Dataset Information

BlaCMY-2-positive IncA/C plasmids from Escherichia coli and Salmonella enterica are a distinct component of a larger lineage of plasmids.

ABSTRACT: Large multidrug resistance plasmids of the A/C incompatibility complex (IncA/C) have been found in a diverse group of Gram-negative commensal and pathogenic bacteria. We present three completed sequences from IncA/C plasmids that originated from Escherichia coli (cattle) and Salmonella enterica serovar Newport (human) and that carry the cephamycinase gene blaCMY-2. These large plasmids (148 to 166 kbp) share extensive sequence identity and synteny. The most divergent plasmid, peH4H, has lost several conjugation-related genes and has gained a kanamycin resistance region. Two of the plasmids (pAM04528 and peH4H) harbor two copies of blaCMY-2, while the third plasmid (pAR060302) harbors a single copy of the gene. The majority of single-nucleotide polymorphisms comprise nonsynonymous mutations in floR. A comparative analysis of these plasmids with five other published IncA/C plasmids showed that the blaCMY-2 plasmids from E. coli and S. enterica are genetically distinct from those originating from Yersinia pestis and Photobacterium damselae and distal to one originating from Yersinia ruckeri. While the overall similarity of these plasmids supports the likelihood of recent movements among E. coli and S. enterica hosts, their greater divergence from Y. pestis or Y. ruckeri suggests less recent plasmid transfer among these pathogen groups.

SUBMITTER: Call DR

PROVIDER: S-EPMC2812137 | biostudies-literature | 2010 Feb

REPOSITORIES: biostudies-literature

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blaCMY-2-positive IncA/C plasmids from Escherichia coli and Salmonella enterica are a distinct component of a larger lineage of plasmids.

Call Douglas R DR Singer Randall S RS Meng Da D Broschat Shira L SL Orfe Lisa H LH Anderson Janet M JM Herndon David R DR Kappmeyer Lowell S LS Daniels Joshua B JB Besser Thomas E TE

Antimicrobial agents and chemotherapy 20091130 2

Large multidrug resistance plasmids of the A/C incompatibility complex (IncA/C) have been found in a diverse group of Gram-negative commensal and pathogenic bacteria. We present three completed sequences from IncA/C plasmids that originated from Escherichia coli (cattle) and Salmonella enterica serovar Newport (human) and that carry the cephamycinase gene blaCMY-2. These large plasmids (148 to 166 kbp) share extensive sequence identity and synteny. The most divergent plasmid, peH4H, has lost sev ...[more]

PMID: 19949054

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Project description: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.

Project description:The multidrug resistance-encoding plasmids belonging to the IncA/C incompatibility group have recently emerged among Escherichia coli and Salmonella enterica in the United States. These plasmids have a unique genetic structure compared to other enterobacterial plasmid types, a broad host range, and propensity to acquire large numbers of antimicrobial resistance genes via their accessory regions. Using E. coli strain DH5M-NM-1 harboring the prototype IncA/C plasmid pAR060302, we sought to define the baseline transcriptome of IncA/C plasmids under laboratory growth and in the face of selective pressure. The effects of ampicillin, florfenicol or streptomycin exposure were compared to cells left untreated at logarithmic phase using Illumina sequencing (RNA-Seq). Under growth in Luria-Bertani broth lacking antibiotics, much of the backbone of pAR060302 was transcriptionally inactive, including its putative transfer regions. A few plasmid backbone genes of interest were highly transcribed, including genes of a putative toxin-antitoxin system and an H-NS-like transcriptional regulator. In contrast, numerous genes within the accessory regions of pAR060302 were highly transcribed, including the resistance genes floR, blaCMY-2, aadA, and aacA. Antibiotic treatment with ampicillin or streptomycin resulted in no genes being differentially expressed compared to controls lacking antibiotics, suggesting that many of the resistance-associated genes are not differentially expressed due to exposure to these antibiotics. In contrast, florfenicol treatment resulted in the up-regulation of floR and numerous chromosomally encoded genes. Overall, the transcriptome mapping of pAR060302 suggests that it mitigates the fitness costs of carrying resistance-associated genes through global regulation with its transcriptional regulators. Bacterial strains and growth conditions. E. coli strain DH5M-NM-1 harboring pAR060302 was grown in 10 mL DifcoTM Luria-Bertani (LB) broth aliquots at 37M-BM-: C with shaking until an OD600 of 0.5. A total of 8 cultures were independently grown representing two biological replicates per condition tested. Six of the cultures were amended, 2 cultures per antibiotic, with ampicillin (50 M-BM-5g/mL final concentration), florfenicol (30 M-BM-5g/mL final concentration), or streptomycin (50 M-BM-5g/mL final concentration) and allowed to incubate at 37M-BM-: C with shaking for an additional 30 min. Two cultures were not amended with any antibiotic. Cells were pelleted and RNA was purified using a commercially available RNA extraction kit (Qiagen). RNA preparations were then subjected to a DNase treatment to eliminate DNA contamination from the sample (Qiagen). A treatment was also included to deplete ribosomal RNA using a commercially available kit (MicrobExpress, Ambion). The two biological replicates for each growth condition were pooled for sequencing. Ilumina sequencing for transcriptome mapping. cDNA libraries were generated with an insert size of 100 bp and sequenced with 76-base cycles of single-end reads using a Genome Analyzer II (Illumina) platform according to manufacturerM-bM-^@M-^Ys protocols at the Biomedical Genomics Center (University of Minnesota, Minneapolis, Minnesota, USA). Approximately 160,000 plasmid-mapped reads each were obtained for the ampicillin and streptomycin treated samples, and 260,000 plasmid-mapped reads each for the control and florfenicol treatment samples. Genome-mapped read counts were as follows: control, ~6.4 million reads, florfenicol treatment, ~5.7 million reads, ampicillin treatment, ~1.7 million reads, and streptomycin treatment, ~5.2 million reads. We only used those reads uniquely mapped on plasmid or chromosomal DNA for global normalization and further analysis. RNAseq data analysis. cDNA reads were trimmed so that the quality at each base position was above 30 (~15-20 bp) and then mapped either to the E. coli K-12 MG1655 published genome sequence (Genbank accession no. NC_000913) or to the pAR060302 published sequence (Genbank accession no. NC_092692) using BOWTIE. The E. coli strain DH5M-NM-1 has an incomplete annotation and for this reason the E. coli K-12 annotation was used, representing an estimation of differentially expressed genes due to exposure of antimicrobials. The reads mapped per kilobase of gene per million (RPKM) reads was calculated using either the E. coli chromosome or the pAR060302 annotation and was used for global normalization. The per kilobase cDNA length normalized the effect of different length of cDNAs such that the sequence reads have a equal chance to map on the long cDNA regions and the short cDNA regions. After RPKM normalization, each sample is comparable to each other. An R package, DEGseq, was used to identify differentially expressed genes between the control and each antibiotic treatment condition. A cutoff of q-value < 0.05 and a fold change of > 3 were used to measure statistical significance.

Dataset Information

BlaCMY-2-positive IncA/C plasmids from Escherichia coli and Salmonella enterica are a distinct component of a larger lineage of plasmids.

Publications

blaCMY-2-positive IncA/C plasmids from Escherichia coli and Salmonella enterica are a distinct component of a larger lineage of plasmids.

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