Center for Biofilm Engineering
Abstract:
"Role of Antibiotic Penetration Limitation in Klebsiella pneumoniae
Biofilm Resistance to Ampicillin and Ciprofloxacin"
00-020 The penetration of two antibiotics—ampicillin and
ciprofloxacin—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. The susceptibilities of planktonic
cultures and biofilms to 10 times the MIC were determined. 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. 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. Biofilms of the same strain were much less susceptible,
yielding log reductions in the number of CFU of – 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.
Anderl, J.N., M.J. Franklin, and P.S. Stewart, "Role of Antibiotic
Penetration Limitation in Klebsiella pneumoniae Biofilm Resistance to
Ampicillin and Ciprofloxacin," Antimicrobiol. Agents Chemother.,
44(7):1818-1824 (2000).
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