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A simple colourimetric method for antimicrobial susceptibility testing of bacteria in biofilms

Abstract number: P584

Objective: Bacteria in biofilms are protected from antibiotics. For some antibiotics, the concentration required to kill biofilm cells may be greater than a thousand times that required to kill planktonic cells of exactly the same strain. Therefore, standard antimicrobial susceptibility test has limited relevance for determining antimicrobial susceptibility of bacteria in biofilms. We here developed a simple colorimetric method for antimicrobial susceptibility testing of bacteria in biofilms using trimethyl tetrazolium chloride (TTC) as an indicator of viable bacteria in biofilms.

Method: Biofilms were formed on 96-well polystyrene microtiter plate for 1 day, treated with antibiotics for additional 1 day and reacted with 0.02% TTC for 1 hr. The optical density at 540nm (OD540) was measured on a microtiter plate colorimeter. The OD540 value of biofilms after treatment of antibiotics was compared with that of biofilms before treatment of antibiotics. Results were expressed as reduction (%) of OD540 value in respect to controls. Using this new method, biofilms formed by Staphylococcus aureus, Escherichia coli and Klebsiella pneumonia were tested for their susceptibility to several antibiotics. Minimum inhibitory concentration (MIC) was determined by the standardized CLSI method.

Results: Reduction of TTC by viable bacteria produced red formazan that could be measured quantitatively by colorimetric absorbance at 540 nm. The minimum biofilm inhibitory concentration (MBIC) was determined as the concentration at which the % of OD540 value is 100%. Minimum biofilm eradication concentration (MBEC) was assessed such as MBEC50 and MBEC90, which was termed as the concentration of drugs that kill 50% and 90% of bacteria in pre-formed biofilms, respectively. MBEC50 and MBEC90 were determined as the concentration at which the % of OD540 value is 50% and 10%, respectively. The results were summarized in Table 1. Estimation of the efficacy of antibiotics for bacterial biofilms was enabled by comparing MBIC, MBEC50 and MBEC90 of drugs. In addition, prominent differences in susceptibility for drugs between biofilm cells and their planktonic counterparts of the same strain (MBIC versus MIC) were observed.

Conclusion: We developed a new simple colorimetric antimicrobial susceptibility testing of bacteria in biofilms using TTC as a viable cell indicator. This method may be useful to screen the effectiveness of antibiotics or biocides at eradicating bacterial biofilms.

  Ampicillin/SulbactamCefotaximeAmikacinCiprofloxacin
E. coli ATCC 25922MIC
MBIC
MBEC50
MBEC90
4
4
16
>512
0.5
0.5
8
>512
0.5
1
8
>512
0.0078
0.0078
1
>8
K. pneumoniae B170MIC
MBIC
MBEC50
MBEC90
2
2
64
256
0.5
0.5
16
128
0.5
0.5
2
>512
0.0625
0.0156
1
>8
K. pneumoniae 172MIC
MBIC
MBEC50
MBEC90
16
64
128
256
0.5
16
64
>512
0.5
8
>512
>512
0.125
0.25
>8
>8
  MethicillinVancomycinTeicoplaninAzithromycin
S. aureus ATCC 25913MIC
MBIC
MBEC50
MBEC90
4
0.5
8
>512
1
4
8
>512
1
0.5
8
>256
2
0.5
>512
>512
S. aureus WS-3MIC
MBIC
MBEC50
MBEC90
64
4
>512
>512
2
4
8
>512
2
0.25
8
>256
4
1
>512
>512
S. aureus WS-8MIC
MBIC
MBEC50
MBEC90
8
2
>512
>512
1
4
16
>512
2
1
16
>256
4
2
>512
>512
S. aureus WS-22MIC
MBIC
MBEC50
MBEC90
8
0.5
4
>512
2
1
8
>512
1
0.5
8
>256
4
0.5
32
>512

Session Details

Date: 10/04/2010
Time: 00:00-00:00
Session name: Abstracts 20th European Congress of Clinical Microbiology and Infectious Diseases
Subject:
Location: Vienna, Austria, 10 - 13 April 2010
Presentation type:
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