Thesis (PhD)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Numerous studies conducted internationally have identified and described several endemic methicillin-susceptible Staphylococcus aureus (MSSA) clones. However, only some of these clones are associated with methicillin resistance (CC5, CC8, CC22, CC30 and CC45). To date, studies reporting on the population structure of S. aureus isolated in South Africa represent limited demographic areas, focus on methicillin-resistant S. aureus (MRSA) only and have displayed little emphasis on virulence. This study was undertaken to elucidate the population structure of S. aureus isolated from specific clinical sources at Tygerberg hospital, and to investigate specific host-pathogen interactions of representative isolates.
Consecutive non-repetitive clinical S. aureus isolates were collected over one year (September 2009/2010) with patient demographics and limited clinical information. Strains were typed by PFGE and molecular markers (spa, multi-locus sequence typing (MLST), agr, Staphylococcal Chromosome Cassette mec and Panton-Valentine leukocidin (PVL)). Representative isolates were selected and investigated for the presence of virulence genes, adherence (to immobilised fibronectin [Fn], fibrinogen [Fg], collagens IV [CnIV] and VI [CnVI]), cellular invasion and cell death induction. Statistical association were determined between all in vitro results and methicillin-resistance, clonality, patient HIV status and bacterial PVL status.
Fifteen percent of the isolates (n = 367) were MRSA. Forty four present of isolates were PVL+. agr I-IV and SCCmec I-V were identified. The MSSA population was diverse: ST22 (dominant), ST1865 and ST121 were PVL+. ST45, ST1863 and ST15 were PVL-. PVL- MRSA were diverse: ST612-MRSA-IV (dominant), ST5-MRSA-I, ST239-MRSA-III, ST36-MRSA-II and ST22-MRSA-IV. The genes fnbA/B (fibronectin-binding protein A/B), clfA/B (clumping factor A/B), eap (extracellular adherence protein), nuc (nuclease), coa (coagulase) and hld (delta toxin) were detected in all representative isolates.
The CC8 and CC6 isolates adhered strongly to all ligands (100-700% of control, ligand dependent), while isolates of CC45, CC22 and CC88 adhered strongly only to Fg and Fn. The CC30, CC15, and CC12 isolates adhered extremely strongly to CnIV (>300%) and CC8, CC15, and C6 to CnVI (>200%). Isolates from CC30, CC8, CC15, CC6, CC12, CC97, CC88 and CC45 were highly invasive (>100%). ST121 was non-invasive (>50%). Isolates of CC5, CC30 and CC121 were non-cytotoxic (<50%), while isolates of CC22, CC8, CC15, CC45 and CC88 were very cytotoxic (>70%). No significant difference was observed in adherence or cell death induction of MRSA vs. MSSA clones or between isolates from HIV+ vs. HIV- persons. PVL- isolates displayed higher cellular invasiveness than PVL+ isolates.
The presence of ST612-MRSA-IV, ST22-MRSA-V and ST8-MRSA-V points to local SCCmec acquisition, as we found MSSA isolates with the same spa types. Numerous MSSA clones were prevalent, but do not appear to have a major common genetic background with MRSA. PVL was highly prevalent among MSSA, indicating acquisition of PVL genes independently of SCCmec. The abilities to adhere to specific immobilised ligands in vitro were diverse and grouped with the genetic background, while the vast majority of isolates were invasive and induced significant cell death.
We can conclude that the population of S. aureus at Tygerberg hospital is composed of a vast number of MSSA and MRSA clones, which display varying patters of adherence to selected ligands and of which, the majority clones are invasive and cytotoxic. / AFRIKAANSE OPSOMMING: Talle internasionale studies het verskeie endemiese metisillien vatbare Staphylococcus aureus (MSSA) klone geïdentifiseer en beskryf. Slegs 'n paar van hierdie klone word geassosieer met metisillien weerstandigheid (Klonale kompleks (KK) 5, KK8, KK22, KK30 en KK45). Studies oor die bevolking struktuur van S. aureus geïsoleer in Suid-Afrika is tot dusver beperk tot demografiese gebiede, fokus slegs op metisillien-weerstande S. aureus (MRSA) en het min klem op virulensie geplaas. Hierdie studie is onderneem om die bevolking struktuur van S. aureus, geïsoleer vanaf spesifieke kliniese bronne, in die pasiëntpopulasie van Tygerberg-hospitaal te ondersoek en om ondersoek in te stel na spesifieke gasheer-patogeen interaksies van verteenwoordigende isolate.
Opeenvolgende, nie-herhalende en suiwer kliniese S. aureus isolate is versamel oor ´n periode van een jaar (September 2009/2010), tesame met pasiënt demografiese- en beperkte kliniese inligting. Stamme is deur PFGE en molekulêre merkers (spa, MLST, agr, SCCmec en PVL) beskryf. Verteenwoordigende isolate is gekies en ondersoek vir die teenwoordigheid van virulensie gene, aanhegting ( aan geïmmobiliseerde fibronektien [Fn], fibrinogeen [Fg], kollageen IV [CnIV] en kollageen VI [CnVI]), sellulêre indringing en die induksie van seldood. Statistiese assosiasies is bepaal tussen alle in vitro resultate en methicillin-weerstandigheid, klonaliteit, pasiënt MIV status en bakteriese PVL status.
Fyftien persent van die isolate (n = 367) was MRSA. Vier-en-veertig van die isolate was PVL+. agrI-IV en SCCmec I-V is geïdentifiseer. Die MSSA bevolking was divers: ST22 (dominant), ST1865 en ST121 PVL +. ST45, ST1863 en ST15 was PVL+. PVL- MRSA was divers: ST612-MRSA-IV (dominant), ST5-MRSA-I, ST239-MRSA-III, ST36-MRSA-II en ST22-MRSA-IV. Die gene fnbA/B (fibronektien A/B), clfA/B (klontings faktor A/B), eap (ekstrasellulêre aanhegtings protein), nuc (nukease), coa (koagulase) en hld (delta toksien) was aangetref in alle verteenwoordigende isolate.
Isolate van KK8 en KK6 het sterk aan alle ligande (100-700% van kontrole, ligand-afhanklike) aangeheg, terwyl isolate van KK45, KK22 en KK88 slegs sterk aand fibronektien en fibrinogeen aangeheg het. Isolate van KK30, KK15, en KK12 het baie sterk aan CnIV (> 300%) aangeheg en KK8, KK15, en KK6 and CnVI (> 200%). Isolate van KK30, KK8, KK15, KK6, KK12, KK97, KK88 en KK45 was hoogs indringend (> 100%). ST121 was nie-indringende (> 50%). Isolate van KK5, KK30 en KK121 was nie-sitotoksiese (<50%), terwyl isolate van KK22, KK8, KK15, KK45 en KK88 baie sitotoksies was (> 70%).
Geen betekenisvolle verskil is waargeneem in die aanhegting of seldood induksie van MRSA teenoor MSSA klone of tussen isolate van MIV+ teenoor MIV- persone nie. PVL- isolate het hoër sellulêre indringing as PVL+ isolate vertoon.
Die teenwoordigheid van ST612-MRSA-IV, ST22-MRSA-V en ST8-MRSA-V verwys na die plaaslike verwerwing van SCCmec, aangesien ons MSSA isolate beskryf het met dieselfde spa-tipes. Talle MSSA klone was algemeen, maar het nie 'n beduidende genetiese agtergrond met MRSA vertoon nie. PVL was baie algemeen onder MSSA isolate en die PVL gene is dalk onafhanklik van SCCmec verkry.
Die vermoë om aan spesifieke geïmmobiliseer ligande in vitro aan te heg was divers en groepeer met die genetiese agtergrond, terwyl die meerderheid van die isolate indringend was en kon betekenisvolle sel dood veroorsaak.
Ons kan aflei dat die bevolking van S. aureus by die Tygerberg hospitaal saamgestel is uit 'n groot aantal van MSSA en MRSA klone, wat verskillende patrone van aanhegting aan geselekteerde ligande vertoon en waarvan die meeste klone indringende en sitotoksies is. / DFG/NRF International Research and Training Group (IRTG) 1522 “HIV and associated infectious diseases in Southern Africa” / National Research Foundation / Medical Research Council, Medi-Clinic / Harry Crossley Fund (Stellenbosch University) / Stellenbosch University
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/85580 |
| Date | 12 1900 |
| Creators | Oosthuysen, Wilhelm Frederick |
| Contributors | Wasserman, Elizabeth, Orth, Heidi, Sinha, Bhanu, Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Type | Thesis |
| Format | xxxiv, 215 p. : ill. |
| Rights | Stellenbosch University |
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