Biocide susceptibility testing of bacteria: Development of a broth microdilution method

Angela R. Schug, Alexander Bartel, Anissa D. Scholtzek, Marita Meurer, Julian Brombach, Vivian Hensel, Séamus Fanning, Stefan Schwarz, Andrea T. Feßler*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

9 Citations (Scopus)

Abstract

Biocide susceptibility testing (BST) of bacteria lacks standardised methods. Based on a recently established broth macrodilution BST method, a broth microdilution method for BST was developed. To establish the respective protocol, four reference strains Staphylococcus aureus ATCC® 6538, Enterococcus hirae ATCC® 10541, Escherichia coli ATCC® 10536 and Pseudomonas aeruginosa ATCC® 15442 were investigated for their minimal inhibitory concentrations (MICs) towards quaternary ammonium compounds (benzalkonium chloride), cationic compounds (chlorhexidine), aldehydes (glutardialdehyde) and alcohols (isopropanol) using tryptic soy broth. All combinations of (i) inoculum preparation according to the German Veterinary Medical Society (DVG) or the Clinical and Laboratory Standards Institute (CLSI) with some modifications, (ii) use of 1st subculture (SC) and 2nd SC, (iii) direct colony suspension (DCS) with/without glass beads, and (iv) incubation at 37 °C for 24 h, 48 h, and 72 h were compared using seven independent replications. Overall, the reproducibility was high for all abovementioned strain/biocide/parameter combinations. In total, 86.9 % – 100 % of the results were within ± one dilution step of the mode value. The proposed method for a standardised BST protocol comprises (i) two different inoculum densities, (ii) the use of a fresh overnight culture (1st SC or 2nd SC), (iii) the preparation of the inoculum suspension by either of the two methods using DCS with or without glass beads, and (iv) the incubation at 37 °C for 24 h. This broth microdilution method will help to harmonize BST of bacterial pathogens in routine diagnostics.

Original languageEnglish
Article number108791
JournalVeterinary Microbiology
Volume248
Early online date10 Aug 2020
DOIs
Publication statusPublished - Sept 2020
Externally publishedYes

Bibliographical note

Funding Information:
This work was funded by the German Federal Ministry of Education and Research (BMBF) under project number 01KI1727D as part of the Research Network Zoonotic Infectious Diseases. Angela R. Schug was supported by a scholarship of the Dres. Bruns-Stiftung.

Funding Information:
This work was funded by the German Federal Ministry of Education and Research (BMBF) under project number 01KI1727D as part of the Research Network Zoonotic Infectious Diseases. Angela R. Schug was supported by a scholarship of the Dres. Bruns-Stiftung.

Publisher Copyright:
© 2020 Elsevier B.V.

Keywords

  • Benzalkonium chloride
  • Chlorhexidine
  • Enterococcus hirae
  • Escherichia coli
  • Glutardialdehyde
  • Isopropanol
  • Minimum inhibitory concentration (MIC)
  • Pseudomonas aeruginosa
  • Staphylococcus aureus

ASJC Scopus subject areas

  • Microbiology
  • veterinary(all)

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