Complete Sequence of the FII Plasmid p42-2, Carrying bla_CTX-M-55, oqxAB, fosA3, and floR from Escherichia coli

Abstract

We sequenced a novel conjugative multidrug resistance IncF plasmid, p42-2, isolated from Escherichia coli strain 42-2, previously identified in China. p42-2 is 106,886 bp long, composed of a typical IncFII-type backbone (∼54 kb) and one distinct acquired DNA region spanning ∼53 kb, harboring 12 antibiotic resistance genes [bla_CTX-M-55, oqxA, oqxB, fosA3, floR, tetA(A), tetA(R), strA, strB, sul2, aph(3′)-II, and Δbla_TEM-1]. The spread of these multidrug resistance determinants on the same plasmid is of great concern and, because of coresistance to antibiotics from different classes, is therapeutically challenging.

TEXT

Widespread antibiotic resistance poses an enormous threat for human and animal health worldwide (1). This problem has been exacerbated in recent years with the emergence of multidrug resistance (MDR) plasmids conferring resistance to most classes of antimicrobials (2). Plasmids of the incompatibility F (IncF) group, representing one of the most frequently encountered plasmid types (3), have frequently been associated with MDR phenotypes, including extended-spectrum β-lactamases (ESBLs) and plasmid-mediated quinolone resistance (PMQR) genes (4, 5). In a previous study of ours, IncF plasmids recovered from Escherichia coli strains of food-producing and companion animals were investigated, and most of them carried numerous resistance determinants, such as bla_CTX-M, rmtB, oqxAB, and floR (4, 6). The spread of these multiresistance plasmids has prompted worldwide concern because of coresistance to multiple antimicrobial agents that facilitates the survival of bacteria under the selective pressure of antibiotics. Herein, we analyzed a common subtype IncF plasmid, F33:A−:B− (FII, FIA, FIB [FAB] formula); this plasmid, designated p42-2, contains 12 different resistance genes and was fully sequenced, and the data were compared with those of other IncF plasmids.

E. coli 42-2 was recovered from the feces of a healthy duck in Guangzhou, China. Conjugation was performed by mixing E. coli 42-2 and E. coli C600 in a liquid medium and isolating for E. coli C600 (p42-2) by selection on MacConkey agar containing streptomycin (1,000 mg/liter) and cefotaxime (2 mg/liter) as previously described (4, 6). p42-2 was extracted from the E. coli C600 transconjugant using a commercial kit (Qiagen midikit; Qiagen, Germany). Sequencing of p42-2 was performed on an Illumina IIx genome analyzer with a 500-bp paired-end library (approximately 100 million available reads, 935-fold genome coverage) and a 2,000-bp paired-end library (∼337 million available reads, 3,150-fold genome coverage). These raw data were assembled by SOAPdenovo (7). Gene prediction and annotation were performed using RAST tools (8). The sequence comparison and map generation were performed using BLAST (http://blast.ncbi.nlm.nih.gov) and Easyfig version 2.1 (9).

Plasmid p42-2 confers resistance to ampicillin, chloramphenicol, kanamycin, streptomycin, sulfonamides/trimethoprim, tetracycline, olaquindox, fosfomycin, cephalosporin, and florfenicol. Because of its broad resistance spectrum, p42-2 was sequenced and fully annotated. p42-2 contains 106,886 bp with 141 predicted open reading frames (ORFs) and is composed of a typical IncFII-type backbone (∼54 kb) that encodes functions for IncF plasmid replication, horizontal transfer, maintenance, and stability functions. The remaining ∼53 kb is an acquisition region carrying various resistance genes and mobile genetic elements (MGEs). The whole sequence of p42-2 is organized in a manner similar to that of other IncF plasmids, including pHNFP460-1 (GenBank accession number KJ020575, from mainland China) (Fig. 1) and pHN7A8 (JN232517, from mainland China) (10), with an ∼61% to 63% coverage and an overall nucleotide identity of 99%. Sequencing analysis confirmed that p42-2 is a chimera plasmid made up of a basic IncF plasmid backbone and an additional ∼53-kb mobile region.

FIG 1.

Comparison of p42-2 MRR with other plasmids containing the same resistance modules. The extents and directions of antibiotic resistance genes (dark arrows) and other selected genes (gray arrows) are indicated by labeled arrows (hypothetical proteins [HPs] are indicated by black boxes). The insertion elements (ISs) are shown as dotted arrows labeled with their number/name, and Δ represents a truncated gene. The plasmid backbone is indicated by dark lines. Light-gray shading and oblique dotted lines between two regions indicate the regions with homology. Diagrams are drawn from sequences available under GenBank accession numbers KT990220 for p42-2, CP009414 for pCFSAN007428_01, KC853434 for pACN001-A, KJ020575 for pHNFP460-1, and JN232517 for pHN7A8.

The p42-2 backbone (replication and transfer regions) is highly conserved with other known IncF plasmids, such as pHNF460-1, pHN7A8, and pHK23a (GenBank accession number JQ432559, from Hong Kong) (see Fig. S1 in the supplemental material). p42-2 contains one replication region (copB, repA3, repA1, and repA4) belonging to IncF with plasmid replicon type F33:A−:B−, according to FAB (FII, FIA, FIB) criteria (11). The p42-2 replication region was flanked by plasmid functional modules (pemK/pemI and snrB), which contributes to plasmid maintenance in the bacterial population (12). This region is identical to that in the other IncFII plasmids pHNFP460-1 and pHN7A8 (see Fig. S1). The transfer region of p42-2 comprises 26 tra genes and 6 trb genes (traM, traJ, traY, traA, traL, traE, traK, traB, traP, traD, traG, traV, traR, traC, trbI, traW, traU, trbC, traN, trbE, traF, traQ, trbB, trbJ, trbF, traH, traG, traS, traT, traD, traI, and traX). The gene order of this region is totally conserved in pHNFP460-1 and pHN7A8 (see Fig. S1).

The ∼53-kb variable region (positions 3996 to 56766) comprises 12 resistance determinants, complete or truncated insertion sequences, and transposons, as well as many genes of unknown function, and was located downstream of the replication region. Compared with pHNFP460-1 and pHN7A8, p42-2 is composed of a larger multiresistance region (MRR) which is flanked by seven differently oriented IS26 elements in five succinct regions harboring antibiotic resistance genes (Fig. 1). The first region comprises six antibiotic resistance genes [floR, tetA(A), tetA(R), strA, strB, and sul2], conferring resistance to florfenicol, tetracycline, streptomycin, and sulfonamide. This region has highest identity (99.9%) with part of pCFSA007428_01 (CP009414, from the United States). p42-2 possesses an additional sequence (∼20 kb) in the second region, with many genes of putative functions, such as plasmid maintenance and stability determinants (stbD/stbE) (13) and genes associated with virulence factors (vagC/vagD). Interestingly, partial replication genes (bis-repX-pir) of the IncX plasmid also have been found in this region, suggesting that only part of an IncX plasmid has been captured or acquired, followed by a partial deletion event. The third region possesses two multidrug efflux pumps, oqxA and oqxB, which are active against fluoroquinolones. IS26-like sequences flank this locus (IS26-oqxA-oqxB-IS26), which is closely related to pACN001-A (KC853434, from China) and pOLA52 (EU370913, from Denmark) (14). There are an additional four resistance determinants, tetA(R), fosA3, Δbla_TEM-1, and bla_CTX-M-55, combined with three IS26 elements. This region, IS26-tetA(R)-fosA3-IS26-Δbla_TEM-1-orf477-bla_CTX-M-55-ΔISEcp1-IS26, was identical to plasmids pHNFP460-1 and pHP588 (AB778291, from Japan). The p42-2 MRR is flanked by fragments of IS1 with the same IS26/IS1 backbone as seen in pHN7A8 (Fig. 1) and was shown to be combined with a diverse range of MGEs (IS5075, IS50, ISCR2, IS26, IS1294, ISEcp1, and Tn21). These MGEs are important in horizontal gene transfer and may provide a favorable genetic environment for plasmid plasticity. For instance, a total of seven IS26 copies are located on p42-2 (Fig. 1), each associated with different antibiotic resistance genes, such as bla_CTX-M-55, oqxAB, and fosA3. This observation provides further evidence that IS26 plays an important role in the dissemination and evolution of IncF antibiotic resistance plasmids by creating regions containing multiple antibiotic resistance genes through stepwise integration and/or recombination events mediated by IS26 (15, 16). Thus, the variable region of p42-2, encoding 12 antibiotic resistance determinants, may have arisen from MRR acquisition by the actions of MGEs from multiple plasmid sources with an IncF plasmid backbone.

In conclusion, p42-2 is a chimera plasmid made up of a basic IncF plasmid backbone with a large multiresistance region. This region appears to have evolved through the integration of multiple resistance determinants from different sources by the action of MGEs and recombination. The association of these important antibiotic resistance genes (such as bla_CTX-M-55, oqxAB, fosA3, and floR) on the same plasmid is therapeutically challenging due to coselection by various drugs and may confer a selection advantage for antibiotic-resistant E. coli clones.

Nucleotide sequence accession number.

The annotated sequence of plasmid p42-2 from strain 42-2 has been submitted to GenBank under the accession number KT990220.