Paired methods to measure biofilm killing and removal: a case study with Penicillin G treatment of Staphylococcus aureus biofilm

Abstract

Biofilms are microbial aggregates that show high tolerance to antibiotic treatments <i>in vitro</i> and <i>in vivo</i>. Killing and removal are both important in biofilm control, therefore methods that measure these two mechanisms were evaluated in a parallel experimental design. Kill was measured using the single tube method (ASTM method E2871) and removal was determined by video microscopy and image analysis using a new treatment flow cell. The advantage of the parallel test design is that both methods used biofilm covered coupons harvested from a CDC biofilm reactor, a well‐established and standardized biofilm growth method. The control <i>Staphylococcus aureus</i> biofilms treated with growth medium increased by 0·6 logs during a 3‐h contact time. Efficacy testing showed biofilms exposed to 400 μmol l⁻¹ penicillin G decreased by only 0·3 logs. Interestingly, time‐lapse confocal scanning laser microscopy revealed that penicillin G treatment dispersed the biofilm despite being an ineffective killing agent. In addition, no biofilm removal was detected when assays were performed in 96‐well plates. These results illustrate that biofilm behaviour and impact of treatments can vary substantially when assayed by different methods. Measuring both killing and removal with well‐characterized methods will be crucial for the discovery of new anti‐biofilm strategies.