High Genetic Plasticity in Multidrug-Resistant Sequence Type 3-IncHI2 Plasmids Revealed by Sequence Comparison and Phylogenetic Analysis

ABSTRACT

We report a novel fusion plasmid, pP2-3T, cointegrating sequence type 3 (ST3)-IncHI2 with an IncFII plasmid backbone mediating multidrug resistance (MDR) and virulence. Phylogenetic analysis and comparative genomics revealed that pP2-3T and other MDR ST3-IncHI2 plasmids clustered together, representing a unique IncHI2 lineage that exhibited high conservation in backbones of plasmids but possessed highly genetic plasticity in various regions by acquiring numerous antibiotic resistance genes and fusing with other plasmids. Surveillance studies should be performed to monitor multiresistance IncHI2 plasmids among Enterobacteriaceae.

TEXT

Multidrug resistance (MDR) in bacteria is a serious problem that threatens human and animal health (1), and this problem has been exacerbated by the emergence of multiresistance plasmids conferring resistance to most classes of antimicrobials (2). Plasmids of the incompatibility HI2 (IncHI2) group, one of the most frequently encountered plasmid types in the Enterobacteriaceae family (3), have frequently been associated with MDR phenotypes and may carry numerous resistance determinants, including extended-spectrum β-lactamases (bla_CTX-M) and plasmid-mediated quinolone resistance determinant genes [oqxAB and aac(6′)-Ib-cr], as well as carbapenemase genes (bla_IMP, bla_VIM, and bla_NDM) and the plasmid-mediated colistin resistance gene (mcr-1) (4–7). In our previous study, IncHI2 plasmids recovered from Escherichia coli strains of food animals were found to play an important role in cospreading oqxAB, bla_CTX-M-9G/1G, and other antibiotic resistance genes (ARGs) (4). The spread of these multiresistance plasmids has prompted great concern because coresistance to antibiotics from different classes is therapeutically challenging. Here, an MDR IncHI2-FII fusion plasmid, pP2-3T, was fully sequenced, and phylogenetic analysis and comparative genomics of IncHI2 plasmids were performed.

Plasmid pP2-3T was identified in E. coli P2-3 isolates from a diseased pig in Guangdong, China, and was conjugated to E. coli strain C600 as previously described (4). It was extracted from the transconjugant using the Qiagen Plasmid Midi kit and subjected to plasmid sequencing using Illumina NextSeq 500 and PacBio RSII single-molecule and real-time (SMRT) platforms. The raw data from plasmid sequencing using Illumina NextSeq 500 (3,302,000 available reads) were assembled by Velvet (8), and these assembled data (20 contigs, N50, 73,817 bp) combined with PacBio data (30,156 available reads) were further assembled by SPAdes (9), resulting in a single contig (549× coverage [Illumina reads]) for pP2-3T that was manually closed into a circle. The sequence of plasmid pP2-3T was annotated with the RAST (10) and ISfinder (https://www-is.biotoul.fr) tools, and sequence comparison and map generation were performed using BLAST (http://blast.ncbi.nlm.nih.gov), Easyfig (11), and the BLAST Ring Image Generator (12).

IncHI2 plasmids with complete sequences were selected from the GenBank database by using the BLASTn algorithm with the repHI2 sequence (1,089 bp) from the IncHI2 reference plasmid R478 (accession no. BX664015) as a search query. All of the selected plasmids and pP2-3T identified in this study were then reanalyzed by PlasmidFinder to validate the IncHI2 replicon and to investigate the possibility of additional replicons (https://cge.cbs.dtu.dk/services/PlasmidFinder-1.0/). To determine the ARGs and plasmid double-locus sequence type (pDLST), IncHI2 plasmids were subjected to ResFinder (https://cge.cbs.dtu.dk/services/PlasmidFinder/) and pMLST (https://pubmlst.org/plasmid/) analyses. pDLST was set up on the basis of the in silico comparative analysis of backbones of the original five fully sequenced IncHI2 plasmids, and two loci (smr0018 and smr0199) that showed significantly different sequences were used to discriminate the IncHI2 plasmids and select for pDLST (5). To date, a total of 11 STs were found among IncHI2 plasmids. To clarify the genetic and evolutionary relationships among IncHI2 plasmids, a phylogenetic tree was generated by Parsnp based on the core genome sequences as previously reported (13).

This plasmid pP2-3T is 392,275 bp in length, and it was composed of ∼380 predicted open reading frames (ORFs) (>200 bp), including backbone elements of IncHI2 and IncFII plasmids that encoded functions for IncHI2 and IncFII plasmid replication, horizontal transfer, maintenance, and stability, respectively (see Fig. S1 in the supplemental material). Sequence comparison analysis showed that pP2-3T was formed by fusion of the IncHI2 plasmid (designated pP2-3T-1) and the IncFII plasmid (designated pP2-3T-2), with sizes of ∼260 and ∼132 kb, respectively (Fig. 1a). Plasmid pP2-3T-1 showed high similarity to oqxAB-carrying IncHI2 plasmid pHXY0809 (accession no. KM877269) from a Salmonella Typhimurium strain of chicken origin. A large multiple resistance region (MRR) (∼60 kb), flanked by IS26, was found in pP2-3T-1 (Fig. 1b). This MRR contained oqxAB, bla_CTX-M-65, and fosA3, as well as another 15 ARGs and a wide range of mobile genetic elements, and closely resembled the MRRs in IncHI2 plasmids pHXY0809 and pA3 (accession no. KX421096), which carried oqxAB, bla_CTX-M-14, and fosA3 in a Salmonella Indiana isolate from chicken.

FIG 1.

Characteristics of the novel fusion plasmid pP2-3T cointegrating ST3-IncHI2 with IncFII plasmid backbone. (a) Comparison with pP2-3T, pHXY0809, and pSMS35_130, and illustration of direct insertion of the IncFII plasmid pP2-3T-2 into the ST3-IncHI2 plasmid pP2-3T-1. Blue, replicon genes; red, two IS26s mediating insertion of pP2-3T-2. (b) Comparison of the pP2-3T MDR region with other plasmids containing the same resistance modules.

Plasmid pP2-3T-2 showed high similarity to IncFII plasmid pSMS35_130 (accession no. CP000971), and a large virulence region (∼42 kb) was identified that contained ompT, hlyF, sitABCD, colicinBM, iroBCDEN, and iss. These virulence genes were previously identified on virulence plasmids pAPEC-O1-ColBM and pAPEC-O2-ColV and associated with virulence in avian-pathogenic E. coli strains (APEC) (14). Plasmid pP2-3T-2 was captured at a site immediately upstream of the IS26-oqxAB-IS26 regions, linearized, and inserted into the space between two IS26s in the same orientation. Two different plasmids could undergo fusion via a Tnp26-catalyzed reaction (15, 16) or IS26-mediated homologous recombination (17). The cointegrate plasmid pP2-3T was probably formed during the conjugation process, as previously reported (18), because the original E. coli P2-3 isolate had lost the typical ∼390-kb plasmids but harbored two additional plasmids of ∼130 and ∼260 kb, respectively (data not shown). This was further confirmed by designing three primer pairs (see Table S1 in the supplemental material) to identify the two putative integrated loci (bp 104,304 to 110,970 and 239,316 to 241,877) between IncHI2 and IncFII plasmids. Of note, this fusion may result in expanding the number of replicons and host ranges of plasmids and facilitate the acquisition of ARGs and other traits that may be advantageous to the host bacterial cell (17, 19). In the present study, clinically significant ARGs and multiple APEC virulence genes were integrated into the same conjugative plasmid pP2-3T via plasmid fusion, highlighting the risk of promoting the spread of virulence through the extensive use of antibiotics and challenging the effective treatment of infectious diseases.

We further explored genetic evolution, ARG distribution, and sequence types (STs) among IncHI2 plasmids. We identified 44 sequences corresponding to fully sequenced IncHI2 plasmids, and their repHI2 genes showed high similarity to those in R478 with only a 0- to 11-bp difference. These plasmids were from various species of Enterobacteriaceae, diverse sources, and distinct geographic areas (see Data Sets S1 and S2 in the supplemental material); and only two plasmids (pGD0503Z13 [accession no. KR653209] and pP2-3T) contained replicons (FII and/or FIB) in addition to HI2. These 44 IncHI2 plasmids comprised ST1 (n = 16), ST3 (n = 11), ST2 (n = 7), and ST4 (n = 3), in addition to untypeable members (n = 7). New pMLST schemes for IncHI2 plasmids probably need to be designed, since the smr0199 locus was not detected among four untypeable IncHI2 plasmids.

The core genome phylogenetic tree for 36 of 44 IncHI2 plasmids revealed two distinct clusters, cluster I and cluster II (Fig. 2), and the remaining 8 plasmids were excluded because their core genome regions were very different from those of the other 36 plasmids. ST2-, ST3-, and ST4-IncHI2 plasmids mostly clustered together, and most ST1-IncHI2 plasmids clustered separately. This phenomenon seemed to be consistent with phylogenetic analysis of replication regions from 24 fully sequenced IncHI2 plasmids (20). Clusters I and II can be further subdivided into seven (groups A1 to A7) and two (groups B1 and B2) subgroups, respectively. Groups A1 and B1 were the largest subgroups in clusters I and II and were composed mainly of ST3-IncHI2 and ST1-IncHI2 plasmids, respectively. Furthermore, the novel fusion plasmid pP2-3T was included in group A1 (Fig. 2). The number and diversity of ARGs were high in groups A1 and B1 but low in the remaining groups. In group B1, diverse clinically relevant and important β-lactam resistance genes were identified, including bla_CTX-M-9G/1G, bla_CMY-8, bla_KPC-3, bla_IMP-4/8, and bla_VIM-1. In group A1, diverse important ARGs were found, including mcr-1, bla_CTX-M-9G/1G, oqxAB, fosA3, and floR; the latter three were prevalent exclusively in this group. Plasmids from group A1 seemed to represent a unique IncHI2 plasmid lineage, as they all belonged to ST3 and were mostly from E. coli and Salmonella isolates from food animals in China. Of note, ST3-IncHI2 plasmids coharboring oqxAB with bla_CTX-M, fosA3, mcr-1, and floR have been increasing among E. coli and Salmonella isolates from poultry and livestock in China (4, 6, 21, 22).

FIG 2.

Phylogenetic phylogram constructed from the core genome sequences of 36 IncHI2 plasmids and important ARG distribution. The repHI2 genes of 44 IncHI2 plasmids showed high similarity to those in the IncHI2 reference plasmid R478, with only a 0- to 11-bp difference. Plasmid pP2-3T was identified in this study, and the additional 43 IncHI2 plasmids with complete sequences were from GenBank. Information about the 44 IncHI2 plasmids can be found in Data Sets S1 and S2 in the supplemental material. ND, untypeable.

Linear comparison was further constructed for 10 MDR ST3-IncHI2 plasmids belonging to group A1 and plasmid pHXY0809 belonging to group A2, and the regions of the putative IncFII plasmid were excluded for fusion plasmid pP2-3T. High conservation and synteny were observed in the core backbones of the 11 plasmids. Differences were observed primarily within the regions between the conserved genes trhG and terF, containing one or more MRRs. Furthermore, a large MRR inserted between umuC and dcm was found in each of the 10 group A1 plasmids except 1 (accession no. KX129949). The MRRs contained several ARGs, including oqxAB, bla_CTX-M, and fosA3, and partial class 1 integrons or gene cassettes that were frequently flanked by one or two copies of IS26. Linear comparisons of these MRRs showed high similarity among plasmids pP2-3T (this study), pHK0653 (accession no. KT334335), pHXY0908 (accession no. KM877289), pHNSHP45-2 (accession no. KU341381), and pA3T (accession no. KX421096) (see Fig. S2 in the supplemental material). This high degree of synteny and conservation in backbones and the diverse ARGs indicated that MDR ST3-IncHI2 plasmids may have diverged from a common ancestor through horizontal acquisition of ARG arrays.

In conclusion, we characterize a novel ST3-IncHI2 hybrid plasmid mediating MDR and virulence. This plasmid and other MDR ST3-IncHI2 plasmids clustered together by phylogenetic analysis, representing a unique IncHI2 lineage that possessed a high genetic plasticity through recruiting numerous ARGs, including clinically common and significant ARGs, and fusing with other plasmids via mobile genetic elements, especially IS26. It is imperative that surveillance studies be carried out on the prevalence and dissemination of IncHI2 plasmids among Enterobacteriaceae in human and veterinary medicine.

Accession number(s).

The complete nucleotide sequences of pP2-3T have been deposited into the GenBank database under accession number MG014722.