Theses and Dissertations (Medical Microbiology)

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Relationship between the molecular diversity of plasmids, antibiotic resistance and virulence genes of Staphylococcus aureus isolates from private hospitals in Gauteng, South Africa
(University of Pretoria, 2024-11) Ehlers, M.M. (Marthie Magdaleen); Hamiwe, Thabo; Kingsburgh, Chanel; tatianangoie17@gmail.com; Ngoie, Kazadi
Objectives: Replication initiation (rep) genes found in plasmids have been linked to antibiotic resistance in bacteria. This study aimed to explore the relationship between plasmid diversity, antibiotic resistance profiles and the presence of virulence genes in S. aureus isolates from private hospitals in Gauteng, South Africa. Methods: Automated antibiotic susceptibility testing was done on 100 taphylococcus aureus isolates. Plasmid DNA extraction and polymerase chain reaction (PCR)-based replicon typing were done on all isolates. Multiplex-PCR was used to screen for virulence genes in plasmid positive isolates. Whole genome sequencing (WGS) was conducted on 10 representative isolates to assess genetic diversity. Results: The most common phenotypic antibiotic resistances observed were erythromycin 29% (29/100), clindamycin 26% (26/100) and ciprofloxacin 15% (15/100). Plasmids were found in 60% (60/100) of the isolates. Associations were observed between: rep7a (pS0385p1) with tetracycline (tetK) and rep7a (pTZ4) with chloramphenicol (cat) resistance, rep10a (pDLK1) with macrolide, lincosamide and streptogramin (ermC) resistance and rep5 (pN315) and rep16 (pSaaS6159) with penicillin (blaZ) resistance. No specific virulence gene could be associated with a particular rep gene. Sequence types (ST) associated with epidemics (ST22 and ST36), foodborne outbreaks (ST6) and zoonosis (ST1 and ST152) were identified by WGS. The ST1, ST6, ST22 and ST36 were identified in one isolate each, while ST152 was identified in two isolates. Conclusions: These findings highlighted an association between certain plasmids (pS0385p1, pTZ4, pDLK1, pN315, and pSaaS6159) and ARG. Enhanced surveillance and antibiotic stewardship programs in South Africa focusing on these plasmids could help better understand the spread of antibiotic resistance in healthcare settings.
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, 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.
Genetic and phenotypic diversity of bloodstream associated Staphylococcus epidermidis isolates obtained from private and public hospitals in South Africa
(University of Pretoria, 2023) Ehlers, M.M. (Marthie Magdaleen) ; KingsBurgh, Chanel; Skosana, Lebogang; fn.makamo@gmail.com; Makamo, Felicity Nosipho
Staphylococcus epidermidis is a commensal bacterium commonly implicated in bloodstream infections. Most S. epidermidis infections are acquired in hospitals and are associated with the use of medical devices. In contrast to its more virulent relative Staphylococcus aureus, S. epidermidis is often overlooked and regarded as a contaminant in diagnostic laboratories. As a result, limited information reporting on the prevalence of antibiotic resistance, virulence genes and clonality of S. epidermidis is available. In attempts to bridge this information gap, this study used phenotypic and genotypic methods to investigate the genetic diversity and genetic relatedness of bloodstream associated S. epidermidis isolates collected from private and public hospitals in Gauteng. A total of 161 bloodstream associated S. epidermidis isolates were collected from private and public hospitals over a period of eight months. The isolates were identified and characterised for the presence of antibiotic resistance and virulence genes using multiplex polymerase chain reaction (M-PCR) assays. The pathogenicity of S. epidermidis was further investigated using the microtitre plate assay to quantify biofilm formation. The genetic relatedness of the isolates was characterised using the staphylococcal cassette chromosome methicillin (SCCmec), pulsed field gel electrophoresis (PFGE) and whole genome sequencing (WGS). All 161 isolates (98 from private and 63 from public hospitals) were identified as S. epidermidis, with 91.9% (148/161) of the isolates being resistant to methicillin (i.e MRSE). The high prevalence of the mecA gene is concerning as this gene can easily be transferred to more pathogenic bacteria such as S. aureus. The most prevalent antibiotic resistance genes in isolates from public and private hospitals were the aac, tetK and msrA genes conferring resistance to tetracyclines, aminoglycosides and macrolides. The following virulence genes were prevalent among the private hospital isolates; EMBP [97.9% (96/98)], agrA, and aae [all at 95.9% (94/98)]. Among public hospitals isolates, the agrA [93.7% (59/63)], EMBP [84.1% (53/63)] and aae [73% (46/63] were the most prevalent genes. These virulence genes are known to play an important role in the pathogenicity of S. epidermidis through strengthening biofilm formation. Using the biofilm quantification method, all [100% (5/5)] the representative isolates from private hospitals were characterised as strong biofilm formers while the isolates from public hospitals were characterised as either weak biofilm formers [60% (3/5)] or non-biofilm formers [40% (2/5)]. A community based SCCmec type, SCCmec type IV was found to be dominant in both the private [11.8% (11/93)] and public [18.2% (10/55)] hospitals. This indicates the possible introduction of community-based strains of S. epidermidis into the hospitals. The PFGE showed a high genetic diversity among the S. epidermidis isolates, with a total of 16 Pulsotypes defined. Whole genome sequencing on selected representative isolates gave the sequence types, ST2, ST5, ST7, ST23 and ST28. The three sequence types, ST2, ST5 and ST23 were reported by other researchers to be endemic and predominantly associated with complicated bloodstream infections in S. epidermidis. The high prevalence of antibiotic resistance and virulence genes in this study supports the notion that S. epidermidis serves as a reservoir for more pathogenic bacteria such as S. aureus. The isolation of endemic strains warrants for comprehensive surveillance of S. epidermidis in South African hospitals.
Resistance, virulence and genetic relatedness of invasive Staphylococcus aureus isolates collected from private and public clinical settings in South Africa
(University of Pretoria, 2022) Ehlers, M.M. (Marthie Magdaleen); Kingsburgh, Chanel; Skosana, Lebogang; theevasha.govender@gmail.com; Govender, Theevasha
Staphylococcus aureus (S. aureus) is a leading cause of healthcare-associated infections (HAIs) and community-associated infections (CAIs) attributed to the harbouring of antibiotic resistance genes (ARGs) and virulence factors. New antibiotics are urgently required to treat the pathogen. The two-tiered healthcare system in South Africa has elicited differences in antibiotic regimens among private and public clinical settings for treatment of bacterial infections resulting in different selective pressures among these settings. Information on molecular characteristics of S. aureus isolates from these settings remains limited. This study investigated the prevalence of ARGs, biocide resistance genes, virulence genes, staphylococcal cassette chromosome mec (SCCmec) types, accessory gene regulator (agr) groups and genetic relatedness of invasive S. aureus isolates collected from private and public clinical settings in South Africa to determine the molecular characteristics and genetic relatedness of invasive S. aureus isolates among these settings. The study included a total of 183 invasive S. aureus isolates (110 isolates from private settings and 73 isolates from public settings). Multiplex polymerase chain reaction (M-PCR) assays were used for species confirmation and to characterise ARGs, biocide resistance genes, virulence genes, SCCmec types and agr groups. Pulsed-field gel electrophoresis (PFGE) was utilised to assess genetic relatedness. Based on M-PCR assay results and PFGE pulsotypes, 11 representative isolates were selected for whole-genome sequencing (WGS). The S. aureus isolates harbouring tetracycline ARGs [20% (22/110) private isolates and 53.4% (39/73) public isolates] and biocide resistance genes [36.4% (40/110) private isolates and 61.6% (45/73) public isolates]; as well as containing at least two virulence genes, were detected by M-PCR assays. The SCCmec types II, IV and V were detected in private settings and SCCmec types I, III, IVd and V were detected in public settings by M-PCR assays and WGS. The agr groups I, II, III and IV were detected among the isolates. The PFGE results revealed two major pulsotypes, 28 minor pulsotypes and 47 singletons, suggesting considerable genetic diversity among the isolates. Several minor pulsotypes clustered according to the clinical setting, however the largest major pulsotype (pulsotype O) contained isolates from both settings. Whole genome sequencing revealed additional ARGs, virulence genes, mobile genetic elements (MGEs), multi-drug resistance (MDR) efflux pumps and mutations detected at high frequencies, confirming the pathogenic significance of S. aureus. In addition, WGS also detected sequence types (STs) and staphylococcal protein A (spa) types. The identification of pulsotype O as ST152 as well as the identification of the epidemic strain ST5 in both settings indicated dominant strains circulating in both settings. The ST1 and epidemic strains ST8 and ST22 were identified in private settings; epidemic strain ST239 and ST2430 and novel STs-ST8011 and ST8012 identified in public settings; and several spa types were identified, indicating diverse strains circulating in each setting. The novel STs in public settings and novel spa types in both settings suggest new S. aureus strains emerging in each setting. Dominant MDR, biocide resistant and virulent epidemic S. aureus strains are circulating in private and public settings in South Africa. The establishment of epidemic strains in these settings emphasises the importance of S. aureus strain surveillance monitoring.
Lung microbiome and antimicrobial resistome of bronchiectasis patients attending a clinic at a tertiary academic hospital in Pretoria, South Africa
(University of Pretoria, 2022) Ehlers, M.M. (Marthie Magdaleen); Goga, Ameena Ebrahim; rashmikanaidoo@gmail.com; Naidoo, Rashmika
Bronchiectasis is a heterogeneous disease with multiple aetiologies and diverse clinical phenotypes. The pathogenesis of bronchiectasis, a growing economic health burden, is poorly understood. While culture-independent technologies have significantly increased the understanding of the lung microbiome, there is limited data on the lung microbiome, resistome and virome from bronchiectasis patients with and without HIV on the African continent. Therefore, the purpose of this study was to identify the lung microbiome, resistome and virome in bronchiectasis patients in Pretoria, South Africa, in order to determine whether HIV status affects the bacterial and viral lung composition and accompanying antimicrobial resistance (AMR) observed.
Lung microbiome and antibiotic resistome of cystic fibrosis patients attending clinics in the Gauteng Province, South Africa
(University of Pretoria, 2021) Ehlers, M.M. (Marthie Magdaleen); Goga, Ameena Ebrahim; thabohamiwe@gmail.com; Hamiwe, Thabo
Cystic fibrosis (CF) is a life-limiting hereditary disease characterised by recurrent polymicrobial lung infections and Pseudomonas aeruginosa lung colonisation is associated with increased morbidity and mortality. The life-long administration of antibiotics to treat lung infections leads to the accumulation of antibiotic resistance genes (ARGs) in CF pathogens. Culture-dependent techniques used in diagnostics resulted in limited pathogens being associated with CF lung disease and antibiotic susceptibility testing does not typically characterise resistance mechanisms employed by the pathogens. This study aimed to use the culture-independent techniques:16S rRNA sequencing and conventional multiplex-polymerase chain reactions (M-PCR) assays for the characterisation of the lung microbiomes and antibiotic resistomes respectively, of the CF patients in Gauteng, South Africa with a special focus on P. aeruginosa. A comparison of the results found with 16S rRNA sequencing and the M-PCR assays was made with shotgun metagenomics, a more comprehensive but costly sequencing technique on selected specimens. Additionally, the genomes of selected P. aeruginosa isolates were analysed using whole genome sequencing (WGS). In total, 22 CF patients (7 to 46 years of age), comprised of 11 P. aeruginosa (PA) colonised and 11 non-P. aeruginosa (NPA) colonised patients provided consent for the collection of sputum specimens at two tertiary academic hospitals in Gauteng, South Africa. More than 77 bacterial genera were found in the total (22) CF lung microbiomes which were dominated by traditional genera: Staphylococcus (32%), Pseudomonas (21.5%), Streptococcus (12%) and Haemophilus (5.1%). Notable genera such as Pseudomonas (43%) and Achromobacter (1%) were unique to PA microbiomes, while Burkholderia-Caballeronia-Paraburkholderia was unique to NPA microbiomes. Clinically relevant mobilizable ARGs were found in the 22 CF resistomes where macrolide [ermB 95% (21/22), ermA 91% (20/22) and ermF 91% (20/22)], sulfonamide [sul2 64% (14/22)], aminoglycoside [ant(2”)-Ia 59% (13/22)], beta lactamase [blaTEM 59% (13/22)] and tetracycline [59% (13/22)] ARGs were prevalent. The ARGs: aph(3’)-IIIa-3 [27% (3/11)] and tetL [27% (3/11)] were exclusive to PA patients, while blaZ [91% (10/11)] was exclusive to NPA patients. A comparison of 16S rRNA and shotgun sequencing on two randomly selected CF sputum specimens found that more bacterial genera were detected with 16S rRNA sequencing [85% (41/48)], than with shotgun sequencing [69% (33/48)]. However, limited agreement was found in the ARGs detected with M-PCR assays and shotgun sequencing. Thirteen P. aeruginosa isolates analysed with WGS showed that unique strains mostly colonised the lungs of CF patients. Efflux pumps (MexAB-OprM and MexCD-OprJ) and acquired mutations in the crpP and gryA genes were important antibiotic resistance mechanisms detected in the isolates, while adaptive mutations detected in genes such as: lasR, mucA, pilA, pilB and pilC enhanced P. aeruginosa virulence and persistence in CF lungs. The study CF lung microbiomes were diverse and harboured mobilisable, clinically relevant ARGs. The use of 16S rRNA sequencing proved superior to shotgun sequencing, however, M-PCR assays and shotgun sequencing gave contrasting results. Unique P. aeruginosa strains colonised patient lungs that carried clinically relevant ARGs, virulence genes and mutations. Future longitudinal studies analysing CF microbiomes and antibiotic resistomes and the expression of functional genes are required from the study settings.
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 P.; Rossouw, Theresa M.; 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.

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