Title page for ETD etd-08022012-131316
Mutepe, Ndiafhi Daphney Email
Effects of cigarette smoke condensate on the growth of and production of biofilm by Streptococcus pneumoniae and on the bioactivity of pneumolysin
Prof R Cockeran
Streptococcus pneumoniae is a common human pathogen, causing severe and often life-threatening respiratory tract infections. Even though most patients receive appropriate antimicrobial chemotherapy, a significant percentage still die. Cigarette smoking is a well-documented risk factor for severe pneumococcal pneumonia; however, the microbiological/ immunological mechanisms which predispose smokers to infection are not yet completely understood. The pneumococcal toxin, pneumolysin, is a major virulence factor of S. pneumoniae; it forms pores in eukaryotic cell membranes, resulting in the influx of extracellular Ca2+. Biofilm is a self-generated polymer matrix, used by microbial pathogens to isolate themselves from both host defences, as well as antibiotics. In this dissertation, the effects of cigarette smoke condensate (CSC) on the bioactivity of pneumolysin, as well as on the growth of, and production of biofilm by Streptococcus pneumoniae are described.
A clinical isolate of S. pneumoniae, strain 172, was used for both growth and biofilm determinations in the presence or absence of concentrations of CSC (20-160 μg/ml), representative of the smoking habit. Growth and biofilm determinations were performed using spectrophotometric procedures, viability by standard colony forming unit procedures, and the effect of the condensate on pneumolysin bioactivity using a fura-2/AM-based spectrofluorometric procedure. Exposure of the pneumococcus to CSC resulted in a dose-dependent increase in biofilm formation which achieved statistical significance (P≤0.05) at concentrations of 80 and 160 μg/ml, in the setting of modest effects on bacterial growth. These findings were not unique to S. pneumoniae since exposure of Staphylococcus aureus, a known biofilm former, to CSC showed similar results. Exposure of pneumolysin to CSC (20 and 40 μg/ml) was accompanied by attenuation of the biological activity of the toxin, resulting in impaired pore-forming ability manifest as a considerable reduction in influx of extracellular Ca2+ following exposure of isolated neutrophils to the toxin.
It is possible that CSC acts as a stressor to the bacteria, thereby enhancing biofilm formation and consequently persistence in the respiratory tract. These effects of the toxin may be complemented by inactivation of pneumolysin, presumably by pro-oxidative mechanisms, affecting innate cellular host defences. These mechanisms may underpin the predisposition of smokers to develop severe pneumococcal infections.
AFRIKAANS : Streptococcus pneumoniae is �n algemene menslike patogeen, wat ernstige en soms lewensbedreigende lugweg infeksies veroorsaak. Alhoewel die meeste pasi�nte geskikte antimikrobiese chemoterapie ontvang, sterf �n betekenisvolle hoeveelheid steeds. Sigaretrook is �n welbekende risikofaktor vir ernstige pneumokokkale pneumonie; die mikrobiologiese/ immunologiese meganisme wat rokers vatbaar maak vir infeksie word egter nog nie heeltemal verstaan nie. Die pneumokokkale toksien, pneumolisien, is �n belangrike virulensie faktor van S. pneumoniae; dit vorm porie� in die eukariotiese selmembrane wat �n invloei van ekstrasellul�re Ca2+ tot gevolg het. Biofilm is �n self-genererende polimeermatriks, wat deur mikrobiese patogene gebruik word om hulself teen gasheerverdedigings meganismes en antibiotika te isoleer. In hierdie verhandeling word die uitwerking van sigaretrook kondensaat (SRK) op die bioaktiwiteit van pneumolisien, asook die effekte op groei en die produksie van biofilm deur S. pneumoniae, beskryf.
�n Kliniese isolaat van S. pneumoniae, stam 172, is gebruik vir beide groei en biofilm bepalings, in die teenwoordigheid en afwesigheid van SRK (20-160 μg/ml), wat verteenwoordigend van die rook gewoonte is. Groei en biofilm bepalings is uitgevoer deur gebruik te maak van spektrofotometriese prosedures, lewensvatbaarheid, deur standard kolonievormende eenheid prosedures, en die effek van die kondensaat op die pneumolisien bioaktiwiteit, deur fura-2/AM-gebaseerde spektrofluorometriese prosedures. Blootstelling van die pneumokokkus aan SRK het in �n dosis-verwante verhoging in biofilm-formasie tot gevolg gehad, wat statistiese betekenisvolheid (P≤0.05) bereik het teen konsentrasies van 80 en 160 μg/ml, teenoor die agtergrond van beskeie effekte op bakteri�le groei. Hierdie bevindinge is nie uniek aan S. pneumoniae nie, aangesien blootstelling van Staphylococcus aureus, �n bekende biofilm-vormer, aan SRK soortgelyke resultate gelewer het. Blootstelling van pneumolisien aan SRK (20-160 μg/ml) was geassosieer met die verswakking van die biologiese aktiwiteit van die toksien, met verminderde porie-vormende vermo�, wat gemanifesteer het met �n ongelooflike verlaging in die invloei van ekstrasellul�re Ca2+ na die blootstelling van ge�soleerde neutrofiele aan die toksien.
Dit is moontlik dat die SRK as �n stressor vir die bakterie� optree, en daardeur biofilm formasie en gevolglik hardnekkige infeksies in die lugweg tot sellul�re gasheervededing be�nvloed. Hierdie effekte op die toksien mag gekomplimenteer word deur die inaktivering van pneumolisien, heelwaarskynlik deur pro-oksidatiewe meganismes, wat die vatbaarheid van rokers om ernstige pneumokokkale infeksies te ontwikkel, beklemtoon.
Mutepe, ND 2011, Effects of cigarette smoke condensate on the growth of and production of biofilm by Streptococcus pneumoniae and on the bioactivity of pneumolysin, MSc dissertation, University of Pretoria, Pretoria, viewed yymmdd < http://upetd.up.ac.za/thesis/available/etd-08022012-131316