Medical Microbiology

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Activation of silent biosynthetic gene clusters and profiling of secondary metabolites secreted by Endophytic Fungi for use as potential anti-HIV agents.
(2022) Makhwitine, John Phuti.; Ndlovu, Sizwe Innocent.; Mkhwanazi, Nompumelelo Prudence.
The continuous burden of Human Immunodeficiency Virus-1 in Sub-Saharan Africa, coupled with the inability of antiretroviral agents to eradicate HIV-1 from viral reservoirs, the potential risks of drug resistance development, and the development of adverse effects, emphasizes the need to develop a new class of HIV-1 inhibitors. Here, we cultivated five endophytic fungi isolated from Albizia adianthifolia with the addition of small epigenetic modifiers, sodium butyrate and valproic acid, to induce the expression of biosynthetic gene clusters encoding active secondary metabolites with probable anti-HIV activities. We identified a non-toxic crude extract of the endophytic fungus Penicillium chrysogenum treated with sodium butyrate to possess significantly greater anti-HIV activity than the untreated extracts. Single-round fractionated extracts of treated P.chrysogenum showed potent anti-HIV activity with an IC₅₀ of 5.90 μg/mL and a 5-fold increase compared to the untreated fraction. The active fractionated extracts were subjected to gas chromatography-mass spectrometry (GCMS), and more bioactive compounds were detected in treated P.chrysogenum fractions than in untreated fractions. These results indicate that treatment of endophytic fungi with small epigenetic modifiers enhances the secretion of secondary metabolites with anti-HIV-1 properties, acknowledging the feasibility of epigenetic modification as an innovative approach for the discovery of cryptic fungal metabolites as therapeutic compounds
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.
Antibiotic resistance in mycobacterium tuberculosis : the role of genetic mutations in resistance conferring genes and efflux transporters.
(2016) Dookie, Navisha.; Moodley, Prashini.
Two decades after the World Health Organisation (WHO) declaration of tuberculosis (TB) as a global emergency, the disease remains a public health crisis of epic proportions. The emergence of drug resistant strains of Mycobacterium tuberculosis, the etiologic agent of TB, and the convergent human immunodeficiency virus (HIV) epidemic places a devastating burden on an already weakened public health care system in South Africa. Rapid and accurate detection of drug resistance to first and second line drugs to guide effective treatment of TB is central to control of the disease and in preventing further dissemination of drug resistant strains. Knowledge of the underlying resistance mechanisms driving drug resistance in M.tuberculosis is pivotal in the design of rapid molecular based assays and will impact of the development of novel drugs and regimens for the disease. The manuscript in chapter 2 of this thesis, entitled Dynamics of antimicrobial resistance in Multi-Drug and Extensively Drug resistant strains of Mycobacterium tuberculosis in KwaZulu-Natal, South Africa, demonstrated the diversity of the resistance mechanisms amongst the multidrug resistant (MDR) TB strains currently circulating in the KwaZulu-Natal province of South Africa by the analysis of the rpoB, katG, inhA, pncA and embB genes associated with resistance to key drugs used in the treatment of TB. Multiple drug resistance mechanisms in the MDR-TB isolates suggests that the strains emerged separately and acquired resistance mutations independently. The findings of this study also confirms the clonality of the XDR-TB epidemic demonstrated by the predominance of the F15/LAM4/KZN strain family and reveals that MDR-TB strains are evolving and spreading via transmission. The manuscript in chapter 3 of this thesis, entitled Streptomycin resistance in the F15/LAM4/KZN strain of Mycobacterium tuberculosis is mediated by lineage-specific alteration of the gidB gene, demonstrated that streptomycin (STR) resistance in the F15/LAM 4/KZN MDR and XDR-TB strains was mediated by a rare, 130bp deletion within the gidB gene of M.tuberculosis leading to a complete disruption of the gene. Classical mutations in the rpsL gene mediated STR resistance in the remaining strain families. Widespread STR resistance has resulted in the exclusion of the drug from current treatment regimens. The findings of this study support the decision of policymakers and cautions the application of the drug in the absence of drug susceptibility testing. The manuscript in chapter 4 of this thesis, entitled Moxifloxacin resistance in the F15/LAM4/KZN extensively drug-resistant strain of Mycobacterium tuberculosis, demonstrated that the F15/LAM4/KZN XDR strain harboured the A90V gyrA mutation associated with high level ciprofloxacin (CPX) and ofloxacin (OFX) resistance and correlated with increased minimum inhibitory concentrations (MIC) for moxifloxacin (MXF). The results of this study cautions the utilization of MXF as part of empiric treatment protocols in the absence of moxifloxacin MIC data of the circulating XDR strains in an area. It also raises concerns regarding the regarding the use of moxifloxacin in KwaZulu-Natal. Furthermore, the current breakpoint defining resistance to MXF is of concern and requires revision. The manuscript in chapter 5 of this thesis, entitled Evaluation of Capreomycin in the treatment of the F15/LAM4/KZN extensively drug-resistant strain of Mycobacterium tuberculosis demonstrated that the A1401G rrs mutation was the main mechanism mediating resistance to the aminoglycosides, kanamycin (KAN) and amikacin (AMIK); and to capreomycin (CAP). CAP was reintroduced into TB treatment protocols without prior drug susceptibility testing. This results of this study demonstrates high level resistance to CAP and urges careful consideration in the application of CAP the KwaZulu-Natal province. Furthermore, concerns regarding the high breakpoint value that defines CAP resistance as compared to wild-type MICs for the drug results in misdiagnosis of resistance that results inadequate patient treatment and amplifies resistance. The manuscript in chapter 6 of this thesis, entitled KZN Multidrug and Extensively drug resistant strains of Mycobacterium tuberculosis remain susceptible to Linezolid and para-Amino salicylic Acid, demonstrated that the mechanisms most commonly associated with resistance to the linezolid (LIN) and para-amino salicylic acid (PAS) were absent in the MDR and XDR-TB strains in this study. Mutations detected in the drug targets were lineage specific markers rather than resistance mechanisms. This study also highlights the poor understanding of resistance to these drugs and the need for further study to allow for resistance detection to be incorporated into diagnostic assays, thus prolonging the utility of these drugs. The manuscript in chapter 7 of this thesis, entitled Efflux mediated drug resistance in clinical isolates of Mycobacterium tuberculosis in KwaZulu-Natal, South Africa, demonstrated the role of efflux pumps in mediating low level resistance. The results of this study supports the hypothesis that efflux activity leads to decreased intracellular antibiotic concentrations, thereby allowing the survival of a sub-population of bacteria under the sub-inhibitory level of the antibiotic, from which resistant mutants emerge, leading to clinically significant levels of resistance. The results of this study strongly supports the application of efflux pump inhibitors as adjunctive to the current treatment protocols. The results emanating from this thesis has contributed to the body of knowledge of drug resistance in M.tuberculosis, especially in the KwaZulu-Natal province of South Africa. Furthermore, the results can be used to guide treatment protocols and contributes to the future development of molecular based assays aimed at detecting resistance.
Antimicrobial properties of traditional medicine used for treatment of HIV/AIDS and its opportunistic infections.
(2012) Jwara, Nhlanhla David L.; Sturm, Adriaan Willem.; Gqaleni, Nceba.
This study was conducted to establish the scientific basis of the reported ethnomedicinal use of Ihlamvu laseAfrika (IHL) against Human Immunodeficiency Virus (HIV) and Acquired Immunodeficiency Virus (AIDS) related infections. IHL is believed to have a positive effect on AIDS however this has neither been clinically nor laboratory proven. Such effect can either be directly due to IHL’s inhibition of the virus causing AIDS or indirectly by the inhibition of organisms causing opportunistic infections. Experiments were carried out to test for the effect of IHL against Cryptococcus neoformans, Candida albicans, Herpes Simplex Virus (HSV), Mycobacterium tuberculosis (MTB) and HIV. The toxicity of IHL was determined by means of three assays. Using the Trypan Blue Dye exclusion test, an aqueous mixture of IHL was tested on Vero cells (African Green Monkey) for acute toxicity at two concentrations. Cell membranes compromised by IHL would take up dye and eventually spill their contents. Vero cells that were exposed to 1μg/mL and 100μg/mL concentrations of IHL for 7 hours resulted in (8.9±0.15) % and (98.7±0.84) % cell viability (n=3), respectively. When the duration of incubation increased to 48 hours, percentage cell viability of 1μg/mL and 100μg/mL concentrations were (98.3±0.50) and (98.2±0.50) respectively. The second cytotoxicity test involved incorporation an aqueous mixture of IHL onto 3- (4,5-Dimethylthiazol-2-Yl)-2,5-Diphenyltetrazolium Bromide (MTT). Cells were incubated in IHL for 24 and 48 hours resulting in a decrease in cell viability in a dose-dependent manner. At the lowest IHL concentration (0.1μg/mL) the cell viability was 80% and 78.5% after 24 and 48 hours incubations, respectively whereas at the highest concentration (1000μg/mL) was used in 24 and 48 hours incubation, cell viability was 50% and 80% respectively. The third cytotoxicity test called glutathione (GSH) focused on antioxidant level. The aim was to determine the highest concentration at which cells starts dying, concentrations used were 0.23; 0.46; 0.94; 1.88; 3.75; 7.50; 15.0 and 30.0 mg/mL. The results showed that the antioxidants levels were reduced in proportions relative to IHL concentration levels. The safe and effective dose of IHL obtained was 1.88mg/mL. The second objective of the study was to determine IHL’s active principle that is capable of inhibiting growth of C. albicans and C. neoformans, HSV, MTB and HIV. Solvents such as methanol, ethanol and acetone were utilized including an aqueous extract to extract it. The most suitable extract to inhibit the proliferation of the aforementioned organisms needed to be established. Upon its establishment, it was then used to determine the minimum inhibitory concentration (MIC). This was done in all susceptibility tests except for HIV whereby a ‘neat substance’ was used. In the case of HSV a causative agent for herpes, its susceptibility towards several IHL extracts was assessed with real-time polymerase chain reaction (RT-PCR). PCR attenuates specific site of DNA and quantifies viral load and the focus was the UL30 position which is targeted by most drugs. When comparing all solvent extracts as well as an aqueous extract of similar concentration, it was found that the methanol extract emerged as the strongest viral inhibitor with the lowest viral yield, and its threshold value, Ct = 18.4± 0.86 while the IHL concentration was 1.88mg/mL. The MIC of the methanol extract was 1.25mg/mL and Ct=18.9±1.14. An acetone extract proved to be the weakest thus its viral load was the highest, its Ct= (8.50±1.33) whilst IHL concentration was 1.88mg/mL. Cryptococcus neoformans known for causing meningitis and encephalitis in AIDS patients and C. albicans a causative agent for vaginal and oral thrush were two opportunistic infections tested for susceptibility towards IHL. The disk diffusion method was used for both fungal organisms. The best suited solvent extract was established and then used to determine the MIC. An aqueous extract showed the best activity with the inhibition zones of (10.5±1.642) mm when tested against C. albicans followed by ethanol extract (9.2±0.676) mm while acetone extract (8.80 ±1.21) mm had the lowest activity. The MIC of IHL’s aqueous extract was 1.0mg/mL and the corresponding zone of inhibition was (10.6±1.34) mm. When C. neoformans was tested for susceptibility against various IHL solvent extracts, the IHL’s aqueous extract had inhibition zones of (21.1±2.40) mm thus emerged as the strongest followed by methanol extract (10.3±0.43) mm while ethyl acetate extract was least active (7.13±0.33) mm. The MIC of the aqueous extract was 1.0mg/mL and its corresponding zone of inhibition was (11.4±0.55) mm. Furthermore, the growth inhibition of both C. neoformans and C. albicans by IHL’s aqueous extract were confirmed in liquid media with broth microdilution method. This technique tends to mimic what is likely to happen in a biological fluid. The results obtained depicted a dose-dependent response and both organisms shared a common MIC of 2.0mg/mL. From the broth microtitre plate aliquots samples were plated onto agar and used to further determine the minimum lethal concentration (MLC). The MLC essentially determines the antifungal concentration of an agent at which no colonies displayed visible growth. The MLC’s of IHL towards C. albicans and C. neoformans were 32 and 8 mg/mL respectively. IHL proved fungicidal at higher concentrations and fungistatic at low concentrations. Further susceptibility tests of IHL extracts were carried out on bacterial pathogens such as the MTB, a causative agent for Tuberculosis with 1% proportion method. This method seeks to determine if isolates are resistant if colonies grown in the presence of drugs are greater or equal to 1% of colonies grown in drug-free control quadrant. The best solvent extract was determined and then used to determine the MIC. Acetone extract results were 0.2% meaning that it strongly inhibited growth of MTB better than ethyl acetate (5%) and others the worst results were that of an aqueous extract (113%). A confirmation exercise was done with an agar dilution method. All extracts were incorporated onto agar and MTB colonies growing relative to negative controls after 21 days of incubation meant resistance while no growth meant susceptible. The MTB strain again proved susceptible towards the acetone extract but resistant towards methanol, ethanol, and aqueous extracts. The dichloromethane and ethyl acetate extracts seemed to have damaged the polypropylene plates rendering results null and void. Using agar dilution method, an MIC of an acetone extract was 16mg/mL. An aqueous extract was used for assessing HIV for susceptibility towards IHL. The quantitation of viral results were carried out on a spectrophotometer and a second generation tetrazolium dye (XTT) was used. The results showed that approximately -3.29 dilution of the aqueous extract did not protect cells. On the contrary, it proved to be toxic to both uninfected and infected cells. Moreover at low doses the extract demonstrated 50% protection towards uninfected cells. The third objective entailed the assessment of reproducibility of IHL that is routinely prepared by the Traditional Health Practitioner (THP). Batch to batch reproducibility is always a concern especially since traditional medicine is manufactured without any traceable set of standards. Two IHL samples that were prepared on different dates were assessed. Using a thin layer chromatography (TLC) a striking resemblance in the two samples was established visually by way of fractions produced. However, since TLC is a qualitative tool, it was incumbent that an instrument that doesn’t separate sample’s chemical constituents was used. The results produced by nuclear magnetic resonance (NMR) confirmed similarities in the two batch of IHL samples produced on different dates as it was the case with TLC. Peak intensity and the number of peaks in the chromatogram was a mirror image of the other thus confirming consistency in IHL preparation. The susceptibility tests of IHL towards viruses, bacteria and fungal pathogens present reasons why IHL is regarded as a non-specific repressor of pathogens people living with AIDS (PLWA) present with. The fourth objective of the study entailed the establishment of active principles responsible for the aforementioned activities. The acquisition of chemical fingerprints and their analysis was carried out on an Ultra Performance Liquid Chromatography Mass Spectrometer (UPLC-MS). The substances thought to be responsible for antimicrobial activities included:- thalebanin B, methyillukumbin A, kuguacin J, mauritine H, 2-methyl-3-(piperidin-1- yl) naphthalene-1,4-dione, isoferuloyllpeol, diosindigo A, kuguacin R, verbascoside, kuguacin B and nuciferin. Further confirmation studies are needed on fractions to identify their chemical makeup as well as their activities on all of the aforementioned microorganisms.
Aspects of sexually transmitted diseases at King Edward VIII hospital.
(1988) Hoosen, Anwar Ahmed.; Van den Ende, Jan.
Abstract available in PDF.
Calymmatobacterium granulomatis: culture, electron microscopic studies and molecular analysis.
(1997) Kharsany, Ayesha Bibi Mahomed.; Hoosen, Anwar Ahmed.; Kiepiela, Photini.
Abstract available in PDF.
Causes of meningitis in the era of HIV.
(2021) Ramjathan, Praksha.; Swe Swe-Han, Khine.
No abstract available.
The characterization of a putative DNA repair protein in Mycobacterium Tuberculosis, encoded rv2414c.
(2023) Maleke, Dieketseng Palesa.; Senzani, Sibusiso.
Mycobacterium tuberculosis (MTB) is a causative agent of the communicable disease tuberculosis (TB), which is regarded as one of the top ten causes of death worldwide. Globally, TB accounts for over 10 million infections and over 1.8 million deaths annually. These statistics are subject to a constant increase due to the emergence of drug resistant strains. Although, the recent use of next generation sequencing technology has generated complete genome sequences and functional genomic data for various organisms (MTB included), to this point, the biological functions of several proteins encoded for in the MTB genome are not known or characterized hence they are called hypothetical proteins. Characterization of these hypothetical proteins is essential, as they could be involved in key regulatory processes of MTB, which is essential for the pathogen to retain a successful life cycle and disease progression. For successful invasion of the host and disease progression, it is important for MTB to retain genomic stability. Therefore, the degree of survival of MTB in the host environment is largely dependent on the bacterium’s ability to retain genomic stability. DNA repair mechanisms protect bacterial DNA from damage that can be induced by numerous stress factors. The hypothetical protein rv2414c encodes a gene amongst the MTB immunogenic protein identified in a study by Chiliza et al., (2019) which is closely associated with genes involved in MTB DNA repair, suggesting a possible role in DNA repair pathways. Therefore, the present study is aimed at characterizing a conserved hypothetical protein encoded rv2414c in Mycobacterium tuberculosis to elucidate the proteins biological function. For in-silico characterization, three bioinformatics tools were used, namely; Mycobroswer, I-TASSER and STRING online tools. Thereafter, a CRISPR-cas9 gene silencing mechanism was developed to elucidate the biological role of rv2414c. CRISPR system entails the co-expression of the silenced form of RNA-guided DNA endonuclease from the type II CRISPR system (dCas9) and a small guide RNA specific to a target sequence, leading to the DNA recognition complex resultant in transcription interference of corresponding DNA sequence. This CRIPSR mechanism was achieved by generating knockdown mutants. Phenotypic characterization of the mutants was accomplished by monitoring the mutants’ growth kinetics and biological assays were done to assign a possible biological function to rv2414c. Bioinformatics analysis suggests rv2414c is involved in DNA repair based on the structural networks it forms with 3 genes (dprA, recA and cinA) involved in MTB DNA repair and 3 proteins (rv3242c, rv3737 and rv2897c) that are involved in mainly aiding in the mechanism of DNA repair. However, the growth kinetics showed that rv2414c has no impact on the MTB growth rate, as all strains grew in a similar growth pattern with no statistical significance (p > 0.05) observed at the different time points. Additionally, UV biological assay showed that rv2414c is not a major role player in DNA repair, as UV exposure did not have an effect on bacterial survival rate even in the knockdown strain. A slight decrease in cell survival rate was noticed after addition of Mitomycin C (MMC) between Δrv2414c (100 ng/ml) + MMC and Δrv2414c + MMC, however, the difference was not significant. This implies that rv2414c is not involved in MTB DNA repair.
Characterization of immunoglobulin (Ig) isotypes, IgG subclasses and cytokines in the blood and genital tracts of HIV infected and healthy women from an observational cohort study (CAPRISA 082)
(2020) Zuma, Mandisa Nokukhanya.; Archary, Derseree.; Sorbia, Parveen.
Background: Heterosexual transmission remains the dominant route of HIV infections in women. Immune responses that predict HIV acquisition during pre-exposure prophylaxis (PrEP) remain undefined. We hypothesized that increased genital tract antibodies and cytokines pre-HIV infection predict HIV acquisition in seroconverters compared to non-seroconverters irrespective of PrEP use. Methods: Plasma and Softcup specimens were collected from n=12 seroconverters (cases) and n=48 non-seroconverters (controls) in the CAPRISA 082 study at five time points. Of 12 cases-, nine took PrEP, while 29 of 48 controls took PrEP. IgG1, IgG2, IgG3, IgG4, IgM and IgA, and nine cytokines: MIP-1􀄮, MIP-1􀈕, IP-10, MCP-1, and IL-8, TNF-􀄮, IL-1􀄮, IL-1􀈕, and IL-6 pre- and post-HIV infection, were measured using multiplexed technology. Results: Baseline levels of IgG subclasses, Ig isotypes, and mucosal cytokines were similar between cases and controls. Over time within the cases, plasma IgA significantly declined, in controls, plasma IgG2, IgG3, and IgM significantly declined over time (p<0.05). In cases and controls on PrEP, plasma IgG3 trended higher compared to no PrEP (p<0.1). Relative to baseline, only within the controls, mucosal IgG1, IgG2, IgG3, IgG4, IgM, and IgA declined significantly. Mucosal IgM significantly predicted four-fold increased HIV risk (p=0.01). Eight of nine cytokines in the genital tract were significantly elevated in the cases compared to controls (all p<0.05). In cases and controls who used PrEP relative to no PrEP, IP-10 was significantly lower (p=0.04 and p=0.009). Baseline mucosal IL-8 significantly correlated with mucosal IgG1, IgG2, total IgG, and IgM (p<0.001 for all). Conclusions: Although no significant elevated genital antibodies or cytokines pre-HIV infection were found, significantly different patterns of antibodies and cytokines were observed in this cohort. Plasma IgG3, one of the most effective of the IgG􀂶s eliciting diverse antibod􀁜 functions, was increased in PrEP users. Mucosal IgM was associated with increased HIV-acquisition risk, while pleiotropic IP-10, a reported risk factor was modulated in PrEP users among cases. Collectively, these data suggest that PrEP use may modulate or preserve specific immune responses that can modify HIV risk. As PrEP uptake increases, its effect on mucosal and systemic immunity is important for informing on prevention strategies where PrEP may be given alone or in combination with HIV vaccine for added efficacy.
Characterization of the function of RV1268c, an ATP binding cassette transporter in Mycobacterium tuberculosis.
(2023) Hallom, Pumelela.; Senzani, Sibusiso.
Tuberculosis (TB) is an epidemic disease that is caused by a bacterium called Mycobacterium tuberculosis (Mtb). This disease infects and kills millions of people globally. Anti-Tuberculosis (anti-TB) drugs such as isoniazid, ethambutol, pyrazinamide, and fluoroquinolones have been discovered and produced for TB treatment but regardless, TB persists because of the resistance to these drugs, leading to the development of multidrug-resistant (MDR) Mtb and extensively-drug resistant (XDR) Mtb. One of the key areas to focus on for the development of new effective anti- TB drugs are efflux systems, because they transport molecules outside cells and have a role in the resistance against TB treatment. This study aimed to identify the biological function of the Mtb protein, Rv1268c. RV1268c was one of the identified proteins in a study that was done by Chiliza et al., 2019 where a few protein biomarkers that are recognized by both active TB and latent TB patient antibodies. Some of these biomarkers that were studied are TreY, Bfr, and TrpG, which are biomarkers that are specific to ATP and play an important role in pathogenesis. Other biomarkers included MoaE, PonA1, and NarG, which are specific to latent TB and play a role in dormancy. The Rv1268c protein of Mtb is classified as a hypothetical membrane protein of the cell envelope and its associated proteins are Rv1267c and RV1269c, which are regulatory protein and a conserved putatively exported protein, respectively. The Rv1268c protein is hypothesised to be an ATP-binding cassette (ABC) transporter. The nucleotide and protein sequences of Rv1268c were v downloaded from the database of Mtb H37Rv using Mycobrowser. To create a knock down strain, annealed oligos were ligated to PLJR965 plasmid and transformed into XL10-Gold ultracompetent cells and grown on kanamycin-containing plates. Extracted plasmids were electroporated into Mtb, and after 4 weeks of incubation, the colonies were screened to check if they carried the knock down plasmid . DNA was then extracted to characterize the function of the Rv1268c Mtb protein. The results showed that Rv1268c had no effect on the in vitro growth of Mtb, while the Ethidium bromide (EtBr) assay displayed a difference on the extrusion of EtBr as the knock down and the knock down with anhydrotetracycline (aTc) had lower fluorescence as compared to the wild type which implies that Rv1268c is not an ABC transporter. The statistical analysis showed that the was no significant difference on the drug susceptibility between the wild type, knock down, and the knock down with aTc strains . The growth of neither the wild type or knock down strains was completely inhibited by either of the drugs tested. Keywords:
Characterizing the function of the Rv3218 gene in Mycobacterium tuberculosis.
(2023) Canham, Khumbuzile.; Senzani, Sibusiso.
Tuberculosis (TB), caused by Mycobacterium tuberculosis, is a primordial affliction that continues to torment humankind since its known history and prehistory. TB is among the major causes of ill-health and death in the world with an estimated 1.8 million cases of death recorded yearly. The situation is worsened by the emergence of the strains of TB that are regarded as resistant. Recently, Mycobacterium bovis bacillus Calmette-Guerin (BCG), has been the only available vaccine for TB. An intense understanding of Mtb’s biology, should reveal new perceptions that can lead to the improved treatment, diagnostics, vaccines and highly needed control measures. Throughout infection, Mtb produces some proteins into the host environment to play critical role in pathogen host interactions. Close to half of the Mtb genome consists of genes with unknown functions. Among those genes is Rv3218 gene which was identified in the study by Chiliza et al., 2019. The Rv3218 gene is hypothesised to have a Diacylglycerol kinase activity. This study aimed at characterising the function of Rv3218 gene in Mtb with the purpose of coming up with ideas of how that can be used in the development of more effective and convenient diagnostic tools, therapeutics, or the total elimination of TB. There is a vast amount of molecular techniques that are currently used to characterise unknown genes. Here we employed a CRISPRi dCas9 system for the silencing of the Rv3218 gene in Mtb. We also used a number of Bioinformatics tools for in silico analysis of the gene and construction of all relevant primers necessary for this molecular cloning. The Rv3218 knockdown repressed by Anhydrotetracycline (ATc) was constructed for assaying the effect of this gene silencing compared to the MtbH37Rv wild type. We then conducted growth curves and MICs (Minimum Inhibitory Concentrations) to check if this gene has an impact on antimicrobial susceptibility and growth of Mtb. We also tested its activity as a diacylglycerol kinase via osmolarity assay as it is said that dgk mutants do not grow well on nutrient media of low osmolarity. On bioinformatics analysis, we found that the gene has cell wall and transcription regulatory functions and possesses a similar structure as diacylglycerol kinase. However, the in vitro analysis was contradictory to these findings. We found that the Rv3218 gene has no impact on the growth of Mtb and it’s susceptibly to the antimicrobial drugs that were used in this study. On the osmolarity assay, there was no observable difference between the growth of the wild type and the knockdown strain in all the concentrations of osmolarity. Judging from these findings, we then concluded that this gene does not function as a diacylglycerol kinase. We then suggested that, more advanced experimental studies still need to be conducted in order to confirm this hypothesis as we were unable to do them due to the short time frame for this study.
Cytokine immune response profiles during 5 intestinal helminths and Mycobacterium 6 tuberculosis coinfection: An in vitro and human ex vivo study in KwaZulu-Natal.
(2023) Bhengu, Khethiwe Nomcebo.; Mkhize-Kwitshana, Zilungile Lynette.; Singh, Ravesh.; Naidoo, Pragalathan.
Background: There is a striking geographic overlap between helminths and tuberculosis (TB), particularly in developing countries like Africa. Underprivileged communities are more susceptible to these illnesses due to poverty, poor sanitation, and other environmental factor Helminth and tuberculosis infections exhibit distinct immune responses, which may be antagonistic in coinfected hosts and lead to poor prognosis. Helminth infections induce anti422 inflammatory Th2/Treg responses contrary to the pro-inflammatory Th1 responses triggered by Mycobacterium tuberculosis (Mtb) infection. Reduced TB protection has been associated with a strong Th2 response. Uncertainty exists on how helminth infection affects the host’s resistance to TB. This necessitates further investigation of immune responses in helminths and TB coinfection cases, particularly in KwaZulu-Natal (KZN). Aim: To determine the cytokine response profiles during intestinal helminth and TB coinfection using lymphocytic Jurkat and monocytic THP-1 cell lines for the in vitro study and TB and helminth coinfected South African adults for the human ex vivo study. Methods: Lymphocytic Jurkat and monocytic THP-1 cell lines were stimulated for 24 and 48 hours with Mtb H37Rv and Ascaris lumbricoides (A. lumbricoides) excretory-secretory protein extracts for the in vitro study. A cross-sectional study on consenting adult participants (≥18 years) (n = 414) recruited from primary health care clinics was conducted between March 2020 and August 2021 in Durban, KwaZulu Natal, for the pilot human ex vivo study. Blood and stool samples were collected from the recruited participants. The Kato-Katz and Mini-Parasep faecal parasite concentration techniques were used to detect intestinal parasite infections in stool samples. Blood samples were analysed to determine A. lumbricoides-specific immunoglobulin E (IgE) and immunoglobulin G4 (IgG4) levels to improve microscopy sensitivity. In this study, cytokine analysis was undertaken for 164 participants; 96 were HIV infected and had to be excluded, leaving 68 eligible participants. The eligible individuals were subdivided into uninfected controls (no helminth and TB infection) (n = 18), helminth only infected (n = TB only infected (n = 6), and TB and helminth co-infected (n = 6) groups. Thereafter, for both the in vitro and ex vivo study, the gene expression profiles of the T helper type 1(Th1) and transcription factors [Interferon-γ (IFN-γ), Tumour necrosis factor-α (TNF-α), Interleukin-2 (ILxvii 2), Nuclear factor of activated T cells 2 (NFATC2), Eomesodermin 446 (eomes), T helper 2 (Th2) and transcription factors (Interleukin-4 (IL-4), Interleukin5 (IL-5, Transforming growth factor-β (TGF-β), T helper type 17 (Th17) (Interleukin-17 (IL-17), immune protein and proteases (Granzyme B, Perforin), Regulatory T cells (Tregs) (Interleukin-10 (IL-10) and Fork head box P3 (FoxP3)] and the uninfected controls, TB alone, helminth alone and coinfected groups were determined using RT-qPCR. Results: (i) In vitro study: TB-stimulated Jurkat cells had significantly higher levels of IFN-γ, TNF-α, Granzyme B, and perforin compared to unstimulated controls, LPS, A. lumbricoides, and A. lumbricoides plus TB costimulated cells (p<0.0001). IL-2, IL-17, Eomes, and NFATC2 levels were also higher in TB-stimulated Jurkat cells (p<0.0001). TB alone stimulated cells had lower IL-5 and IL-4 levels compared to A. lumbricoides alone stimulated and TB plus A. lumbricoides costimulated Jurkat and THP-1 cells (p<0.0001. A. lumbricoides alone stimulated cells had higher IL-4 levels compared to TB plus A. lumbricoides costimulated Jurkat and THP- 1 cells (p<0.0001). TGF-β levels were also lower in TB alone stimulated cells compared to TB plus A. lumbricoides costimulated cells. IL-10 levels were lower in TB stimulated Jurkat and THP-1 cells compared to TB plus A. lumbricoides costimulated cells (p<0.0001. (ii) Ex vivo study: Similar results were noted for both the in vitro and the ex vivo study, although the human study had a smaller sample size. Conclusion: Data suggest that helminths induce a predominant anti-inflammatory Th2 and Treg response which may downregulate critical proinflammatory Th1 responses crucial for TB protection.
Cytokine production by ME-180 cells and VK2 E6/E7 cells on exposure to Neisseria gonorrhoeae, HIV, N. gonorrhoeae and HIV.
(2018) Govindasami, Merusha.; Sturm, Adriaan Willem.
Gonorrhoea is a sexually transmitted disease caused by Neisseria gonorrhoeae. Women are more at risk in developing secondary complications due to asymptomatic infections. In 2001, a study was done on the different responses of epithelial cells from three different regions of the lower female genital tract exposed to N. gonorrhoeae. Upregulation of cytokines found in cervical and vaginal secretions has been linked with human immunodeficiency virus 1 infection. In vitro studies of the immune response following exposure to multiple STI pathogens are relevant as mixed infections are common and not many studies have been done. The aim of this study was to determine the cytokine response in a co-infection model with N. gonorrhoeae and HIV using two genital epithelial cell lines. ME-180 cervical cells and VK2 E6/E7 vaginal cells were infected with N. gonorrhoeae and HIV only and with both organisms in different sequence. Infected cells were incubated at 37 °C in 5 % CO2 for 72 h. The supernatant was assayed for cytokines TNF-α, RANTES, IL-1β, IL-4, IL-6, IL-8, and IL-10 by means of the Bio-Plex Pro Cytokine, Chemokine, and Growth Factor Assay kit. The spontaneous cytokine release was higher in VK2 E6/E7 cells than in the ME-180 cells. On exposure to single organisms the response to N. gonorrhoeae was stronger than to HIV in both cells for IL-10, IL-8 and IL-6. For infection with N. gonorrhoeae the VK2 E6/E7 cells had a stronger cytokine response than ME-180 but this was not so for HIV. The response the cells had to exposure to both organisms was independent of the sequence of exposure. Further studies should be done on mixed infections of N. gonorrhoeae and HIV with additional STI pathogens.
The cytotoxic effects of fumonisin B1 in human kidney cells and the ability of allicin to ameliorate these effects.
(2019) Mahlalela, Nomalungelo Nothando Felicity.; Khan, Rene Bernadette.
Fumonisin B1 (FB1) is a widespread contaminant of crops and is produced as a secondary metabolite of fungi. It has been found to disrupt sphingolipid metabolism, cause epigenetic modifications and induce cellular toxicity that can manifest through oxidative stress and apoptosis. Although implicated in animal toxicity including kidney cancer in rats, FB1 effects in the human kidney have not been explored. Allicin is a biological component of garlic that has been widely studied for its health benefits including its anti-cancer and antioxidant properties. This study evaluated allicin as a possible therapeutic measure for FB1-induced cytotoxicity in Hek293 cells. Both FB1 and allicin decreased cellular viability (MTT assay) in a dose-dependent manner and generated IC50 values of 215 μM and 3.905 μM respectively. Three 24 h treatments (FB1, allicin and combined FB1+allicin) were compared to untreated cells for induction of apoptosis and oxidative stress. Luminometry was used to determine cytotoxicity (lactate dehydrogenase assay), caspase activity and mitochondrial toxicity (apoptosis), and quantify intracellular ROS (iROS) and glutathione (GSH) (oxidative stress). Free radical production was estimated by the TBARS and NOS assays respectively, while DNA damage was evaluated using the comet assay. Western blotting confirmed the expression of various antioxidant and apoptotic proteins, and superoxide dismutase 2 (SOD2) transcripts were quantified using qPCR. ATP concentration was increased for all treatments, but mitochondrial toxicity was increased in the allicin treatment. While lipid peroxidation decreased, luminometry results indicate that iROS was increased and was accompanied by a corresponding decrease in SOD2 and catalase protein expression. Depletion of GSH was consistent with increased GPx1, HSP70 and Nrf2 protein expression suggesting the presence of oxidative stress. SOD2 transcripts were only increased in the allicin treatment. Apoptosis was initiated as indicated by increased caspases 8 and 9, and pro-apoptotic Bax protein expression, but caspase 3/7 was not activated for the FB1 treatment. However, DNA fragmentation and cPARP were increased for all treatments suggesting that apoptosis was executed. Overall, FB1 and allicin individually and combined induced oxidative stress by increasing ROS and decreasing antioxidants. Apoptosis was also induced, although in a caspase independent manner in the FB1 treatment. Overall, allicin did not ameliorate the effects of FB1 in Hek293 cells.
Defining the role of high-dose isoniazid in the treatment of multi-drug resistance tuberculosis: isoniazid resistant profiling.
(2021) Ngema, Senamile Lale.; Dookie, Navisha.; Naidoo, Kogieleum.
Background: High-dose isoniazid is recommended in short-course regimens for multidrug-resistant tuberculosis (MDR-TB). However, there is no substantial evidence supporting its use in the presence of INH resistant mutations. Therefore, this study aimed to establish the efficacy of INH in the presence INH resistance associated mutations. Methods: We selected 94 clinical isolates obtained from 65 patients from the IndEX (CAP020) study specimen biorepository. Isolates were selected based on whole genome sequencing results showing evidence of INH resistant conferring mutations. Twenty-one isolates had inhA promoter gene and/ inhA coding region mutations, 35 had katG mutations, and 20 had both inhA promoter and/ inhA coding region plus katG mutations. Additionally, 18 INH susceptible clinical isolates were included in this analysis. Minimum inhibitory concentrations (MICs) were done in different concentration ranges depending on the mutation present. INH susceptible and H37Rv (0.016-0.256) μg/ml, inhA (0.256-4.0) μg/ml, katG (1.0-16.0) μg/ml and inhA plus katG (4.0-16) μg/ml. Results: Among 94 isolates, 36 were excluded: 11 MPT64 antigen negative, 23 non-growers and two were contaminated. Fifty-eight isolates from 55 patients were left for analysis. Eleven isolates had inhA mutations, 23 katG mutations, 12 had double mutations in inhA and katG, and 12 were INH susceptible. MICs obtained varied within isolates ranging from 0.016 to >64.0 μg/ml. InhA, katG, inhA plus katG mutations and INH susceptible isolates had median INH MIC of 8.0 (4.0-64.0), 4.0 (95% CI, 4.0-8.0), 64.0 (95% CI, 64.0-64.0), and 0.48 (95% CI, 0.32-1.0) μg/ml, respectively, confirming the association between INH MICs and genotypic profile. The MDR-TB and pre/XDR-TB had median INH MIC of 8.0 (95% CI, 8.0-32.0) and 48.0 (4.0-64.0) μg/ml, respectively. We found association between cavitary disease and increase in INH MICs for inhA mutants, median of 64.0 (64.0-64.0) μg/ml, and previous TB history and increased INH MICs (8.0[95% CI, 8.0-64]. Conclusion: This study demonstrated highly variable MIC range with significant overlap in MIC range among the mutant groups. Furthermore, inhA mutants demonstrated unexpectedly high MICs raising a concern for the ongoing use of the high-dose INH in our setting. Our findings suggest that the current one-size-fits all approach to MDR-TB short-course regimen requires urgent review.
A descriptive analysis of the routine use of genotype MTBDRsl in a high HIV/TB prevalent region in South Africa.
(2020) Lutchminarain, Keeren.; Mvelase, Nomonde Ritta.; Swe Swe-Han, Khine.
Background. Since the implementation of shortened drug regimens for the management of drug resistant tuberculosis (TB), there is a growing need for rapid detection of resistance to second line antimycobacterial drugs. The GenoType MTBDRsl Version 2 allows for the rapid molecular detection of resistance conferring mutations to fluoroquinolones (FQ) and second line injectable drugs (SLIDs). Although the GenoType MTBDRsl is recommended for use directly on smear positive and smear negative clinical specimens, the feasibility of using this assay routinely within a programmatic setting in high HIV/TB endemic areas requires exploration. Objectives. To assess the feasibility of routinely using the GenoType MTBDRsl in a high HIV/TB prevalent region and to describe the various circulating resistance patterns detected by this test in KwaZulu-Natal. Methods. A retrospective data analysis of all GenoType MTBDRsl results in newly diagnosed rifampicin resistant (RR-TB)/multidrug resistant TB (MDR-TB) specimens was performed. The assays performance on direct testing of smear positive and smear negative specimens was compared. The various mutation patterns for FQ and SLIDs identified by this test was observed. 21 Results. Of 1873 RR-TB/MDR-TB, 37,4% were smear negative and 62,5% was smear positive. In smear negative specimens, the GenoType MTBDRsl showed an inconclusive rate of 61,2%, whilst amongst the smear positive specimens, an inconclusive rate of only 6,6% was observed. The commonest mutation pattern observed for FQ occurred at the gyrA gene at codon 90 (A90V) 61/158(38,6%) followed by the D94G mutation 31/158 (19,6%). Heteroresistance for FQ was observed in the gyrA gene for 6/158 (10,1%) isolates. For SLIDs, the commonest mutation occurred in the rrs region specifically A1401G, 71/108 (65,7%) followed by C1402T at 20/108 (18.5%). Conclusion. The routine use GenoType MTBDRsl Version 2 assay is more feasible in smear positive specimens as compared to smear negative specimens in high HIV/TB settings. To improve future development of the assay, further studies looking at the various resistance patterns are required.
The developement of a Real-Time Polymerase Chain Reaction using TaqMan probes to determine the burden of multi-drug resistant tuberculosis (MDR-TB) in KwaZulu-Natal.
(2008) Ganas, Anura.; Pym, Alexander S.
Abstract available in PDF.
The development and application of a high throughput methodology to determine MICs of Mycobacterium tuberculosis isolates against antimicrobial agents.
(2014) Rampersad, Tashmin.; Sturm, Adriaan Willem.
Chapter 1 of this dissertation entails the introduction, aims, objectives and the literature review. Drug susceptibility testing of Mycobacterium tuberculosis is time consuming and expensive. Multi-point inoculation offers the advantage of testing multiple isolates on a series of solid media with a single breakpoint concentration of a drug in each plate or a series of different drug concentrations of one drug. We aimed to determine the reproducibility of MIC determination for anti-TB drugs of M. tuberculosis isolates using agar dilution with multi-point inoculation and thereafter validating the results by comparing it to classic agar dilution on quadrant plates and the MTT assay. Chapter 2 contains the manuscript that has been submitted for publication. This manuscript contains a brief introduction with the aim and objectives, and a detailed description of the methodology, results and discussion. Thirty M. tuberculosis isolates were grown in Middlebrook 7H9 broth with 20% Tween until mid-log phase was reached. Agar dilution MICs were determined on Middlebrook 7H10 agar for 11 anti-TB drugs at concentrations ranging from 128 to 0.125 mg/L. The agar plates were inoculated using a multi-point inoculation device with 36 points each delivering 1μL of a suspension of 1×104 cfu/ml. For the quadrant plate method and the MTT assay 100 μl of the same suspension was used. All tests were done 3 times in triplicate. Agar dilution with multi-point inoculation was found to be reproducible within the 11 anti-TB drugs tested and correlated well with agar dilution on quadrant plates and the MTT assay for the three anti-TB drugs tested. Chapter 3 entails a brief summary (synthesis) of the discussion found in the above-mentioned manuscript. The multi-point inoculation method has potential for wide scale application in breakpoint drug susceptibility testing as well as MIC testing of M. tuberculosis isolates. Lastly this dissertation contains the required references and appendices.
The development of a stratified keratinocyte model for chlamydia trachomatis pathogenesis studies.
(2017) Jadoo, Sasha.; Sturm, Adriaan Willem.; Joubert, Bronwyn C.
A number of different methods to generate stratified keratinocyte layers have been published. These involved the use of normal human epidermal keratinocytes (NHEKs/NEKS), which have a better ability to stratify compared to HaCaT keratinocytes, which usually require supplemented growth factors or stromal interactions with fibroblasts to do so. This study aimed to generate a model of stratified keratinocytes, closely resembling in vivo skin, using HaCaT cells and to demonstrate the effect that C. trachomatis has on these layered keratinocytes, allowing us to gain insight on the pathophysiology of this organism. All cells and bacteria were propagated and titrated according to conventional protocols. HaCaT cells were subcultured upon confluence, seeded (1x106 cells/ml) onto collagen-coated PTFE Transwell membrane inserts and incubated at 33°C and 37°C for 24 days to allow differentiation and stratification. Once cells became confluent they were exposed to the air-liquid interface and fed with KGM Gold (Lonza) supplemented with 10% FBS and additional calcium. Thereafter, cells were fixed in 3.7% phosphate-buffered formaldehyde, embedded in a paraffin block, sectioned, stained and viewed. Hematoxylin and Eosin (H&E) staining was used to determine the resemblance to in vivo human skin. Immunofluorescence was used to detect keratin 10, keratin 14 and involucrin which are markers of keratinocyte differentiation. Stratified keratinocyte layers were infected with C. trachomatis and this was confirmed using the MicroTrak ® C. trachomatis Culture Confirmation Test Kit. Subsequent changes to the layers were also observed and recorded. It was shown that HaCaT cells grown at the air-liquid interface on collagen-coated PTFE Transwell membrane inserts were able to stratify at 33°C. However, more layers of keratinocytes were seen at 37°C after the same duration of incubation (24 days). Keratin 10, keratin 14 and involucrin were all detected in the layers grown at both temperatures, suggesting that the keratinocytes had committed to differentiation. However, the fluorescence seen at 33°C for keratin 10 and involucrin was more intense as compared to that seen at 37°. This suggests that although stratification was faster at 37°C, differentiation was quicker at 33°C. C. trachomatis was able to infect layered keratinocytes grown at both temperatures although not all layers formed at 33°C were infected. Degradation of keratinocyte layers after infection with C. trachomatis was more prominent in those grown at 37°C, which is in keeping with previous findings that the optimum growth temperature of the C. trachomatis LGV biovar is 37°C. This study provided a novel insight in suggesting the manner in which C. trachomatis is able to infect and migrate through in vivo skin, leaving room for further studies in which similar methods of generating stratified keratinocytes may be used to better understand the pathophysiology of various other organisms that affect keratinocytes.
Development of an antigen detection based point-of-care test for the diagnosis of primary syphilis.
(2011) Naicker, Meleshni.; Sturm, Adriaan Willem.
Aim: To develop an antigen detection based, point-of-care test that will rapidly exclude syphilitic infection in patients presenting with genital ulcers. Materials and Method: T. pallidum subsp pallidum, Nichols strain, was propagated by intra-testicular inoculation of rabbits. T. pallidum DNA was obtained by suspending the testicular extract in ProbeTec lysis buffer followed by heating. Crude DNA was purified and concentrated. Specific primers were used for the amplification of the gene encoding the 31 kDa T. pallidum rare outer membrane porin protein (termed “Tromp1”). The amplified gene was cloned in frame with the pET100/D-TOPO vector that carries the N-terminal Xpress epitope and polyhistidine fusion tags. A screening PCR, restriction digest and DNA sequencing were used to confirm the presence of the tromp1 insert. Isolated plasmid DNA, pET100/D/tromp1 and the pET100/D/lacZ (positive control) were transformed into BL21 (DE3) pLysS E. coli cells for expression of recombinant Tromp1 and β-galactosidase as fusion proteins. SDS-PAGE and Western blot analysis were applied for detection of the recombinant proteins. Results: The gene encoding the 31 kDa Tromp protein was successfully cloned and sequenced. Multiple sequence alignment showed 100% homology between the cloned tromp1 gene sequence and its reference sequence. In addition, a screening PCR for transformation products and restriction digest of isolated plasmid DNA confirmed the presence of the tromp1 insert. Following gene expression, SDS-PAGE gel analysis showed no difference in the banding pattern between IPTG induced and uninduced lysates. The positive control however, showed a bright and distinct band at its expected size range of ~121 kDa. A Western blot and ELISA using specific antibodies to the N-terminal Xpress epitope fusion tag confirmed the absence of recombinant Tromp1 protein. Discussion and Conclusion: The results show that the tromp1 insert was successfully cloned and maintained up until the expression level. However expression of recombinant Tromp1 in BL21 (DE3) pLysS E. coli cells, for use as antigen in the serodiagnosis of primary syphilis was not achieved, despite several attempts to optimize gene expression. Expression of the positive control gene confirmed that growth and induction were properly performed.

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