Formation of Pseudomonas aeruginosa inhibition zone during tobramycin disk diffusion is due to 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

doi:10.1016/j.ijantimicag.2018.12.015

Formation of Pseudomonas aeruginosa inhibition zone during tobramycin disk diffusion is due to 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

12 Citations (Scopus)

Abstract

Pseudomonas aeruginosa PAO1 (tobramycin MIC = 0.064 µg/mL) was used to perform agar diffusion tests employing tobramycin-containing tablets. Bacterial growth and 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. Diffusion of tobramycin from the deposit was modelled using a 3D geometric version of Fick's second law of diffusion. The time-dependent gradual increase in the minimum biofilm eradication concentration (MBEC) was studied using a Calgary Biofilm Device. The early inhibition zone was visible after 5 h of incubation. The corresponding calculated tobramycin concentration at the border was 1.9 µg/mL, which increased to 3.2 µg/mL and 6.3 µg/mL after 7 h and 24 h, respectively. The inhibition zone increased to the stable final zone after 7 h of incubation. Bacterial growth and small aggregate formation (young biofilms) took place inside the inhibition zone until the small aggregates contained less than ca. 64 cells and production of polysaccharide matrix including PSL had begun; thereafter, the small bacterial aggregates were killed by tobramycin. 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. Formation of the inhibition zone during agar diffusion antimicrobial susceptibility testing is due to a switch from a planktonic to biofilm mode of growth and gives clinically important information about the increased antimicrobial tolerance of biofilms.

Original language	English
Journal	International Journal of Antimicrobial Agents
Volume	53
Issue number	5
Pages (from-to)	564-573
ISSN	0924-8579
DOIs	https://doi.org/10.1016/j.ijantimicag.2018.12.015
Publication status	Published - May 2019

Keywords

Agar diffusion
Antimicrobial susceptibility testing
Biofilm
Pseudomonas aeruginosa
Tobramycin

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Høiby, N., Henneberg, K. Å., Wang, H., Stavnsbjerg, C., Bjarnsholt, T., Ciofu, O., Johansen, U. R., & Sams, T. (2019). Formation of Pseudomonas aeruginosa inhibition zone during tobramycin disk diffusion is due to transition from planktonic to biofilm mode of growth. International Journal of Antimicrobial Agents, 53(5), 564-573. https://doi.org/10.1016/j.ijantimicag.2018.12.015

Formation of Pseudomonas aeruginosa inhibition zone during tobramycin disk diffusion is due to transition from planktonic to biofilm mode of growth. / Høiby, Niels; Henneberg, Kaj Åge; Wang, Hengshuang et al.
In: International Journal of Antimicrobial Agents, Vol. 53, No. 5, 05.2019, p. 564-573.

Research output: Contribution to journal › Journal article › Research › peer-review

Høiby, N, Henneberg, KÅ, Wang, H, Stavnsbjerg, C , Bjarnsholt, T , Ciofu, O, Johansen, UR & Sams, T 2019, 'Formation of Pseudomonas aeruginosa inhibition zone during tobramycin disk diffusion is due to transition from planktonic to biofilm mode of growth', International Journal of Antimicrobial Agents, vol. 53, no. 5, pp. 564-573. https://doi.org/10.1016/j.ijantimicag.2018.12.015

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title = "Formation of Pseudomonas aeruginosa inhibition zone during tobramycin disk diffusion is due to transition from planktonic to biofilm mode of growth",

abstract = "Pseudomonas aeruginosa PAO1 (tobramycin MIC = 0.064 µg/mL) was used to perform agar diffusion tests employing tobramycin-containing tablets. Bacterial growth and 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. Diffusion of tobramycin from the deposit was modelled using a 3D geometric version of Fick's second law of diffusion. The time-dependent gradual increase in the minimum biofilm eradication concentration (MBEC) was studied using a Calgary Biofilm Device. The early inhibition zone was visible after 5 h of incubation. The corresponding calculated tobramycin concentration at the border was 1.9 µg/mL, which increased to 3.2 µg/mL and 6.3 µg/mL after 7 h and 24 h, respectively. The inhibition zone increased to the stable final zone after 7 h of incubation. Bacterial growth and small aggregate formation (young biofilms) took place inside the inhibition zone until the small aggregates contained less than ca. 64 cells and production of polysaccharide matrix including PSL had begun; thereafter, the small bacterial aggregates were killed by tobramycin. 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. Formation of the inhibition zone during agar diffusion antimicrobial susceptibility testing is due to a switch from a planktonic to biofilm mode of growth and gives clinically important information about the increased antimicrobial tolerance of biofilms.",

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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 - 2019/5

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N2 - Pseudomonas aeruginosa PAO1 (tobramycin MIC = 0.064 µg/mL) was used to perform agar diffusion tests employing tobramycin-containing tablets. Bacterial growth and 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. Diffusion of tobramycin from the deposit was modelled using a 3D geometric version of Fick's second law of diffusion. The time-dependent gradual increase in the minimum biofilm eradication concentration (MBEC) was studied using a Calgary Biofilm Device. The early inhibition zone was visible after 5 h of incubation. The corresponding calculated tobramycin concentration at the border was 1.9 µg/mL, which increased to 3.2 µg/mL and 6.3 µg/mL after 7 h and 24 h, respectively. The inhibition zone increased to the stable final zone after 7 h of incubation. Bacterial growth and small aggregate formation (young biofilms) took place inside the inhibition zone until the small aggregates contained less than ca. 64 cells and production of polysaccharide matrix including PSL had begun; thereafter, the small bacterial aggregates were killed by tobramycin. 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. Formation of the inhibition zone during agar diffusion antimicrobial susceptibility testing is due to a switch from a planktonic to biofilm mode of growth and gives clinically important information about the increased antimicrobial tolerance of biofilms.

AB - Pseudomonas aeruginosa PAO1 (tobramycin MIC = 0.064 µg/mL) was used to perform agar diffusion tests employing tobramycin-containing tablets. Bacterial growth and 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. Diffusion of tobramycin from the deposit was modelled using a 3D geometric version of Fick's second law of diffusion. The time-dependent gradual increase in the minimum biofilm eradication concentration (MBEC) was studied using a Calgary Biofilm Device. The early inhibition zone was visible after 5 h of incubation. The corresponding calculated tobramycin concentration at the border was 1.9 µg/mL, which increased to 3.2 µg/mL and 6.3 µg/mL after 7 h and 24 h, respectively. The inhibition zone increased to the stable final zone after 7 h of incubation. Bacterial growth and small aggregate formation (young biofilms) took place inside the inhibition zone until the small aggregates contained less than ca. 64 cells and production of polysaccharide matrix including PSL had begun; thereafter, the small bacterial aggregates were killed by tobramycin. 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. Formation of the inhibition zone during agar diffusion antimicrobial susceptibility testing is due to a switch from a planktonic to biofilm mode of growth and gives clinically important information about the increased antimicrobial tolerance of biofilms.

KW - Agar diffusion

KW - Antimicrobial susceptibility testing

KW - Biofilm

KW - Pseudomonas aeruginosa

KW - Tobramycin

U2 - 10.1016/j.ijantimicag.2018.12.015

DO - 10.1016/j.ijantimicag.2018.12.015

M3 - Journal article

C2 - 30615928

AN - SCOPUS:85063209164

SN - 0924-8579

VL - 53

SP - 564

EP - 573

JO - International Journal of Antimicrobial Agents

JF - International Journal of Antimicrobial Agents

IS - 5

ER -

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

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Keywords

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