Thesis (MSc)--University of Stellenbosch, 2004. / ENGLISH ABSTRACT: Little is known about the impact of nutrient conditions on antimicrobial resistance in biofilms grown under continuous flow conditions. Furthermore, community-level response of biofilms to antimicrobial substances and different nutrient regimes are poorly described. A better understanding of the influence of environmental conditions on biofilm behavior and antimicrobial susceptibility may contribute to the efforts, addressing the problems associated with increased antimicrobial resistance. Therefore, the aim of this study was to evaluate the survival and population dynamics in a defined mixed-species biofilm community grown under different nutrient conditions and when subjected to biocide treatment. Epi-fluorescence microscopy in conjunction with the LIVE/DEAD® BacLight™ viability kit, a conventional cultivation technique (plate counts), and culture-independent techniques (terminal restriction fragment length polymorphism and fluorescent in situ hybridization) were applied to observe biofilm and planktonic antimicrobial susceptibility, as well as population dynamics. A defined mixed-species community, consisting of four bacterial strains, was cultivated and monitored in a flow cell system. Two nutrient types were used: 1) a complex growth medium [tryptone soy broth (TSB)] and 2) a defined synthetic medium [minimal salts supplemented with glucose (MSM + Glucose)]. In addition, these two nutrient types were applied in different concentrations. Biofilm and planktonic community behavior was influenced by the nutrient type and concentration. Species evenness in the planktonic community was influenced by the nutrient conditions, while species richness changed in response to biocide treatment and nutrient conditions. TSB-grown microbial communities were more susceptible directly after biocide treatment than those grown in MSM + Glucose, however, biofilm viability in the latter nutrient condition decreased within 24 h after biocide treatment. Furthermore, a surprising difference in the recovery rate between biofilm and associated planktonic communities was observed. A conceptual model was developed that aimed to explain the observed biofilm-planktonic interactions. This model proposes that the cells found in the outer regions of a biofilm are the primary source of the associated planktonic cells, and that this phenomenon is independent from overall biofilm activity. / AFRIKAANSE OPSOMMING: Daar IS tans mm bekend oor die invloed van nutriënte op die antirnikrobiese weerstandbiedendheid van biofilms onder kontinue vloeitoestande. Verder is daar 'n groot leemte in die literatuur oor die invloed van nutriënte op die antimikrobiese sensitiwiteit van bakteriese gemeenskappe in multispesie-biofilms. 'n Beter begrip van die invloed van omgewingsfaktore op biofilmgedrag en antimikrobiese sensitiwiteit sal bydra om die probleem van verhoogde antimikrobiese weerstandbiedendheid te beheer. Die doel van hierdie studie was gevolglik om die oorlewingsvermoë, asook die gemeenskapverskuiwings binne 'n bekende biofilmgemeenskap te evalueer, nadat dit blootgestel was aan biosiedbehandeling en verskillende nutriëntkondisies. Epifluoressensie-mikroskopie in kombinasie met 'n lewensvatbaarheidskleurstof (LIVEIDEAD® BacLight™ viability kit), konventioneie kweektegnieke (spreiplaattegniek) en kweekonafhanklike identifikasietegnieke (terminale restriksie fragment lengte polimorfisme en fluoressensie in situ hibridisasie) is gebruik om biofilm- en planktoniese lewensvatbaarheid, asook gemeenskapverskuiwings te bestudeer. 'n Gedefinieerde biofilmgemeenskap, bestaande uit vier verskillende bakteriese rasse, is gekweek en gemonitor in 'n vloeiselsisteem. Twee tipes nutriënte is gebruik: (1) 'n komplekse groeimedium [Tryptone Soy Broth (TSB)] en (2) 'n chemies-gedefinieerde medium [minimale soutoplossing met glukose (MSM + Glucose)]. Beide nutriënttipes is in verskillende konsentrasies toegedien. Die gedrag van biofilm- sowel as planktoniese populasies is beïnvloed deur beide die nutriënttipe en konsentrasie. Planktoniese populasiegetalle is beïnvloed deur nutriëntkondisies, terwyl die aantal populasies beïnvloed is deur biosiedbehandeling en nutriënttoestande. Bakteriese gemeenskappe wat in komplekse medium (TSB) gekweek is, was meer sensitief teenoor die biosied direk na behandeling as die wat in MSM + Glukose gekweek is. Die lewensvatbaarheid van die biofilm wat in MSM + Glukose gekweek is, het egter eers 24 uur na biosiedbehandeling begin daal. Daar was verder 'n opvallende verskil in die tempo van herstel van die biofilmgemeenskap in vergelyking met die planktoniese gemeenskap. Die planktoniese gemeenskap het vinniger herstel as die biofilm gemeenskap. 'n Model is ontwikkel om die biofilm-planktoniese interaksies te verklaar. In die model word voorgestel dat selle wat in die buitenste lae van 'n biofilm groei, die primêre bron is van planktoniese selle in 'n vloeisisteem, en dat hierdie verskynselonafhanklik is van die algehele biofilm-aktiwiteit.

This study was undertaken in order to determine whether the extracts of common garden vegetables could be incorporated into simple, economical culture media which might be used for the growth and cultivation of at least some of the more commonly used microorganisms.