Using a Sequential Regimen to Eliminate Bacteria at Sub-Lethal Antibiotic Dosages

Escherichia coli K12 (AG100) was targeted with two antibiotics, erythromycin (a macrolide, ERY) and doxycycline (a tetracycline, DOX) that bind to different ribosomal RNA subunits, thereby inhibiting translation. Two treatment of 8 seasons of 12 hours at dosages corresponding to the IC50 of each drug were sequenced; IC50 denotes the antibiotic concentration that reduces bacterial density by a factor of 50% relative to that produced without antibiotic in one season. We studied one 50-50 combination using a half-dose of both drug for each season, and the EDEDEDED treatment, writing 'E' for a season of ERY and D for DOX. Library preparation method: DNA was fragmented by sonication using a Biorupter for 30s on, 90s off, using low power for 10 minutes on ice. Libraries were prepared using SPRIworks cartridges for Illumina (Beckman Coulter) and Nextflex indexed adapters, with 300-600 bp size selection, amplified with 8 cycles PCR using Kapa HiFi DNA polymerase and purified using GeneRead kit (Qiagen). Concentrations were determined using a Bioanalyser 7500 DNA chip. Libraries were pooled in equimolar amounts, denatured, diluted to 6.5 pMol and clustered on a flowcell using a cBot (Illumina). 100 paired end sequencing with a custom barcode read was completed on a HiSeq 2500 using Truseq SBS v3 reagents Illumina).