D R Call1, M-S Kang, J Daniels, T E Besser. 1. Department of Veterinary Microbiology and Pathology, Washington State University, WA 99164-7040, USA. drcall@wsu.edu
Abstract
AIMS: To compare genetic composition of plasmids using microarrays composed of randomly selected fragments of plasmid DNA. METHODS AND RESULTS: Separate shotgun libraries were constructed from plasmid DNA pooled from Escherichia coli and Salmonella enterica. Cloned fragments were used as probes for microarrays. Plasmid targets were labelled, hybridized overnight, and bound targets were imaged after enzymatic signal amplification. Control hybridizations demonstrated significantly higher signal when probes and targets shared >95% sequence identity. Diagnostic sensitivity and specificity of the assay was 95 and 99%, respectively. Cluster analysis showed close matches for replicate experiments with a high correlation between replicates (r = 0.91) compared with the correlation for nonreplicates (r = 0.09). Analysis of hybridization data from 43 plasmids generated five distinct clusters, two for known serovar-specific plasmids, one for enterohemorrhagic E. coli plasmids, and two for plasmids harboring a recently disseminated antibiotic resistance gene (bla(CMY-2)). CONCLUSION: Mixed-plasmid microarrays are suitable for comparing genetic content of wild-type plasmids and hybridization results from this study suggest several novel hypotheses about plasmid gene exchange between E. coli and S. enterica. SIGNIFICANCE AND IMPACT OF STUDY: Mixed-plasmid microarrays permit rapid, low cost analysis and comparison of many plasmids. This ability is critical to understanding the source, fate, and transport of plasmids amongst commensal and pathogenic bacteria.
AIMS: To compare genetic composition of plasmids using microarrays composed of randomly selected fragments of plasmid DNA. METHODS AND RESULTS: Separate shotgun libraries were constructed from plasmid DNA pooled from Escherichia coli and Salmonella enterica. Cloned fragments were used as probes for microarrays. Plasmid targets were labelled, hybridized overnight, and bound targets were imaged after enzymatic signal amplification. Control hybridizations demonstrated significantly higher signal when probes and targets shared >95% sequence identity. Diagnostic sensitivity and specificity of the assay was 95 and 99%, respectively. Cluster analysis showed close matches for replicate experiments with a high correlation between replicates (r = 0.91) compared with the correlation for nonreplicates (r = 0.09). Analysis of hybridization data from 43 plasmids generated five distinct clusters, two for known serovar-specific plasmids, one for enterohemorrhagic E. coli plasmids, and two for plasmids harboring a recently disseminated antibiotic resistance gene (bla(CMY-2)). CONCLUSION: Mixed-plasmid microarrays are suitable for comparing genetic content of wild-type plasmids and hybridization results from this study suggest several novel hypotheses about plasmid gene exchange between E. coli and S. enterica. SIGNIFICANCE AND IMPACT OF STUDY: Mixed-plasmid microarrays permit rapid, low cost analysis and comparison of many plasmids. This ability is critical to understanding the source, fate, and transport of plasmids amongst commensal and pathogenic bacteria.
Authors: C Verdet; V Gautier; E Chachaty; E Ronco; N Hidri; D Decré; G Arlet Journal: Antimicrob Agents Chemother Date: 2009-07-13 Impact factor: 5.191
Authors: Min-Su Kang; Thomas E Besser; Dale D Hancock; Steffen Porwollik; Michael McClelland; Douglas R Call Journal: Appl Environ Microbiol Date: 2006-09-08 Impact factor: 4.792
Authors: LaShanda M Glenn; Rebecca L Lindsey; Jason P Folster; Gary Pecic; Patrick Boerlin; Mathew W Gilmour; Heather Harbottle; Shaohua Zhao; Patrick F McDermott; Paula J Fedorka-Cray; Jonathan G Frye Journal: Microb Drug Resist Date: 2013-01-25 Impact factor: 3.431
Authors: Joshua B Daniels; Douglas R Call; Dale Hancock; William M Sischo; Katherine Baker; Thomas E Besser Journal: Appl Environ Microbiol Date: 2009-04-17 Impact factor: 4.792