Phylogenomic Investigation of IncI1-Iγ Plasmids Harboring bla_CMY-2 and bla_SHV-12 in Salmonella enterica and Escherichia coli in Multiple Countries

The objective of this study was to elucidate the genetic and evolutionary relatedness of bla_CMY-2- and bla_SHV-12-carrying IncI1-Iγ plasmids. Phylogenomic analysis based on core genome alignments and gene presence/absence was performed for different IncI1-Iγ sequence types (STs).

ABSTRACT

The objective of this study was to elucidate the genetic and evolutionary relatedness of bla_CMY-2- and bla_SHV-12-carrying IncI1-Iγ plasmids. Phylogenomic analysis based on core genome alignments and gene presence/absence was performed for different IncI1-Iγ sequence types (STs). Most IncI1-Iγ/ST12 and IncI1-Iγ/ST231 plasmids had near-identical core genomes. The data suggest that widely occurring bla_CMY-2-carrying IncI1-Iγ/ST12 plasmids originate from a common ancestor. In contrast, bla_SHV-12 was inserted independently into different IncI1-Iγ/ST231-related plasmids.

TEXT

Plasmid-encoded extended-spectrum and AmpC β-lactamases (ESBL/pAmpC) are the dominant causes of resistance to extended-spectrum cephalosporins in Enterobacteriaceae (1–3). Poultry and poultry products have been considered reservoirs of ESBL/pAmpC-producing Salmonella enterica and Escherichia coli (2–9). ESBL/pAmpC-carrying plasmids can be classified in different incompatibility groups, including IncI1-Iγ (10, 11). IncI1-Iγ plasmids harboring ESBL/pAmpC are dominant in S. enterica and E. coli originating from poultry in multiple countries (4, 12–17). Using plasmid multilocus sequence typing (pMLST) (18, 19), specific ESBL/pAmpC variants were found to be associated with particular IncI1-Iγ STs (12, 13, 16, 17). bla_CMY-2 carriage has been associated with IncI1-Iγ/ST12 in isolates from poultry (12–14, 16, 17, 20). In contrast, bla_SHV-12 has been described in multiple IncI1/STs in isolates originating from humans, animals (mainly poultry), and the environment (20–23). However, a resolution higher than the nucleotide sequences of the five housekeeping genes in the pMLST scheme is required to identify the evolutionary relatedness of plasmids belonging to the same ST (4, 17, 24). The objective of the present study was to elucidate the genetic and evolutionary relatedness of bla_CMY-2- and bla_SHV-12-carrying IncI1-Iγ plasmids within the same pMLSTs using whole-genome sequence (WGS)-based phylogenetic analysis.

Sequences of IncI1-Iγ plasmids originating from previous characterization of ESBL/pAmpC-carrying strains from Colombian baseline studies in poultry were selected. All sequences of bla_CMY-2-carrying (n = 20) and bla_SHV-12-carrying (n = 4) IncI1-Iγ plasmids from Salmonella (17) and all available bla_CMY-2-carrying (n = 15) and bla_SHV-12-carrying IncI1-Iγ plasmids (n = 4) from E. coli (16) were included. Plasmid sequences from Salmonella were characterized using Illumina WGS and electroporation of reference plasmids as previously described (17). For E. coli, previously transformed E. coli DH10B cells harboring bla_CMY-2 and bla_SHV-12 on IncI1-Iγ plasmids were subjected to Illumina WGS for the present study (16). Chromosomal contigs were detected and removed using BLAST as previously described for Salmonella (17). In addition, the allele sequences of IncI1-Iγ STs (https://pubmlst.org/plasmid/) encountered more than once in the selection of plasmids described above were concatenated as separate sequences for each allele in a single FASTA file and used as a query for the nucleotide database using BLAST (last accessed 29 May 2018). E. coli-derived plasmid sequences of two publications were used to include additional IncI1-Iγ/ST12 plasmids (n = 12) from Europe (see Table S1C in the supplemental material) (12, 13). Overall, ESBL/pAmpC gene variants and plasmids were characterized in silico with ResFinder 2.1 (25), PlasmidFinder 1.3, and pMLST 1.4 (19). A summary of all included plasmids is given in Table 1. The plasmid STs that were found repeatedly in Salmonella and E. coli from Colombian poultry were IncI1-Iγ/ST12 and ST231. From GenBank, 28 plasmids belonged to IncI1-Iγ/ST12 or ST12 single-locus variants (SLVs), and 5 plasmids belonged to IncI1-Iγ/ST231 or ST231 SLVs. Plasmids from GenBank originated from different S. enterica serovars and E. coli. Information regarding the source, isolation year, and in silico characterization of all plasmids (19, 25) and strains (26, 27) is shown in Table S1. The genome sequences of transformed E. coli DH10B strains harboring plasmids from Salmonella and E. coli from Colombia, which were used for reference, were submitted to the European Nucleotide Archive (ENA) under project numbers PRJEB23610 and PRJEB29690, respectively.

TABLE 1.

Inventory of sequenced IncI1-Iγ plasmids from Colombian poultry and previous reports used for detailed phylogenetic comparisons and analysis of the genetic environment

ESBL/pAmpC	n and pMLST of sequenced IncI1-Iγ plasmids from:
	Colombian poultry	GenBank or previously publishedd
blaCMY-2	32 ST12, 2 SLV ST12,c 1 ST231	29 ST12, 8 ST2,e 5 SLV ST12,c 3 ST23,e 1 ST20,e 1 ST265e
blaSHV-12	4 ST231, 1 ST12, 1 ST26, 1 SLV ST26,c 1 ST230f	1 ST95,e 1 ST178,g 1 ST231
Other bla genesa		5 ST12, 1 ST107,f 1 ST131,f 1 ST270f
No bla genesb		1 ST12, 1 SLV ST12,c 1 ST230f
Total	43	61

^abla genes other than bla_CMY-2 and bla_SHV-12.

^bNo bla genes detected with ResFinder.

^cSLV due to incomplete match or missing 1 allele from the pMLST scheme.

^dSequences of plasmids listed in this column were obtained based on the allele sequences of the highly prevalent IncI1-Iγ/ST12 and ST231 from GenBank and publications from Europe.

^eSelected for analysis of the genetic environment of bla_CMY-2/bla_SHV-12.

^fSLV of ST231.

^gSLV of ST12.

Phylogenomic reconstruction was based on core plasmid genome alignments using Parsnp v1.2 (28). Phylogenetic maximum-likelihood (midpoint-rooted) trees were constructed using FastTree2 v2.1.8 (29). Gene presence/absence maximum-likelihood trees were built by annotating the plasmid genomes using Prokka v.1.13 (30) followed by orthology predictions using Roary (31). The resulting gene presence/absence data were encoded as binary values, and trees were constructed using RAxML v.8.2.4 (32) with the BINCAT model. Genome annotations were used to describe the genetic environment of bla genes. Visualization of the trees was made with FigTree (http://tree.bio.ed.ac.uk/software/figtree/). The core genome of the resulting tree based on IncI1-Iγ/ST12-related plasmids was 40,056 bp (∼40% of the plasmid genome) (see Fig. S1 in the supplemental material). The sublineage of IncI1-Iγ/ST12 and ST12 SLVs is shown in Fig. 1A. Most IncI1-Iγ/ST12 plasmids carried bla_CMY-2 and originated from samples from poultry (Fig. 1A). Although frequently reported (12–14, 24, 33, 34), detailed genomic relatedness of bla_CMY-2-carrying IncI1-Iγ/ST12 plasmids originating from multiple countries and sources has not been assessed. In this study, several plasmids with an identical core genome were identified (clusters I to IX, Fig. 1A). Cluster IX included plasmids from European and American countries, which showed high similarity between Salmonella- and E. coli-derived plasmids. The gene presence/absence phylogeny grouped most of the plasmids from ST12 and SLVs in a sublineage within the tree (MG825376.1 to ERR915116) (see Fig. S2 in the supplemental material). bla_CMY-2-carrying IncI1-Iγ/ST12 plasmids from nonpoultry sources, such as other livestock species and humans, were also found (Fig. 1A). These findings underscore the potential of IncI1-Iγ plasmids to be transferred in strains from Salmonella and E. coli outside the poultry environment (13, 35–38). The genetic environment of bla_CMY-2 in most IncI1-Iγ/ST12 plasmids and ST12 SLVs was similar and characterized upstream by insertion sequence ISEcp1 and downstream by blc and sugE (Fig. 1A and Fig. S3A in the supplemental material). IS1294 (39) and IS26 were found upstream of bla_CMY-2 in non-ST12 plasmids (see Fig. S4 in the supplemental material).

FIG 1.

Phylogenetic tree based on core genome of closely related bla_CMY-2-carrying IncI1-Iγ/ST12 plasmids and its SLVs (A) and bla_SHV-12-carrying IncI1-Iγ/ST231 and its SLVs (B). ^aSLVs of IncI1-Iγ/ST12. ^bSLVs of IncI1-Iγ/ST231. ^cTransformed plasmids from Colombian Salmonella included as reference. ^dCarrying bla_SHV-12. ^eCarrying bla_TEM1B-like. ^fCarrying bla_CMY-2 together with bla_CTX-M-1. ^gCarrying bla_CMY-2-like. ^hCarrying bla_CMY-2. ⁱCarrying bla_TEM1A. ^jCarrying no bla genes. ^kThe patterns of the genetic environment (GE) with their designated numbers can be found in Fig. S3 in the supplemental material. ⧫, unique pattern; ●, genetic environment of CMY-2 was characterized by bla_CMY-2-blc-sugE and for SHV-12 by bla_SHV-12-deoR (Fig. S3); -, carrying no bla_CMY-2 or bla_SHV-12 genes. ^lData from information accompanying the sequence submission in GenBank but not specifically found in the metadata fields. ^mDetails of the source listed in this column are available in Table S1 in the supplemental material. ⁿN/A, data not available. ^oStrain MLST was added when information or complete sequence of strains was available. ^pClusters I to X of plasmids referred to in the manuscript are grouped in shaded boxes. Scale bars at the bottom of the phylogenetic trees represent nucleotide substitutions per site.

The core genome of the tree based on IncI1-Iγ/ST231-related plasmids was 32,789 bp (∼32% of the plasmid genome) (see Fig. S5 in the supplemental material). The sublineage of IncI1-Iγ/ST231 and related ST231 SLVs is shown in Fig. 1B. The phylogeny based on gene presence/absence of ST231-related plasmids confirmed phylogenetic distance between the plasmids from Colombian Salmonella and E. coli. Thus, no evidence of the exchange of bla_SHV-12-carrying plasmids between these bacterial species was observed (see Fig. S6 in the supplemental material). In contrast, the plasmids from Colombian Salmonella and one from E. coli from a human in the United States were found to be closely related, at both the core genome and gene content levels. In this case, these plasmids may be derived from a common ancestor. Despite differences in core genome and gene presence/absence, the genetic environment of bla_SHV-12 in all IncI1-Iγ/ST231 and SLVs was characterized upstream by IS26 and downstream by deoR (see Fig. S7 in the supplemental material). This pattern of genetic environment was found repeatedly (Fig. 1B and Fig. S3B). However, the results of ST231-related plasmids have to be interpreted with care, given the limited number of plasmids available for phylogenetic analysis.

In conclusion, WGS-based analysis supports the hypothesis that bla_CMY-2-carrying IncI1-Iγ/ST12 plasmids in Salmonella and E. coli likely originated from a common ancestor. As previously suggested, the source of the contamination with these plasmids may be related to similar practices in poultry trade and farming (40, 41). bla_SHV-12 in association with IS26 was likely introduced independently in different lineages within IncI1-Iγ/ST231. More observations are needed to better understand the transmission of bla_SHV-12 in ST231 plasmids.