Automated spiral plater

To determine AZT frequency of mutation, ST131c, ST131 pCTgfp, and ST131 mcherry were plated onto LB agar plates containing 16 μg/ml AZT and LB agar plates containing no drug. Plates were incubated for 24 h at 37°C, and the colonies were enumerated. The Don Whitley Scientific automated spiral plater was used, per the manufacturer’s instructions. For each strain, 4 biological and 5 technical replicates were used. Mutation frequency was calculated by dividing the number of mutants by the viable count. From the AZT plates, mutant colonies were selected, and the MIC of AZT was determined as indicated above. Candidates with an AZT MIC of >32 μg/ml were selected, and the thymidine kinase gene was sequenced to determine if mutations had occurred (primers listed in Table S3). Mutants with deletions in thymidine kinase were then used in the AZT flow cytometry assay.

Enumeration of L. monocytogenes from fresh brine samples was carried out by plating appropriate dilutions on BHI agar (Oxoid Ltd., Basingstoke, UK) supplemented with rifampicin (200 µg/mL), incubating for four days at 30 °C. For determination of L. monocytogenes on fish samples, pieces of 10 g were added to 90 mL of peptone water (0.1% peptone and 0.02% Tween-80) and homogenized for 2 min in a stomacher (AESAP1064 Smasher, Cheminex, Combourg, France). Ten milliliters of the homogenate was concentrated by centrifugation at 12,000× g for 5 min. The supernatant containing phage P100 was removed, and the pellets containing L. monocytogenes cells were resuspended in 1 mL of peptone water before plating on BHI agar. Total anaerobic bacterial counts were determined by plating brine samples from one of the biological replicates collected on days zero, seven, 14, 28, 42, and 91 on plate count agar (PCA; Oxoid Ltd., Basingstoke, UK). For logistic reasons, this analysis used frozen brine samples, and all samples were processed at the same time. This procedure was previously shown not to affect the number of bacteria in rakfisk brine [13 (link)]. The plates were incubated anaerobically (AnaeroGen Atmosphere Generation System, Oxoid Ltd., Basingstoke, UK) for three days at 20 °C. All plating was done using an automated spiral plater (Don Whitley Scientific Limited, Shipley, UK).