The role of the accessory gene regulator system on biofilm formation and stress response in Staphylococcus aureus

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dc.contributor.advisorMatukane, Siphiween_ZA
dc.contributor.advisorShima, Abdulgaderen_ZA
dc.contributor.authorMaleka, Kgomotso Franken_ZA
dc.contributor.otherStellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology: Division of Medical Microbiology.en_ZA
dc.date.accessioned2023-02-23T13:40:08Z
dc.date.accessioned2023-05-18T07:15:36Z
dc.date.available2023-02-23T13:40:08Z
dc.date.available2023-05-18T07:15:36Z
dc.date.issued2023-03
dc.descriptionThesis (MSc)--Stellenbosch University, 2023.en_ZA
dc.description.abstractENGLISH SUMMARY: Background: Biofilm formation is a key contributor to Staphylococcus aureus virulence and pathogenicity. It is regulated by the accessory gene regulator (agr) operon, which may become dysfunctional due to genetic mutations. These mutations may affect the expression of key genes like RNAIII and icaA that are involved in key pathogenesis pathways. Previous studies have associated agr dysfunctional strains with strong biofilm formation, persistent infections, and treatment failure. Therefore, this study aimed to determine the impact of agr functionality status on biofilm development and antibiotic stress tolerance in clinical S. aureus isolates. Methods: Twelve previously characterized (phenotypically and genotypically) blood culture S. aureus isolates, collected from February 2015 to March 2017 at Tygerberg Hospital were selected for this study. Crystal Violet biofilm assay was then performed to assess biofilm formation over a 24-hour period at different time points in the presence and absence of oxacillin, vancomycin, and rifampicin at sub-minimum inhibitory concentrations [sub-MIC: 0.25 μg/ml (oxacillin and vancomycin), 0,005 μg/ml rifampicin] and clinically relevant concentrations (10 μg/ml). The minimum inhibitory concentration (MIC) was determined using the gradient diffusion assay (E-test). Reverse-transcription real-time PCR was used to measure the expression of RNAIII and icaA genes. Whole genome sequence data were analyzed for genetic differences in the agr locus including the bap, icaA, and icaD regions for the 12 isolates, using online platforms (Prokka, Artemis, and BioEdit 7.2). Result: There was statistically an insignificance increase in the overall biofilm formation levels in agr dysfunctional isolates than in agr functional isolates in the absence and presence of antibiotics, except for when exposed to sub-MIC of oxacillin (p=0.007). Similarly, an increase in the overall biofilm formation level in agr I isolates was observed when compared to agr II and agr III isolates in the absence and presence of antibiotics. Furthermore, overall methicillin-resistant S. aureus (MRSA) isolates produced more biofilm, especially at time point 6 and 8 hours after incubation in the absence of antibiotics; while methicillin-sensitive S. aureus (MSSA) isolates formed more biofilm in the presence of antibiotics overall time points. Furthermore, a significant increase in the expression levels of both RNAIII (p=0.041) and icaA (p=0.020) was observed in agr dysfunctional isolates when compared to agr functional isolates. A significant increase in the expression of icaA (p=0.008) was observed in MRSA isolates; and dysfunctional isolates had more mutations on the agr-related gene than functional isolates. Conclusion: An increase in biofilm formation based on phenotypic agr functionality, agr type, and methicillin susceptibility profile in the absence or presence of antibiotics was not statistically significant. Additionally, mutations observed on the agr locus in agr dysfunctional isolates confirmed the role mutations play on agr functionality. The study analyzed 12 isolates, which may decrease statistical power. Therefore, future studies with a larger sample size should confirm or refute these study findings about the role agr functionality, agr type, and methicillin susceptibility profile have on the ability of clinical S. aureus isolates to produce biofilm in the absence and in the presence of antibiotics.en_ZA
dc.description.abstractAFRIKAANSE OPSOMMING: Inleiding: Biofilmvorming is 'n belangrike bydraende faktor vir Staphylococcus aureus virulensie en patogenisiteit. Dit word gereguleer deur die geassosieerde geen reguleerder (agr) lokus, wat disfunksioneel kan raak as gevolg van genetiese mutasies. Hierdie mutasies beinvloed die uitdrukking van belangrike gene soos RNAIII en icaA wat betrokke is by patogenisiteit. Vorige studies het agr-disfunksionele stamme geassosieer met sterk biofilmvorming, aanhoudende infeksies en behandelingsmislukking. Hierdie studie het gepoog om die impak van agr-funksionaliteit status op biofilm ontwikkeling en antibiotika-stresverdraagsaamheid in kliniese S. aureus isolate te bepaal. Metodes: Twaalf voorheen gekarakteriseerde (fenotipies en genotipies) S. aureus isolate is vir hierdie studie geselekteer. Die isolate is van bloedkulture vanaf Februarie 2015 tot Maart 2017 by Tygerberg Hospitaal versamel. ‘n Kristal Violet biofilm-toets is uitgevoer om biofilmvorming oor 'n 24-uur periode op verskillende tydpunte te bepaal in die teenwoordigheid en afwesigheid van oksassilien, vankomisien en rifampisien by sub-minimum inhiberende konsentrasies [sub-MIK: 0.25 μg/ml (oksasillien en vankomisien), 0,005 μg/ml rifampisien] en klinies relevante konsentrasies (10 μg/ml). Die minimum inhiberende konsentrasie (MIK) is bepaal met behulp van die gradientdiffusietoets (E-toets). Omgekeerde transkripsie intydse PKR is gebruik om die uitdrukking van die RNAIII en icaA gene te meet. Heelgenoomvolgordedata van die 12 isolate is ontleed met behulp van aanlyn platforms (Prokka, Artemis en BioEdit 7.2) om genetiese verskille in die agr-lokus, insluitend die bap-, icaA- en icaD-streke, te identifiseer. Resultate: Daar was 'n statisties onbeduidende toename in die algehele biofilmvormingsvlakke in agr-disfunksionele isolate in vergelyking met agr-funksionele isolate in die afwesigheid en teenwoordigheid van antibiotika, met die uitsondering van blootgestelling aan sub-MIK van oksasillien (p=0.007). 'n Toename in die algehele biofilmvormingsvlak in agr I isolate in vergelyking met agr II en agr III isolate is waargeneem in die afwesigheid en teenwoordigheid van antibiotika. Verder, metisillien-weerstandige S. aureus (MWSA) isolate het meer biofilms geproduseer, veral by tydpunte 6 en 8 uur na inkubasie in die afwesigheid van antibiotika, terwyl metisillien-sensitiewe S. aureus (MSSA) isolate meer biofilms in die teenwoordigheid van antibiotika by alle tydpunte gevorm het. Verder is 'n betekenisvolle toename in die uitdrukkingsvlakke van beide RNAIII (p=0.041) en icaA (p=0.020) in agr disfunksionele isolate waargeneem in vergelyking met agr funksionele isolate. 'n Betekenisvolle toename in die uitdrukking van icaA (p=0.008) is in MWSA-isolate waargeneem; en disfunksionele isolate het meer mutasies op die agr-verwante gene gehad as funksionele isolate. Gevolgtrekking: 'n Toename in biofilmvorming gebaseer op fenotipiese agr funksionaliteit, agr tipe en metisillien-vatbaarheidsprofiel in die afwesigheid of teenwoordigheid van antibiotika was nie statisties betekenisvol nie. Boonop, mutasies wat op die agr-lokus in agr-disfunksionele isolate waargeneem is, het die rol van mutasies op agr-funksionaliteit bevestig. Die studie het 12 isolate ontleed wat statistiese krag kan verminder, daarom is toekomstige studies met 'n groter steekproefgrootte nodig om hierdie studiebevindinge te bevestig of te weerle.af_ZA
dc.description.versionMasters
dc.format.extentxv, 88 pages : illustrations, includes annexures
dc.identifier.urihttp://hdl.handle.net/10019.1/127314
dc.language.isoen_ZAen_ZA
dc.publisherStellenbosch : Stellenbosch University
dc.rights.holderStellenbosch University
dc.subject.lcshStaphylococcus aureus -- Genetic aspectsen_ZA
dc.subject.lcshMethicillin resistanceen_ZA
dc.subject.lcshDrug resistance in microorganismsen_ZA
dc.subject.nameUCTD
dc.titleThe role of the accessory gene regulator system on biofilm formation and stress response in Staphylococcus aureusen_ZA
dc.typeThesisen_ZA
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