Title: Molecular analysis of plasmid encoded multi-drug resistance (MDR) in Salmonella enterica animal isolates by PFGE, replicon typing, and DNA microarray screening followed by high-throughput DNA sequencing Authors
Submitted to: American Society for Microbiology
Publication Type: Abstract Only
Publication Acceptance Date: May 23, 2010
Publication Date: June 8, 2010
Citation: Lindsey, R.L., Frye, J.G., Bono, J.L., Smith, T.P., Harhay, D.M., Cray, P.J., Meinersmann, R.J. 2010. Molecular analysis of plasmid encoded multi-drug resistance (MDR) in Salmonella enterica animal isolates by PFGE, replicon typing, and DNA microarray screening followed by high-throughput DNA sequencing. American Society for Microbiology. June 8-11, 2010. Toronto, Canada. B16. p41-42. Technical Abstract: Background: The development of Multi-Drug Resistant (MDR) Salmonella is of global concern. MDR Salmonella genes can be transmitted in a number of ways including transfer of plasmids. To understand how MDR plasmids develop and are transmitted, their genetics must be thoroughly described. To achieve this, a set of 437 Salmonella isolates obtained from clinically ill animals in 2005 were subjected to AR phenotypic analysis. Representative subsets of these isolates were selected for genetic analyses including PFGE, plasmid replicon typing, microarray hybridization, and high-throughput sequencing to determine their genetic structure and evolution. Methods: All 437 Salmonella enterica isolates were tested for antimicrobial susceptibility using a semi-automated broth micro-dilution custom panel of antimicrobials (SensititerTM, TREK Diagnostic Systems, Inc., Westlake, OH, USA). Isolates were also assayed for the presence of 18 plasmid replicon regions by multiplex PCR as described by Johnson et al. (2007 Appl. Environ. Microbiol. 73:1976–1983) and Carattoli et al. (2005 J. Microbiol. Methods 63:219–228). A subset of 216 isolates was subjected to PFGE analysis as described by PulseNet (CDC, Atlanta, GA, USA). A subset of 59 isolates PCR positive for IncA/C plasmids were hybridized to a DNA microarray constructed of 70mer probes designed to detect 775 AR genes and 493 IncA/C and H1 plasmid genes using standard methods (Corning Inc., Acton, MA, USA). From these results, plasmid preparations from 32 isolates were selected for sequence analysis by 454 sequencing (Roche, Branford, CT, USA). Results: Cluster analysis based on PFGE paterns showed that Salmonella positive for IncA/C plasmids grouped together based on serotype and MDR profile. Microarray analysis of IncA/C plasmids further defined a core “backbone” of plasmid genes and also identified variable regions associated with resistance to heavy metals. Preliminary sequencing data has confirmed the microarray data and has identified and assembled contiguous sequences encoding IncA/C plasmid core genes as well as AR genes. Conclusions: The cluster analysis showed that IncA/C plasmids and MDR has been associated with Salmonella previous to the introduction of the therapeutic use of antimicrobials and that the spread of resistance is likely due to the expansion of resistant clones rather than the accretion of new resistances. Microarray hybridization identified regions of variability associated with metal resistance that could explain the stability of MDR IncA/C plasmids in the absence of antimicrobial selective pressure. Analysis of high-throughput DNA sequencing has increased the resolution of this information and will enable the accurate determination of the history and evolution of IncA/C plasmids and the MDR genes they carry.