Our study was initiated by the recovery of a multidrug-resistant E. coli isolate from a urine sample of a 67-year-old man who had been hospitalized at St. George Hospital, Sydney, Australia, following a medical transfer from Bangladesh, where he had been hospitalized for pneumonia over a period of 12 days. MICs were determined by Etest (AB bioMérieux, Solna, Sweden) on Mueller-Hinton (MH) agar plates at 37°C, and results of susceptibility testing were recorded according to CLSI guidelines (3) (Table ​(Table1).1). E. coli isolate 271 was resistant to all β-lactams (including carbapenems), all aminoglycosides, fluoroquinolones, nitrofurantoin, and sulfonamides, remaining susceptible only to tetracycline, tigecycline, fosfomycin, and colistin. MBL detection performed by using Etest MBL strips (AB Biodisk, Solna, Sweden) was positive.

Shotgun cloning experiments performed as described previously (14), followed by sequencing, revealed that the genetic structures surrounding the bla_NDM-1 gene diverged significantly from those observed in K. pneumoniae 05-506 (20). A novel insertion sequence element, namely, ISEc33 (http://www-is.biotoul.fr), was identified upstream of the bla_NDM-1 gene. ISEc33 shared 88% nucleotide identity with the most closely related element IS630 (belonging to the IS630 family and previously identified in Shigella sonnei), with their respective transposases sharing 93% amino acid identity. ISEc33 was bracketed by a 2-bp duplication (TA), as observed for other IS630-like elements, therefore suggesting an independent transposition of that mobile element.

Detailed analysis of the 194 bp separating the bla_NDM-1 start codon from ISEc33 revealed a promoter that was made of −35 (TTGAAT) and −10 (TACAGT) sequences separated by an optimal 17-bp distance. It is noteworthy that no obvious promoter that could play a role in bla_NDM-1 expression was identified in ISEc33 (Fig. ​(Fig.11 A). The locations of these promoter sequences were further analyzed by mapping the bla_NDM-1 transcription start site using 5′ rapid amplification of cDNA ends (5′ RACE) (version 2.0; Invitrogen/Life Technologies, Cergy-Pontoise, France), as described previously (9). The +1 transcription site of bla_NDM-1 was identified 7 bp downstream of the putative −10 sequence indicated above, thus confirming our in silico analysis. This result indicated that the expression of bla_NDM-1 was not driven by a promoter provided by ISEc33. The same promoter sequences were identified upstream of the bla_NDM-1 gene in K. pneumoniae 05-506 (Fig. 1A and B). The bla_NDM-1 upstream sequences diverged exactly at the ISEc33 location. Downstream from the bla_NDM-1 gene, another novel IS element, namely, ISSen4 (belonging to the IS3 family, subgroup IS407), previously identified in Salmonella enterica serovar Choleraesuis (GenBank no. EU219534) and not bracketed by any target site duplication, was identified in E. coli 271 (Fig. ​(Fig.1A).1A). This ISSen4 element was absent in the sequence identified from K. pneumoniae 05-506. It seems, therefore, that the mobilization events that were at the origin of acquisition of the bla_NDM-1 gene in E. coli 271 and K. pneumoniae 05-506 differed significantly.

Since E. coli 271 was resistant to all β-lactams, including aztreonam, which is not a substrate for MBLs, an additional extended-spectrum β-lactamase (ESBL) was sought. PCR followed by sequencing using specific primers for bla_TEM, bla_SHV, bla_PER-1, bla_VEB-1, bla_GES-1, and bla_CTX-M ESBL genes (12) identified the ESBL CTX-M-15, together with penicillinase TEM-1. In addition, screening of 16S rRNA methylase-encoding genes was performed by using a multiplex PCR approach as described previously (1) and identified two methylase genes, namely, armA and rmtB.

Plasmid analysis performed using the Kieser technique (7) revealed that E. coli 271 harbored four plasmids of ca. 160, 130, 80, and 50 kb. By using a PCR-based replicon typing (PBRT) method as described previously (2), we showed that these plasmids belong to incompatibility groups IncI1 and IncF, respectively. Transfer of the β-lactam resistance markers from E. coli 271 to E. coli J53 (azide resistant) was performed by mating assays, with selection based on different and amoxicillin, 100 μg/ml) (9). The E. coli TOP10(p271A) transconjugant showed an MBL phenotype and was susceptible to non-β-lactam antibiotics. Plasmid analysis revealed that E. coli TOP10(p271A) harbored a bla_NDM-1-positive 50-kb plasmid, which could not be typed. The bla_CTX-M-15 gene was carried on an 80-kb IncF plasmid, although the rmtB gene together with bla_TEM-1 was carried on a 130-kb IncFII plasmid. Since the incompatibility group of plasmid p271A carrying bla_NDM-1 was not determinable, attempts to evaluate a possible broad host range were performed. For that purpose, DNA of plasmid p271A was electrotransformed in Pseudomonas aeruginosa and Acinetobacter baumannii recipient strains, and selection was performed with ticarcillin (50 μg/ml)-containing Mueller-Hinton (MH) plates, as described previously (11, 15), but no transformant was obtained in either species, suggesting that plasmid p271A might have a narrow host range.

Since the worldwide spread of CTX-M-15-producing E. coli isolates has been demonstrated to be associated with the clonal dissemination of an E. coli strain belonging to sequence type 131 (ST131) (5, 10), multilocus sequence typing (MLST) was performed as described previously (10, 18) to identify the genotype of E. coli 271. PCR and sequencing of the seven different alleles followed by computer analysis on the MLST website (www.mlst.net) revealed that E. coli 271 belonged to ST101, which corresponds to a phylogenetic lineage different from that of ST131 (data not shown).