The epidemiology and virulence characteristics of Staphylococcus aureus isolates from bacteraemic patients at Tygerberg Hospital, South Africa

Van Rijswijk, Amike (2019-04-02)

Thesis (MSc)--Stellenbosch University, 2019.

Thesis

ENGLISH ABSTRACT: Introduction: Staphylococcus aureus is a versatile pathogen that produces multiple virulence factors which work together to establish and maintain infections. The accessory gene regulator (agr) locus is a quorum sensing two-component system which regulates at least 23 virulence factors. There are four different agr types, I-IV, and mutations within the agr locus may result in a dysfunctional agr. These can result in altered gene expression which may affect disease presentation and outcome. Data on the molecular epidemiology of S. aureus and its association with clinical outcome in South Africa is limited. This study aimed to determine the effect of epidemiology and agr-associated virulence characteristics on the clinical outcome of bacteraemic patients at Tygerberg Hospital. Methods: S. aureus isolates were collected from blood cultures from February 2015 to March 2017. Genotyping was performed using staphylococcal protein A (spa) typing and multi-locus sequence typing (MLST); and staphylococcal cassette chromosome mec (SCCmec) typing was performed on all methicillin resistant S. aureus (MRSA) isolates. Agr typing was performed by PCR and agr functionality was assessed using a phenotypic δ-haemolysin assay and matrix assisted laser desorption ionisation-time of flight mass spectrometry (MALDI-TOF MS). Associations between patient- and strain- characteristics, and the final outcomes mortality, methicillin resistance and length of stay were investigated by means of regression models. Results and discussion: Of the 199 S. aureus isolates collected, 27% were MRSA. Seventy three spa types were identified; reflecting a diverse population. MRSA isolates were more clonal than methicillin susceptible S. aureus (MSSA) isolates. A previously described novel variant SCCmec type (NV) and SCCmec type IV were most common among MRSA isolates. Agr type I was the dominant agr type, while agr type IV was least prevalent; consistent with the literature. The dominant clone in this study was an MRSA outbreak strain, t045-ST5-MRSA-NV, agr type II (spa-CC 002, CC5), which appears to be circulating in multiple hospital settings in South Africa. The most prevalent MSSA strain/clone was t318-ST1865, agr type III. Pandemic clones such as t037-ST239-MRSA-III, t032-ST22-MRSA-IV and t012-ST36-MRSA-II were also identified. A previously described association between MRSA and spa-CC 002 (CC5) was confirmed in this study; however this association may have been driven by the MRSA outbreak. Agr dysfunctionality was low at 12.6% and 6% using the phenotypic assay and MALDI-TOF MS respectively. Agr dysfunctionality was not clone specific and there was no difference in agr dysfunctionality between MRSA and MSSA isolates. A borderline association between agr dysfunction and shorter length of stay was identified, but needs further investigation. The overall mortality rate was 29% and older age was associated with increased mortality. Hospital acquired (HA) infections were also associated with a higher mortality, which could be explained by the complicated nature of these infections, leading to death. An association between HA infections and MRSA was identified, which is consistent with previous studies and not surprising considering antibiotic selective pressure is higher in hospitals. Conclusion: This study provides insights into the associations between S. aureus epidemiology and agr related virulence characteristics and clinical outcome, despite the limited clinical data available.

ENGLISH ABSTRACT: Inleiding: Staphylococcus aureus is 'n veelsydige patogeen wat verskeie virulensie-faktore produseer wat saamwerk om infeksies te veroorsaak en onderhou. Die geassosieerde geen reguleerder (agr) lokus is 'n dubbele-komponent kworumwaarnemingstelsel wat minstens 23 virulensie-faktore reguleer. Daar is vier verskillende agr tipes, I-IV. Mutasies in die agr lokus lei dikwels tot ‘n disfunksionele agr wat daaropvolgend lei tot wisselende geen uitdrukking, wat die voorkoms en uitkoms van siektes kan beïnvloed. Data oor die molekulêre epidemiologie van S. aureus en die assosiasie daarvan met kliniese uitkoms in Suid-Afrika is beperk. Hierdie studie het gepoog om die effek van epidemiologiese- en agr-verwante virulensie eienskappe op die kliniese uitkoms van pasiënte met bakteremie by Tygerberg Hospitaal te bepaal. Metodes: S. aureus isolate van bloedkulture was vanaf Februarie 2015 tot Maart 2017 versamel. Genotipering was uitgevoer met behulp van stafilokokkale proteïn A (spa) tipering en multi-lokus volgordebepaling (MLST). “Staphylococcal chromosome complex mec” (SCCmec) tipering was op alle metisillienweerstandige Staphylococcus aureus (MRSA) isolate uitgevoer. Agr tipering was deur polimerasekettingreaksie (PKR) uitgevoer en agr funksionaliteit was met behulp van 'n fenotipiese δ-hemolisien toets en “matrix assisted laser desorption ionisation-time of flight mass spectrometry” (MALDI-TOF MS) ondersoek. Assosiasies tussen pasiënt- en stam-eienskappe met die finale uitkomstes, mortaliteit, metisillienweerstandigheid en lengte van hospitaalverblyf, was ondersoek. Resultate en bespreking: Sewe-en-twintig persent van die 199 S. aureus-isolate wat ingesamel is, was metisillienweerstandig. Drie-en-sewentig spa-tipes is geïdentifiseer; wat 'n diverse bevolking weerspieël. MRSA-isolate was meer klonaal as metisillien-vatbare S. aureus (MSSA) isolate. 'n Nuwe variant SCCmec-tipe (NV), voorheen beskryf, en SCCmec-tipe IV was die algemeenste tipe in die MRSA groep. Agr tipe I was die dominante agr tipe, terwyl agr tipe IV die minste voorgekom het; dit stem ooreen met wat in literatuur beskryf word. Die dominante kloon in hierdie studie was 'n MRSA-uitbraakstam, t045-ST5-MRSA-NV, agr tipe II (spa-CC 002, CC5), wat in verskeie hospitale in Suid-Afrika voorkom. Die algemeenste MSSA kloon was t318-ST1865, agr tipe III. Pandemiese klone soos t037-ST239-MRSA-III, t032-ST22-MRSA-IV en t012-ST36-MRSA-II is ook geïdentifiseer. 'n Voorheen beskryfde assosiasie tussen MRSA en spa-CC 002 (CC5) is in hierdie studie bevestig; dit is egter moontlik dat dit deur die MRSA-uitbraak gedryf kon word. Agr disfunksie was laag, 12.6% en 6%, soos bepaal deur die fenotipiese δ-hemolisien toets en MALDI-TOF MS onderskeidelik. Agr disfunksionaliteit was nie kloon spesifiek nie en daar was geen verskil in agr disfunksionaliteit tussen MRSA en MSSA isolate nie. Die assosiasie tussen agr disfunksie en ‘n korter hospitaalverblyf het gegrens aan statistiese betekenisvolheid, maar dit benodig verdere ondersoek. Die totale sterftesyfer was 29% en ouer ouderdom is geassosieer met verhoogde sterftes. Hospitaalverworwe (HA) infeksies is ook geassosieer met 'n hoër sterftesyfer, wat verklaar kan word deur die ingewikkelde aard van hierdie infeksies wat tot die dood lei. 'n Assosiasie tussen HA-infeksies en MRSA is geïdentifiseer; dit stem ooreen met vorige studies en is nie verbasend nie, aangesien antibiotika-selektiewe-druk in hospitale hoër is. Gevolgtrekking: Hierdie studie bied insig oor die assosiasies tussen S. aureus epidemiologie en agr-verwante virulensie eienskappe en kliniese uitkoms, ten spyte van die beperkte kliniese data wat beskikbaar was.

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