Doctoral Degrees (Medical Microbiology)

Permanent URI for this collectionhttps://hdl.handle.net/10413/9619

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The molecular mechanisms of mycobacterium tuberculosis curli pili (MTP) in regulation of fatty acid metabolism, carbon metabolism, and bioenergetics of the pathogen and host during infection.
(2024) Ashokcoomar, Shinese.; Pillay, Manormoney.
Background/Aim: The initial contact between pathogen and host is a vital step in the establishment of infection. Mycobacterium tuberculosis (M. tuberculosis) mediates adhesion to target hosts cells such as macrophages via various proteinaceous molecules known as adhesins, found on the cell surface. A distinguished adhesin of M. tuberculosis is M. tuberculosis curli pili (MTP). Previous functional genomics studies have described the involvement of MTP in bacterial aggregation and biofilm formation in addition to its role as an adhesin and invasin of epithelial cells and macrophages. Transcriptomic studies elucidated the importance of MTP in modulating host immune response in vitro and in vivo. Moreover, MTP was found to be present exclusively in the M. tuberculosis complex, and to bind to antibodies in patient sera. The use of metabolomics, bioenergetics and molecular biology will further improve current understanding of MTP as a virulence factor, thereby corroborating its role as a biomarker for the development of better tuberculosis diagnostics and therapeutics. Therefore, this study aimed to elucidate the effect of MTP on M. tuberculosis metabolism and expression of selected genes involved in fatty acid transport, β-oxidation, lactate oxidation, and gluconeogenic carbon flow. Furthermore, this study aimed to investigate the metabolomic and bioenergetic role that MTP plays in the pathogen-host infection model. Methods: Confirmed wildtype (WT) M. tuberculosis, mtp-deletion mutant (Δmtp) and mtp-complemented strains were used throughout this study. Bacterial cultures were standardised prior to harvesting of wet cell mass for metabolite and ribonucleic acid (RNA) extraction. The bacterial metabolite extraction was performed using a whole metabolome extraction method and the metabolites were detected using untargeted two-dimensional gas chromatography time-of-flight mass spectrometry (GC×GC-TOFMS) (Chapter 2). The RNA was extracted via the TriZol method for reverse transcription quantitative polymerase chain reaction (RT-qPCR) on selected genes to support metabolomic data (Chapters 2 and 6). Host THP-1 monocytes were differentiated into macrophages prior to infection. Each of the bacterial strains were used to infect macrophages at a multiplicity of infection (MOI) of five. After four hours of incubation, host cells were used for: a) metabolomics via whole metabolome extraction for either detection with untargeted GC×GC-TOFMS (Chapters 3 and 4) or targeted liquid chromatography tandem mass spectrometry (LC-MS/MS) with stable isotope carbon-13 (13C6)-glucose tracing (Chapter 5); b) bioenergetics through cell mitochondrial stress tests (CMSTs) and glycolytic rate assays (GRAs) performed in an extracellular flux analyser (Chapter 5); and c) lysis to harvest intracellular bacteria for RNA extraction to perform RT-qPCR (Chapter 6). All experiments were repeated to ensure reproducibility of the data, which were analysed by the appropriate statistical tests to highlight significant differences or similarities amongst the models, groups and samples. Results/Discussion: Bacterial RT-qPCR and functional metabolomics through GC×GC-TOFMS revealed that the Δmtp was associated with an altered cell wall composition. Of the 28 metabolites shortlisted, 19 of them were detected in elevated concentrations in the Δmtp compared to the WT, thus indicating that the lack of MTP reduces the overall ability of the pathogen to utilise these metabolites for biological processes. This was deduced from the high concentrations of carbohydrates involved in cell wall biogenesis and functions, and fatty acid metabolism. Additionally, alterations to amino acid concentrations further explained the previously reported growth retardation compared to the WT. Mycobacterium tuberculosis infected THP-1 macrophage models revealed significant alterations to central carbon metabolism (CCM), fatty acid metabolism and amino acid metabolism in the absence of MTP. In particular, the Δmtp infected THP-1 macrophages demonstrated that nine of the GC×GCTOFMS detected metabolites were significantly higher relative to the WT infected THP-1 macrophages. These altered concentrations were observed for metabolites involved in glycolysis/gluconeogenesis, tricarboxylic acid (TCA) cycle, fatty acid and amino acid metabolic pathways that feed into or are fed by CCM. Given that M. tuberculosis establishes contact with both macrophages and epithelial cells during infection, a comparison of two different host cells were performed. Metabolite concentrations compared between the THP-1 macrophage infection model and A549 epithelial infection model demonstrated that the macrophages are more likely to advocate for pathogen clearance through a more efficient pro-inflammatory response, particularly in the absence of MTP, relative to the epithelial cells. Data presented here demonstrated that the MTP adhesin plays a role in securing a protective environment for M. tuberculosis during initial stages of infection. Therefore, the THP-1 macrophage infection model was further investigated by targeted LC-MS/MS and bioenergetics to ascertain a more comprehensive understanding of the carbon flux, energy profiles and macrophage polarity in the absence and presence of MTP. The Δmtp infection model mimicked the oxygen consumption rate (OCR) for basal respiration, adenosine triphosphate (ATP) production, maximal respiration, and spare respiratory capacity of the uninfected THP-1 macrophages and had the highest compensatory glycolytic rate. Furthermore, the tracing experiments revealed that MTP has no effect on the flux of glucose, but rather has an impact on the total concentrations of the macrophage infection model. These findings indicate that MTP plays a role in modulating the macrophage respiratory parameters and dampens the pro-inflammatory response of the host. Hence, the macrophage host will be more proficient at launching an immune response if the adhesin is absent. The RT-qPCR performed on selected genes from pure bacterial culture and intracellular bacteria demonstrated that removal of MTP from M. tuberculosis alters lipid import, β-oxidation, and lactate oxidation of M. tuberculosis. Moreover, in all models in which the mtp-complement was used, partial restoration of the phenotype was observed as a result of the overexpressing episomal plasmid. Cumulatively, MTP was evidenced to play a multifunctional role for M. tuberculosis. Conclusion: The surface-located adhesin, MTP, is a virulence factor of M. tuberculosis as it initiates and facilitates attachment to, and invasion of hosts in addition to providing structural integrity to the pathogen. This adhesin enhances bacterial survival as it reprograms the M. tuberculosis and THP-1 macrophage metabolism to manipulate host effector functions. Collectively, this study provided metabolomic, bioenergetic and molecular evidence that MTP significantly contributes to the success of M.tuberculosis as an intracellular pathogen. Hence, the focus of TB intervention strategies should include intercepting the MTP interaction with the host to offset M. tuberculosis pathogenicity and confer an advantage to the host.
Cloning, expression and purification of mycobacterium tuberculosis RV0309 adhesin protein.
(2023) Deyal, Nikita.; Pillay, Manormoney.
Tuberculosis (TB) remains a global burden despite major advances in the design of rapid diagnostics and therapeutics. Mycobacterium tuberculosis (Mtb) adhesin proteins are key in the pathogenicity and virulence of Mtb and are potential biomarkers for diagnostics, drug targets or vaccine candidates. Numerous adhesin proteins have been considered; however, they have not proven to be both highly sensitive and specific in point-of-care tests. The increased emergence of drug-resistant strains, lack of suitable treatment regimens and poor compliance to treatments demonstrate the need for novel targets for therapeutic intervention. Research has shown that the relatively unknown Mtb Rv0309 adhesin membrane protein is essential in the growth, biofilm development, and cellular morphology of Mtb. The Rv0309 gene also enhances mycobacterial intracellular survival after infection, and deletion of the gene may decrease the infecting potential of the resultant mutant strain. Hence, the Rv0309 adhesin encoding L-D transpeptidase was investigated in the current study for its ability to be cloned in glutathione S-transferase (GST)- and polyhistidine- tagged (His-tag) vectors, expressed and purified efficiently for future downstream processing. The Rv0309 gene was amplified using polymerase chain reaction (PCR). The plasmid DNA of pGEX-6P-1 (GST-tagged) and pET28a (His-tagged) vectors was extracted. The Rv0309 DNA and vector DNA were restricted with the appropriate restriction endonucleases, ligated and transformed into E. coli BL21 cells. Transformants were confirmed by colony PCR and plasmid DNA sequencing. Recombinant protein expression was optimised using various isopropyl β-D-1-thiogalactopyranoside (IPTG) concentrations, time intervals and visualised using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. Protein lysis and purification trials were performed and visualised using SDS-PAGE and Western blotting. The target band found in eluent was excised and sent for peptide mass fingerprinting for protein confirmation. The Rv0309 gene was successfully cloned into both vectors and optimally expressed in Escherichia coli (E. coli). Despite employing an array of lysis and purification techniques, obtaining a pure form of the recombinant protein remained elusive. There was some success with pure protein being obtained during the study; however, the concentration was low and results were not reproducible. The main problems were sub-optimal lysis of the Rec-protein and ineffective binding to the purification column. Based on the bioinformatics analysis performed and information from GenScript, the reason for these problems was the high hydrophobicity of this protein. The insights gained from the lysis, purification and bioinformatic analysis of Rv0309 in this study contribute to the understanding of membrane protein biochemistry and the intricacies associated with Mtb protein purification.
Triangulating the molecular epidemiology of carbapenem-resistant enterobacterales from humans, food Animals, and the environment.
(2021) Ramsamy, Yogandree.; Mlisana, Koleka Patience.; Essack, Sabiha Yusuf.
Introduction Antimicrobial resistance (AMR) is largely a consequence of selection pressure, from indiscriminate antimicrobial use in humans and animals, however release of other resistance-driving chemicals such as metals and biocides also play a role in development of AMR. Disposal of these drivers of AMR into the environment, requires One Health approach towards its understanding and containment. The ongoing dissemination of carbapenem-resistant Enterobacterales (CRE), particularly carbapenemase-producing Enterobacterales (CPE), represents a significant public health issue threatening the lives of millions globally. Carbapenems, in human health, are antibiotics of last resort, and conserving them for the future is of utmost importance. Therefore, it is critical to conduct surveillance of CRE and CPE in a One Health context using molecular techniques to determine a representative picture of the overall problem of CRE, its evolution and dissemination. This point prevalence study ascertained the carriage of CPE in humans, livestock animals (pigs), and environmental sources within the same geographical area of the uMgungundlovu district, KwaZulu-Natal, South Africa and triangulated the molecular epidemiology of CPE in humans, food animals, and the environment. Methodology The point prevalence study involved collecting rectal swabs from pigs and humans along with environmental water samples collected from a wastewater treatment plant that received water from both the hospital and abattoir. All samples were processed at the accredited National Health Laboratory Service (NHLS) Public Health Laboratory in KwaZulu-Natal. Selective chromogenic agar was used to isolate CPE from all samples obtained across the three sectors. Microbiological processing and analysis of samples were undertaken as per standard operating procedures of the NHLS. Bacterial identification and antibiotic susceptibility testing were performed using the VITEK® 2 automated system. Pure isolates were then subjected to whole genome sequencing (WGS), and generated sequence data were analysed using different bioinformatic tools, to determine the resistomes, virulomes, mobilomes, clonality, and phylogenomics of these isolates. Results Of 587 rectal swab samples screened for CPE, 230 (39.1%) were from humans, 345 (58,7%) were from pigs with 12 (2%) water samples. A total of 19/587 (3.2%) isolates i.e., 15 from humans and four from the environment, were CRE. All the environmental isolates (4) and 12/15 human isolates were carbapenemase producers. The three non-carbapenemase producing human isolates were resistant to ertapenem but susceptible to meropenem and imipenem. No CPE were isolated from the pig samples. Sixteen of the nineteen isolates were CPE. The most common CPE was Klebsiella pneumoniae 9/16 (56%), followed by Enterobacter hormaechei 3/16 (19%), Klebsiella quasipneumoniae 2/16 (13%), a novel ST498 Citrobacter freundii 1/16 (6%), and Serratia marcescens 1/16 (6%). Carbapenem xx resistance was attributed to plasmid-mediated carbapenemase encoding genes: blaOXA-181, blaOXA-48, blaOXA-484, blaNDM-1, and blaGES-5. Notably blaOXA-181 and blaNDM-1 were found in both human and environmental isolates. Common MGEs were found in different bacterial species/clones across humans and the environment. The IncFIB(K) plasmid replicon was found in all isolates of K. quasipneumoniae (2) from the environment and the majority of the K. pneumoniae strains (7/9) from humans. The majority of the K. pneumoniae isolates were OXA-181 (5/9) producers. The vast majority of β–lactamase encoding genes were associated with class 1 integrons IntI1, insertion sequences (IS) (IS91, IS5075, IS30, IS3000, IS3, IS19, ISKpn19, IS5075) and transposons (Tn3). The Col440I plasmid replicon the most common and identified in 11 (26.82%) isolates, mostly E. hormaechei (n = 6). The IncL/M(pMU407) and IncL/M(pOXA48) plasmid replicons were found exclusively in Klebsiella pneumoniae, with all but one of these isolates being OXA-181 producers. Virulence determinants were predicted for the eleven Klebsiella spp. as the most common species isolated where a total of 80 virulence genes were delineated. Phylogenomic analysis with other South African carbapenemase-producing K. pneumoniae, E. hormaechei, S. marcescens, and C. freundii from different sources (animals, environmental sources, and humans) revealed that some species from this study clustered with clinical isolates, some clustered according to sequence type and other species belonged to the same clonal node as other clinical isolates. Phylogenetics linked with metadata revealed that some isolates clustered according to the source. Notably, five Aeromonas spp. isolates, part of a novel sequence type – ST657, and habouring the blaCPHA-3 and blaOXA-12 genes were obtained from pigs during the screening process of this One Health point prevalence study. Although these isolates were resistant to imipenem, they were not CPE. Two ARGs were noted, blaCPHA3 and blaOXA-12, conferring the resistance to imipenem and penicillin (ampicillin and amoxicillin). No MGEs were identified in these isolates. Conclusion This One Health Study delineated the resistome, mobilome, virulome, and phylogeny of CPE in human and the environment sectors, highlighting the potential propagation of carbapenemase antibiotic resistance genes via diverse MGEs across the sectors. Such genomic fluidity highlights the need for comprehensive, integrated genomic surveillance in a One Health context to address AMR successfully.
Single cell ribonucleic acid sequencing in Tuberculosis research.
(2021) Mbano, Ian Maheti.; Leslie, Alasdair.
Tuberculosis (TB) remains a global challenge, with approximately 1,5 million deaths annually. Addressing deficits in our understanding of disease pathology and treatment is needed for the development of new treatment modalities. Despite much effort, prevalence of this disease remains high in resource limited regions, where research capacity is not sufficient to successfully combat the endemic. Research in developed countries has generally been constrained to animal models due lack of access to clinical samples from the site of TB disease, the human lung. Although these animal models have their utility, it is essential that findings from these systems be tested and validated in human tissue. In this thesis, I leveraged a relatively new technology called Seq-well, which is highly portable and low-tech single cell ribonucleic acid sequencing (scRNAseq) platform and access to TB infected lung tissue obtained from lung resections, to generate a single cell atlas of TB affected lung tissue. This involved processing the human tissue immediately post-surgery and loading unprocessed/neat cells or FACS sorted cells (tissue resident t cells) onto a microarray that allowed capture and subsequent sequencing of the cell transcriptomes. In the first part of the thesis, I identified and profiled cellular subsets from TB infected tissue, focussing on a subset of FAP+PDPN + fibroblasts associated with the organisation of tertiary lymphoid organs. I also demonstrated that this dataset can be useful in evaluating current and future TB biomarkers, by superimposing signatures from the literature onto the cellular subsets and localizing them to different parenchymal, stromal and immune cell types. I also profiled tissue resident CD4 T cells from the same lung tissue, identifying canonical marker genes (ITGA1, PRF1) in one specific cluster, together with naive (CCR7, SELL), regulatory (RORA) and activated/myeloid-like T cells (LYZ, S100A9) in separate clusters. Finally, I demonstrated the applicability of this dataset in research involving other pulmonary diseases, by identifying ACE2+ TMPRSS2+ type 2 pneumocytes, a target of the SARS-CoV-2. Taken together, these findings provide new insights into the immunopathology of TB in the human lung together with the impact of HIV on specific immune subsets. It serves as a resource for cross validation of lung immune signatures generated in experimental infections of both mice and non-human primates, which is beneficial for scientists lacking access to the technology and/or tissue. Iqoqa Isifo sofuba (i-TB) silokhu siyinselelo emhlabeni jikelele, ngokufa okuhlobene naso okucishe kufike esigidini esi-1.5 njalo ngonyaka. Ukubhekana nokushoda ekuqondeni kwethu umumosakhiwo wesifo bese kuncishiswa ukufa. Ngaphandle kwemizamo emikhulu, ukudlanga kwalesi sifo kusalokhu kuphezulu ezifundeni ezintula imithombokusiza, lapho umthamokwenza wocwaningo unqindekile. Ucwaningo emazweni asethuthukile, ngakolunye uhlangothi, belwenzeka kuphela kumamodeli asebenzisa izilwane ngenxa yokuntuleka kokufinyelela amasampuleni okwelapha engxenyeni okuqubuke kuyo isifo sofuba, okuyiphaphu lomuntu. Nakuba kunamamodeli ezilwane anomsebenzi, kubalulekile ukuba okutholakele kulezo zinhlelo kuyohlolwa bese kuqinisekiswa ngesigqa somuntu ukuqinisekisa ubunjalo. Kule thesisi, ngiveze ubuchwepheshe obusha obungenayo obubizwa nge-Seq-well, iseli eyodwa e-low-tech ephathekayo ene-ribonucleic acid sequencing (scRNASeq) okuyindawo kanye nokufinyelela esicutshini sephaphu esitheleleke ngesifo sofuba esitholakale ekuhlukanisweni kabusha kwamaphaphu okukhonjwe ngokokwelapha, ukwakha iseli eyodwa yesicutshana sephaphu elitheleleke ngesifo sofuba. Lokhu kwafaka ukusebenzakuhlola isicubu somuntu ngokushesha emva kokuhlinza nokufaka amaseli ahlanzekile angasetshenziwe noma amaseli ahleliwe angama-FACS (ama-T cells asesicutshini) ohlelweni lolibofuzo olwavumela ukufaka ohlwini nokulandelanisa okulandelayo womumofuzo oqondene nezicubu. Engxenyeni yokuqala yethesisi, amaqoqwana ahlonziwe nafakwe kwiphrofayli esicubini esitheleleke ngesifo sofuba kugxilwe eqoqweni le FAP+PDPN + amafayibhroplasti ahlobene nokuhlelwa kwezingxenye zomzimba ezinkulu zamalimfoyidi kanye nemichilwana yamafayibhrodi kanye noma igranyuloma yesifo sofuba. Ngivezile ukuthi lamadathasethi angaba nomsebenzi omkhulu ekuhlaziyeni amabhayomakha amanje nawasesikhathini esizayo esifo sofuba, ngokufaka izinkombabunjalo emaqoqweni amancane nokuwabeka ezinhlotsheni ezehlukene zamaseli angamapharenikhayma nangamastroma. Ngiphinde ngachaza esizindeni sezicutshana ze-CD4 T esicutshini sephaphu elifanayo okuchaza ulibofuzo olukala amakhenoni (i-ITGA1, PRF1) eqoqweni elilodwa eliqondile, kanye namaseli angachazi lutho (CCR7, SELL), alawulayo (RORA) nama-T cell aqaliswe ukusebenza/efana ne-myeloid (LYZ, S100A9) emaqoqweni aseceleni. Okokugcina, ngiveze ukungena kwedathasethi ocwaningweni olufaka izifo zamaphaphu nokuphefumula ngokuhlonza i- ACE2+ TMPRSS2+ type 2 wama-pneumocytes, okuhlosiwe kwe-SARS-CoV- 2. Uma kuhlanganisiwe, lokhu okutholakele kuletha imibono emisha yomumobugciwane bokutheleleka ngesifo sofuba ephashini lomuntu, umthelela we-HIV kokutholakele emumwenikuphila kwephaphu ekuthelelekeni okuyilinga kwakho kokubili amagundane kanye nalokho okungebona abantu.
Effect of hybrid immunity on the neutralizing antibody response to emerging SARS-CoV-2 variants including in people living with HIV=Umphumela wamasotsha omzimba ahlobongxube ekutheneni amandla impendulosenzo yezivikelamzimba ekuhlanganiseni izinhlobokuguquka zamagciwane kubandakanya nabantu abaphila ne-HIV.
(2022) Khan, Bibi Khadija.; Sigal, Alexander.
This work focused on the effect of SARS-CoV-2 variants and co-infection with HIV on the neutralizing antibody response elicited by SARS-CoV-2 infection and vaccination. The first study described the effect of HIV status and suppression on antibody neutralization capacity against the Delta variant elicited by previous infection, the Janssen adeno vectored Ad26.CoV2.S vaccine, or hybrid immunity from infection followed by Ad26.CoV2.S vaccination. It was determined that while neutralizing immunity elicited by infection decreased in people living with HIV (PLWH) and particularly in those with unsuppressed HIV viremia, this effect was counteracted with hybrid immunity from infection and Ad26.CoV2.S vaccination, where with hybrid immunity neutralization levels increased and differences between PLWH and HIV negative individuals became non-significant. With the emergence of the Omicron variant, we investigated whether Omicron infection boosts cross-neutralizing antibody levels against other variants. It was determined that this cross-protection does occur but is considerably stronger in people with hybrid immunity consisting of Pfizer BNT162b2 mRNA or the Ad26.CoV2.S vaccination followed by breakthrough infection, implying that hybrid immunity from Omicron variant infection and vaccination may prevent infection with other, more pathogenic variants. Lastly, we demonstrated the escape of the BA.4 and BA.5 subvariants from Omicron BA.1 subvariant elicited immunity. It was found that BA.4 and BA.5 showed substantial escape from BA.1 elicited immunity in the absence of vaccination, but the escape was more moderate in individuals with hybrid immunity from BA.1 infection combined with vaccination. Overall, these studies show the capacity of hybrid immunity, consisting of vaccination and infection, to reduce the negative effects of HIV viremia and emerging variants on the ability of the pre-existing immunity to neutralize SARS-CoV-2. Iqoqo Lo msebenzi wawugxile emiphumeleni yezinhlobokuguquka zamagciwane e-SARS-CoV-2 kanye nesifo eziphila neHIV ekutheneni amandla impendulosenzo yezivikelamzimba ezidalwa isifo i-SARS-CoV-2 kanye nokugomakuhlungisa. Ucwaningo lokuqala lwachaza ngomumo nokucindezela kwesilinganiso sokuthena amandla isivikelimagciwane kuhlobokuguquka iDelta ezidalwa isifo esedlule, iJanssen adeno ithwala isifo somgomo we-Ad26.CoV2.S noma amasotsha omzimba anhlobongxube asuka esifweni esilandelwa ukugomakuhlungisa kwe-Ad26.CoV2.S. Kwanqunywa ukuthi ngenkathi amasotsha omzimba ethenwa amandla okudalwa ukwehla kwesifo ebantwini abaphila ne-HIV (PLWH) ikakhulukazi kulabo elingacindezelwe egazini igciwane le-HIV, lo mphumela wawuphikisana namasotsha anhlobongxube asuka esifweni kanye nakumgomokuhlungisa we-Ad26.CoV2.S, lapho amazinga okuthena amandla amasotsha anhlobongxube ekhula kanye nomehluko phakathi kwe-PLWH kanye nalabo bantu abangenayo i-HIV abavele babe ngabangekho mqoka. Ukuqhamuka kohlobokuguquka kwegciwane i-Omicron, saphenya ukuba ngabe isifo i-Omicron ikhuthaza amazinga esivikelamzimba esithena amandla kwezinye izinhlobokuguquka zamagciwane. Kwanqunywa ukuthi ukuvikelana akwenzeki kodwa kuthathwa ngokunamandla kubantu abanamasotsha anhlobongxube equkethe i-Pfizer BNT162b2 mRNA noma umgomokuhlunga i-Ad26.CoV2.S okulandelwa isifo esehlula umgomo, okusho ukuthi amasotsha anhlobongxube asuka esifweni sohlobokuguquka kwegciwane i-Omicron kanye nokugomakuhlunga kungasivimba isifo nezinye eziningi zinhlobokuguquka zemindeni yamagciwane. Okokugcina, sabonisa wukuphunyuka kwezinhlokuguquka zamagciwane ezincane i-BA.4 ne-BA.5 kusuka hlobokuguquka kwegciwane okuncane i-Omicron BA.1 eveza amasotsha omzimba. Kwatholakala ukuthi i-BA.4 ne-BA.5 ikhombisa ukuphunyuka okubonakalayo ukusuka ku-BA.1 eveza amasotsha omzimba uma kungenziwanga ukugomakuhlungisa kodwa ukuphunyuka kwakungakukhulu kulabo abanamasotsha omzimba anhlobongxube kusuka esifweni i-BA.1 ihlanganiswe nokugomakuhlungisa. Phezu kwakho konke, lolu cwaningo lwakhombisa isilinganiso samasotsha omzimba anhlobongxube, aqukethe abunjwe umgomokuhlungisa nesifo, ukuze kuncishiswe imiphumela engemihle yamagciwane asegazini e-HIV kanye nezinhlobokuguquka zamagciwane ekukwazini ukuthi amasotsha omzimba abevele ekhona ngaphambilini athene amandla i-SARS-CoV-2.
Effect of HIV status and suppression on SARS-CoV-2 disease severity, vaccine response, and evolution=Umthelela wesifo sesandulela-ngculazi nokucindezelwa kwaso yi-SARS-CoV-2 ubunzima besifo, ukusebenza kwekhambi kanye nokuthuthuka.
(2023) Karim, Farina.; Sigal, Alexander.; Moosa, Mahomed Yunus Suleman.
Abstract SARS-CoV-2 is a global pandemic that has infected 672,115,430 people globally (https://coronavirus.jhu.edu/map.html, accessed 08.02.2023). SARS-CoV-2 is continuously evolving, and new variants pose a continuous threat to curbing this pandemic. Simultaneously, South Africa still struggles to control and manage an enduring HIV pandemic. The synergistic interplay between these two pandemics has necessitated an understanding of how these two viruses interact with each other to tailor an intervention. This thesis investigated the effect of SARS-CoV-2 infection on disease dynamics, differential disease outcomes, vaccine response, as well as SARSCoV- 2 evolution in people living with HIV (PLWH) with different levels of HIV suppression and differing HIV suppression history. The first study investigated the difference in disease severity amongst PLWH in the first and second infection waves in South Africa. COVID-19. Thereafter, we explored persistent SARS-CoV-2 infection in immunocompromised individuals and intra-host evolution in a case study of a participant with advanced HIV infection. Here, we illustrated that advanced HIV disease may lead to prolonged SARS-CoV-2 infection and shedding of infectious virus and results in intra-host evolution of variant mutations, making intra-host evolution in advanced HIV individuals a particular concern within the South African context. Finally, we observed that effectively controlling HIV through ART facilitates SARS-CoV-2 clearance. The last study widened the observations to five participants with advanced HIV disease and showed that vaccination does induce a potent neutralizing antibody response in this group, but only if HIV viremia is first effectively suppressed with antiretroviral therapy. These findings highlight the importance of suppressing HIV infection in eliciting an effective immune response against, and preventing evolution of SARS-CoV-2. Iqoqa I-SARS-CoV-2 yisifo esesigulise umhlaba wonke sase sigulisa inani elingama 672,115,430 abantu emhlabeni jikelele (https://coronavirus.jhu.edu/map.html, accessed 08.02.2023). I-SARS-CoV-2 isaqhubeka nokusabalala kanti izinhlobo zayo ezinye zisaqhubeka nokuba yingozi ukuba isifo singanqandeka. Khona-manjalo, iNingizimu Afrikha isathwele kanzima ukulawula isifo sesandulela-ngculazi. Ukuthelelana nokuxhumana phakathi kwalezi zifo kwenze kwanesidingo sokuqonda ukuthi zidlelana kanjani ukwenzela ukuphuma nekhambi. Lolu cwaningo lucwaninge umthelela we SARS-CoV-2 nezinkinga ezikhona ngenxa yazo, imiphumela ehlukene yesifo, ukusebenza kwekhambi, kanjalo nokukhula kwe SARS-CoV-2 ebantwini abaphila naso lesi sifo bebe benesandulela-ngculazi benezinga lesandulela-ngculazi elehlukile kanye nomlando owehlukile. Ucwaningo lokuqala lucubungule umehluko wesankahlu sesifo ezifweni ze-PLWH esiwombeni sokuqala nesesibili salesi sifo eNingizimu Afrikha. Ngakho-ke iye yahlolwa kwalendela umthelela we-SARS-CoV-2 kubantu abanamasosha aphansi omzimba kanye nalokhu esikubiza nge-intra-host evolution ocwaningweni lwesimo kubabambi-qhaza abanesandulela-ngculazi esisezingeni eliphezulu. Lapha kutholakale ukuthi isandulela-ngculazi esisezingeni eliphezulu singaholela ekuguleni isikhathi eside uma unesifo seSARS-CoV-2 kanye nokuthola ezinye izifo ezithelelanayo kanti imiphumela ye-intra-host evolution of variant mutations, okwenza ukukhula kwe-intra-host kulabo abanesandulela-ngculazi ephezulu kusabise isimo saseNingizimu Afrikha. Isiphetho, kutholakale ukuthi ukulawula isandulela-ngculazi nge-ART kwenza iSARS-CoV-2 idambe. Ucwaningo lokugcina luveze ngokubanzi obekubukelwe kubabambiqhaza abanesandulela-ngculazi esiphezulu ngokuthi kuvele ukuthi ikhambi lenza kudambe ukugula kuleli qoqo, kodwa kuphela uma isandulela-ngculazi siqale sacindezelwa nge-antiretroviral therapy. Lemiphumela iveza ukubaluleka kokucindezelwa kwesandulela-ngculazi ukulwa kanye nokucindezela isifo seSARS-CoV-2.
Epidemiology and alternative approaches for SARS-CoV-2 testing within limited resources settings=Isifundo ngembangela nokusabalala kwezifo i-epidemiology nezindlela ezahlukile zokuhlolwa kwe-SARS-CoV-2 esimeni sokwesweleka kwezinsiza.
(2023) Duma, Zamathombeni.; Mkhize-Kwitshana, Zilungile Lynette.; Chuturgoon, Anil Amichund.; Ramsuran, Veron.
Background: In the context of the global battle to contain the rapidly mutating SARS-CoV-2, diagnostic testing for SARS-CoV-2 infection remains a challenge, particularly in low-middleincome countries (LMICs) due to low socioeconomic backgrounds. Concerningly, because less attention is paid to asymptomatic cases, particularly in LMICs with limited resources for SARSCoV- 2 testing, the virus is spreading silently in communities, and the majority of these individuals could be contributing to the resurgence of SARS-CoV-2 infection. This study aimed to determine the epidemiology and alternative approaches for SARS-CoV-2 testing within limited resources settings. Methods: A total sample size of 1335 residual patient samples from the Global Health Innovation (GHI) laboratory was used for the epidemiology study and methods comparison. Results and Discussion: Literature review showed that high income countries (HICs) test more frequently for SARS-CoV-2 infection, with a range of 113% to 146% higher than LMICs (1% to 43%). The present study demonstrated a higher proportion of asymptomatic cases (68%) among SARS-CoV-2 infected patients. Regarding the methods comparison for the detection of SARSCoV- 2, the evaluated alternative methods [three RNA extraction (Lucigen QuickExtract™ RNA Extraction Kit, Bosphore EX-Tract Dry Swab RNA Solution, Sonicator method and four commercial SARS-CoV-2 RT-PCR assay kits (Nucleic Acid COVID-19 Test Kit (SARS-CoV-2), abTESTM COVID-19 qPCR I Kit, PCL COVID19 Speedy RT-PCR Kit, and PCLMD nCoV One- Step RT-PCR Kit)] were found to be cheaper and faster. Conclusion: Notably LMICs are undertesting for SARS-CoV-2 infection compared to HICs, and there was a higher proportion of asymptomatic cases among SARS-CoV-2 infected patients in South Africa. This study suggests that using the above-mentioned cost-effective, quick, and accurate evaluated alternative methods for mass SARS-CoV-2 testing in routine diagnostic laboratories with limited resources can help to increase testing capacity for SARS-CoV-2 infection in LMICs. This means that the sooner SARSCoV- 2 infection control and prevention measures can be implemented to reduce community transmission. IQOQA Isendlalelo: Esimeni somzabalazo womhlaba jikelele wokuvimba i-SARS-CoV-2 eguquguquka ngokushesha, uvivinyo oluhlolayo lokutheleleka nge-SARS-CoV-2 luselokhu luyinselelo, ikakhulu emazweni anemiholo ephansi kuya kwephakathi nendawo (low-middle income countries - MICs) ngenxa yesizinda senhlalomnotho ephansi. Ngokuxakayo-ke, ngenxa yokunganakwa kokutheleleka okungenazimpawu, ikakhulu ama-LMIC anezinsiza zokuhlolela i-SARSCoV-2, igciwane liyanda buthule emiphakathini, futhi iningi lalaba bantu lingase libe nomthelela yokuvembuka kabusha kokuthelelana nge-SARSCoV-2. Lolu cwaningo lwaluhlose ukuthola imbangela nokusabalala kwezifo i-epidemiology nezinye izindlela zokuhlola i-SARSCoV-2 esimeni sokwesweleka kwezinsiza. Izindlela: Kwasetshenziswa isampula eliphelele lamasampula eziguli ezisilele eziyi-1332 avela emalaborethri akwaGlobal Health Innovation (GHI) ngokocwaningo lwe-ephidemiyoloji nokuqhathaniswa kwezindlela. Imiphumela nengxoxo: Imibhalo eyabuyekezwa yakhombisa ukuthi amazwe anemiholo ephezulu (high income countries - HICs) ahlolela ukutheleleka kwe-SARS-CoV-2 kaningana ngokwebanga eliyi-113% kuya kweliyi-146% ngaphezu kwama-LMICs (1% kuya kuma-43%). Lolu cwaningo lwakhombisa isibalo esiphezulu lotho olungenazimpawu (68%) phakathi kweziguli ezitheleleke nge-SARS-CoV-2. Mayelana nezindlela zoqhathaniso lokuhlolwa kwe-SARS-CoV-2, izindlela ezahlukile ezihloliwe zokukhishwa kokuthathu (Lucigen QuickExtract™ RNA Extraction Kit, Bosphore EX-Tract Dry Swab RNA Solution, nendlela iSonicator) nezinsiza ze-assayi i-SARS-CoV-2 RT-PCR (Nucleic Acid COVID-19 Test Kit (SARS-CoV-2), abTESTM COVID-19 qPCR I Kit, PCL COVID19 Speedy RT-PCR Kit, ne-PCLMD nCoV OneStep RT-PCR Kit)] okwatholwa kushibhile futhi kushesha. Isiphetho: Ngokuqaphelekayo ama-LMIC akuhlolela kancane ukutheleleka nge-SARS-CoV-2 uma kuqhathaniswa nama-HIC, futhi kwakunesibalo esiphezulu sotho olungenazimpawu phakathi kweziguli ezitheleleke nge-SARS-CoV-2 eSouth Africa. Lolu cwaningo lukhomba ukuthi ukusebenzisa izindlela ezibalwe ngenhla ezishibhile, ezisheshayo, futhi ezinembayo nezihloliwe futhi ezingezinye izindlela zokuhlola isisindo se-SARS-CoV-2 engukuhlola okwejwayelekile emalabhorethri anezinsiza ezingeziningi kungasiza ekukhuliseni ukukwazi ukuhlola kokutheleleka nge-SARS-CoV-2 ema-LMIC. Lokhu kuchaza ukuthi uma kungashesha ukulawulwa kokutheleleka nge-SARS-CoV-2 nezinyathelo zokuvikeleka kungenziwa ukunciphisa ukuthelelana emphakathini.
Prevalence, phenotypic and genotypic characterization of resistant clinical gram-negative isolates at Kamuzu Central Hospital, Lilongwe Malawi.
(2022) Choonara, Faheema Ebrahim.; Essack, Sabiha Yusuf.; Sundsfjord, Arnfinn Staale.; Lampiao, Fanuel.
nterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp. (ESKAPE) pathogens are the leading cause of nosocomial infections worldwide. They are highly virulent, multi-drug resistant (MDR) and therefore difficult to treat posing major public health and clinical challenges globally, particularly in developing countries where resources and microbiology diagnostic services are limited or not available. The aim was to investigate prevalence, phenotypic and genotypic resistant profiles of selected ESKAPE and other important bacterial pathogens isolated from adult patients admitted at Kamuzu Central Hospital (KCH) Escherichia coli and Staphylococcus aureus were the dominant species isolated. Multi-drug resistance and extended spectrum β-Lactamase -production was evident in K. pneumoniae (n=20/29; 69%) and E. coli (49/92; 53%). Pseudomonas aeruginosa was resistant to meropenem but none were carbapenemase producers. MRSA was detected in 10.5% (n=9/86) of S. aureus. These MDR isolates were mostly isolated from pus specimens from the surgical department Genotypically, the CTX-M type (55/60; 92%) and CMY type (16/21) were most prevalent among phenotypically-positive ESBL and pAmpC β-lactamases respectively. Both CTX-M and CMY were most prevalent in E.coli with 71% (15/21) carrying both CTX-M and CMY The most common sequence type in the CTX-M group 1 and CTX-M group 9 positive E.coli was ST410 (n=14/29; 48%) and ST131 (n=5/7; 71%) respectively; all of which contained the blaCTX-M-15 resistance gene. In CMY positive E. coli, ST410 was the most prevalent and all contained blaCMY- 2 resistance gene. All the E.coli isolates carrying both CTX-M and CMY were ST410 and contained both blaCMY- 2 and blaCTX-M-15 resistance genes. All phenotypically confirmed methicillin resistant Staphylococcus aureus (MRSA) contained mecA gene and t064 was most common spa type. Spa type t355 was most common in S. aureus that were negative for mecA gene Findings demonstrate the need for continuous antibiotic resistance surveillance at the hospital to inform antibiotic treatment options. There is also a need for the establishment of antibiotic stewardship programs to sustain the efficacy of antibiotics in Malawi
Diversity of rodent-borne zoonotic pathogens at the human-animal-environment interface in Qatar.
(2021) Islam, Md. Mazharul.; Mkhize-Kwitshana, Zilungile Lynette.; Farag, Elmoubashar Abubaker Abd.
Rodents are the most diversified terrestrial mammals in the world. These animals assist with maintaining a healthy ecosystem through the soil structure modification, aeration, and hydration, although 5-10% are regarded as pests and carry zoonotic pathogens. Besides consumption and damage of our food and property, they are responsible for the transmission of several diseases, including plague, typhus, and leishmaniasis. Commensal rodents are the primary source of these pathogens because of their close proximity to humans. Qatar is a small country in the Arabian Peninsula. Four rodent species have been recorded in this country, that includes three commensal (Mus musculus, Rattus norvegicus, and Rattus rattus) and one wild (Jaculus jaculus) species. The zoonotic importance of rodents is yet to be explored. Knowing the pathogens originating from rodents is essential for early preparedness, prevention, and control. Therefore, the current study was undertaken on commensal rodents, rodentborne zoonotic pathogens, and the factors that are associated with pathogen prevalence among rodents, such as rodent sex, age, and trapping location in Qatar. A cross-sectional study was conducted between August 2019 and February 2020, which trapped rodents from different facilities, such as livestock and agricultural farms, bachelor and family accommodations, and industrial and commercial areas of Qatar. After studying the morphological and morphometric characters, blood samples, ectoparasites and visceral samples were collected from the captured rodents. Parasitic, bacterial, and viral pathogens were identified and characterized using gross, necropsy, microscopic, culture, biochemical, immunologic, and molecular methods. Descriptive statistics and univariate analysis were conducted to detect rodents, rodent-borne pathogens abundance, and the related risk factors. The study trapped 148 rodents, most of which were adults (n = 138, 93.2%, 95% CI: 87.92–96.71), and from livestock farms (n = 79, 49%, 95% CI: 41.02–57.65). R. norvregicus was the most prevalent (n = 120, 81%, 95% CI: 73.83–87.05), followed by R. rattus (n=24, 16%, 95% CI:10.68–23.16) and M. musculus (n=4, 3%, 95% CI: 0.74– 6.78) with an average body weight of 18.8 ± 2.2 gm, 264.3 ± 87.5 gm, and 130 ± 71.3 gm, respectively. This is the first morphologic and morphometric study of commensal rodents in Qatar and the Arabian Peninsula that detected the Qatari rodents are relatively smaller than those of Turkey, Tunisia, and Iran. About 63.5% of the rodents were infected with at least one of the 9 species of parasites, viz. Xenopsylla astia, Ornithonyssus bacoti, Hymenolepis diminuta, Taenia taeniaeformis, Capillaria annulosa, Strongyloides spp., Giardia spp., Toxoplasma gondii, Trypanosoma lewisi, and Leishmania spp. Helminths were the most prevalent (46.0%), followed by ectoparasites (31.8%) and protozoa (29.1%). Going by individual species prevalence, X. astia ranked the highest (31.8%), where the lowest prevalent parasite was C. annulosa (0.7%). The prevalence of H. diminuta was positively correlated (OR=4.13; p = 0.00) with the prevalence of X. astia. The study also identified thirteen bacterial species, namely Acinetobacter baumannii, Aeromonas salmonicida, Citrobacter freundii, Citrobacter koseri, Enterobacter aerogenes, Enterobacter cloacae, Escherichia coli, Hafnia alvei, Klebsiella pneumoniae, Providencia stuartii, Proteus mirabilis, Pseudomonas aeruginosa, and Salmonella enterica, from the intestine samples. The majority of the bacteria were E. coli (54.63%, 95% CI: 44.76-64.24), followed by P. mirabilis (17.59%, 95% CI: 10.94-26.10), and K. pneumoniae (8.33%, 95% CI: 3.88-15.23). The study detected 31.58% (6/19, 95% CI: 12.58-56.55) of the flea pools, and one (1/1) mite pool was positive with Rickettsia spp. S. enterica showed the highest antimicrobial resistance (100% resistant to 8 antimicrobials). The top resistant antimicrobials were from cephalosporin, followed by penicillin and tetracycline groups. E. coli (26.92%, 95% CI:11.57-47.97) and K. pneumonia (50%, 95% CI: 6.76- 93.24) were ESBL (extended-spectrum beta-lactamases) producers. The studied rodents are indicators of the presence and dispersal of zoonotic pathogens in Qatar. Urgent action is needed to prevent future spillover of these pathogens at the human-animal-environment interface. It is essential to understand the biology, epidemiology, and transmission dynamics of these pathogens. Farm biosecurity and integrated pest management approach should be implemented in the farm premises. Implementing the One Health approach to combat rodent-borne zoonoses in order to reduce the risk of the future epidemic in Qatar is strongly recommended.
Using neutrophil-specific biomarkers as a measure of Mycobacterium tuberculosis burden, lung damage and treatment response kinetics.
(2021) Ndlovu, Lerato Noluthando.; Leslie, Alasdair.
The spectrum of TB disease remains poorly characterized impacting case identification and linkage to care. Furthermore, the absence of simple, sensitive and easy to measure biomarkers of disease severity and treatment response makes it difficult to distinguish patients at risk of treatment failure from those who would benefit from shorter treatment regimens. Ultimately, these challenges contribute to perpetuating the global TB epidemic. This thesis explores the potential of blood neutrophils as biomarkers for delineating TB disease, characterizing disease severity and monitoring TB treatment response. Neutrophils are short lived and rapidly responsive phagocytes that have been shown to have a strong TB associated signature. Using longitudinal samples, collected during a 2-year observational TB treatment response study, I analyzed the neutrophil response over the course of standard TB drug therapy. This included the novel assessment of a panel of neutrophil surface markers by flow cytometry. Overall, I show that an active TB infection is associated with elevated blood neutrophil counts that resolved over the course of TB treatment and is associated with disease severity, shown by direct correlation with bacterial load and extent of lung involvement. Importantly, these associations were not impacted by the high background of HIV infection present in this cohort. In addition, I identified the expression level of surface CD15 as a novel, rapid, robust and highly sensitive marker of TB disease. This correlated strongly with clinical characteristics and, uniquely, baseline CD15 expression predicted TB treatment response, as shown by solid culture conversion at 2 months. To extend these findings I measured a panel of inflammatory cytokines and neutrophil specific soluble factors by Luminex and ELISA. As shown in other studies, many plasma cytokines are elevated in subjects with active TB and reduced in response to treatment. Baseline levels of the neutrophil associated markers, S100A8/9 and TREM-1 associated most strongly with disease severity, again supporting the hypothesis that neutrophils are a good sentinel immune cell in TB. Hierarchical clustering indicated a coordinated inflammatory response to TB infection, which resolved over the course of treatment. However, these did not associate with disease severity or predict treatment outcome. Finally, I tested the potential of simple neutrophil characteristics (abundance and neutrophil:lymphocyte ratio) to identify clinical and subclinical TB identified in the community, using blood smears obtained during a recent community wide TB screen conducted in rural Kwa-Zulu Natal. Despite reasonable performance in the clinic, however, these metrics failed to distinguish symptomatic TB from sub-clinical TB, and either of these from TB uninfected community controls. Unfortunately, it was not possible to evaluate neutrophil phenotype, particularly CD15 expression level, in this setting. Taken together, these data suggest that detail measuring of the neutrophil response to TB in the clinic may provide useful information about disease severity, offer a sensitive measure of treatment response and, potentially, identify patients at baseline who may be at risk of poor treatment outcomes.
The impact of semen exposure on the immune and microbial environments of the female genital tract.
(2021) Jewanraj, Janine.; Liebenberg, Lenine Julie.; Ngcapu, Sinaye.
Background: Semen is an immunomodulatory fluid that induces mucosal changes at the female genital tract (FGT) for sperm survival and conception. Semen-induced alterations necessary for reproduction may also modulate the inflammatory environment related to HIV risk in women. This thesis investigated the impact of semen exposure on biomarkers of female genital inflammation (GI) and the persistence of these associations over time. Methods: Stored genital specimens were assessed from HIV-negative women participating in the CAPRISA 008 trial. Cervicovaginal lavage (CVL) samples were screened for Y-chromosome DNA (YcDNA) by real-time PCR as a biomarker of semen exposure within 15 days of genital sampling. Prostate-specific antigen (PSA) detection by ELISA stratified CVLs into semen exposure within 48 hours (PSA+YcDNA+) and between 3-15 days (PSA-YcDNA+). Vaginal cytokine concentrations, matrix metalloproteinases (MMPs), and tissue inhibitors of metalloproteinases (TIMPs) were assessed in CVLs using multiplexed ELISA. Endocervical T-cell frequencies were measured in cytobrushes by flow-cytometry. Vaginal microbes and sexually transmitted infections (STIs) were detected in vulvovaginal swabs by PCR. Results: Self-reported condom use as a measure of semen exposure was not associated with changes in the FGT microenvironments. Conversely, YcDNA detection predicted significant increases in several cytokines, barrier-related proteins, and Prevotella bivia detection (p=0.001). Since YcDNA detection alone was not associated with the immune environment linked to HIV risk, this thesis further investigated the contribution of more recent sex to female GI. PSA detection (semen exposure within 48 hours) was associated with higher YcDNA concentrations (p<0.0001), suggesting a relationship between the timing of semen exposure and vaginal YcDNA concentrations after condomless sex. In support of this, both PSA detection and higher YcDNA concentrations predicted significant increases in several cytokines, barrier-related proteins (MMP-2, TIMP-1, TIMP-4), and higher frequencies of activated CD4+HLA-DR+ T-cells (p=0.032) and CD4+CCR5+HLA-DR+ HIV targets (p=0.046). PSA detection was also associated with increased detection of several bacterial vaginosis (BV)-associated microbes and reduced Lactobacillus jensenii detection. Conclusion: Recent semen exposure contributes to the inflammatory environment associated with HIV risk in women. These studies highlight the need for clinical and immunological studies of STIs and their biomedical interventions to consider semen’s contribution to the immune and microbial microenvironments of the FGT.
Molecular epidemiology of antibiotic-resistant Escherichia coli and Enterococcus spp. from agricultural soil fertilized with chicken litter in uMgungundlovu district, KwaZulu-Natal Province, South Africa.
(2021) Fatoba, Dorcas Oladayo.; Abia, Akebe Luther King.; Essack, Sabiha Yusuf.; Amoako, Daniel Gyamfi.
The application of animal manure contaminated with antibiotic-resistant bacteria (ARB) and antibiotic-resistance genes (ARGs) represents a major route by which antibiotic resistance is transmitted into the soil environment. The introduction and persistence of ARB in agricultural soil may pose a risk to public health via the consumption or handling of contaminated farm produce. Understanding the impact of animal manure application on the agricultural soil resistome and the risk it poses on public health is critical. However, such information is limited in South Africa as most antibiotic resistance research focuses on humans and food animals. This study, therefore, describes the prevalence and the genomic profiles of antibiotic-resistant Escherichia coli and Enterococcus spp. isolated from agricultural soil fertilized with chicken litter and the chicken litter. A total of 237 samples were examined and included soil before litter application, the litter-amended soil, and the chicken litter. Isolation and quantification of Escherichia coli and Enterococci were carried out using the Colilert® -18 / Quantiti-Tray® 2000 system and the Enterolert® -18® Quanti-Tray®/2000 system, respectively. The antibiotic susceptibility profiles of the isolates was determined using the Kirby-Bauer disk diffusion method. Whole-genome sequencing (WGS) and bioinformatics tools were used to determine the resistome, virulome, mobilome, clonal lineages, and phylogenies of the isolates circulating between the soil and the chicken litter. The application of chicken litter to the soil statistically significantly increased Enterococci count and the number of antibiotic-resistant enterococci in the litter-amended soil. A total of 835 enterococci (680 from soil and 155 from litter) isolates recovered from the samples was dominated by E. casseliflavus (56%), followed by E. faecalis (22%), E. faecium (8%), E. gallinarum (2%) and other Enterococcus spp 102 (12%). Overall, 55.8% (466/835) of the enterococci isolates were resistant to one or more antibiotics with the highest rate in the litter-amended soil (68.9%, 321/466), followed by chicken litter (19.9%, 93/466) and the least in the soil samples collected before the litter amendment (11.2%; 52/466). The enterococci isolates were mostly resistant to tetracycline (33%), erythromycin (25%), and trimethoprim-sulfamethoxazole (23%), among others, intimating the high usage of these antibiotics in poultry farms in South Africa. Additionally, multidrug resistance (MDR) was recorded in 27.8% (130/466) of the enterococci isolates with MAR indices ranging from 0.13 (resistance to two antibiotics) to 0.44 (resistance to seven antibiotics). A total of 63 different resistance patterns were recorded in the MDR enterococci isolates. Notably, enterococci count and the number of antibiotic-resistant enterococci in the litter-amended soil were reduced to levels comparable to the unamended soil at 50 and 28 days after soil amendment respectively. The whole-genome analysis of the few selected enterococci isolates revealed eight novel sequence types (STs) (ST1700, ST1752, ST1753, ST1754, ST1755, ST1756, ST1004, and ST1006). Several resistance genes that confer resistance to aminoglycosides (aac(6’)-Ii, aac(6’)-Iih, ant(6)-Ia, aph(3’)-III, ant(9)-Ia), macrolide-lincosamide-streptogramin AB (MLSAB) [erm(B), lnu(B), lnu(G), lsaA, lsaE, eat(A), msr(C)], trimethoprim-sulfamethoxazole (dfrE, and dfrG), tetracycline (tet(M), tet(L), and tet(S)), fluoroquinolones (efmA, and emeA), vancomycin (VanC {VanC-2, VanXY, VanXYC-3, VanXYC-4, VanRC}), and chloramphenicol (cat) were detected in the isolates. The bioinformatics analysis further revealed that the chicken litter amendment increased the number and diversity of ARGs in the soil, resulting in increased detection of tetracycline resistance genes (tet(M), tet(L)), and the macrolide resistance gene erm(B) and appearance of some ARGs (ant(6)-Ia, aph(3’)-III, lnu(G), dfrG)) that were not detected in the unamended soil. ARGs were mostly associated with diverse insertion sequences (ISs) (IS982, ISL3, IS6, IS5, IS3, IS256, IS30) and/or transposons (Tn3, Tn916, Tn6009) on plasmids or chromosome. The tet(M) and erm(B) were also co-located on Tn916-like transposons (Tn644, Tn645, and Tn659) in the three sample groups. Some of the isolates also harboured virulence genes that encoded adherence/biofilm formation (ebpA, ebpB, ebpC), anti-phagocytosis (elrA), and bacterial sex pheromones (Ccf10, cOB1, cad, and camE). Phylogenomic analysis showed that few isolates from litter-amended soil clustered with the chicken litter isolates. The isolates from this study also clustered with clinical and animal isolates from South Africa (Pretoria, Pietermaritzburg), Angola, and Tunisia. There was also an increase (albeit statistically insignificant) in E. coli count and the number of antibiotic-resistant E. coli in the soil following chicken litter amendment. A total of 126 E. coli was recovered from the soil and chicken litter samples. In total, 76% (96/126) of the E. coli isolates displayed resistance to at least one antibiotic, with the highest prevalence in the litter-amended soil (71.9%, 69/96) and the least (1%, 1/96) in soil samples collected before the litter amendment. The E. coli isolates displayed a high percentage resistance to tetracycline (78.1%), chloramphenicol (63.5%), ampicillin (58.3%), trimethoprim-sulfamethoxazole (39.6%), cefotaxime (30.2%), ceftriaxone (26.0%), and cephalexin (20.8%). Lower percentages of XVI resistance to cefepime (11.5%), amoxicillin-clavulanic acid (11.5%), cefoxitin (10.4%), nalidixic acid (9.4%), amikacin (6.3%), ciprofloxacin (4.2%), imipenem (3.1%), tigecycline (3.1%), and gentamicin (3.1%) were also recorded in the isolates. All the isolates were completely susceptible to meropenem and ceftazidime. Approximately 54% (52/96) of the resistant isolates were MDR, and the MAR indices of the isolates ranged between 0.11 (resistance to two antibiotics) and 0.56 (resistance to ten antibiotics). Overall, 38.5% (37/96) of all the resistant isolates had a MARI > 0.2, with the highest rate (51.4%) in the litter-amended soil and the least in the soil before litter amendment (2.7%). Twenty-one multidrug resistance patterns were observed among the isolates. These results show that the soil resistome was augmented by chicken litter application. Agricultural soil and chicken litter are rich reservoirs of multidrug-resistant E. coli and Enterococcus spp. that could threaten public health through contamination of food products and the surrounding water bodies. There is therefore a need for urgent and stringent measures to mitigate the spread of antibiotic resistance in the environment via prudent use of antibiotics in food animal production and treatment of animal manure before its application onto agricultural soil.
Hydrogen sulfide (H2S) production by mycobacteria.
(2020) Kunota, Tafara Takunda Remigio.; Steyn, Andrie J. C.
The gasotransmitter hydrogen sulfide (H2S) has been recognized as a physiological mediator with a variety of functions across all domains of life. Many prokaryotic bacterial species endogenously generate H2S in their natural environments. However, to date, it is not known whether Mycobacterium tuberculosis (Mtb) is an endogenous producer of H2S. In this study, we tested the hypothesis that Mtb endogenously produces H2S to modulate respiration, central metabolism, oxidative stress, and drug susceptibility. We demonstrated that fast-growing non-pathogenic, slow-growing pathogenic mycobacterial species, as well as drug resistant clinical strains of Mtb species produce H2S. Here we demonstrate that fast-growing non-pathogenic M. smegmatis produces barely detectable quantities of H2S, whereas MDR Mtb produces large quantities of H2S. We have also developed a native PAGEbased assay for the rapid screening of H2S producing enzymes in the lysates of mycobacterial species. Using LC-MS/MS, we identified the protein, Rv3684 as an H2S-producing enzyme in Mtb. Disruption of rv3684, demonstrated using the genetic knock out of rv3684, reduces, but does not eliminate, H2S production, suggesting the involvement of multiple genes in H2S production. Whole Mtb cell-based and lysate assays showed reduced levels of H2S production in the Mtb knockout strain compared to the wild-type strain. Noticeably, we demonstrated that the Mtb mutant is resistant to oxidative stress and the anti-TB drugs clofazimine and rifampicin. We also found that endogenous H2S is an effector molecule that maintains bioenergetic homeostasis by regulating Mtb respiration, and that H2S also plays a key role in central metabolism by modulating the balance between oxidative phosphorylation (OXPHOS) and glycolysis. In summary, our findings reveal previously unknown concepts of Mtb physiology with respect to Mtb-derived H2S and energy metabolism which has significant implication for routine laboratory culturing, understanding susceptibility and TB disease.
Enterococcus sp. contamination surveillance in different levels of healthcare in eThekwini District, KwaZulu-Natal (KZN) South Africa.
(2021) Shobo, Christiana Omowunmi.; Bester, Linda Antionette.; Essack, Sabiha Yusuf.
Hospital-acquired infections (HAIs) have been identified as long-standing setbacks affecting hospitals' quality of health care. While one of the major challenges related to HAIs is controlling cross-transmission, the role and significance of the inanimate hospital environment chain of transmission are yet to be unequivocally elucidated. Therefore, this study investigated the functional profile and diverseness of bacteria from various inanimate environmental sources, from two different wards in public hospitals at various healthcare levels in eThekwini District, KwaZulu-Natal, South Africa. True to the study focus on investigating the dissemination of bacteria from equipment within the hospital, the study further used Enterococcus as well-known HAI as target bacteria and described the molecular and genomic profiles of this specie isolated from the hospital environments. Samples were collected for a period of three months (September – November 2017) from the four levels of healthcare in eThekwini district, KwaZulu-Natal. The intensive care unit and peadiatic ward were employed in this study. An overall of 620 swabs were collected from areas frequently touched by healthcare workers (HCWs) and patients. These sites include the occupied bed linen, unoccupied bed linen, drip stands, patient files, ward phones, ventilators, nurses' tables, blood pressure apparatus, sinks, linen room door handle and mops. Swabs were placed in Amies transport medium and transported in a cooler box to the laboratory facility to be processed within four hours. The collected swabs (n=620) were pooled and incubated in tryptone soya broth containing 6.5% NaCl at 36.5oC for 24 hrs and subsequently plated on enterococci chromogenic media. The microbial diversity and functional profiles from the sites were identified using 16S rRNA metagenomics. Positive colonies were sub-cultured on bile esculin azide agar, and screened using standard microbiological methods, including haemolytic, oxidase and catalase, and API. Identifications were confirmed with polymerase chain reaction (PCR) with the added genus-specific tuf-gene and species-specific sodA-gene. Antibiotic resistance patterns in the Enterococcus spp. isolates were determined by the Kirby-Bauer disk diffusion method against 14 antibiotics as recommended by the Clinical and Laboratory Standard Institute (CLSI) guidelines. Thirty-seven samples from E. faecalis showed intermediate Resistance to vancomycin and were further analyzed using molecular tools viz. whole-genome sequencing (WGS) and bioinformatics analyses. This enabled determining the resistome, mobile genetic elements (MGEs), and clonal lineages circulating across the sites, wards, and hospitals. Metagenomics identified a total of 288 species, 190 genera, 105 families, 50 orders, 29 classes and 11 phyla from the samples analyzed. The dominant functional metabolic pathways implicated in causing human infection discovered were the signal transduction mechanisms, citrate cycle (TCA), transcription-factor bisphenol degradation, tyrosine metabolism. A total of 295 Enterococcus spp. isolates were recovered from the hospitals` environmental sites, 83% (n=245) were identified as Enterococcus faecium, 13% (n=38) as Enterococcus faecalis, 2% (n=6) Enterococcus gallinarum and another 2% (n=6) Enterococcus casseliflavus. Notably, the pediatric wards had the highest isolation rate compared to ICU, 64% and 36%, respectively. Overall, the sites with the highest isolation rate were occupied beds and mops (to clean ward floors) with 14.9% (n=44) each. The tertiary hospital were the most affected. The most prominent MDR antibiogram for E. faecium was CIP-RIF-NIT-TET-ERY and for WGS analysis of the E. faecalis samples confirmed that the tet(M) and erm(C) genes were the prevalent antibiotic resistance genes found in hospitals. The isolates harboured mobile genetic elements consisting of plasmids (n =11) and prophages (n=14), predominantly clonally specific. The 37 isolates analyzed consisted of 15 clonal lineages with six major sequence types (ST). Phylogenomic analysis showed that major lineages were mostly conserved within specific hospital environments. This study highlighted the inanimate hospital environment as a possible source of opportunistic nosocomial pathogens using Enterococcus as an illustrative example and emphasized the urgent necessity to optimize infection prevention and control measures to intercept/moderate the spread of bacteria in the hospital environments.
Roles of single nucleotide polymorphisms in the promoter regions of tumour necrosis factor-α and interleukine-1o genes in Schistosoma haematobium infection susceptibility.
(2020) Marume, Amos.; Mduluza, Takafira.; Mann, Jaclyn Kelly.
Background: Schistosomiasis remains a public health threat in sub-Saharan Africa which carries 85% of the global burden. With effective vaccines a distant future away, and no one allround intervention, research is still required to ensure that only effective programmes are introduced and implemented as well as evaluated and/or monitored. It is therefore key for researchers, policy makers and implementers to understand the epidemiology, immunology, immunopathology, immunogenetics, chemotherapy, management and control of Schistosoma haematobium for optimal elimination strategies to be implemented. A research study was therefore instituted to determine the prevalence, risk factors and host immunogenetic factors in S. haematobium infections among pre- and school going children living in endemic regions of Manicaland and Mashonaland central provinces in rural Zimbabwe. The relationship between single nucleotide polymorphisms (SNPs) of the promoter regions of tumor necrosis factor alpha (TNF-α or rs1800629) and interleukin- 10 (IL-10 or rs1800871) and susceptibility to Schistosoma haematobium was investigated. In addition, the relationship between these SNPs and cytokine levels, as well as the relationship between actual cytokine levels and susceptibility to Schistosoma haematobium was assessed. Methods: In this cross-sectional immune-epidemiological study Schistosoma haematobium was diagnosed by the microscopic examination of urine specimens for the presence of parasite eggs using the urine filtration technique. DNA for the genotyping was extracted from approximately 300μl whole blood using the QiagenFlexiGene DNA extraction kit, following the manufacturer’s protocol. IL-10 and TNF-α promoter region single nucleotide polymorphisms were genotyped using amplification refractory mutation system-polymerase chain reaction (ARMS-PCR).The allele frequencies and genotype distribution of S. haematobium infected and uninfected participants were then analysed using the chi-square test. All analyses were performed using SPSS (version 21) and p-values <0.05 were considered statistically significant. The levels of the cytokines (IL-10 and TNF-α) were measured by indirect enzyme linked immunosorbent assay (ELISA) using MABTECH, 3510-1H-6 kits, according to the manufacturer’s instruction. Results: The overall prevalence of S. haematobium among children in endemic rural and farming communities of the two provinces of Zimbabwe assessed was 17.1% (158/924). Gender specific prevalences were similar (17.5% in girls and 16.7% in boys; p = 0.735). Age and location were significant risk factors for schistosomiasis in children living in endemic regions surveyed. The older the child the higher the risk of getting infected by S. haematobium xvii (10.5% in 0-5 year olds; 24.0% in 6-10 year olds and 30.7% in 11-15 year olds; p < 0.001). IL-10 -1082 G/A, IL-10 -819 C/T and TNF-α -308 G/A single nucleotide polymorphisms were not significantly associated with susceptibility to S. haematobium infection. TNF- α genotypes AA, GA and GG were associated with high, moderate to high and low production of TNF-α respectively. IL-10 TT, CT and CC genotypes were associated with low, moderate and high IL-10 plasma levels respectively. Higher TNF-α and lower IL-10 serum levels were negatively associated with schistosomiasis infection. Praziquantel treatment reduced prevalence among the study participants as reinfections were only recorded in 6 of the 59 (10.2%) who were infected at baseline of children. Discussion and Conclusion: The determined schistosomiasis prevalence puts the regions of Zimbabwe studied within the moderate range as described by the World Health Organisation (10 – 49% prevalence), hence more concerted efforts are required to fight schistosomiasis. Although cytokine genotypes were associated as expected with cytokine levels, genotypes did not directly correlate with schistosomiasis infection while cytokine levels did. This indicates that circulating TNF-α and IL-10 levels are a result of many factors apart from genotypes. Taken together with previous work, this study suggests that high TNF-α and low IL-10 serum levels confer protection against schistosomiasis infection. Since schistosomiasis prevalence was similar between boys and girls and prevalence was high in all age groups (increasing with age), all programmes aimed at eliminating schistosomiasis should include both genders and children of all age groups. Specific locations could be targeted in resource limited settings as location was a significant risk factor for infection. Praziquantel is effective, with few reinfections observed, and therefore remains central in schistosomiasis management. To clearly understand the role host genetic factors in infection and to inform effective control, elimination and eradication programmes, more research on risk factors and host immunogenetics is necessary
Molecular and genomic analysis of clinical multidrug-resistant coagulase-negative staphylococci from the uMgungundlovu District in the KwaZulu-Natal Province, South Africa.
(2020) Asante, Jonathan.; Essack, Sabiha Yusuf.; Amoako, Daniel Gyamfi.; Abia, Akebe Luther King.
Coagulase-negative staphylococci (CoNS) are among the most commonly recovered bacteria in clinical specimens. They are usually colonisers (commensals) of the skin and nasal passages and considered contaminants of microbial cultures. However, they have been recognised as emerging pathogens, frequently causing opportunistic infections. The frequent use of indwelling medical devices and long-term hospitalisation present an increased risk of exposure to CoNS, resulting in infections usually caused by multidrug-resistant pathogens. Few studies focus on CoNS, including characterisation of their mechanisms of resistance, virulence, and persistence. Therefore, this study describes the molecular and genomic profiles of clinical CoNS from public sector hospitals in the uMgungundlovu District in KwaZulu-Natal, South Africa. Eighty-nine clinical CoNS isolates collected from three hospitals within the uMgungundlovu District between October 2019 and February 2020, constituted the sample. Isolates were speciated using the Vitek 2 system. Antibiotic susceptibility testing was done against a panel of 20 antibiotics according to Clinical and Laboratory Standards Institute (CLSI) guidelines using the Kirby-Bauer disk-diffusion method and minimum inhibitory concentration (MIC) was determined using the broth microdilution method for penicillin G, cefoxitin, ceftaroline, ciprofloxacin, moxifloxacin, azithromycin, erythromycin, gentamicin, amikacin, chloramphenicol, tetracycline, doxycycline, teicoplanin, tigecycline, linezolid, clindamycin, rifampicin, sulphamethoxazole/trimethoprim, nitrofurantoin and vancomycin. PCR was used to detect the presence of the mecA gene to confirm phenotypic methicillin resistance. Based on their resistance profiles, a sub-sample of isolates were subjected to wholegenome sequencing (Illumina MiSeq) to ascertain the resistome, virulome, mobilome, clonality and phylogenomic relationships using bioinformatic tools. The SPAdes software was used for the assembly of the raw reads. ResFinder 4.1 and CARD were used to identify antibiotic resistance genes in the isolates, while the virulence factor database (VFDB), Center for Genomic Epidemiology‘s MLST 2.0 server and MobileElementFinder v1.0.3 were used to identify virulence genes, sequence types and mobile genetic elements, respectively. Mutations in fluoroquinolone and rifampicin resistance genes were identified by manual curation using BLASTn alignment which was also used to determine the genetic environment of the resistance genes.S. epidermidis was the most abundant CoNS species isolated. Phenotypic methicillinresistance was detected in 76.4% (n=68) of isolates, 92.6% (n=63) of which were genotypically confirmed by PCR. Multidrug resistance (MDR) was observed in 76.4% (n=68) of isolates, with 51 antibiograms observed. The resistance genes mecA, blaZ, erm(A), erm(B), erm(C), msr(A), aac(6')-aph(2'') and fosB, among others, were detected and corroborated the observed phenotypes. Molecular mechanisms of resistance to tigecycline, teicoplanin, linezolid and nitrofurantoin were not detected even though some isolates were resistant to them. There was no association between ARG type and hospital/department. The ica operon known to facilitate biofilm formation was detected in 7/16 isolates sequenced. Known and putatively novel mutations in the gyrA, parC, parE and rpoB genes were also detected for fluoroquinolone- and rifampicin-resistant isolates. Prediction of isolates’ pathogenicity towards human hosts yielded a high average probability score (Pscore ≈ 0.936), which, together with the several virulence genes detected (including atl, ebh, clfA, ebp, icaA, icaB,icaC), support their pathogenic potential to humans. Seven MLST types were found, while the community-acquired SCCmec type IV was the most common SCCmec type detected. Mobile genetic elements (MGEs) haboured by isolates included plasmid replicon Rep10 and insertion sequence IS256. Defense systems such as arginine catabolic mobile element (type I and III), CRISPR system (16), and the restriction-modification system (type II) were detected. Genetic analysis showed that resistance genes were frequently bracketed by MGEs such as transposons (such as Tn554) and insertion sequences (such as IS257 and IS1182) that facilitated their mobility. Phylogenetic studies showed that the distribution of genes did not coincide with the phylogenetic clades. Despite the relatedness of isolates (clades A and B), there is still considerable variation within individual strains that can facilitate adaptation to local environments. The isolates exhibited several permutations and combinations of ARGs, virulence genes and MGEs, pointing to a complex milieu of mobilized antibiotic resistance and pathogenic characteristics in clonal and multiclonal strains. The study necessitates surveillance of CoNS as emerging pathogens.
Calymmatobacterium granulomatis: culture, electron microscopic studies and molecular analysis.
(1997) Kharsany, Ayesha Bibi Mahomed.; Hoosen, Anwar Ahmed.; Kiepiela, Photini.
Abstract available in PDF.
The activity of nybomycin against mycobacterium tuberculosis.
(2018) Niehaus, Abraham Johannes.; Moodley, Prashini.; Sturm, Adriaan Willem.
Nybomycin was discovered in 1955, but was never developed for clinical use. The compound was noticed again in recent years when it displayed bactericidal activity against certain fluoroquinolone-resistant bacterial species. The work presented here aims chiefly at describing the effect of nybomycin on Mycobacterium tuberculosis. The study is made up of three parts. In the first part, in vitro nybomycin susceptibility testing was conducted with various fluoroquinolone-susceptible and fluoroquinolone-resistant bacterialspecies. All M. tuberculosis isolates displayed low nybomycin inhibitory concentrations regardless of fluoroquinolone resistance. Similar susceptibility results were obtained for N. gonorrhoeae isolates, but results obtained with other bacterial species were less promising. In the second part, in silico investigations were conducted to elucidate the mechanism of action of nybomycin in M. tuberculosis. Results show that nybomycin binds to M. tuberculosis gyrase enzyme with an affinity at least similar to that of fluoroquinolones. No clear differences in binding affinity were observed when gyrA mutations, commonly associated with fluoroquinolone resistance, were considered. The results suggest that the mechanism of action of nybomycin against M. tuberculosis involves inhibition of gyrase enzyme. In the third part, M. tuberculosis mutants with increased nybomycin minimum inhibitory concentrations were selected and compared with the wild type organism through whole genome sequencing. None of the isolates harbored any mutations commonly linked to known drug resistance mechanisms. This indicates that M. tuberculosis likely employs a novel mechanism of resistance against nybomycin. This may further signify that nybomycin has an additional mechanism of action against M. tuberculosis, besides the action on gyrase enzyme, as suggested by the in silico results from this study. Twenty-two genes were identified through whole genome sequencing that may potentially be linked to the mechanism of resistance and possibly an additional mechanism of action.
The role of heparin binding haemagglutinin adhesin and curli pili on the pathogenicity of Mycobacterium tuberculosis.
(2018) Moodley, Suventha.; Pillay, Manormoney.
Background: Phagocytic host cells drive both the innate and adaptive arms of the host immune response during Mycobacterium tuberculosis (M. tuberculosis) infection. M. tuberculosis modulates the host immune responses and is able to proliferate in macrophages. The structures that mediate M. tuberculosis adherence (Adhesins) to macrophages are of particular interest for therapeutic development due to their cell surface localisation and immunogenic characteristics. M. tuberculosis produces numerous antigens that display adhesin functionality, including heparin-binding haemagglutinin adhesin (HBHA) and M. tuberculosis curli pili (MTP) that are critical for adherence to host cells. Recently, the independent elucidation of the immunogenic potential of each suggested that HBHA and MTP may represent a novel combination as a biomarker for future therapeutic development. This study aimed to elucidate the effect of HBHA and MTP in combination on adhesion, invasion, replication, cytokine production and transcription regulation of macrophages infected with HBHA and MTP proficient and deficient strains in an attempt to assess their immunogenic capacity. Materials and methods: THP-1 monocytic cells were differentiated into macrophages and infected at a multiplicity of infection of 5 with single mutants (ΔhbhA and Δmtp), single complements of double mutant (hbhA comp and mtp comp), MTP and HBHA deficient double mutant ΔhbhA-mtp (DM) and MTP and HBHA proficient wild-type (WT) strain. The relative percentage adhesion/ invasion of the mutant and complemented strains was calculated at 1 h and 2 h post-infection respectively and compared to wild-type. Intracellular replication was quantified by colony forming units at 4 h, day 3 and day 6 post-infection. To assess host transcriptomic changes elicited during early infection of THP-1 differentiated macrophages by WT and DM, RNA was extracted from host cells at 4 h post-infection. For the biological adhesion data set, raw data were filtered for genes in common with the Gene Ontology biological adhesion dataset sourced from EntrezGeneIds using the molecular signatures database with a False Discovery Rate q-value <1 (Chapter 1). Significantly differentially expressed genes with a p value <0.05 were used for further enrichment analysis (Chapter 2 and 3). Ingenuity Pathway Analysis (IPA) software (Ingenuity Systems, USA) upstream regulator, canonical pathway and biofunctions enrichment analysis were used to further investigate the differential regulation of molecular signatures by MTP and HBHA proficient and deficient strains. Macrophage cytokine/chemokine production was quantified at 24, 48 and 72 h post-infection using the Bio-Plex Pro Human Cytokine Multi-Plex Panel (Bio-Rad). Real-time quantitative RT-PCR was used to validate RNA sequencing findings and investigate transcriptional regulation of HBHA and MTP of following genes: CD80, DLX3, NLRP3, TGM5 and TLR2 at 1 h, 2 h and 4 h post-infection Results: During adhesion, DM induced a similar decrease in percentage adhesion (33.16%) to Δmtp (39.4%), ΔhbhA (22.78%), mtp comp (24.72%), but statistically lower decrease in percentage adhesion than hbhA comp (53.85%). During invasion, DM displayed a significant decrease in percentage invasion (36.49%) compared to Δmtp (61.49%) and hbhA comp (53.85%); and significantly higher decrease in percentage invasion than ΔhbhA (22.29%) and mtp comp (24.72%). Δmtp demonstrated a 39.4% and 61.49% decrease in percentage adhesion and invasion compared to WT respectively. The HBHA-MTP proficient strain induced greater transcriptional changes resulting in enhanced adhesion to phagocytes and invasion of cells. Furthermore, the HBHA-MTP proficient strain displayed the sole ability to induce activation of phagocytosis. Further investigation of canonical pathway differential regulation by HBHA-MTP proficient strain demonstrated greater induction of canonical pathways. The most differentially regulated pathway was Gαq signalling canonical pathway, which is vital for migration of phagocytes. In addition, the HBHA-MTP proficient strain also enhanced activation of the acute phase response, role of pattern recognition receptors in recognition of bacteria and viruses, and production of nitric oxide and reactive oxygen species in macrophages canonical pathways. RNA sequencing analysis showed that the M. tuberculosis adhesins, HBHA and MTP, elicited differential transcriptional regulation in macrophages, and demonstrated that predicted upstream regulators were associated with cytokine production. Further investigation of canonical pathways associated with these upstream regulators and cytokine quantification revealed that HBHA and MTP activate NF-κB, toll-like receptor, p38 MAPK and PI3-K/AKT canonical signalling pathways. HBHA and MTP elicited greater production of IL-4 and IL-10 at 24 h; G-CSF, GM-CSF, IL-2, IL-4, IL-5, IL-10, IL-12, IL-17, IFN-γ and TNF-α at 48 h and G-CSF, GM-CSF, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-17, IFN-γ and TNF-α at 72 h respectively, compared to DM infection. IL-1β, IL-2, IL-6, IL-12(p70), IL-17, TNF-α, IFN-γ, colony-stimulating factors G-CSF, GM-CSF and chemokines MCP-1 and MIP-1β were produced in higher concentrations by M. tuberculosis infection than anti-inflammatory cytokines IL-4, IL-5, IL-10 and IL-13. The bacillary load of M was significantly less than WT at all time intervals and similar to DM. The decreased replication ability of the HBHA-MTP mutant was attributed to MTP and not HBHA, suggesting that MTP facilitates replication during infection of macrophages. A transcriptional response common to both WT and DM, independent of HBHA-MTP, as well as unique responses induced by HBHA-MTP presence and deficiency were observed. The common transcriptional pattern exhibited the most enrichment for granulocyte adhesion and diapedesis canonical pathway, TNF upstream regulation and migration of cells biological function. The HBHA-MTP uniquely induced transcripts were associated with the most significant enrichment of the Adipogenesis pathway, whilst HBHA-MTP deficiency induced the most significant enrichment of T helper cell differentiation. Unique transcripts elicited by HBHA-MTP deficiency induced less enrichment of NF-κB upstream regulator and were associated with migration of cells. The top 10 canonical pathways enriched by all transcripts were similar between both infections, but differed in molecules involved and their significance. HBHA-MTP enriched the TREM1 signalling pathway to a greater degree than HBHA-MTP deficiency in macrophages. HBHA-MTP deficiency, but not presence, enriched Th1 and Th2 Activation, Th1, Th2, Melatonin degradation, Sumoylation, Methylglyoxal degradation III, Granzyme A signalling, PCP pathways. Discussion and conclusion: MTP played a greater role in adhesion and invasion during independent knockout and complementation in the double knockout strain than HBHA. HBHA and MTP together induced transcriptional changes that favour adhesion and invasion of macrophages. In addition, these 2 adhesins serve as pathogen-associated molecular patterns that enable host immune recognition during early infection of macrophages. HBHA and MTP activate intracellular signalling pathways that result in the longitudinal enhancement of a pro-inflammatory response during M. tuberculosis infection of macrophages. HBHA and MTP predominately induced a pro-inflammatory cytokine profile instead of an anti-inflammatory cytokine profile. This suggests that HBHA and MTP play a role in protective immunity and immunopathology as a consequence of pro-inflammatory cytokines such as TNF-α and minimal anti-inflammatory cytokines during M. tuberculosis infection. HBHA and MTP deficiency led to advanced immune activation and decreased intracellular growth. This suggests in the absence of HBHA and MTP, the presence of multiple, alternate antigens stimulate the intracellular signalling and transcriptional regulation in vitro. This advanced immune activation would potentially be detrimental to M. tuberculosis establishing a successful infection and would suggest that HBHA and MTP play a role in host immune response modulation as a protective measure during initial infection. Further investigation into the identity of these antigens would possibly result in a more successful, novel therapeutic target combination in addition to HBHA and MTP.
The effect of HIV and Neisseria gonorrhoeae on the tight junctions of cervical epithelial cells.
(2020) Maharaj, Shevani.; Sturm, Adriaan Willem.; Moodley, Prashini.
Introduction: Neisseria gonorrhoeae and HIV are major public health concerns globally. The interaction between these diseases is unclear. To determine the effect that N. gonorrhoeae and HIV have on the tight junctions of cervical epithelial cells, a cervical epithelial cell line was infected with N. gonorrhoeae only, HIV only and with N. gonorrhoeae and HIV simultaneously. Methods: The ME180 cervical cell line was grown to confluence and infected withN. gonorrhoeae only, HIV only and with N. gonorrhoeae and HIV simultaneously. Following infection, N. gonorrhoeae and HIV tansmigration assays and the blue dextran permeability assay were also performedto determine the effect that exposure to the microbes would have on the intact cervical epithelial layer. The tight junction gene expression assays, blue dextran permeability assay and immunofluorescence staining was performed to determine the effect that exposure to the different microbes had on the tight junctions. Results: The results of this study showed that exposure of the cervical epithelial layer to N. gonorrhoeae alone, HIV alone and to N. gonorrhoeae and HIV simultaneously did not affect the paracellular permeability of the epithelial layer. The results showed that a small percentage of N.gonorrhoeaeand HIV was able to migrate across the epithelial layer. With the simultaneous infection of N.gonorrhoeae and HIV, the presence of HIV did not seem to influence the migration of N. gonorrhoeae, as compared to infection with N. gonorrhoeae only, while the presence of N.gonorrhoeae seemed to cause the HIV to pass through the epithelial layer less efficiently than with exposure to HIV only. Discussion: The overall results suggest that since exposure to these microbes does not seem to affect the tight junctions of the intact epithelial layer and does not affect the paracellular permeability, the migration of the microbes across the epithelial layer was possibly through transcytosis.

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