Differential response of sessile and planktonic bacterial populations following exposure to antimicrobial treatment

Bester, Elanna (2004-04)

Thesis (MSc)--University of Stellenbosch, 2004.

Thesis

ENGLISH ABSTRACT: The ability of biofilms to resist antimicrobial treatment, when planktonic microbes cannot, is of not only fundamental scientific interest, but also a concern in industrial and medical fields. The inability to control biofouling of water distribution networks and products, as well as recurrent infections of implanted medical devices, is not only costly, but also potentially lethal. Several mechanisms whereby biofilms are able to evade antibiotic and biocidal agents have been proposed and investigated, but no universally relevant characteristic has been identified. . Initial investigation, involving BacLightTh ! LIVEIDEAD viability probes, epifluorescence microscopy and image analysis into the ability of natural biofilm and planktonic populations, .cultured in situ in a cooling tower, to survive treatment with a commercial biocide was not conclusive. Subsequent laboratory experimentation with a bacterial isolate from the cooling tower water revealed that the ability of attached biofilms to resist antimicrobial treatment exceeded that of planktonic cells shed from the biofilm. The reduced ability of suspended cells to survive antimicrobial treatment was not statistically significant, compared to that of the biofilm (P = 0.05). This is in contrast to the wealth of literature published on the subject of biofilm antimicrobial resistance The dilution rate in the flowcells in which biofilms were cultivated was more than 100 times higher than the maximum specific growth rate of the test organism. Nevertheless, there was typically more than I x 108 cells/ml in the effluent, suggesting that a metabolically active, rapidly dividing layer of cells existed at the biofilm bulk-liquid interface, from where daughter cells continuously detached. Treatment with an antimicrobial agent resulted in a significant reduction in the viability and number of cells detached from the biofilm, suggesting that this metabolically active layer of the biofilm was more sensitive to antimicrobial treatment, possibly due to a higher specific growth rate. Antimicrobial resistance was shown to be affected by the growth rate for planktonic bacterial populations, with an increased ability to survive, correlated with a decrease in specific growth rate. This supports the contention that growth rate plays a role in the susceptibility of the active layer. The bacterial cells in the layers closest to the attachment surface of the biofilm has frequently been shown to be slow growing, due to nutrient and oxygen limitation, while the outer biofilm layer is more susceptible to unfavourable environmental conditions. It is possible that such differentiation, which results in a responsive outer biofilm layer, provides a mechanism for the protection of the cells in the deeper layers, and thus survival over time. The results presented here support several hypotheses put forth in literature to account for the increased resistance of biofilms towards antimicrobial agents. Future work will include an investigation into changes in the patterns of gene expression when a bacteria becomes attached to a surface, upon subsequent release from the biofilm, and the influence this has on the ability to resist antimicrobial treatment.

AFRIKAANSE OPSOMMING: Die vermoë van aangehegte mikrobes, in teenstelling met vrydrywende mikroorganismes, om behandeling met antimikrobiese middels te oorleef, is nie net van belang vanuit 'n fundamenteel wetenskaplike oogpunt nie, maar ook betekenisvol vir die industriële en mediese velde. Die beheer van bio-bevuiling van waterverspreidingsnetwerke en produkte, sowel as herhaalde infeksies van mediese inplantings, is nie net van kostebelang nie, maar ook potensieël lewensgevaarlik. Verskeie meganismes wat biofilms in staat stelom antimikrobiese behandeling te oorleef, IS voorgestel en ondersoek, maar geen alomteenwoordige eienskap is tot dusver geïdentifiseer nie. Aanvanklike ondersoeke na die vermoë van natuurlike biofilms en planktoniese 'gemeenskappe, om biosiedbehandeling in situ in 'n lugversorgingskoeltoring se water te oorleef, was onbeslis. Die eksperimentele metodes het gebruik gemaak van BacLight™ LIVE/DEAD lewensvatbaarheidkleurstof, epifluoressensie-mikroskopie en beeldanalise. Daaropvolgende ondersoeke met 'n bakteriese isolaat vanuit die koeltoring het daarop gedui dat biofilms beter in staat is om antimikrobiese behandeling te oorleef as selle wat vrygelaat word vanuit die biofilm. Die afname in the lewensvatbaarheid van vrydrywende selle, na afloop van biosiedbehandeling, was nie statisties beduidend in vergelyking met die van die biofilm nie (P = 0.05). Die bevinding is in teenstelling met wat algemeen aanvaar word in die literatuur. Die verdunningstempo waaronder die biofilms in die vloeiselle gekweek is, was meer as 100- voudig hoër as die maksimum spesifieke groeitempo van die toetsorganisme. Ten spyte hiervan was daar tipies meer as 1 x 108 selle/ml in die uitvloeisel teenwoordig. Dit dui op 'n metabolies aktiewe, vinnig verdelende laag selle in die boonste laag van die biofilm, naaste aan die vloeistof fase, waarvandaan dogterselle voortdurend vrygestel word. Behandeling met die antimikrobiese agent het 'n beduidende afname in die lewensvatbaarheid en aantal dogterselle tot gevolg gehad, wat lei tot die gevolgtrekking dat die metabolies aktiewe laag van die biofilm meer sensitief is vir antimikrobiese behandeling, moontlik weens 'n hoër spesifieke groeitempo. Daar is verder bewys dat die vermoë om die werking van die antimikrobiese middel teen te staan, afhanklik is van die spesifieke groeitempo van planktoniese populasies. 'n Afname in groeitempo word geassosieer met 'n toename in oorlewing na antimikrobiese behandeling, wat die voorstel dat die groeitempo van die aktiewe laag 'n rol speel in die vatbaarheid daarvan, ondersteun. Dit is bekend dat die metaboliese aktiwiteit van bakteriese selle nader aan die aanhegtingsoppervlak van die biofilm verlaag is, weens 'n afname in diffusie van suurstof en nutriente in daardie deel van die biofilm. Dit is moontlik dat hierdie differensiasie, wat lei tot die vatbaarheid van die buitenste laag van die biofilm vir ongunstige omgewingstoestande, 'n oorlewingsmeganisme daarstel wat die onderliggende selle beskerm. Die resultate wat hier voorgelê word, ondersteun verskeie hipoteses wat die verhoogde weerstandbiedendheid van biofilms teen antimikrobiese middels beskryf. Toekomstige werk sluit ondersoeke in na veranderende patrone van geenuitdrukking wat plaasvind wanneer 'n bakterie in aanraking kom met 'n oppervlak, vasheg en ook weer vrygestel word, asook die invloed hiervan op die vermoë om antimikrobiese behandeling te oorleef.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/49931
This item appears in the following collections: