First Description of an Escherichia coli Strain Producing NDM-1 Carbapenemase in Spain

Abstract

A carbapenem-resistant Escherichia coli strain (DVR22) was recovered from a stool specimen from a patient with traveler's diarrhea who had traveled to India. Molecular screening led to the first identification of NDM-1 in Spain. The bla_NDM-1 gene was located in a conjugative plasmid of ca. 300 kb that also contained the bla_CTX-M-15, bla_TEM-1, Δbla_DHA-1, and armA genes. In addition, bla_NDM-1 was preceded by an ISAba125 insertion element only found in Acinetobacter spp.

TEXT

The emergence of carbapenem resistance among Enterobacteriaceae is a major cause of concern since carbapenems currently represent the treatment of choice for severe infections caused by multidrug-resistant strains producing extended-spectrum β-lactamases (ESBLs) (8).

In addition to commonly known carbapenem-hydrolyzing enzymes in Enterobacteriaceae (IMP, VIM, KPC, and OXA-48), a novel class B metallo-β-lactamase (NDM-1) has recently been described. This enzyme, first identified in Klebsiella pneumoniae and Escherichia coli clinical isolates recovered in Sweden from a traveler returning from India, confers resistance to all β-lactams except aztreonam (22). Since then, several reports have identified bla_NDM genes worldwide that have typically been associated with multidrug-resistant strains (1, 5, 12, 16–18, 23).

A 40-year-old Spanish Caucasian male reported intermittent abdominal discomfort, fever, and bloody diarrhea about 5 days before returning from India. He visited a local dispensary in India where treatment with ofloxacin and ornidazole tablets (twice a day) was prescribed for 5 days. The patient reported to the Hospital Clinic of Barcelona 1 day after his return, still complaining of bloody diarrhea, although with fewer unformed stools. He was afebrile, without any sign of dehydration, and the rest of the physical examination was normal. The diarrhea resolved spontaneously over the next 9 days.

A carbapenem-resistant E. coli (DVR22) strain was recovered from the stool samples of the patient, and after isolation and identification, antimicrobial susceptibility profiling analysis performed with both BD Phoenix (Becton Dickinson, Franklin Lakes, NJ) and Etest strips (AB bioMérieux, Solna, Sweden) indicated that strain DVR22 was resistant to all the antibiotics tested except tigecycline (MIC of 0.75 μg/ml), fosfomycin (MIC of 32 μg/ml), and colistin (MIC of 0.5 μg/ml) (Table 1), presenting MICs of 8 μg/ml and 16 μg/ml for imipenem and meropenem, respectively, 24 μg/ml for ertapenem, and 6 μg/ml for doripenem (CLSI breakpoints from broth microdilution tests were used to classify the MICs obtained by Etest [7]). Screening for carbapenemase/MBL production yielded positive results when using either the cloverleaf test (modified Hodge test) or imipenem-EDTA Etest strips. PCR screening for β-lactamase genes followed by DNA sequencing using specific primers (NDM-1 F, 5′-CCAATATTATGCACCCGGTCG-3′, and NDM-1 R 5′-ATGCGGGCCGTATGAGTGATTG-3′) (2, 14, 21) identified the presence of bla_NDM-1, bla_CTX-M-15, bla_TEM-1, and a partial sequence of the bla_DHA-1 gene. In addition, screening for aminoglycoside resistance genes (21) identified the armA gene, encoding a 16S rRNA methylase conferring resistance to aminoglycosides.

Table 1.

In vitro susceptibilities of E. coli DVR22 and E. coli transconjugant expressing NDM-1 carbapenemase

Antibiotic(s)	MIC (μg/ml) in:
	E. coli DVR22	E. coli J53 DVR22T	E. coli J53
Amoxicillin	>256	>256	4
Amoxicillin + clavulanatea	32	32	4
Piperacillin + tazobactamb	>256	>256	1
Cefoxitin	>256	>256	2
Cefotaxime	>256	>256	0.094
Ceftazidime	>256	>256	0.125
Cefepime	256	>256	0.25
Imipenem	8	16	0.19
Meropenem	16	12	0.023
Doripenem	6	4	0.032
Ertapenem	24	18	0.008
Aztreonam	>256	>256	64
Gentamicin	>8	>8	<1
Amikacin	>32	>32	<4
Tobramycin	>8	>8	<2
Ciprofloxacin	>32	0.008	0.008

^aClavulanate was used at a fixed concentration of 2 μg/ml.

^bTazobactam was used at a fixed concentration of 4 μg/ml.

In order to study the transferability of the resistance phenotype, a biparental mating between DVR22 and the E. coli strain J53AziR was conducted and transconjugants were selected on LB agar plates containing 1 μg/ml imipenem and 100 μg/ml sodium azide (Sigma Chemical Co., St. Louis, MO). PCR and susceptibility profiling showed that all transconjugants had become resistant to all the β-lactams and aminoglycosides tested (Table 1) and had also acquired the bla_NDM-1, bla_CTX-M-15, bla_TEM-1, Δbla_DHA-1, and armA genes. Plasmid analysis by S1 nuclease–pulsed-field gel electrophoresis (PFGE) (20) was then performed on both the DVR22 strain and selected transconjugants, revealing the presence of a single plasmid of ca. 300 kb. Digoxigenin-labeled probes for the bla_NDM-1, bla_CTX-M-15, bla_TEM-1, bla_DHA-1, and armA genes were hybridized against blotted nylon membranes from the S1-PFGE gels. All probes matched the band corresponding to the 300-kb plasmid. Altogether, these results indicate that all β-lactamases plus the armA gene are located in a single conjugative plasmid. Replicon typing classified this plasmid within the incompatibility group IncHI1 (3).

Previous reports have described the concurrence of bla_NDM-1 together with additional ESBLs, mainly CTX-M-15 (1, 16–19), but this is the first time that they seem to be located on the same plasmid. Then again, this is also the first time that a bla_NDM-1 gene has been found on such a large plasmid (NDM enzyme genes are typically found in plasmids ranging from 50 to 200 kb) (1, 5, 16–18) and could reflect the formation of a cointegrate from two or more smaller plasmids carrying individual β-lactamase genes, such as bla_CTX-M-15 or bla_NDM-1. On the other hand, the armA gene appears to be commonly linked to bla_NDM-1 genes (1, 10, 13, 16, 19).

Multilocus sequence typing (MLST) (http://mlst.ucc.ie/mlst/dbs/Ecoli) and PCR-based phylogroup analysis (6) revealed that E. coli DVR22 belonged to sequence type 156 (ST156) and phylogroup B1, respectively, differing from previously described NDM-carrying E. coli sequence types (16, 19, 22). PCR analysis to detect the presence of heat-stable (ST) and heat-labile (LT) toxins, verotoxins (VT), and enteroaggregative E. coli virulence factors were all negative.

In order to characterize the genetic environment of the bla_NDM-1 gene, outward NDM primers (NDMinv-R2, 5′-GGTCGCCAGTTTCCATTTGC-3′, and NDMinv-F2, 5′-TGCCGACACTGAGCACTAC-3′) were used to perform an inverse PCR over genomic DNA from strain DVR22 partially digested with Sau3AI and ligated with T4 DNA ligase (New England BioLabs, Ipswich, MA). Sequencing of the PCR products revealed that the region flanking the 3′ end of bla_NDM-1 was very similar to that described for plasmid pNDM-HK, with a downstream trpF gene, encoding the N-(5′-phosphoribosyl)anthranilate isomerase, followed by a truncated bla_DHA-1 gene (10). The published sequence of pNDM-HK was then used as a template to design specific primers to further span the downstream region. The PCR amplicons obtained with these primers always concurred with the expected size from the pNDM-HK sequence and allowed the bla_NDM-1 gene to link up to the armA gene (Fig. 1). Sequencing of the bla_NDM-1 upstream region identified the conserved putative promoter sequence described by Poirel et al. (16), as well as the presence of an ISAba125 insertion sequence (http://www-is.biotoul.fr). Although the presence of insertion sequences upstream from the bla_NDM-1 gene has already been reported in Enterobacteriaceae (10, 16, 22), DVR22 is unique in the sense that ISAba125 (IS30 family) has only been described in Acinetobacter spp. (9, 11, 13, 15, 24) and has not been found to be linked to bla_NDM-1 before, suggesting horizontal transfer between Acinetobacter and Enterobacteriaceae. To further support this hypothesis, sequence comparison of the bla_NDM-1 upstream sequences from E. coli strain 271 (16) and plasmids pNDM-HK and pKpANDM-1 (10, 22) identified a fragment of variable length containing the right-end repeat from ISAba125 in between the bla_NDM-1 gene and the corresponding IS element as a remnant of ISAba125 insertion in all of these sequences. Moreover, the insertion sequence element ISEc33 from E. coli strain 271 is bracketed by the sequence upstream from the ISAba125 right end (Fig. 1). Reports describing NDM enzymes in Acinetobacter have already been published, but there is no information available regarding their genetic surroundings (5, 12, 13). In view of these results and also taking into account current investigations by our group concerning the identification of a chromosomally encoded ISAba125-bla_NDM-2 in A. baumannii (Paula Espinal, personal communication), it may be speculated that Acinetobacter is the source of the NDM enzymes found in Enterobacteriaceae that were originally spread by ISAba125-mediated mobilization.

Fig. 1.

Schematic drawing showing the genetic elements surrounding the bla_NDM-1 genes in E. coli 271, pkpANDM-1, pNDM-HK, and E. coli DVR22. Adapted from reference 10. The lengths of the arrows are proportional to the lengths of the genes or open reading frames (ORFs) except for the region spanning hypA to armA in both pNDM-HK and DVR22, which has been compressed to fit in the figure. The partial downstream region of ISAba125 containing the right inverted repeat (highlighted in black) found in all four sequences is shown in the lower alignment. The upper alignment shows the 42 missing base pairs from the downstream region of ISAba125 that are located upstream from the ISEc33 insertion element in the sequence from E. coli 271. Δ, truncated gene; ampR, LysR family bla_DHA-1 regulator; armA, 16S rRNA methylase gene; bla_DHA-1, class C β-lactamase gene; bla_NDM-1, New Delhi metallo-β-lactamase gene; ble, bleomycin resistance gene; hypA, putative hydrogenase nickel-incorporating gene; IRL, inverted repeat left; IRR, inverted repeat right; IS, insertion sequence; ldh, lactate dehydrogenase gene; sul1, sulfonamide resistance gene; Tn, transposon; trpF, phosphoribosylanthranilate isomerase gene. The GenBank accession numbers of the sequences are as follows: plasmid encoding NDM-1 in E. coli 271 (HQ162469), pkpANDM-1 (FN396877), pNDM-HK (HQ451074), and plasmid encoding NDM-1 in E. coli DVR22 (JF922606.1).

It is also worth mentioning that after a 4-month period, another stool specimen from the same patient was screened for bla_NDM-1 carriage, but neither the DVR22 strain nor additional carbapenem-resistant strains could be isolated. These findings suggest that while carriage of multiple resistance determinants in a single plasmid might be beneficial under certain circumstances, in the absence of selective pressure, the burden associated with the replication and expression of extrachromosomal DNA involves a fitness cost that is not affordable, leading to the eradication of NDM-carrying bacteria (4). However, the simple clearance of DVR22 unrelated to its plasmid burden cannot be excluded.

This study reports the first identification of an E. coli strain producing NDM-1 in Spain and highlights the tremendous plasticity and disseminating potential of the bla_NDM-1 gene.

Nucleotide sequence accession number.

The sequence spanning from the ISAba125 to the ampR gene from the E. coli strain DVR22 has been submitted to GenBank and assigned sequence accession number JF922606.1.