HB10 and HB11 were bactericidal to E. coli and Streptococcus salivarius at low micromolar concentration, and both had the reverse effect to bacterial drug resistance.
Preparation of bacterial strains and E. coli strains producing ESBLs. Four bacterial strains from the ATCC were used,including Staphylococcus aureus ATCC 12600, Streptococcus salivarius ATCC 13419, Streptococcus epidermidis ATCC 14990, and Escherichia coli ATCC 25922. All dilutions were performed in PBS and cells were spread on
Luria agar (LA) for viable counts. Seventy E. coli strains producing ESBLs were selected from strain samples in our laboratory based on Performance Standard for Antimicrobial Susceptibility Testing proposed by Clinical And Laboratory Standards Institute, USA,strongest resistant strain was selected for reversal assay.
MIC assay. Minimum Inhibitory Concentration (MIC) assays
were performed in 10 mM SPB supplemented with 0.1% TSB in 96-microwell plates (round bottom; Nunc A/S, Roskilde, Denmark). Bacteria were grown overnight (10 mM SPB/0.1%
TSB) and diluted to 5×105 cfu/mL. Then, 90 mL of bacterial solution was mixed with 10 mL of peptide solution of different concentrations (the highest concentration of each peptide was 1 mM). The microwell plates were incubated with continuous shaking at 37°C. The assay medium allowed sufficient growth of bacterium to visually determine the MIC of the different peptides. Experiments were performed with
duplicates of each sample on at least two separate occasions and the average MIC was calculated.
MIC of HB10 against E.coli,S.salivarius,S.epidermidis,S.aureus is 1.5μM, 2.1μM, >100μM, >100μM.
MIC of HB11 against E.coli,S.salivarius,S.epidermidis,S.aureus is 1.8μM, 2.2μM, >100μM, >100μM.