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. 2018 Apr 26;62(5):e00006-18. doi: 10.1128/AAC.00006-18

Complete Sequence of the IncA/C1 Plasmid pCf587 Carrying blaPER-2 from Citrobacter freundii

Melina Ruggiero a,b, Delphine Girlich c,d, Laura Dabos c,d, Pablo Power a,b, Thierry Naas c,d,e, Gabriel Gutkind a,b,✉
PMCID: PMC5923148  PMID: 29463531

ABSTRACT

The blaPER-2-harboring plasmid pCf587 (191,541 bp) belongs to lineage IncA/C1 and is closely related to pRA1. It contains a large resistance island including the blaPER-2 gene between two copies of ISKox2-like elements, the toxin-antitoxin module pemK-pemI, several other resistance genes inserted within a Tn2 transposon, a Tn21-like structure, and a class 1 integron. pCf587 belongs to sequence type 13 (ST13), a new plasmid multilocus sequence typing (pMLST) ST.

KEYWORDS: IncA/C, PER-2, ST13, oxyimino-cephalosporinase, pMLST

TEXT

Since the initial report of PER-2 (1), blaPER-2 has been detected in different species, including Klebsiella pneumoniae, Enterobacter cloacae, Enterobacter aerogenes, Vibrio cholerae, and community-acquired enteropathogenic Escherichia coli. PER-2 has been the second most prevalent extended-spectrum β-lactamase (ESBL) (after the pandemic CTX-Ms) in Argentina (and probably Uruguay), accounting for nearly 10% and 5% of the oxyimino-cephalosporin resistance in K. pneumoniae and E. coli, respectively (2–4); it has also been sporadically observed in a few other countries (5–10). PER-1 and PER-2 are the most frequently reported members of the 9-variant PER family in clinical settings (2).

In contrast to blaPER-1-containing plasmids (11–13), little is known about the genetic organization of the blaPER-2 gene. We previously reported the immediate flanking sequences in plasmid pCf587 from Citrobacter freundii 33587, which was recovered from a urine sample in 1999 (14). To further delineate the genetic background of blaPER-2, the aim of this study was to analyze the complete sequence of pCf587, which was recovered almost 20 years ago.

E. coli 33587-TC9 is an E. coli CAG12177 transconjugant clone harboring the pCf587 plasmid from C. freundii 33587 (14). The plasmid sequence was determined with the Illumina MiSeq platform. Full genomic DNA was sequenced with a MinION analyzer (Oxford Nanopore Technologies). A hybrid de novo assembly was performed with SPAdes v3.9.0, using both generated libraries (15). Gaps were closed by using a PCR-based strategy and Sanger sequencing. Gene predictions and annotation were performed with the classic RAST tool (16, 17) and were manually curated with BLAST online tools. Sequence comparisons were performed with Mauve software (18). Identification of acquired antibiotic resistance genes and the incompatibility group determination were conducted with ResFinder 3.0 (19) and PlasmidFinder (20), respectively. Alignments were constructed using ClustalW, with default settings. The phylogenetic tree was produced with MEGA 7.0.26 (21), using maximum likelihood methods with default settings and 1,000 bootstraps.

The overall genetic organization of pCf587 is shown in Fig. 1. The plasmid (191,541 bp, with an average G+C content of 49.97%) contains 253 coding sequences, of which 141 have no assigned annotation. The genes involved in a type IV secretion system, the master regulators acaD and acaC, the traI gene (encoding the MOBH121 relaxase), and the putative maintenance genes parAB, stbA (parM), and kfrA were easily recognized (Fig. 1). A large resistance island (RI), RI-pCf, was also identified (Fig. 2).

FIG 1.

FIG 1

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Schematic representation of the genetic organization of pCf587. The two outer rings show the coding sequences on the forward and reverse strands of the plasmid. Each coding sequence is color coded according to its predicted function, as shown in the figure. The gray arc depicts the resistance island (RI-pCf), with the insertion sequences identified on the plasmid. The two inner rings represent the GC plot and the GC skew graph, respectively. For both plots, magenta and olive green indicate the measures below and above the average, respectively.

FIG 2.

FIG 2

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Schematic representation of the pCf587 resistance island (RI-pCf). Color codes match those used in Fig. 1. The elements highlighted in the figure are discussed further in the text. The box indicates the close environment of the blaPER-2 gene. Direct repeats bracketing the postulated ISKox2-like-mediated transposition of blaPER-2 and its surrounding genes are shown in capital letters. Inverted repeats from ISKox2-like are marked with yellow and orange rectangles.

The in silico analysis established that pCf587 belongs to the IncA/C group. In a nucleotide BLAST search for closely related plasmids in the NCBI database, only three IncA/C plasmids with query coverage of >65% were found, namely, Enterobacter hormaechei subsp. steigerwaltii strain 34998 plasmid p34998 (73%) (GenBank accession number CP012168.1), Aeromonas hydrophila plasmid pRA1 (66%) (GenBank accession number FJ705807.1), and K. pneumoniae subsp. pneumoniae strain KP4898 plasmid pIncAC-KP4898 (67%) (GenBank accession number KY882285.1). The plasmid backbone comparison revealed that pCf587 is more closely related to pRA1 (Fig. 3); they have similar overall genetic arrangements and backbone lengths, with almost 99% nucleotide identity.

FIG 3.

FIG 3

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Local colinear block comparison between pCf587, pRA1, p34998, and pIncAC-KP4898 by Mauve software. Each local colinear block represents regions with homologous sequences without rearrangements. The same local colinear blocks are identified with the same color. Within each collinear block, the height of the similarity profile corresponds to the average level of conservation in that region. Gray and red lines indicate the plasmid backbone and the resistance islands, respectively. The dashed gray line indicates an insertion in the p34998 backbone. The black arrow indicates the position of a deletion in the pIncAC-KP4898 backbone. The repA gene of each plasmid is identified.

IncA/C backbones are highly conserved, and it has been postulated that they are derived from a common ancestor (22). However, two different lineages, A/C1 and A/C2, were established based on repA gene sequence similarities (23); IncA/C2 plasmids were further split into two types (24). To date, only the primitive plasmid pRA1 (sequence type 11 [ST11]) (25) and the recently incorporated VIM-encoding pIncAC-KP4898 (ST12) (26) belong to the first lineage.

The plasmid pCf587 repA gene has 99% nucleotide identity with pRA1 repA, with only two nucleotide changes not resulting in amino acid changes; the tra genes are 99 to 100% identical to the corresponding tra genes from pRA1. Therefore, pCf587 belongs to IncA/C1, along with pRA1 and related plasmids. Other features shared with pRA1 are: (i) an open reading frame (ORF) between traA and dsbC, encoding an 1,828-amino-acid protein (orf1828) with 99% amino acid identity; (ii) the toxin-antitoxin (TA) system genes hipA and hipB; (iii) the absence of tad and ata genes from other putative TA systems typical of all IncA/C2 plasmids; and (iv) a lack of the ssb gene (present in IncA/C2 plasmids).

In most A/C2 type 1 plasmids, antimicrobial resistance island A (ARI-A) is found either embedded in or upstream of the rhs1 gene. It contains a class 1 integron, multiple transposons, a Tn21-tnp module, and a Tn21-mer module generally interrupted by IS4321, which docks the resistance island at that site (24). RI-pCf (Fig. 2) has similar characteristics and location, compared to ARI-A, although rhs1 is absent, as expected. RI-pCf includes (i) a blaTEM-1B-containing Tn2 whose tnpA is interrupted by an ISAs1-like element; (ii) the TA system pemKI, followed by a large region with three IS1R copies, an ISAba125-like element, and the aph(3′)-VIa-like and catA1-like resistance genes; and (iii) a Tn21-like structure including Tn21-tnp and Tn21-mer modules whose tnp inverted repeat is interrupted by a IS4321-like element, a class 1 integron carrying the aadB and sul1 resistance genes, and a zone delimited by two similar copies of ISKox2-like elements (sharing 99% nucleotide identity) carrying the blaPER-2 gene, a truncated IS1326 element, and an IS6100 element. The two ISKox2-like elements were found 26 bp upstream of abct and 3,703 bp downstream of ISPa12 elements previously found as part of the blaPER-2 environment (ISPa12-blaPER-2-gst-like-abct) (14). The region between ISKox2-like and ISPa12, with no homologues in the NCBI database, contains 8 ORFs, including a traW gene encoding a putative conjugal transfer pilus assembly protein. We postulate that ISKox2-like elements could have been involved in the recruitment of blaPER-2 and its surrounding genes from a still unknown reservoir to an ancient RI-pCf (Fig. 2). A recent publication describes blaPER-2 in the chromosome of a clinical Shewanella sp. isolate, Shew256 (27). The abct in Shew256 was larger than that in pCf587, which may suggest that the ISKox2-like element partially interrupted pCf587-abct during recruitment.

Interestingly, there are no copies of IS26 in pCf587. This insertion sequence was found to be associated with most IncA/C plasmids described previously, including pRA1 (22), and is considered to be implicated in the evolution of ARI-A in type 1 A/C2 plasmids (24).

For pCf587, the plasmid multilocus sequence typing (pMLST) database (https://pubmlst.org/plasmid) could not recognize a specific ST, according to the pMLST scheme for IncA/C plasmids developed by Hancock et al. (28). We built a phylogenetic tree using concatenates of the four pMLST genes (Fig. 4) and observed that, as expected, pCf587 was related to ST11, which includes pRA1 and other IncA/C1 plasmids, such as the recently described pIncAC-KP4898 (26) and p34998, all separate from the rest of the STs, including the IncA/C2 lineage (type 1 and type 2). The pMLST alleles of pCf587 were assigned to ST13 and core gene pMLST (cgPMLST) ST13.1.

FIG 4.

FIG 4

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Phylogenetic relationships between pCf587, p34998, and the different IncA/C STs identified to date, based on maximum likelihood and Bayesian methods. The tree was created based on the alignment of concatenated gene sequences of repA, parA, parB, and 053 (putative DNA binding protein-encoding gene). Numbers at the branch points indicate bootstrap values.

IncA/C plasmids are high-molecular-weight, low-copy-number plasmids that were initially described, around 1970, in fish pathogens such as A. hydrophila and Vibrio spp. (25, 29, 30) and now are disseminated among Enterobacteriaceae species (28, 31). Currently, IncA/C plasmids are considered an important health care problem (32), being responsible for dissemination of blaCTX-M, blaCMY, blaNDM, blaIMP, blaVIM, and blaKPC genes, among others (28, 31).

It is noteworthy that IncA/C1 plasmids such as pCf587 might have been circulating among pathogens in Argentina since at least the late 1990s and, even so, their dissemination seems to be not as proficient as that of other resistance plasmids, such as IncA/C2, involved in mobilization of CTX-M or metallo-β-lactamases, which are much more widespread enzymes. The presence of efficient TA systems in their backbones may provide some stability even in the absence of selective pressure. This plasmid lineage may also have a role in the mobilization of other (still unrecognized) resistance markers, as shown by the recent finding of some metallo-β-lactamases associated with similar backbones in recent isolates (26) (Alan Elena, Daniela Cejas, Francisco Magariños, Virginia Jewtuchowicz, Andrea Facente, Gabriel Gutkind, José Di Conza, and Marcela Radice, unpublished results). Although further studies on the mechanisms involved in blaPER-2 mobilization are still needed, this study provides some insights regarding the genetic elements that might have facilitated the recruitment of blaPER-2 in IncA/C1 plasmids.

Accession number(s).

The complete annotated sequence of the pCf587 plasmid has been deposited in GenBank under accession number MG053108.

ACKNOWLEDGMENTS

This work was funded by grants from the University of Buenos Aires (grant UBACyT 2014–2017 to P.P. and grant UBACyT 2013–2015 to G.G.) and the Agencia Nacional de Promoción Científica y Tecnológica (grant BID PICT 2015-1925 to G.G. and grant PICT 2014-0457 to P.P.), as well as by the Assistance Publique-Hôpitaux de Paris through a grant from the Université Paris Sud (grant EA 7361) and by the LabEx LERMIT, supported by a grant from the French National Research Agency (grant ANR-10-LABX-33). This work was also funded in part by a grant from the Joint Programme Initiative on Antimicrobial Resistance (grant ANR-14-JAMR-0002). M.R. is a postdoctoral fellow of CONICET, Argentina. P.P. and G.G. are members of Carrera del Investigador Científico, CONICET, Argentina.

We have no conflicts of interest to declare.

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