Ibiotics other than blactams were very likely present in their parental strains. Complete sequencing showed that although the parent isolates were unrelated based on molecular typing using PFGE and MLST, the 370-86-5 biological activity plasmid carrying the blaNDM-1 is the same. Overall, the blaNDM-1 arrying plasmid pTR3/4 is very similar to the blaNDM-1-encoding plasmid p271A from E. coli strain 271 collected in 2009 from a patient from Bangladesh [22]. A recently reported blaCTX-M-62-containing plasmid, pJIE137, also possesses a similar backbone to p271A, but carries a 5.2-kb CUP regulon region in addition [23]. These plasmids are referred to as an IncN2 subgroup which have a backbone similar to the IncN plasmid R46 but a repA gene unrelated to incN plasmids [22,23]. Plasmid pTR3 and pTR4 also possesses the CUP regulon region and are closely related to these plasmids, especially with respect to the pJIE137 backbone. The discovery of pTR3/4 adds to the IncN2 subgroup of plasmids that cannot be classified using current PCR-based surveys. It appears that the resistance genes were I-BRD9 site acquired by this plasmid backbone and have been spreading to different locations in the world. Comparison with pJIE137 revealed that the 9,180-bp blaNDM-1-containing insert region in pTR3/4, as depicted in Figure 2, was bounded by the outermost IR of Tn5403 and the 257-bp element at the other end. It had been proposed that in p271A the formation of this insert region was probably a result of progressive insertions and deletions of transposons in the fipA gene in the pJIE137 backbone or insertion of the entire region as a hybrid transposon created elsewhere [23]. The loss of the 5.2-kb CUP regulon region in p271A, on the other hand, may be explained by recombination between CUP repeats [23,27]. It is likely the 9,180-bp bp blaNDM-1 containing region may have been inserted and settled in the pJIE137-like backbone form pTR3/4, while subsequently loss of the CUP regulon region in pTR3/4 resulted in p271A. The unknown IR-associated elements associated with blaNDM-*43320 and 44951, clinical isolates from patient 1 and 2 respectively; TCJ-P1 and TCJ-P2, transconjugants from 43320 and 44951 respectively. { The MIC is presented according to the concentration of trimethoprim. doi:10.1371/journal.pone.0048737.tplasmid pJIE137, a 58,107-kb blaCTX-M-62-encoding plasmid from K. pneumoniae JIE137 identified in Australia [23]. Similar to p271A, and JIE137, pTR3/4 also have a backbone organization similar to the IncN plasmid R46. In addition, the conserved repA in these plasmids are unrelated to the IncN plasmids [23]. Since the 5.2-kb CUP region is missing in p271A, the backbone of pTR3/4 is more closely related to pJIE137. Comparative genomics studies revealed that, apart from a 9-kb region containing the blaNDM-1 gene in pTR3/4 (Figure 2) 1527786 and two resistance regions (a class 1 integron/ Tn and a complex ISEcp1-blaCTX-M-62 transposition unit) in pJIE137, the backbone sequences of pTR3/4 and pJIE137 are 97 identical (Figure 1).Sequence Comparison of the Immediate Region Near blaNDM-The immediate flanking regions of blaNDM-1, including the bleMBL bleomycin-resistance protein gene, the trpF pseudogene, the nearby ISAba125 (interrupted), ISEc33, ISSen4 and Tn5403 are identical in pTR3/4 and p271A (Figure 2). Upstream of blaNDM-1 is a short fragment corresponding to the left extremity of an ISAba125. When compared with the E. coli DVR22 sequence from Spain [GeneBank accession no. JF922606; [24]], it is apparen.Ibiotics other than blactams were very likely present in their parental strains. Complete sequencing showed that although the parent isolates were unrelated based on molecular typing using PFGE and MLST, the plasmid carrying the blaNDM-1 is the same. Overall, the blaNDM-1 arrying plasmid pTR3/4 is very similar to the blaNDM-1-encoding plasmid p271A from E. coli strain 271 collected in 2009 from a patient from Bangladesh [22]. A recently reported blaCTX-M-62-containing plasmid, pJIE137, also possesses a similar backbone to p271A, but carries a 5.2-kb CUP regulon region in addition [23]. These plasmids are referred to as an IncN2 subgroup which have a backbone similar to the IncN plasmid R46 but a repA gene unrelated to incN plasmids [22,23]. Plasmid pTR3 and pTR4 also possesses the CUP regulon region and are closely related to these plasmids, especially with respect to the pJIE137 backbone. The discovery of pTR3/4 adds to the IncN2 subgroup of plasmids that cannot be classified using current PCR-based surveys. It appears that the resistance genes were acquired by this plasmid backbone and have been spreading to different locations in the world. Comparison with pJIE137 revealed that the 9,180-bp blaNDM-1-containing insert region in pTR3/4, as depicted in Figure 2, was bounded by the outermost IR of Tn5403 and the 257-bp element at the other end. It had been proposed that in p271A the formation of this insert region was probably a result of progressive insertions and deletions of transposons in the fipA gene in the pJIE137 backbone or insertion of the entire region as a hybrid transposon created elsewhere [23]. The loss of the 5.2-kb CUP regulon region in p271A, on the other hand, may be explained by recombination between CUP repeats [23,27]. It is likely the 9,180-bp bp blaNDM-1 containing region may have been inserted and settled in the pJIE137-like backbone form pTR3/4, while subsequently loss of the CUP regulon region in pTR3/4 resulted in p271A. The unknown IR-associated elements associated with blaNDM-*43320 and 44951, clinical isolates from patient 1 and 2 respectively; TCJ-P1 and TCJ-P2, transconjugants from 43320 and 44951 respectively. { The MIC is presented according to the concentration of trimethoprim. doi:10.1371/journal.pone.0048737.tplasmid pJIE137, a 58,107-kb blaCTX-M-62-encoding plasmid from K. pneumoniae JIE137 identified in Australia [23]. Similar to p271A, and JIE137, pTR3/4 also have a backbone organization similar to the IncN plasmid R46. In addition, the conserved repA in these plasmids are unrelated to the IncN plasmids [23]. Since the 5.2-kb CUP region is missing in p271A, the backbone of pTR3/4 is more closely related to pJIE137. Comparative genomics studies revealed that, apart from a 9-kb region containing the blaNDM-1 gene in pTR3/4 (Figure 2) 1527786 and two resistance regions (a class 1 integron/ Tn and a complex ISEcp1-blaCTX-M-62 transposition unit) in pJIE137, the backbone sequences of pTR3/4 and pJIE137 are 97 identical (Figure 1).Sequence Comparison of the Immediate Region Near blaNDM-The immediate flanking regions of blaNDM-1, including the bleMBL bleomycin-resistance protein gene, the trpF pseudogene, the nearby ISAba125 (interrupted), ISEc33, ISSen4 and Tn5403 are identical in pTR3/4 and p271A (Figure 2). Upstream of blaNDM-1 is a short fragment corresponding to the left extremity of an ISAba125. When compared with the E. coli DVR22 sequence from Spain [GeneBank accession no. JF922606; [24]], it is apparen.
