Medical Microbiology

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Antibiotic-resistant Serratia marcescens infection in a hospital
(HMPG, 1979-04) Ambrosio, R. E.; Van Wyk, A .J.; De Klerk, H. C.
Over a 12-month period, 74 isolates of Serratia marcescens were obtained from various sources at Tygerberg Hospital. The majority of these isolates were from catheterized patients with urinary tract infections, and were non-pigmented and resistant to all antibiotics tested, excepting amikacin and neomycin. All isolates transferred resistance to tobramycin, gentamicin and tetracycline by conjugation to Escherichia coli recipients as separate markers at low frequency. A non-self-transmissible plasmid conferring resistance to kanamycin, ampicillin and gentamicin was mobilized from Serratia species to E. coli, and became fully self-transmissible in subsequent matings.
Aspects of the pharmacokinetics of hypoxoside in rodents
(Stellenbosch : Stellenbosch University, 1993) Bester, Christoffel Carolus; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Medical Microbiology.
ENGLISH ABSTRACT: Certain aspects of the pharmacokinetics of a novel diglucoside, hypoxoside, were investigated in mice and rats. After intragastric dosage of the mouse, hypoxoside was hydrolysed to its aglycone, rooperol. Most enzyme activity occurred in the mouse caecum with intestinal bacteria playing a major role in producing the hydrolytic enzymes. Caecalase extracts hydrolysed hypoxoside in vitro but this activity ceased after the mice were treated with antibiotics, suggesting bacteria as the enzyme source. However, in vivo tests with similar antibiotic-treated mice seemed to demonstrate a minor second, possibly mucosal source of hypoxoside hydrolysing B-glucosidase. An attempt to isolate a single strain of faecal bacteria capable of this activity was unsuccessful although hypoxoside was hydrolysed by pure cultures of some non-intestinal clinical isolates
Association between clinical and environmental factors and the gut microbiota profiles in young South African children
(Nature Research (part of Springer Nature), 2021) Nel Van Zyl, Kristien; Whitelaw, Andrew C.; Hesseling, Anneke C.; Seddon, James A.; Demers, Anne‑Marie; Newton‑Foot, Mae
ENGLISH ABSTRACT: Differences in the microbiota in populations over age and geographical locations complicate cross-study comparisons, and it is therefore essential to describe the baseline or control microbiota in each population. This includes the determination of the influence of demographic, clinical and environmental factors on the microbiota in a setting, and elucidates possible bias introduced by these factors, prior to further investigations. Little is known about the microbiota of children in South Africa after infancy. We provide a detailed description of the gut microbiota profiles of children from urban Cape Town and describe the influences of various clinical and environmental factors in different age groups during the first 5 years of life. Prevotella was the most common genus identified in the participants, and after infancy, the gut bacteria were dominated by Firmicutes and Bacteroidetes. In this setting, children exposed to antibiotics and indoor cooking fires were at the most risk for dysbiosis, showing significant losses in gut bacterial diversity.
Association between fluoroquinolone resistance and MRSA genotype in Alexandria, Egypt
(Nature, 2021-02-19) Alseqely, Mustafa; Newton-Foot, Mae; Khalil, Amal; El-Nakeeb, Mostafa; Whitelaw, Andrew; Abouelfetouh, Alaa
Antimicrobial stewardship isn’t strictly observed in most Egyptian hospitals, raising antibiotic resistance. Epidemiology of Egyptian MRSA isolates, or associations with resistance to other antibiotics remain largely unknown. We identified MRSA genotypes in Alexandria Main University Hospital (AMUH) and investigated rates of moxifloxacin resistance, an alternative MRSA treatment, among different genotypes. Antibiotic susceptibility of 72 MRSA clinical isolates collected in 2015 from AMUH was determined by disc diffusion and broth microdilution. spa- and Staphylococcal Cassette Chromosome mec (SCCmec) typing were performed; with multi-locus sequence typing conducted on isolates representing major genotypes. Resistance to moxifloxacin, levofloxacin and ciprofloxacin were 69%, 78% and 96%, respectively. spa type t037 (57%) was commonest, followed by t127 (12.5%), t267 (8%) and t688 (6%). SCCmec III predominated (57%), all of these were moxifloxacin resistant and 97.6% t037 (ST241). SCCmec IV, IV E and V represented 15%, 7% and 11% of the isolates, respectively, 79% of these were moxifloxacin susceptible and of different spa types. t127 (ST-1) was associated with SCCmec V in 56% of the isolates, mostly moxifloxacin susceptible. Moxifloxacin resistance was high, most resistant isolates belonged to t037 and SCCmec III, suggesting local dissemination and antibiotic pressure. We recommend caution in treating MRSA infections with moxifloxacin.
The association between pathogen factors and clinical outcomes in patients with Staphylococcus aureus bacteraemia in a tertiary hospital, Cape Town
(Elsevier on behalf of International Society for Infectious Diseases, 2019-11-29) Abdulgadera, Shima M.; Van Rijswijk, Amike; Whitelawa, Andrew; Newton-Foota, Mae
Background: Staphylococcus aureus is a serious pathogen, able to cause life-threatening infections such as bacteraemia. The association between S. aureus microbial characteristics and clinical outcomes is under- investigated in African settings. This study aimed to determine the molecular epidemiology and virulence characteristics of S. aureus isolates from bacteraemic patients at Tygerberg Hospital, South Africa, and to investigate the associations between pathogen characteristics and clinical outcomes. Methods: This study included 199 S. aureus isolates collected from blood cultures between February 2015 and March 2017. Methicillin resistance was determined using disc diffusion and all resistant isolates were further characterized by staphylococcal cassette chromosome mec (SCCmec) typing. Genotyping was done using spa and agr typing, and agr functionality was assessed using the phenotypic d-haemolysin assay. Logistic regression models were performed to describe the associations between strain characteristics and the clinical outcomes methicillin resistance, in-hospital mortality, and length of stay (LOS). Results: Of the 199 S. aureus isolates collected, 27% were MRSA, and the overall crude in-hospital mortality rate was 29%. Seventy-three different spa types were identified, including seven new types. Agr I was the most common type, in 99 (49.7%) isolates, followed by agr II, III, and IV in 57 (28.6%), 37 (18.6%), and six (3%) isolates, respectively. Agr dysfunctionality was observed in 25 (13%) isolates, mostly belonging to spa-clonal complex (CC) 012. Methicillin resistance was significantly associated with hospital-acquired infection (odds ratio (OR) 4.77, 95% confidence interval (CI) 2.09–10.87). A significant increase in mortality was observed with increasing age (OR 7.48, 95% CI 2.82–19.8) and having a hospital-acquired infection (OR 2.26, 95% CI 1.12–4.55). S. aureus strains with a functional agr system showed an association with longer duration of stay (OR 1.66, 95% CI 0.93–2.99). Conclusions: We report the lowest MRSA prevalence at Tygerberg Hospital for the past 10 years, and agr dysfunctionality was shown to be driven by a certain genotype, spa-CC012. Despite the limited available clinical data, the study provided insights into associations between S. aureus epidemiology and agr-related virulence characteristics, and clinical outcomes.
B-cell and T-cell activation in South African HIV-1-positive non-Hodgkin’s lymphoma patients
(Medpharm Publications, 2018) Flepisi, Brian T.; Bouic, Patrick; Sissolak, Gerhard; Rosenkranz, Bernd
Background: Altered immune mechanisms play a critical role in the pathogenesis of non-Hodgkin’s lymphoma (NHL). HIV-1 (HIV) infection is associated with a state of excessive T-cell activation, which can lead to increased T-cell turnover and lymph node fibrosis. Objectives: This study aimed to determine the serum levels of circulating B-cell activation markers, and the expression of T-cell activation and regulatory markers in HIV-positive NHL patients. Method: The serum levels of circulating soluble(s) sCD20, sCD23, sCD27, sCD30 and sCD44 molecules, all of which are biomarkers of B-cell activation, were determined by enzyme-linked immunosorbent assays (ELISA), while biomarkers of T-cell activation (CD8+CD38+) and regulation (FoxP3) were determined by flow cytometry in 141 subjects who were divided into five groups: Combination antiretroviral therapy (ART)-naïve HIV-positive patients; ART-treated HIV-positive patients; HIV-negative NHL patients; HIV-positive NHL patients on ART; and healthy controls. Results: HIV-positive NHL patients had significantly higher serum levels of sCD20, sCD23, sCD30 and sCD44 than HIV-negative NHL patients, while all five biomarkers were significantly elevated in HIV-positive NHL patients when compared with ART-treated HIV-positive patients. HIV-positive NHL patients had higher CD8+CD38+ and lower FoxP3 expression than HIV-negative NHL and ART-treated HIV-positive patients. Conclusion: B-cell activation is increased in HIV-positive NHL patients and is associated with reduced regulatory T-cell populations and increased CD8+ T-cell activation.
Bacterial infection, antibiotic use and COVID-19 : lessons from the intensive care unit
(Health & Medical Publishing Group, 2021-04-14) Moolla, M. S.; Reddy, K.; Fwemba, I.; Nyasulu, P. S.; Taljaard, J. J.; Parker, A.; Lalla, U.; Koegelenberg, C. F. N.; Allwood, B. W.
Background. Empirical broad-spectrum antibiotics are frequently prescribed to patients with severe COVID-19, motivated by concern about bacterial coinfection. There is no evidence of benefit from such a strategy, while the dangers of inappropriate antibiotics are well described. Objectives. To investigate the frequency, profile and related outcomes of infections by bacterial pathogens in patients admitted to an intensive care unit (ICU) with severe COVID-19 pneumonia. Methods. This was a prospective, descriptive study in a dedicated COVID-19 ICU in Cape Town, South Africa, involving all adult patients admitted to the ICU with confirmed COVID-19 pneumonia between 26 March and 31 August 2020. We collected data on patient comorbidities, laboratory results, antibiotic treatment, duration of admission and in-hospital outcome. Results. We included 363 patients, who collectively had 1 199 blood cultures, 308 tracheal aspirates and 317 urine cultures performed. We found positive cultures for pathogens in 20 patients (5.5%) within the first 48 hours of ICU admission, while 73 additional patients (20.1%) had positive cultures later during their stay. The most frequently isolated pathogens at all sites were Acinetobacter baumannii (n=54), Klebsiella species (n=13) and coagulase-negative staphylococci (n=9). Length of ICU stay (p<0.001) and intubation (p<0.001) were associated with positive cultures on multivariate analysis. Disease severity (p=0.5), early antibiotic use (p=0.5), diabetes mellitus (p=0.1) and HIV (p=0.9) were not associated with positive cultures. Positive cultures, particularly for tracheal aspirates (p<0.05), were associated with longer ICU length of stay and mortality. Early empirical antibiotic use was not associated with mortality (odds ratio 2.5; 95% confidence interval 0.95 - 6.81). Conclusions. Bacterial coinfection was uncommon in patients at the time of admission to the ICU with severe COVID-19. Avoiding early empirical antibiotic therapy is therefore reasonable. Strategies to avoid coinfection and outbreaks in hospital, such as infection prevention and control, as well as the strict use of personal protective equipment, are important to improve outcomes.
Bedaquiline, moxifloxacin, pretomanid, and pyrazinamide during the first 8 weeks of treatment of patients with drug-susceptible or drug-resistant pulmonary tuberculosis: a multicentre, open-label, partially randomised, phase 2b trial
(Elsevier, 2019) Tweed, Conor D.; Dawson, Rodney; Burger, Divan A.; Conradie, Almari; Crook, Angela M.; Mendel, Carl M.; Conradie, Francesca; Diacon, Andreas H.; Ntinginya, Nyanda E.; Everitt, Daniel E.; Haraka, Frederick; Li, Mengchun; Van Niekerk, Christo H.; Okwera, Alphonse; Rassool, Mohammed S.; Reither, Klaus; Sebe, Modulakgotla A.; Staples, Suzanne; Variava, Ebrahim; Spigelman, Melvin
Background: New anti-tuberculosis regimens that are shorter, simpler, and less toxic than those that are currently available are needed as part of the global effort to address the tuberculosis epidemic. We aimed to investigate the bactericidal activity and safety profile of combinations of bedaquiline, pretomanid, moxifloxacin, and pyrazinamide in the first 8 weeks of treatment of pulmonary tuberculosis. Methods: In this multicentre, open-label, partially randomised, phase 2b trial, we prospectively recruited patients with drug-susceptible or rifampicin-resistant pulmonary tuberculosis from seven sites in South Africa, two in Tanzania, and one in Uganda. Patients aged 18 years or older with sputum smear grade 1+ or higher were eligible for enrolment, and a molecular assay (GeneXpert or MTBDR plus ) was used to confirm the diagnosis of tuberculosis and to distinguish between drug-susceptible and rifampicin-resistant tuberculosis. Patients who were HIV positive with a baseline CD4 cell count of less than 100 cells per uL were excluded. Patients with drug-susceptible tuberculosis were randomly assigned (1:1:1) using numbered treatment packs with sequential allocation by the pharmacist to receive 56 days of treatment with standard tuberculosis therapy (oral isoniazid, rifampicin, pyrazinamide, and ethambutol; HRZE), or pretomanid (oral 200 mg daily) and pyrazinamide (oral 1500 mg daily) with either oral bedaquiline 400 mg daily on days 1–14 then 200 mg three times per week (B load PaZ) or oral bedaquiline 200 mg daily (B 200 PaZ). Patients with rifampicin-resistant tuberculosis received 56 days of the B 200 PaZ regimen plus moxifloxacin 400 mg daily (BPaMZ). All treatment groups were open label, and randomisation was not stratified. Patients, trial investigators and staff, pharmacists or dispensers, laboratory staff (with the exception of the mycobacteriology laboratory staff), sponsor staff, and applicable contract research organisations were not masked. The primary efficacy outcome was daily percentage change in time to sputum culture positivity (TTP) in liquid medium over days 0–56 in the drug-susceptible tuberculosis population, based on non-linear mixed-effects regression modelling of log 10 (TTP) over time. The efficacy analysis population contained patients who received at least one dose of medication and who had efficacy data available and had no major protocol violations. The safety population contained patients who received at least one dose of medication. This study is registered with ClinicalTrials.gov , NCT02193776 , and all patients have completed follow-up. Findings: Between Oct 24, 2014, and Dec 15, 2015, we enrolled 180 patients with drug-susceptible tuberculosis (59 were randomly assigned to B load PaZ, 60 to B 200 PaZ, and 61 to HRZE) and 60 patients with rifampicin-resistant tuberculosis. 57 patients in the B load PaZ group, 56 in the B 200 PaZ group, and 59 in the HRZE group were included in the primary analysis. B 200 PaZ produced the highest daily percentage change in TTP (5·17% [95% Bayesian credibility interval 4·61–5·77]), followed by B load PaZ (4·87% [4·31–5·47]) and HRZE group (4·04% [3·67–4·42]). The bactericidal activity in B 200 PaZ and B load PaZ groups versus that in the HRZE group was significantly different. Higher proportions of patients in the B load PaZ (six [10%] of 59) and B 200 PaZ (five [8%] of 60) groups discontinued the study drug than in the HRZE group (two [3%] of 61) because of adverse events. Liver enzyme elevations were the most common grade 3 or 4 adverse events and resulted in the withdrawal of ten patients (five [8%] in the B load PaZ group, three [5%] in the B 200 PaZ group, and two [3%] in the HRZE group). Serious treatment-related adverse events affected two (3%) patients in the B load PaZ group and one (2%) patient in the HRZE group. Seven (4%) patients with drug-susceptible tuberculosis died and four (7%) patients with rifampicin-resistant tuberculosis died. None of the deaths were considered to be related to treatment. Interpretation: B 200 PaZ is a promising regimen to treat patients with drug-susceptible tuberculosis. The bactericidal activity of both these regimens suggests that they have the potential to shorten treatment, and the simplified dosing schedule of B 200 PaZ could improve treatment adherence in the field. However, these findings must be investigated further in a phase 3 trial assessing treatment outcomes. Funding: TB Alliance, UK Department for International Development, Bill & Melinda Gates Foundation, US Agency for International Development, Directorate General for International Cooperation of the Netherlands, Irish Aid, Australia Department of Foreign Affairs and Trade, and the Federal Ministry for Education and Research of Germany.
Beta-lactam resistance mechanisms in Enterobacter species isolates from Tygerberg Hospital.
(Stellenbosch : Stellenbosch University, 2018-10-31) Okyere, Dora; Newton-Foot, Mae; Stellenbosch University. Faculty of Health Sciences. Dept. of Pathology. Medical Microbiology.
Background Resistance to carbapenem antibiotics in Gram-negative bacteria is a major public health problem due to limited treatment options. In Enterobacter species, carbapenemase production and reduced membrane permeability, resulting from reduced expression of outer membrane proteins OmpF and OmpC, in combination with extended-spectrum β-lactamase (ESBL) production, hyper-production of chromosomal blaAmpC β-lactamases and / or over-expression of the AcrAB/TolC efflux pump have been speculated to promote carbapenem resistance. This study investigated the mechanisms that promote ertapenem resistance in clinical Enterobacter cloacae isolates from Tygerberg Hospital. Materials and methods Twenty ertapenem non-susceptible clinical E. cloacae isolates, four ertapenem susceptible controls and five wild-type controls were selected based on the VITEK2®AES. Ertapenem MICs were determined using broth microdilution (BMD) and gradient diffusion. Resistance mechanisms were characterized using the phenotypic assays VITEK2®AES, Mastdiscs D68C, D69C and D63C combination sets, Rapidec® Carba NP kit and a synergy assay using disc diffusion in the presence of an efflux pump inhibitor (EPI). Molecular assays included multiplex PCR to identify carbapenemases, multiplex PCR and sequencing to identify ESBLs, SDS-PAGE to characterize OmpC and OmpF abundance and RT-qPCR to quantify expression of blaAmpC, ompC, ompF and acrB. Results and Discussion Seventeen (85%) ertapenem non-susceptible isolates were confirmed non-susceptible by BMD and six (30%) by gradient diffusion, suggesting possible undercalling of ertapenem resistance by gradient diffusion and overcalling by VITEK2®AES. Seven ertapenem non-susceptible isolates were predicted to be carbapenemase producers by the VITEK2®AES and one by the Rapidec® Carba NP kit, however no carbapenemases were detected by PCR. Nineteen ESBL producers were identified by the VITEK2®AES, eight by the D68C combination set and eleven by the D63C combination set. ESBLs were detected in 12 (60%) isolates by PCR and sequencing; of which eight were blaCTX-M, three were blaSHV-12 and one isolate contained both genes. Twelve (60%) ertapenem non-susceptible isolates were predicted to be derepressed blaAmpC producers by the VITEK2®AES, thirteen (65%) by the D68C combination set and six (30%) by the blaAmpC RT-qPCR.
Biofilm formation and adherence characteristics of an Elizabethkingia meningoseptica isolate from Oreochromis mossambicus
(BioMed Central, 2011-05-05) Jacobs, Anelet; Chenia, Hafizah Y.
ABSTRACT: Background. Elizabethkingia spp. are opportunistic pathogens often found associated with intravascular device-related bacteraemias and ventilator-associated pneumonia. Their ability to exist as biofilm structures has been alluded to but not extensively investigated. Methods. The ability of Elizabethkingia meningoseptica isolate CH2B from freshwater tilapia (Oreochromis mossambicus) and E. meningoseptica strain NCTC 10016T to adhere to abiotic surfaces was investigated using microtiter plate adherence assays following exposure to varying physico-chemical challenges. The role of cell-surface properties was investigated using hydrophobicity (bacterial adherence to hydrocarbons), autoaggregation and coaggregation assays. The role of extracellular components in adherence was determined using reversal or inhibition of coaggregation assays in conjunction with Listeria spp. isolates, while the role of cell-free supernatants, from diverse bacteria, in inducing enhanced adherence was investigated using microtitre plate assays. Biofilm architecture of isolate CH2B alone as well as in co-culture with Listeria monocytogenes was investigated using flow cells and microscopy. Results E. meningoseptica isolates CH2B and NCTC 10016T demonstrated stronger biofilm formation in nutrient-rich medium compared to nutrient-poor medium at both 21 and 37°C, respectively. Both isolates displayed a hydrophilic cell surface following the bacterial adherence to xylene assay. Varying autoaggregation and coaggregation indices were observed for the E. meningoseptica isolates. Coaggregation by isolate CH2B appeared to be strongest with foodborne pathogens like Enterococcus, Staphylococcus and Listeria spp. Partial inhibition of coaggregation was observed when isolate CH2B was treated with heat or protease exposure, suggesting the presence of heat-sensitive adhesins, although sugar treatment resulted in increased coaggregation and may be associated with a lactose-associated lectin or capsule-mediated attachment. Conclusions. E. meningoseptica isolate CH2B and strain NCTC 10016T displayed a strong biofilm-forming phenotype which may play a role in its potential pathogenicity in both clinical and aquaculture environments. The ability of E. meningoseptica isolates to adhere to abiotic surfaces and form biofilm structures may result from the hydrophilic cell surface and multiple adhesins located around the cell.
Biofilm formation in invasive Staphylococcus aureus isolates is associated with the clonal lineage
(Stellenbosch : Stellenbosch University, 2016-01) Hoek, Kim G. P.; Harvey, Justin; Wasserman, Elizabeth; Naicker, Preneshni Rochelle; Karayem, Karayem
ENGLISH ABSTRACT: Objectives: The ability to form biofilms contributes significantly to the virulence of Staphylococcus aureus. Virulence factors may be associated with certain S. aureus lineages. However, the contribution of the genetic background of S. aureus to biofilm formation is poorly understood. This study investigated the association between the genetic background and the biofilm forming ability of clinical invasive S. aureus isolates. Secondary objectives included investigating any correlation with biofilm formation and methicillin resistance or the source of bacteraemia. Methods: The study was conducted at a 1300-bed tertiary hospital in Cape Town, South Africa. S. aureus isolates obtained from blood cultures between January 2010 and January 2012 were included. Genotypic characterization was performed by PFGE, spa typing, SCCmec typing and MLST. Thirty genotypically unique strains were assessed for phenotypic biofilm formation with the microtitre plate assay. All isolates were tested in triplicate and an average optical density, measured at a wavelength of 490nm, was determined. The biofilm forming ability of isolates with A490 > 0.17 was considered ‘weak positive’ and A490 > 0.34 ‘strong positive’. Isolates with A490 ≤ 0.17 were considered non-adherent. ANOVA with Bonferroni-adjusted post-hoc tests and t-tests were used for statistical analysis of the association between biofilm forming ability and strain characteristics. Results: Fifty seven percent of isolates were capable of forming biofilms. Weak biofilm formation occurred in 40% (n=12) and strong biofilm formation in 17% (n=5) of isolates. Thirteen (43%) of the isolates were classified as non-adherent. All 5 isolates capable of strong biofilm formation belong to one spa clonal complex (spa-CC 064). Strains from spa-CC 064 were capable of higher biofilm formation than other spa clonal complexes (p=0.00002). These 5 strains belonged to MLST CC5 and CC8. Biofilm formation did not correlate with methicillin resistance and was not related to the source of bacteraemia. Conclusion: Biofilm formation correlates with the spa clonal lineage in our population of invasive S. aureus strains. High biofilm formation was associated with spa-CC 064. MLST CC5 and CC8 strains are capable of strong biofilm formation.
Biomarkers of HIV-associated cancer
(Libertas Academica, 2014) Flepisi, Brian Thabile; Bouic, Patrick; Sissolak, Gerhard; Rosenkranz, Bernd
Cancer biomarkers have provided great opportunities for improving the management of cancer patients by enhancing the efficiency of early detection, diagnosis, and efficacy of treatment. Every cell type has a unique molecular signature, referred to as biomarkers, which are identifiable characteristics such as levels or activities of a myriad of genes, proteins, or other molecular features. Biomarkers can facilitate the molecular definition of cancer, provide information about the course of cancer, and predict response to chemotherapy. They offer the hope of early detection as well as tracking disease progression and recurrence. Current progress in the characterization of molecular genetics of HIV-associated cancers may form the basis for improved patient stratification and future targeted or individualized therapies. Biomarker use for cancer staging and personalization of therapy at the time of diagnosis could improve patient care. This review focuses on the relevance of biomarkers in the most common HIV-associated malignancies, namely, Kaposi sarcoma, non-Hodgkin’s lymphoma, and invasive cervical cancer.
Can the Xpert MRSA/SA BC assay be used as an antimicrobial stewardship tool? A prospective assay validation and descriptive impact assessment study in a South African setting
(BMC (part of Springer Nature), 2021-02-15) Reddy, Kessendri; Whitelaw, Andrew
Background: Positive blood cultures showing Gram positive cocci in clusters signifies either Staphylococcus aureus or the less-virulent coagulase-negative staphylococci. Rapid identification and methicillin susceptibility determination with the Xpert MRSA/SA BC assay can improve management of S. aureus bloodstream infection and reduce inappropriate antibiotic use. Methods: We prospectively evaluated the Xpert MRSA/SA BC assay in comparison with culture, on samples referred to our laboratory in the Western Cape, South Africa. We interviewed attending clinicians upon culture result availability, to assess antibiotic choices and estimate potential impact of the assay. Results: Of the 231 samples included, there was 100% concordance between the Xpert MRSA/SA BC assay and culture (methicillin-resistant S. aureus 15/15, methicillin-susceptible S. aureus 42/42, coagulase-negative staphylococci 170/170). Time to final result could be reduced by approximately 30 h with the assay. Of the 178 patients with adequate antibiotic history, optimisation of antistaphylococcal therapy could have occurred more than 1 day sooner in 68.9% with S. aureus bloodstream infection (31/45, 95% CI 53.2–81.4%). Six of the 11 patients with methicillin-resistant S. aureus bloodstream infection (54.5%) could have received anti-MRSA cover sooner. Fifty-four days of antibiotic therapy could have been spared, equating to 0.3 days (95% CI, 0.2–0.4) saved per patient, driven by broad-spectrum beta-lactams (32 days, in 18.0% of the cohort). Conclusion: This assay has potential as an antimicrobial stewardship tool; costing and impact on clinical outcome in patients with S. aureus bloodstream infection should be assessed.
Carbapenemase-producing Enterobacteriaceae colonisation in adult inpatients : a point prevalence study
(AOSIS, 2019) Nel, Pieter; Paterson, Lauren A.; Hoffmann, Rena
Background: Infections caused by carbapenemase-producing Enterobacteriaceae (CPE) have been increasing worldwide in recent years, but data regarding the prevalence and clinical significance of CPE colonisation in South Africa is not well documented. Local private hospital groups have implemented routine screening programmes for selected high-risk patients as endorsed by the South African Society for Clinical Microbiology. This practice is not routinely performed in the public sector. Methods: A point prevalence study was performed at Tygerberg Hospital (TBH) by screening patients of all the adult inpatient wards to investigate the current prevalence of CPE colonisation. Common risk factors associated with CPE colonisation were also investigated. Results: From a total of 439 patient samples collected, only one patient was colonised with a Klebsiella pneumoniae organism harbouring blaNDM-1. The identified patient had none of the common risk factors associated with CPE colonisation. Conclusion: Based on these findings, screening for CPE colonisation in adults on admission to TBH is currently not recommended.
The carriage of antibiotic resistant Gram-negative organisms in children in the Cape Town community and the impact of antibiotic exposure on the development of resistance (a TB-CHAMP sub-study)
(Stellenbosch : Stellenbosch University, 2019-12) Ocloo, Remous; Whitelaw, Andrew; Newton-Foot, Mae; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology. Medical Microbiology.
Introduction Antibiotic resistance has become a major issue across the globe and the situation is worsening in low- and middle-income countries. In sub-Saharan Africa and the world at large, antibiotic resistance research is localized and focused on hospitalized individuals. There is, therefore, little or no data on antibiotic resistance in the community; especially in children. This study described the carriage of resistant isolates in children in Cape Town and investigated the effects of antibiotic exposure on the development of resistance in stool using an in-vitro model. Materials and Methods Stool samples from fifty participants of the Tuberculosis Child Multidrug-resistant Preventive Therapy Trial (TB-CHAMP) were cultured onto McConkey agar (MCC) with the addition of ertapenem and cefpodoxime discs to select for carbapenem and cephalosporin-resistant and susceptible E. coli and Klebsiella isolates. Antibiotic susceptibility testing was performed using Kirby Bauer disk diffusion. Carbapenem-, quinolone- and cephalosporin-resistance genes were detected by PCR and resistance-conferring mutations were detected using Sanger sequencing. Ten stool samples were exposed to two sub-clinical concentrations of amoxicillin, ciprofloxacin and colistin for 48 hours, whereafter they were plated onto MCC with the addition of various antibiotic discs (amoxicillin, ertapenem, ciprofloxacin, colistin, cefotaxime and nalidixic acid). The impact of antibiotic exposure on the development of resistance was assessed by enumeration of presumptive resistant E. coli and Klebsiella colonies within the zones of inhibition around the antibiotic discs. Results Twenty-one (42%) of the participants were colonized by quinolone-resistant isolates and 18 (36%) by cephalosporin-resistant isolates (predicted ESBL-producing organisms). Of the 21 quinolone-resistant E. coli isolates, 5 (24%) harbored qnrS while of the 6 quinolone-resistant Klebsiella isolates, 4 (67%) had qnrB. The most common quinolone resistance mutations were S83L in gyrA and S80I, A141V and S129A in parC. blaCTX-M was the only ESBL gene detected. All of the blaSHV and blaTEM genes detected were β-lactamases without extended spectrum activity. One of the participants was colonized by a carbapenem resistant Klebsiella isolate, which carried the blaNDM carbapenemase gene. Exposure of the stool samples to ciprofloxacin selected for resistant bacteria, however exposure to amoxicillin and colistin did not. Conclusion Children in Cape Town are frequently colonized by resistant bacteria and are at risk of becoming infected by these resistant organisms. The presence of plasmid-mediated resistance genes is concerning because they can be transferred between bacteria of the same and different species. There is also a need to further investigate what might be driving the high prevalence of quinolone resistance in the community. This study is the first to report the carriage of carbapenemase resistant bacteria in healthy children in South Africa. Although the in-vitro antibiotic exposure model was crude, the approach provides some evidence for the development of resistance during exposure to sub-clinical concentrations of antibiotics (especially ciprofloxacin); and notably, to agents other than those to which the sample had been exposed. This highlights the need for further investigations into the impact of sublethal antibiotic concentrations on the selection of resistance.
The carriage of antimicrobial resistance in community children – a TB-CHAMP sub-study.
(Stellenbosch : Stellenbosch University., 2019-12) Brand, Chante; Whitelaw, Andrew Christopher; Newton-Foot, Mae; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology: Medical Microbiology.
Introduction: The world-wide rise in antimicrobial resistance (AMR) is threatening the effectivity of antibiotics and the control of infectious diseases. The challenge of AMR is considerably exacerbated by the presence of mobile genetic elements harbouring various antibiotic resistance genes, which can easily spread to other species by horizontal gene transfer. This poses serious risks for clinical infections, however, reports on the carriage of these plasmid-mediated resistance genes are still rare in South Africa. This study aimed to describe the rates of carriage and mechanisms of antimicrobial resistance at baseline, as well as the effect of levofloxacin exposure on these rates in children in communities in Cape Town. Methods: Stool samples were collected from 100 children enrolled in the Tuberculosis Child Multidrug-resistant Preventive Therapy Trial (TB-CHAMP) at baseline and at 16- and 24-week follow up visits between November 2017 and November 2019. The stool samples were cultured onto MacConkey agar plates with cefpodoxime and ertapenem disks and in some cases, nalidixic acid and ciprofloxacin disks, in order to select for cephalosporin, carbapenem and quinolone resistant and susceptible E. coli and Klebsiella isolates. Kirby Bauer disk diffusion was used to determine the susceptibility profiles of the organisms and PCR and Sanger sequencing were used for subsequent detection of cephalosporin and quinolone resistance mechanisms. DNA was extracted directly from the stools and targeted molecular detection and quantification of plasmidmediated quinolone resistance (PMQR) genes using real-time PCR. Results: High levels of antibiotic resistance were detected at baseline, with 81% of participants carrying an organism resistant to at least one antibiotic and 49% and 33% carrying quinolone and cephalosporin resistant organisms respectively. These rates increased over time, with significant increases in quinolone resistance after 16 weeks (69.8%). The presence of the extended spectrum -lactamase gene, blaCTX-M, in cephalosporin resistant E. coli and Klebsiella spp. remained relatively constant over time, ranging between 19.7 – 26.8% and 33.3 – 37.5% respectively over 24 weeks. However, this gene was observed in higher proportions in Klebsiella spp. compared to E. coli. We saw high rates of carriage of qnrB (53.3%) and aac(6’)-Ib-cr (66.7%) in Klebsiella spp. at baseline and significant increases in qnrS and aac(6’)-Ib-cr, as well as mutations in gyrA and parC after 16 and, in some cases, 24 weeks. The presence of aac(6’)-Ib-cr also increased significantly in E. coli from baseline (3.8%) to 16 weeks (21.3%). qnrS was detected in 86% of stools in the targeted molecular analysis, while qnrB was only detected in 14%, although it was more abundant than qnrS in the stool samples. Conclusions: We report high rates of resistance to various antibiotics, as well as the presence of -lactamase and PMQR genes, in commensal gut bacteria in children in Cape Town communities before and over 24 weeks of levofloxacin treatment. The high rate of quinolone resistance at baseline is especially worrying as roughly half of these children started levofloxacin treatment after the baseline stool samples were collected. The increase in rates of resistance and presence of PMQR genes is interesting, however, the TB-CHAMP trial is still ongoing and we do not yet know which participants are receiving levofloxacin or placebo. Once the TB-CHAMP study has been completed and the blind has been broken, the results can be stratified according to treatment group to determine the impact of levofloxacin on resistance carriage.
Carriage of colistin-resistant Gram-negative bacteria in children from communities in Cape Town (Tuberculosis child multidrug-resistant preventive therapy trial sub-study)
(AOSIS, 2021) Snyman, Yolandi; Whitelaw, Andrew C.; Maloba, Motlatji R. B.; Hesseling, Anneke C.; Newton-Foot, Mae
ENGLISH ABSTRACT: Colistin is a last-resort antibiotic against multidrug-resistant, Gram-negative bacteria. Colistin resistance has been described in the clinical settings in South Africa. However, information on carriage of these bacteria in communities is limited. This study investigated gastrointestinal carriage of colistin-resistant Escherichia coli and Klebsiella spp. and mcr genes in children from communities in Cape Town. Colistin-resistant E. coli was isolated from two participants (4%, 2/50), and mcr-1-mcr-9 genes were not detected. Gastrointestinal carriage of colistin-resistant Enterobacterales was rare; however, continuous extensive surveillance is necessary to determine the extent of carriage and its contribution to resistance observed in clinical settings.
The Characterisation of Antibiotic Resistance Plasmids in Escherichia coli and Klebsiella pneumoniae Isolates from Hospital and Community Settings
(Stellenbosch : Stellenbosch University, 2021-03) Stein, Lisa; Newton-Foot, Mae; Whitelaw, Andrew; Pienaar, Colette; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology.
ENGLISH ABSTRACT: Antimicrobial resistance has become one of the biggest challenges and threats to public health systems worldwide. Widespread distribution of resistance is commonly due to horizontal gene transfer, which includes mobile genetic elements (MGE) such as plasmids, insertion sequences, transposons, and integrons. This study aimed to characterise plasmids conferring antibiotic resistance in extended-spectrum β-lactamase (ESBL) positive Escherichia coli and Klebsiella pneumoniae isolates from bloodstream infections to determine whether ESBL and plasmid-mediated quinolone resistance (PMQR) genes were mobilised on the same plasmids and whether the same plasmids are disseminated in healthcare and community settings. Methods Illumina MiSeq whole-genome sequencing (WGS) was previously performed on 112 E. coli and 66 K. pneumoniae isolates from blood cultures submitted to the National Health Laboratory Service Microbiology Laboratory at Tygerberg Hospital during 2017 and 2018. Assembled genomes were interrogated for the presence of ESBL and PMQR genes and plasmid replicon types. Based on the results, eight E. coli and nine K. pneumoniae isolates were selected for plasmid sequencing on the Oxford Nanopore Technologies MinION platform. Unicycler assembler was used for hybrid assembly of Illumina short-reads and Nanopore plasmid long-reads. In silico analyses were performed using ResFinder, PlasmidFinder and ISsaga to identify ESBL and PMQR genes, plasmid replicon types, and MGEs. Results Based on Illumina WGS, the ESBL and PMQR containing isolates contained multiple resistance genes and IncF plasmid replicons, individually or in combination with additional plasmid types. The IncF replicons and resistance genes were on separate contigs, therefore associations between different IncF replicons and with resistance genes could not be confirmed. Nanopore sequencing resolved plasmids from several E. coli and K. pneumoniae isolates; however, chromosomal genes could not be visualised and misassembly resulted in fragmented plasmids. Hybrid assembly fully resolved plasmids and chromosomal genes in several E. coli and K. pneumoniae isolates. Amongst the E. coli isolates, three F-type multireplicon plasmids and two single replicon plasmids IncI1-γ and IncB/O/K/Z, which contained resistance genes, were described. Novel multireplicon plasmid FII(FIC)-FIB-X was detected and harboured ESBL blaTEM-135 and PMQR qnrS1. The blaCTX-M genes were confirmed to be chromosomally located in three E. coli isolates and plasmid-mediated on F-type plasmids in two E. coli isolates. K. pneumoniae isolates harboured single replicon F-type plasmids, multireplicon FIB-HI1B fusion plasmids, and a single replicon IncC plasmid. The FIB-HI1B plasmids were associated with blaCTX-M-15, aac(6’)-Ib-cr, and qnrS1. The blaCTX-M-15 was plasmid-mediated in all K. pneumoniae isolates. Conclusion Amongst the E. coli isolates, ESBL and PMQR genes were present both on the same plasmid and on separate plasmids. In K. pneumoniae, ESBLs and PMQRs were found collectively on the same plasmids, and the F-type plasmids harbouring ESBL and PMQR genes differed from those in E. coli. As only two community-acquired K. pneumoniae isolates were selected for Nanopore plasmid sequencing, conclusions regarding the dissemination of K. pneumoniae plasmids in healthcare and community settings could not be made. Plasmids of the same FAB-types were detected amongst E. coli isolates of various sequence types and from both hospital- and community-settings, which is indicative of spread between these settings.
Characterisation of follicular helper T (Tfh) cells in early treated HIV-infected children: relationship to immune activation and inflammation.
(Stellenbosch : Stellenbosch University, 2022-11-24) Olifant, Paulina; Glashoff, Richard Helmuth; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology: Division of Medical Microbiology.
ENGLISH ABSTRACT: Background: South Africa has a high burden of Human Immunodefiency Virus (HIV) infection. Babies of HIV-positive pregnant women can become HIV-infected or exposed through vertical transmission. Follicular helper T (Tfh) cells have been of particular interest in HIV infection due to their preferential expansion, contribution to the HIV reservoir and dysregulation within HIV-infected individuals. The aim of this study was to investigate the Tfh cell population within children from the Children with HIV Early Antiretroviral Therapy (CHER) trial who started treatment within the first six months of life to determine whether the numbers of these cells is altered as compared to uninfected children and also whether persistent immune activation and inflammation in these children is associated with Tfh cell dysregulation. Methodology: This retrospective cross-sectional observational study consisted of three groups, i.e., early antiretroviral-treated HIT (HIV Infected Treated), HEU (HIV Exposed Uninfected) and HUU (HIV Uninfected Unexposed), of children. Cryopreserved peripheral mononuclear blood cells (PBMCs) were stained with an 11-colour antibody panel designed and optimised for phenotypic identification and quantification of T cell populations using flow cytometry. Tfh cells populations were characterised as CD4+CXCR5+PD-1+ with/without ICOS+. CD4, CD8 and Treg cells were defined as follicular/ follicular-homing based on CXCR5+ expression and activated based on CD38+ and/or CD69+ expression. Secondary data of clinical parameters and inflammatory cytokines for each group were evaluated. Statistical comparisons between groups were made using the Mann-Whitney test to identify significant differences. Significant correlations between Tfh cells and clinical parameters, other T cell populations and inflammatory cytokines were identified using Spearman’s rank order test. Results: Phenotypic results generally indicated significantly increased proportions of CD38+ subsets in HIT group and CD69+ subsets in HEU group. Although there was no significant difference in median CD4+CXCR5+PD-1+ Tfh cell percentage between groups, the ICOS-expressing subset namely CD4+CXCR5+PD-1+ICOS+ Tfh cells was significantly higher in the HIT (33.6% vs 19.2%; p = 0.016) and HEU group (31.6% vs 19.2%; p = 0.006) compared to the HUU group. In the HIT group, CD4+CXCR5+PD-1+ Tfh cells shared significant negative correlations with a majority of activated T cell subsets. A significant positive correlation between CD4+CXCR5+PD-1+ Tfh and CD8+PD-1+ Tc cells, general indicator of immune exhaustion, was demonstrated. Lastly, the HIT group showed the highest level of INF-α and hsCRP inflammatory cytokines and levels of IL-1β and hsCRP significantly correlated with CD4+CXCR5+PD-1+ICOS+ Tfh cells within this group. Conclusions: Overall levels of immune activation were significantly higher in HIT and HEU groups. Several activated T cell subsets inversely correlated with CD4+CXCR5+PD-1+ Tfh cells, suggesting high levels of immune activation can lead to decreased proportions of circulating Tfh cells. Even though no significant difference in the proportion of CD4+CXCR5+PD-1+ Tfh cells was found between groups, the ICOS+ subset was significantly expanded in HIT and HEU children in comparison to HUU children. The significant positive correlation between IL-1β and ICOS-expressing Tfh cells, within the entire study population and HIT group, suggested that increased inflammation resulted in an Tfh cell increase.
Characterisation of fosfomycin resistance in urinary pathogens from the Western Cape, South Africa.
(Stellenbosch : Stellenbosch University, 2021-03) Mosime, Lesedi Bridget; Nel, Pieter; Newton-Foot, Mae; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology.
ENGLISH ABSTRACT: Introduction: Urinary tract infections (UTI) are the most commonly acquired bacterial infections worldwide. The South African Department of Health advised that fosfomycin, nitrofurantoin and gentamicin be used for the treatment of uncomplicated UTI due to other antibiotics showing adverse side effects. Fosfomycin has effectively been utilised in the management of UTI, however resistance has been detected in urinary pathogens at the Tygerberg Hospital National Health Laboratory Service (NHLS) Medical Microbiology diagnostic laboratory. This study aimed to determine the prevalence of fosfomycin resistance among community-acquired urinary pathogens in the Western Cape and to characterise fosfomycin resistance mechanisms in fosfomycin resistant Escherichia coli and Klebsiella pneumoniae isolates. Methods and Materials: Two-hundred urinary isolates (Enterobacterales and Enterococcus spp.) from antenatal clinics in the Western Cape were collected from the Tygerberg Hospital NHLS Medical Microbiology laboratory during 2019 and 2020 and used to determine the prevalence of fosfomycin resistance. Fosfomycin susceptibility was determined using disc diffusion and Etest®. Fosfomycin resistant E. coli and K. pneumoniae isolates from the prevalence study and another set of fosfomycin resistant isolates (5 E. coli and 19 K. pneumoniae) collected from urine samples submitted to the NHLS at Tygerberg Hospital in 2017 (Ethics #: U17/05/026) were used to characterise fosfomycin mechanisms. FosA mediated resistance was determined using a phenotypic assay and fosA genes were detected by PCR. Mutations in the fosfomycin target gene murA and transporter genes, glpT and uhpT, were characterised by polymerase chain reaction (PCR) and Sanger sequencing. Results: Fosfomycin resistance was detected in 3.5% of community-acquired urinary pathogens. Fosfomycin resistance rates were 2.2% in E. coli (3/139) and 12.9% in other Enterobacterales. All Enterococcus spp. isolates were susceptible to fosfomycin. In the combined sample set of 31 fosfomycin resistant isolates, the phenotypic assay detected FosA in only 7 isolates, while fosA genes were detected by PCR in 25. Chromosomal mutations were identified in 6 isolates, of which three isolates (1 K. pneumoniae and 2 E. coli) had deletions in the uhpT gene, which has previously been reported to confer fosfomycin resistance. The role of other mutations found in the glpT gene of E. coli and the murA and glpT of K. pneumoniae isolates has not been determined. Conclusion: The fosfomycin resistance rate in community-acquired UTI was low, which supports the careful ongoing use of fosfomycin for the treatment of uncomplicated community-acquired UTI. FosA mediated resistance was the most common mechanism of fosfomycin resistance identified in this population.

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