The resistome of the multidrug-resistant Escherichia coli strain 271 carrying the plasmid-mediated bla NDM-1 carbapenemase gene was analyzed by high-throughput genome sequencing. The p271A plasmid carrying the bla NDM-1 gene was 35.9 kb in size and possessed an IncN-type backbone that harbored a novel replicase gene. Acquisition of the bla NDM-1 gene on plasmid p271A had been likely the result of a cointegration event involving the transposase of Tn5403. The expression of bla NDM-1 was associated with the insertion sequence ISAba125 likely originating from Acinetobacter baumannii. E. coli 271 accumulated multiple resistance determinants, including five -lactamase genes (comprising the extended-spectrum -lactamase CTX-M-15), two 16S RNA methylase ArmA-and RmtB-encoding genes, and the qepA gene encoding an efflux pump involved in resistance to fluoroquinolones. These resistance genes were located on three additional plasmids, of 160 kb (IncA/C), 130 kb (IncF), and 110 kb (IncI1). In addition, several chromosomally encoded resistance determinants were identified, such as topoisomerase mutations, porin modifications and truncations, and the intrinsic ampC gene of E. coli that was weakly expressed. The multidrug resistance pattern observed for E. coli 271 was therefore the result of combined chromosome-and plasmid-encoded mechanisms.Recent reports show unambiguously that the bla NDM-1 gene encoding the metallo--lactamase (MBL) NDM-1 is spreading worldwide (31,35,36). The bla NDM-1 gene was initially identified in Klebsiella pneumoniae and Escherichia coli isolates but has been recently reported to occur in Citrobacter freundii, Morganella morganii, Providencia spp., and Enterobacter cloacae isolates (24,32,40). It is noteworthy that the bla NDM-1 gene was most often reported to be located on plasmid supports. Additionally, the bla NDM-1 and bla NDM-2 genes have been identified recently on the chromosome of Acinetobacter baumannii and, very recently, in the genome of Pseudomonas aeruginosa isolates (14,18,19). Spread of bla NDM-1 is considered a serious threat, because the enzyme it encodes possesses a broad spectrum of activity.Our study was initiated by isolation of a multidrug-resistant E. coli strain (strain 271) in Australia from a patient transferred from Bangladesh (38). The E. coli 271 isolate was resistant to all -lactams, including carbapenems, all aminoglycosides, fluoroquinolones, nitrofurantoin, and sulfonamides, remaining susceptible only to tetracycline, tigecycline, fosfomycin, and colistin (38).We previously showed that E. coli 271 harbored the bla NDM-1 gene on a plasmid (p271A) that does not carry other resistance determinants and that was not typeable by using the PCR-based replicon typing (PBRT) technique (4).In order to identify which were the resistance mechanisms involved in the multidrug resistance pattern of E. coli 271, and to identify the plasmid backbone that carried the bla NDM-1 gene, the whole genome of this isolate was determined.
MATERIALS AND METHODSHigh-density pyrosequencing an...