Antimicrobial Tolerance in Biofilms

Abstract

The tolerance of microorganisms in biofilms to antimicrobial agents is examined through a meta-analysis of literature data. A numerical tolerance factor comparing the rates of killing in the planktonic and biofilm states is defined to provide a quantitative basis for the analysis. Tolerance factors for biocides and antibiotics range over three orders of magnitude. This variation is not explained by taking into account the molecular weight of the agent, the chemistry of the agent, the substratum material, or the speciation of the microorganisms. Tolerance factors do depend on the areal cell density of the biofilm at the time of treatment and on the age of the biofilm as grown in a particular experimental system. This suggests that there is something that happens during biofilm maturation, either physical or physiological, that is essential for full biofilm tolerance. Experimental measurements of antimicrobial penetration times in biofilms range over orders of magnitude, with slower penetration (>12 min) observed for reactive oxidants and cationic molecules. These agents are retarded through the interaction of reaction, sorption, and diffusion. The specific physiological status of microbial cells in a biofilm contributes to antimicrobial tolerance. A conceptual framework for categorizing physiological cell states is discussed in the context of antimicrobial susceptibility. It is likely that biofilms harbor cells in multiple states simultaneously (e.g., growing, stress-adapted, dormant, inactive) and that this physiological heterogeneity is an important factor in the tolerance of the biofilm state.

FIGURE 1

Tolerance factors versus antimicrobial agent molecular weight for the data on (A) biocides and antiseptics from Table 1 and (B) antibiotics from Table 2. doi:10.1128/microbiolspec.MB-0010-2014.f1

FIGURE 2

Tolerance factors grouped and compared by substratum material. doi:10.1128/microbiolspec.MB-0010-2014.f2

FIGURE 3

Tolerance factor versus biofilm cell density for the data in Table 1. The line is the least squares regressed fit. doi:10.1128/microbiolspec.MB-0010-2014.f3

FIGURE 4

Efficacy of chlorine treatment against biofilms as a function of the untreated control biofilm areal cell density. The y-axis is the reported log reduction divided by the product of dose concentration and duration (C_Bt_B). The line is the least squares regressed fit. Sources: references , –. doi:10.1128/microbiolspec.MB-0010-2014.f4

FIGURE 5

Antibiotic efficacy against Pseudomonas aeruginosa biofilms as a function of the untreated control biofilm areal cell density. Dashed lines connect data points from the same investigation. The antibiotics used include tobramycin, cipro-floxacin, and gentamicin. Sources: references , –. doi:10.1128/microbiolspec.MB-0010-2014.f5

FIGURE 6

Antimicrobial efficacy as a function of biofilm age. Dashed lines connect data points from the same investigation. Sources: references , , , , –. doi:10.1128/microbiolspec.MB-0010-2014.f6

FIGURE 7

(A) Maturation of S. aureus biofilm and (B) change in gentamicin susceptibility with age. The dashed line in panel A connects the mean values at each time point. The solid line in panel B is the least squares regressed fit. Source: reference . doi:10.1128/microbiolspec.MB-0010-2014.f7

FIGURE 8

Tolerance factors for biocides and antibiotics for four bacterial phyla and a fungus. doi:10.1128/microbiolspec.MB-0010-2014.f8

FIGURE 9

Medium effects on biofilm susceptibility to antibiotics. The different bar fills denote various media: LB (gray); LB + glucose (triangles); TSA, aerobic (white); TSA, anaerobic (hatched); noble agar, aerobic (black); noble agar, anaerobic (honeycomb). Sources: reference for E. coli and unpublished data of Borriello and Stewart for P. aeruginosa. doi:10.1128/microbiolspec.MB-0010-2014.f9

FIGURE 10

Experimentally measured antimicrobial penetration times in biofilms versus molecular weight of the antimicrobial. The penetration time was determined as the time to attain, at the base or center of the biofilm, 50% of the equilibrium concentration of the antimicrobial agent either through a direct measurement of the antimicrobial agent (solid circles) or by loss of membrane integrity detected with a fluorescent probe (open circles). Penetration times greater than 12 min are circled. Sources: references , , –. doi:10.1128/microbiolspec.MB-0010-2014.f10

FIGURE 11

Experimentally measured antimicrobial penetration times in biofilms versus dose concentration. The line is the least squares regressed fit. Symbols indicate data for chlorine (cross, 55), chlorine (gray, 54), tobramycin (white, 62), peracetic acid (black, 53). doi:10.1128/microbiolspec.MB-0010-2014.f11

FIGURE 12

Comparison of antimicrobial susceptibility of exponential phase planktonic (solid symbols) or stationary phase planktonic (open symbols) to biofilm cells. The solid line is the line of equality. Points below the line indicate that biofilm cells were less susceptible than planktonic cells. Points above the line indicate that planktonic cells were less susceptible than biofilm cells. Sources: references , –. doi:10.1128/microbiolspec.MB-0010-2014.f12

FIGURE 13

Conceptual diagram of distinct cell states important for antimicrobial sensitivity. The dead cell state can presumably be accessed from any of the other states. doi:10.1128/microbiolspec.MB-0010-2014.f13