Characterization of bacterial populations within three-dimensional biofilm environments

Stephanie Ann Sincock, Purdue University

Abstract

Biofilms in medical devices such as catheters are a source of recurring infections in chronically ill patients. Eradication of microorganisms within biofilms is very difficult using standard antimicrobial treatments. Examination of bacteria growing as biofilms revealed that these matrix-enclosed populations can survive exposure to antimicrobial agents at higher concentrations than can their planktonic counterparts. In order to study this process, hydrogel-biofilm constructs were employed. Using a thermoreversible hydrogel, we have shown that it is possible to recover individual biofilm cells and visualize the spatial and physiological heterogeneity within biofilm communities. Klebsiella pneumoniae and Escherichia coli cells seeded within the hydrogel formed dense biofilms characterized by an exopolysaccharide matrix, complex structural features (i.e., microcolonies, channels), and decreased antibiotic susceptibility. Quantitative comparison of biofilm structures revealed little variation between bacterial strains. Over time (24 to 48 hours), mean biomass and thickness of biofilms increased significantly; however, the mean roughness, an indicator of biofilm structural heterogeneity, deceased significantly. Multiple equivalent biofilms were prepared and exposed to imipenem, ceftazidime, or gentamicin. Plate-count data showed that exposure to these antibiotics had little effect on established biofilms, whereas planktonic control cultures were eradicated. Transport limitation was not a significant factor in reduced biofilm susceptibility. Imipenem and ceftazidime easily penetrated through the hydrogel-biofilm constructs and retained sufficient activity to inhibit growth of susceptible organisms; gentamicin penetration was slowed but not halted. None of the antibiotics studied was capable of completely eradicating biofilms, but all caused significant damage at high concentrations. At the single-cell level, subpopulations of viable, injured, and dead cells were identified and enumerated based on membrane permeability and differential uptake of nucleic-acid dyes. Results showed a dose-dependent decrease in viable cells and an increase in injured and dead cells. At the community level, in situ viability staining in conjunction with confocal microscopy was used to visualize and quantify the physiological responses within intact, hydrated biofilms. Biofilms treated with oxygen-dependent or growth-rate dependent antibiotics demonstrated that cell location plays a major role in biofilm resistance to killing. Patterns of antibiotic-induced damage within microcolonies reflected the physiological heterogeneity of biofilms.

Degree

Ph.D.

Advisors

Robinson, Purdue University.

Subject Area

Microbiology

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