Role of antibiotic penetration limitation in Klebsiella pneumoniae biofilmresistance to ampicillin and ciprofloxacin

The penetration of two antibiotics, ampicillin and ciprofloxacin, through b iofilms developed in an in vitro model system was investigated. The suscept ibilities of biofilms and corresponding freely suspended bacteria to killin g by the antibiotics were also measured. Biofilms of Klebsiella pneumoniae were developed on microporous membranes resting on agar nutrient medium. Th e susceptibilities of planktonic cultures and biofilms to 10 times the MIC were determined, Antibiotic penetration through biofilms was measured by as saying the concentration of antibiotic that diffused through the biofilm to an overlying filter disk Parallel experiments were performed with a mutant R. pneumoniae strain in which beta-lactamase activity was eliminated. For wild-type K. pneumoniae grown in suspension culture, ampicillin and ciprofl oxacin MICs were 500 and 0.18 mu g/ml, respectively. The log reductions in the number of CFU of planktonic wild-type bacteria after 4 h of treatment a t 10 times the MIC were 4.43 +/- 0.33 and 4.14 +/- 0.33 for ampicillin and ciprofloxacin, respectively. Biofilms of the same strain were much less sus ceptible, yielding log reductions in the number of CFU of -0.06 +/- 0.06 an d 1.02 +/- 0.04 for ampicillin and ciprofloxacin, respectively, for the sam e treatment. The number of CFU in the biofilms after 24 h of antibiotic exp osure was not statistically different from the number after 4 h of treatmen t. Ampicillin did not penetrate wild-type K. pneumoniae biofilms, whereas c iprofloxacin and a nonreactive tracer (chloride ion) penetrated the biofilm s quickly. The concentration of ciprofloxacin reached the MIC throughout th e biofilm within 20 min. Ampicillin penetrated biofilms formed by a beta-la ctamase-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 3 h of exposure and a 1.64 +/- 0.33 log reducti on in the number of CFU after 24 h of exposure. Poor penetration contribute d to wild-type biofilm resistance to ampicillin but not to ciprofloxacin. T he increased resistance of the wild-type strain to ciprofloxacin and the mu tant strain to ampicillin and ciprofloxacin could not be accounted for by a ntibiotic inactivation or slow diffusion since these antibiotics fully pene trated the biofilms. These results suggest that some other resistance mecha nism is involved for both agents.