The characterization of antimicrobial resistance and virulence in Coagulase-Negative Staphylococci isolated from neonatal blood cultures in the Western Cape

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dc.contributor.advisorWhitelaw, Andrew Christopheren_ZA
dc.contributor.advisorMatukane, Siphiwe Ruthyen_ZA
dc.contributor.authorCloete, Stephanie Simoneen_ZA
dc.contributor.otherStellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology: Division of Medical Microbiology.en_ZA
dc.date.accessioned2023-02-23T10:31:19Z
dc.date.accessioned2023-05-18T07:09:19Z
dc.date.available2023-02-23T10:31:19Z
dc.date.available2023-05-18T07:09:19Z
dc.date.issued2023-03
dc.descriptionThesis (MSc)--Stellenbosch University, 2023.en_ZA
dc.description.abstractENGLISH SUMMARY: Introduction: Coagulase-Negative Staphylococci (CoNS) are frequently isolated from the neonatal intensive care unit. However, it is difficult to discriminate between invasive CoNS and coincident contaminants since they are commensals of skin, and the nonspecific clinical signs of neonatal sepsis may be even more subtle due to the low virulence of CoNS. The extensive range of virulence factors in CoNS, some of which may be regulated by mobile genetic elements, contributes to their pathogenicity. This study aims to describe the species distribution, antimicrobial resistance, and molecular virulence markers in CoNS from neonatal blood cultures to identify potential pathogenicity markers. Methods: Between February and July 2021, 127 CoNS isolates were collected from neonatal blood cultures submitted to Tygerberg Hospital National Health Laboratory Services microbiology laboratory. Species identification was performed on all isolates using MALDI-TOF mass spectrometry. Antimicrobial susceptibility testing (AST) using Kirby Bauer disc diffusion was performed on 82 isolates representing the two predominant CoNS species from the most represented hospitals. Twenty isolates, representing a range of susceptibility patterns, were chosen for Oxford Nanopore whole genome sequencing (WGS). The assembled genomes were analysed using the Center for Genomic Epidemiology, pubMLST, Virulence Factor Database, ISsaga, and Resistance Gene Identifier. Reverse-transcription real-time PCR was used to explore the potential association of insertion sequence (IS)256 with the expression of the biofilm associated virulence gene icaA. The icaA normalised cycle threshold (ΔCt) was calculated relative to that of the house-keeping gene (tpi). Results: The most common species identified were Staphylococcus epidermidis (80/127, 63 %) and Staphylococcus hominis (29/127, 23 %). Among the 62 S. epidermidis and 20 S. hominis isolates selected for AST, 81.7 % (67/82) were non-susceptible to at least one antibiotic, and 54 % (44/82) were resistant to three or more antibiotic classes (multidrug resistant). The highest rates of resistance were to erythromycin (64.6%), trimethoprim-sulfamethoxazole (56.1 %), and cloxacillin (54.9 %). There was considerable concordance between the observed phenotypes and the predicted genotype, especially for cefoxitin, linezolid and vancomycin. The WGS data predicted all 20 isolates (14 S. epidermidis and six S. hominis isolates) as possible human pathogens, with a probability pathogen score higher for S. epidermidis (=0.93) than S. hominis (=0.89) isolates (p<0.001). In-silico MLST revealed substantial diversity, reflected by nine S. epidermidis sequence types identified. The ica operon was present in 10/14 S. epidermidis isolates, and 7/14 isolates contained the IS256. None of the S. hominis isolates contained the ica operon. The ΔCt values in isolates with and without IS256 were not significantly different, suggesting that IS256 was not related to ica operon expression. Conclusion: There was a high-level of genetic diversity among CoNS isolates, enriched with virulence factors, antimicrobial resistance genes and mobile genetic elements. S. epidermidis harboured a greater array of virulence factors than S. hominis isolates, which require further investigation to be used as markers of clinical significance. Antimicrobial resistance was common among these isolates, which may complicate treatment strategies. There was no effect of IS256 on ica gene expression, which may relate to the distance of IS256 from the ica operon.en_ZA
dc.description.abstractAFRIKAANSE OPSOMMING: Inleiding: Koagulase-Negatiewe Stafilokokke (KoNS) word dikwels in die neonatale intensiewesorgeenheid gevind. Dit is egter moeilik om tussen indringende KoNS en toevallige kontaminante te onderskei, aangesien dit ook natuurlik op die vel kan voorkom. As gevolg van die lae virulensie van KoNS, kan die algemene kliniese tekens van neonatale sepsis selfs meer subtiel wees. Die uitgebreide reeks virulensiefaktore in KoNS, waarvan sommige deur mobiele genetiese elemente gereguleer kan word, dra by tot hul patogenisiteit. Hierdie studie het gemik om die spesieverspreiding, antimikrobiese weerstand en molekulere virulensiemerkers in KoNS vanaf neonatale bloedkulture te beskryf, ten doel om potensiele patogenisiteitsmerkers te identifiseer. Metodes: Tussen Februarie en Julie 2021 is 127 KoNS-isolate vanaf neonatale bloedkulture, wat by Tygerberg Hospitaal Nasionale Gesondheidslaboratoriumdienste se mikrobiologie-laboratorium ingedien is, versamel. Die identifisering van spesies is op alle isolate uitgevoer, met behulp van MALDI-TOF massaspektrometrie. Antimikrobiese vatbaarheidstoetsing (AVT) is deur Kirby Bauer skyfdiffusie uitgevoer op 82 isolate wat deel was van die twee oorheersende KoNS spesies in die mees verteenwoordigde hospitale. Twintig isolate, wat 'n reeks vatbaarheidspatrone verteenwoordig, is gekies vir Oxford Nanopore heelgenoomvolgordebepaling (WGS). Die saamgestelde genome is met behulp van die Sentrum vir Genomiese Epidemiologie, pubMLST, Virulence Factor Database, ISsaga en Resistance Gene Identifier ontleed. Omgekeerde-transkripsie intydse PKR is gebruik om die potensiele assosiasie van invoegvolgorde (IS)256 met die uitdrukking van die biofilm-geassosieerde virulensiegeen icaA te verken. Die genormaliseerde icaA “cycle threshold” (ΔCt) is relatief tot die van 'n huishoudelike geen (tpi) bereken. Resultate: Die algemeenste spesies was Staphylococcus epidermidis (80/127, 63 %) en Staphylococcus hominis (29/127, 23 %). Tussen die 62 S. epidermidis- en 20 S. hominis-isolate wat vir AVT geselekteer is, was 81.7 % (67/82) nie vatbaar vir ten minste een antibiotikum nie, en 54 % (44/82) was weerstandbiedend teen drie of meer klasse van antibiotika (multi-middel weerstandbiedend). Die hoogste koerse van weerstand was teen eritromisien (64.6%), trimetoprim-sulfametoksasool (56.1%) en kloksasillien (54.9%). Die waargenome fenotipes en die voorspelde genotipes het aansienlik ooreengestem, veral vir kefoksitien, linezolid en vankomisien. Die WGS data het voorspel dat al 20 isolate (14 S. epidermidis en ses S. hominis isolate) moontlike menslike patogene is, met 'n hoer patogeenwaarskynlikheidstelling vir S. epidermidis (=0.93) teenoor S. hominis (=0.89) isolate (p<0.001). In-silico MLST het aansienlike diversiteit geopenbaar, weerspieel deur nege S. epidermidis-volgordetipes wat geidentifiseer is. Die ica operon was teenwoordig in 10/14 S. epidermidis isolate, en 7/14 isolate het die IS256 bevat. Nie een van die S. hominis-isolate het die ica-operon bevat nie. Die ΔCt waardes in isolate met en sonder IS256 was nie betekenisvol verskillend nie, wat daarop dui dat IS256 nie verband hou met ica operon uitdrukking nie. Gevolgtrekking: Daar was 'n hoe vlak van genetiese diversiteit tussen KoNS-isolate, verryk deur virulensiefaktore, antimikrobiese weerstandsgene en mobiele genetiese elemente. S. epidermidis het 'n groter verskeidenheid virulensiefaktore as S. hominis-isolate gehuisves, wat verdere ondersoek vereis om as merkers van kliniese betekenis gebruik te kan word. Antimikrobiese weerstand het algemeen voorgekom, wat behandelingstrategiee kan bemoeilik. Daar was geen effek van IS256 op ica geenuitdrukking nie, wat moontlik verband hou met die afstand tussen IS256 en die ica operon.af_ZA
dc.description.versionMasters
dc.format.extentxv, 109 pages : illustrations, includes annexures
dc.identifier.urihttp://hdl.handle.net/10019.1/127198
dc.language.isoen_ZAen_ZA
dc.publisherStellenbosch : Stellenbosch University
dc.rights.holderStellenbosch University
dc.subject.lcshDrug resistance in microorganisms -- Western Cape (South Africa)en_ZA
dc.subject.lcshVirulence (Microbiology) -- Western Cape (South Africa)en_ZA
dc.subject.lcshAnti-infective agents -- Western Cape (South Africa)en_ZA
dc.subject.lcshStaphylococcal infections -- Western Cape (South Africa)en_ZA
dc.subject.lcshStaphylococcus -- Western Cape (South Africa)en_ZA
dc.subject.nameUCTD
dc.titleThe characterization of antimicrobial resistance and virulence in Coagulase-Negative Staphylococci isolated from neonatal blood cultures in the Western Capeen_ZA
dc.typeThesisen_ZA
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