Clinton Moodley

The molecular epidemiology of carbapenem-resistant Enterobacterales in Cape Town remains largely unknown. This study aimed to describe the molecular epidemiology, resistome, virulome and mobilome of carbapenem-resistant Klebsiella pneumoniae (CRKP) within Cape Town to guide therapy, antimicrobial stewardship and infection prevention and control practices. Eighty-five CRKP isolates from hospitalized patients underwent WGS as part of a prospective, multicentre, cross-sectional study, conducted between 1 November 2020 and 30 November 2022, across public-sector and private-sector hospitals in Cape Town, South Africa. MLST revealed three novel types, ST6785, ST6786 and ST6787, while the most common were ST219, ST307, ST17, ST13 and ST2497. Different predominant clones were noted in each hospital. The most common carbapenemase gene was bla_OXA-48-like, detected in 71% of isolates, with bla_NDM detected in 5%. Notably, co-detection of two carbapenemase genes (bla_OXA-48-like and bla_NDM) occurred in 13% of isolates. The yersiniabactin siderophore was detected in 73% of isolates, and was most commonly associated with the ICEKp5 mobile element. All carbapenemases were located on plasmids. The genes bla_OXA-181 and bla_OXA-232 colocalized with a ColKP3 replicon type on assembled contigs in 83% and 100% of cases, respectively. CRKP epidemiology in Cape Town reflects institutionally dominant, rather than regional, clones. The most prevalent carbapenemase gene was bla_OXA-48-like, in keeping with CRKP epidemiology in South Africa in general. Emerging clones harbouring both bla_OXA-48-like and bla_NDM, such as ST17, ST2497 and the novel ST6787, are a concern due to the limited availability of appropriate antimicrobial agents in South Africa.

Carbapenem-resistant Enterobacterales (CRE) are a substantial problem in Cape Town. CRE epidemiology is largely unknown and mortality remains high. To describe and characterize the clinical and microbiological epidemiology of CRE within Cape Town hospitals to better inform therapy with regard to current and novel antibiotics, as well as improve antimicrobial stewardship (AMS), and infection prevention and control (IPC). This prospective, multicentre study performed between 1 November 2020 and 30 November 2022, across three public and three private hospitals included hospitalized participants with CRE from clinical cultures. Participant demographics, clinical information and microbiology results were collected and analysed. Ninety percent of participants were from public hospitals. The age distribution ranged from 7 days to 88 years. Notable risk factors for CRE infection included recent exposure to antibiotics, medical devices and surgery. The most prevalent species was Klebsiella pneumoniae. However, a higher proportion of Serratia marcescens compared with previous reports was identified. The detected carbapenemases were bla_OXA-48-like (80%) and bla_NDM (11%). With the exception of amikacin (63%), tigecycline (65%), colistin (95%) and ceftazidime/avibactam (87%), susceptibility to antibiotics was low. This study identified common risk factors for CRE infection and generated a description of carbapenemase enzymes, species distribution and antibiograms, enabling a better understanding of CRE epidemiology. This provides insights into transmission patterns and resistance determinants of CREs, beneficial to informing data-driven regional patient management, AMS and IPC strategies.

Background: Sexually transmitted infections are among the most commonly occurring infections globally, with countries in sub-Saharan Africa exhibiting disproportionately higher prevalence rates. Numerous reports indicate the need for accurate detection, epidemiological characterisation, and appropriate management of these infections. This prospective observational laboratory study sought to determine the occurrence of STI, using a validated molecular assay as a diagnostic and surveillance tool in our setting. Methods: Urogenital swabs from symptomatic and asymptomatic patients, submitted to the National Health Laboratory Service, at Groote Schuur Hospital, from 04 August 2021–03 February 2022, for routine microbiological investigations, were subjected to the Allplex™ STI Essential Assay (Seegene Inc, South Korea) to determine the distribution of STI pathogens in our setting. This multiplex assay includes C. trachomatis, Mycoplasma genitalium, Mycoplasma hominis, N. gonorrhoeae, Trichomonas vaginalis, Ureaplasma parvum, and Ureaplasma urealyticum. Correlations between detected organisms and participant age and clinical indications for testing were determined using Stata® software. Results: A total of 148 urogenital swabs (91.2% from women) were included in the analysis, of which 56/148 (37.84%) were from symptomatic patients. Up to 83.8% of the samples tested positive for ≥1 organism, with all seven target organisms detected in at least one sample. Ureaplasma parvum was the most common organism detected, followed by N. gonorrhoeae, M. hominis, U. urealyticum, T. vaginalis, C. trachomatis, with M. genitalium being the least detected. All 25 samples submitted for routine antenatal Group B Streptococcal screening were positive for at least one STI organism, and one sample from sexual non-accidental injury tested positive for five different organisms. Conclusions: STIs comprise a variety of organisms in our setting, with many patients exhibiting coinfection with multiple organisms. This suggests the need for a critical evaluation of current syndromic testing and treatment guidelines so as to stem inadvertent spread of STI organisms and the development of resistance. The use of molecular testing methods may improve detection, especially in resource limited settings, providing speedy results, and thus allowing for guided therapy in only infected patients.

Background: Culture remains the diagnostic standard for Streptococcus pneumoniae bacteraemia but is limited by time to identification, prior antibiotics and bacterial autolysis. Culture-independent methods for detecting S. pneumoniae include PCR and antigen tests. We evaluated an antigen test on blood culture broth for the rapid detection of S. pneumoniae bacteraemia. Method: We collected 212 signal-positive blood cultures, with gram-positive cocci in pairs, chains or with uncertain morphology. The BinaxNOW S. pneumoniae urinary antigen test, Gram stain, culture and lytA PCR were performed on all samples. Diagnostic accuracy of the antigen test and Gram stain with gram-positive cocci in pairs were compared with culture, polymerase chain reaction (PCR) and the composite of culture and PCR. Results: Streptococcus pneumoniae was isolated in 26% of samples, 66% cultured other gram-positive organisms and 8% of samples had no growth. Sensitivity and negative predictive values of the antigen test were 100%, specificity and positive predictive values were 87% – 88% and 76% – 81%, but increased to 93% – 96% and 96% – 98% when applied to subsets with gram-positive cocci in pairs, or history compatible with respiratory illness or meningitis. Sensitivity (69% – 75%) and specificity (81%) of Gram stain (gram-positive cocci in pairs) were lower than the antigen test even when applied to the same subsets. Conclusion: Accurate and rapid diagnosis of S. pneumoniae bacteraemia is challenging. Specificity of this antigen test is limited by cross-reactivity with other gram-positive organisms, but could be improved if Gram stain morphology and clinical history are available. The antigen test is a useful adjunct for rapid diagnosis of S. pneumoniae bacteraemia.

Introduction: There are few studies describing colonisation with extended spectrum beta-lactamase-producing Enterobacterales (ESBL-PE) and carbapenem-resistant Enterobacterales (CRE) among children in sub-Saharan Africa. Colonisation often precedes infection and multi-drug-resistant Enterobacterales are important causes of invasive infection. Methods: In this prospective cross-sectional study, conducted between April and June 2017, 200 children in a tertiary academic hospital were screened by rectal swab for EBSL-PE and CRE. The resistance-conferring genes were identified using polymerase chain reaction technology. Risk factors for colonisation were also evaluated. Results: Overall, 48% (96/200) of the children were colonised with at least one ESBL-PE, 8.3% (8/96) of these with 2 ESBL-PE, and one other child was colonised with a CRE (0.5% (1/200)). Common colonising ESBL-PE were Klebsiella pneumoniae (62.5%, 65/104) and Escherichia coli (34.6%, 36/104). The most frequent ESBL-conferring gene was blaCTX-M in 95% (76/80) of the isolates. No resistance- conferring gene was identified in the CRE isolate (Enterobacter cloacae). Most of the Klebsiella pneumoniae isolates were susceptible to piperacillin/tazobactam (86.2%) and amikacin (63.9%). Similarly, 94.4% and 97.2% of the Escherichia coli isolates were susceptible to piperacillin/tazobactam and amikacin, respectively. Hospitalisation for more than 7 days before study enrolment was associated with ESBL-PE colonisation. Conclusion: Approximately half of the hospitalised children in this study were colonised with ESBL-PE. This highlights the need for improved infection prevention and control practices to limit the dissemination of these microorganisms.

Nasopharyngeal (NP) colonization with antimicrobial-resistant bacteria is a global public health concern. Antimicrobial-resistance (AMR) genes carried by the resident NP microbiota may serve as a reservoir for transfer of resistance elements to opportunistic pathogens. Little is known about the NP antibiotic resistome. This study longitudinally investigated the composition of the NP antibiotic resistome in Streptococcus-enriched samples in a South African birth cohort. As a proof of concept study, 196 longitudinal NP samples were retrieved from a subset of 23 infants enrolled as part of broader birth cohort study. These were selected on the basis of changes in serotype and antibiogram over time. NP samples underwent short-term enrichment for streptococci prior to total nucleic acid extraction and whole metagenome shotgun sequencing (WMGS). Reads were assembled and aligned to pneumococcal reference genomes for the extraction of streptococcal and non-streptococcal bacterial reads. Contigs were aligned to the Antibiotic Resistance Gene-ANNOTation database of acquired AMR genes. AMR genes were detected in 64% (125/196) of the samples. A total of 329 AMR genes were detected, including 36 non-redundant genes, ranging from 1 to 14 genes per sample. The predominant AMR genes detected encoded resistance mechanisms to beta-lactam (52%, 172/329), macrolide-lincosamide-streptogramin (17%, 56/329), and tetracycline antibiotics (12%, 38/329). MsrD, ermB, and mefA genes were only detected from streptococcal reads. The predominant genes detected from non- streptococcal reads included bla_OXA-60, bla_OXA-22, and bla_BRO-1. Different patterns of carriage of AMR genes were observed, with only one infant having a stable carriage of mefA, msrD and tetM over a long period. This study demonstrates that WMGS can provide a broad snapshot of the NP resistome and has the potential to provide a comprehensive assessment of resistance elements present in this niche.

Background: Phenotypic detection of extended-spectrum beta-lactamases (ESBLs) is based on the inhibition of ESBL enzymes by β-lactamase inhibitors and on the comparison of cephalosporin activity with or without a β-lactamase inhibitor. Many South African diagnostic laboratories rely on the Vitek 2 for automated susceptibility testing and for ESBL detection. However, the Gram-negative susceptibility card currently used locally (AST-N255) has been modified and its accuracy for ESBL detection is not known.Methods: We randomly selected 50 isolates of Klebsiella pneumoniae and Escherichia coli from a collection of clinical bloodstream isolates from Groote Schuur Hospital from 2015 to 2016, including ESBL-producing and non-ESBL-producing strains. We used standardised phenotypic (disc diffusion and broth microdilution) and genotypic (conventional polymerase chain reaction (PCR) for blaCTX-M, blaSHV and blaTEM) methods for detection of ESBLs. We compared ESBL detection by Vitek 2 to a composite reference standard comprising ESBL detection either by both phenotypic methods or by one phenotypic method together with genotypic detection.Results: The sensitivity of Vitek 2 system for detection of ESBLs was 33/36 or 92% (78% – 97%) for E. coli, and 40/40 or 100% (91% – 100%) for K. pneumoniae, whilst specificity was 10/10 or 100% (72% – 100%) and 9/10 or 90% (60% – 98%), respectively. This is comparable with previous studies.Conclusion: Using a composite reference standard of the phenotypic and genotypic methods employed in this study, no Vitek-categorised ESBL E. coli or K. pneumoniae was found to be a non-ESBL with the exception of possible misinterpretation with K. pneumoniae SHV-hyper-producing isolates.