- ItemThe role of the accessory gene regulator system on biofilm formation and stress response in Staphylococcus aureus(Stellenbosch : Stellenbosch University, 2023-03) Maleka, Kgomotso Frank; Matukane, Siphiwe; Shima, Abdulgader; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology: Division of Medical Microbiology.ENGLISH SUMMARY: Background: Biofilm formation is a key contributor to Staphylococcus aureus virulence and pathogenicity. It is regulated by the accessory gene regulator (agr) operon, which may become dysfunctional due to genetic mutations. These mutations may affect the expression of key genes like RNAIII and icaA that are involved in key pathogenesis pathways. Previous studies have associated agr dysfunctional strains with strong biofilm formation, persistent infections, and treatment failure. Therefore, this study aimed to determine the impact of agr functionality status on biofilm development and antibiotic stress tolerance in clinical S. aureus isolates. Methods: Twelve previously characterized (phenotypically and genotypically) blood culture S. aureus isolates, collected from February 2015 to March 2017 at Tygerberg Hospital were selected for this study. Crystal Violet biofilm assay was then performed to assess biofilm formation over a 24-hour period at different time points in the presence and absence of oxacillin, vancomycin, and rifampicin at sub-minimum inhibitory concentrations [sub-MIC: 0.25 μg/ml (oxacillin and vancomycin), 0,005 μg/ml rifampicin] and clinically relevant concentrations (10 μg/ml). The minimum inhibitory concentration (MIC) was determined using the gradient diffusion assay (E-test). Reverse-transcription real-time PCR was used to measure the expression of RNAIII and icaA genes. Whole genome sequence data were analyzed for genetic differences in the agr locus including the bap, icaA, and icaD regions for the 12 isolates, using online platforms (Prokka, Artemis, and BioEdit 7.2). Result: There was statistically an insignificance increase in the overall biofilm formation levels in agr dysfunctional isolates than in agr functional isolates in the absence and presence of antibiotics, except for when exposed to sub-MIC of oxacillin (p=0.007). Similarly, an increase in the overall biofilm formation level in agr I isolates was observed when compared to agr II and agr III isolates in the absence and presence of antibiotics. Furthermore, overall methicillin-resistant S. aureus (MRSA) isolates produced more biofilm, especially at time point 6 and 8 hours after incubation in the absence of antibiotics; while methicillin-sensitive S. aureus (MSSA) isolates formed more biofilm in the presence of antibiotics overall time points. Furthermore, a significant increase in the expression levels of both RNAIII (p=0.041) and icaA (p=0.020) was observed in agr dysfunctional isolates when compared to agr functional isolates. A significant increase in the expression of icaA (p=0.008) was observed in MRSA isolates; and dysfunctional isolates had more mutations on the agr-related gene than functional isolates. Conclusion: An increase in biofilm formation based on phenotypic agr functionality, agr type, and methicillin susceptibility profile in the absence or presence of antibiotics was not statistically significant. Additionally, mutations observed on the agr locus in agr dysfunctional isolates confirmed the role mutations play on agr functionality. The study analyzed 12 isolates, which may decrease statistical power. Therefore, future studies with a larger sample size should confirm or refute these study findings about the role agr functionality, agr type, and methicillin susceptibility profile have on the ability of clinical S. aureus isolates to produce biofilm in the absence and in the presence of antibiotics.
- 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.
- 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 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.