Bacteriophage cell lysis of Shiga toxin-producing Escherichia coli for top-down proteomic identification of Shiga toxins 1 & 2 using matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry.

摘要: RATIONAL Analysis of bacteria by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) often relies upon sample preparation methods that result in cell lysis, e.g. bead-beating. However, Shiga toxin-producing Escherichia coli (STEC) can undergo bacteriophage-induced lysis triggered antibiotic exposure may allow greater selectivity the proteins extracted. METHODS We have developed a method for selective extraction bacteriophage-encoded and specifically toxins 1 2 (Stx1 & 2) expressed from STEC strains induced DNA-damaging antibiotics. were cultured overnight on agar supplemented with ciprofloxacin, mitomycin-C or an iron chelator to induce bacteriophage lytic cycle concomitant expression release Stx1 and/or Stx2. Sample relied exclusively Stx into solution. RESULTS Three clinical analyzed tandem (MALDI-TOF-TOF-MS/MS) top-down proteomics analysis: E. O157:H7 strain EDL933, O91:H21 B2F1 O26:H11 ECRC #05.2217. The B-subunit Stx1a EDL933 was detected identified even though it ~100-fold less abundant than Stx2a had been previously this strain. Two also identified: L0117 L0136. B-subunits Stx2d #05.2217 identified. CONCLUSIONS Bacteriophage appeared enhance detection sensitivity these compared previous work using mechanical lysis. Detection/identification other (beyond Stx) tends support hypothesis