Penemase genes tested, only blaKPC-2 was detected. Repeated conjugation experiments failed to transfer the blaKPC-2 marker from R31 to P. aeruginosa PAO1 (induced rifampin resistance) or Escherichia coli EC600 (rifampin resistance). 2.three. Overview of pR31-KPC The R31 isolate harbors only a single extrachromosomal closed circular DNA sequence, designated as pR31-KPC, which was determined to become 29,402 bp in length and include a imply GC content material of 57.five and 44 predicted open reading frames (ORFs) (Figure S1). The backbone of pR31-KPC includes a modular structure, with the insertion of two accessory modules: The IS26-blaKPC-2 -IS26 unit and IS26-Tn6376-IS26 area. The accessory modules had been defined as acquired DNA regions linked with and bordered by mobile components. two.four. The Backbone of pR31-KPC The backbone of pR31-KPC is 16.9-kb in length and contains the following components: The RepA and its iterons (repeat region for the RepA binding website), that are accountable for TFC 007 supplier plasmid replication initiation. The iterons are 137 bp in size, within which 12-bp web pages are positioned, somewhat conserved, and repeated six times; parA for plasmid partition; higBA, which encodes the toxin-antitoxin technique for post-segregational killing; and also a traMLI conjugation program remnant. The identified RepA protein of pR31-KPC showed a one hundred amino acid similarity to the homologs in the four other blaKPC-2 -carrying Pseudomonas plasmids of the identical incompatibility group, which are obtainable in public sequence databases, namely p1011-KPC2, p14057A, YLH6_P3 (accession quantity MK882885), and pP23-KPC (accession number CP065418). The backbone of pR31-KPC showed 8300 coverage and one hundred identity for the abovementioned plasmids (Supplementary Table S1). A linear comparison on the backbones of these 5 plasmids revealed the following: (1) The regions in between the iterons and orf207 are hot spots for the acquisition of resistance genes, and all of the blaKPC-2 genes reside in these regions; (2) p1011-KPC2 may be the most total plasmid of this incompatibility group, having a comprehensive conjugative area along with a fairly intact upkeep area, even though pR31-KPC is the smallest plasmid of this group (Figure 1). Though most of its conjugative region is missing and is unable to conjugate experimentally, pR31-KPC and hence, blaKPC-2 can stay in its host.Antibiotics 2021, ten, 1234 Antibiotics 2021, 10, x FOR PEER REVIEW4 of 10 4 ofFigure 1. Linear comparison of plasmid genome sequences. Genes are denoted by arrows. The plasmid backbone replicaFigure 1. Linear comparison of plasmid genome sequences. Genes are denoted by arrows. The plasmid backbone replication, tion, maintenance, and conjugation regions are colored in green, dark blue, and orange, AQX-016A PI3K respectively. The accessory modmaintenance, and conjugation regions are colored in green, dark blue, and orange, respectively. The accessory module ule regions are colored in red. Shading denotes homology (nucleotide identity 90) of the plasmid backbone regions, but regions are colored PEER Critique not Antibiotics 2021, ten, x FOR in red. Shading denotes homology (nucleotide identity 90) of your plasmid backbone regions, but 5 of 11 not the accessory modules. RepA and orf207 represent the names of your labeled genes, respectively. The GenBank accession the accessory modules. RepA and orf207 represent the namespR31-KPC are MH734334, KY296095, MK882885, CP065418, on the labeled genes, respectively. The GenBank accession numbers of p1011-KPC2, p14057A, YLH6_P3,.