High-throughput method for rapid induction of prophages from lysogens and its application in the study of Shiga Toxin-encoding Escherichia coli strains.
A high-throughput 96-well plate-based method for the rapid induction of endogenous prophages from individual bacterial strains was developed. The detection of endogenous prophages was achieved by the filtration of the culture liquor following norfloxacin induction and subsequent PCRs targeting bacteriophage-carried gene markers. The induction method was tested on 188 putative Shiga toxin (Stx)-producing Escherichia coli (STEC) strains and demonstrated the ability to detect both lambdoid and stx-carrying bacteriophages in strains for which plaques were not observed via plaque assay. Lambdoid bacteriophages were detected in 37% of the induced phage preparations via amplification of the Q gene, and Stx1- and Stx2-encoding phages were detected in 2 and 14% of the strains, respectively. The method therefore provided greater sensitivity for the detection of Stx and other lambdoid bacteriophage populations carried by STEC strains than that for the established method of plaque assay using bacterial indicator strains, enabling, for the first time, large-scale bacteriophage population and diversity studies.
Microbiology Research Group, School of Biological Sciences, BioSciences Building, University of Liverpool, Crown St., Liverpool L69 7ZB, United Kingdom.
James E
JE
McDonald
Darren L
DL
Smith
Paul C M
PC
Fogg
Alan J
AJ
McCarthy
Heather E
HE
Allison
D000900:Anti-Bacterial Agents / Q000494:pharmacology; D003090:Coliphages / Q000235:genetics / Q000254:growth & development*; D009643:Norfloxacin / Q000494:pharmacology; D039002:Prophages / Q000235:genetics / Q000254:growth & development*; D012680:Sensitivity and Specificity; D022621:Shiga Toxin / Q000096:biosynthesis* / Q000235:genetics*; D054323:Shiga-Toxigenic Escherichia coli / Q000187:drug effects / Q000821:virology*; D014775:Virus Activation
D000900:Anti-Bacterial Agents; D022621:Shiga Toxin; D009643:Norfloxacin