Comparative Sequence Analysis of Multidrug-Resistant IncA/C Plasmids from Salmonella enterica

Abstract

Determinants of multidrug resistance (MDR) are often encoded on mobile elements, such as plasmids, transposons, and integrons, which have the potential to transfer among foodborne pathogens, as well as to other virulent pathogens, increasing the threats these traits pose to human and veterinary health. Our understanding of MDR among Salmonella has been limited by the lack of closed plasmid genomes for comparisons across resistance phenotypes, due to difficulties in effectively separating the DNA of these high-molecular weight, low-copy-number plasmids from chromosomal DNA. To resolve this problem, we demonstrate an efficient protocol for isolating, sequencing and closing IncA/C plasmids from Salmonella sp. using single molecule real-time sequencing on a Pacific Biosciences (Pacbio) RS II Sequencer. We obtained six Salmonella enterica isolates from poultry, representing six different serovars, each exhibiting the MDR-Ampc resistance profile. Salmonella plasmids were obtained using a modified mini preparation and transformed with Escherichia coli DH10Br. A Qiagen Large-Construct kit™ was used to recover highly concentrated and purified plasmid DNA that was sequenced using PacBio technology. These six closed IncA/C plasmids ranged in size from 104 to 191 kb and shared a stable, conserved backbone containing 98 core genes, with only six differences among those core genes. The plasmids encoded a number of antimicrobial resistance genes, including those for quaternary ammonium compounds and mercury. We then compared our six IncA/C plasmid sequences: first with 14 IncA/C plasmids derived from S. enterica available at the National Center for Biotechnology Information (NCBI), and then with an additional 38 IncA/C plasmids derived from different taxa. These comparisons allowed us to build an evolutionary picture of how antimicrobial resistance may be mediated by this common plasmid backbone. Our project provides detailed genetic information about resistance genes in plasmids, advances in plasmid sequencing, and phylogenetic analyses, and important insights about how MDR evolution occurs across diverse serotypes from different animal sources, particularly in agricultural settings where antimicrobial drug use practices vary.

Transferable Plasmids of Salmonella enterica Associated With Antibiotic Resistance Genes

“…The most common AR genes carried by IncA/C are str AB (aminoglycosides), sul 2 (sulfonamides), tet AR (tetracycline), bla CMY-2 (β-lactams), and flo R (chloramphenicols). Other genes for resistance to aminoglycosides, tetracyclines, trimethoprim, chloramphenicols, and cephalosporins have also been identified ( Welch et al, 2007 ; Hoffmann et al, 2017 ; Cao et al, 2018 ). In Salmonella isolated in the United States, IncA/C plasmids have been found in several different serotypes and animal commodities, especially cattle ( Lindsey et al, 2009 ; Folster et al, 2017 ; Mollenkopf et al, 2017 ).…”

Analysis of COMPASS, a New Comprehensive Plasmid Database Revealed Prevalence of Multireplicon and Extensive Diversity of IncF Plasmids

“…Even though several bioinformatics tools can be applied to reconstruct plasmid sequences from short reads, a contiguous assembly is still difficult to obtain (Zhou and Xu, 2010;Lanza et al, 2014;Antipov et al, 2016;Rozov et al, 2017). This pitfall can be overcome by using long read sequencing technology such as Oxford Nanopore or PacBio Technologies (Hoffmann et al, 2017;Margos et al, 2017;Li et al, 2018).…”

Genomic comparison of diverse Salmonella serovars isolated from swine

“…and can be transferred among multiple bacterial genera [7,8]. A wide range of plasmids that carry AR and virulence genes have been reported in Salmonella [9][10][11]. Here we catalog all the antibiotic resistance genes and their organization in a diverse set of Salmonella from swine.…”