Medical Microbiology
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- ItemAspects of the pharmacokinetics of hypoxoside in rodents(Stellenbosch : Stellenbosch University, 1993) Bester, Christoffel Carolus; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Medical Microbiology.ENGLISH ABSTRACT: Certain aspects of the pharmacokinetics of a novel diglucoside, hypoxoside, were investigated in mice and rats. After intragastric dosage of the mouse, hypoxoside was hydrolysed to its aglycone, rooperol. Most enzyme activity occurred in the mouse caecum with intestinal bacteria playing a major role in producing the hydrolytic enzymes. Caecalase extracts hydrolysed hypoxoside in vitro but this activity ceased after the mice were treated with antibiotics, suggesting bacteria as the enzyme source. However, in vivo tests with similar antibiotic-treated mice seemed to demonstrate a minor second, possibly mucosal source of hypoxoside hydrolysing B-glucosidase. An attempt to isolate a single strain of faecal bacteria capable of this activity was unsuccessful although hypoxoside was hydrolysed by pure cultures of some non-intestinal clinical isolates
- ItemBeta-lactam resistance mechanisms in Enterobacter species isolates from Tygerberg Hospital.(Stellenbosch : Stellenbosch University, 2018-10-31) Okyere, Dora; Newton-Foot, Mae; Stellenbosch University. Faculty of Health Sciences. Dept. of Pathology. Medical Microbiology.Background Resistance to carbapenem antibiotics in Gram-negative bacteria is a major public health problem due to limited treatment options. In Enterobacter species, carbapenemase production and reduced membrane permeability, resulting from reduced expression of outer membrane proteins OmpF and OmpC, in combination with extended-spectrum β-lactamase (ESBL) production, hyper-production of chromosomal blaAmpC β-lactamases and / or over-expression of the AcrAB/TolC efflux pump have been speculated to promote carbapenem resistance. This study investigated the mechanisms that promote ertapenem resistance in clinical Enterobacter cloacae isolates from Tygerberg Hospital. Materials and methods Twenty ertapenem non-susceptible clinical E. cloacae isolates, four ertapenem susceptible controls and five wild-type controls were selected based on the VITEK2®AES. Ertapenem MICs were determined using broth microdilution (BMD) and gradient diffusion. Resistance mechanisms were characterized using the phenotypic assays VITEK2®AES, Mastdiscs D68C, D69C and D63C combination sets, Rapidec® Carba NP kit and a synergy assay using disc diffusion in the presence of an efflux pump inhibitor (EPI). Molecular assays included multiplex PCR to identify carbapenemases, multiplex PCR and sequencing to identify ESBLs, SDS-PAGE to characterize OmpC and OmpF abundance and RT-qPCR to quantify expression of blaAmpC, ompC, ompF and acrB. Results and Discussion Seventeen (85%) ertapenem non-susceptible isolates were confirmed non-susceptible by BMD and six (30%) by gradient diffusion, suggesting possible undercalling of ertapenem resistance by gradient diffusion and overcalling by VITEK2®AES. Seven ertapenem non-susceptible isolates were predicted to be carbapenemase producers by the VITEK2®AES and one by the Rapidec® Carba NP kit, however no carbapenemases were detected by PCR. Nineteen ESBL producers were identified by the VITEK2®AES, eight by the D68C combination set and eleven by the D63C combination set. ESBLs were detected in 12 (60%) isolates by PCR and sequencing; of which eight were blaCTX-M, three were blaSHV-12 and one isolate contained both genes. Twelve (60%) ertapenem non-susceptible isolates were predicted to be derepressed blaAmpC producers by the VITEK2®AES, thirteen (65%) by the D68C combination set and six (30%) by the blaAmpC RT-qPCR.
- ItemThe carriage of antibiotic resistant Gram-negative organisms in children in the Cape Town community and the impact of antibiotic exposure on the development of resistance (a TB-CHAMP sub-study)(Stellenbosch : Stellenbosch University, 2019-12) Ocloo, Remous; Whitelaw, Andrew; Newton-Foot, Mae; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology. Medical Microbiology.Introduction Antibiotic resistance has become a major issue across the globe and the situation is worsening in low- and middle-income countries. In sub-Saharan Africa and the world at large, antibiotic resistance research is localized and focused on hospitalized individuals. There is, therefore, little or no data on antibiotic resistance in the community; especially in children. This study described the carriage of resistant isolates in children in Cape Town and investigated the effects of antibiotic exposure on the development of resistance in stool using an in-vitro model. Materials and Methods Stool samples from fifty participants of the Tuberculosis Child Multidrug-resistant Preventive Therapy Trial (TB-CHAMP) were cultured onto McConkey agar (MCC) with the addition of ertapenem and cefpodoxime discs to select for carbapenem and cephalosporin-resistant and susceptible E. coli and Klebsiella isolates. Antibiotic susceptibility testing was performed using Kirby Bauer disk diffusion. Carbapenem-, quinolone- and cephalosporin-resistance genes were detected by PCR and resistance-conferring mutations were detected using Sanger sequencing. Ten stool samples were exposed to two sub-clinical concentrations of amoxicillin, ciprofloxacin and colistin for 48 hours, whereafter they were plated onto MCC with the addition of various antibiotic discs (amoxicillin, ertapenem, ciprofloxacin, colistin, cefotaxime and nalidixic acid). The impact of antibiotic exposure on the development of resistance was assessed by enumeration of presumptive resistant E. coli and Klebsiella colonies within the zones of inhibition around the antibiotic discs. Results Twenty-one (42%) of the participants were colonized by quinolone-resistant isolates and 18 (36%) by cephalosporin-resistant isolates (predicted ESBL-producing organisms). Of the 21 quinolone-resistant E. coli isolates, 5 (24%) harbored qnrS while of the 6 quinolone-resistant Klebsiella isolates, 4 (67%) had qnrB. The most common quinolone resistance mutations were S83L in gyrA and S80I, A141V and S129A in parC. blaCTX-M was the only ESBL gene detected. All of the blaSHV and blaTEM genes detected were β-lactamases without extended spectrum activity. One of the participants was colonized by a carbapenem resistant Klebsiella isolate, which carried the blaNDM carbapenemase gene. Exposure of the stool samples to ciprofloxacin selected for resistant bacteria, however exposure to amoxicillin and colistin did not. Conclusion Children in Cape Town are frequently colonized by resistant bacteria and are at risk of becoming infected by these resistant organisms. The presence of plasmid-mediated resistance genes is concerning because they can be transferred between bacteria of the same and different species. There is also a need to further investigate what might be driving the high prevalence of quinolone resistance in the community. This study is the first to report the carriage of carbapenemase resistant bacteria in healthy children in South Africa. Although the in-vitro antibiotic exposure model was crude, the approach provides some evidence for the development of resistance during exposure to sub-clinical concentrations of antibiotics (especially ciprofloxacin); and notably, to agents other than those to which the sample had been exposed. This highlights the need for further investigations into the impact of sublethal antibiotic concentrations on the selection of resistance.
- ItemThe carriage of antimicrobial resistance genes in children from Cape Town communities.(Stellenbosch : Stellenbosch University, 2022-12) Venter, Amanda-Jo; Newton-Foot, Mae; Whitelaw, Andrew; Stellenbosch University. Faculty of Science. Dept. Department of Pathology. Medical Microbiology.ENGLISH ABSTRACT: Introduction: The human microbiota is a rich source of microbial diversity and an accessible reservoir of antibiotic resistance genes (ARGs). The collection of ARGs present in the microbiota has been termed the resistome. Antibiotic use is a major driver of antibiotic resistance (ABR) and selection of ARGs. There is limited surveillance data on ABR in undercharacterized populations, particularly in children, in the community and in developing countries. A collection of stool specimens collected from healthy children as part of the Tuberculosis Child Multidrug-resistant Preventive Therapy Trial, which evaluates the efficacy of levofloxacin as prophylaxis in multidrug-resistant tuberculosis exposed children, provides an opportunity to describe the carriage of ARGs in a population of healthy children in the community. Methods: Fifty baseline stool samples from children in Cape Town communities that are part of the TB CHAMP trail, collected from November 2017 to July 2018, were used in this substudy for resistome analysis. Both targeted and functional metagenomics (FM) approaches were used to investigate the resistome. The blaCTX-M-1, -9 and -B (2, -8 and -25) genes were detected with real-time PCR in fifty stool samples. Conventional PCR was used to identify blaSHV and blaTEM genes. A FM approach was developed to detect ARGs from stool samples. DNA was extracted from five stool samples, fragmented and end repaired. Fragmented DNA between 1500 - 8000 bp was selected and ligated into the Eco53KI site in plasmid pHSG298, a plasmid vector with kanamycin as selectable marker. The ligation mix was transformed into chemically competent DH5α Escherichia coli cells, blue-white colony screening was performed, and all white colonies were stored, creating a metagenomic library from each stool sample. The FM libraries were plated on selected antibiotics and Sanger sequencing was used to identify the inserts from resistant colonies. Results: blaCTX-M-1 (72%) was present in more samples than blaCTX-M-9 (8%). SHV and TEM were present in 64% and 100% of the samples, respectively. The FM libraries created ranged from 1.7 x 106 to 2.5 x 107 bp in size. Resistant colonies were detected on media containing ampicillin, amikacin and cefotaxime. Sequencing results identified known ARGs, including an aminoglycoside adenylyl transferase gene selected on the amikacin-containing agar plates and a MATE family efflux pump from the ampicillin-containing agar plates. Various resistance associated and potential resistance proteins were also detected. Conclusion: blaCTX-M-1 was the commonest ESBL, in line with global trends. When compared to a culture-based study on bacterial isolates from the same samples; beta-lactamase genes were detected more commonly using targeted PCR than by culture The high prevalence of ESBL genes in samples from the community is concerning and supports the concept of microbiota acting as a reservoir of resistance genes. We have also developed a FM approach to detect ARGs from stool samples, which is able to identify known and potentially novel resistance mechanisms. This study has contributed to our knowledge of ARGs in under characterized populations in Cape Town.
- ItemThe carriage of antimicrobial resistance in community children – a TB-CHAMP sub-study.(Stellenbosch : Stellenbosch University., 2019-12) Brand, Chante; Whitelaw, Andrew Christopher; Newton-Foot, Mae; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology: Medical Microbiology.Introduction: The world-wide rise in antimicrobial resistance (AMR) is threatening the effectivity of antibiotics and the control of infectious diseases. The challenge of AMR is considerably exacerbated by the presence of mobile genetic elements harbouring various antibiotic resistance genes, which can easily spread to other species by horizontal gene transfer. This poses serious risks for clinical infections, however, reports on the carriage of these plasmid-mediated resistance genes are still rare in South Africa. This study aimed to describe the rates of carriage and mechanisms of antimicrobial resistance at baseline, as well as the effect of levofloxacin exposure on these rates in children in communities in Cape Town. Methods: Stool samples were collected from 100 children enrolled in the Tuberculosis Child Multidrug-resistant Preventive Therapy Trial (TB-CHAMP) at baseline and at 16- and 24-week follow up visits between November 2017 and November 2019. The stool samples were cultured onto MacConkey agar plates with cefpodoxime and ertapenem disks and in some cases, nalidixic acid and ciprofloxacin disks, in order to select for cephalosporin, carbapenem and quinolone resistant and susceptible E. coli and Klebsiella isolates. Kirby Bauer disk diffusion was used to determine the susceptibility profiles of the organisms and PCR and Sanger sequencing were used for subsequent detection of cephalosporin and quinolone resistance mechanisms. DNA was extracted directly from the stools and targeted molecular detection and quantification of plasmidmediated quinolone resistance (PMQR) genes using real-time PCR. Results: High levels of antibiotic resistance were detected at baseline, with 81% of participants carrying an organism resistant to at least one antibiotic and 49% and 33% carrying quinolone and cephalosporin resistant organisms respectively. These rates increased over time, with significant increases in quinolone resistance after 16 weeks (69.8%). The presence of the extended spectrum -lactamase gene, blaCTX-M, in cephalosporin resistant E. coli and Klebsiella spp. remained relatively constant over time, ranging between 19.7 – 26.8% and 33.3 – 37.5% respectively over 24 weeks. However, this gene was observed in higher proportions in Klebsiella spp. compared to E. coli. We saw high rates of carriage of qnrB (53.3%) and aac(6’)-Ib-cr (66.7%) in Klebsiella spp. at baseline and significant increases in qnrS and aac(6’)-Ib-cr, as well as mutations in gyrA and parC after 16 and, in some cases, 24 weeks. The presence of aac(6’)-Ib-cr also increased significantly in E. coli from baseline (3.8%) to 16 weeks (21.3%). qnrS was detected in 86% of stools in the targeted molecular analysis, while qnrB was only detected in 14%, although it was more abundant than qnrS in the stool samples. Conclusions: We report high rates of resistance to various antibiotics, as well as the presence of -lactamase and PMQR genes, in commensal gut bacteria in children in Cape Town communities before and over 24 weeks of levofloxacin treatment. The high rate of quinolone resistance at baseline is especially worrying as roughly half of these children started levofloxacin treatment after the baseline stool samples were collected. The increase in rates of resistance and presence of PMQR genes is interesting, however, the TB-CHAMP trial is still ongoing and we do not yet know which participants are receiving levofloxacin or placebo. Once the TB-CHAMP study has been completed and the blind has been broken, the results can be stratified according to treatment group to determine the impact of levofloxacin on resistance carriage.
- ItemThe Characterisation of Antibiotic Resistance Plasmids in Escherichia coli and Klebsiella pneumoniae Isolates from Hospital and Community Settings(Stellenbosch : Stellenbosch University, 2021-03) Stein, Lisa; Newton-Foot, Mae; Whitelaw, Andrew; Pienaar, Colette; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology.ENGLISH ABSTRACT: Antimicrobial resistance has become one of the biggest challenges and threats to public health systems worldwide. Widespread distribution of resistance is commonly due to horizontal gene transfer, which includes mobile genetic elements (MGE) such as plasmids, insertion sequences, transposons, and integrons. This study aimed to characterise plasmids conferring antibiotic resistance in extended-spectrum β-lactamase (ESBL) positive Escherichia coli and Klebsiella pneumoniae isolates from bloodstream infections to determine whether ESBL and plasmid-mediated quinolone resistance (PMQR) genes were mobilised on the same plasmids and whether the same plasmids are disseminated in healthcare and community settings. Methods Illumina MiSeq whole-genome sequencing (WGS) was previously performed on 112 E. coli and 66 K. pneumoniae isolates from blood cultures submitted to the National Health Laboratory Service Microbiology Laboratory at Tygerberg Hospital during 2017 and 2018. Assembled genomes were interrogated for the presence of ESBL and PMQR genes and plasmid replicon types. Based on the results, eight E. coli and nine K. pneumoniae isolates were selected for plasmid sequencing on the Oxford Nanopore Technologies MinION platform. Unicycler assembler was used for hybrid assembly of Illumina short-reads and Nanopore plasmid long-reads. In silico analyses were performed using ResFinder, PlasmidFinder and ISsaga to identify ESBL and PMQR genes, plasmid replicon types, and MGEs. Results Based on Illumina WGS, the ESBL and PMQR containing isolates contained multiple resistance genes and IncF plasmid replicons, individually or in combination with additional plasmid types. The IncF replicons and resistance genes were on separate contigs, therefore associations between different IncF replicons and with resistance genes could not be confirmed. Nanopore sequencing resolved plasmids from several E. coli and K. pneumoniae isolates; however, chromosomal genes could not be visualised and misassembly resulted in fragmented plasmids. Hybrid assembly fully resolved plasmids and chromosomal genes in several E. coli and K. pneumoniae isolates. Amongst the E. coli isolates, three F-type multireplicon plasmids and two single replicon plasmids IncI1-γ and IncB/O/K/Z, which contained resistance genes, were described. Novel multireplicon plasmid FII(FIC)-FIB-X was detected and harboured ESBL blaTEM-135 and PMQR qnrS1. The blaCTX-M genes were confirmed to be chromosomally located in three E. coli isolates and plasmid-mediated on F-type plasmids in two E. coli isolates. K. pneumoniae isolates harboured single replicon F-type plasmids, multireplicon FIB-HI1B fusion plasmids, and a single replicon IncC plasmid. The FIB-HI1B plasmids were associated with blaCTX-M-15, aac(6’)-Ib-cr, and qnrS1. The blaCTX-M-15 was plasmid-mediated in all K. pneumoniae isolates. Conclusion Amongst the E. coli isolates, ESBL and PMQR genes were present both on the same plasmid and on separate plasmids. In K. pneumoniae, ESBLs and PMQRs were found collectively on the same plasmids, and the F-type plasmids harbouring ESBL and PMQR genes differed from those in E. coli. As only two community-acquired K. pneumoniae isolates were selected for Nanopore plasmid sequencing, conclusions regarding the dissemination of K. pneumoniae plasmids in healthcare and community settings could not be made. Plasmids of the same FAB-types were detected amongst E. coli isolates of various sequence types and from both hospital- and community-settings, which is indicative of spread between these settings.
- ItemCharacterisation of follicular helper T (Tfh) cells in early treated HIV-infected children: relationship to immune activation and inflammation.(Stellenbosch : Stellenbosch University, 2022-11-24) Olifant, Paulina; Glashoff, Richard Helmuth; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology: Division of Medical Microbiology.ENGLISH ABSTRACT: Background: South Africa has a high burden of Human Immunodefiency Virus (HIV) infection. Babies of HIV-positive pregnant women can become HIV-infected or exposed through vertical transmission. Follicular helper T (Tfh) cells have been of particular interest in HIV infection due to their preferential expansion, contribution to the HIV reservoir and dysregulation within HIV-infected individuals. The aim of this study was to investigate the Tfh cell population within children from the Children with HIV Early Antiretroviral Therapy (CHER) trial who started treatment within the first six months of life to determine whether the numbers of these cells is altered as compared to uninfected children and also whether persistent immune activation and inflammation in these children is associated with Tfh cell dysregulation. Methodology: This retrospective cross-sectional observational study consisted of three groups, i.e., early antiretroviral-treated HIT (HIV Infected Treated), HEU (HIV Exposed Uninfected) and HUU (HIV Uninfected Unexposed), of children. Cryopreserved peripheral mononuclear blood cells (PBMCs) were stained with an 11-colour antibody panel designed and optimised for phenotypic identification and quantification of T cell populations using flow cytometry. Tfh cells populations were characterised as CD4+CXCR5+PD-1+ with/without ICOS+. CD4, CD8 and Treg cells were defined as follicular/ follicular-homing based on CXCR5+ expression and activated based on CD38+ and/or CD69+ expression. Secondary data of clinical parameters and inflammatory cytokines for each group were evaluated. Statistical comparisons between groups were made using the Mann-Whitney test to identify significant differences. Significant correlations between Tfh cells and clinical parameters, other T cell populations and inflammatory cytokines were identified using Spearman’s rank order test. Results: Phenotypic results generally indicated significantly increased proportions of CD38+ subsets in HIT group and CD69+ subsets in HEU group. Although there was no significant difference in median CD4+CXCR5+PD-1+ Tfh cell percentage between groups, the ICOS-expressing subset namely CD4+CXCR5+PD-1+ICOS+ Tfh cells was significantly higher in the HIT (33.6% vs 19.2%; p = 0.016) and HEU group (31.6% vs 19.2%; p = 0.006) compared to the HUU group. In the HIT group, CD4+CXCR5+PD-1+ Tfh cells shared significant negative correlations with a majority of activated T cell subsets. A significant positive correlation between CD4+CXCR5+PD-1+ Tfh and CD8+PD-1+ Tc cells, general indicator of immune exhaustion, was demonstrated. Lastly, the HIT group showed the highest level of INF-α and hsCRP inflammatory cytokines and levels of IL-1β and hsCRP significantly correlated with CD4+CXCR5+PD-1+ICOS+ Tfh cells within this group. Conclusions: Overall levels of immune activation were significantly higher in HIT and HEU groups. Several activated T cell subsets inversely correlated with CD4+CXCR5+PD-1+ Tfh cells, suggesting high levels of immune activation can lead to decreased proportions of circulating Tfh cells. Even though no significant difference in the proportion of CD4+CXCR5+PD-1+ Tfh cells was found between groups, the ICOS+ subset was significantly expanded in HIT and HEU children in comparison to HUU children. The significant positive correlation between IL-1β and ICOS-expressing Tfh cells, within the entire study population and HIT group, suggested that increased inflammation resulted in an Tfh cell increase.
- ItemCharacterisation of fosfomycin resistance in urinary pathogens from the Western Cape, South Africa.(Stellenbosch : Stellenbosch University, 2021-03) Mosime, Lesedi Bridget; Nel, Pieter; Newton-Foot, Mae; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology.ENGLISH ABSTRACT: Introduction: Urinary tract infections (UTI) are the most commonly acquired bacterial infections worldwide. The South African Department of Health advised that fosfomycin, nitrofurantoin and gentamicin be used for the treatment of uncomplicated UTI due to other antibiotics showing adverse side effects. Fosfomycin has effectively been utilised in the management of UTI, however resistance has been detected in urinary pathogens at the Tygerberg Hospital National Health Laboratory Service (NHLS) Medical Microbiology diagnostic laboratory. This study aimed to determine the prevalence of fosfomycin resistance among community-acquired urinary pathogens in the Western Cape and to characterise fosfomycin resistance mechanisms in fosfomycin resistant Escherichia coli and Klebsiella pneumoniae isolates. Methods and Materials: Two-hundred urinary isolates (Enterobacterales and Enterococcus spp.) from antenatal clinics in the Western Cape were collected from the Tygerberg Hospital NHLS Medical Microbiology laboratory during 2019 and 2020 and used to determine the prevalence of fosfomycin resistance. Fosfomycin susceptibility was determined using disc diffusion and Etest®. Fosfomycin resistant E. coli and K. pneumoniae isolates from the prevalence study and another set of fosfomycin resistant isolates (5 E. coli and 19 K. pneumoniae) collected from urine samples submitted to the NHLS at Tygerberg Hospital in 2017 (Ethics #: U17/05/026) were used to characterise fosfomycin mechanisms. FosA mediated resistance was determined using a phenotypic assay and fosA genes were detected by PCR. Mutations in the fosfomycin target gene murA and transporter genes, glpT and uhpT, were characterised by polymerase chain reaction (PCR) and Sanger sequencing. Results: Fosfomycin resistance was detected in 3.5% of community-acquired urinary pathogens. Fosfomycin resistance rates were 2.2% in E. coli (3/139) and 12.9% in other Enterobacterales. All Enterococcus spp. isolates were susceptible to fosfomycin. In the combined sample set of 31 fosfomycin resistant isolates, the phenotypic assay detected FosA in only 7 isolates, while fosA genes were detected by PCR in 25. Chromosomal mutations were identified in 6 isolates, of which three isolates (1 K. pneumoniae and 2 E. coli) had deletions in the uhpT gene, which has previously been reported to confer fosfomycin resistance. The role of other mutations found in the glpT gene of E. coli and the murA and glpT of K. pneumoniae isolates has not been determined. Conclusion: The fosfomycin resistance rate in community-acquired UTI was low, which supports the careful ongoing use of fosfomycin for the treatment of uncomplicated community-acquired UTI. FosA mediated resistance was the most common mechanism of fosfomycin resistance identified in this population.
- ItemThe characterization of antimicrobial resistance and virulence in Coagulase-Negative Staphylococci isolated from neonatal blood cultures in the Western Cape(Stellenbosch : Stellenbosch University, 2023-03) Cloete, Stephanie Simone; Whitelaw, Andrew Christopher; Matukane, Siphiwe Ruthy; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology: Division of Medical Microbiology.ENGLISH SUMMARY: Introduction: Coagulase-Negative Staphylococci (CoNS) are frequently isolated from the neonatal intensive care unit. However, it is difficult to discriminate between invasive CoNS and coincident contaminants since they are commensals of skin, and the nonspecific clinical signs of neonatal sepsis may be even more subtle due to the low virulence of CoNS. The extensive range of virulence factors in CoNS, some of which may be regulated by mobile genetic elements, contributes to their pathogenicity. This study aims to describe the species distribution, antimicrobial resistance, and molecular virulence markers in CoNS from neonatal blood cultures to identify potential pathogenicity markers. Methods: Between February and July 2021, 127 CoNS isolates were collected from neonatal blood cultures submitted to Tygerberg Hospital National Health Laboratory Services microbiology laboratory. Species identification was performed on all isolates using MALDI-TOF mass spectrometry. Antimicrobial susceptibility testing (AST) using Kirby Bauer disc diffusion was performed on 82 isolates representing the two predominant CoNS species from the most represented hospitals. Twenty isolates, representing a range of susceptibility patterns, were chosen for Oxford Nanopore whole genome sequencing (WGS). The assembled genomes were analysed using the Center for Genomic Epidemiology, pubMLST, Virulence Factor Database, ISsaga, and Resistance Gene Identifier. Reverse-transcription real-time PCR was used to explore the potential association of insertion sequence (IS)256 with the expression of the biofilm associated virulence gene icaA. The icaA normalised cycle threshold (ΔCt) was calculated relative to that of the house-keeping gene (tpi). Results: The most common species identified were Staphylococcus epidermidis (80/127, 63 %) and Staphylococcus hominis (29/127, 23 %). Among the 62 S. epidermidis and 20 S. hominis isolates selected for AST, 81.7 % (67/82) were non-susceptible to at least one antibiotic, and 54 % (44/82) were resistant to three or more antibiotic classes (multidrug resistant). The highest rates of resistance were to erythromycin (64.6%), trimethoprim-sulfamethoxazole (56.1 %), and cloxacillin (54.9 %). There was considerable concordance between the observed phenotypes and the predicted genotype, especially for cefoxitin, linezolid and vancomycin. The WGS data predicted all 20 isolates (14 S. epidermidis and six S. hominis isolates) as possible human pathogens, with a probability pathogen score higher for S. epidermidis (=0.93) than S. hominis (=0.89) isolates (p<0.001). In-silico MLST revealed substantial diversity, reflected by nine S. epidermidis sequence types identified. The ica operon was present in 10/14 S. epidermidis isolates, and 7/14 isolates contained the IS256. None of the S. hominis isolates contained the ica operon. The ΔCt values in isolates with and without IS256 were not significantly different, suggesting that IS256 was not related to ica operon expression. Conclusion: There was a high-level of genetic diversity among CoNS isolates, enriched with virulence factors, antimicrobial resistance genes and mobile genetic elements. S. epidermidis harboured a greater array of virulence factors than S. hominis isolates, which require further investigation to be used as markers of clinical significance. Antimicrobial resistance was common among these isolates, which may complicate treatment strategies. There was no effect of IS256 on ica gene expression, which may relate to the distance of IS256 from the ica operon.
- ItemColistin resistance in gram-negative pathogens in the Western Cape, South Africa(Stellenbosch : Stellenbosch University, 2021-12) Snyman, Yolandi; Newton-Foot, Mae; Whitelaw, Andrew Christopher; Maloba, Motlatji Reratilwe Bonnie; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology: Division of Medical Microbiology.ENGLISH ABSTRACT: Background Antimicrobial resistance is a public health concern and injudicious antibiotic prescribing and inadequate infection control practices have left the global community with untreatable multidrugresistant (MDR) bacteria. Colistin is a last resort antibiotic used to treat infections with MDR Gramnegative bacteria (GNB), especially carbapenem-resistant GNB. Therefore, the emergence of colistin resistance is a serious problem. This study from the Western Cape, South Africa, describes colistin resistance mechanisms in colistin-resistant GNB isolates from clinical specimens from various hospitals, stool samples from healthy children in the community, and river and storm water. Methods Colistin-resistant GNB isolates from clinical specimens from different healthcare facilities were collected from the NHLS microbiology laboratory at Tygerberg Hospital during 2016 and 2017. Fifty stool samples from healthy children (≤ 5 year of age) in the Cape Town metropolitan were collected between November 2017 and August 2018, and three surface water sources and stormwater were collected in 2019 and 2020. Selective media was used to isolate colistin-resistant GNB from the stool and water samples. Colistin resistance was confirmed using broth microdilution (BMD). The mobile colistin resistance genes, mcr-1-9, were detected by PCR and whole-genome sequencing (WGS). In selected mcr-negative isolates chromosomal colistin resistance mutations were identified by WGS. Strain typing was performed by WGS (MLST and SNP analyses) and repPCR. The functionality of mcr genes with unknown colistin resistance profiles was determined by BMD following recombinant expression or plasmid curing. Results mcr-1 was present in 55% (12/22) of Escherichia coli and 71% (5/7) of Klebsiella spp. isolates from patients at various hospitals during 2016-2017. pmrB mutations were identified in 8/10 mcrnegative E. coli and mgrB was disrupted in the two mcr-negative Klebsiella spp. isolates. Most colistin-resistant GNB isolated from hospitalised patients in 2016 and 2017 were unrelated, however, some clonal relatedness was observed in the 2017 E. coli population and a clonal expansion of an emerging colistin-resistant MDR Acinetobacter baumannii strain was noted among isolates from 2017. No previously described colistin resistance mechanism was detected in the A. baumannii isolates, but a possible novel mechanism was described. mcr-4.3 was detected in a Stellenbosch University https://scholar.sun.ac.za iii single Acinetobacter nosocomialis isolate, although recombinant mcr-4.3 did not confer colistin resistance in E. coli, plasmid curing of the mcr-4.3-containing plasmid restored colistin susceptibility. Colistin-resistant E. coli were isolated from the stools of two healthy children from the community (4%, 2/50) during 2017-2018; however, mcr genes were not detected. Colistin-resistant GNB, mainly Aeromonas spp., and mcr-5.1 and/or various mcr-3 variants were detected in the Plankenburg river, Eerste river, and Berg river and stormwater from Muizenberg and Fish Hoek in 2019 and 2020. Of the colistin-resistant Aeromonas spp. isolated from the Berg river, 25% (6/24) contained five novel mcr-3 variants, which were confirmed to confer colistin resistance. Conclusion The emergence of colistin resistance mechanisms in diverse strains obtained from hospital patients, with the limited gastrointestinal carriage of colistin-resistant Enterobacterales in community children and the disparate colistin-resistant species and mechanisms in the environment, suggest that selective pressure, and not community transmission, is the main driver of colistin resistance in clinical settings.
- ItemDetermination of the permeability of biological membranes to various chemical markers, including anti-HIV drugs(Stellenbosch : University of Stellenbosch, 2009-12) Pretorius, Erina; Bouic, Patrick J. D.; University of Stellenbosch. Faculty of Health Sciences. Dept. of Pathology. Medical Microbiology.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.
- ItemDevelopment and validation of an in vitro model of dendritic cell identification and activation(Stellenbosch : Stellenbosch University, 2008-03) Clark, Anel; Bouic, Patrick J. D.; Stellenbosch University. Faculty of Health Sciences. Dept. of Pathology. Medical Microbiology.ENGLISH ABSTRACT: The aim of this study was to investigate the effect of MBV and Coley’s Toxin on dendritic cells in vitro. The dendritic cell system of antigen presenting cells is the initiator and modulator of the immune response. The principle function of the dendritic cells is to present antigens to resting naïve T lymphocytes: these cells are the only APCs that prime naïve T cells and only mature DCs can carry out this function.Previous studies done on dendritic cells showed that bacterial peptides can induce the maturation of dendritic cells. With the results of these studies in mind we hypothesized that these two vaccines will also induce the maturation of dendritic cells. Chapter 1 is a literature review on the immune system explaining the organs and cells of the immune system. Chapter 2 includes a full description of DCs, the MBV and Coley’s toxin. Also included in this chapter is a short explanation of the principle of the technique being used for the identification and maturation of both mDCs and pDCs, namely the technique of flow cytometry. Chapter 3 describes the method for the phenotypic identification of DCs: the subsets are distinguished by their absence of expression of several lineage markers for lymphocytes, monocytes and NK cells and the expression of CD11c (in the case of myeloid DCs) and CD123 (in the case of plasmacytoid DCs). The inclusion of HLA-DR in addition to the previous described markers allows the discrimination of CD123+ DCs from basophils. The assay requires three tubes per sample which enables quick analysis of these rare subsets with a small sample volume. This assay was applied to peripheral blood samples obtained from healthy individuals and individuals with cancer, HIV and HIV and TB co-infected patients. Our results showed that the maturation status of DCs in HIV and lymphoma were low but those measured in the case of HIV + TB patients were even higher than in the control group. Chapter 4 and 5 describe the in vitro activation and maturation status of DCs following their incubation with bacterial-derived products. Interactions between DCs and microbial pathogens are fundamental to the generation of innate and adaptive immune responses and upon contact with bacteria or bacterial components such as lipopolysaccharide (LPS), immature DCs undergo a maturation process that involves expression of costimulatory molecules, HLA molecules, and cytokines and chemokines, thus providing critical signals for lymphocyte development and differentiation. In this study, we investigated the response of human DCs to MBV and Coley’s Toxin. Previous studies showed DCs can be activated with killed Streptococcus pyogenes. With this study in mind it was hypothesized that the MBV and Coley’s Toxin used in this study might modulate DC maturation. The results of this study showed that the MBV and Coley’s toxin did induce the maturation of both pDCs and mDCs as measured by increased surface expression of costimulatory molecules such as CD80 and CD83. Chapter 6 presents the measurement of cytokines released after the PMBCs had been were incubated with Coley’s Toxin and Mixed Killed bacteria. The BD™ Cytometric Bead Array (CBA) flex set was used for the simultaneous detection of multiple soluble analytes. The results indicated that both Coley’s Toxin and the MBV activated the DCs and subsequently induced TH1 as well as a TH2 responses in the T cells present in the cell cultures. Finally, a general conclusion discussing the significance and implications of our results as well as possible future research required is discussed in Chapter 7. DCs are potent antigen presenting cells (APCs) which play a critical role in the regulation of the immune response. There is great interest in exploiting DCs to develop immunotherapies for cancer, chronic infections, immunodeficiency diseases and autoimmune diseases.
- ItemDevelopment and validation of stabilized whole blood samples expressing T-cell activation markers as quality control reference material(Stellenbosch : Stellenbosch University, 2008-03) Louw, Anne-Rika; Bouic, Patrick J. D.; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Medical Microbiology.ENGLISH ABSTRACT: Introduction: Flow cytometry has progressively replaced many traditional laboratory tests due to its greater accuracy, sensitivity and rapidity in the routine clinical settings especially clinical trails. It is a powerful tool for the measuring of chemical (the fluorochrome we add) and physical (size and complexity) characteristics of individual cells. As these instruments became major diagnostic and prognostic tools, the need for more advanced quality control, standardized procedures and proficiency testing programs increased as these instrumentations and their methodology evolve. Minor instrument settings can affect the reliability, reproducibility and sensitivity of the cytometer and should be monitored and documented in order to ensure identical conditions of measurement on a daily basis. This can be accomplished by following an Internal Quality Assurance (IQA) and/ or External Quality Assurance (EQA) program. Currently there are no such programs available in South Africa and poorer Africa countries. HIV is a global concern and the laboratories and clinics in these places are in need of such IQA programs to ensure quality of their instrumentation and accurate patient results. Quality assurance programs such as CD Chex® and UK Nequas are available but due to bad sample transport, leave the receiving laboratories with nightmares. It would be best if there was a laboratory in South Africa that could provide the surrounding laboratories with stabilized whole blood samples that can be utilized as IQA. The transport of these samples can be more efficient due to shorter distance and thus the temperature variations limited. Aims and Objectives: The aim of Chapter one is to familiarize the reader with general terminology and concepts of immunology. Chapter two describes in detail the impact stabilized whole blood had on clinical immunology concerning Quality Control and Quality Assurance. The objective of this study is to stabilize whole blood with a shelf life of greater than 30 days to serve as reference control material for South African Immunophenotyping. It is further an objective to use these in-house stabilized control samples for poorer African countries as Internal Quality Assurance reference material. It is a still further objective to stimulate various lymphocyte subsets to express activation antigens and then stabilize these cells for more specialized immunological test and can serve as a QC for those required samples. Study design: In Chapter three, the method currently used to stabilize whole blood was modified. The stability of different concentrations of a first stabilizing agent (Chromium Chloride hexahydrate) was investigated. Incubation periods and concentrations of paraformaldehyde as second stabilizing agent were investigated. Blood samples from healthy individuals (n=10) were stabilized and monitored for the routine HIV phenotypic surface antigens over a period of 40 days. These samples (n=10) were compared on the Becton Dickinson Biosciences (BD) FACSCalibur™ versus BD FACSCount™ instrumentation. Blood samples (n=3) were stabilized and monitored to identify phenotypic cell surface molecules for as long as possible. They were quantified on both flow cytrometric instruments. In addition, these stabilized samples (n=3) were investigated as control blood for calibration purposes on the BD FACSCount™ instrument. In Chapter four, lymphocytes were isolated and activated with various stimuli to express sufficient activation antigens such as CD25, CD69, HLA-DR and CD40 Ligand on the T helper cell surfaces. These activated antigens were analyzed on the BD FACSCalibur™ and further stabilized to serve as possible IQA samples in future. Results: In Chapter three, the ten individual stabilized samples had non-significant P values (P > 0.05) for CD3, CD4 and CD8 percentages and absolute values comparing day 3 until day 40. Comparing the BD FACSCalibur™ versus BD FACSCount™, resulted in a R2 = 0.9848 for CD4 absolute values and a R2 = 0.9636 for CD8 absolute values. Stabilized blood samples (n=3) were monitored for routine HIV phenotypic markers until day 84. The cells populations were easily identifiable and could be quantified on both BD FACSCalibur™ and BD FACSCount™ instruments. In Chapter four; for the activation study purposes, activated T helper lymphocytes expressed approximately 25 to 35% CD40 Ligand cell surface molecules. The stimulant of choice was Ionomycin at a 4μM concentration. Cells were incubated for four hours at 37 degree Celsius in a 5% CO2 environment. For CD69 surface expression, 6 hour incubation was optimum. The stimulus of choice in this case was 4μM Ionomycin which induced 84.21% CD69 expression in the test samples. For CD25 expression; 6 hour incubation with PHA resulted in approximately 43% of CD25 expression. For HLA-DR surface expression; 6 hour incubation with PHA resulted in approximately 43.32% of HLA-DR expression. Activated lymphocytes expressing CD40 Ligand showed stability until day 23. Activated Lymphocytes expressing CD69, CD25 and HLA-DR were stabilized in the same manner and stability could be achieved until day 16. Conclusion: This thesis was related to the preparation of control samples (IQA) designed to simulate whole blood having defined properties in clinical laboratory situations. In future kits can be developed with a low, medium and high control sample for the various immunological phenotypic determinants. Another kit can be compiled where various activation markers can be identified, quantified with a “zero”, low and high control. These whole blood IQA kits and “activation IQA kits” can be implemented for training of newly qualified staff, competency testing of staff, method development, software testing, panel settings and instrument setting testing. Control samples ideally must have a number of properties in order to be effective. For instance stability during storage times, preferably lasting more than a few weeks, reproducibility and ease of handling. These will provide the information on day-to-day variation of the technique or equipment which will enhance accuracy and improve patient care.
- ItemDevelopment of functional immune readouts for the confirmation of mendelian susceptibility to mycobacterial disease and related primary immunodeficiencies(Stellenbosch : Stellenbosch University, 2019-04) Van Coller, Ansia; Glashoff, Richard; Esser, Monika; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology. Medical Microbiology.Background: Mendelian Susceptibility to Mycobacterial Disease (MSMD) is a primary immunodeficiency (PID) characterised by a predisposition to infection by weakly-pathogenic mycobacteria. In countries with a high prevalence of tuberculosis, individuals with MSMD are also prone to severe, persistent, unusual or recurrent infections by pathogenic Mycobacterium tuberculosis. Several MSMD-associated genes have been described, including IFNGR1, IFNGR2, IL12RB1, IL12B, STAT1, NEMO, ISG15, IRF8, TYK2, and CYBB, many resulting in a disruption of IL-12 and IFN-γ cytokine axis, which is essential for control of mycobacterial infections. This genetic heterogeneity results in many distinct disorders, which vary in their mode of inheritance and clinical presentation. An accurate molecular diagnosis, confirmed by immune functional studies, is essential to ensure that the patient receives optimal treatment and prophylaxis for infections. The aim of this study was to implement and optimise a set of immune phenotyping and functional validation tests for the key pathway, the IFN-γ and IL-12 cytokine axis, involved in MSMD, and to use these assays to assess immune function in a cohort of suspected MSMD patients. Methodology: Blood was collected from 17 participants with MSMD-like clinical phenotypes. DNA was extracted and PBMCs were isolated from the patients’ blood. Whole exome sequencing (WES) was performed and the resulting data was processed using an in-house bioinformatics pipeline, TAPER™. A set of flow cytometry and ELISA-based functional assays were implemented and optimised to assess the integrity of the IL-12-IFN-γ pathway. IFN-γR1 and IL-12Rβ1 expression were assessed by means of standard surface flow cytometry, and IFN-γ and IL-12 signalling was assessed by the detection of pSTAT1 and pSTAT4 respectively through intracellular phospho-specific flow cytometry. IFN-γ-induced IL-12 production as well as IL-12-induced IFN-γ production was also assessed by ELISA after 48-hour in vitro stimulation. The functional and genetic data were then reconciled in order to confirm the extent of functional impairment associated with each genetic variant. Results: Plausible disease-causing variants were identified through genetic investigations for 11 of the 17 participants. Variants in MSMD-associated genes were found in 8 of these patients, although only one of the identified variants, IFNGR1 (c.818del4), has been described before. Variants in genes not previously associated with MSMD were also found, including variants in IKZF1, NOD2, IRAK1, IKBKB, and NFKB2. All the functional assays were optimised and the combination of the three assays for the assessment of the integrity of the IL-12-IFN-γ pathway was successful in identifying immune deficits in essentially all of the participants included in this study. Conclusions: The current study led to the implementation of functional immune readouts that allowed for the evaluation of the functional impact of both novel and previously described genetic variants on the IL-12-IFN-γ pathway. The results generated from the functional assays were highly variable and often defects within the same gene lead to different phenotypes, which emphasises the importance of in vitro functional confirmation of all PIDs. Hence it would be beneficial to apply these assays routinely for patients with suspected PID relating to mycobacterial susceptibility. A molecular diagnosis with confirmed functional impairment paves the way for targeted treatment and improved disease management options for these patients.
- ItemDie effek van musiek op die immuunsisteem, emosies en longfunksie tydens die standaard fisioterapeutiese behandeling van spesifieke longpatologie(Stellenbosch : University of Stellenbosch, 2005-03) Le Roux, Frances Hendriehetta; Bouic, Patrick J. D.; University of Stellenbosch. Faculty of Health Sciences. Dept. of Pathology. Medical Microbiology.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.
- ItemElucidation of the substrates of mycosin 3, an essential protease of Mycobacterium tuberculosis(Stellenbosch : University of Stellenbosch, 2011-03) Fang, Zhuo; Gey van Pittius, Nicolaas Claudius; Warren, Robert Mark; University of Stellenbosch. Faculty of Health Sciences. Dept. of Biomedical Sciences.ENGLISH ABSTRACT: Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), infects one third of the world’s population and kills 1.7 million people per year. The increasing prevalence of multi- and extensively drug resistant M. tuberculosis strains means that there is an urgent need to develop new anti-TB drugs. The genome of M. tuberculosis has five copies of the ESAT-6 gene clusters (ESX-1, -2, -3, -4 and -5), which are essential for the survival (ESX-3) and pathogenicity (ESX-1 and ESX-5) of the bacterium. The ESX clusters encode for proteins which form a novel secretion system which has been shown to secreted small T-cell antigens of the esx gene family, as well as other proteins such as the PE and PPE’s. The mycosins are a family of genes situated in the ESX clusters which encode for putative subtilisin-like serine proteases. These proteins are the most conserved proteins within the five clusters. Apart from their conserved protein sequence, mycosin-3 is also an essential protein specific to the mycobacteria, which makes it an attractive potential drug target. Identifying the substrate(s) of mycosin-3 could help to understand the function of this enzyme and discover novel inhibitors from which new drugs could be designed. We hypothesize that the secreted products of the ESX system could be potential substrates for the mycosins. Specifically, we hypothesize that PE5, PPE4, esxG and esxH (all found in ESX-3) might be the substrates for mycosin-3. Mycosin-3, PE5, PPE4, esxG and esxH were thus cloned, expressed and purified respectively. The four substrates were used for protease assays using mycosin-3 as the protease. The protease-substrate mixture were subsequently separated on 2-D SDS-PAGE gels to check whether there were any cleavage of the four substrates. Although all the target fusion proteins were cloned and expressed successfully, the protease assay results showed no cleavage for any of the four substrates. Possible explanations for the failure of cleavage were: (1) impure enzyme and substrate(s); (2) inappropriate buffer conditions; (3) the hypothesized substrates might not be the substrates of mycosin-3; and (4) incorrect folding or modification of the target fusion proteins might have taken place. Future research will aim to address these possible limitations in order to fully elucidate the function and substrate specificity of mycosin-3 and to use this information for the design of novel drugs against M. tuberculosis.
- ItemEpidemiology and antibiotic susceptibility patterns of mycoplasma sp. and ureaplasma urealyticum(Stellenbosch : University of Stellenbosch, 2010-12) Govender, Sharlene; Chalkley, Lynda J.; Wasserman, Elizabeth; University of Stellenbosch. Faculty of Health Sciences. Dept. of Pathology. Medical Microbiology.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.
- ItemThe epidemiology and virulence characteristics of Staphylococcus aureus isolates from bacteraemic patients at Tygerberg Hospital, South Africa(Stellenbosch : Stellenbosch University, 2019-04-02) Van Rijswijk, Amike; Newton-Foot, Mae; Abdulgader, Shima; Whitelaw, Andrew; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology: Medical Microbiology.ENGLISH ABSTRACT: Introduction: Staphylococcus aureus is a versatile pathogen that produces multiple virulence factors which work together to establish and maintain infections. The accessory gene regulator (agr) locus is a quorum sensing two-component system which regulates at least 23 virulence factors. There are four different agr types, I-IV, and mutations within the agr locus may result in a dysfunctional agr. These can result in altered gene expression which may affect disease presentation and outcome. Data on the molecular epidemiology of S. aureus and its association with clinical outcome in South Africa is limited. This study aimed to determine the effect of epidemiology and agr-associated virulence characteristics on the clinical outcome of bacteraemic patients at Tygerberg Hospital. Methods: S. aureus isolates were collected from blood cultures from February 2015 to March 2017. Genotyping was performed using staphylococcal protein A (spa) typing and multi-locus sequence typing (MLST); and staphylococcal cassette chromosome mec (SCCmec) typing was performed on all methicillin resistant S. aureus (MRSA) isolates. Agr typing was performed by PCR and agr functionality was assessed using a phenotypic δ-haemolysin assay and matrix assisted laser desorption ionisation-time of flight mass spectrometry (MALDI-TOF MS). Associations between patient- and strain- characteristics, and the final outcomes mortality, methicillin resistance and length of stay were investigated by means of regression models. Results and discussion: Of the 199 S. aureus isolates collected, 27% were MRSA. Seventy three spa types were identified; reflecting a diverse population. MRSA isolates were more clonal than methicillin susceptible S. aureus (MSSA) isolates. A previously described novel variant SCCmec type (NV) and SCCmec type IV were most common among MRSA isolates. Agr type I was the dominant agr type, while agr type IV was least prevalent; consistent with the literature. The dominant clone in this study was an MRSA outbreak strain, t045-ST5-MRSA-NV, agr type II (spa-CC 002, CC5), which appears to be circulating in multiple hospital settings in South Africa. The most prevalent MSSA strain/clone was t318-ST1865, agr type III. Pandemic clones such as t037-ST239-MRSA-III, t032-ST22-MRSA-IV and t012-ST36-MRSA-II were also identified. A previously described association between MRSA and spa-CC 002 (CC5) was confirmed in this study; however this association may have been driven by the MRSA outbreak. Agr dysfunctionality was low at 12.6% and 6% using the phenotypic assay and MALDI-TOF MS respectively. Agr dysfunctionality was not clone specific and there was no difference in agr dysfunctionality between MRSA and MSSA isolates. A borderline association between agr dysfunction and shorter length of stay was identified, but needs further investigation. The overall mortality rate was 29% and older age was associated with increased mortality. Hospital acquired (HA) infections were also associated with a higher mortality, which could be explained by the complicated nature of these infections, leading to death. An association between HA infections and MRSA was identified, which is consistent with previous studies and not surprising considering antibiotic selective pressure is higher in hospitals. Conclusion: This study provides insights into the associations between S. aureus epidemiology and agr related virulence characteristics and clinical outcome, despite the limited clinical data available.
- ItemThe epidemiology of Gram negative bacteraemia at Tygerberg Hospital(Stellenbosch : Stellenbosch University, 2018-03) Paterson, Lauren Ann; Whitelaw, Andrew; Newton-Foot, Mae; Stellenbosch University. Faculty of Faculty of Medicine and Health Sciences. Dept. of Pathology. Medical Microbiology.Background Escherichia coli and Klebsiella pneumoniae are common causes of Bloodstream Infections (BSI). β-lactam antibiotics, such as cephalosporins and carbapenems, are commonly used to treat these infections. Increasing resistance has been noted, usually due to plasmid mediated β-lactamases such as Extended-Spectrum β-lactamases (ESBLs) and carbapenemases. This study describes the antibiotic resistance profiles, outcomes and epidemiology of Gram negative BSIs in a tertiary hospital in Cape Town, South Africa. Methods Patients with E. coli (n=70) and K. pneumoniae (n=70) bacteraemia identified at Tygerberg Hospital between April 2015 and March 2016 were included. Identification and Antibiotic Susceptibility Testing (AST) were performed as part of routine testing. Patient data was obtained through record review. ESBL and carbapenemase genes were characterised by Polymerase Chain Reaction (PCR) and DNA sequencing. Isolates were typed using rep-PCR and Pulsed Field Gel Electrophoresis (PFGE). Chi-square and Mann-Whitney tests were used to estimate significance of correlation. Results and discussion 45% of patients were male, and 30.7% were paediatric. 66.4% of BSI were hospital-acquired. K. pneumoniae accounted for 61.3% of hospital-acquired isolates; 72.3% of community-acquired isolates were E. coli. 55.7% of K. pneumoniae and 15.7% of E. coli were cephalosporin resistant (presumed ESBL); one K. pneumoniae isolate was carbapenem resistant. Increased antibiotic resistance and ESBL production was seen in hospital-acquired isolates. ESBL genes were harboured in 35.7% of isolates; 50.7% contained β-lactamase genes and 13.6% no β-lactamase genes. Most TEM genes (98%) were β-lactamases; 47.4% of SHV genes were β-lactamases, 7% were ESBLs and 45.6% were SHV genes whose spectrum is uncertain. Isolates containing SHV genes with uncertain spectrum were phenotypically susceptible to cephalosporins, suggesting these enzymes do not have extended-spectrum activity. Multiple β-lactamase genes were present in 60% of K. pneumoniae isolates, and only 5.7% of E. coli isolates. CTX-M genes were the most common ESBL genes, with most (91.3%) of these belonging to group 1. CTX-M genes were found in combination more often than not (84.8%). No carbapenemase genes were detected. Molecular and phenotypic resistance agreed in 95.3% of isolates. The 30-day mortality rate was 30%, with no association between mortality and hospital-acquired infection, or with ESBL production (phenotypic or molecular). Molecular and phenotypic resistance was associated with hospital-acquired isolates (P=0.001, P<0.001). Both strain typing techniques showed substantial diversity among isolates, with minimal clustering; which suggests multiple clones in the hospital, precluding any possibility of assessing associations. Conclusion Increased resistance was observed in hospital-acquired isolates, and the association between hospital-acquired isolates and ESBL presence was significant, which is not unexpected. Isolates were genetically diverse and showed minimal clustering, suggesting that resistance may be due to horizontal transmission. Continuous efforts towards surveillance of the epidemiology and resistance patterns of circulating strains are required to monitor and guide antimicrobial stewardship, infection prevention and control (IPC) practises and empiric therapy.
- ItemAn evaluation of the diagnostic utility of bone marrow and peripheral blood cultures in patients with suspected disseminated Mycobacterium tuberculosis infection(Stellenbosch : Stellenbosch University, 2015-03) Abrahams, Riezaah; Wasserman, Elizabeth; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology.ENGLISH ABSTRACT: Background Disseminated tuberculosis is defined as the involvement of two or more non-contiguous organ sites simultaneously. The aim of this study was to optimize, evaluate and compare the diagnostic utility of bone marrow aspirate culture and peripheral blood culture (PBC) for the diagnosis of disseminated tuberculosis. Methods This prospective descriptive, laboratory based study recruited patients suspected of having disseminated tuberculosis and who had both study specimens (peripheral blood and bone marrow aspirate) submitted for mycobacterial culture. Study specimens were processed according to standard routine laboratory practice with an additional tube of solid media (Löwenstein-Jensen agar) containing 0.5ml of growth supplement inoculated during sub-culture from the positive blood cultures. HIV status and bone marrow trephine histology results were obtained from the Laboratory Information System. Results A total of 96 patients were recruited. Mycobacterium tuberculosis complex was isolated from bone marrow culture and/or PBC in eight (8.3%) of the study patients. The yield of M. tuberculosis complex from bone marrow aspirate culture was 7% vs. 3% from PBC. The mean time to detection (TTD) of M. tuberculosis complex from sub-cultured solid media was more rapid for PBC (n=3) compared to bone marrow aspirate culture (n=7). The addition of growth supplement to solid media did not enhance amount of growth nor improved the TTD of M. tuberculosis complex. Conclusion Our findings support bone marrow as the specimen of choice for the diagnosis of disseminated tuberculosis, but could not indicate any advantage in sub-culturing positive specimens on enriched media.
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