Browsing by Author "Anderl, Jeff N."

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Role of antibiotic penetration limitation in Klebsiella pneumoniae biofilm resistance to ampicillin and ciprofloxacin
(2000) Anderl, Jeff N.; Franklin, Michael J.; Stewart, Philip S.
The penetration of two antibioticsâ€”ampicillin and ciprofloxacinâ€”through biofilms developed in an in vitro model system was investigated. The susceptibilities of biofilms and corresponding freely suspended bacteria, to killing by the antibiotics were also measured. Biofilms of Klebsiella pneumoniae were developed on microporous membranes resting on agar nutrient medium.Â The susceptibilities of planktonic cultures and biofilms to 10 times the MIC were determined.Â Antibiotic penetration through biofilms was measured by assaying the concentration of antibiotic that diffused through the biofilm to an overlying filter disk. Parallel experiments were performed with a mutant K. pneumoniae strain in which B-lactamase activity was eliminated.Â For wild-type K. pneumoniae grown in suspension culture, ampicillin and ciprofloxacin MICs were 500 and 0.18 ug/ml, respectively. The log reductions in the number of CFU of planktonic wild-type bacteria after 4 h of treatment at 10 times the MIC were 4.43 + 0.33 and 4.14 + 0.33 for ampicillin and ciprofloxacin, respectively.Â Biofilms of the same strain were much less susceptible, yielding log reductions in the number of CFU of â€“ 0.06 + 0.06 and 1.02 + 0.04 for ampicillin and ciprofloxacin, respectively, for the same treatment. The number of CFU in the biofilms after 24 h of antibiotic exposure was not statistically different from the number after 4 h of treatment. Ampicillin did not penetrate wild-type K. pneumoniae biofilms, whereas ciprofloxacin and a nonreactive tracer (chloride ion) penetrated the biofilms quickly. The concentration of ciprofloxacin reached the MIC throughout the biofilm within 20 min. Ampicillin penetrated biofilms formed by a B-lactamase-deficient mutant. However, the biofilms formed by this mutant were resistant to ampicillin treatment, exhibiting a 0.18 + 0.07 log reduction in the number of CFU after 4 h of exposure and a 1.64 + 0.33 log reduction in the number of CFU after 24 h of exposure. Poor penetration contributed to wild-type biofilm resistance to ampicillin but not to ciprofloxacin. The increased resistance of the wild-type strain to ciprofloxacin and the mutant strain to ampicillin and ciprofloxacin could not be accounted for by antibiotic inactivation or slow diffusion since these antibiotics fully penetrated the biofilms. These results suggest that some other resistance mechanism is involved for both agents.
Role of nutrient limitation and stationary-phase existence in klebsiella pneumoniae biofilm resistance to ampicillin and ciprofloxacin
(2003-04) Anderl, Jeff N.; Zahller, Jeff; Roe, Frank L.; Stewart, Philip S.
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 μm. 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.