ABSTRACT: 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.
