Characterization of KPC-Encoding Plasmids from Enterobacteriaceae Isolated in a Czech Hospital

ABSTRACT

Ten Enterobacteriaceae isolates collected in a Czech hospital carried bla_KPC-positive plasmids of different sizes (∼30, ∼45, and ∼80 kb). Sequencing revealed three types of plasmids (A to C) with the Tn4401a transposon. Type A plasmids comprised an IncR backbone and a KPC-2-encoding multidrug resistance (MDR) region. Type B plasmids were derivatives of type A plasmids carrying an IncN3-like segment, while type C plasmids were IncP6 plasmids sharing the same KPC-2-encoding MDR region with type A and B plasmids.

TEXT

KPC-type β-lactamases comprise a distinct group of plasmid-borne enzymes, with carbapenemase activity mainly occurring in Klebsiella pneumoniae. KPC-producing Enterobacteriaceae have emerged as challenging pathogens causing health care-associated infections, due to their extremely drug-resistant phenotypes and ability to cause infections associated with high mortality (1). KPC producers have disseminated worldwide and currently constitute an important public health problem (2). In Europe, Greece and Italy are the most affected countries, with high proportions of KPC-producing K. pneumoniae (3). In the Czech Republic, however, the occurrence of KPC producers has been rare. A sporadic case of KPC-2-producing K. pneumoniae recovered from a patient, who had been previously hospitalized in Greece, was detected in the Czech Republic in 2009 (4). Additionally, in 2011, an outbreak of KPC-3-producing K. pneumoniae was observed in another Czech hospital (5), with the index case being a patient repatriated from Italy.

In the present study, we describe the molecular characterization of KPC-2-producing Enterobacteriaceae isolates, mainly of the species Citrobacter freundii, recovered in the University Hospital of Hradec Kralove (Czech Republic).

From 2014 until 2016, a total of 10 nonrepetitive Enterobacteriaceae isolates showing carbapenemase activity on matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) meropenem hydrolysis assay (6) were detected. Among them, 7 of the isolates were identified to be C. freundii, 1 was identified to be K. pneumoniae, 1 was identified to be Escherichia coli, and 1 was identified to be Morganella morganii (Table 1). Phenotypic testing, PCR screening, and sequencing (7) showed that all isolates were positive for the presence of the bla_KPC-2 gene. The 10 KPC-2-producing isolates were recovered from 7 patients, 6 of which were hospitalized in the same unit (Table 1). In addition, 5 of the patients had overlapping stays in several combinations, suggesting transmission of KPC-2 producers.

TABLE 1.

Characteristics of KPC-2-producing Enterobacteriaceae isolates

Species and isolate	Hospital departmenta	Isolation date	Clinical material	STb	Size of blaKPC plasmid (kb)	Type of plasmid sequence (replicon)	MIC (μg/ml) ofc:
							Ctx	Caz	Fep	Atm	Imp	Mem	Etp	Gen	Amk	Tob	Sxt	Cip	Col	Tgc	Fos
C. freundii
Cfr-27569	ICU	11/2014	Urine	18	46.826	A (IncR)	>8	>16	>16	>16	16	>16	>4	>32	16	>8	>64	>8	0.25	0.25	≤2
Cfr-31260	ICU	09/2015	Urine	18	46.826	A (IncR)	>8	>16	>16	>16	>32	>16	>4	>32	32	>8	>64	>8	0.12	0.25	≤2
Cfr-31816	ICU	10/2015	Urine	18	46.826	A (IncR)	>8	>16	>16	>16	>32	16	4	>32	>32	>8	>64	>8	0.12	0.12	≤2
Cfr-33038d	ICU	11/2015	Rectal swab	18	46.826	A (IncR)	>8	>16	>16	>16	>32	>16	>4	>32	16	>8	>64	>8	4	1	8
Cfr-33795	ICU	04/2016	Urine	142	30.051	C (IncP6)	>8	>16	>16	>16	4	8	4	4	32	>8	>64	4	0.12	0.5	≤2
Cfr-36049	ICU	09/2016	Wounds	87	81.348	B (IncR and IncN3)	>8	>16	>16	>16	8	8	>4	1	16	>8	1	≤0.06	1	0.06	≤2
Cfr-36808	HD	11/2016	Rectal swab	18	46.826	A (IncR)	>8	16	16	>16	12	16	>4	>32	>32	>8	>64	>8	0.25	0.12	≤2
K. pneumoniae
Kpn-35786d	ICU	09/2016	Catheter	11	46.826	A (IncR)	>8	>16	>16	>16	>32	>16	2	>32	16	>8	>64	>8	>16	1	>128
E. coli
Eco-36682d	ICU	11/2016	Catheter	216	81.348	B (IncR and IncN3)	4	4	2	>16	32	4	2	2	>32	>8	0.06	≤0.06	≤0.06	0.25	≤2
M. morganii
Mmo-37590d	ICU	12/2016	Urine	NA	30.051	C (IncP6)	>8	8	>16	>16	16	8	1	2	32	8	>64	8	>16	0.5	>128

^aICU, intensive care unit; HD, hematology department.

^bST, sequence type; NA, not applicable.

^cCtx, cefotaxime; Caz, ceftazidime; Fep, cefepime; Atm, aztreonam; Imp, imipenem; Mem, meropenem; Etp, ertapenem; Gen, gentamicin; Amk, amikacin; Tob, tobramycin; Sxt, trimethoprim-sulfamethoxazole; Cip, ciprofloxacin; Col, colistin; Tgc, tigecycline; Fos, fosfomycin.

^dKPC-2-like-producing isolates recovered from the same patient.

Susceptibility to various antimicrobial agents was determined by the broth dilution method (8). MICs, interpreted according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) criteria (http://www.eucast.org/), showed that all KPC-2 producers exhibited resistance to aminopenicillins, aminopenicillin-inhibitor combinations (data not shown), cephalosporins, and aztreonam, and were nonsusceptible to carbapenems. Additionally, KPC-2 producers also exhibited resistance to several non-β-lactam antibiotics, whereas all isolates remained susceptible to tigecycline (Table 1).

The population structure of KPC-2-producing isolates studied by multilocus sequence typing (MLST) (9–11) is shown in Table 1. The C. freundii isolates comprised three sequence types (STs). ST18 was the most prevalent, accounting for five isolates. ST18 was previously found among NDM-1-producing isolates from Denmark and VIM-1-producing isolates from Spain (12, 13). The K. pneumoniae isolate was assigned to the high-risk clone ST11, previously associated with the production of several carbapenemases (14), while the E. coli isolate belonged to ST216.

None of the clinical isolates was capable of transferring the bla_KPC-2 gene to the E. coli A15 laboratory strain by conjugation. Plasmid DNAs from clinical isolates were extracted using a Qiagen maxikit (Qiagen, Hilden, Germany) and used to transform E. coli DH5α cells. Transformants were selected on Luria-Bertani agar plates with ampicillin (50 μg/ml), confirmed to be KPC-2 producers by PCR (7), and MALDI-TOF MS meropenem hydrolysis assay (6), and tested for antimicrobial susceptibility (see Table S1 in the supplemental material). The plasmid location of the bla_KPC-2 genes was demonstrated by S1 nuclease analysis of clinical and recombinant strains (15), followed by hybridization with a digoxigenin-labeled bla_KPC probe. Plasmid analysis revealed the transfer of plasmids, most of which (n = 6) were ∼45 kb in size. The remaining plasmids were ∼80 kb (n = 2) or ∼30 kb (n = 2) in size. Replicon typing showed that eight of the plasmids, including those ∼45 kb and ∼80 kb in size, were positive for the IncR replicon (16) (Table 1), whereas the two remaining plasmids were nontypeable by the PCR-based replicon typing (PBRT) method (17, 18).

Plasmid DNAs from all KPC-2-producing transformants were extracted using a Qiagen large-construct kit (Qiagen, Hilden, Germany). Multiplexed plasmid DNA libraries were prepared using the Nextera XT library preparation kit, and 300-bp paired-end sequencing was performed on the Illumina MiSeq platform (Illumina Inc., San Diego, CA, USA) using the MiSeq v3 600-cycle reagent kit. Initial paired-end reads were quality trimmed using the Trimmomatic tool v0.33 (19) with a sliding window size of 4 bp, required average base quality ≥17, and minimum read length of 48 bases. For assembly of the plasmids, reads were mapped to the reference E. coli strain K-12 substrain MG 1655 genome (GenBank accession no. U00096) using the BWA-MEM algorithm (20), in order to filter out the chromosomal DNA. Then, all of the unmapped reads were assembled by use of the de Bruijn graph-based de novo assembler SPAdes v3.9.1 (21), using k-mer sizes 21, 33, 55, and 77. De novo assembly resulted in sets of contigs with length-weighted average k-mer coverage ranging from 23× to 95×. The sequence gaps were filled by a PCR-based strategy and Sanger sequencing. For sequence analysis and annotation, the BLAST algorithm (www.ncbi.nlm.nih.gov/BLAST), the ISfinder database (www-is.biotoul.fr/), and the ORF (open reading frame) Finder tool (www.bioinformatics.org/sms/) were utilized. Comparative genome alignments were performed using the Mauve (version 2.3.1) program (22).

Plasmid analysis revealed three types of bla_KPC-2-carrying plasmid sequences (types A to C; Table 1), with type A being the most prevalent. All plasmids contained the Tn4401a isoform of the Tn4401 transposon, which is similar to that described in plasmid pNYC, lacking 100 bp upstream of bla_KPC-2 (23).

All bla_KPC-2-carrying plasmids that were ∼45 kb in size belonged to type A and showed high degrees of similarity to each other. The plasmids included a contiguous segment of 12,036 bp (nucleotide [nt] 1 to 10294 and 45085 to 46826; GenBank accession number MF497780) sharing extensive similarity with the backbone of the recently described IncR plasmids (24). This segment was composed of regions responsible for the replication (repB gene and iteron region), maintenance (resD gene), and stability (parAB, vagCD, and umuDC operons) of the plasmids (Fig. 1).

FIG 1.

Linear maps of the bla_KPC-2-carrying plasmids. For each plasmid, the type of plasmid sequence is indicated in red next to the plasmid name. Arrows show the direction of transcription of open reading frames (ORFs), while truncated ORFs appear as rectangles (arrows within rectangles indicate the direction of transcription). Resistance genes, insertion sequence (IS) elements, and transposases are shown in red, yellow, and green, respectively. intI1 genes are shaded blue. Gray arrows or rectangles indicate plasmid scaffold regions; the replication genes are shown in purple. The remaining genes are shown in white. Homologous segments (representing ≥99% sequence identity) are indicated by light gray shading. Thin lines above and below the maps correspond to highly similar sequences from other plasmids.

In the remaining 34,790-bp sequence (nt 10295 to 45084; GenBank accession number MF497780) adjoining the boundaries of the IncR backbone, a multidrug resistance (MDR) region containing the KPC-2-encoding transposon Tn4401a was identified. The Tn4401a transposon was localized within a copy of insertion sequence IS26 (ΔIS26). Target site duplications of 5 bp (ATGCA) at the boundaries of Tn4401a indicated insertion by transposition. Upstream from ΔIS26*1, an ISEc21-like element and a 916-bp fragment of an ISEc15-like element (ΔISEc15) were found. The ISEc21-ΔISEc15 structure was at the boundary of the plasmid backbone, downstream of vagCD, in the same configuration previously described in the IncR MDR plasmid pKP1780 (24).

In the remaining part of the MDR region, an IS1 that was followed by a 674-bp segment of the IncN replication region (ΔrepA) (25) was found at the boundary of the plasmid backbone, downstream of retA. The IS1-ΔrepA structure was also identified in pKP1780 at a similar position (24). Next to this sequence, an intact IS903.B-like element and a second copy of a IS903.B-like element truncated at the 3′ end (ΔIS903.B) were found. The deleted part of IS903.B was occupied by a Tn1721-like fragment (ΔTn1721-like) consisting of the 38-bp inverted repeat (IRtnp) of the transposon, tnpA, tnpR, and tnpM. The Tn1721-like sequence also included an integron similar to In37 from pHSH2, the variable region of which comprised the aacA4, bla_OXA-1, catB3, and arr-3 cassettes (26). The IRi of In37 was located within the tnpM gene of the Tn1721-like transposon, while the 3′CS of In37 was truncated 174 bp after the start codon of orf5 (Δorf5). Immediately downstream of Δorf5, a Tn501-like sequence including the 38-bp inverted repeat (IR) of the transposon, a chrA-like gene encoding a chromate ion transporter, an IS6100, a macrolide resistance operon [mph(A)], and the remaining part of IS26 (ΔIS26*2) was found. A similar structure, which confers resistance to ampicillin, streptomycin, sulfonamides and mercury, has also been observed in plasmid pLEW517 from the primate intestinal E. coli strain 517-2H1 (27).

The type B plasmids pCfr-36049cz and pEco-36682cz appeared to be derivatives of type A IncR KPC-2-encoding plasmids characterized during the present study. Type B plasmids differed from type A plasmids by the presence of an additional 34,522-bp sequence (nt 41399 to 75920; GenBank accession number MF497781) upstream of ΔIS26*1. This sequence comprised two fragments of the Tn1331 transposon flanking a central sequence (Fig. 1). The central sequence (nt 46232 to 74780; GenBank accession number MF497781) shared extensive similarity with the sequence of pN-Cit (96% coverage and 95% identity), an IncN3-type plasmid originally described from C. freundii STE strain collected in France from a patient who had been transferred from India (28). The IncN3-derived sequence possessed genes encoding a transfer locus, and a repA gene that was 98% similar to the respective region of pN-Cit. However, a part of the IncN3 transfer system was missing, explaining the inability of pCfr-36049cz and pEco-36682cz to transfer via conjugation.

Plasmids pCfr-33795cz and pMmo-37590cz, which were assigned to type C, included a contiguous segment of 4,062-bp (nt 261 to 4322; GenBank accession number MF497782) containing the partitioning genes, parA, parB, and parC, and the replication gene repA (Fig. 1). The parABC operon of pCfr-33795cz and pMmo-37590cz was identical to those of IncP6-type plasmids like pCOL-1 described from the KPC-2-producing Pseudomonas aeruginosa COL-1 strain isolated in Colombia (29) and to pLNU-11 (GenBank accession number KX863568), which was identified from a C. freundii ATetA strain captured from the sediments of an urban coastal wetland. The putative repA product of pCfr-33795cz and pMmo-37590cz showed high amino acid sequence similarity (99%) with the replication initiation protein of pLNU-11. Additionally, type C plasmids included a 3,835-bp segment (nt 1 to 260 and 26477 to 30051; GenBank accession number MF497782) consisting of genes encoding a DNA invertase/recombinase (int), a deoxymethyltransferase (dmt), and a DNase (drn) of type II restriction module. The int-dmt-drn region has also been observed in IncQ1 bla_GES-5-carrying plasmids isolated from E. coli and Serratia marcescens strains persisting in Canada (30). The remaining 22,154-bp sequence of pCfr-33795cz and pMmo-37590cz (nt 4323 to 26476; GenBank accession number MF497782), which contained the KPC-2-encoding transposon Tn4401a, was identical to the MDR region of type A and B plasmids (Fig. 1). In contrast, in plasmid pCOL-1, the bla_KPC-2 gene was part of the Tn4401b isoform of the transposon and was located in a different insertion site.

In conclusion, the present study reports the “hidden outbreak” of ST18 KPC-2-producing C. freundii isolates in a Czech hospital. However, the bla_KPC-2 gene was also identified in other STs of C. freundii and other species of Enterobacteriaceae. In one of the patients, four different KPC-2 producers were identified during the hospitalization, implying in vivo horizontal transfer of the bla_KPC-2-carrying plasmid. Sequencing data confirmed the presence of the same bla_KPC-2-carrying plasmid in two of these isolates (Table 1), further supporting this hypothesis. Of note was that, in the remaining two isolates recovered from the same patient, two different types of bla_KPC-2-carrying plasmids were identified, indicating the ability of enterobacterial plasmids to further evolve through reshuffling.

Illumina analysis results showed that, in 6 out of the 10 isolates, the KPC-2-encoding transposon Tn4401a was localized on an IncR-type plasmid (type A). To our knowledge, this is the first report on complete sequences of IncR plasmids carrying Tn4401a transposon. However, previous studies have reported the presence of multireplicon IncFII_K2-IncR KPC-2-encoding plasmids from ST101 K. pneumoniae isolated in Italian hospitals (31, 32). In addition, type B plasmids were derivatives of type A IncR bla_KPC-2-positive plasmids carrying an IncN3-derived segment. Type C plasmids belonged to the IncP6 group and shared the same KPC-2-encoding MDR region with type A and B plasmids. Therefore, en bloc acquisition of the KPC-2-encoding MDR region by an InpP6-type replicon from type A or B plasmids is a plausible hypothesis regarding the formation of type C bla_KPC-2-carrying plasmids. All three types of plasmids were noncapable of transferring the bla_KPC-2 gene via conjugation, due to partial deletion or absence of the transfer system genes. Thus, the hypothesis of mobilization in trans of the bla_KPC-2-carrying plasmids by a coresident plasmid cannot be excluded.

The data presented here contribute to the current knowledge of KPC-2-producing Enterobacteriaceae. In agreement with the results of previous studies (16, 24, 31, 32), our findings underline the increasing clinical importance of the IncR plasmid family as well as the spreading potential of large MDR segments through reshuffling of enterobacterial plasmids.

Accession number(s).

The nucleotide sequences of pCfr-31816cz, pCfr-36049cz, pMMO-37590cz, pCfr-27569cz, pCfr-31260cz, pCfr-33038cz, pCfr-36808cz, pKpn-35786cz, pEco-36682cz, and pCfr-33795cz have been deposited in GenBank under the accession numbers MF497780, MF497781, MF497782, MG557994, MG557995, MG557996, MG557997, MG557998, MG557999, and MG558000, respectively.