Transcriptome mapping of pAR060302, a blaCMY-2-positive broad-host-range IncA/C plasmid

Abstract

The multidrug resistance-encoding plasmids belonging to the IncA/C incompatibility group have recently emerged among Escherichia coli and Salmonella enterica strains in the United States. These plasmids have a unique genetic structure compared to other enterobacterial plasmid types, a broad host range, and a propensity to acquire large numbers of antimicrobial resistance genes via their accessory regions. Using E. coli strain DH5α 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 those on cells left untreated at logarithmic phase using Illumina platform-based RNA 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, bla(CMY-2), aadA, and aacA. 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 upregulation of floR and numerous chromosomal 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.

Fig 1

Heat map displaying transcriptome map of pAR060302. Blue indicates no or low expression (0 to 75 reads aligned per nucleotide position), green indicates moderate expression (250 to 500 reads aligned per nucleotide position), and red indicates high expression (>2,500 reads aligned per nucleotide position). In the pAR060302 (pAR) map, the shaded regions indicate the plasmid backbone, as follows: red, IncA/C replicon and hypothetical genes; blue, Tra1 region; and green, hypothetical genes and Tra2 region. Open boxes depict the accessory regions, as follows: pink, flo-tet-sul-containing region (enlarged in Fig. 4); green, bla_CMY-2-containing region (enlarged in Fig. 5); and blue, Tn21-like region, which contains a class 1 integron (enlarged in Fig. 6). ^a, antibiotics are abbreviated as follows: ampicillin, Amp; florfenicol, Ffl; and streptomycin, Str.

Fig 2

Transcription of regions not previously annotated. (a) Region between two IS26 elements upstream of the ISCR2 elements; (b) intergenic transcript downstream of the class I integron. Antibiotic treatment conditions are labeled along the x axis. The x axis has a scale of 0 to 2,500 reads aligned per base. Arrows indicate direction of transcription. ^a, antibiotics are abbreviated as follows: ampicillin, Amp; florfenicol, Ffl; and streptomycin, Str.

Fig 3

Transcriptome maps of plasmid backbone genes of interest. (a) pAR060302_0025 and pAR060302_0026 are transcribed at extremely high levels. The x axis has a scale of 0 to 30,000 reads aligned per nucleotide position. Arrows indicate direction of transcription. (b) pAR060302_0188 is transcribed as a single gene transcript. The x axis has a scale of 0 to 2,500 reads aligned per nucleotide position. Arrows indicate direction of transcription. ^a, antibiotics are abbreviated as follows: ampicillin, Amp; florfenicol, Ffl; and streptomycin, Str.

Fig 4

Transcriptome map of the sul2 containing region of pAR060302. Antibiotic treatment conditions are labeled along the y axis. The x axis has a scale of 0 to 2,500 reads aligned per base. Arrows indicate direction of transcription. ^a, antibiotics are abbreviated as follows: ampicillin, Amp; florfenicol, Ffl; and streptomycin, Str. *, a statistically significant greater or less than log₂ (fold change) in expression (q < 0.05).

Fig 5

Transcriptome map of the bla_CMY-2-containing Tra1 region of pAR060302. Antibiotic treatment conditions are labeled along the x axis. The x axis has a scale of 0 to 2,500 reads aligned per base. Arrows indicate direction of transcription. ^a, antibiotics are abbreviated as follows: ampicillin, Amp; florfenicol, Ffl; and streptomycin, Str.

Fig 6

Transcriptome map of the class I integron-containing region of pAR060302. Antibiotic treatment conditions are labeled along the y axis. The x axis has a scale of 0 to 2,500 reads aligned per base. Arrows indicate direction of transcription. ^a, antibiotics are abbreviated as follows: ampicillin, Amp; florfenicol, Ffl; and streptomycin, Str.