRSA. PVL positive strains were found in 22.7% of all MSSA isolates with no detection in MRSA isolates. It was noted that MRSA strains clustered in spa CC-701 and CC-012, whereas CC-002 only contained MSSA strains. Likewise HA-strains representing the majority of MRSA strains also clustered in spa CC-701 and CC-012. Forty nine spa types were identified in 89.3% of all isolates, whereas 9.7% of these strains were non-typeable. Five novel spa types were revealed. We detected a diverse number of spa-types that correlated to international clones. The most predominant spa type found in our setting was t037 (only in MRSA), followed by t891. According to the literature, t037 is associated to the Brazilian/Hungarian clone (SCCmec type III; ST 239). Our findings, as well as other South African studies, indicate that t037 has been identified in clinical strains from numerous provinces in South Africa. Interestingly, all isolates from spa type t891 were PVL positive MSSA. Bacteraemia cases were predominantly related to catheter sepsis, followed by skin and soft tissue infections (SSTI). Only one persistent bacteraemia case was identified related to a HA-SSTI. Recurrent bacteraemia cases were found in patients on dialysis for chronic renal failure and in burns patients related to intravascular catheter infections. The local epidemiology of S. aureus and the prevalence rate of different strains are important to investigate. The information provided contributes to the epidemiology of staphylococcal strains causing bacteraemia in our setting. These insights are useful for optimal diagnostic and therapeutic measures. The techniques developed can be used to identify outbreaks and recurrent infections. / AFRIKAANSE OPSOMMING: S. aureus veroorsaak ernstige infeksies in die hospitaalomgewing en in die gemeenskap. Wêreldwyd, neem metisillien-weerstandige S. aureus (MRSA) infeksies vinnig toe. Huidiglik by Tygerberg hospitaal is ongeveer ‘n derde van S. aureus isolate MRSA. Hierdie is die eerste epidemiologiese studie by Tygerberg hospitaal wat prospektiewe kliniese data van pasiënte met S. aureus bakteremie saam met spa tipering en aantoning van die mecA en pvl gene in ‘n multipleks PKR insluit. Klonale groepe (spa-CC) van MRSA en MSSA isolate is deur BURP analise verkry, en vergelyk met internasionaal belangrike klone. Die molekulêre epidemiologie van hospitaalverworwe (HA), gesondheidsorgverworwe (HCA) en gemeenskapsverworwe (CA) S. aureus bakteremie by hierdie hospitaal is ondersoek. Laastens, oorspronklike en daaropvolgende herhaal isolate is gekollekteer om moontlike organisme- faktore geassosieerd met persisterende en herhalende bakteremiese episodes te analiseer. Ons het in totaal 113 S. aureus isolate van 104 pasiënte ondersoek (70% MSSA, 30% MRSA). Nege isolate (van 5 pasiënte) was herhaal isolate. Alle isolate was afkomstig vanaf bloedkulture wat gedurende die periode Maart 2008 tot Mei 2009 gekollekteer is. Fenotipiese en genotipiese aantoning van metisillien weerstandigheid het goed gekorreleer. Volgens die literatuur kan die meeste CA-MRSA isolate van HA-MRSA isolate onderskei word op grond van die teenwoordigheid van die PVL toksien. Geen CA-MRSA is egter in ons studie gevind nie, dus kon die assosiasie tussen HA-MRSA en CA-MRSA isolate nie ondersoek word nie. CA-MSSA was in 22% van alle MSSA geidentifiseer teenoor 0% CA-MRSA. PVL is in MSSA isolate gevind (22.7% van alle MSSA) maar glad nie in MRSA nie. Dit is opgemerk dat MRSA isolate hoofsaaklik in spa CC 701 en CC-012 kloongroepe voorkom, teenoor kloongroep CC-002 wat slegs MSSA isolate bevat het. Soortgelyk het HA-isolate wat die meerderheid van MRSA isolate verteenwoordig het ook in kloongroepe 1 & 2 gegroepeer. Nege-en-veertig spa tipes is geïdentifiseer in 89.3% of alle isolate en 9.7% was nie-tipeerbaar. Vyf nuwe spa tipes is getoon. Ons het ‘n diverse aantal spa-tipes geïdentifiseer wat met internasionale klone gekorreleer het. Die mees dominante spa tipe in ons omgewing was t037 (slegs in MRSA), gevolg deur t891. Volgens die literatuur word t037 met die Brasiliaanse/Hongaarse kloon geassosieer (SCCmec tipe III; ST 239). Ons bevindings, asook ander Suid Afrikaanse studies, dui aan dat t037 in kliniese isolate vanaf talle provinsies in Suid-Afrika aangetoon is. Van belang is dat al die isolate van spa tipe t891 MSSA en PVL positief was. Bakteremiese gevalle was hoofsaaklik geassosieer met kateter-sepsis, gevolg deur vel en sagteweefsel infeksies (SSTI). Slegs een persisterende bakteremiese geval was geïdentifiseer geassosieer met HA-SSTI. Herhalende bakteremiese episodes is in pasiënte op dialise vir kroniese nierversaking en in brandwonde pasiënte met intra-vaskulêre kateter infeksies aangetoon. Die lokale epidemiologie van S. aureus en die prevalensie koers van verskillende stamme is van belang. Hierdie inligting dra by tot kennis van die epidemiologie van stafilokokkale stamme wat in ons omgewing bakteremie veroorsaak. Hierdie insigte is nuttig vir optimale diagnostiese en terapeutiese riglyne. Die tegnieke wat ontwikkel is, kan gebruik word om uitbrake en herhalende infeksies te identifiseer.

Thesis (PhD (Pathology. Medical Microbiology))--University of Stellenbosch, 2005. / Pneumocystis jirovecii is a significant contributor to the burden of disease in immunocompromised patients. The polymerase chain reaction (PCR) is more sensitive and specific than microscopy. Cotrimoxazole prophylactic breakthrough and treatment failures have been reported, and associated with mutations at codons 55 and 57 of P. jirovecii dihydropteroate synthase (DHPS). No phylogenetic or population genetic models have been successful in elucidating P. jirovecii intraspecies strain relatedness. Aims: 1) Compare detection rates of nine PCR techniques and immunofluorescence microscopy (IF); 2) Determine the extent of co-infecting pathogens associated with Pneumocystis Pneumonia (PcP); 3) Determine local P. jirovecii ITS1-5.8S-ITS2 rDNA strain types, and model lineage evolution employing a coalescent-theory based statistical parsimony network analysis; 4) Investigate the possible emergence of cotrimoxazole-resistant strains Methods: PCR was evaluated on clinical specimens employing: ITS nested; DHPS single and nested; DHFR nested; major surface glycoprotein (MSG) heminested; mitochondrial large subunit rRNA (mtLSUrRNA) single and nested; 18S rRNA onetube nested, and real-time 5S rRNA PCR. Retrospective analysis of co-infecting pathogens seen in PcP patients was conducted. ITS regions were amplified, cloned and sequenced. Statistical parsimony was applied for coalescence based network genotype analysis. DHPS genome walking was attempted and DHPS and DHFR primer annealing sites explored. Amplified DHPS and DHFR genes were cloned and sequenced. Results: Most sensitive PCR technique was mtLSUrRNA nested followed by 5S realtime PCR. A poor correlation exist between mtLSUrRNA PCR and IF. Review of clinical records suggested a high rate of false-positive IF results. P. jirovecii was detected in 4.3% M. tuberculosis-positive HIV-positive, and 2.5% M. tuberculosispositive HIV-negative patients. P. jirovecii was detected in 45% HIV-negative patients. The most prevalent ITS type was Eg. Four new combinations: Eo, Je, Ge, No; 11 new ITS1 and 13 new ITS2 sequences were identified. A new ITS2 type was detected in three patients and designated u. More than one strain type was detected in 15/19 patients. Analysis of 5.8SrDNA region revealed 13 clones containing 1-2 nucleotide polymorphisms. Of 85 mtLSUrRNA PCR-positive specimens, currently employed primers amplified DHPS and DHFR genes from 53 and 27 specimens, respectively. Newly designed DHPS primers increased detection in 3 / 28 previously DHPS-negative mtLSUrRNA-positive specimens. Of 56 DHPS genes amplified and sequenced, one contained the double mutation (Thr55Aa; Pro57Ser). DHFR Ala67Val was detected in three specimens and a new DHFR genotype (Arg59Gly; C278T) was demonstrated. Conclusions: The study emphasises the need to evaluate PCR primers against local strains. It is recommended that mtLSUrRNA PCR be performed in parallel to IF and discordant results resolved with clinical evaluation. Co-infection with P. jirovecii and M. tuberculosis occurs in South Africa, and treatment for both pathogens is recommended when demonstrated by the laboratory. ITS genotyping employing statistical parsimony network analysis suggests type Eg as major ancestral haplotype, and supports recombination contributing to strain diversity worldwide. DHPS mutations may signal emergence of resistance to cotrimoxazole in South Africa, however, low sensitivity of primers limits surveillance efforts.

Thesis (MScMedSc (Pathology. Medical Microbiology))--University of Stellenbosch, 2005. / The aim of this study was to investigate the anti-inflammatory and immunosuppressive effects of the synthetic progestins, MPA and NET-A on human cells in vitro. These injectable contraceptives are used extensively throughout the world, including Africa. The potential of these two synthetic hormones to have certain immunosuppressive and GC properties have previously been shown. Therefore, it was of concern to us to investigate whether these two hormones could possibly demonstrate any of these GC-like properties at contraceptive doses. This was achieved by determining the effects of these two synthetic hormones in vitro on certain immunologic parameters. Chapter 1 is a literature review on MPA, NET and GCs. This chapter starts with a short introduction that sets the scene. The mode of action, effectiveness, sideeffects as well as previously reported relevant data on both MPA and NET-A is portrayed in this review. Research on the known GC, Dex, is also included in the section dealing with GCs, because this synthetic hormone was used as a comparative GC in all our experiments. This chapter soon makes the reader realize how much evidence exists that indicate the possible immunosuppressive effects these two contraceptive hormones, in particular MPA, could have. The possible anti-inflammatory or pro-inflammatory effects of MPA and NET-A are investigated in Chapter 2. This was done in vitro by measuring the effects of these two synthetic hormones on the inflammatory markers, IL-6 and TNFα, by means of ELISA. In this chapter we demonstrate that MPA, even at contraceptive doses, exhibits significant anti-inflammatory properties on both cytokines tested, while NETA displayed considerably less anti-inflammatory tendencies. In its true antiinflammatory manner, we found that Dex significantly inhibited the release of both inflammatory markers from human monocytes. In Chapter 3, we investigated the effects of MPA and NET-A on the activation of human lymphocytes. This was achieved by flow cytometric measurement of the expression of the activation membrane marker CD69 by CD4 and CD8 T cells. Here we discovered that MPA had a very significant inhibitory effect on the activation of both CD4+ and CD8+ T cells, while NET-A only significantly inhibited the activation of CD8+ T cells. In addition, we found that the inhibition of CD4+ and CD8+ T cell activation by MPA was more or less the same as the known GC, Dex, and in some cases even more potent. Chapter 4 consists of an investigation of the effects of MPA and NET-A on the cytokines belonging to TH1 and TH2 subsets of CD4 T cells. This was achieved by determining whether MPA and/or NET-A targeted specific subsets of T helper cells by measuring the distinct regulatory cytokines, IFNγ and IL-4. The mechanism and role of the T helper subsets are discussed in the introduction of this chapter. Our results were portrayed as a ratio of TH2: TH1 on which the statistical analysis was done. In addition to the analysis done on the ratio, we analyzed the helper subsets separately in order to determine which subset(s) were influenced. The results of this chapter showed that neither MPA nor NET-A significantly affected either one of the helper subsets, while Dex significantly decreased this ratio. After our observed effects of MPA and NET-A on CD8 T cells, it became of interest in Chapter 5 to investigate the effects of these two synthetic hormones on the CD8 T cell-specific chemokine, RANTES. This was achieved by measuring the effects MPA and NET-A had on RANTES production in vitro by means of ELISA. Surprisingly, we discovered in this chapter that MPA and NET-A enhanced RANTES production before and after activation of CD8 T cells. We also found that Dex had the same effect on RANTES production, but to a lesser degree. Finally, a general conclusion depicting the significance and implications of our results as well as possible future research that is required is presented in Chapter 6. It was of great importance to discuss and interpret the magnitude of data generated out of all our experiments to the utmost of our capabilities. We found that MPA, even at contraceptive doses, displayed significant immunosuppressive as well as anti-inflammatory properties. NET-A, on the other hand, demonstrated weaker immunosuppressive properties in our research and no significant anti-inflammatory properties. These findings could have clinical implications in females being treated with these synthetic contraceptives. We also demonstrated significant variation found amongst genders in response to MPA, NET-A and Dex.

Thesis (PhD (Pathology. Medical Microbiology))--University of Stellenbosch, 2005. / There has recently been a significant transformation in the medical world, in particular regarding the relation between the mind/health and mind/illnesses. The changes are briefly a revolution whereby the new approach sees the development of an illness as an interaction between the psychological, biochemical and physiological factors. Music, which is used as a clinical intervention, is perceived first through the brain, affirms this interaction between the body systems, as well as having the capacity to modify the mind and thus the biochemistry of the body. The aim of this study was essentially to supply empirical data by measuring selective parameters while the patients were receiving music intervention during the physiotherapeutic treatment for pneumonia and bronchitis. Forty adult patients who were divided into an experimental and control group, according to a random scale, participated in the research. The dependant variables that had shown significant changes amongst the experimental group after three days of physiotherapeutic treatment were as follows: the cortisol, the cortisol: DHEA ratio plasma levels, the POMS scale (that measures different moods), the peak flow measurements of the lung functions and the immune parameters, namely, CD4+ : CD8+ ratio and B-cells. The results showed that the experimental group that was exposed to the acoustic stimuli of the Magnificat in D, BWV 243 of JS Bach, experienced a more positive mood and lower cortisol levels, while the immune markers as well as the peak flow of the lungs had improved. The results of the control group showed significant implications, in that its cortisol levels increased and the POMS subscale of anger and depression showed no significant change, while the tension decreased significantly. This research provided sufficient scientific evidence to confirm the concept of a bidirectional communication between the brain and the immune system. It also showed clearly that music had the capacity to modify emotional conditions, which again influenced the endocrine and autonomic nervous system and modulated the immune systems.

Thesis (PhD (Pathology. Medical Microbiology))--University of Stellenbosch, 2009. / ENGLISH ABSTRACT: Due to modern high-throughput technologies, large numbers of compounds are produced by parallel synthesis and combinatorial chemistry. The pharmaceutical industry therefore requires rapid and accurate methods to screen new drugs leads for membrane permeability potential in the early stages of drug discovery. Around 50 % of all investigational new drugs fail in pre-clinical and clinical phases of development due to inadequate absorption/permeation, distribution, metabolism, excretion and/or unacceptable toxicity. This may be decreased by applying in vitro screening methods early in the discovery process. Reliable in vitro models can be applied to determine permeation of the test compounds, which will help avoid the wasting of valuable resources for the development of drugs that are destined to fail in preclinical and clinical phases due to insufficient permeability properties. It is important to decide as early as possible on the most promising compound and physical formulation for the intended route of administration. With awareness of the increasing importance of in vitro models in the investigations of the permeability properties of drug compounds, this research project was specifically devoted to determine the suitability of our in vitro model to evaluate and predict drug permeability. A continuous flow-through diffusion system was employed to evaluate the permeability of nine different compounds/drugs with different chemical properties, across three biological membranes. The biological membranes chosen for the present study were human vaginal mucosa, human skin tissue and human small intestine mucosa. The continuous flow-through diffusion system was furthermore utilised to investigate the effects of de-epithelialisation of mucosal surfaces, chemical enhancers, temperature, permeant concentration and formulation on the permeability of the test compounds/drugs. The in vitro permeability information and data from the flow-through diffusion model were compared to in vitro and in vivo literature studies and drug profile. An in vitro model that is able to reliably predict in vivo data will shorten the drug development period, economise resources and may potentially lead to improved product quality. In this thesis research results are reported on the permeability of the mentioned biological membranes to the various chemical markers, including anti-HIV (human immunodeficiency virus) drugs. The permeability studies will be discussed in three sections: vaginal mucosa, skin tissue, small intestine mucosa. The results of the vaginal permeability studies showed that the three peptides (MEA- 5, MDY-19 and PCI) readily penetrated the vaginal mucosa. MDY-19 had a higher flux rate than MEA-5, commensurate with its smaller molecular size (weight). The surfactant enhanced the flux rate of MDY-19 approximately 1.3 times and decreased the lag time of the peptide. Removal of the vaginal epithelium increased the flux rates of the peptides across the mucosa and may have implications for a more rapid uptake of these and other microbicides in vivo. The permeability of 1 mM MDY-19 and PCI at 37 °C were significantly (p<0.05) higher than at 20 °C. At 37 °C the AUCs of the overall mean flux values of MDY-19 and PCI increased with concentration according to well-established diffusion theory. The experiments on the permeability of different terbinafine hydrochloride formulations through human skin demonstrated that the terbinafine hydrochloride formulations used in this study, readily diffused into the skin tissue. However, no flux values for any of the terbinafine hydrochloride formulations through the skin into the acceptor fluid were found. The mean terbinafine concentrations in the skin after 24 h exposure to the three commercial, terbinafine hydrochloride formulations were 3.589, 1.590 and 4.219 μg/ml respectively. The mean terbinafine concentration in the skin exposed to the 10 mg/ml PBS/Methanol solution was higher than those from the three commercial formulations. The results of the temperature study demonstrated that an increase of 5 ºC caused a significant increase in flux values of tritiated water across skin. The flux values for tritiated water across skin at 37 ºC were on average double those at a temperature of 32 ºC. The permeability of excised human small intestine mucosa to different oral dosage drugs was investigated over a 24 h period. The four drugs selected were zidovudine, propranolol hydrochloride, didanosine and enalapril maleate. They were selected as representative model compounds of drug classes 1 (high solubility, high permeability) and 3 (high solubility, low permeability) according to the Biopharmaceutics Classification System. The flux rates of the four chosen test drugs were influenced by the length of the experiment. Between the time periods 2-4 h and 4-6 h, zidovudine’s mean flux values across small intestine tissue were respectively 1.8 and 2.0 times higher than didanosine and 2.3 and 2.2 times higher than enalapril. Propranolol’s mean flux values were respectively 1.2 and 1.4 times higher than didanosine and 1.6 higher than enalapril during both the 2-4 and 4-6 h time periods. Between both the time periods 2-4 and 4-6 h AZT’s mean flux values were 1.4 times higher than propranolol and didanosine’s mean flux values were respectively 1.3 and 1.1 times higher than enalapril during the mentioned time periods. Class 1 drugs showed a significantly higher flux rate across the jejunal mucosa compared to the class 3 drugs and these results are in line with their Biopharmaceutics Classification System classification. The in vitro model has proved to be reliable to predict permeability of class 1 and 3 drugs and also showed correlation with human in vivo data. It seems that the in vitro flow-through diffusion model used in the present study have the potential to overcome some of the problems and limitations demonstrated by other in vitro techniques and may potentially serve as a future tool for pharmaceutical companies to predict the diffusion characteristics of new drugs and different formulations, across different biological membranes. Furthermore, it may serve as a prospective method for assessing the bioequivalence of alternative (generic) vehicles or formulations containing the same drug/compound. / AFRIKAANSE OPSOMMING: As gevolg van moderne hoë spoed tegnologie kan groot hoeveelhede middels vervaardig word deur ooreenkomende sintese en kombinasieleer chemie. Die farmaseutiese industrie benodig dus vinnige en akkurate metodes om nuwe geneesmiddels te evalueer t.o.v. membraan deurlaatbaarheid. Hierdie evaluasie moet verkieslik so vroeg moontlik in die geneesmiddel se ontwikkelingsproses geskied. Ongeveer 50 % van alle potensiële geneesmiddels misluk in pre-kliniese en kliniese fases van geneesmiddelontwikkeling. Die mislukte pogings kan toegskryf word aan onvoldoende absorbsie/deurlaatbaarheid, distribusie, metabolisme, ekskresie en/of onaanvaarbare middel toksisiteit. Dit is daarom belangrik om so vroeg moontlik in die geneesmiddelontwikkelingsproses te besluit op die mees belowende middel, asook die geskikte formulasie vir die spesifieke roete van toediening van die middel. Die farmaseutiese industrie benodig tans in vitro modelle met die potensiaal om die deurlaatbaarheid van geneesmiddels te bepaal en te voorspel. Betroubare in vitro modelle kan aangewend word om die deurlaatbaarheid van potensiële geneesmiddels te toets. Sodoende sal die onnodige uitgawes op die ontwikkkeling van geneesmiddels wat in elk geval later gaan faal in pre-kliniese en kliniese fases van geneesmiddelproewe a.g.v. deurlaatbaarheidseienskappe, vermy word. Hierdie navorsingsprojek was dus spesifiek onderneem om die waarde en toepaslikheid van ‘n in vitro deurlopende-vloei perfusie model te ondersoek. Die model se potensiaal om geneesmiddels se deurlaatbaarheid en absorpsie te voorspel was geëvalueer. Die deurlopende-vloei perfusie apparaat was gebruik om die deurlaatbaarheidsvloede van drie verskillende biologiese membrane t.o.v. nege chemiese stowwe (MEA-5, MDY-19, PCI, terbinafien hidrochloried, getritieerde water, zidovudien, propranolol, hidrochloried, didanosien, enalapril maleaat) te bepaal. Die drie biologiese membrane wat gebruik was, was vaginale weefsel, vel en klein intestinale weefsel. Al drie weefsel tipes was van menslike oorsprong. Die deurlopende-vloei perfusie apparaat was ook gebruik om die effek wat verwydering van die mukosa se epiteellaag op deurlaatbaarheidsvloede het, te ondersoek. Verder was navorsing gedoen op die effek van temperatuur en die konsentrasie en formulasie van die toetsmiddels op hulle diffusie vloedwaardes. Daar was ook gekyk na die invloed van ander chemiese stowwe op die toetsmiddels se diffusie vloedwaardes. Die in vitro deurlaatbaarheidsinformasie en -gegewens was vergelyk met ander in vitro en in vivo literatuurstudies en geneesmiddel databasisse. ‘n In vitro model wat in staat is om in vivo resultate betroubaar te voorspel, het die potensiaal om die tyd wat dit neem om geneesmiddels te ontwikkel, te verkort, finansiële uitgawes te besnoei en om geneesmiddelkwaliteit te verseker. In die tesis word dan die resultate gerapporteer van die deurlaatbaarheidsvloede van die verskillende tipes weefsel ten op sigte van verskeie chemiese stowwe, insluitende anti-MIV (menslike immuniteitsgebreksvirus) middels. Die deurlaatbaarheidstudies word bespreek in drie afdelings: vaginale mukosa, vel en klein intestinale mukosa. Die resultate van die deurlaatbaarheidstudies op die vaginale weefsel dui daarop dat die drie peptiede (MEA-5, MDY-19 and PCI) die vaginale mukosa goed penetreer. Soos verwag, het MDY-19 hoër diffusie vloedwaardes as MEA-5 gehad. Dit kan toegeskryf word aan MDY-19 se kleiner molekulere grootte (gewig). Surfaktant het die diffusie vloedwaardes van MDY-19 1.3 keer vergroot en het ook die tyd na vaste vlak verminder. Die verwydering van die vaginale epiteel het die diffusie vloedwaardes van die peptiede verhoog en mag dus dui op die vinniger opname van peptiede en moontlike ander mikrobisiede in vivo, wanneer die belyning van die epiteel onderbreek. Die deurlaatbaarheid van 1 mM MDY-19 en PCI by 37 °C was satisties beduidend (p<0.05) hoer as teem 20 °C. Die area onder die kurwe (AOK) van die gemiddelde vloedwaardes van MDY-19 en PCI by 37 °C, het toegeneem met ‘n toename in die konsentrasie van hierdie peptiede. Die toename vloedwaardes ondersteun dus die alombekende diffusie teorie. Die transdermale diffusie eksperimente van verskillende terbinafien formulasies het getoon dat terbinafien geredelik vrygestel word vanuit hierdie formulasies na die vel. Geen terbinafien vloedwaardes, van enige van die formulasies, was egter gevind in die ontvangselle van die deurlopende-vloei perfusie apparaat nie. Die gemiddelde terbinafien konsentrasies in die vel na 24 h se blootstelling aan drie kommersiële terbinafien hidrochloried formulasies was onderskeidelik 3.589, 1.590 en 4.219 μg/ml. Die gemiddelde terbinafien konsentrasie in die vel wat aan 10 mg/ml PBS/metanol blootgestel was, was hoër as die konsentrasies in die vel wat aan die drie kommersiële formulasies blootgestel was. Die resultate van die temperatuurstudie op vel het aangetoon dat ‘n temperatuur toename van 5 ºC ‘n statisties beduidende toename in vloedwaardes van getritieerde water oor vel veroorsaak. Die vloedwaardes van die getritieerde water oor vel teen ‘n temperatuur van 37 ºC was gemiddeld dubbeld so veel as teen 32 ºC. Die deurlaatbaarheidsvloede van klein intestinale mukosa ten opsigte van verskillende geneesmiddels (wat oraal toegedien word) was ondersoek gedurende ‘n 24 h eksperiment. Die vier geneesmiddels wat gebruik was, was zidovudine, propranolol hidrochloried, didanosien en enalapril maleaat. Hierdie geneesmiddels is verteenwoordigers van die Biofarmaseutiese Klassifikasie Sisteem se klas 1 (hoë oplosbaarheid, hoë deurlaatbaarheid) en klas 3 (hoë oplosbaarheid, lae deurlaatbaarheid) geneesmiddels. Die vloedwaardes van die vier geneesmiddels het gewissel na aanleiding van die tydsverloop in die eksperiment. Zidovudien se gemiddelde vloedwaardes tussen 2-4 en 4-6 h was onderskeidelik 1.8 en 2.0 keer hoër as didanosien se gemiddelde vloedwaardes vir hierdie tyd periodes en onderskeidelik 2.3 en 2.2 keer hoër as enalapril se gemiddelde vloedwaardes. Tydens hierdie selfde periodes was propranolol se gemiddelde vloedwaardes 1.2 en 1.4 keer hoër as didanosien en vir beide periods 1.6 keer hoër as enalapril se gemiddelde vloedwaardes. Gedurende beide genoemde tyd periodes was zidovudien se gemiddelde vloedwaardes 1.4 keer hoer as propranolol en didanosien se gemiddelde vloedwaardes was onderskeidelik 1.3 en 1.1 keer hoër as enalapril tydens 2-4 en 4-6 h. Die klas 1 geneesmiddels het statisties beduidende hoër vloedwaardes gehad as die klas 3 geneesmiddels. Hierdie resultate stem ooreen met die geneesmiddels se Biofarmaseutiese Klassifikasie Sisteem klassifikasie. Dit wil dus voorkom asof die in vitro model wat gebruik was in die studie, gebruik kan word om die deurlaatbaarheidsvloede van klas 1 en 3 te voorspel. Die resultate van hierdie studie stem ooreen met ander in vivo studies. Dit wil voorkom asof die in vitro deurlopende-vloei perfusie apparaat die potensiaal het om sommige van die probleme en tekortkominge van ander in vitro modelle te oorkom en dat dit moontlik die potensiaal het om die diffusie-eienskappe van nuwe geneesmiddels en verskillende formulasies oor verskillende biologiese membrane te voorspel. Die model kan verder moontlik dien as ‘n potensiële toestel om biogelykbaarheid van alternatiewe (generiese) formulasies, wat dieselfde geneesmiddel/chemiese stof bevat, te bepaal.

Thesis (MScMedSc (Pathology. Medical Microbiology))--University of Stellenbosch, 2009. / Water is used for various purposes and contamination can have severe implications if untreated. One of the most common and cost effective water disinfectants, especially used in developing countries, is chlorine. However, microorganisms have developed different mechanisms in response to environmental stress conditions, such as the viable but nonculturable (VBNC) effects possibly displayed in this study, enabling them to survive. Chlorine may also exert several effects on microorganisms, such as the expression of multi-substrate efflux pumps, decreased membrane permeability and transport inhibition that may lead to chlorine tolerance and antimicrobial resistance. In a descriptive and comparative study, the molecular characteristics of E. coli strains isolated from environmental waters in the Western Cape and the possible relationship between chlorination and antimicrobial resistance were investigated. Water and biofilm samples were exposed to chlorine, as well as efflux pump inhibitor (EPI) concentrations, and surviving E. coli strains were tested for their phenotypic characteristics including antimicrobial susceptibility profiles and morphological types. Candidate genes possibly involved in resistance to antimicrobials, disinfection and efflux pumps were detected with polymerase chain reaction (PCR) and sequenced. Sequencing analysis and homology searches were done and E. coli strains were typed as either Enteropathogenic E. coli strains (EPEC) or Enterotoxigenic E. coli strains (ETEC) on the presence of virulence genes. All water and biofilm sources examined were heavily polluted with E. coli, and a high enumeration level of this indicator organism of faecal contamination was recorded. Chlorine tolerance was found to be associated with antimicrobial resistance. Addition of EPI with exposure to chlorine decreased enumeration levels of these organisms, suggesting that efflux pumps may play a role in tolerance to chlorine. Several morphological patterns were described amongst the E. coli strains and a change in this was recorded after exposure to chlorine. Highly resistant antibiograms displayed by the isolated strains included ampC β-lactamase producing E. coli strains and extended spectrum β-lactamases (ESBLs). Amplification of the candidate genes selected for heatshock, oxidative stress genes and efflux pump were most frequently detected while the structural genes involved in fluoroquinolones (FQs) resistance were detected less frequently in the selected strains. Sequencing of these amplified candidate genes demonstrated various changes in amino acid sequences, including one common mutational pathway taken by E. coli when exposed to stress conditions. Further homology searches of the sequenced candidate genes illustrated similarities in 19 pathogenic and 14 non-pathogenic E. coli as well as 3 Shigella strains. Detection of virulence genes found three EPEC strains (bfpA, eaeA), two EPEC (eaeA), ten EPEC (bfpA) and one ETEC strain (st) amongst the isolates. This study underlines the need for monitoring our water sources, which poses a public health risk due to incomplete chlorination, antimicrobial resistance and the spread of clinically relevant pathogenic strains.

Bibliography / Thesis (MMed (Pathology. Medical Microbiology))--University of Stellenbosch, 2011. / ENGLISH ABSTRACT: Acinetobacter baumannii has emerged as one of the most troublesome nosocomial pathogens globally. This organism causes infections that are often extremely difficult to treat because of the widespread resistance to the major antibiotic groups. Colonization or infection with multidrugresistant A. baumannii is associated with the following risk factors: prolonged hospital stay, admission to an intensive care unit (ICU), mechanical ventilation, and exposure to broad spectrum antibiotics, recent surgery, invasive procedures, and severe underlying disease. A. baumannii has been isolated as part of the skin flora, mostly in moist regions such as axillae, groin and toe webs. It has also been isolated from the oral cavity and respiratory tract of healthy adults. Debilitated hospitalized patients have a high rate of colonization, especially during nosocomial Acinetobacter outbreaks. This organism is an opportunistic pathogen as it contains few virulence factors. Clinical manifestations of A. baumannii include nosocomial pneumonia, nosocomial bloodstream infections, traumatic battlefield and other wound infections, urinary tract infections, and post-neurological surgery meningitis. Fulminant community-acquired pneumonia has recently been reported, indicating that this organism can be highly pathogenic. The number of multidrug-resistant A. baumannii strains has been increasing worldwide in the past few years. Therefore the selection of empirical antibiotic treatment is very challenging. Antibiotic combinations are used mostly as empirical therapy in critically ill patients. One rationale for the use of combination therapy is to achieve synergy between agents. The checkerboard and time-kill methods are two traditional methods that have been used for synergy testing. These methods are labor intensive, cumbersome, costly, and time consuming. The E-test overlay method is a modification of the E-test method to determine synergy between the different antibiotics. This method is easy to perform, flexible and time efficient. The aim of this study was to assess the in vitro activity of different combinations of colistin, rifampicin, imipenem, and tobramycin against selected clinical strains of A. baumannii using the checkerboard and the E-test synergy methods. The MICs obtained with the E-test and broth microdilution method were compared. The results of the disk diffusion for imipenem and tobramycin as tested in the routine microbiology laboratory were presented for comparison. Overall good reproducibility was obtained with all three methods of sensitivity testing. The agreement of MICs between the broth dilution and E-test methods was good with not more than two dilution differences in MIC values for all isolates, except one in which the rifampicin E-test MIC differed with three dilutions from the MIC obtained with the microdilution method. However, the categorical agreement between the methods for rifampicin was poor. Although MICs did not differ with more than two dilutions in most cases, many major errors occurred because the MICs clustered around the breakpoints. The combinations of colistin + rifampicin, colistin + imipenem, colistin + tobramycin, rifampicin + tobramycin, and imipenem + tobramycin all showed indifferent or additive results by the E-test method. No results indicating synergy were obtained for all the above-mentioned combinations. There was one result indicating antagonistic effect for the combination of colistin + tobramycin. The results of the checkerboard method showed results indicating synergy in four of the six isolates for which the combination of colistin and rifampicin was tested. The other two isolates showed indifferent/additive results. All the other combinations showed indifferent/additive results for all isolates except isolate 30 (col + tob) and isolate 25 (rif + tob) which showed synergism. No antagonistic results were observed by the checkerboard method. When the results obtained with the E-test and checkerboard methods were compared, it was noted that for most antibiotic combinations an indifferent/additive result was obtained. However, for the colistin + rifampicin combination, the checkerboard method showed synergism for 4 of 6 isolates, whereas the E-test method showed indifference and an additive result in one. For the rifampicin + tobramycin, and colistin + tobramycin combinations, synergism was also shown with the checkerboard method in one isolate for each combination. The E-test method however showed an indifferent and additive result respectively. . The E-test method was found to be a rapid, reproducible, easy-to-perform, and flexible method to determine synergistic antibiotic activity. This study was however limited by low numbers of isolates. This might explain why no synergistic results were obtained with the E-test method and few synergistic results with the checkerboard method. Genotypic analysis using pulse-field gel electrophoresis (PFGE) may be considered in future studies to determine relatedness of the isolates which will facilitate the selection of different strains for synergy testing. Furthermore, clinical studies are needed to establish whether in vitro synergy testing is useful in the clinical setting and whether the results of synergy testing will have any bearing on the clinical outcome of patients infected with multidrug resistant A. baumannii. / AFRIKAANSE OPSOMMING: Acinetobacter baumannii het wêreldwyd as een van die mees problematiese nosokomiale patogene verskyn. Hierdie organisme veroorsaak infeksies wat dikwels baie moeilik is om te behandel weens wydverspreide weerstandigheid teen major antibiotikagroepe. Kolonisasie of infeksie met multi-weerstandige A. baumannii word geassosieer met die volgende riskofaktore: verlengde hospitaalverblyf, toelating tot ‘n intensiewe sorgeenheid (ICU), meganiese ventilasie, blootstelling aan breëspektrum antibiotika, onlangse chirurgie, indringende prosedures en ernstige onderliggende siekte. A. baumannii kan deel vorm van die normale velflora, veral in die axillae, inguinale area en tussen die tone. Dit is ook al vanuit die mondholte en die respiratoriese traktus van gesonde volwassenes geïsoleer. Verswakte gehospitaliseerde pasiënte word veral gekoloniseer gedurende nosokomiale Acinetobacter uitbrake. Hierdie organisme is ‘n opportunistiese patogeen en bevat min virulensie faktore. Kliniese manifestasies van A. baumannii sluit nosokomiale pneumonie, nosokomiale bloedstroom infeksies, troumatiese slagveld- en ander wondinfeksies, urienweginfeksies en meningitis wat volg op neurologiese chirurgie in. Fulminerende gemeenskapsverworwe pneumonie is onlangs beskryf en dui aan dat hierdie organisme hoogs patogenies kan wees. Die aantal multi-weerstandige A. baumannii stamme het wêreldwyd toegeneem oor die laaste paar jare. Daarom is die seleksie van empiriese antibiotiese behandeling ‘n uitdaging. Antibiotika kombinasies word meestal as empiriese behandeling in ernstige siek pasiënte gebruik. Die beginsel hiervan is om sinergistiese werking tussen agente te verkry. Die “checkerboard” en “time-kill” metodes is twee tradisionele metodes van sinergisme toetsing. Hierdie metodes is werksintensief, duur en tydrowend. Die E-toets sinergisme metode is gebaseer op die E-toets metode. Hierdie metode is maklik, buigbaar en tydseffektief. Die doel van hierdie studie was om die in vitro aktiwiteit tussen verskillende antibiotika kombinasies van colistin, rifampisien, imipenem, en tobramisien teen geselekteerde kliniese A. baumannii isolate te toets met die “checkerboard” en E-toets sinergisme toetsing metodes. Die minimum inhibitoriese konsentrasies (MIKs) verkry met die E-toets en “broth microdilution” metode is ook vergelyk. Die resultate van die skyfie diffusie metode (die metode wat in die roetiene mikrobiologie laboratorium gebruik word) vir imipenem en tobramisien word ook verskaf vir vergelyking van die resultate van verskillende sensitiwiteitsmetodes. In oorsig is goeie herhaalbaarheid van resultate verkry met al drie metodes van sensitiwiteitstoetsing. Die ooreenstemming van MIKs tussen die “broth dilution” en E-toets metodes was goed en resultate het met nie meer as twee verdunnings in MIK waardes verskil nie. Daar is een uitsondering waar die rifampisien E-toets MIK waarde met drie verdunnings van die MIK waarde verkry met die “microdilution” metode verskil. Die ooreenstemming tussen die sensitiwiteitskategorie resultate tussen die twee metodes was egter swak vir rifampisien. Alhoewel die MIKs in die meeste gevalle met nie meer as twee verdunnings in waarde verskil het nie, was daar baie major foute aangetoon omdat die MIKs rondom die breekpunte geval het. Die kombinasies van colistin + rifampisien, colistin + imipenem, colistin + tobramisien, rifampisien + tobramisien, en imipenem + tobramisien het oorwegend slegs matige interaksie met die E-toets metode getoon. Geen sinergisme is verkry met enige van die antibiotika kombinasies met hierdie metode nie. Daar was egter een resultaat wat antagonisme getoon het vir die kombinasie van colistin + tobramycin. Die resultate van die “checkerboard” metode het sinergisme getoon in vier van die ses isolate wat vir die kombinasie van colistin en rifampisien getoets was. Die ander twee isolate het slegs matige interaksie getoon. Al die ander kombinasies het ook slegs matige interaksie getoon, behalwe in isolaat 30 (col + tob) en isolaat 25 (rif + tob) waar die spesifieke kombinasies sinergisme getoon het. Geen antagonisme is waargeneem met die “checkerboard” metode nie. Met vergelyking van die E-toets en “checkerboard” metodes, is dit opmerklik dat vir die meeste van die antibiotika kombinasies slegs matige interaksie verkry is. Vir die colistin + rifampisien kombinasie toon die “checkerboard” metode egter sinergisme vir 4 uit 6 isolate, terwyl die E-toets metode slegs matige interaksie toon. Vir rifampisien + tobramisien, en colistin + tobramisien kombinasies is sinergisme getoon met die “checkerboard” metode in een isolaat vir elke kombinasie. Die E-toets metode het slegs matige interaksie getoon. Die E-toets sinergisme metode was vinnig, herhaalbaar en maklik om uit te voer. Hierdie studie word egter beperk deur lae getalle van isolate. Dit mag verklaar waarom geen sinergistiese resultate met die E-toets metode verkry is nie en die min sinergistiese resultate met die “checkerboard” metode. Genotipiese analiese met “pulse-field gel electrophoresis” mag in aanmerking geneem word in toekomstige studies om die verwantskap tussen isolate te bepaal wat die seleksie van verskillende stamme vir sinergisme toetsing sal vergemaklik. Verder, kliniese studies is nodig om te bepaal of in vitro sinergisme toetsing van waarde is en of die resultate van sinergisme toetsing ‘n rol speel in die kliniese uitkoms van pasënte geïnfekteer met multiweerstandige A. baumannii. / The National Health Laboratory Serivice

Bibliography / Thesis (PhD (Pathology. Medical Microbiology))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: Overview: Mycoplasmas and ureaplasmas are not routinely diagnosed and are under researched in South Africa. Prevalence, population shifts especially concerning genital flora and implications in infection or other conditions are unknown. Information pertaining to Mycoplasma pneumoniae in respiratory disease is similarly lacking. There is little information on antimicrobial susceptibilities and resistance development against Sexually Transmitted Infections (STI) syndromic management approaches. Aims: a) Elucidate mycoplasmal and ureaplasmal prevalence and contributing factors concerning cervical colonisation or preterm delivery in conjunction with HIV and Chlamydia trachomatis b) Investigate prevalence of M. pneumoniae in respiratory infections in conjunction with HIV, Mycobacterium tuberculosis and Pneumocystis jiroveci. c) Determine antimicrobial susceptibilities of mycoplasmas and ureaplasmas and analyse resistance genes. d) Assess the inter-generic transfer potential of resistance gene (tetM) between Ureaplasma spp. and Neisseria gonorrhea. Genital setting: The prevalence of genital mycoplasmas, ureaplasmas and Chlamydia on women attending their first prenatal visit, in conjunction with preterm labour or HIV status was investigated. For preterm labour (2003), 199 women were monitored for preterm delivery (<37 weeks); for colonisation and HIV (2005), 219 women were screened. Microbial detection was performed on DNA extracted from endocervical swabs employing PCR techniques. Colonisation was seen to be highest in the 14-20 year group from 2003. In women aged ±21 years, co-colonisation was 13% although there was a shift from co-colonisation with Mycoplasma hominis and Ureaplasma spp. in 2003 to other dual/triple combinations in 2005. Overall major trends from both collection periods were that the prevalence of Ureaplasma spp. tended to be higher in women ±26 years, whilst prevalence of C. trachomatis and M. hominis were lower. No association was evident between colonisation with M. hominis, U. urealyticum, Ureaplasma parvum and labour outcome. HIV status had no effect on the prevalence/co-colonisation of M. hominis, Ureaplasma spp. or C. trachomatis. Respiratory setting: Studies were conducted to determine the prevalence of community acquired atypical pneumonias in adults (M. pneumoniae and P. jiroveci) and neonates (mycoplasmas, ureaplasmas and Chlamydia trachomatis) in order to improve treatment management programmes in the Port Elizabeth region. Sputum specimens from 102 adult patients presenting with pneumonia/symptoms of pneumonia admitted to hospitals were assessed by PCR. Details of patient’s gender, age, HIV and Mycobacterium tuberculosis status were provided by the hospitals. Women were seen to be at high risk for community-acquired P. jiroveci colonisation. Overall, prevalence of P. jiroveci was 52.9% (54/102 patients). P. jiroveci was mainly associated with HIV (25/74) (P. jiroveci and HIV positive patients in patient sample for which clinical data and HIV status was available) and co-infection with M. tuberculosis was observed in 12 HIV cases and one HIV negative patient. No DHPS (20) or DHFR (17) resistance associated mutations were found in P. jiroveci. M. pneumoniae was detected in one patient. For prevalence studies (2007-2008) on atypical pneumonia in neonates, 69 endotracheal aspirates were obtained. PCR detection of M. hominis, U. urealyticum and C. trachomatis was performed and U. parvum detected in two specimens. Antibiotic susceptibilities and resistance genes: The following investigations on clinical isolates of U. parvum and U. urealyticum were conducted (i) antibiotic susceptibility profiles, (ii) detection of drug target gene mutations, or gene acquisitions and (iii) inter-generic resistance gene transfer potential to Neisseria gonorrhoeae. Culture techniques applied to 132 endocervical specimens provided 66 Ureaplasma cultures (35 U. parvum, 9 U. urealyticum, 22 U. parvum + U. urealyticum). MIC determinations to ofloxacin, erythromycin, tetracycline, doxycycline, azithromycin and josamycin were performed. Thirty-seven ureaplasma cultures were fully susceptible to all antibiotics tested; 21 showed intermediate resistance to erythromycin, azithromycin and ofloxacin; while seven were resistant to tetracycline, three of which were also resistant to doxycycline and one also resistant to azithromycin. Concerning ofloxacin resistance directed at quinolone resistance determining regions, a substitution of Ser83Leu in ParC was demonstrated in one intermediately-resistant Ureaplasma (MIC 4 µg/ml) while a triple substitution of Asp112Glu in GyrA along with Ala125Thr and Ala136Thr in ParC was found in six further intermediately-resistant strains. No mutations were found in strains with MICs 1 µg/ml. No mutations were detected in 23S rRNA operons, L4 or L22 proteins. TetM and int-Tn genes were found in seven tetracycline-resistant strains. On screening 59 tetracycline-susceptible and -intermediate strains, eleven whilst possessing an int-Tn gene lacked a large region of tetM and 48 only contained small regions of tetM. The tetM genes of the seven tetracycline-resistant strains were sequenced and comparisons performed against GenBank sequences of Neisseria gonorrhoeae, Streptococcus pneumoniae and U. urealyticum. For five strains tetM was seen to be highly mosaic in structure containing regions that were similar to those of the GenBank strains and others that were unique. In the tetM leader region, four hot spot recombination sites were identified that could certainly influence the formation of the mosaic structures, upstream insertion sequences/open reading frames and transposon regions that regulate expression. On characterising the int-Tn genes of the seven tetracycline-resistant strains, three types were present indicating transposons from different origins had integrated into ureaplasma genomes. Reciprocal tetracycline resistance gene transfer between ureaplasmas and N. gonorrhoeae were unsuccessful. However, low-level tetracycline resistance (MICs 4-8 µg/ml) was transferred to a U. parvum recipient from one U. urealyticum and three U. parvum donors that carried tetM with MICs 16-64 µg/ml. On tetM PCR analysis, tetM was not detected in the transformants. Conclusions: The importance of genital mycoplasmas, ureaplasmas and C. trachomatis in long term aetiologies requires further investigations, certainly in relation with syndromic management regimens that fail to reduce colonisation rates. The high prevalence of P. jiroveci, the presence of M. pneumoniae in cases of pneumonia and detection of U. parvum in two cases of neonatal pneumonia investigated emphasises that in the absence of definitive diagnoses, it is crucial to monitor treatment responses carefully, especially when first line antibiotic preferences are ß-lactams, in order to ensure adequate and informed delivery of medical care. The finding of transposon and/or tetM regions in all ureaplasmas investigated with or without full expression of tetracycline resistance, in conjunction with tetM gene diversity, certainly places ureaplasmas strongly in the picture for intra- and inter-generic exchange of antibiotic resistance genes. / AFRIKAANSE OPSOMMING: Oorsig: Mikoplasma en ureaplasma word nie roetineweg gediagnoseer nie en in Suid Afrika is nog min navorsing daaroor gedoen. Prevalensie, populasie verskuiwings, veral in genital flora, en die impliksies van infeksie en ander toestande is onbekend. Inligting rakende Mycoplasma pneumoniae in respiratoriese siekte is ook gebrekkig. Daar is min inligting beskikbaar rakende die antimikrobiale vatbaarheid en die ontwikkeling van weerstandigheid gesien teen die benadering tot sindromiese hantering van seksueel oordraagbare siektes. Doelwitte: a) Om inligting te verskaf oor die prevalensie van mikoplasma en ureaplasma en bydraende faktore betreffende voortydige kraam tesame met MIV en Chlamydia trachomatis. b) Ondersoek van die prevalensie van M. pneumoniae in respiratoriese infeksies tesame met MIV, Mycobacterium tuberculosis en Pneumocystis jiroveci. c) Bepaling van die antimikrobiale vatbaarheid van mikoplasma en ureaplasma en analisevan weerstandigheids gene. d) Bereken die inter-genetiese oordrag potensiaal van weerstandigheids gene (tetM) tussen Ureaplasma spp. en Naisseria gonorrhoeae. Genitale omgewing: Die prevalensie van genitale mikoplasma, ureaplasma en Chlamydia in vroue tydens hul eerste prenatale besoek, tesame met vroegtydige kraam en MIV status is ondersoek. In voortydige kraam (2003), is 199 vroue gemonitor vir voortydige kraam (<37 weke); vir kolonisasie en MIV (2005), is 219 vroue getoets. Mikrobiale toetsing is gedoen deur DNS te win vanaf endoservikale deppers met PKR tegnieke. Kolonisasie was die hoogste in die ouderdomsgroep 14.20 jaar, in 2003. In vroue van ±21 jaar was medekolonisasie 13% alhoewel daar en verskuiwing was van mede-kolonisasie met Mycoplasma hominis en Ureaplasma spp. in 2003 tot ander dubbel/trippel kombinasies in 2005. Die oorkoepelende tendens in altwee die tydperke van waarneming was dat die prevalensie van Ureoplasma spp. geneig was om hoër te wees in vroue ±26 jaar, terwyl prevalensie van C. trachomatis en M. hominis laer was. Geen assosiasie kon getoon word tussen koloniesasie met M. hominis, U. urealyticum, Ureaplasma parvum en uitkoms van kraam nie. MIV status het geen effek gehad op die prevalensie/mede-kolonisasie van M. hominis, Ureaplasma spp. of C. Trachomatis nie. Respiratories: Studies is gedoen om die prevalensie van gemeenskaps verworwe atipiese pneumonie in volwassenes (M. pneumoniae en P. jiroveci) en neonate (mikoplasma, ureaplasma en Chlamydia trachomatis) te bepaal om behandeling en hantering programme in die Port Elizabeth area te verbeter. Sputum monsters van 102 volwasse pasiënte wat presenteer het met pneumonie of simptome van pneumonie en wat tot hospitale toegelaat was, is ontleed. Besonderhede van die pasiënte se geslag, ouderdom, MIV en Mycobacterium tuberculosis status is deur die hospitale verskaf. PKR is gedoen met inleiers gerig teen die volgende gene: P. jiroveci vir die aantoning van mitokondriale groot subeenheid RNS en vir die analise van mutasies vir ko-trimoksasool weerstandigheid dihydropteroaat sintetase (DHPS) en dihydrofolaat reduktase (DHFR); M. pneumoniae vir die aantoning van P1 adhesien en 16S rRNS. Vroue het ‘n hoë risiko vir gemeenskapsverworwe P. jiroveci kolonisasie gehad. In die algemeen was die prevalensie van P. jiroveci 52.9% (54/102 pasiënte). P. jiroveci was hoofsaaklik geassosieerd met MIV (25/74) (P. jiroveci en MIV positiewe pasiënte in die pasiënt monster waarvoor daar kliniese data en MIV status bekend was) en mede-infeksie met M. tuberculosis is gesien in 12 MIV gevalle en een MIV negatiewe pasiënt. Geen DHPS (20) of DHFR (17) weerstandigheids geassosieerde mutasies is gevind in P. Jiroveci nie. M. pneumoniae was aangetoon in een pasiënt. Vir prevalensie studies (2007-2008) op atipiese pneumonie in neonate is 69 endotrageale aspirate verkry. PKR toetsing vir M. hominis, U. urealyticum en C. trachomatis is gedoen met ‘primers’ soos voorheen gepubliseer. Ureaplasma parvum is aangetoon in twee neonate met PKR met negatiewe kultuur resultate. Antibiotika sensitiwiteite en weerstandigheids gene: Die volgende toetse is gedoen op kliniese isolate van U. parvum en U. urealyticum (i) antibiotika sensitiwiteits profiele, (ii) aantoning van teiken geen mutasies, of geen aanwinste en (iii) potensiaal vir inter-generiese weerstandigheids geen oordrag na Neisseria gonorrhoeae. Kultuur tegnieke toegepas op 132 endoservikale monsters het 66 Ureaplasma kulture gelewer (35 U. parvum, 9 U. urealyticum, 22 U. parvum + U. urealyticum). MIK bepaling vir ofloksasien, eritromisien, tetrasiklien, doksisiklien, azitromisien en josamisien is gedoen. Sewe-en-dertig kulture was ten volle sensitief vir alle antibiotika wat getoets is; een-en twintig het intermediere weerstandigheid teenoor eritromisien, azitromisien en ofloksasien getoon, terwyl sewe weerstandig was vir tetrasiklien, drie daarvan was ook weerstandig vir doksisiklien. Wat betref ofloksasien weerstandigheid gemik teen kwinoloon weerstandigheids bepalende gebiede, is vervanging van Ser83Leu in ParC gedemonstreer in een intermedier weerstandige Ureaplasma (MIK 4 µml) terwyl en trippel vervanging van Asp112Glu in GyrA saam met Ala125Thr en Ala136Thr in ParC gevind is in ses ander intermedier weerstandige stamme. Geen mutasies is gevind in stamme met MIKs van MICs 1 µg/ml nie. Geeneen van die ureaplasma was weerstandig vir eritromisien/azitromisien nie en geen mutasies is gevind in 23S rRNA operons , L4 of L22 proteine nie. TetM en int- Tn gene is gevind in sewe tetrasiklien weerstandige stamme. 58 Tetrasiklien sensitiewe en .intermediere stamme is getoets, waarvan elf en int-Tn geen gekort het sowel as en groot deel van tetM, terwyl 48 slegs klein dele van TetM bevat het. Die tetM gene van die sewe tetrasiklein-werstandige stamme se geenvolgorde is bepaal en vergelykings is getref teenoor die GenBank volgordes van Neisseria gonorrhoeae, Streptococcus pneumoniae en U. urealyticum. In vyf stamme is gevind dat die tetM geen hoogs mosaiek in struktuur was met areas wat ooreenstem met die in GenBank stamme, en ander areas wat uniek is. In die tetM leier area, is vier ehot spot f herkombinasie areas geidentifiseer wat sekerlik die vorming van die mosaiiek strukture kon beinvloed, asook transposon areas wat geenuitdrukking bepaal. Met karakterisering van die int-Tn gene van die sewe tetrasikleinweerstandlige stamme, was drie tipes teenwoordig waarin transposons vanaf verskillende oorsprong aangedui was, geintegreerd met die ureaplama genome. Resiprokale tetrasiklien weerstandigheids geen oordrag tussen ureaplasma en n. gonorrhoea was nie suksesvol nie. Lae-vlak tetrasiklien weerstandigheid (MIK fs van 4 . 8 µg/ml) is wel suksesvol oorgedra na en U. parvum ontvanger vanaf een U. urealyticum en drie U. parvum ontvangers wat tetM gedra het met MIKs van 16-64 µg/ml. Met die analise van tetM met PKR, kon tetM nie aangetoon word in die transformante nie. Gevolgtrekkings: Die belang van genitale mykoplasma, ureaplasma en C. trachomatis in langtermyn etologie benodig verdere ondersoek, veral in die lig van die sindromiese behandeling regimes wat nie kolonisasie verminder nie. Die hoe prevalensie van P. jiroveci, die teenwoordigheid van M. pneumoniae in gevalle van pneumonie en die aantoning van U. parvum in twee gevalle van neonatale pneumonie benadruk dat, in die afwesigheid van en definitiewe diagnose, dit noodsaaklik is om respons tot behandeling sorgvuldig te moniteer, veral indien die eerste lyn antibiotika keuse ß-laktam antimikrobiale middels of kefalosporiene is, sodat behoorlike en ingeligde gesondheidsorg gelewer kan word. Die bevinding van transposon en/of tetM gebiede in alle ureaplasma wat ondersoek is met of sonder volle uitdrukking van tetrasiklien weerstandigheid, in samehang met tetM diversiteit, plaas verseker ureaplasma sterk in die prentjie vir intra- en inter-generiese uitruiling van antibiotika weerstandigheids gene. / Nelson Mandela Metropolitan University / National Research Foundation (NRF Thuthuka) / Medical Research Council