Genetic Features of bla_NDM-1-Positive Enterobacteriaceae

Population structure of NDM-1 producers.Multilocus sequence typing (MLST) was performed as described previously (7, 26) 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 (Table 1). 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) bla_CTX-M-15 gene worldwide (4, 13). Similarly, four MLST types were identified among the seven K. pneumoniae isolates (Table 1). 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 bla_KPC 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 bla_NDM-1 gene. This interesting feature is highlighted in Fig. 1, which includes data from our study but also from other studies in which information about MLST typing was available.

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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 in the present study (underlined STs) and strains reported in other studies only when the results of MLST typing were available (6, 11, 15–20, 22, 25, 29).

Antibiotic resistance gene content.PCR was used with primers specific for many resistance genes (Table 2) (1, 3, 23). Most of the bla_NDM-1-positive isolates coharbored an ESBL gene, mostly the bla_CTX-M-15 gene. The bla_TEM-1, bla_OXA-1, and bla_OXA-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, rmtA, rmtB, 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 (Table 1). 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.

Genetic support of the bla_NDM-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 (Table 1). 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 (Table 1). No bla_NDM-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 bla_NDM-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 PS1.

Plasmid incompatility groups were then assigned by using the PCR-based replicon typing method (2), identifying a variety of plasmid scaffolds carrying the bla_NDM-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 and Pseudomonas species.

PCR mapping of the bla_NDM-1-surrounding sequences.We previously identified insertion sequences (IS) located on both extremities of the bla_NDM-1 gene in E. coli 271 (19). In that isolate, the bla_NDM-1 gene was bracketed by ISEc33 and ISSen4 (19) (Fig. 2). Further analysis performed on that isolate identified that part of ISAba125, an IS element that had been previously identified in bla_NDM-1-negative Acinetobacter strains (8, 12) was present upstream of bla_NDM-1. Primers specific for ISAba125 were designed, targeting its right end comprising the promoter sequence as well as its left end (Table 2; Fig. 2). We also identified a gene we named ble_MBL which encoded a 122-amino-acid-long protein and conferred putative resistance to bleomycin immediately downstream of the bla_NDM-1 gene in E. coli GUE. Thus, primers specific for this ble_MBL gene were also designed (Table 2), and their locations are indicated in Fig. 2.

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Fig. 2.

Schematic map of bla_NDM-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 bla_NDM-1 gene (19); (B)structure in which ISAba125 is present as a truncated element, but without ISEc33 (K. pneumoniae Kp7 and 601); (C) structure in which ISAba125 is present as a full element and the ble_MBL gene is present (E. coli GUE, I5, RKI, and 5649; K. pneumoniae K2, IBN, and 419; Citrobacter freundii STE, and P. stuartii PS1); (D) structure initially identified in K. pneumoniae 05-506 (29); (E) structure in which ISAba125 is present as a full element but the bleomycin resistance ble_MBL gene is absent or truncated (E. coli RIC, K. pneumoniae 6642 and 6759, Proteus mirabilis 7892). Genes and their corresponding transcription orientations are indicated by horizontal arrows. Positions of the primers as well as the bla_NDM-1 promoter are indicated.

Genetic structures surrounding the bla_NDM-1 gene showed that at least a remnant of insertion sequence ISAba125 was systematically present upstream of the bla_NDM-1 gene. The entire ISAba125 element was identified upstream of the bla_NDM-1 gene in most isolates, except in three cases (E. coli 271 and K. pneumoniae Kp7 and 601) (Table 1). However, the ISEc33 identified in E. coli 271 was absent in K. pneumoniae Kp7 and 601 (Fig. 2). These results suggest that ISAba125 was likely at the origin of the original mobilization of the bla_NDM-1 from its progenitor.

Downstream of the bla_NDM-1 gene, the ble_MBL gene was quite systematically identified in most isolates; it was not amplified by PCR in four isolates only (Table 1). 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 bla_NDM-1 and ble_MBL genes. Therefore, our working hypothesis is that both the bla_NDM-1 and the ble_MBL gene originated from the same progenitor.

Concluding remarks.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 ble_MBL gene identified upstream and downstream, respectively, of the bla_NDM-1 gene. The diversity of genetic features associated with the bla_NDM-1 gene may explain its current high rate of spread worldwide.