Formation of Pseudomonas aeruginosa inhibition zone during Tobramycin disk diffusion is due to a transition from planktonic to biofilm mode of growth

Niels Høiby*, Kaj-Åge Henneberg, Hengshuang Wang, Camilla Stavnsbjerg, Thomas Bjarnsholt, Oana Ciofu, Ulla Rydal Johansen, Thomas Sams

*Corresponding author for this work

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Abstract

Pseudomonas aeruginosa PAO1 (MIC 0.064µg/ml) was used to perform agar diffusion tests employing tobramycin containing tablets. The growth of the bacteria and the formation of inhibition zones were studied by stereomicroscopy and by blotting with microscope slides and staining with Methylene blue, Alcian blue and a fluorescent lectin for the P. aeruginosa PSL which was studied by confocal laser scanning microscopy. The diffusion of tobramycin from the deposit was modelled by using a 3D geometric version of Fick's 2nd law of diffusion. The time-dependent gradual increase of Minimal Biofilm Eradication Concentration (MBEC) was studied by the Calgary Biofilm Devise. The early inhibition zone was visible after 5 h incubation. The corresponding calculated tobramycin concentration at the border was 1.9µg/ml and increased to 3.2µg/ml and 6.3µg/ml after 7 and 24 h incubation. The inhibition zone increased to the stable, final zone after 7 h incubation. Bacterial growth and small aggregate formation (young biofilms) took place inside the inhibition zone until the small aggregates contained ≤≈64 cells and production of polysaccharide matrix including PSL had begun, thereafter the small bacterial aggregates were killed by tobramycin. The bacteria at the border of the stable inhibition zone and beyond continued to grow to a mature biofilm and produced large amount of polysaccharide containing matrix. The formation of the inhibition zone during the agar diffusion antibiotic susceptibility test is due to a switch from the planktonic to the biofilm mode of growth and gives clinical important information about the increased antibiotic tolerance of biofilms.

Original languageEnglish
JournalInternational Journal of Antimicrobial Agents
Volume53
Issue number5
Pages (from-to)564-573
ISSN0924-8579
DOIs
Publication statusPublished - 2019

Keywords

  • Pseudomonas aeruginosa
  • Biofilm
  • Agar diffusion
  • Antibiotic susceptibility test
  • Tobramycin

Cite this

Høiby, Niels ; Henneberg, Kaj-Åge ; Wang, Hengshuang ; Stavnsbjerg, Camilla ; Bjarnsholt, Thomas ; Ciofu, Oana ; Johansen, Ulla Rydal ; Sams, Thomas. / Formation of Pseudomonas aeruginosa inhibition zone during Tobramycin disk diffusion is due to a transition from planktonic to biofilm mode of growth. In: International Journal of Antimicrobial Agents. 2019 ; Vol. 53, No. 5. pp. 564-573.
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title = "Formation of Pseudomonas aeruginosa inhibition zone during Tobramycin disk diffusion is due to a transition from planktonic to biofilm mode of growth",
abstract = "Pseudomonas aeruginosa PAO1 (MIC 0.064µg/ml) was used to perform agar diffusion tests employing tobramycin containing tablets. The growth of the bacteria and the formation of inhibition zones were studied by stereomicroscopy and by blotting with microscope slides and staining with Methylene blue, Alcian blue and a fluorescent lectin for the P. aeruginosa PSL which was studied by confocal laser scanning microscopy. The diffusion of tobramycin from the deposit was modelled by using a 3D geometric version of Fick's 2nd law of diffusion. The time-dependent gradual increase of Minimal Biofilm Eradication Concentration (MBEC) was studied by the Calgary Biofilm Devise. The early inhibition zone was visible after 5 h incubation. The corresponding calculated tobramycin concentration at the border was 1.9µg/ml and increased to 3.2µg/ml and 6.3µg/ml after 7 and 24 h incubation. The inhibition zone increased to the stable, final zone after 7 h incubation. Bacterial growth and small aggregate formation (young biofilms) took place inside the inhibition zone until the small aggregates contained ≤≈64 cells and production of polysaccharide matrix including PSL had begun, thereafter the small bacterial aggregates were killed by tobramycin. The bacteria at the border of the stable inhibition zone and beyond continued to grow to a mature biofilm and produced large amount of polysaccharide containing matrix. The formation of the inhibition zone during the agar diffusion antibiotic susceptibility test is due to a switch from the planktonic to the biofilm mode of growth and gives clinical important information about the increased antibiotic tolerance of biofilms.",
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Formation of Pseudomonas aeruginosa inhibition zone during Tobramycin disk diffusion is due to a transition from planktonic to biofilm mode of growth. / Høiby, Niels; Henneberg, Kaj-Åge; Wang, Hengshuang; Stavnsbjerg, Camilla; Bjarnsholt, Thomas; Ciofu, Oana; Johansen, Ulla Rydal; Sams, Thomas.

In: International Journal of Antimicrobial Agents, Vol. 53, No. 5, 2019, p. 564-573.

Research output: Contribution to journalJournal articleResearchpeer-review

TY - JOUR

T1 - Formation of Pseudomonas aeruginosa inhibition zone during Tobramycin disk diffusion is due to a transition from planktonic to biofilm mode of growth

AU - Høiby, Niels

AU - Henneberg, Kaj-Åge

AU - Wang, Hengshuang

AU - Stavnsbjerg, Camilla

AU - Bjarnsholt, Thomas

AU - Ciofu, Oana

AU - Johansen, Ulla Rydal

AU - Sams, Thomas

PY - 2019

Y1 - 2019

N2 - Pseudomonas aeruginosa PAO1 (MIC 0.064µg/ml) was used to perform agar diffusion tests employing tobramycin containing tablets. The growth of the bacteria and the formation of inhibition zones were studied by stereomicroscopy and by blotting with microscope slides and staining with Methylene blue, Alcian blue and a fluorescent lectin for the P. aeruginosa PSL which was studied by confocal laser scanning microscopy. The diffusion of tobramycin from the deposit was modelled by using a 3D geometric version of Fick's 2nd law of diffusion. The time-dependent gradual increase of Minimal Biofilm Eradication Concentration (MBEC) was studied by the Calgary Biofilm Devise. The early inhibition zone was visible after 5 h incubation. The corresponding calculated tobramycin concentration at the border was 1.9µg/ml and increased to 3.2µg/ml and 6.3µg/ml after 7 and 24 h incubation. The inhibition zone increased to the stable, final zone after 7 h incubation. Bacterial growth and small aggregate formation (young biofilms) took place inside the inhibition zone until the small aggregates contained ≤≈64 cells and production of polysaccharide matrix including PSL had begun, thereafter the small bacterial aggregates were killed by tobramycin. The bacteria at the border of the stable inhibition zone and beyond continued to grow to a mature biofilm and produced large amount of polysaccharide containing matrix. The formation of the inhibition zone during the agar diffusion antibiotic susceptibility test is due to a switch from the planktonic to the biofilm mode of growth and gives clinical important information about the increased antibiotic tolerance of biofilms.

AB - Pseudomonas aeruginosa PAO1 (MIC 0.064µg/ml) was used to perform agar diffusion tests employing tobramycin containing tablets. The growth of the bacteria and the formation of inhibition zones were studied by stereomicroscopy and by blotting with microscope slides and staining with Methylene blue, Alcian blue and a fluorescent lectin for the P. aeruginosa PSL which was studied by confocal laser scanning microscopy. The diffusion of tobramycin from the deposit was modelled by using a 3D geometric version of Fick's 2nd law of diffusion. The time-dependent gradual increase of Minimal Biofilm Eradication Concentration (MBEC) was studied by the Calgary Biofilm Devise. The early inhibition zone was visible after 5 h incubation. The corresponding calculated tobramycin concentration at the border was 1.9µg/ml and increased to 3.2µg/ml and 6.3µg/ml after 7 and 24 h incubation. The inhibition zone increased to the stable, final zone after 7 h incubation. Bacterial growth and small aggregate formation (young biofilms) took place inside the inhibition zone until the small aggregates contained ≤≈64 cells and production of polysaccharide matrix including PSL had begun, thereafter the small bacterial aggregates were killed by tobramycin. The bacteria at the border of the stable inhibition zone and beyond continued to grow to a mature biofilm and produced large amount of polysaccharide containing matrix. The formation of the inhibition zone during the agar diffusion antibiotic susceptibility test is due to a switch from the planktonic to the biofilm mode of growth and gives clinical important information about the increased antibiotic tolerance of biofilms.

KW - Pseudomonas aeruginosa

KW - Biofilm

KW - Agar diffusion

KW - Antibiotic susceptibility test

KW - Tobramycin

U2 - 10.1016/j.ijantimicag.2018.12.015

DO - 10.1016/j.ijantimicag.2018.12.015

M3 - Journal article

VL - 53

SP - 564

EP - 573

JO - International Journal of Antimicrobial Agents

JF - International Journal of Antimicrobial Agents

SN - 0924-8579

IS - 5

ER -