Browsing by Author "Maleka, Kgomotso Frank"

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    The role of the accessory gene regulator system on biofilm formation and stress response in Staphylococcus aureus
    (Stellenbosch : Stellenbosch University, 2023-03) Maleka, Kgomotso Frank; Matukane, Siphiwe; Shima, Abdulgader; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology: Division of Medical Microbiology.
    ENGLISH 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.