Recent reports showed that the plasmid-mediated blaNDM-1
gene encoding the metallo-β-lactamase (MBL) NDM-1 is spreading worldwide, mainly in members of the Enterobacteriaceae
originating primarily from the Indian subcontinent (10
Our aim was to compare the genetic features of NDM-1-producing strains recovered from unrelated countries by analyzing a collection of 16 multidrug-resistant and blaNDM-1-pos-itive enterobacterial isolates (). The genetic context and plasmid support of the blaNDM-1 gene were investigated, and the main broad-spectrum resistance determinants to non-β-lactam antibiotics were also examined.
Genetic features associated with the blaNDM-1 gene
Population structure of NDM-1 producers.
Multilocus sequence typing (MLST) was performed as described previously (7
) for Escherichia coli
and Klebsiella pneumoniae
isolates. Five MLST types were identified among the six E. coli
isolates, showing that diverse NDM-1-positive clones were circulating (). Only one out of the six E. coli
isolates was an ST131 type, which is the major clone carrying the extended-spectrum ß-lactamase (ESBL) blaCTX-M-15
gene worldwide (4
). Similarly, four MLST types were identified among the seven K. pneumoniae
isolates (). The ST14 type that corresponds to that of the first described NDM-1-producing K. pneumoniae
isolate recovered in Sweden from an Indian patient (29
) was also identified in three isolates, one each from India, Oman, and Kenya. Pulsed-field gel electrophoresis (PFGE) performed as described previously (4
) and interpreted according to the criteria of Tenover et al. (27
) showed that isolates from Oman, Kenya, and Sweden were clonally related, whereas the remaining ST14 isolate from India had a significantly different pattern (data not shown). In addition, the two ST147 K. pneumoniae
isolates, one each from Switzerland and Iraq, were clonally related. The ST258 type known to carry the blaKPC
genes worldwide was not identified here (5
Just by analyzing strains isolated in India or elsewhere but from patients previously hospitalized on the Indian subcontinent, a large variety of ST types was evidenced, emphasizing the diversity of clones carrying the blaNDM-1 gene. This interesting feature is highlighted in , which includes data from our study but also from other studies in which information about MLST typing was available.
Fig. 1. Worldwide distribution of NDM-1-producing E. coli and K. pneumoniae isolates. The color codes for the isolates are used to indicate the country where the isolate was recovered. Circles, E. coli; stars, K. pneumoniae. Isolates include those identified (more ...) Antibiotic resistance gene content.
PCR was used with primers specific for many resistance genes () (1
). Most of the blaNDM-1
-positive isolates coharbored an ESBL gene, mostly the blaCTX-M-15
gene. The blaTEM-1
, and blaOXA-10
genes were also frequently detected. Cooccurrence of 16S RNA methylase genes encoding broad-spectrum aminoglycoside resistance was often observed, being of different types (armA
, and rmtC
). Among the plasmid-mediated quinolone resistance genes, a qnr
-type gene was identified in a few cases, as well as the aac(6′)-Ib-cr
gene encoding reduced susceptibility to ciprofloxacin and the qepA
efflux pump-encoding gene (). These results indicate that the NDM-1 producers have gathered uncommon and unrelated broad-spectrum resistance genes, suggesting that they have been selected by broad-spectrum antibiotics. These high levels of resistance did not result from single genetic events.
Oligonucleotides used in this study
Genetic support of the blaNDM-1 gene.
Mating-out assays performed as described previously (24
) with NDM-1 producers as the donors and azide-resistant E. coli
J53 as the recipient and using a selection based on cefoxitin (10 μg/ml) (cefoxitin being a substrate of NDM-1 but not of ESBLs) and azide (100 μg/ml) produced transconjugants except for two strains (). Analysis of plasmid DNAs as reported previously (9
) identified a variety of plasmid supports in those transconjugants according to their sizes, which ranged from 35 to 170 kb (). No blaNDM-1
-carrying plasmid was identified in two isolates (E. coli
RKI and Providencia stuartii
PS1), suggesting the presence of a chromosomal support. Some transconjugants possessed two plasmids, indicating that several blaNDM-1
-positive plasmids were likely not self-conjugative and required a helper plasmid for their mobilization. This was confirmed by electrotransformation experiments using plasmid extracts from donors and electrocompetent E. coli
TOP10 as the recipient and a selection based on cefoxitin (10 μg/ml). Transformants were obtained for all strains except for E. coli
RKI and P. stuartii
Plasmid incompatility groups were then assigned by using the PCR-based replicon typing method (2
), identifying a variety of plasmid scaffolds carrying the blaNDM-1
gene. Our data indicate that its current spread is therefore not related to a single plasmid. Several plasmids were of narrow host range, such as the IncF plasmids, and thus able to disseminate among Enterobacteriaceae
only; others were of broad host range, such as the IncA/C types, able to also replicate in Acinetobacter
PCR mapping of the blaNDM-1-surrounding sequences.
We previously identified insertion sequences (IS) located on both extremities of the blaNDM-1
gene in E. coli
). In that isolate, the blaNDM-1
gene was bracketed by ISEc33
) (). Further analysis performed on that isolate identified that part of ISAba125
, an IS element that had been previously identified in blaNDM-1
) was present upstream of blaNDM-1
. Primers specific for ISAba125
were designed, targeting its right end comprising the promoter sequence as well as its left end (; ). We also identified a gene we named bleMBL
which encoded a 122-amino-acid-long protein and conferred putative resistance to bleomycin immediately downstream of the blaNDM-1
gene in E. coli
GUE. Thus, primers specific for this bleMBL
gene were also designed (), and their locations are indicated in .
Fig. 2. Schematic map of blaNDM-1-associated genetic structures identified among enterobacterial isolates. (A) Structure identified from E. coli 271 in which the ISEc33 and ISSen4 elements have been identified on both ends of the blaNDM-1 gene (19); (B)structure (more ...)
Genetic structures surrounding the blaNDM-1 gene showed that at least a remnant of insertion sequence ISAba125 was systematically present upstream of the blaNDM-1 gene. The entire ISAba125 element was identified upstream of the blaNDM-1 gene in most isolates, except in three cases (E. coli 271 and K. pneumoniae Kp7 and 601) (). However, the ISEc33 identified in E. coli 271 was absent in K. pneumoniae Kp7 and 601 (). These results suggest that ISAba125 was likely at the origin of the original mobilization of the blaNDM-1 from its progenitor.
Downstream of the blaNDM-1
gene, the bleMBL
gene was quite systematically identified in most isolates; it was not amplified by PCR in four isolates only (). Those isolates were from France and Switzerland, and the source of contamination could be traced back to India, Pakistan, and Serbia (21
; this study). This would suggest that an ISAba125
-related initial mobilization mechanism had been responsible for an acquisition en bloc
of both the blaNDM-1
genes. Therefore, our working hypothesis is that both the blaNDM-1
and the bleMBL
gene originated from the same progenitor.
This study showed that almost all NDM-1 producers coexpressed plasmid-mediated AmpCs or ESBLs, which may have initially contributed to the frequent use of carbapenems for treating related infections. As opposed to what has been observed for CTX-M-15 or KPC-2, NDM-1 is not associated with certain clones, plasmids, or transposons. The only common features identified here correspond to an ISAba125 element and a bleMBL gene identified upstream and downstream, respectively, of the blaNDM-1 gene. The diversity of genetic features associated with the blaNDM-1 gene may explain its current high rate of spread worldwide.