Theses and Dissertations (Medical Microbiology)

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Characterization of plasmids mediating carbapenem resistance in Klebsiella pneumoniae in Pretoria, South Africa
(University of Pretoria, 2020-01) Mbelle, Nontombi; Osei-Sekyere, John; kopotsakatlego@gmail.com; Kopotsa, Katlego
pneumoniae is a Gram-negative bacterium belonging to the Enterobacteriaceae family. This pathogen is implicated in community- and hospital-acquired infections, particularly in neonates, the elderly, and immunocompromised patients. Risk factors such as extended hospital stay, being immunocompromised, and excessive antibiotic use lead to disease severity and potential acquisition of resistance to antibiotics. Carbapenems are usually the treatment of choice in multidrug-resistant K. pneumoniae infections. Since the last decade, carbapenems has become less effective as K. pneumoniae stains develop mechanisms of resistance against them. Among the various mechanisms of carbapenem resistance, enzyme (carbapenemases, ESBLs, AmpCs) production is the most dominant. Carbapenemases are classified into three classes viz., class A, B and D, and have the ability to hydrolyse β-lactams antibiotics, including “last resort” carbapenems. The most commonly reported carbapenemases worldwide in K. pneumoniae strains include the blaKPC, blaNDM, blaVIM, blaIMP and blaOXA genes. In South Africa, blaOXA-48/181 and blaNDM-1 have been reported in K. pneumoniae in almost all provinces, with outbreaks being reported in other provinces within the past six years. Plasmids are shuttles that mediate the acquisition and dissemination of carbapenemases. This is because plasmids are mobile and may be transferred from one specie to another via horizontal gene transfer. Conjugative plasmids such as IncF, A/C, IncL/M, IncN and IncX plasmids have been associated with commonly reported carbapenemases worldwide. In South Africa, little is known about plasmid types associated with carbapenemase genes and their molecular characteristics. This study aimed to identify and characterise plasmids mediating carbapenem resistance in Pretoria, South Africa. Sixty K. pneumoniae clinical isolates were collected from the National Health Laboratory service (NHLS, Pretoria) in 2018. Carbapenem resistance and carbapenemase production was determined using MicroScan automated system and PCR assays, respectively. The isolates’ plasmids were characterized to determine their size, number and replicon types using gel electrophoresis, and PCR-based replicon typing techniques, respectively. Molecular characteristics of the isolates’ carbapenemases and plasmids were analysed using both PCR assays and whole-genome sequencing (WGS). All K. pneumoniae isolates were multidrug resistant. Carbapenemase production was identified in 65% (blaOXA-48-like) and 29% (blaNDM-1) of the isolates. Multi-locus typing revealed five sequence types: ST307, ST607, ST17, ST39, and ST3559. Both PCR and WGS revealed multiple plasmid replicons associated with the carbapenem-resistant K. pneumoniae (CRKP) isolates viz., IncF, A/C, IncL/M and IncX3 plasmids. WGS proved to be more useful in characterising plasmids over the PCR-based replicon typing (PBRT). The PBRT could not identify the IncX3 replicons, which were detected by WGS. Identified plasmids could be transferred from donor CRKP strains to recipient E. coli strains. Phylogenomic analysis showed that strains in this study were closely related to stains from the United States, China, Thailand, and South Korea more than other countries. These strains shared similar antimicrobial resistance mechanisms, however, they belonged to different sequence types including ST14, ST11, ST147, and ST152. This study shows an ongoing plasmid-mediated dissemination of carbapenemase genes in 2018 in Pretoria, with blaOXA-48-like and blaNDM-1 genes being the major resistance determinants. This study has also shown the important role conjugative or mobile plasmids play in the acquisition and dissemination of carbapenemase genes from one specie to another via conjugation.
Molecular characterisation of methicillin-resistant Staphylococcus aureus isolates associated with outbreaks in burn wound and neonatal ward patients at healthcare centres in Gauteng, South Africa
(University of Pretoria, 2019-12) Ehlers, M.M. (Marthie Magdaleen); Kock, Marleen Magdalena; Strydom, Kathy-Anne; keitumetseg8@gmail.com; Gama, Keitumetse B.
Methicillin-resistant Staphylococcus aureus (MRSA) is one of the leading causes of nosocomial infections worldwide. It is an ESKAPE pathogen and is known for causing difficult-to-treat infections due to its antibiotic resistance. In addition to its antibiotic resistance, this bacterium has a large arsenal of virulence factors that allow this pathogen to cause disease and to evade the host immune system. An increase in the number of reports of MRSA isolates from the burn unit and neonatal wards from various hospitals across the Gauteng province prompted this study. The aim of this study was to molecularly characterise the MRSA isolates associated with outbreaks in the burn and neonatal wards at four hospitals in Gauteng, South Africa using multiplex polymerase chain reaction (M-PCR) assays, pulsedfield gel electrophoresis (PFGE) and whole genome sequencing (WGS). The study also aimed to determine the antibiotic and virulence gene profiles associated with these MRSA strains. viii To identify MRSA, a M-PCR assay was performed to confirm the presence of the Staphylococcus 16S rRNA gene, the S. aureus-specific nuclease (nuc) gene and the methicillin A (mecA) gene that confers resistance to beta-lactam antibiotics. The isolates were also screened for the Panton-Valentine Leukocidin (pvl) gene, which encodes a pore forming toxin associated with severe disease. All 85 isolates were confirmed to be MRSA and none of the isolates were pvl-positive. Susceptibility testing of the MRSA isolates revealed that the isolates were resistant to antibiotics such as penicillin (100%), cloxacillin (100%), gentamicin (98%), clindamycin (97%), erythromycin (97%), ciprofloxacin (91%) and tetracycline (84%). Susceptibility to vancomycin, teicoplanin, linezolid and fusidic acid was observed. The dendrogram constructed based on the PFGE banding profiles revealed that the MRSA isolates clustered into three major pulsotypes. The largest pulsotype, Pulsotype A, consisted of 32 MRSA isolates that were recovered from burn and neonatal wards. Pulsotypes B and C were made up of five isolates each and only consisted of isolates from the neonatal wards. All three pulsotypes were composed of MRSA isolates from different hospitals, recovered between 2015 and 2019. Five representative isolates were selected based on their clustering and antibiotic resistance and sent for WGS. Three clones, ST239-MRSA-III, ST5-MRSA-I and ST612-MRSA-IV were identified using WGS data. These clones were associated with spa types t037, t045 and t1257 respectively. The clone ST239-MRSA-III-t037 was detected in three different hospitals. The virulence factors detected in the five isolates included staphylococcal enterotoxins A (SEA), SEB, SEG, SEK, SEN, SEO, and SEQ and the bicomponent pore-forming leukocidins, gamma-hemolysin and leucocidin ED. The immune evasion complex (IEC) genes identified were the staphylococcal complement inhibitor, staphylokinase and SEA. The movement of patients and healthcare workers between wards and hospitals may have resulted in intra- and inter-hospital spread of MRSA. The study emphasises the importance of having infection control programs in place and adhering to them. The importance of continuous surveillance is also emphasised.
Antimicrobial resistance mechanisms of linezolid resistant staphylococci and enterococci collected in Gauteng, South Africa
(University of Pretoria, 2019) Ehlers, M.M. (Marthie Magdaleen); Strydom, Kathy-Anne; karenladdison@gmail.com; Addison, Karen
Staphylococci and enterococci are key human pathogens responsible for infections associated with healthcare settings. Linezolid is crucial for managing multidrug-resistant (MDR) infections. The monitoring of resistance to antimicrobial agents is a global effort. Linezolid has two surveillance programs which endeavour to monitor linezolid resistance, namely: Zyvox® Annual Appraisal of Potency and Spectrum (ZAAPS) and Linezolid Experience and Accurate Determination of Resistance (LEADER). In contrast to the ZAAPS and LEADER surveillance programs, linezolid resistance data is lacking in South Africa. This study aimed to determine linezolid resistance 23S ribosomal ribonucleic acid (rRNA) gene mutations and the acquired chloramphenicol-florfenicol resistance (cfr) gene of staphylococci and enterococci obtained from public and private hospitals in Gauteng, South Africa. A total of 79 staphylococcal isolates (43 Staphylococcus capitis, 27 Staphylococcus epidermidis and nine Staphylococcus haemolyticus) and 32 enterococcal isolates (28 Enterococcus faecalis and four Enterococcus faecium) were obtained for investigation. Initial linezolid susceptibility was evaluated using the VITEK® 2 automated system (bioMérieux, France). Staphylococcal and enterococcal isolates showing intermediate resistant and resistant according to the 2019 Clinical and Laboratory Standards Institute (CLSI) guidelines were selected for this study. The minimum inhibitory concentration (MIC) values were confirmed using the ETEST® (bioMérieux, France). Confirmatory identification multiplex polymerase chain reaction (M-PCR) assays and cfr gene detection by polymerase chain reaction (PCR) was used, followed by evaluation of relatedness using pulsed-field gel electrophoresis (PFGE). Whole-genome sequencing (WGS) of seven S. epidermidis isolates and three S. capitis isolates determined the 23S rRNA gene mutations and confirmed the presence of the cfr gene. The ETEST® (bioMérieux, France) MIC values of the staphylococcal isolates ranged between 8 μg/mL and > 256 μg/mL and the enterococcal isolates MIC values ranged between 2 μg/mL and 4 μg/mL. All the staphylococcal isolates were resistant to linezolid and the enterococcal isolates showed susceptibility and intermediate resistance, according to the 2019 CLSI guidelines. The cfr gene was found in eight S. epidermidis isolates. The S. capitis isolates and S. haemolyticus isolates were all cfr negative. The dominant sequence type (ST) among the S. epidermidis isolates was ST23 (n = 4), followed by ST2 (n = 2) and ST22 (n = 1), all of which are clinically relevant STs having been extensively reported among nosocomial infections. Several 23S rRNA gene mutations were observed in this study among the S. epidermidis isolates and the S. capitis isolates. Known and previously reported mutations found in this study were C2190T, G2603T and C2561T among S. epidermidis and S. capitis. However, several unknown and previously unreported 23S rRNA gene mutations were observed among S. capitis, namely: T2157A, T2346C, C2287G, A2295G, A2296G, C2302G, A2305G, C2308G and A2314C. The S. capitis isolate that showed all previously unreported 23S rRNA mutations had a significantly higher MIC value, thus indicating that 23S rRNA gene mutations are a significant contributing factor in linezolid resistance. These novel 23S rRNA gene mutations are not previously reported in the literature and are therefore important for future research. The PFGE results showed diversity among the staphylococcal isolates between hospitals, suggesting a wide spread of the strains. Linezolid resistance is concerning for antimicrobial management efforts and the data generated from this study provides valuable information regarding the prevalence of linezolid resistant strains circulating in the Gauteng region of South Africa.
Molecular epidemiology of Klebsiella pneumoniae isolates causing invasive disease from a private laboratory in South Africa
(University of Pretoria, 2024-06-07) Kock, Marleen; Pitout, Johann; Dreyer, Andries; sarah.abro@gmail.com; Liknaitzky, Sarah
Klebsiella pneumoniae (K. pneumoniae) is the most clinically significant species within the Klebsiella genus. The main burden of K. pneumoniae infections are healthcare-associated infections (HAIs), generally caused by the classical multidrug resistant (MDR) strains that can resist the activity of multiple antibiotics. The most clinically significant antibiotic resistance in K. pneumoniae is toward the beta-lactam antibiotics mediated by beta-lactam hydrolysing enzymes, specifically the extended-spectrum beta-lactamases (ESBLs), AmpC beta-lactamases and carbapenemases. Resistance is constantly spreading and evolving, predominantly attributed to several high-risk clonal lineages. High-risk clonal lineages are those with an enhanced ability to disseminate and survive in multiple settings and cause antibiotic resistant infection. The aim of this study was to characterise and investigate the molecular epidemiology and beta-lactam resistance of invasive K. pneumoniae isolates from a private laboratory in Gauteng, South Africa. A total of 154 K. pneumoniae isolates isolated from invasive specimens including blood, tissue and fluid were collected between 2021 and 2022 from a private laboratory in Pretoria. Specimens originated from patients in hospitals in Johannesburg, Pretoria and Emalahleni. Antibiotic susceptibility profiles for isolates were obtained using Vitek 2. Disc xv diffusion tests phenotypically confirmed the production of ESBLs, AmpCs and carbapenemases by K. pneumoniae isolates. Phenotypically confirmed isolates underwent polymerase chain reaction (PCR) assays for the detection of specific resistance genes. All K. pneumoniae isolates were genotyped by pulsed field gel electrophoresis (PFGE) and subsequently a dendrogram was constructed using the PFGE profile of each isolate. Major and minor PFGE clusters were identified, and one representative isolate per cluster was chosen from six of the clusters to undergo whole genome sequencing (WGS). The majority of the K. pneumoniae isolates originated from specimens taken from patients in intensive care unit hospital wards, predominantly representing bloodstream infections. High levels of resistance toward multiple antibiotic classes were observed, with 107 (70%) isolates classified as MDR. Amongst all K. pneumoniae isolates, 99 (64%) were phenotypically confirmed ESBL producers, 18 (12%) AmpC producers and 55 (36%) carbapenemase producers. Resistance genes detected included beta-lactamase (bla) sulfhydryl variant (SHV) in 95 isolates (49%), Temoneira (blaTEM) in 60 isolates (39%), the ESBL cefotaximase-Munich (blaCTX-M-15) in 68 isolates (44%), Dhahran (blaDHA) AmpC in seven isolates (5%), and the carbapenemases, oxacillinase-48-like (blaOXA-48-like) in 65 isolates (42%), New Delhi metallo beta-lactamase (blaNDM) in 21 isolates (14%) and K. pneumoniae carbapenemase (blaKPC) in seven isolates (5%). A rare genotype of triple carbapenemase production was detected in two isolates. Within the dendrogram, nine minor PFGE clusters were identified. The six representative isolates from clusters Minor A, Minor C, Minor F, Minor G, Minor H and Minor I that underwent WGS belonged to sequence types (ST) 307 (n = 3), ST2497 (n = 2) and ST3559 (n = 1). The ST307 isolates carried the blaOXA-181 variant. The ST2497 isolates were positive for yersiniabactin, as well as MDR, characterised by the blaOXA-232 variant. The ST3559 isolate was unique in carrying the blaDHA-1 AmpC. The high-risk clones detected in this study are evidence to the local epidemiology of K. pneumoniae in the private healthcare sector of South Africa and represent a major healthcare crisis. Surveillance and molecular epidemiological studies allow insight into current state of affairs regarding HAIs, and are vital in implementing measures to curb spread and outbreaks, as well as developing effective treatment models for highly resistant strains.
Molecular epidemiology of carbapenemase-producing Klebsiella pneumoniae isolates obtained from community-acquired urinary tract infections and rectal carriage isolates
(University of Pretoria, 2023-11) Kock, Marleen Magdalena; Pitout, Johann; kafilattai@gmail.com; Salvador-Oke, Kafilat Taiwo
Antimicrobial resistance (AMR) due to Enterobacterales is a worldwide public health threat. The continuous emergence of multidrug resistant (MDR) bacteria in developing countries with poor sanitation programmes, inactive antimicrobial policies and inadequate infection control infrastructures can affect the health of community members with significant financial burden. South Africa has a high burden of infectious diseases and AMR. Contributing factors to these problems include the use of antimicrobials as growth promoters in food-producing animals and the overuse and misuse of antimicrobials. Among Enterobacterales, Klebsiella pneumoniae (K. pneumoniae) has emerged as the major driver of AMR globally. Klebsiella pneumoniae strains expressing carbapenem-hydrolysing enzymes have been associated with several hospital outbreaks in South Africa. These enzymes are disseminated by K. pneumoniae high-risk clones [sequence type (ST)-307 and non-ST307] and epidemic plasmid [incompatibility group (Inc)-X3]. There is paucity of information on the circulating K. pneumoniae high-risk clones isolated from urine and rectal carriage of patients in South Africa. Active surveillance of K. pneumoniae clones isolated from urine of patients is necessary to implement treatment strategies to manage urinary colonisation or infection. Early screening of at-risk-patients for carbapenem-resistant K. pneumoniae (CRKp) colonisation in hospitals will aid epidemiological monitoring to combat the spread of K. pneumoniae high-risk clones globally. Aim of this study was to investigate the molecular epidemiology of K. pneumoniae obtained from urine and rectal carriage isolates. A total of 446 carbapenemase-producing K. pneumoniae isolates were recovered from urine (n=194; 43%) and rectal carriage (n=252; 57%). Isolates were collected from the Ampath National Reference Laboratory (Ampath-MDRC), Pretoria, between February 2021 and May 2022. Patients’ demographic data were collected. This study employed phenotypic and molecular methods. The molecular methods included polymerase chain reaction (PCR) to detect ST307, IncX3 plasmid and carbapenemase genes [beta-(β)-lactamase genes encoding oxacillinase (blaOXA)-181, New Delhi metallo-β-lactamase (blaNDM), K. pneumoniae carbapenemase (blaKPC), Verona integron-encoded metallo-β-lactamase (blaVIM)]. Repetitive extragenic palindromic (REP)-PCR was used to determine the genetic relatedness among ST307 and non-ST307 isolates. Twenty-three carbapenemase-producing K. pneumoniae isolated from urine (n=10) and rectal carriage (n=13), selected based on the presence of carbapenemase genes were subjected to whole genome sequencing (WGS). The sequenced isolates exhibited MDR, extensively drug resistant and pandrug resistant phenotypes. Ten STs were detected. Among the 23 isolates, three were ST307. The non-ST307 clones include ST2497 (n=5) and ST17 (n=4). The ST17 strains were exclusively isolated from rectal carriage. Co-existence of carbapenemase genes were observed in 10 isolates. The predominant carbapenemase genes among the sequenced isolates were blaOXA-181 (n=11) and blaNDM (n=11). Most isolates were associated with plasmids, bacteriophages and acquired virulence determinants. In this study, K. pneumoniae high-risk clones acquired additional AMR and virulence traits. The high-risk clones were associated with several mobile genetic elements, including the IncX3 plasmid. This study provides insights into the epidemiology of CRKp in South Africa, highlighting the importance of infection prevention and control and antimicrobial therapeutic strategies based on the prevailing high-risk clones in different patient populations. This is essential for successful treatment and prevention of infectious diseases associated with AMR in South Africa.
One Health approach to investigate antimicrobial resistance and public health risk of foodborne pathogens in Gauteng and Limpopo provinces, South Africa
(University of Pretoria, 2023-12) Ehlers, M.M. (Marthie Magdaleen); Fasina, Folorunso Oludayo; stogundare@gmail.com; Ogundare, Samuel Tolulope
Globally, antimicrobial resistance (AMR) in foodborne pathogenic bacteria of the family Enterobacteriaceae has continued to rise. There is an increase in public health concerns about the spread of multi-drug resistant (MDR) foodborne pathogenic E. coli (PEC) and Salmonella enterica of zoonotic importance. This study aimed to characterise and investigate the profiles and resistome of zoonotic foodborne PEC and S. enterica using a One Health approach. Between May 2019 and August 2020, faecal samples from swine and poultry, hand swabs from workers and environmental run-off water samples were collected from commercial abattoirs and farms in Gauteng and Limpopo provinces of South Africa. Similarly, effluents from wastewater treatment plants (WWTPs) and a tertiary hospital setting were collected. The PEC and S. enterica isolates detected were screened for phenotypic antimicrobial susceptibility testing (AST) for extended-spectrum beta-lactamases (ESBL), carbapenem and colistin resistance. Additionally, all PEC and S. enterica isolates were characterised using M-PCR assays to detect AMR genes for ESBL, carbapenems and colistin genes. Genetic relatedness of isolates was determined using pulsed-field gel electrophoresis (PFGE) and representative isolates were sequenced using whole genome sequencing (WGS). A pilot study compared resistomes from run-off water effluent samples from swine and poultry abattoirs and a WWTP using culture-dependent method and shotgun metagenomics. A total of 542 samples were collected from swine (n = 198), poultry (n = 220), human hand swabs (n = 108), abattoir/farm run-off water (n = 11) and effluents samples (n = 5) from WWTPs and a hospital setting. The M-PCR assays detected a prevalence of 26.2% (142/542) and 2.2% (12/542) of PEC and S. enterica, respectively. The AST results detected resistance to ESBLs and colistin in PEC and S. enterica, respectively. No Carbapenem or colistin resistance was observed among PEC and S. enterica isolates. Dendrogram showed both PEC and S. enterica isolates clustered together according to the sampling site indicating genetic relatedness among isolates from the same site. Whole genome sequencing detected an array of virulence genes including: eae, stx1 and stx2 and AMR genes such as blaCTX-M-14 and blaTEM-1B. Mobile genetic elements associated with virulence and AMR were also reported such as the IncF variants and IncQ1 replicon types in PEC and S. enterica respectively. This study reported for the first time in Africa, the stx2k subtype in a PEC strain and the S. Typhimurium monophasic variant I 4,[5],12:i:- clonal group ST34 strain from animal and food sources. The culture-dependent method identified PEC in only the poultry wastewater effluent. Shotgun metagenomics did not detect the presence of PEC and S. enterica at genus and species levels in all three wastewater effluents which may indicate a low abundance of these bacterial pathogens in samples collected. The sul1 AMR gene was detected simultaneously with the culture-dependent method and shotgun metagenomics. Shotgun metagenomics identified Aliarcobacter cryaerophilus, an emerging foodborne pathogen, from the WWTP sample. The detection of diverse PEC and S. enterica strains in swine, poultry, human hands and environmental run-off water in this study emphasises the need for continuous monitoring of foodborne pathogenic bacteria in abattoirs and farms across South Africa using a One Health Approach.
Phenotypic and genotypic characterisation of clinically relevant Staphylococcus aureus isolates from cystic fibrosis patients from private clinics in South Africa
(University of Pretoria, 2024) Ehlers, M.M. (Marthie Magdaleen); Strydom, Kathy-Anne; awsilen@yahoo.co.za; Ndlovu, Neliswa
Introduction. Cystic fibrosis is a hereditary disease that results in ineffective mucocilliary clearance of secretions, creating an environment that allows Staphylococcus aureus to thrive. The aim of this study was to determine the phenotypic and genotypic characteristics of S. aureus isolates obtained from cystic fibrosis patients attending private clinics in South Africa. Materials and Methods. A total of 87 S. aureus isolates were collected. Phenotypic susceptibility testing was performed using the Vitek®2 automated system (bioMérieux, France) according to EUCAST guidelines. Multiplex-PCR assays were used to target antibiotic resistance genes and a selection of biofilm formation, haemolysins and cytoxin genes. Pulsed-field gel electrophoresis was conducted and a dendrogram constructed to assess the genetic relatedness of isolates. Whole genome sequencing was performed on four isolates to determine sequence types, the resistome and virulome of the isolates. Results. Phenotypic antibiotic susceptibility testing showed the highest resistance against erythromycin in 62.1% (54/87) of isolates. Multiplex-PCR assay results showed 11.4% (10/87) of the S. aureus isolates were MRSA; and the most prevalent macrolide and tetracycline resistance genes was ermC 36.8% (32/87) and tetK 2.3% (2/87). The biocide resistance gene, qacC was detected in 4.5% (4/87) of the isolates. The most prevalent virulence gene was: hla 48.2% (42/87). Sequence types detected were pandemic strains ST5 and ST36, and livestock associated strain ST398 as well as a novel ST. The S. aureus isolates from CF patients in this study were highly diverse, indicating limited spread from clinical settings. Multidrug resistant isolates were detected which limits treatment options for these patients; the isolates also harbour virulence genes that can increase the severity of the disease and potentially increase the morbidity and mortality among these patients.
Molecular epidemiology and treatment outcomes of vulvovaginal candidiasis in Namibian women
(University of Pretoria, 2023-11-03) Peters, Remco P.H.; Kock, Martha Magdalena; duncarmia@gmail.com; Dunaiski, Cara Mia
Vulvovaginal candidiasis (VVC) is a common condition in women of childbearing age worldwide and usually presents as vaginal discharge syndrome (VDS). Other important causes of VDS are bacterial vaginosis (BV) and sexually transmitted infections (STIs). The most common STI causing VDS include Trichomonas vaginalis. Vaginal dischare syndrome (VDS) can also be caused by Neisseria gonorrhoeae, Chlamydia trachomatis and Mycoplasma genitalium. However, these are more commonly associated with endocervical infections, and may not present with VDS. Syndromic treatment is the standard of care for VDS, i.e. empirical antibiotics are provided without diagnostic testing. Performance of the syndromic approach depends on the aetiology of VDS and presence of antimicrobial resistance but there is little information from sub-Saharan Africa. The aim of this PhD study was to describe the aetiology of VDS and determine outcome of syndromic treatment of VDS in Namibian women. This PhD study had three components: first, a cross-sectional evaluation was completed to determine the aetiology of VDS. De-identified vaginal swabs (n=253) sent to the routine laboratory at the Namibia Institute of Pathology in Windhoek were tested for STIs using real-time PCR, BV by smear microscopy and VVC by culture and drug susceptibility testing. Second, a prospective cohort study of women (n=109) with VDS at Katutura Intermediate Hospital in Windhoek was conducted to determine incidence, risk factors and microbial aetiology of treatment failure. Last, comprehensive molecular microbiological analysis of phenotypic and genotypic resistance including multi-locus sequence typing was conducted through whole genome sequencing analysis of all Candida glabrata isolates (n=21) obtained in the two studies. Vulvovaginal candidiasis was the main aetiology (43%) of VDS in the cross-sectional study followed by BV (39%) and STIs (36%); multiple infections were present in 34% of women. Candida albicans was the most common fungal species (79%); most isolates (98%) were susceptible to fluconazole. In contrast, no non-albicans Candida species were susceptible to fluconazole. Vulvovaginal candidiasis aetiology at baseline was similarly high in the prospective cohort study with 41% with VVC, 43% of women diagnosed with BV and 40% with STI. At 30 days follow-up, treatment failure was reported by 31% of women, with 18% reporting recurrent and 13% persistent VDS after syndromic treatment. Incidence of treatment failure was 3.6 per 100 person-days at 7 days follow-up and 1.0 per 100 person-days at 30 days follow-up. Vulvovaginal candidiasis was the main risk factor for treatment failure (OR 4.3, 95% CI 1.7‒11, p=0.002). Microbial evaluation of treatment failure attributed most cases to untreated (31%) and azole-resistant (23%) VVC. Twenty-one fluconazole-resistant C. glabrata isolates were obtained from the two studies. Whole genome sequencing analysis showed non-synonymous single nucleotide polymorphisms in antifungal resistance genes in 95% of isolates. Single nucleotide polymorphisms were also detected in genes associated with polyene, echinocandin and multiple class antifungal resistance despite full phenotypic susceptibility to these drug classes. Multilocus sequence typing classified isolates in eight sequence types. The results show that VVC is an important cause of VDS in Namibian women, with C. albicans as the main species followed by C. glabrata. Untreated and fluconazole resistant VVC constitute an important risk factor for VDS treatment failure. Therefore, clinical consideration of VVC in women with VDS and access to fungal culture and susceptibility testing is warranted, especially among women with treatment failure.
One Health approach investigating Shiga toxin-producing Escherichia coli serotypes from human stool specimens and cattle run-off water samples in South Africa
(University of Pretoria, 2023) Ehlers, M.M. (Marthie Magdaleen); Smith, Anthony; Kingsburgh, Chanel; Said, Mohamed; shanerie06@gmail.com; Bronkhorst, Shanerie
Shiga toxin-producing Escherichia coli (STEC) are known for producing cytotoxins called Shiga toxins and are prevalent in cattle. Data regarding the prevalence of STEC serotypes in South Africa is lacking. This study investigated the prevalence and genomic characteristics of clinical and environmental serotypes of STEC in South Africa, using the One Health approach. Forty-four STEC isolates were collected from a private health diagnostic laboratory and 30 run-off water samples from 10 beef abattoirs and 20 cattle feedlots. Genomic analysis involved multiplex polymerase chain reaction assays for screening Shiga toxin, O-antigen, and virulence genes. Genetic relatedness of isolates was investigated using a repetitive extragenic palindromic polymerase chain reaction assay, guiding the selection of isolates for WGS. The stx2 gene [43.18% (19/44)] was most prevalent, followed by the stx1 gene [34.09% (15/44)]. One isolate tested positive for both stx1 and stx2. The most prevalent serotype was O26 [29.55% (13/44)], followed by O157 [11.36% (5/44)]; both implicated in past outbreaks. Genes associated with severe illness in humans, including: stx2a, stx2c, stx2d, stx2f, eae and ehxA, were detected. Genetic diversity was apparent among isolates, except for two closely related isolates from human stool specimens. Hybrid strains containing extraintestinal pathogenic E. coli and other diarrhoeagenic E. coli virulence genes were detected in two isolates. Sequence type (ST) 14855, ST300, ST730 and ST5989, previously unreported in South Africa, were identified. Forty percent (4/10) of isolates harboured antimicrobial resistance (AMR) genes, including: strA, strB, sul2, tetA, tetB and dfrA. All isolates harboured multidrug-resistance-associated plasmids from the Inc-family. These results highlight the heterogeneity, genomic plasticity and propensity STEC to acquire AMR and virulence traits, increasing the bacteria’s potential to cause severe illness in humans. Farm-to-plate-to-hospital surveillance systems need to be implemented in South Africa to develop strategies to curb the spread of AMR and virulent strains of STEC.
Antimicrobial, synergistic and autophagic effects of medicines for Malaria venture pathogen box compounds on resistant strains of Mycobacterium tuberculosis and Neisseria gonorrhoeae
(University of Pretoria, 2023) Fourie, P.B. (Petrus Bernardus); Peters, Remco P.H.; u19244283@tuks.co.za; Mensah, Eric
Antimicrobial resistance in Mycobacterium tuberculosis and Neisseria gonorrhoeae is emerging globally. Due to the limited treatment options, the World Health Organization has designated M. tuberculosis and N. gonorrhoeae as two critical and high-priority pathogens for research and development of novel antibiotic drugs. Particularly for M. tuberculosis, new agents that can induce autophagy processes directed at clearing intracellular M. tuberculosis from host cells is highly needed. To speed up the discovery and development of novel agents, the Medicines for Malaria Venture (MMV) group developed the Pathogen Box, containing a collection of 400 novel drug compounds. The Pathogen Box was originally assessed primarily for anti-malarial properties but, in the initial screen, has been shown to contain compounds potentially also effective against several other microorganisms, including M. tuberculosis. The aim of this study was to explore the antibiotic potential, including synergistic and autophagic effects, of this diverse compound library of the MMV Pathogen Box. As a first step, the identities and resistance profiles of clinical strains of M. tuberculosis and N. gonorrhoeae selected for use in this study were confirmed, using GeneXpert MTB/RIF and MTBDRplus assays, followed by whole genome sequencing (WGS). Broth microdilution assay was used to determine the pathogen-specific minimum inhibitory and minimum bactericidal concentrations (MICs/MBCs) of the Pathogen Box compounds (PBCs) against reference strains of M. tuberculosis and N. gonorrhoeae. Finally, a checkerboard assay approach was used to determine synergy between the active compounds if used in combination with reference drugs. Time-kill kinetics was performed to determine bactericidal or bacteriostatic activity. Selecting priority compounds for further investigation was based on the following criteria: (1) MIC and MBC for N. gonorrhoeae ≤ 10 µM; and (2) MIC and MBC for M. tuberculosis ≤ 0.625 μM. Five PBCs, MMV676603, MMV687146, MMV687696, MMV687180, and MMV153413, showed potent activity against both susceptible and multidrug-resistant M. tuberculosis strains at MIC and MBC below 0.625 μM. Except for MMV687696, the remaining four PBCs were clearly bactericidal. Combining the PBCs with isoniazid or rifampicin demonstrated either synergistic or additive activity, with fractional inhibitory concentration indexes ranging between 0.18 and 2.60. The five selected anti-TB PBCs recorded low cytotoxicity in murine-derived macrophages and effectively suppressed the growth of intracellular M. tuberculosis. Western blotting analysis was used to assess the potential of the five selected PBCs to induce autophagy against intracellular M. tuberculosis in host cells. All compounds induced some level of LC3 lipidation and LC3II/LC3I, although this was not statistically significant compared to controls. Notably, inhibition of the autophagic flux reversed the anti-mycobacterial activity of MMV676603, MMV687146, and MMV687180. Eight PBCs, MMV676501, MMV002817, MMV688327, MMV688508, MMV024937, MMV687798 (Levofloxacin), MMV021013, and MMV688978 (Auranofin), demonstrated potent activity against resistant strains of N. gonorrhoeae at a MIC and MBC of ≤ 10 µM. All the compounds showed potent bactericidal activity between 4 and 24 hrs, with time-kill kinetics similar to that of ceftriaxone. The N. gonorroheae active PBCs in combination with ceftriaxone showed either synergistic or additive activity with fractional inhibitory concentration indexes ranging between 0.40 to 1.8. Conclusion. This study has identified novel compounds with potent activity against both resistant and susceptible strains of N. gonorrhoeae and M. tuberculosis. The study has also identified compounds that can suppress the growth of intracellular M. tuberculosis with the potential to induce autophagy at high concentrations. Overall, the study results point to promising anti-gonococcal and anti-TB drug leads worthy of further exploration.
Genotypic profiles of emerging multidrug-resistant Staphylococcus capitis isolates from an ongoing outbreak in critically ill patients
(University of Pretoria, 2023) Ehlers, M.M. (Marthie Magdaleen); Hoosien, Ebrahim; bmynhardt@gmail.com; Mynhardt, Barend J.
An increasing number of linezolid resistant (LZR), multidrug-resistant (MDR) Staphylococcus capitis infections have been observed in private hospitals in the greater Gauteng area since 2014. The aim of this study was to investigate the genotypic profiles of emerging LZR MDR S. capitis isolates from an ongoing outbreak in critically ill patients in South Africa’s private sectors. A total of 119 S. capitis isolates from 29 private hospitals were identified and reported as linezolid resistant. The antimicrobial resistance patterns of the LZR MDR S. capitis isolates were: erythromycin 99.2% (118/119), amoxycillin/clavulanate 98.3% (117/119), cloxacillin 98.3% (117/119), clindamycin 97.5% (116/119), fucidic acid 84% (100/119), gentamycin 74.8% (89/119), cotrimoxazole 27.2% (33/119), rifampicin 16.8% (20/119), daptomycin 2.5% (3/119), vancomycin 1.7% (2/119) and teicoplanin 0.8% (1/119). The cfr gene was found in one isolate, while the optrA and poxtA genes were not detected with multiplex (M)-PCR. The pulsed-field gel electrophoresis (PFGE) dendrogram showed 1 major pulsotype consisting of 76 isolates, 3 minor pulsotypes with nine, five and three isolates respectively and 10 singletons. Fifteen isolates were classified as untypeable. Whole genome sequencing analysis of five representative S. capitis isolates showed a less known point mutation at G2604T on the rRNA gene conferring resistance to linezolid. Antimicrobial resistant genes identified included: tetK, aac(6’)-le-aph(2”)-la, fusB, sepA, sdrM, mupA, mdeA, mecA, blaZ, ermC, dfrC. Phenotypic antibiotic susceptibility did not show expression of all the genotypic genes detected. The results showed that highly resistant LZR MDR S. capitis isolates are circulating in these private hospitals among adult patients in ICUs. This emphasizes the importance of continious surveillance (with the inclusion of molecular epidemiological investigations) to monitor the transmission and spread of these circulating LZR MDR S. capitis strains in clinical settings in South Africa.
Molecular characterisation of β-lactamase producing Klebsiella pneumoniae isolates
(University of Pretoria, 2015) Kock, Martha Magdalena; Ehlers, M.M. (Marthie Magdaleen); marleen.kock@up.ac.za; De Jesus, Marissa Batista
Genetic typing of Klebsiella pneumoniae is used for epidemiological referencing. In the clinical setting it can be useful in outbreak investigations, understanding transmission and managing hospital infections. Multi-drug resistant bacteria exist and proliferate either due to natural selection of clonal lineages or the transfer of mobile genetic elements, sometimes in response to antibiotic-use selective pressure. Pulsed-field gel electrophoresis (PFGE) is highly discriminatory and the gold standard typing method for the characterisation of K. pneumoniae isolates. The aim of the study was to genetically characterise K. pneumoniae isolates by PFGE and multilocus sequence typing (MLST). One hundred unrepeated ESBL-producing K. pneumoniae isolates were collected from the National Health Laboratory Service (NHLS). The PFGE was performed on a Rotaphor VI system (Biometra, Germany). Clonal representatives were further characterised by MLST. All the strains were typeable by PFGE using XbaI, which discerned multiple pulsotypes and MLST identified 10 different STs including a novel sequence type, ST1632. The diverse pulsotypes of K. pneumoniae isolates are not suggestive of clonal spread of particular strains. The MLST results further confirmed the variability among isolates tested and elucidated several STs, some of which have been identified internationally and often associated with carbapenem-resistance. Data on K. pneumoniae STs is still limited in the South African clinical setting, although the close monitoring of resistance profiles and characterisation of isolates is imperative for outbreak analysis, identification of prominent STs in clinical settings as compared to international counterparts and surveillance of expanding resistance.
Lung microbiome of chronic obstructive pulmonary disease patients with and without HIV infection in Pretoria, South Africa
(University of Pretoria, 2020) Ehlers, M.M. (Marthie Magdaleen); Peters, Remco P.H.; Goolam Mahomed, Tanweer
Chronic obstructive pulmonary disease (COPD) is a leading cause of death and is highly prevalent in South Africa (19% in adults over the age of 40 years). Inflammation of the lungs in COPD impairs the immune response and allows colonisation and infection with bacteria and viruses, that may cause exacerbations of the disease. Culture-independent technologies have greatly increased the understanding of the lung microbiome. The most widely used method for targeted metagenomics is 16S rRNA sequencing. The IS-Pro (intergenic spacer profiling) method provides an alternative targeted metagenomics approach; however, the two methods have not been compared. There is limited data on the microbiome in the lungs of COPD patients in Africa. Due to local environmental conditions, immunological differences and clinical comorbidities, such as HIV, the microbiome may be different from that reported in studies from other countries. The purpose of this study was to identify the lung microbiome and lung virome in COPD patients in South Africa and to determine if the COPD disease states result in differences in its composition. Next-generation sequencing was used to determine the microbiome and virome of COPD patients from hospitals in Pretoria, South Africa and the IS-Pro method was compared to targeted metagenomics. Twenty-four patients over the age of 40 years with a confirmed COPD diagnosis and no Mycobacterium tuberculosis infection were included; eighteen were in the stable state of diseases and six were in the exacerbation state of disease. Sputum specimens were collected from all consenting participants and DNA and RNA were extracted directly from the specimens using commercial kits. The extracted bacterial DNA was sent for targeted metagenomics and the IS-Pro method and the extracted viral DNA and RNA were sent for shotgun metagenomics sequencing. The lung of the COPD participants showed a diverse microbiome with over 77 genera identified and the Firmicutes phylum predominating. When the stable and exacerbation states of COPD disease were compared, no significant differences in the alpha and beta diversity between the disease states were observed. However, during exacerbation state of the disease, the abundance of key phyla had decreased. Analysis of the virome showed a high prevalence of BeAn 58058, a close relative of the smallpox virus, with bacteriophages being the second most prevalent viruses. When comparing the IS-Pro method to targeted metagenomics, an increased relative abundance of Proteobacteria with the IS-Pro method was observed, which was attributed to known lung pathogens, such as Burkholderia. The IS-Pro method was able to classify more operational taxonomic units (OTUs) to a species level, however, the unclassified OTUs from the IS-Pro method could only be classified to a phylum level. To conclude, a diverse COPD microbiome was observed, with a virome that was dominated by the BeAn 58058 virus. The COPD disease states showed no variations in terms of diversity, however, the relative abundances of key phyla differed between disease states for the bacterial microbiome. Future studies should focus on longitudinal studies of the sputum microbiome in an African setting as well as functional metatranscriptomics studies with a focus on antibiotic resistance and virulence factors.
Molecular epidemiology and mechanisms of colistin and carbapenem resistance in Enterobacteriaceae from clinical isolates, the environment and porcine samples in Pretoria, South Africa
(University of Pretoria, 2020) Mbelle, Nontombi; Osei-Sekyere, John; Naidoo, Vinny; u12236102@tuks.co.za; Bogoshi, Dineo
Introduction: Carbapenems and colistin are the last-line antibiotics for treating Gram-negative bacterial infections. However, increasing reports of resistance to these antibiotics is being reported in clinical settings, the environment and in animals. In this paper, we describe the molecular epidemiology and resistance mechanisms of colistin and carbapenem resistance in clinical, veterinary, and environmental Enterobacterales isolates in Pretoria, South Africa. Method: One hundred VITEK®-2-confirmed colistin and carbapenem-resistant clinical isolates were collected from the departmental isolate bank at the National Health Laboratory Service. A total of 88 porcine (stool) and 11 environmental (effluents) samples were collected in November 2018 and again in March 2019 from a farm in Pretoria. Both the porcine and environmental samples were screened using Eosin methylene blue agar with colistin and ertapenem disks. All isolates were identified and a minimum inhibitory concentration of colistin and carbapenems was determined using the MicroScan® WalkAway system. Isolates resistant to colistin were confirmed by the broth microdilution method. Isolates phenotypically resistant to colistin and carbapenems were selected for whole genome sequencing to determine the resistome and phylogenetic trees were drawn to determine the relatedness of isolates. Results: A total of 275 Gram-negative isolates were identified from the clinical (100), environmental (57) and veterinary (118) samples using the MicroScan® WalkAway system. The MicroScan® WalkAway system’s minimum inhibitory concentration results for clinical isolates revealed 88% and 93% resistance to colistin and carbapenems, respectively. BMD was found to be more reliable in all isolates, and it recorded higher MICs (increased resistance) than the MicroScan® WalkAway system. Overall, colistin susceptibility was higher among animal isolates compared to the clinical and environmental samples. Genomic analysis identified several resistance genes associated with resistance among the isolates and the CTX-M family were the dominant resistance genes. Phylogenomic analysis demonstrated closer evolutionary relationship between EB008 (environment), SW10B (animals), and C080 and C084 (both humans) strains as well as with strains from the United States of America, Canada, China, Russia and Durban (South Africa). Conclusion: The study established multiple resistance genes from different antibiotics to mediate resistance in Enterobacterales isolates from humans, animals and the environment. The presence of carbapenemases in animals is alarming and poses a public health concern. Strains EB008 (environment), SW10B (animals) and C080 and C084 (both human) were phylogenetically related with strains from the United States of America, China and Durban (South Africa) more commonly. Therefore, One Health approach studies are significant to ascertain colistin and carbapenem transmission from human to animals/the environment and vice versa to combat increasing resistance in Enterobacterales.
Assessment of macrophage and granulocyte cell-enhanced phagocytosis of Mycobacterium tuberculosis
(University of Pretoria, 2020) Fourie, Bernard; Rossouw, Theresa; sbongakee@yahoo.com; Shey, Bong-Akee
This study demonstrates the ability of a selected novel IgG1 mouse monoclonal antibody, JG7 III D3 I F9 (JG7), to enhance the phagocytic elimination (opsonophagocytosis) of susceptible M. tuberculosis strains at both their mid-logarithmic and stationary growth phases, by human U-937 macrophage and HL-60 granulocyte cell lines which are involved in intracellular killing of bacteria, and to assess the cytokine response to this process. The assays showed JG7-enhanced phagocytosis to all M. tuberculosis strains used, though at different levels. There was a relative increase in OPKA with increase in mAb concentration, at each bacterial dilution assessed (1:100 and 1:1000), and this increase was demonstrated mostly with the cMtb strain by the U-937 macrophage cell line. For the multiplex suspension array assays, the JG7 concentration of 25 µg/mL was selected, since it was used in both binding and OP assays. Results showed that the mAb induced a possible pro-inflammatory effect, driven by IL-8, in neutrophils and a significant anti-inflammatory response in MNL cells, as demonstrated by the significant decrease in pro-inflammatory IL-12p70 and increase in anti-inflammatory IL-10.
Phenotypic and genotypic characterization of isoniazid resistance mutations in Mycobacterium tuberculosis isolates from new and previously treated patients in the Tshwane region
(University of Pretoria, 2020) Maningi, Nontuthuko Excellent; Sekyere, John Osei; u13258402@tuks.co.za; Sinthumule, Ndivhudzannyi Peace
Background: Isoniazid (INH) is one of the most potent anti-tuberculosis (TB) drugs and a key component of the multi-drug regimen for the treatment of TB. Tuberculosis cases with initial INH resistance are more at risk of poor clinical outcomes than those susceptible to INH among cases receiving standard first-line chemotherapy. Drug resistance arises mainly from spontaneous mutations in the Mycobacterium tuberculosis (M. tuberculosis) genome. Resistance to INH is more complex mainly because it may involve mutations in one or more of several genes, such as katG, inhA, ndh, kasA and ahpC. However, resistance in the katG and inhA genes is responsible for approximately 65% of all instances of INH resistance. Continued usage of INH has been compromised by the increasing prevalence of INH resistance among M. tuberculosis strains. Furthermore, accurate molecular diagnosis of drug resistance depends on knowledge of mechanisms conferring resistance to anti-TB drugs. Early detection of INH resistance is essential to allow proper initial drug therapy and a decrease in MDR-TB. Different M. tuberculosis lineages have been reported to be associated with different levels of pathogenicity, are thought to drive resistance and to play a role in virulence characteristics and transmission. Seven M. tuberculosis lineages have been identified, are geographically distributed in diverse but specific regions and have been associated with specific resistance-conferring mutations in the M. tuberculosis genome. The East Asian lineage (Beijing) has been shown to be the foremost virulent and most dominant among the known lineages. It is the predominant lineage in Far East Asia and causes more than 60% of TB cases in this region. The lineage has a worldwide distribution and is also dominant in some parts of South Africa. The aim of this study was to characterize INH resistance mutations in M. tuberculosis isolates from new and previously treated patients and determine the association of mutations with TB lineages in the Tshwane region, South Africa. Methods: A total of 150 M. tuberculosis resistant isolates were obtained (2016 to 2019) from the National Health Laboratory Service, Tshwane Academic Division. The study population included: 118/150 (79%) new cases and 32/150 (21%) previously treated cases. Of the new cases, 47/118 (40%) were females and 71/118 (60%) were males. Of the previously treated cases, 11/32 (34%) were females and 21/32 (67%) were males. Genomic DNA was extracted using the GenoLyse® kit (Hain Lifescience, Germany). Characterization of INH resistance-conferring mutations was done using GenoType MTBDRplus (Hain Lifescience, Germany). Thereafter, screening of XDR- TB was done using GenoType MTBDRsl (Hain Lifescience, Germany). Genotyping was performed using spoligotyping, a PCR-based method. Association of M. tuberculosis lineages and INH mutations was evaluated. Results: Overall results showed that 117/150 (78%) were both RIF and INH resistant and 33/150 (22%) were RIF mono-resistant. Of the 33 pre-MDR-TB isolates, 12/33 (36%) were phenotypically discrepant in RIF and INH resistance. Of these, 10/12 (83%) were new and 2/12 (17%) were previously treated TB cases. Mutation S315T of the katG gene was found in 75/118 (64%) of new TB cases and 13/32 (41%) in previously treated cases. The sensitivity and specificity of LPA for detecting INH resistance was between 29% to 71% and 84% to 100% respectively. Screening of MDR-TB isolates for XDR-TB showed that 139/150 were sensitive to second-line injectable drugs (SLIDs) and 11/150 were resistant. Only 90/150 were susceptible to fluoroquinolones (FLQs) and 60/150 were resistant. Spoligotyping identified 9 major distinct TB families including East African Indian (EAI) (14%), Latin American and Mediterranean (LAM) (13%), Beijing (27%), T-family (13%), S-family (7%), H-family (1%), X-family (7%), Manu2- family (4%) and CAS-family (1%). Remaining isolates 13% of the isolates were orphans. Mutation S315T of the katG gene in new TB cases was mostly associated with Beijing (18/118 or 15%) and with T1-family (9/118 or 8%). In previously treated cases, mutation S315T was associated with Beijing 5/32 (16%) and with X2 (3/32 or 9%). Furthermore, mutation C-15T in new TB cases was mostly associated with Beijing (9/118 or 8%). However, the was no significant association between INH resistance-conferring mutations and M. tuberculosis lineage in new and previously treated cases. Conclusion: This study showed that male gender is more prominently associated with resistance to INH within the Tshwane region. This shows that male gender is prone to disease exposure than females. The line probe assay MTBDRplus 2.0 is commonly used as a rapid diagnostic for drug resistance to first-line drugs, including RIF and INH resistance. However, in comparison with phenotypic methods, around 8% of isolates produced discrepant results, i.e. INH resistance missed by the LPA. This indicates that a more discriminatory technique should be used to further analyse INH susceptible isolates. A high genetic diversity of M. tuberculosis lineages was observed in both new and previously treated cases. Furthermore, M. tuberculosis lineages associated with S315T mutation within the Tshwane region appears to be spreading rapidly compared to previous studies done in the region.
Evaluation of single nucleotide polymorphisms in virulence genes of Mycobacterium tuberculosis as markers of lineages and sub-lineages in Tshwane region
(University of Pretoria, 2020) Maningi, Nontuthuko Excellent; Sekyere, John Osei; Mutshembele, Awelani M.; u18392840@tuks.co.za; Matodzi, Unarine
Tuberculosis (TB) is one of the top ten leading causes of death worldwide with millions of new TB cases reported every year. Understanding the genetic diversity of Mycobacterium tuberculosis (M. tuberculosis) is very crucial for rapid diagnosis and to reduce transmission of TB. Various diagnostic techniques, anti-tuberculosis reagents and vaccination are available, however, the disease is far from being eradicated (Brudey et al., 2006). Mycobacterium tuberculosis is classified into seven major lineages that are key to the most research areas. Recently, multidrug M. tuberculosis have been reported as the most dangerous strains that cause a life-threatening TB. However, the M. tuberculosis with modified virulence and transmissibility, particularly those that are caused by mutations leading to genetic variation and increased pathogenicity are highly reported (Zaychikova et al., 2015). Genetic markers such as variable number tandem repeats, insertion sequence element and direct repeats have been used to identify lineages. However, the techniques (such as spoligotyping, IS6110-RLFP and MIRU- VNTR) that use these genetic markers have a lot of drawbacks and some have low discriminatory power (Mikheecheva et al., 2017). Recently, single nucleotide polymorphisms (SNPs) are regarded as the most promising genetic markers for genotyping M. tuberculosis because they have low-level homoplasy and high discriminatory power (Zaychikova et al., 2015). The present study proposed that genotyping M. tuberculosis using polymorphisms in virulence genes may be an alternative approach to determine lineages and may help to detect the M. tuberculosis strains that are epidemiologically dangerous and have adapted to specific geographic regions. This study aimed to identify and evaluate a set of virulence gene SNPs as markers of M. tuberculosis strains circulating in the Tshwane region. A total of 150 susceptible and resistant M. tuberculosis cultures stored in Mycobacteria growth indicator tubes (MGIT) tubes were collected from May to October 2018 at the National Health Laboratory Service, Tshwane Academic Division (NHLS/TAD) to conduct this study. The DNA was extracted using hexadecyltrimethylammonium bromide (CTAB) method and spoligotyping was done to screen for M. tuberculosis lineages. The Beijing and LAM genotypes detected by spoligotyping were sequenced using the Illumina Miseq platform. The bioinformatic analysis of virulence genes in 56 genomes of M. tuberculosis belonging to Beijing and LAM genotypes was performed to detect lineage-specific SNPs markers. Of the 150 M. tuberculosis collected, 57.3% were susceptible M. tuberculosis strains while 42.7% were drug-resistant TB. Spoligotyping of 150 isolates resulted to 86.7% previously shared type (ST) and 13.3% orphans yielding a clustering rate of 63.3%. The Beijing family was found to be the most predominant lineage by 26.7%, followed by T family (16%), LAM (13.3%), East Africa Indian (EAI) (8.7%), S (6%), Manu (4.7%), H (4.7%), CAS (4.0%) and X3 (2.7%). The number of susceptible M. tuberculosis isolates per lineages was higher than drug-resistant TB with isolates detected as Beijing contributing 17.3% of all susceptible isolates, followed by isolates classified as orphans (10%), T family (9.3%), LAM family (8%) and CAS (2.67%). The association between anti-tuberculosis drug-resistant TB and lineages was found in EAI lineage (6.7%), Manu (4%) and S family (3.3%). The family with a high number of isolates which were drug-resistant TB was the EAI1-SOM sub-lineage belonging to the EAI family. This study successfully identified 29 Beijing and 6 LAM signature SNPs that can be used to classify clinical M. tuberculosis isolates. Within these signature SNPs, fadD28 (1521 C>T), eccCb1 (1479 G>A), pks5 (6210 G>A), and ponA2 (372 G>T) were identified in the Beijing strains and fadD28 (1392 C>G) within the LAM strains that were not reported in previous studies. Furthermore, this study detected the lineage-specific SNPs: mce3B (145 T>G), eccCb1 (1556 G>T), vapC12 (95 A>G) in Beijing BO/W148 and cyp125 (1076 T>C), mce3B (44 T>C), vapC25 (221 A>C), vapB34 (140 C>A) F15/LAM4/KZN sub-lineages which have been reported to be virulent and associated with drug resistance. This study showed a high genetic diversity of M. tuberculosis strains circulating within the Tshwane region. The Beijing lineage identified in this study was found to be more predominant than the rest of the identified genotypes. This study proposed the alternative method for genotyping M. tuberculosis strains using SNPs in virulence genes of M. tuberculosis. Observations from this study also highlight the advantage of using WGS technique over other genotyping methods such as IS6110-RFLP that has more drawbacks, as most genotypic methods discriminate M. tuberculosis strains using specific genes or regions in the genome of M. tuberculosis while WGS uses the complete genome of M. tuberculosis to determine different M. tuberculosis lineage.
Molecular epidemiology and antimicrobial resistance of Neisseria gonorrhoeae in men at risk in Gauteng South Africa
(University of Pretoria, 2020-02) Peters, Remco P.H.; Kock, Marleen Magdalena; litebohomaduna@gmail.com; Maduna, Liteboho Daniel
Antimicrobial resistance (AMR) in Neisseria gonorrhoeae has emerged worldwide and treatment failures of ceftriaxone and azithromycin; the last remaining empirical first-line therapy for gonorrhoea, are reported. However, there is little information about the situation in South Africa where syndromic management is used to treat sexually transmitted infections (STIs). The purpose of this PhD study was to investigate the occurrence of AMR and molecular epidemiology of N. gonorrhoeae infections in high-risk men from the public and private healthcare sectors in South Africa. The study included specimens from two study groups of participants: (1) core transmission groups of men-who-have-sex-with-men and men with recurrent discharge accessing sexual health services in Johannesburg; (2) N. gonorrhoeae and Mycoplasma genitalium isolates from patients accessing the private healthcare services. Urine and urethral swabs were collected from men for N. gonorrhoeae culture followed by antimicrobial susceptibility testing (AST). Molecular diagnostics for curable STIs was performed, including M. genitalium as an important coinfection, and whole genome sequencing (WGS) of gonococcal isolates to identify genetic resistance mutations and describe gonococcal populations. Antimicrobial susceptibility testing was performed on N. gonorrhoeae isolates obtained from private sector followed by genotyping using the N. gonorrhoeae multiantigen sequence typing (NG-MAST) method. Melting curve and sequence analysis were performed on M. genitalium strains for detection of macrolide resistance-associated mutations in the 23S rRNA. The quinolone resistance-determining regions of the parC and gyrA genes were also sequenced. Neisseria gonorrhoeae was the main aetiology (82%) of urethral discharge in male core transmission groups. High rates of AMR to tetracycline, ciprofloxacin and penicillin were detected among gonococcal isolates and azithromycin resistance was identified in 15% of the gonococcal isolates obtained from public but not in the private sector. There was no resistance to spectinomycin and cephalosporins found. Resistance to azithromycin was associated with an A39T alteration in mtrR and A deletions in the mtrR promoter. Amino acid alterations in GyrA (S91F, D95G, D95A) and ParC (D86N, S87N, E91G) were associated with ciprofloxacin resistance. Tetracycline resistant isolates harboured a tetM plasmid and had mutations in the rpsJ gene. Whole genome sequencing analysis of the gonococcal isolates revealed a wide diverse epidemic with a substantial number of novel N. gonorrhoeae sequence typing for antimicrobial resistance (NG-STAR) (70%) and NG-MAST (70%) sequence types (STs) identified. Neisseria gonorrhoeae strains from the private sector were genetically diverse and a substantial number of novel NG-MAST STs (83%) were identified. A high rate of azithromycin resistance was detected (19%) in the private sector but not in the public sector in M. genitalium strains harbouring mutations in the 23S rRNA. Fluoroquinolone resistance (2%) was detected in M. genitalium isolates harbouring mutations in the gryA and parC genes. The results show AMR in N. gonorrhoeae, and in the important coinfection M. genitalium, has emerged in South Africa. This work highlights that WGS can be successfully implemented in a resource-constraint setting for microbiological characterisation of gonococcal populations and their mechanisms of resistance. There is a need to urgently introduce diagnostics for STI care and scale-up surveillance for early detection of emerging AMR in STIs, in both public and the private sector in South Africa.
Genotypic and phenotypic diversity of Mycobacterium tuberculosis strains in patients with concomitant pulmonary and extra-pulmonary tuberculosis
(University of Pretoria, 2019) Peters, Remco P.H.; Mbelle, Nontombi Marylucy; sibandzedoctor@gmail.com; Sibandze, Busizwe Busizwe
future, more robust or higher-resolution genomic typing tools, such as target Next Generation Sequencing (t-NGS) or metagenomic analysis are recommended to improve our understanding of the complexity of infections and for detection of both minority and major variants within extrapulmonary and pulmonary specimens for better estimation of the extent of M. tuberculosis genotypic and phenotypic heterogeneity.
The association between genital mycoplasmas and bacterial vaginosis in pregnant women with or without genital symptoms
(University of Pretoria, 2014) Kock, Martha Magdalena; Ehlers, M.M. (Marthie Magdaleen); shanered72@gmail.com; Redelinghuys, Mathys J.
Bacterial vaginosis (BV) and genital mycoplasmas are infections of the reproductive tract that play important roles in maternal and foetal health. Genital mycoplasmas include Mycoplasma genitalium, M. hominis, Ureaplasma parvum and U. urealyticum. Infection may increase a woman’s susceptibility to infection with the human immunodeficiency virus (HIV). Bacterial vaginosis associated bacteria may form biofilms that are responsible for antimicrobial resistance and about 30% of affected women will relapse within three months of treatment. Genital mycoplasmas are prone to develop point mutations, which are responsible for increased antimicrobial resistance. Infections with these bacteria become prominent during pregnancy as infection may lead to infertility and foetal death. The purpose of the study was to determine the association between genital mycoplasmas and BV in pregnant women. Pregnant women attending the antenatal and Maternal and Foetal Unit (MAFU) clinics of a tertiary academic hospital in Pretoria, South Africa were included in the study. Self-collected vaginal swab specimens were obtained from consenting women older than 18 years of age. With the aid of microscopy, the Nugent scoring system was used to diagnose BV. Genital mycoplasmas were cultured on A2 agar and were diagnosed and speciated with a multiplex polymerase chain reaction (mPCR) assay. In addition, genital mycoplasmas were diagnosed and the antimicrobial susceptibility profiles determined with the Mycofast Revolution assay. A quantitative real-time polymerase chain reaction (qPCR) was employed to quantify the BV associated bacteria Atopobium vaginae and Gardnerella vaginalis. The prevalence of BV in this study was found to be 17.7% in 220 recruited pregnant women. Threshold concentrations between 106 to 107 copies/reaction of A. vaginae and G. vaginalis were found to be the best predictors of BV. Genital mycoplasmas were poorly recovered from A2 agar media, which had a contamination rate of 54.9%. An mPCR assay revealed that genital mycoplasmas were prevalent in 2.3% to 71.4% of specimens with U. parvum being the most prevalent species. The resistance of Ureaplasma species to tetracycline and erythromycin was 73% and 80%, respectively. Minor resistance to the fluoroquinolones, levofloxacin and moxifloxacin was recorded. This study found that only the genital mycoplasmas, namely M. hominis and U. parvum, were significantly associated with BV, while M. hominis was also significantly isolated from HIV positive women. This study found that there is an association between BV and genital mycoplasmas. The high prevalence of BV and genital mycoplasmas suggests that current management and/or intervention strategies are insufficient. Bacterial vaginosis associated bacteria can form a polymicrobial biofilm, which confer protection against antimicrobial agents and host immune responses. These biofilms are present on genital sites like the endometrium, which is located close to the amniotic membranes, posing health risks for the pregnancy. Future research must focus on the study of in vitro BV biofilm models and effective treatment strategies to minimise antimicrobial resistance. In the meantime, low-cost point-of-care (POC) tests that can accurately diagnose RTIs are needed to prevent excessive and unnecessary administration of antimicrobial agents and improve maternal and foetal health in the South African health care system.

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