Browsing by Author "Sturm, Adriaan Willem."

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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.
Analysis of a multidrug resistant acinetobacter SPP. outbreak in the intensive care unit of King Edward VIII Hospital.
(2000) Deedat, Fathima.; Sturm, Adriaan Willem.
The study arose out of a need to investigate and control a nosocomial outbreak caused by multidrug resistant Acinetobacter spp in the fifteen-bed intensive care unit of King Edward VIII Hospital. Following the discovery of the index case, four other patients were found to have a similar strain of Acinetobacter spp. All fifteen patients in the ward were subsequently screened for the organism. Forty-seven isolates were obtained from 12 patients. Eight of the patients were infected with the organism and six of these eight patients subsequently died. Swabs from the ward environment were also screened for the organism, which was found in patients' baths, suction water and urine collection jars. The outbreak was aborted by the use of strict infection control techniques. Minimum inhibitory concentrations (MICs) of 20 of the 47 isolates were determined for the following antimicrobials: imipenem, ciprofloxacin, gentamicin, amikacin, netilmycin,cefotaxime, ceftazidime and tetracycline. The same 20 isolates were further typed using ribotyping. Seven different antibiogram patterns were obtained using the MIC data. The majority of isolates (11) fit into a Single type, and showed resistance to all drugs tested, except for susceptibility to tetracycline and netilmycin only. Ribotyping revealed 5 different types. There were 9 isolates of ribotype a, 2 of ribotype b, 3 of ribotype c, 5 of ribotype d and 1 of ribotype e. In conclusion, this study describes a nosocomial outbreak with a multidrug resistant Acinetobacter spp. in an intensive care unit. The results showed that there was no correlation between the two typing methods used, ribotyping was more discriminatory than antibiogram types, with the majority of strains belonging to two different ribotypes.
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.
The assessment of humoral immunity in the vaginal mucosa of pregnant and non-pregnant women.
(2003) Omar, Momeen.; Sturm, Adriaan Willem.
Mucosal surfaces are prominent in the gastrointestinal, urogenital, and respiratory tracts and provide portals of entry for pathogens. The mucosal immune system consists of molecules, cells, and organised lymphoid structures intended to provide immunity to pathogens that impinge upon mucosal surfaces. The aim of this study was to assess humoral immunity in the vaginal mucosa and compare this immune response to a systemic response. The use of commercially available tampons provided a self-administered, pain free method for the collection of vaginal secretions. To standardise specimens, a total protein determination was performed on vaginal secretions and on sera. All subjects were screened for sexually transmitted infections (STIs) using conventional and deoxyribonucleic acid (DNA) amplification tests. Immunoglobulin levels in vaginal secretions and in sera were quantitated using a quantitative sandwich enzyme- linked- immunosorbent assay (ELISA). The immunoglobulin levels quantitated were analysed on the basis of pregnancy status and the presence or absence of an STI. Immunoglobulin results for serum showed a significant increase in IgG and IgA in women with an STI regardless of pregnancy (p< 0.001). This study showed a decrease in vaginal IgG and IgA in women with an STI. Non-pregnant women with an STI had significantly lower levels of IgG and IgA in the cervico-vaginal secretions as compared to the controls (p=0.002 and p=0.0002 respectively). This was also observed in pregnant women (p= 0.03 and p< 0.001 respectively). IgM levels were mostly too low to be detectable but showed a tendency to increase in vaginal secretions of women with an STI. Pregnancy did not have an effect on immunoglobulin levels except for IgA. The effects observed were due to the presence of an STI. All the STI pathogens studied displayed a similar effect on immunoglobulin levels. Bacterial vaginosis, however, appears to exert an effect specifically on lowering IgG (p=0.008) in vaginal fluid and increasing IgG levels (p=0.008) in serum. Once a more complete understanding of the mechanisms associated with the host defence of the vaginal mucosa is obtained, specific immunotherapeutic strategies can be developed. A greater knowledge of host defence factors specific to the vagina will provide insights into understanding susceptibility to opportunistic infections and STIs.
Beta-lactamase mediated resistance in Escherichia Coli isolated from state hospitals in KwaZulu-Natal.
(2008) Mocktar, Chunderika.; Essack, Sabiha Yusuf.; Sturm, Adriaan Willem.
Escherichia coli, one of the most common pathogens causing urinary tract infections, has shown increased resistance to commonly used antibiotics. In this study we analyzed the β-lactamase profiles of 38 inhibitor-resistant E. coli isolates obtained from public hospitals at three different levels of healthcare in KwaZulu-Natal, selected on the basis of their resistance profiles to the three antibiotic/inhibitor combinations, viz., amoxicillin/clavulanate, ampicillin/ sulbactam and piperacillin/ tazobactam. The isolates were subjected to MIC determinations, IEF analysis, plasmid profile analysis, PCR of the different β-lactamase genes and sequencing thereof to detect the possible mechanism/s of resistance. A range of β-lactamases including two novel inhibitor-resistant TEM β-lactamases, TEM-145 and TEM-146 were detected in two isolates whilst a novel plasmid-mediated AmpC-type β-lactamase, CMY-20 was detected in three isolates. Other β-lactamases included OXA-1, TEM-55, SHV-2, CTX-M-l and TEM-1. Changes were detected in the chromosomal AmpC promoter/attenuator regions in one isolate. Diverse β-lactamase genes and plasmid profiles inferred extensive mobilization of β-lactamase genes causing the concern of limited therapeutic options in the face of increasing resistance.
Beta-lactamase mediated resistance in Salmonella spp. at a tertiary hospital in KwaZulu-Natal.
(2008) Govinden, Usha.; Essack, Sabiha Yusuf.; Sturm, Adriaan Willem.; Moodley, Prashini.
Extended spectrum (3-lactamases (ESBLs) were characterized in Salmonella spp. isolates from a pediatric ward of a hospital in Durban. Forty one Salmonella spp. were subjected to serotyping, antibiotic susceptibility testing, E-Tests for ESBL detection, iso-electric focusing, polymerase chain reaction for detection of genes and sequencing. Isolates were screened for the presence of WaTEM, WaSHV, WaCTX-M, WaOXA , WaCMY, WaDHA and WaACC genes. The most common serotype was Salmonella Typhimurium. Isolates were multi-drug resistant with 100% susceptibility only to meropenem and ciprofloxacin. Tazobactam was the most effective inhibitor. Forty-one percent of the isolates were resistant to ceftriaxone, thus limiting therapeutic options for Salmonella infections.TEM-1 was the most predominant (3-lactamase found in 51% of isolates while SHV-12 found in 39 % was the most common ESBL. TEM-63 was evident in 29 %, TEM-116 in 10 % and TEM-131 was found in one isolate. The high ceftazidime MICs of isolates expressing only TEM-63 were indicative of R164S substitution which widens the binding cavity to accommodate the bulky side chains of oxyiminoaminothiazolyl cephalosporins. The identification of TEM-131 which differs from TEM-63 by 1 amino acid reiterates the evolutionary potential of the TEM-type plactamase. Other ESBLs identified included SHV-2, CTX-M-3, CTX-M-15 and CTX-M-37. CMY-2 and the OXA-1 p-lactamase were also detected. This is the first report of TEM-116, CTX-M-3, -15 and -37 in Salmonella spp. in South Africa. All isolates with nalidixic acid MICs > 48 ug/ml had the mutation D87N, or D87G in the QRDR of the gyrA gene. This study showed that Salmonella spp. may be multi-drug resistant with the propensity to harbour p-lactamases in unique combinations. The diversity of ESBLs and the co-expression of quinolone resistance suggests that their incidence in salmonellae needs to be monitored.
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 develolpment of a rapid diagnostic test for the detection of haemophilus ducrey.
(2010) Pillay, Mona.; Sturm, Adriaan Willem.
Aim: To develop an antigen detection test that would quickly exclude H. ducreyi infection in individuals with genital ulcers. Materials and Methods: H. ducreyi strains A54 and A68 were grown on Modified Bieling (MB) agar plates and in MB broth under microaerophilic conditions. The 58.5 kDa GroEL Heat Shock Protein (HSP) was extracted from H. ducreyi strain A54 by means of sonication. The purified HSP was used to raise antibodies in rabbits. HSP determination and separation was done on SDS PAGE gels and protein was eluted by means of a passive elution process. Antibody was purified by affinity chromatography and a fraction of the antibody was conjugated to a chromogen to be used as a detection antibody. An ELISA was developed to evaluate the antibody response to the HSP. A second ELISA was developed to evaluate test parameters. Results: A good immune response was achieved with the crude serum of one of the three rabbits when tested against the antigen by means of ELISA. However, after purification of the IgG from the serum of the same rabbit no antigen-antibody binding was observed. Anti-rabbit IgG was able to recognise the antibodies. Discussion and Conclusion: While the Fc portion of the purified IgG remained active, the Fab portion of the antibody had lost biological activity. This loss of biological activity of antibody can be attributed to the low pH of the elution buffers used during the purification steps. Alternative antibody purification systems need to be explored. The use of monoclonal antibodies also needs to be considered.
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 and implementation of a mycobacterium tuberculosis rapid diagnostic assay, using reporter mycobacteriophages.
(2013) Makume, Mantha Thandiwe.; Sturm, Adriaan Willem.; Jacobs, William R.
Abstract available in PDF file.
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.
Differences in the susceptibility of mycobacterium tuberculosis to the 1st and 2nd line antituberculosis drugs under aerobic and anaerobic conditions.
(2015) Ngcobo, Zethembiso Brightness.; Botha, Stephanus Johannes.; Sturm, Adriaan Willem.
Although Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is now considered a facultative anaerobe, and bacilli isolated from sputum specimen possess morphologies identified from bacilli growing aerobically and under oxygen deprived conditions, most of the targets for the antituberculosis drugs are readily found on bacilli that are thriving aerobically. This raises questions on the efficiency of antituberculosis drugs on eradicating the pathogen from the host during treatment. In this study to determine whether the antituberculosis drugs that are used currently for the treatment of TB have similar effect of these different populations of this mycobacterium, we grew this organism under aerobic and oxygen deprived environments and then subjected them to the antimicrobial agents. The minimum inhibitory concentration (MICs) of these isolates against nine antituberculosis drugs were determined under aerobic conditions for the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay and under both aerobic and anaerobic conditions using the Microscopic Observation Drug Susceptibility (MODS) assays. In addition the bactericidal activities of isoniazid, rifampicin, kanamycin and ofloxacin were tested and compared amongst MDR isolates that were growing aerobically and anaerobically. There were some differences in the MICs determined by the MTT assay and the MODS assay for some isolates. For the susceptible isolates the MICs from the MTT assay were higher than the MICs from the MODS assay. The reverse was true for the drug resistant isolates. The reference strain H37Rv was resistant to some of the antimicrobial agents that were tested in this study. This was under both methods. However, MICs measured under anaerobic conditions with anaerobic bacilli did not yield viable results due to absence of growth as the bacilli are known to replicate at a negligible rate under anaerobic conditions. The bacilli in the inoculum were viable as following 40 days of anaerobic incubation but upon aerobic incubation of these cultures, growth was observed. And again with the bactericidal assays that were conducted on the multidrug resistant (MDR) isolates proved this. Rifampicin was the most potent antimicrobial agent against the anaerobic M. tuberculosis as susceptibility to this antimicrobial agent increased under anaerobic conditions.
The effect of antimicrobials used for genital discharge disease on trichomonas vaginalis.
(2017) Mabaso, Nonkululeko Gladness.; Sturm, Adriaan Willem.
Trichomoniasis is the most common sexually transmitted infection caused by the protozoan, Trichomonas vaginalis. T. vaginalis infection is often asymptomatic. This infection causes vaginal discharge in women and urethritis in men. It has been reported that trichomoniasis is associated with serious health complications and it increases the risk of HIV acquisition and transmission. The gold standard for trichomoniasis diagnosis is culture, however various point of care tests have been approved by the US FDA. Metronidazole is the standard treatment for trichomoniasis. Multiple cases of metronidazole-resistance have been reported since 1962. Syndromic management of STIs is used to treat multiple infections simultaneously based on the signs and symptom with which the patient presents. In South Africa, the vaginal discharge syndrome is managed with ceftriaxone, azithromycin and metronidazole. Ten T. vaginalis isolate were tested. Each isolate was tested against six combinations of two antimicrobials by the checkerboard method, four combinations of three antimicrobials and two combinations of four antimicrobials. The results obtained from the checkerboard of two antimicrobials were used to design the experiments for three and four antimicrobials combinations. The MICs for metronidazole ranged between 0.25 – 1 μg/ml and for doxycycline, they ranged between 64 – 128 μg/ml. Ceftriaxone and fluconazole showed no antitrichomonal activity. All combinations tested has an indifferent effect. Combinations of metronidazole and antimicrobials used in syndromic management including fluconazole has no effect against T. vaginalis. However, in the combination of metronidazole and doxycycline a decrease in the MICs for these antibiotics was observed. Further studies are required to test this combination on a larger sample size.
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.
The effect of self-generated hypoxia on the expression of target genes coding for electron transport related products in mycobacterium tuberculosis.
(2010) Ramchandra, Prathna Harrikaran.; Sturm, Adriaan Willem.
The work presented here aims at identifying whether the genes identified in the genome of Mycobacterium tuberculosis that code for products involved in anaerobic metabolism are active or inactivated genes. The study consists of three distinct parts. In part one, serial dilutions of sputum of patients with pulmonary tuberculosis (PTB) were grown on agar surface and in high columns of un-agitated broth. The highest dilution from which mycobacteria was grown was for all patients significantly higher in the broth cultures than on the plates suggesting the presence of anaerobically metabolizing mycobacteria in the lungs of patients with PTB. Part two of the study identified gene expression by measuring the concentration of transcripts for 5 genes involved in aerobic or anaerobic pathways. This was done over a period of 15 weeks using un-agitated broth cultures (the Wayne method). Undulating patterns of gene expression were found with the genes coding for anaerobic metabolic pathway components expressed at higher levels than those coding for aerobic pathway components while the cultures grew older. Part three aimed at measuring transcription products of the same set of genes directly in sputum specimens. Although quantitation at bacterial cell level in the sputum could not be achieved, expression of all genes was established with on average larger quantities of transcripts of genes coding for the anaerobic pathway components.
Effect of the capsular material of cryptococcus neoformans on the interplay between Mmicroglial cells and Neutrophils.
(2019) Bhola, Prathna.; Sturm, Adriaan Willem.
Cryptococcal meningitis is an important opportunistic infection in immunocompromised patients. It has been well established that a distinguishing feature of this form of meningitis is a relatively low neutrophil count in the cerebrospinal fluid (CSF) compared to bacterial meningitis. There has been speculation and research undertaken previously to understand this phenomenon, however, little information is available in human studies. Furthermore, there is insufficient information on expression and function of Toll-like receptors (TLR) in the human central nervous system (CNS). The work presented here investigated the effect of the capsular material of a series of clinical isolates of Cryptococcus neoformans on neutrophil recruitment at the site of infection and determined whether downregulation occurs at the level of TLR expression. This was done in a multiple component study. Clinical information was collected from patients with cryptococcal meningitis and baseline blood and CSF investigations were performed, which included the quantification of neutrophils in CSF and blood specimens. The size of the Cryptococcus capsule was measured in each isolate and shed capsular material was quantified in individual CSF specimens. The extent of neutrophil chemotaxis inhibition by individual strains of C. neoformans was determined by using a Transwell migration assay. Toll-like receptor (TLR)2 and TLR4 gene expression induced by individual C. neoformans isolates in human microglial cells was quantified. The possible associations among these experiments were subsequently evaluated. As anticipated, a paucity of neutrophils in the CSF was observed. The cryptococcal capsule was larger in isolates of patients with lower CSF neutrophil counts. In addition, patients with lower CSF neutrophil counts shed more capsular material in the CSF. Chemotaxis inhibition occurred in close to 70% of tested isolates. The concentration of shed capsular material in this group was higher compared to the group with no chemotaxis inhibition. Patients presenting with fever had higher CSF neutrophil counts as well as elevated intracranial pressures. The majority of isolates expressed downregulation for TLR2 and TLR4 in microglial cells exposed to C. neoformans. CSF neutrophil counts were lower in this group. These findings imply that the capsular components of C. neoformans downregulated recruitment of neutrophils into the CSF. Downregulation of neutrophil recruitment was observed at the level of TLR expression.
Evaluation of time to detection of mycobacterium tuberculosis in broth culture as a determinant for end points in treatment trials.
(American Society for Microbiology., 2010) Weiner, Marc.; Prihoda, Thomas J.; Burman, William.; Johnson, John L.; Goldberg, Stefan.; Padayatchi, Nesri.; Duran, Paula.; Engle, Melissa.; Muzanye, Grace.; Mugerwa, Roy D.; Sturm, Adriaan Willem.
Development of new treatments for tuberculosis is hampered by the lack of an accurate surrogate end point and the high degree of efficacy of current 6-month regimens. Sputum culture status after 2 months of therapy, a binary test, is widely used for phase IIB trials but has only moderate accuracy for predicting failure/relapse (12) and requires large sample sizes (4, 8). Changes in the number of colonies found in dilutions of sputum applied to solid medium is an end point that has been used to assess activities of single drugs and doses in phase IIA (early-bactericidal-activity) studies (10) and has also been suggested as an end point for phase IIB trials (15). Though promising, quantitative culture on solid medium involves prolonged sputum collections and intensive laboratory techniques and has been difficult to standardize at multiple sites. Time to detection in broth culture (TTD) is a potential end point that has a good correlation with quantitative culture on solid medium (11, 13). An initial small study had suggested a correlation between a shorter time to detection (an indication of higher numbers of viable bacilli) and poor treatment outcomes (9). In this study, TTD was evaluated as a marker of regimen potency. Preliminary results have been reported elsewhere (16).
In vitro culture and isoenzyme analysis of giardia lamblia.
(1999) Kwitshana, Zilungile L.; Jackson, Terry F. H. G.; Sturm, Adriaan Willem.
Giardia lamblia, an enteric protozoan parasite, infects a large number of individuals worldwide. In South Africa prevalences ranging between 4 and 63% are documented, however, the impact of giardiasis is underreseached in this country. Giardia infections vary from asymptomatic carriage or a self-limiting acute symptomatic illness to chronic, debilitating malabsorption syndrome. The factors responsible for development of symptomatic versus asymptomatic infection are poorly understood. It is believed by some that host factors determine the clinical outcome of infection. On the other hand, the possibility of the existence of pathogenic and non-pathogenic strains (a situation akin to Entamoeba spp.) remains to be explored. One requirement for investigation of the potential contribution of strain differences to pathogenecity of infection is establishment of laboratory cultures of different strains isolated from symptomatic and asymptomatic patients. The present study was undertaken to develop and modify existing methods for: (i) establishment of laboratory cultures of Giardia trophozoites from excystation of faecal cysts, (ii) long-term maintenance and cryopreservation of the cultures and (iii) preliminary characterisation methodology. One thousand and twenty-three stool specimens were collected from day care centres, hospital wards and Hlabisa hospital laboratory. A further 6246 were retrieved from the Microbiology Laboratory at King Edward VIII Hospital and screened by direct wet preparation. Giardia was detected by light microscopy following formol-ether concentration (127 of 1023 samples) or direct examination of wet preparations (78 of 6246 samples). Cysts were purified from the positive specimens by sucrose gradient separation. Viability was assessed by a dye-exclusion method (eosin). Three in vitro excystation techniques were employed in an attempt to obtain trophozoites for initiation and establishment of viable cultures thereof. Culture conditions were optimised using two reference strains of Giardia, WB & H7 (obtained from the National Institutes of Health, USA). The percentage excystation ranged between 0-42% with all the in vitro methods of excystment. Excysted trophozoites remained viable in TYI-S-33 culture medium for periods ranging between 12-72 hours or up to 9 days, and gradually died, hence viable trophozoite cultures could not be established. Some culture initiates (overall 65%) were lost through overwhelming bacterial and!or fungal contaminants. An animal model was subsequently set up in which C57BL/6 and Praomys (Mastomys) coucha mice were used for in vivo excystation experiments. 1-3 day old suckling mice were intragastrically injected with 10,5 -cysts/ ml in 0,1 ml distilled water. Trophozoites were retrieved from the stomachs of infected mice 7-10 days after inoculation and cultivated in TYI-S-33 medium. Six local isolates were axenised using the in vivo excystation method. They have been maintained for more than 15 months in culture after stabilates and Iysates of confluent growths had been cryopreserved in Liquid Nitrogen. Successful (100%) retrieval of the cryopreserved cultures has been achieved. Seven isoenzyme electrophoresis systems have been set up and optimised. Reproducible results were obtained in six of the enzymes. Some differences in banding patterns of the enzymes were demonstrated.
Interaction between Mycobacterium tuberculosis and pulmonary epithelium.
(2013) Ashiru, Olubisi T.; Sturm, Adriaan Willem.
Background Mycobacterium tuberculosis isolates such as the Beijing and F15/LAM4/KZN families dominate in patients. The emergence of extensively drug resistant (XDR) M. tuberculosis isolates raises concern. The need to better understand the pathogenesis of M. tuberculosis isolates resulted in this work. Methods M. tuberculosis clinical isolates that belonged to the Beijing and F15/LAM4/KZN families, isolates with unique DNA fingerprints and laboratory strains were used. Isolates were grown in the presence of oxygen and then exposed to A549 alveolar and BBM bronchial epithelial cells. The number of bacilli that adhered to the epithelial cells were viewed and counted using light microscopy. Isolates grown in the presence of oxygen and under oxygen deprivation were used for subsequent assays. Invasion of A549 and BBM cells by isolates grown under these different circumstances was investigated. Based on the results, the remaining assays were performed with A549 cells only. Cytotoxicity was quantified using the Cyto Tox96 Non-Radioactive Cytotoxicity Assay kit. Morphological changes in A549 cells after exposure to the isolates were observed using the scanning electron microscopy (SEM). Real-time quantitative PCR was performed to assess the relative expression levels of four genes potentially associated with virulence (hbhA; mdp1; fdxA; hspX). Results were normalized against 16S rRNA and ftsZ gene transcription and reported as fold difference as compared to H37Rv. Results All isolates adhered to and invaded A549 cells in significantly higher numbers than BBM cells (P<0.0029). Isolates grown under oxygen deprivation displayed higher levels of virulence than their aerobic phenotype. Grouped together, the isolates belonging to the Beijing and F15/LAM4/KZN families of strains showed greater adhesion capacity (28%) than isolates with unique DNA fingerprints (5%) (P<0.05%). Three F15/LAM4/KZN isolates (two XDR-variants), were at least twice as invasive (>33%) as the most invasive Beijing isolate (15%) (P<0.05). The highest cytotoxicity level (35.7%) was produced by an XDR-F15/LAM4/KZN strain. SEM revealed bleb-like structures on bacterial cells grown under oxygen deprivation. Beijing and XDR-F15/LAM4/KZN isolates had the highest number of projections (16+5 per bacillus. The expression levels of all four genes were highest in Beijing and F15/LAM4/KZN isolates grown under oxygen deprivation and exposed to A549 cells. Conclusions Beijing and F15/LAM4/KZN strains are more virulent and their successful spread might be related to their interaction with alveolar epithelium. M. tuberculosis pathogenesis studies should include isolates grown under oxygen deprivation.