Browsing by Department "Division of Medical Microbiology"

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    A study of the immune response to human papillomavirus types causing cervical cancer
    (2000) Marais, Dianne Jean; Anna-Lise Williamson
    This study constitutes the first reported assessment in Africa of the antibody responses to human papillomavirus (HPV) proteins and the association of these responses with cervical disease. Immunoglobulin G (IgG) antibodies to HPV antigens were measured by enzymelinked immunosorbent assay (ELISA) in the serum of children, female blood donors, and patients with cervical intraepithelial neoplasia (CIN). A significant association with CIN was found for IgG antibodies to an E2 peptide (E2-16, P = 0.039) and HPV-16 virus-like particles (VLP-16, P = 0.002). This association was found to be age-dependant in women with CIN, with a higher antibody seropositivity rate to E2-16 noted in women over 40 years and a lower antibody seropositivity rate to VLP-16 found in the older women. This implied that in women with CIN, VLP-16 antibodies were markers of CIN in younger women and E2-16 antibodies markers of CIN in older women. A high antibody seroprevalence rate was noted in the children's sera to E2-16 (44.5%), with a decreased seroprevalence in older children. The assessment of seroresponses to VLPs ofHPV types 16, 18, 31, 33 and 45 in the women with cervical cancer (CaCx) as well as women with CIN, blood donors, San people and children is the first study of antibody responses to 5 oncogenic HPV VLP types. The prevalence rate to the 5 HPV VLP types was lowest amongst the children but 27.3% of Cape Town children and 22.6% Df the San children were seropositive to at least one VLP type, as were 58% of all adult women tested. The San adults displayed high seroprevalence rates to · VLP-18 and VLP-45, corroborating other evidence of the high prevalence rates ofHPV-18 and HPV-45 in parts of Africa. The antibody prevalence rates among the CIN patients' sera were elevated above that of blood donors, for all VLP types. VLP antibody prevalence rates in the CaCx patient sera were higher than prevalence rates of the CIN patients for all VLP types, with the exception of antibodies to VLP-16. Association with disease was found for IgG antibodies to VLP-16 and VLP-45 in women with CIN, compared with blood donor controls. This is the first report of an association of antibodies to VLP-45 with cervical disease. Seroprevalence to multiple VLP types was evident in all the groups studied, especially so in the women with cervical disease and was most common in older women with CaCx (55%). XVl The antibody responses appeared type-specific, but some cross reactivity was apparent, although not confirmed. Cross reactivity between antibodies to VLP-16 with antibodies to VLP-31, as well as antibodies to VLP-18 with VLP-45 antibodies appeared likely in some women. Anti-VLP-16 IgA antibody levels in oral fluid of women with CIN and CaCx were compared with those of normal healthy control individuals. Prevalence rates were 55.7% compared with 8% in the controls (P = 0.000002). A group of 26 women with CIN 2 or CIN 3 (CIN2/3) were examined for IgA and IgG antibodies to VLP-16 in serum and cervical mucus as well as oral fluid. The percentage of women with VLP-16 antibodies was highest for serum IgA (65.4%), with 61.5% of these women having detectable serum IgG and 42% and 38.5% IgA and IgG antibodies, respectively in cervical mucus. The serum IgG responses correlated best with the presence of HPV-16 deoxyribonucleic acid (DNA) at the cervix, with an 87.5% IgG antibody prevalence in women with CIN2/3 who were HPV-16 DNA positive. Commercial sex workers, at high risk of cervical disease, were examined for their HPV DNA presence and cervico-vaginal lavage and serum samples from these women were examined for anti-VLP-16 antibodies. Many of these women were infected with human immunodeficiency virus (HIV-positive) and others were not infected with HIV (HIV-negative). The HIV-positive women displayed a significant increase in the prevalence of HPV DNA (85%) compared with HIV-negative women (42%, P = 0.00001) and a significant increase in cervical anti-VLP-16 IgG antibodies (P = 0.002) and a significant decrease in anti-VLP-16 serum IgA (P = 0.012) . compared with HIV-negative women. This was the first published report of cervical antibodies to VLP-16 in HIV-positive women. A novel recombinant vaccinia virus (rVv)-HPV-16 Ll challenge model was developed to evaluate the efficacy of the cell mediated immune response following HPV-16 VLP immunisation in mice. Intra-peritoneal vaccination of BALB/c mice with HPV-16 VLPs afforded protection against viral challenge from rVv expressing HPV 16 Ll (VvLl1z:.-l6). Protection was demonstrated by a 4.6log10 reduction in Vv-LlR-16 ovarian titres in vaccinated BALB/c mice, compared with unvaccinated mice. The inability to infect laboratory animals xvu with HPV has in the past abrogated the testing of HPV vaccines in animal systems. Mice immunised intragastrically with VLP-16 were also protected from VvLlR-16 challenge. Three rBCG constructs expressing different lengths of the L 1 protein of HPV-16 were also tested for their ability to protect mice from VVLlR-16 challenge, and all induced >4log10 levels of protection. The mouse challenge model demonst_rated a quantitative measure of the protection induced by potential HPV vaccines.
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    Antimicrobial susceptibility of Acinetobacter isolates from Groote Schuur Hospital region : an investigation of the appropriateness/validity of the NCCLS zone size criteria
    (2002) Ben-Ismaeil, B I; Roditi, D; Oliver, S
    Since the 1970s, relatively uncommon species of non-enteric gram-negative bacilli have emerged as nosocomial colonizers and pathogens. Among them, one of the most significant genera is Acinetobacter, which has given rise to an increasing number of reports of nosocomial infections. The introduction of new broad-spectrum antimicrobials in the hospitals has been one of the main factors responsible for this development.In addition leading to a move from more susceptible towards more resistant pathogens. This occurred both between and within genera. Owing to the unpredictable antimicrobial susceptibility of Acinetobacter spp., it is prudent to test each isolate for its susceptibility profile to guide in the proper treatment of infections caused by Acinetobacter.The disk diffusion method is the technique most widely used by microbiology laboratories for the routine assessment of antimicrobial susceptibility patterns.The zone diameters obtained by this technique for individual antimicrobials are reported as susceptible, intermediate or resistant by referring to an interpretative chart.Most laboratories use interpretative charts (zone size criteria) supplied by the National Committee for Clinical Laboratory Standards (NCCLS) for this purpose.In our laboratory we have noted discrepancies between sensitivities as determined by the NCCLS zone size criteria and the local minimum inhibitory concentration (MIC) results obtained for Acinetobacter species, especially for cefepime (CPIM) and ceftazidime (CT AZ). Since Acinetobacter species contribute significantly to the increased morbidity and mortality of debilitated patients ( especially v entilated ICU patients, and the fact that clinicians rely on the antimicrobial susceptibility results in the management and treatment of these patients, it was felt necessary to investigate the appropriateness/validity of the NCCLS zone size criteria used to interpret the susceptibility test results of cefepime (CPIM), ceftazidime (CT AZ), trimethoprim-sulfamethoxazole (TMP-SULF A), and piperacillin-tazobactam (PIP-T AZO) against Acinetobacter spp.
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    Antimicrobial susceptibility of organisms causing community-acquired urinary tract infections in Gauteng Province, South Africa
    (2013) Lewis, David A; Gumede, Lindy Y E; Van der Hoven, Louis A; De Gita, Gloria N; De Kock, Elsabe J E; De Lange, Telsa; Maseko, Venessa; Kekana, Valentia; Smuts, Francois P; Perovic, Olga
    BACKGROUND: Patients with community-acquired urinary tract infections (UTIs) frequently present to healthcare facilities in South Africa (SA). AIM: To provide information on UTI aetiology and antimicrobial susceptibility of pathogens. METHODS: We recruited women with UTI-related symptoms, who tested positive for ≥2 urine dipstick criteria (proteinuria, blood, leucocytes or nitrites) at 1 public and 5 private primary healthcare facilities in 2011. Demographic and clinical data were recorded and mid-stream urine (MSU) specimens were cultured. UTI pathogens were Gram-stained and identified to species level. Etest-based antimicrobial susceptibility testing was performed for amoxicillin/clavulanic acid, cefixime, cefuroxime, ciprofloxacin, fosfomycin, levofloxacin, nitrofurantoin, norfloxacin and trimethoprim/sulphamethoxazole. RESULTS: Of the 460 women recruited, 425 MSU samples were processed and 204 UTI pathogens were identified in 201 samples. Most pathogens were Gram-negative bacilli (GNB) (182; 89.2%) and 22 (10.8%) were Gram-positive cocci (GPC). Escherichia coli was the most frequent GNB (160; 79.6%), while Enterococcus faecalis was the predominant GPC (8; 4.0%). The UTI pathogens had similar susceptibility profiles for fosfomycin (95.5%; 95% confidence interval (CI) 92.6 - 98.4), the 3 fluoroquinolones (94.1%; 95% CI 90.8 - 97.4), nitrofurantoin (91.7%; 95% CI 87.8 - 95.6), cefuroxime (90.1%; 95% CI 86.0 - 94.3) and cefixime (88.2%; 95% CI 83.7 - 92.6). UTI pathogens were less susceptible to amoxicillin/clavulanic acid (82.8%; 95% CI 77.5 - 88.0) when compared with fluoroquinolones and fosfomycin. Trimethoprim/ sulphamethoxazole was the least efficacious antimicrobial agent (44.3% susceptible; 95% CI 37.4 - 51.2). CONCLUSION: This study provides relevant data for the empirical treatment of community-acquired UTIs in SA.
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    Antimycobacterial, Cytotoxic, and Antioxidant Activities of Abietane Diterpenoids Isolated from Plectranthus madagascariensis
    (2021-01-19) Ndjoubi, Kadidiatou O; Sharma, Rajan; Badmus, Jelili A; Jacobs, Ayesha; Jordaan, Audrey; Marnewick, Jeanine; Warner, Digby F; Hussein, Ahmed A
    Medicinal plants of the Plectranthus genus (Lamiaceae) are well known for their ethnomedicinal applications. Plectranthus madagascariensis, which is native to South Africa, is traditionally used in the treatment of respiratory conditions, scabies, and cutaneous wounds. The phytochemical studies of P. madagascariensis led to the isolation of five known royleanone abietanes, namely, 6β,7α-dihydroxyroyleanone (1), 7α-acetoxy-6β-hydroxyroyleanone (2), horminone (3), coleon U quinone (4), and carnosolon (5). The relative configuration of compound 2 was established by X-ray analysis. Compounds 1–4 showed antimycobacterial activity (Minimum inhibitory concentration for 90% inhibition, MIC90 = 5.61–179.60 μM) against Mycobacterium tuberculosis H37Rv. Compound 4 and 5 showed comparable toxicity (Concentration for 50% inhibition, IC50 98.49 μM and 79.77 μM) to tamoxifen (IC50 22.00 μg/mL) against HaCaT cells. Compounds 1–5 showed antioxidant activity through single-electron transfer (SET) and/or hydrogen-atom transfer (HAT) with compound 5 being the most active antioxidant agent. Compounds 3 and 5 were isolated for the first time from P. madagascariensis. The observed results suggest P. madagascariensis as an important ethnomedicinal plant and as a promising source of diterpenoids with potential use in the treatment of tuberculosis and psoriasis.
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    The association of IS1133 with an aminoglycoside resistance gene, aacC2a, in Acinetobacter baumannii isolates
    (2007) Jacobson, Rachael Kiera; Segal, Heidi
    Includes bibliographical references (leaves 93-103).
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    Characterisation of promoter sequences in a Capripoxvirus genome
    (1992) Fick, Wilhelmina Christina; Dumbell, K R; van Dijk, A A
    Capripoxviruses are of particular interest as live recombinant vectors for use in the veterinary field, since their host-range is restricted to cattle, goats and sheep. The work presented in this thesis is a preliminary study undertaken on the South African Neethling vaccine strain of lumpy skin disease virus (LSDV). As a departure point towards the eventual identification of strong promoter areas in the 143 kb genome of LSDV, a portion of its genome was cloned. Three methods for purification of LSDV DNA were compared, to determine which yielded the best quality DNA for cloning. DNA extracted directly from infected cells was excessively contaminated with bovine host-DNA, complicating the cloning of LSDV DNA. The use of pulsed field gel electrophoresis solved the contamination problem, by separating viral DNA from bovine DNA. However, insufficient amounts of viral DNA for cloning purposes, could be recovered from the gel. Sufficient amounts of good quality LSDV DNA was obtained by extraction from purified virions. Purified LSDV DNA was digested with various restriction enzymes to identify those which yielded several 4-1 0 kb fragments, for cloning into the Bluescribe plasmid transcription vector. Enrichment for large fragments (8-1 0 kb) was achieved by sucrose density centrifugation. Cloned fragments were analysed by Southern blot hybridisation to verify their viral origin. Hybridisation studies indicated that several unique regions of the LSDV genome were cloned as Pst I and Bam HI fragments respectively, i.e. the cloned fragments contained no overlapping regions. In total, 71.25 kb of the DNA of the LSDV Neethling vaccine strain has been cloned, representing approximately 50% of the viral genome. The availability of these clones now paves the way for further molecular investigations of the LSDV Neethling genome, including identification of promoter regions. A trial gene, which will be cloned and expressed in LSDV, namely the cloned VPS-gene of bluetongue virus serotype 4, was prepared and its nucleotide sequence determined. Homopolymer sequences present at the terminal ends of the gene as a result of the original cloning strategy, are known to interfere with expression and were removed by means of the polymerase chain reaction (PCR). The nucleotide sequence of the resulting PCR-tailored BTV4 VPS-genewas determined and used to deduce the amino acid sequence of the protein. The gene is 1638 bp in length and encodes a protein of 526 aa. Conserved sequences, 6 bp in length and unique to the 5'- and 3'terminal ends of all BTV genes, were detected at the termini of the tailored gene, confirming that the original clone was a full-length copy of the gene. Amplification by PCR did not mutate the open reading frame (OAF) of the gene, since it was of similar length to that reported for 5 other BTV serotypes. With a view to future investigations, including the identification of promoter sequences in the LSDV genome, a preliminary investigation of LSDV protein synthesis was undertaken, to acquire some knowledge of the growth cycle of the virus. Eighteen putative virus-specific proteins were identified by radio-labelling infected cells with [³⁵S]-methionine. By pulse-labelling infected cells with [³⁵S]methionine at various times post infection (p.i.), viral proteins were first detected at 16 hr p.i. It is, however, unlikely that the early phase of viral replication commences as late as 16 hr p.i. and these results might be attributed to various problems, such as the low multiplicity of infection used and that host protein shut-down was inefficient, thus masking the presence viral proteins. In conclusion, this investigation resulted in the cloning of 71,25 kb of the LSDV genome, the tailoring and sequencing of the BTV4 VPS gene and the identification of 18 putative LSDV proteins. This now paves the way for further research to develop LSDV as a vaccine vector.
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    Characterisation of STEC and other diarrheic E. coli isolated on CHROMagar™STEC at a tertiary referral hospital, Cape Town
    (BioMed Central, 2018-06-08) Kalule, John B; Keddy, Karen H; Nicol, Mark P
    Abstract Background Shiga toxin producing E. coli (STEC) is an emerging zoonotic pathogen that can cause acute renal failure, especially in children. Clinical microbiology laboratories may fail to detect STEC and other diarrhoeic E. coli unless purposive rigorous screening procedures are followed using appropriate diagnostic technology; CHROMagar™STEC has rarely been used for isolation of African diarrhoeic E. coli hence characteristics of isolates on this medium are not yet fully understood. This study aimed to determine the prevalence and characteristics of STEC and other diarrhoeic E. coli isolated on CHROMagar™STEC from stool samples submitted to the microbiology laboratory of a South African public sector tertiary care hospital. Results In total, 733 stool samples were tested. Of these, 4.5% (33/733) possessed diarrhoeic E. coli. Of the diarrheic E. coli, 5/33 (15.2%) were STEC, 15/33 (45.5%) EAggEC, 6/33 (18.2%) atypical EPEC, 5/33 (15.2%) typical EPEC, and 1/33 (3%) DAEC. None of the STEC isolates had been identified by routine testing (based on using sorbitol media to test for E. coli O157: H7 strains and not the other STEC) in the laboratory. Of the 33 strains, 55% (95% CI = 40.8–72.7) showed resistance to ampicillin. Conclusions CHROMagar™STEC enabled detection of tellurite - resistant diarrhoeic E. coli that would be missed using routine methods. Further studies are needed to determine the proportion and characteristics of those which might have been missed using this approach.
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    Characterisation of the cold-shock response in Mycobacterium smegmatis
    (1999) Shires, Karen Lesley; Steyn, Lafras M; Zappe, Harold
    The response of Mycobacterium smegmatis to a cold shock was investigated in order to gain insight into the stress responses of members of the genus Mycobacterium. Mycobacterium smegmatis cultures were shocked from 37°C to 30°C, 25°C, 15°C, and 10°C and the effects on both growth (ATP concentration, culture turbidity, colony-forming units) and metabolism (incorporation of ¹⁴C-leucine and ³H-uracil) were investigated. The magnitude of the cold-shock response was found to be dependent upon the degree of the cold shock. A cold shock to 10°C had the greatest effect and resulted in a "lag period" of 24 hours in both the growth and metabolism of the culture. The synthesis of proteins was reduced 20-fold during this period, indicating at block in translation. The cold-shock response in Mycobacterium smegmatis was an adaptive response with growth eventually being resumed at the colder temperature, but at a reduced rate. Using the techniques of one-dimensional sodium-dodecyl-sulphate polyacrylamide gel electrophoresis and two-dimensional protein gel electrophoresis, ³⁵S-methiononine-labelled proteins that were synthesised during the cold shock were analysed. At least fourteen radio-labelled proteins were induced during the first 24-hour period and these demonstrated two distinct patterns of cold-shock induced expression: transient and continuous. Depending upon the pattern of expression and size, the cold-shock proteins were classified as "cold-induced proteins", "cold-shock proteins" or "cold-acclimation proteins". CipM, a 27kDa protein, was identified as the major cold-shock protein through one-dimensional protein electrophoresis. From N-terminal sequence data generated from a protein (CipM.1) within this band, a corresponding degenerate DNA probe was used to isolate cipM.1. This gene was cold-inducible, with mRNA levels transiently increasing 5-7 fold after a 37°C to 10°c cold-shock. Homologues of this cold-shock gene are found in the genomes of Mycobacterium tuberculosis and Mycobacterium leprae. The corresponding mycobacterial proteins showed homology at the N-terminus to the HU~ subunit of HU of Escherichia coli and possessed similar C-terminal praline, lysine and alanine degenerate repeats to the mycobacterial heparin-binding hemagglutinin. The response of several mycobacterial cold-shock gene homologues to a cold shock was also investigated, by northern-hybridisation and S1 nuclease analysis. The cspA homologue of Mycobacterium smegmatis demonstrated a 16-24 fold transient induction in mRNA levels following a 37°C to 10°C temperature-shift, while gyrA mRNA levels were maintained at a constant level throughout the cold shock. Although some similarities were demonstrated between the cold-shock response of Escherichia coli and Mycobacterium smegmatis, definite differences occur in the proteins that are involved in the adaptive stages of the response.
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    Chronic otorrhoea: Spectrum of microorganisms and antibiotic sensitivity in a South African cohort
    (2013) Meyer, E; Whitelaw, A; Edkins, O; Fagan, J J
    BACKGROUND: Chronic otorrhoea is difficult to treat, with treatment in South Africa (SA) being protocol driven and generally initiated at the primary healthcare level. There is a lack of local studies that focus on the bacteriology and antimicrobial sensitivities of chronic otorrhoea, which underpins the management advice offered. AIMS: To determine the microbiological profile and antimicrobial susceptibility of patients with chronic otorrhoea and the validity of the Department of Health's (DoH) current guideline. METHODS: We conducted a prospective study at Groote Schuur Hospital from 2005 to 2009. We included patients with chronic otorrhoea classified as either otitis media or otitis externa, according to our definitions. Pus swabs were taken, from which microorganisms were cultured and tested for antimicrobial susceptibility. RESULTS: Of 79 patients with otorrhoea, 50 had otitis media, 21 had otitis externa and the condition was not determined in 8 patients. The most common organism isolated with otitis media was Proteus mirabilis (18/50; 36%) and with otitis externa, Pseudomonas aeruginosa (7/21; 33%). Otorrhoea had a different microbial spectrum compared with international reports, with methicillin-resistant Staphylococcus aureus infection in a single patient. The organisms isolated were susceptible mainly to fluoroquinolones (96%) and aminoglycosides (81%). CONCLUSION: Amoxicillin is a poor choice of antibiotic due to its low sensitivity, which calls into question the current DoH guideline for otorrhoea. Antimicrobial treatment protocols should be based on local data and be revisited from time to time. This study suggests that, should first-line treatment fail, an antibiotic with Gram-negative cover, e.g. a topical fluoroquinolone, should be considered.
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    Cleavage of the moaX-encoded fused molybdopterin synthase from Mycobacterium tuberculosis is necessary for activity
    (BioMed Central, 2015-02-06) Narrandes, Nicole C; Machowski, Edith E; Mizrahi, Valerie; Kana, Bavesh D
    Background: Molybdopterin cofactor (MoCo) biosynthesis in Mycobacterium tuberculosis is associated with a multiplicity of genes encoding several enzymes in the pathway, including the molybdopterin (MPT) synthase, a hetero tetramer comprising two MoaD and two MoaE subunits. In addition to moaD1, moaD2, moaE1, moaE2, the M. tuberculosis genome also contains a moaX gene which encodes an MPT-synthase in which the MoaD and MoaE domains are located on a single polypeptide. In this study, we assessed the requirement for post-translational cleavage of MoaX for functionality of this novel, fused MPT synthase and attempted to establish a functional hierarchy for the various MPT-synthase encoding genes in M. tuberculosis. Results: Using a heterologous Mycobacterium smegmatis host and the activity of the MoCo-dependent nitrate reductase, we confirmed that moaD2 and moaE2 from M. tuberculosis together encode a functional MPT synthase. In contrast, moaD1 displayed no functionality in this system, even in the presence of the MoeBR sulphurtransferase, which contains the rhodansese-like domain, predicted to activate MoaD subunits. We demonstrated that cleavage of MoaX into its constituent MoaD and MoaE subunits was required for MPT synthase activity and confirmed that cleavage occurs between the Gly82 and Ser83 residues in MoaX. Further analysis of the Gly81-Gly82 motif confirmed that both of these residues are necessary for catalysis and that the Gly81 was required for recognition/cleavage of MoaX by an as yet unidentified protease. In addition, the MoaE component of MoaX was able to function in conjunction with M. smegmatis MoaD2 suggesting that cleavage of MoaX renders functionally interchangeable subunits. Expression of MoaX in E. coli revealed that incorrect post-translational processing is responsible for the lack of activity of MoaX in this heterologous host. Conclusions: There is a degree of functional interchangeability between the MPT synthase subunits of M. tuberculosis. In the case of MoaX, post-translational cleavage at the Gly82 residue is required for function.
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    Conditions affecting ergothioneine levels in Mycobacterium smegmatis & the attempted isolation of α-N, N, N-Histidine methyltransferase, the first enzyme in ergothioneine biosynthesis
    (2007) Williams, Monique J; Steyn, Lafras M; Steenkamp, D J
    Ergothioneine and mycothiol are the two major low molecular weight thiols present in mycobacteria. The generation of mycothiol-deficient mutants has demonstrated its role in protecting M tuberculosis against oxidative and nitrosative stress. To date, no ergothioneine-deficient mutants have been identified and the role of ergothioneine in mycobacteria remains unknown. The work in this thesis was performed with the aim of better understanding the function of ergothioneine in mycobacteria, by studying its biosynthesis and the conditions affecting its production.
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    CRISPRI-based high-throughput functional genomic approaches for use in mycobacteria
    (2021) De Wet, Timothy; Warner, Digby
    In the 20 years since the pioneering publication of the genome of Mycobacterium tuberculosis, significant efforts have been made to complete functional annotation of the genome. However, these efforts have generally been performed on a single-gene basis, ensuring slow progress and leaving large portions of the genome unannotated. High-throughput approaches to understanding the functional genome, such as transposon-insertion sequencing, have been developed and applied to mycobacteria in a variety of conditions; however, they have several limitations, particularly in their ability to study genes essential for viability. The recent optimisation of inducible CRISPR-interference for mycobacteria offers the potential to expand the high-throughput functional genomic toolkit. This thesis utilises CRISPR-interference for the development and validation of two high-throughput functional genomic approaches in the model mycobacterium M. smegmatis. The first approach combines large-scale pooled oligonucleotide synthesis and nextgeneration sequencing, and is termed CRISPRi-Seq. A pooled library of 11 367 mutants, targeting 2 385 M. smegmatis genes with M. tuberculosis homologues, was constructed and used to infer gene essentialities which were compared with corresponding predictions from transposon-insertion sequencing data. This process validated the CRISPRi-Seq technique and identified practical considerations for its future use. The second approach utilises data derived from CRISPRi-Seq to create an arrayed library of 263 individual M. smegmatis inducible CRISPRi mutants targeting essential genes. This library is applied to a quantitative imaging pipeline to produce detailed data-driven profiles of the morphological impact of essential gene suppression. These morphological profiles are used to statistically predict genetic function, as well as antimicrobial mechanism-of-action. The two novel approaches developed in this work represent valuable technical advances and produce large datasets of functional genomic data which are available interactively online. Taken individually, or in combination, these methodologies can be utilised to increase fundamental understanding of mycobacteria, including the pathogenic M. tuberculosis
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    Detection of mixed Mycobacterium tuberculosis infections in South African TB Patients
    (2009) Stead, Michael Craig; Evans, Joanna; Grewal, Harleen
    Recently, the widely accepted theory that TB disease resulted from one infecting M. tuberculosis strain leading to heightened immune protection against subsequent infections, has been revised. Epidemiological studies and the advances in molecular genotyping techniques have highlighted the rapidly frequent isolation of several different M. tuberculosis strain lineages in single disease episodes, often with differing drug susceptibilities. This has important implications on drug susceptibility testing and the treatment of patients. It has also highlighted the relative contributions of exogenous reinfection and endogenous reactivation in TB disease progression. However, our understanding of the nature and frequency of mixed infections is lacking. This study investigated the frequency and detection of mixed TB infections in the Delft region of the Western Cape, as part of a larger clinical trial on the effects of multi-nutrient supplementation and standard treatment on the TB bacteriological response. Newly diagnosed, adult TB patients (n=154) produced a single weekly sputum sample over an 8-week period. Genomic DNA was extracted from colonies grown from MGIT cultures on LJ slopes. Spoligotyping was used as an initial screen to detect mixed infections as well as to assess the epidemiology of M. tuberculosis in these serial isolates (n=686). In addition, clonal relatedness of the isolates was assessed by MIRU-VNTR analysis. Thereafter PCR assays to detect infections of W-Beijing and non-W-Beijing isolates, as well as to differentiate mixed non-W-Beijing isolates were carried out. Phenotypic and genotypic drug susceptibility was carried out. Spoligotyping indicated that W-Beijing isolates constituted a large proportion (47.8%) of circulating M. tuberculosis in this region, with other strains detected including LAM (17.1%), T (14.7%), X (6.4%), H (7.9%), S (4.3%), and F33 (2.1%) strains. Using both spoligotyping and PCR assays, mixed infections were detected 21 (16.3%) of 129 patients screened. Phenotypic and genotypic DST confirmed that all isolates identified in patients as harbouring mixed strains by spoligotyping were fully susceptible to both RIF and INH. MIRU-VNTR 2 analysis for genetic relatedness identified 1 clonal cluster in the mixed samples identified by spoligotyping, consisting of the T1, T4, W-Beijing and W-Beijing + X3 isolates. The frequency of mixed infections, particularly in high disease burdened areas, is high, and warrants further attention. This finding has great implications with regards to the interpretation of epidemiological and DST data, and the subsequent treatment of patients.
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    Development of novel anti-tuberculosis drugs from African medicinal plants
    (2006) Ngwane, Andile H; Steyn, Lafras M; Skepu, Zoleka
    Includes bibliographical references (leaves 55-63).
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    Disabling the intrinsic resistome of Mycobacterium tuberculosis: elucidating hierarchies of DNA repair and mutagenesis that undermine current antibiotic efficacy
    (2021) Gobe, Irene; Warner, Digby; Mizrahi, Valerie; Iorger, Thomas R
    DNA damage repair mechanisms are critical to the adaptive evolution of Mycobacterium tuberculosis as obligate human pathogen, including the emergence of drug-resistance during anti-tuberculosis (TB) chemotherapy. In experimental models, DnaE2-dependent translesion synthesis (TLS) and UvrB-dependent nucleotide excision repair (NER) have been identified as major mediators of DNA damage tolerance and repair, respectively. Given the inferred dominance of these pathways, this thesis aimed to elucidate otherwise cryptic repair mechanisms which might buffer loss of DnaE2 and UvrB in bacilli exposed to genotoxic stress. Using dnaE2 and uvrB deletion mutants of the model mycobacterium, M. smegmatis (MSM) mc2 155, we applied genome-wide transposon (Tn) mutagenesis to identify conditionally essential repair pathways under treatment with genotoxins of different mechanistic classes. To this end, the DNA crosslinking agent, mitomycin C (MMC), and the gyrase inhibitor and clinically relevant TB drug, moxifloxacin (MOX), were used. The goal was to reveal potential targets for co-drugs that might shorten treatment duration and reduce the risk of drug resistance by severely limiting the intrinsic capacity of MTB to tolerate lethal drugs for extended periods. Among others, our analysis identified GlgB, which is involved in glycogen biosynthesis, and mycothiol biosynthesis proteins, MSMEG_0933 and MSMEG_5261, as compensating the absence of UvrB during MMC treatment. Under MOX treatment, the absence of UvrB was compensated by the RecC/Single-strand Annealing pathway. In contrast, DnaE2 deficiency revealed the conditional essentiality of the PadR family transcriptional regulator, MSMEG_2868, under MMC exposure. Importantly, in all cases, results from the Tn screen were validated using CRISPR interference targeting the identified genes. Of particular interest, we observed that UvrB was essential to compensate loss of DnaE2, whereas the reciprocal was less definitive: while DnaE2 appeared dispensable in MMC-treated uvrB, Tn analyses suggested that dnaE2 might be essential in the untreated ∆uvrB mutant. This result, which is consistent with very recent results suggesting the co-ordination of NER and DnaE2 functions in Caulobacter crescentus, is intriguing in potentially revealing a previously unappreciated role for DnaE2 in mycobacterial NER function. Taken together, these results support the utility of Tn-based whole-genome screens in revealing unexpected genegene interaction networks, and provide additional impetus to explore ancillary, non-essential metabolic functions as alternative targets for novel combination therapies designed to cripple intrinsic mechanisms of mycobacterial resistance.
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    The epidemiology & molecular basis of fluoroquinolone resistant & susceptible isolates of Campylobacter coli
    (2001) Cooper, Rhett; Elisha, B Gay; Lastovica, Albert
    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.
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    Evolution of sensory neuropathy after initiation of antiretroviral therapy
    (2018) Centner, Chad; Heckmann, Jeannine M
    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.