Bacterial resistance to antibiotics is a common and important clinical problem. Beta lactam resistance in Gram negative bacilli is mediated predominantly by beta lactamases, enzymes able to hydrolyse the beta lactam ring. The commonest plasmid mediated beta lactamases in the Enterobacteriaceae are those related to either TEM-1 or SHY-1. Although TEM-1, TEM-2 and SHY-1 do not have activity against extended spectrum beta lactams, their derivatives (TEM-3 and SHY-2 onwards) are able to confer resistance to one or more of these antibiotics. A problem encountered in clinical microbiology laboratories is the lack of a reliable method for the detection of ESBLs, along with the lack of a quick, reliable method of differentiating TEM-related genes from SHY -related genes. The primary aim of this study was to evaluate two molecular techniques for the detection of SHY and TEM-related genes in clinical isolates. The study sample consisted of 209 clinical isolates of enteric Gram negative bacilli, isolated at Groote Schuur Hospital microbiology laboratory. The isolates had all been selected on the basis of resistance to one or more of the extended spectrum beta lactams. These isolates were all identified, and the susceptibility of each to a variety of beta lactam antibiotics determined. Using this information, 45 isolates, belonging to different genera and with differing antimicrobial sensitivity patterns, were selected for this pilot study. These 45 isolates consisted of 24 Klebsiella spp., 14 Enterobacter spp., 3 Citrobacter spp., 2 Salmonella spp., 1 Pantoea agglomerans and 1 Serratia marcescens.

Introduction: The indoor home environment comprises many niches that are occupied by bacterial communities. The composition of these bacterial communities may be influenced by numerous factors such as number of occupants, pets, season and location. Understanding the house dust microbial community is vital to understanding its' influence on human respiratory health. Aims: The aims of the studies described in this MSc dissertation were to: 1) evaluate the performance of ten commercial nucleic acid extraction kits on dust samples; 2) optimise dust removal from electrostatic dustfall collectors (EDC); 3) determine the bacterial composition of house dust using 16S rRNA gene sequencing and 4) determine those factors influencing the bacterial composition of house dust by performing bioinformatic and data analysis on the sequenced dust samples. Methods: In order to study the microbial content of house dust, an efficient DNA extraction protocol was required. Ten commercial nucleic acid purification protocols were evaluated on their ability to efficiently extract good quality DNA from very low quantities (20 mg) of wet bulk house dust. For the purpose of this study, EDCs were used to collect settled dust from homes of participants in the Drakenstein Child Health Study (DCHS). Electrostatic Dustfall Collectors were placed twice within the same household, approximately 6 months apart, spanning two seasons. The Z/R Fungal/Bacterial DNA MicroprepTM (ZMC) protocol was used to extract DNA from dust removed from EDCs. The V4 region of the 16S rRNA gene was amplified and sequenced using the Illumina MiSeq platform to determine the bacterial taxonomic composition of the house dust samples. A custom python wrapper that meshes a set of tools integrated into a computationally efficient workflow, known as the YAP pipeline was used to classify 16S rRNA sequences into bacterial taxonomies. Based on 97% sequence similarity, the pre-processed sequences were assigned to Operational Taxonomic Units (OTU). R software together with RStudio software was used for all statistical analysis and graphical representations of the data.

With conventional methods of diagnosis, substantial overlaps are common due to an absence of the expected CSF findings clearly aligning to bacterial or viral infection. Reduced sensitivity is commonly observed chiefly due to empiric antibiotic treatment leading to bacterial culture negative results. This leads to costly hospital admissions and unnecessarily prolonged treatment for the unexplained aetiology, further compounded by routine viral diagnostics not being commonly implemented for meningitis diagnosis. We developed and validated in-house quantitative real-time (qPCR) multiplex assays to test for bacterial causes namely: Neisseria meningitidis (ctrA gene), Haemophilus influenzae (hpd gene) and Streptococcus pneumoniae (lytA gene) and viral causes namely: enterovirus (5' UTR), herpes simplex (UL30 gene) and mumps virus (Fusion protein gene). The qPCR assays were carried out on the Biorad CFX 96 real-time instrument. These validated assays were used to screen a cohort of suspected meningitis cases. The retrospective study included 300 paediatric patients aged from 60 days-12 years, over a 1-year period (November1, 2012 to November 30, 2013) with suspected meningitis presented to the outpatient departments of the Red Cross War Memorial Children‟s Hospital (RCCH) in Cape Town. Cerebrospinal fluid with abnormal chemistry and cell counts was selected and total nucleic acid was extracted with the QIAsymphony virus/bacterial DSP kit (QIAGEN, Valencia, CA). The median age of children was 19 months (IQR: 6-65 months). Among the screened 291 CSF samples, 7 (2.4%) cases Gram stain results were obtained along with relatively few cases with positive bacterial culture growth 4/291 (1.4%). Based on bacterial qPCR results, 8 (2.7%), 3 (1%) and 1 (0.3%) were positive for S. pneumoniae, N. meningitidis and H. influenzae respectively. A majority of cases were viral positives with enteroviruses being the dominant at 91/291 (31.3%) and mumps virus 3/291 (1%). No herpes simplex DNA was detected. The bacterial qPCR showed a sensitivity and specificity of 85.7% and 97.7% respectively when compared against a composite reference standard (CRS). We report an improvement with additional detected causes of bacterial meningitis and highlight the burden of the common viral causes. However, a large proportion of cases (63.6 %) have aetiology still unknown. PCR shows valuable in concluding viral aetiology in routine diagnosis.

Background: Meconium microbiota have recently gained great interest; however very few studies have included meconium specimens when longitudinally characterizing the infant GIT microbiota. This study therefore aimed to longitudinally characterize meconium microbiota profiles during the first seven months of life and to compare these profiles with those from maternal faecal specimens using quality controlled Illumina MiSeq sequencing data. Methods: We sampled infant meconium and maternal faecal specimens at birth, as well as two subsets of infant faecal specimens at 4-12 and 20-28 weeks of life. We extracted nucleic acid from faecal specimens using the automated QIAsymphony ® SP instrument. Using Illumina MiSeq technology, we sequenced the V4 region of the bacterial 16S rRNA gene. We determined whether sufficient reads were sequenced using accumulation curves; whether any contamination occurred; and whether our sequencing approach was reproducible. The relative abundances of taxonomically classified operational taxonomic units (OTUs), and the Shannon diversity and Bray Curtis dissimilarity indices served to characterize faecal specimens from participants. Log ratio biplots and generalized linear mixed models served to statistically determine differences between faecal bacterial profiles. Results: Faecal specimens were collected from 90 mothers and 107 infants at birth, 72 infants at 4-12 and 36 infants at 20 28 weeks of age. We classified OTUs from two non-template controls which were indicative of potential contamination. Correcting for contamination resulted in a loss of 10 % of OTUs classified. Our reproducibility analysis correlated with increased concentrations of template used during library preparation. Based on diversity measures, meconium specimens harboured the most diverse bacterial profiles. The highest proportions of OTUs classified from meconium belonged to the phylum Proteobacteria (60 %), while the phylum Firmicutes was most abundant at 4-12 weeks (49 %) and 20-28 weeks (64 %) of life. The phylum Actinobacteria was at its highest at 4-12 weeks of age (26 %) and its increased proportions were associated with breastfeeding at 6-10 weeks of life. Firmicutes constituted the majority (79 %) of bacteria from maternal faecal specimens. No mother- infant pairs clustered at any of the time points studied, but infant bacterial profiles became more adult-like with increased age. An increase in infant age significantly affected bacterial proportions of 87 OTUs. Interestingly, we observed that infants exposed to HIV had higher proportions of the genus Leuconostoc and higher diversity indices compared to HIV unexposed infants at 4-12 weeks of age. Conclusion: Our study highlights that reproducibility may be worsened by the use of low template concentrations during library preparation, which may also skew diversity measures. We conclude that meconium is not sterile and that infant faecal bacterial profiles become more adult-like with increased age.

There is a clear need for a rapid, inexpensive point-of-care diagnostic test for tuberculosis (TB). The aim of this study was to analyze the performance of a novel rapid diagnostic test for TB, GeneXpert MTB/RIF, in symptomatic adults and to compare it to other commercially available nucleic acid amplification assays and to standard microbiological smear and culture. The GeneXpert system performs real time, nested PCR from sputum and provides a result within two hours of sampling. The result includes a semi-quantitative assessment of bacillary load in the sample and simultaneously detects rifampicin resistance. This study was part of a cross-sectional, multi-centre clinical trial. The Cape Town component of this study was conducted at three sites, one hospital based and other two community clinics, all with high TB/HIV coinfection rate. Among 43.2% of patients diagnosed with TB during the evaluation study, GeneXpert detected TB in 95.5% of all culture positive cases. In smear positive patients, sensitivity was 99.0% and in smear-negative, culture positive patients, 86.1%. Specificity in patients who were culture negative and clinically diagnosed as non-TB after follow up was 98.4%. Sensitivity and specificity of GeneXpert in detecting rifampicin resistance was 100% comparing to phenotypically detected drug resistance. GeneXpert is a highly promising novel tool for the rapid diagnosis of adult TB. Future studies are needed to establish the performance and impact of GeneXpert when performed at the level of the microscopy centre.

Includes bibliographical references (leaves 60-78).

Acinetobacter baumannii is responsible for an increasing number of nosocomial infections in patients receiving intensive care and comprehensive antibiotic resistance of these organisms hampers treatment of infections due to A.baumannii. The molecular basis of antibiotic resistance in A.baumannii has not been extensively investigated. A few studies have demonstrated the role of plasmids and transposons in resistance in this organism, but there is little data on the role of integrons and integron-associated antibiotic resistance. This study was undertaken to determine the incidence of integrons in clinical isolates of Acinetobacter from Groote Schuur Hospital (GSH), Cape Town and Universitas Hospital (UH), Bloemfontein, and to characterise the resistance genes carried in the variable regions of these integrons.

Fluoroquinolone susceptible and resistant Campylobacter coli were isolated from pigs on two separate pig farms. C. coli are enteric pathogens of humans and animals and although diarrhoea resulting from C. coli and C. jejuni is generally a self-limiting disease, in severe cases, fluoroquinolones are the choice antibiotic for treatment. The presence of fluoroquinolone resistant C. coli strains in the food chain is cause for concern as this may be a source of resistant strains in humans. Sixty-one isolates were included in the study: 26 were susceptible to nalidixic acid and ciprofloxacin and 35 were resistant to these antibiotics. Fifty-five strains were obtained from pigs on farm A, while 6 strains were obtained from pigs on farm B, the source farm of pigs to farm A. Serotyping and flaA typing were carried out to study the epidemiology of the isolates. Serotyping identified 0:24 (11/61) as the most frequent serotype isolated, followed by 0:5 (7/61). Common serotypes 0:48, 0:54 and 0:59 were identified in strains from both farms. A high number of the strains were non-typeable (23/61) but were distinguished by flaA typing. RFLP analysis of the flaA gene revealed 13 distinct profiles in strains from farm A, and 4 profiles in strains from farm B, of which only 1 was unique to farm B. Profile 1 was the commonest profile observed with 31 % (17 /55) of flaA typed strains in this profile. There was an association between 0:24, profile 6, and resistance. Resistant and sensitive pairs were isolated from 15 pigs; flaA profiles of each of 4 pairs were identical, suggesting selection of resistant mutants from previously sensitive populations. An investigation of the molecular basis of the fluoroquinolone resistance identified a Thr-86 to Ile mutation in GyrA, the primary target of these antibiotics.

Introduction: We studied the evolution of sensory neuropathy after antiretroviral therapy (ART) in human immunodeficiency virus–infected South Africans. Methods: Enrolment commenced before ART with 6-monthly follow-ups for 24 months. Symptomatic distal sensory polyneuropathy (SDSP) was defined as one symptom and sign. Symptom/sign scores were compared between visits. Results: We enrolled 184 participants. Pre-ART, 16% had SDSP. After 18 months of ART, pain prevalence decreased in those with pre-ART SDSP (odds ratio [OR], 0.09; 95% confidence interval [95%CI], 0.03-0.29). Symptoms improved in 50% ever experiencing pain (mean improvement=-4.5 on 11-point scale). Participants SDSP-free pre-ART developed SDSP at a rate of 18 per 100 person-years. After 24 months, 18% had SDSP. Stavudine (60% of cohort) did not predict incident SDSP, but associated with increased prevalence of reduced/absent reflexes at 18 months (OR, 2.24; 95% CI, 1.08-4.65). Conclusions: Painful symptoms improved during ART. Evolving sensory neuropathy was due to increasing small and large fiber dysfunction.

Includes bibliographical references (leaves 85-103). / T helper (Th) 17 cells have recently been implicated in regulating gut mucosal immunity during HIV infection by sustaining gut mucosal barrier integrity, although they do not respond to HIV directly. Depletion of Th17 cells from the gut mucosa during HIV infection has been suggested to contribute to elevated microbial translocation and immune activation. The role of Th17 cells in regulating genital mucosal immunity during HIV infection is less well described. The aims of this study were (1) to compare the frequency and phenotype of Th17 cells in the female genital tract and blood in uninfected compared to HIV-infected women; and (2) to investigate the role of inflammatory/regulatory cytokines and bacterial burden in modulating Th17 cell frequencies in genital secretions and plasma.