First Description of bla NDM-7 Carried on an IncX4 Plasmid in Escherichia coli ST679 Isolated in Spain

Abstract

This study describes the molecular characterization of an NDM-7 carbapenemase-producing Escherichia coli strain Ec188, recovered from a rectal swab of a male patient who had travelled to Pakistan before his hospitalization at the Hospital del Mar in Barcelona, Spain. The Ec188 isolate, assigned to a new multilocus sequence type ST679, was resistant to all beta-lactams, aminoglycosides (gentamicin, tobramycin, and with reduced susceptibility to amikacin), and ciprofloxacin. The bla_NDM-7 gene was located on a 50 kb IncX4 plasmid (pEc188-NDM7), both in the original and transconjugant strains. In addition, bla_CTX-M-15 was located on a 150 kb IncFIA plasmid and bla_CMY-2 on a 95 kb undetermined plasmid type, only in the wild-type strain. The immediate genetic surroundings of bla_NDM-7 included the bleo, trpf, and dsbC genes, and it was flanked by the insertion sequences IS26 and ISAba125, which appeared interrupted by IS5. The res and parA genes were found in the same orientation downstream of the IS26 element. To our knowledge, this is the first report of an NDM-7-carbapenemase carried on an IncX4 plasmid, as well as the first E. coli strain belonging to ST679 harboring an NDM β-lactamase, possibly associated with previous travel to Pakistan. In addition, this study highlights the dissemination of NDM variants accompanied by IncX-type plasmids.

Introduction

The increase in carbapenem-resistant Enterobacteriaceae is a worldwide concern. The emergence of the New Delhi metallo-β-lactamase-1 (NDM-1) has attracted considerable attention because it confers resistance to almost all β-lactam antibiotics, except for the monobactam aztreonam. Since the first report of NDM-1 in 2008 in a Klebsiella pneumoniae recovered from a Swedish patient,¹ 16 variants of this enzyme have been described (NDM-1 to -16) (www.lahey.org/studies) and it has spread among other Enterobacteriaceae and Gram-negative bacteria.² Despite its wide dissemination, a link between NDM-producing Enterobacteriaceae and the Indian subcontinent, Pakistan, and secondary reservoirs such as the Balkan states and the Middle East has been established.^3,4 In Spain, few reports of NDM-1-producing Enterobacteriaceae have been associated with travel to India,^5–7 although recent publications describe cases of Escherichia coli and K. pneumoniae producing NDM-5 and NDM-7, respectively, from nontraveler patients.^8,9 Furthermore, bla_NDM has been described as having a plasmid or chromosome location.^10,11 The majority of NDM variants have been found on IncF type plasmids,^9,12 but the NDM-7 variant, now widely distributed in E. coli and K. pneumoniae, has appeared in A/C and IncF, but mostly in IncX3 type plasmids.^10,12–17 In this report, we describe the genetic context of the bla_NDM-7 gene in an E. coli clinical strain isolated in Spain.

Materials and Methods

Bacterial strain

E. coli Ec188 was isolated from a 49-year-old male patient native to Pakistan, who has lived in Spain since 2005, but had travelled to Pakistan a month before his hospitalization at the Hospital del Mar in Barcelona in September 2013. He was admitted with a history of a chronic necrotizing pulmonary aspergillosis and superinfection caused by a methicillin-resistant Staphylococcus aureus strain. A month after treatment with voriconazole, piperacillin-tazobactam, and vancomycin, the patient deteriorated and was transferred to the intensive care unit, where the carbapenem-resistant E. coli Ec188 strain was recovered from a rectal swab in a routine screening. The isolate was identified as E. coli by the Vitek-2 automated system (bioMérieux, Marcy l'Etoile, France).

Multilocus sequence typing

Multilocus sequence typing (MLST) of the E. coli Ec188 was performed according to the Pasteur Institute scheme (www.pasteur.fr/recherche/genopole/PF8/mlst/EColi.html).

Antimicrobial susceptibility testing and detection of carbapenemase production

Antimicrobial susceptibility testing was performed by the broth microdilution Sensititre GNX2F (Thermo Fisher Scientific, Inc.) method, and the results were interpreted following the clinical break points as defined by the European Committee on Antimicrobial Susceptibility Testing (EUCAST).¹⁸ Carbapenemase activity was determined by the modified Hodge test (MHT) using an imipenem disc.¹⁹

Characterization of the antimicrobial resistance genes

Total DNA was extracted by the GenElute Bacterial Genomic DNA Kit (Sigma-Aldrich, Spain) from an overnight culture in Luria Bertani (LB) broth at 37°C. PCR for detection of extended-spectrum β-lactamases (ESBL) TEM, SHV, and CTX-M and acquired AmpC (acAmpC) β-lactamases was performed as previously described.^20,21 Detection of genes encoding carbapenemases NDM, VIM, IMP, SIM, GIM, SPM, OXA-48, and KPC was performed as previously described.^22,23 The aac(3)-IIa, aac(6′)-Ia y, and ant(2")-Ia genes conferring aminoglycoside resistance were determined, together with 16S rRNA methyltransferase family genes armA and rmtA, B, C and D, as described by Doi and Arakawa.²⁴ Resistance to quinolones was studied by the detection of mutations in the gyrA and parC genes and of plasmid-mediated quinolone resistant qnrA, qnrB, and qnrS genes.^25,26 Amplified DNA fragments were purified using the High Pure PCR Product Purification Kit (Roche) and were sent for sequencing to Macrogen, Inc. Nucleotide and deduced amino acid sequences were analyzed and compared by means of the BLAST programs from the National Center for Biotechnology Information web site (http://blast.ncbi.nlm.nih.gov/Blast.cgi).

Conjugation experiments and PCR-based replicon typing

Transferability of the resistance phenotype was studied by conjugation assays using a broth mating method at 37°C without shaking. The E. coli Ec188 strain was used as a donor and the modified E. coli Hb101 (UA6190) strain (rifampicin- and aminoglycoside resistant, lactose negative, and green fluorescent protein producing) as a recipient strain. Transconjugants were selected based on fluorescence production and grown on LB agar plates supplemented with 10 mg/L imipenem and 100 mg/L rifampicin. Transconjugants were evaluated for antimicrobial susceptibility and the presence of bla_NDM, bla_CTX-M, and bla_CMY determinants by PCR.

Ec188 and its derivative transconjugant strains were assigned to plasmid incompatibility (Inc) groups by PCR-based replicon typing (PBRT) as described by Carattoli et al.^27,28 and Johnson et al.²⁹

Genetic localization of the bla_NDM-7 gene

The genetic location of bla_NDM-7 was analyzed by pulsed-field gel electrophoresis (PFGE) and Southern Blot in the Ec 188 wild-type strain and two of the transconjugants. First, the genomic DNA was digested with the S1 nuclease (Promega) to define plasmid localization. After Southern transfer to a Hybond-N+ membrane (GE Healthcare) the fragments were hybridized with PCR-generated probes marked with the PCR DIG Probe Synthesis Kit (Roche). The different probes used were obtained from the purified product amplicons of the bla_NDM, bla_CTX-M, bla_CMY-2, IncFIA, and IncX4 genes.

Genetic context of the bla_NDM-7 gene

The genetic context of the bla_NDM-7 gene in E. coli Ec188 was analyzed by inverse PCR and sequencing as previously described.³⁰ Briefly, the genomic DNA of Ec188 was digested with XbaI (Promega). The fragments obtained were autoligated using T4 DNA ligase (Promega). The fragment of DNA containing the bla_NDM-7 gene was used as a template for an inverse PCR with primers designed in this study from the bla_NDM-7 gene sequence (NDM-7-inv-F, 5′-GCCAGCATGATCGTGATGAG-3′; NDM-7-inv-R, 5′-CTAATGCGGTGCTCAGCTTC-3′). Additional PCRs were performed to detect the IncX structure res and parA genes based on the sequence of pOM26-1 (KP776609),^4,13 pSD11 (KM21269),³¹ and pJIE143 (JN194214.1).²⁹

The schematic representation of the plasmid sequences, including their comparison and visualization, was generated by Easyfig.³²

Nucleotide sequence accession number

The sequence of bla_NDM-7 and its genetic environment from the E. coli Ec188 strain has been deposited in the GenBank database under accession number KY091260.

Results

Susceptibility testing and MLST

E. coli Ec188 was resistant to all β-lactams tested (including imipenem, meropenem, and ertapenem), as well as aminoglycosides (gentamicin, tobramycin, and with a reduced susceptibility to amikacin) and fluoroquinolones, but remained susceptible to colistin, fosfomycin, and tigecycline (Table 1). Carbapenemase activity was positive by MHT, suggesting the production of an metallo-β-lactamase (MBL). MLST indicated that Ec188 belonged to the new sequence type 679 (ST679).

Table 1.

Antibiotic Susceptibility of Escherichia coli Ec188 Strain and Its Transconjugant

	Transconjugant
Antimicrobial agent	Ec188	MIC (mg/L)
Amikacin	<4	<4
Amoxicillin-clavulanate	>16/8	>16/8
Ampicillin	>16	>16
Aztreonam	>16	<1
Cefepime	>16	16
Cefotaxime	>32	>32
Cefoxitin	>16	>16
Ceftazidime	>32	>32
Ciprofloxacin	>2	<0.12
Colistin	<1	<1
Doripenem	>4	4
Ertapenem	>2	>2
Fosfomycin	<32	<32
Gentamicin	>8	8
Imipenem	8	8
Levofloxacin	>4	<1
Meropenem	8	4
Nalidixic acid	>16	<16
Pip/tazobactam^a	>64/4	>64/4
Tetracycline	>8	<4
Ticarcillin	>64	>64
Tigecycline	<0.5	<0.5
Tobramycin	32	<2
Trimet/sulph^b	>4/76	<2/38

Pip/tazobactam: Piperacillin/Tazobactam.

Trimet/sulph: Trimethoprim/Sulfamethoxazole.

MIC, minimal inhibitory concentration.

Antimicrobial resistance genes and transfer experiments

PCR detection and sequencing analysis for ESBL, acAmpC β-lactamases, and carbapenemases showed that Ec188 harbored bla_CTX-M-15, bla_CMY-2, and bla_NDM-7 genes.

The bla_NDM-7 gene differed from bla_NDM-1 by two-point mutations at nucleotide positions 388 (G→A) and 460 (A→C), which correspond to amino acid substitutions Asp130Asn and Met154Leu, respectively.³³ Aminoglycoside resistance was associated with the acetyltransferase aac(3)-IIa gene. The16S rRNA methyltransferase family genes and quinolone resistance qnr genes tested were not present.

Transconjugants were obtained from strain E. coli Ec188 with a conjugation frequency of 1.66 × 10⁻⁷ transconjugants per recipient. All transconjugants showed resistance to all β-lactams except aztreonam, remaining susceptible to aminoglycosides and fluoroquinolones (Table 1). The presence of the bla_NDM gene in these transconjugants was confirmed by PCR, while bla_CTX-M and bla_CMY were not detected.

Plasmid and genetic localization of the bla_NDM-7 gene

S1-PFGE analysis revealed three plasmids in Ec188 of ∼50-, 97-, and 145-kb. Interestingly, the transconjugants only contained the 50-kb plasmid, which showed a signal with the IncX4 and bla_NDM probes, suggesting that bla_NDM-7 was located in a conjugative IncX4 plasmid of 50-kb (Fig. 1), designated pEc188-NDM7 (KY091260). Otherwise, bla_CTX-M-15 and IncFIA showed a positive signal in the same 145-kb plasmid and bla_CMY-2 in the 97-kb plasmid, which was nontypeable (Fig. 1).

FIG. 1.

Genetic location of bla_NDM-7, bla_CTX-M-15, and bla_CMY-2. (a) S1-nuclease digestion and pulsed-field gel electrophoresis (PFGE); (b) hybridization with bla_NDM probe; (c) hybridization with IncX4 probe; (d) hybridization with bla_CTX-M-15 probe; (e) hybridization with IncFIA; (f) hybridization with bla_CMY-2 probe. M, lambda ladder PFE marker; 1, Escherichia coli Ec188 wild-type strain; 2 and 3, transconjugant strains.

Genetic context of the bla_NDM-7 gene

The region flanking bla_NDM-7 in pEc188-NDM7 was similar to that described for other bla_NDM genes.³⁴ Apart from the NDM module -ISAba125-bla_NDM-7-bleo-trpF-dsbC, the insertion sequence ISAba125, upstream of bla_NDM-7, appeared disrupted by the IS5 element, and IS26 was found downstream of the NDM module (Fig. 2). PCR mapping using the IncX-type plasmid pOM26-1 (KP776609) and pSD11 (KM212169) sequences as a reference allowed the identification of the umuD (UV repair), parA (plasmid partition), and res (serine resolvase) genes downstream of the IS26 element. Interestingly, contrary to what is usually found in IncX3 plasmids, res and parA genes were located in the same orientation as in the IncX4 plasmid pSD11 (Fig. 2). A comparison of the region comprising the IS5 and parA genes among plasmids carrying the bla_NDM-5 (pNDM-MGR194) and bla_NDM-7 genes (pOM26-1, pKpN01-NDM7, and pEc188-NDM-7) showed that the immediate genetic surroundings of the bla_NDM were identical in all of them (Fig. 2). pOM26-1 and pKpN01-NDM7 were the most similar to pEc188-NDM7 in this study, with 88% and 85% of identity, respectively, with the exception of the res gene.

FIG. 2.

Schematic representation of the IncX4-type plasmid pEc188-NDM7 carrying bla_NDM-7 in the E. coli Ec188 and comparative structures with IncX3 plasmids: pNDM_MGR196 (GenBank accession number KF220657), pOM26.1 (GenBank accession number KP776609), pKpN01-NDM7 (GenBank accession number CP012990.1), and the IncX4 plasmid pSD11 (GenBank accession number KM212169). Regions of homology are shaded in gray scale.

Discussion

The aim of this study was to characterize the genetic surroundings of the bla_NDM-7 gene found in the E. coli Ec188 clinical isolate. The growing resistance to carbapenems among Enterobacteriaceae is associated with the NDM-1 β-lactamase, which is well known to have a wide dissemination and increasing number of novel variants, indicating a rapid evolution and spread.^6,12,35

Worryingly, dissemination of NDM-7 in E. coli isolates is increasing and in all reported cases, isolates have belonged to different ST. Since the first identification in France,³³ it has also been found in Germany,¹⁴ Japan,³⁵ India,¹² Canada,¹³ China,¹⁷ United States,¹⁵ Spain,⁷ and in the Arabian Peninsula⁴ and very recently, its occurrence has been described without any links with hospitalization and/or international travel.³⁶ Interestingly, there have been cases of infection by K. pneumoniae harboring NDM-7 in Spain,^6,8 Denmark,³⁷ United States,¹⁵ Canada,^13,16 and Philippines.¹⁰ Notably, NDM-7-harboring strains from different countries, including the Ec188 described herein, have shown similar genetic environments. The NDM module -ISAba125-bla_NDM-7-bleo-trpF-dsbC-, as named by Bonnin et al.,³⁴ remains conserved, in addition to the insertion sequences IS5 and IS26 flanking the module. It is important to highlight that IS26 has been reported as an element in the IS26-related recombination events and the acquisition of bla_NDM.³⁴

Otherwise, the combined presence of genes encoding ESBL (mainly CTX-M-15) and AmpC (CMY-2) is common among NDM producers, including Ec188.^6,12,14,33 However, while 16S rRNA methylases and qnr genes are frequent in strains harboring bla_NDM,^6,17 there are some exceptions, as in our Ec188 strain and the E. coli COU from France.³³

Although the majority of NDM variants have been found combined with CTX-M enzymes and linked with IncF plasmids,^9,12 it should be noted that NDM-7 has appeared in A/C, IncF, and mostly in IncX3 type plasmids.^10,12–17 To the best of our knowledge, this is the first described case of bla_NDM-7 in an IncX4-type plasmid. IncX plasmids are known to have a narrow host range and an ability to disseminate several resistance genes, including bla_TEM-52, other bla_TEM-type, bla_SHV-11, bla_CTX-M-15, bla_NDM, qnrS1, aphA1, oqxAB, and blmS.^13,29 Among IncX subgroups, IncX3 has been found in isolates carrying NDM carbapenemases other than NDM-7, including NDM-1, NDM-4, and NDM-5.^38–40

Interestingly, parA and res genes were found downstream of the IS26 element in the same orientation, which is characteristic of IncX4-type plasmids such as pSD11³¹ and pJIE143.²⁹ In contrast, in IncX3 plasmids such as pOM26-1, res and parA genes appear in opposite orientation.^4,13 Comparison with GenBank sequences revealed that the immediate NDM genetic environment of our plasmid pEc188-NDM7 was highly similar to that of IncX3 plasmids previously described in Enterobacteriaceae: pNDM_MGR194 from Australia,³⁸ pEC1 from Germany,¹⁴ pNDM7 from China,¹⁷ pOM26-1 from Oman,⁴ and pKpN01-NDM-7 from Canada.¹⁶ This suggests that the NDM genetic context in IncX4 could have arisen from IncX3 plasmids as a result of very effective homologous recombination events mediated particularly by the IS26 element, which has been previously described as critical for the mobilization of resistance genes and a frequent recombinational junction between plasmid ancestors.^31,41 Lo et al.,⁴² who have found IncX4 plasmids carrying bla_CTX-M from different host sources, reported the efficient transfer of IncX4 plasmids at different temperatures and underscored the ability of this plasmid to disseminate a variety of antimicrobial resistant determinants.^29,42

To date, the few reports of NDM-1-producing Enterobacteriaceae in Spain have been mainly associated with travel to India.^5–7 However, Pitart et al.⁹ described the first case of an NDM-5-producing E. coli isolate from a nontraveler patient, and Seara et al.⁶ reported the first outbreak of NDM-7-producing K. pneumoniae in three different hospitals, without any proven connection with India or Pakistan. Although our NDM-7-producing E. coli was likely related with previous travel to Pakistan, it should be emphasized that the immediate genetic context of the bla_NDM-7 in Ec188 also resembled that of the bla_NDM-containing module detected in the other studies.^6,9 This might indicate a conserved bla_NDM genetic environment in both E. coli and K. pneumoniae, as well as the spread of sporadic strains rather than a specific clone.

There are also several reports of IncF plasmids co-expressing bla_CTX-M-15,^9,12,34,43 including in Ec188 in the current study. Significantly, IncF-type plasmids have been involved in bla_NDM-1 acquisition in isolates from different countries, as well as the worldwide dissemination of bla_CTX-M-15.³⁴

To the best of our knowledge, this study constitutes the first description of bla_NDM-7 carried on an IncX4 plasmid in an E. coli isolate in Spain, as well as the first E. coli strain belonging to ST679 harboring an NDM β-lactamase related with previous travel to Pakistan. In addition, this study highlights the dissemination of NDM variants accompanied by IncX-type plasmids across country borders, as well as among different bacterial genera. Permanent surveillance to contain carbapenem resistance and implement appropriate treatments against multidrug-resistance microorganisms is required.

Footnotes

Acknowledgments

This study was supported by the Plan Nacional de I+D+i and Instituto de Salud Carlos III, Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Economía y Competitividad, Spanish Network for Research in Infectious Diseases (REIPI RD12/0015/0017)—co-financed by European Development Regional Fund “A way to achieve Europe” ERDF. P.E. is funded by the Instituto de Salud Carlos III “Sara Borrell” contract number CD15/00017.

Disclosure Statement

No competing financial interests exist.

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First Description of bla NDM-7 Carried on an IncX4 Plasmid in Escherichia coli ST679 Isolated in Spain

Abstract

Introduction

Materials and Methods

Bacterial strain

Multilocus sequence typing

Antimicrobial susceptibility testing and detection of carbapenemase production

Characterization of the antimicrobial resistance genes

Conjugation experiments and PCR-based replicon typing

Genetic localization of the blaNDM-7 gene

Genetic context of the blaNDM-7 gene

Nucleotide sequence accession number

Results

Susceptibility testing and MLST

Antimicrobial resistance genes and transfer experiments

Plasmid and genetic localization of the blaNDM-7 gene

Genetic context of the blaNDM-7 gene

Discussion

Footnotes

Acknowledgments

Disclosure Statement

References

Genetic localization of the bla_NDM-7 gene

Genetic context of the bla_NDM-7 gene

Plasmid and genetic localization of the bla_NDM-7 gene

Genetic context of the bla_NDM-7 gene