Role of nutrient limitation and stationary-phase existence in Klebsiella pneumoniae biofilm resistance to ampicillin and ciprofloxacin

Abstract

Biofilms formed by Klebsiella pneumoniae resisted killing during prolonged exposure to ampicillin or ciprofloxacin even though these agents have been shown to penetrate bacterial aggregates. Bacteria dispersed from biofilms into medium quickly regained most of their susceptibility. Experiments with free-floating bacteria showed that stationary-phase bacteria were protected from killing by either antibiotic, especially when the test was performed in medium lacking carbon and nitrogen sources. These results suggested that the antibiotic tolerance of biofilm bacteria could be explained by nutrient limitation in the biofilm leading to stationary-phase existence of at least some of the cells in the biofilm. This mechanism was supported by experimental characterization of nutrient availability and growth status in biofilms. The average specific growth rate of bacteria in biofilms was only 0.032 h(-1) compared to the specific growth rate of planktonic bacteria of 0.59 h(-1) measured in the same medium. Glucose did not penetrate all the way through the biofilm, and oxygen was shown to penetrate only into the upper 100 micro m. The specific catalase activity was elevated in biofilm bacteria to a level similar to that of stationary-phase planktonic cells. Transmission electron microscopy revealed that bacteria were affected by ampicillin near the periphery of the biofilm but were not affected in the interior. Taken together, these results indicate that K. pneumoniae in this system experience nutrient limitation locally within the biofilm, leading to zones in which the bacteria enter stationary phase and are growing slowly or not at all. In these inactive regions, bacteria are less susceptible to killing by antibiotics.

FIG. 1.

Comparison of susceptibility of suspended and biofilm K. pneumoniae to 5,000 μg of ampicillin per ml (A) and 1.8 μg of ciprofloxacin per ml (B). Symbols: ○, untreated planktonic control; •, treated planktonic cells; □, untreated biofilm control; ▪, treated biofilm. Biofilms were 48 h old when tested. Error bars show standard deviations. Positive values of the log reduction represent killing, and negative values represent growth.

FIG. 2.

Susceptibility of K. pneumoniae biofilms to 5,000 μg of ampicillin per ml or 1.8 μg of ciprofloxacin per ml during prolonged exposure. Symbols: □, untreated β-lactamase-positive control; ▪, ampicillin-treated β-lactamase-positive strain; •, ciprofloxacin-treated β-lactamase-positive strain; ▴, ampicillin-treated β-lactamase-negative strain. Data from duplicate experiments are shown; error bars show standard deviations.

FIG. 3.

Typical accumulation curve of a K. pneumoniae colony biofilm.

FIG. 4.

Transmission electron microscope images of ampicillin-treated, β-lactamase-negative K. pneumoniae colony biofilm. The biofilm was treated for 12 h. Enlargement and destruction of bacterial cells are evident near both boundaries of the colony biofilm (A, air interface; C, membrane interface) but not in the most central part of the colony interior (B). Bar, 5 μm.

FIG. 5.

Penetration of glucose through K. pneumoniae biofilms. Symbols: ▪, intact biofilm; ○, control with no biofilm; □, formaldehyde-killed biofilm.

FIG. 6.

Penetration of oxygen into K. pneumoniae biofilms. Symbols: •, β-lactamase-positive biofilm; ○, β-lactamase-negative biofilm; ▪, glutaraldehyde-killed biofilm; □, 0.5% sterile agar control.