ABSTRACT

blaNDM genes confer carbapenem resistance and have been identified on transferable plasmids belonging to different incompatibility (Inc) groups. Here we present the complete sequences of four plasmids carrying a blaNDM gene, pKP1-NDM-1, pEC2-NDM-3, pECL3-NDM-1, and pEC4-NDM-6, from four clinical samples originating from four different patients. Different plasmids carry segments that align to different parts of the blaNDM region found on Acinetobacter plasmids. pKP1-NDM-1 and pEC2-NDM-3, from Klebsiella pneumoniae and Escherichia coli, respectively, were identified as type 1 IncA/C2 plasmids with almost identical backbones. Different regions carrying blaNDM are inserted in different locations in the antibiotic resistance island known as ARI-A, and ISCR1 may have been involved in the acquisition of blaNDM-3 by pEC2-NDM-3. pECL3-NDM-1 and pEC4-NDM-6, from Enterobacter cloacae and E. coli, respectively, have similar IncFIIY backbones, but different regions carrying blaNDM are found in different locations. Tn3-derived inverted-repeat transposable elements (TIME) appear to have been involved in the acquisition of blaNDM-6 by pEC4-NDM-6 and the rmtC 16S rRNA methylase gene by IncFIIY plasmids. Characterization of these plasmids further demonstrates that even very closely related plasmids may have acquired blaNDM genes by different mechanisms. These findings also illustrate the complex relationships between antimicrobial resistance genes, transposable elements, and plasmids and provide insights into the possible routes for transmission of blaNDM genes among species of the Enterobacteriaceae family.

INTRODUCTION

In Gram-negative bacteria, especially the Enterobacteriaceae family, β-lactamases are the major mechanism of resistance against β-lactams. In particular, β-lactamases known as carbapenemases are becoming a key concern in the effective administration of antimicrobial therapy, as they can confer resistance to carbapenems, a major last-line antimicrobial. The NDM carbapenemase was first reported in 2009, produced by a Klebsiella pneumoniae isolated from a Swedish patient recently returned from India (1). There are currently 16 known NDM variants, and blaNDM genes have now been reported in strains sourced from every inhabitable continent and in multiple species of Enterobacteriaceae, including Escherichia coli, K. pneumoniae, and Enterobacter cloacae (2).
Plasmids are important vehicles for the capture, accumulation, and spread of various antimicrobial resistance determinants. Several different types of plasmids associated with the Enterobacteriaceae family have been reported to harbor blaNDM genes, including IncA/C, IncFII subtypes, IncH types, IncL/M, IncN (24), and IncX (5). Some of these plasmids coharbor additional antimicrobial resistance genes, including the 16S rRNA methylase genes armA and rmtC (conferring high-level aminoglycoside resistance), quinolone resistance genes (qnrB1 and qnrS1), and/or other β-lactamase genes (such as blaCMY-2 and variants and blaCTX-M-15) (6).
The original source of blaNDM is not known, but Acinetobacter spp. may have acted as an intermediate between this organism and the Enterobacteriaceae family (79). In Acinetobacter spp., blaNDM genes have often been observed within the 10,099-bp composite transposon Tn125 that is bounded by two copies of ISAba125 (912). The blaNDM gene starts 93 bp downstream of the right-hand end (IRR) of ISAba125, which provides the −35 region of a promoter (13, 14), and is followed by several genes, including bleMBL (bleomycin resistance), trpF (involved in tryptophan biosynthesis), and the mobile element ISCR27. In several Acinetobacter species plasmids (e.g., pNDM-BJ01; GenBank accession no. NC_019268 [15]), ISAba14 and an aphA6 gene (amikacin resistance) are present upstream of the ISAba125 adjacent to blaNDM-1 (Fig. 1A). In plasmids from the Enterobacteriaceae, blaNDM genes are generally found in this immediate genetic context, with at least a fragment of ISAba125 containing the −35 promoter region present upstream, within different length fragments matching Acinetobacter plasmids and associated with different mobile elements (3, 1621).
FIG 1
FIG 1 ARI-A of type 1 IncA/C2 plasmids carrying blaNDM and potential routes for blaNDM insertion. IS are shown as block arrows labeled with their name or number. DR are represented by flags of the same color. Triangles indicate the insertion sites of IS elements flanked by DR. Vertical black bars represent the transposon IR of ARI-A and IRi of class 1 In/Tn. Horizontal green and black lines represent Acinetobacter and IncA/C2 plasmid backbones, respectively. Vertical dotted lines indicate the boundaries of closely related sequences. Vertical black arrows and dotted diagonal lines indicate possible deletion and insertion events. (A) Tn125 in Acinetobacter lwoffii plasmid pNDM-BJ01. (B) ARI-A of type 1 IncA/C2 plasmids closely related to pKP1-NDM-1 and pEC2-NDM-3. (C) Possible derivation of the circular molecule inserted in pEC2-NDM-3. (D) Insertion of circular molecule carrying blaNDM into pEC2-NDM-3 and a P. mirabilis genomic island. The sequences used to draw these diagrams are from the GenBank accession numbers listed in Table 1 plus the following: pNDM-BJ01, NC_019268; pSAL-1, AJ237702; pKP048, NC_014312; SGI1-V, HQ888851; PGI1-PmPEL, KF856624.
We previously reported locally identified K. pneumoniae (22) and E. cloacae (23) clinical isolates carrying blaNDM-1, E. coli carrying blaNDM-3 (G283A, Asp95Asn) (23), and E. coli carrying blaNDM-6 (C698T, Ala233Val) (24). The blaNDM gene could be transferred from all four isolates by transformation and/or conjugation, indicating a plasmid location in each case, but replicon types were not determined (2224). In this study, we present the complete sequences of these four plasmids and a comparison of the genetic contexts of blaNDM with those in closely related plasmids.

MATERIALS AND METHODS

Bacterial isolates and plasmids.

K. pneumoniae KP1 (22) and E. cloacae ECL3 carrying blaNDM-1 (23) were isolated in Australia, as was E. coli EC2 carrying blaNDM-3 (23), while E. coli EC4 carrying blaNDM-6 (previously designated ARL10/167 [24]) was isolated in New Zealand. All isolates were from patients recently returned from India. Transconjugants in sodium azide-resistant E. coli J53Azir were available and/or were obtained by conjugation on solid medium, as previously described (17).

DNA preparation and sequencing.

Genomic DNA (gDNA) was extracted from all four isolates using the UltraClean Microbial DNA isolation kit (Mo Bio Laboratories, Inc., Carlsbad, CA, USA). DNA from KP1, ECL3, and EC4 was sequenced by Illumina HiSeq 2000 technology (Illumina, San Diego, CA, USA). Illumina sequences were de novo assembled using CLC genomic workbench v8.0 (CLC Bio, Aarhus, Denmark). Initial annotation of contigs was performed using RAST (25). IS finder (https://www-is.biotoul.fr/) and the Repository of Antibiotic-resistance Cassettes (RAC; http://rac.aihi.mq.edu.au/rac/) were used to identify insertion elements (IS) and integron components, respectively. BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) searches were used to identify related plasmids carrying blaNDM to guide PCR-based gap closure and Sanger sequencing (Macrogen, South Korea) to assemble contigs into complete plasmids.
gDNA from EC2 was sheared using a g-TUBE (Covaris) into fragment sizes targeted at 20 kb. Following purification, SMRTbell template libraries were prepared using the commercial Template Preparation kit (Pacific Biosciences Inc., Menlo Park, CA, USA) and sequenced on a Pacific Biosciences (PacBio) RSII instrument (University of Queensland Centre for Clinical Genomics [UQCCG]) using the P6 polymerase and C4 sequencing chemistry. The raw PacBio sequence data were assembled de novo using the hierarchical genome assembly process (HGAP version 2) and Quiver (26) from the SMRT Analysis software suite (version 2.3.0; PacBio) with default parameters and a seed read cutoff of 17,000 bp. Following assembly, contigs were examined for overlapping 5′ and 3′ ends (a characteristic feature of the HGAP assembly process) using Contiguity (27) and were manually trimmed to generate circular contigs. Raw sequence reads were then mapped back onto the assembled circular plasmid contig (BLASR [28] and Quiver) to validate the assembly and resolve any remaining errors.
RAST, IS finder, RAC, CLC genomic workbench v8.0, Geneious R9 (Biomatters Ltd., New Zealand, including Mauve [29]), and BLAST were used for manual annotation, alignment, single nucleotide polymorphism (SNP) detection, and other analysis and comparisons of complete plasmid sequences.

Nucleotide sequence accession numbers.

Existing GenBank entries for partial sequences of all four plasmids were updated to include the complete sequences, as follows: pKP1-NDM-1, KF992018; pEC2-NDM-3, KC999035; pECL3-NDM-1, KC887917; pEC4-NDM-6, KC887916.

RESULTS AND DISCUSSION

General features of plasmids carrying blaNDM.

Isolates KP1, EC2, ECL3, and EC4 each transferred a plasmid carrying blaNDM to E. coli J53Azir by conjugation. Plasmids carrying blaNDM assembled from whole-genome sequences (at least 50-fold coverage) were designated pKP1-NDM-1, pEC2-NDM-3, pECL3-NDM-1, and pEC4-NDM-6, respectively. pKP1-NDM-1 (137,552 bp) and pEC2-NDM-3 (160,989 bp) were identified as type 1 IncA/C2 (Table 1). The backbones of pKP1-NDM-1 and pEC2-NDM-3 are very closely related to those of several other type 1 IncA/C2 plasmids (see Table S1 in the supplemental material) and include characteristic IncA/C2 core regions, such as the conjugative transfer (tra) region and parA-parB, required for plasmid partitioning (30). They have identical replication regions, with a repA gene and 14 19-bp direct repeat sequences (iterons), which are binding sites for the RepA protein (30). Both pKP1-NDM-1 and pEC2-NDM-3 have the same ISEcp1 transposition unit carrying a blaCMY-2 variant, in this case blaCMY-6, inserted in the same location as in many other type 1 IncA/C2 plasmids, between traA and traC, flanked by 5-bp direct repeats (DR). Neither carries Tn6170, present in some type 1 IncA/C2 plasmids (31).
TABLE 1
TABLE 1 General features of IncA/C2 and IncFIIY plasmids studied here and close relativesa
PlasmidNDMSize (bp)SpeciesSTCountrybYrSourceGenBank accession no.Reference
A/C2         
    pKP1-NDM-11137,552K. pneumoniae147India/Australia2010HumanKF992018This study
    pEC2-NDM-33160,989E. coli443India/Australia2010HumanKC999035This study
    pNDM-EcoGN5681166,750E. coli1289India/CanadanaHumanKJ80240450
    pNDM-PstGN5761147,886P. stuartiiN/AIndia/CanadanaHumanKJ80240550
    pNDM1023371165,974E. colinaCanadananaNC_019045 
    pNDM105051166,744E. colinaCanadananaNC_019069 
    pNDM104691137,813K. pneumoniaenaCanadananaNC_019158 
    pNDM-KN1162,746K. pneumoniae14Kenya2009HumanJN15780451, 52
    pNDM-US1140,825K. pneumoniae11India/USA2010HumanCP00666153
    pNDM-US-21140,821K. pneumoniaena   KJ588779c 
FIIY         
    pECL3-NDM-1199,435E. cloacae265India/Australia2011HumanKC887917This study
    pEC4-NDM-66110,786E. coli101India/New Zealand2010HumanKC887916This study
    pKOX_NDM11110,781Klebsiella oxytocanaChina/Taiwan2010HumanNC_02150118
    pNDM1_EC146531109,353E. cloacae177China2014HumanKP86864743
    pNDM-EclGN5741110,786E. cloacaenaIndia/CanadanaHumanKJ81299850
    pP10164-NDM199,276Leclercia adecarboxylataN/AChina2012HumanKP90001619
    pRJF8661110,786K. pneumoniae11China2011HumanNC_02518454
    pK3511106,844K. pneumoniae147Iran/USA2014HumanKR35129041
a
Plasmids with names in bold typeface were sequenced in this study. Abbreviations: na, not available; N/A, not applicable (no multilocus typing schemes for these species); ST, sequence type.
b
Travel history is given if available, e.g., India/Australia indicates isolation in Australia from a patient recently returned from India.
c
GenBank accession no. KJ588779 implies that pNDM-US-2 was extracted in China from the same strain (ATCC BAA-2146) as pNDM-US.
Both pECL3-NDM-1 (99,435 bp) and pEC4-NDM-6 (110,786 bp) are IncFIIY type plasmids (Table 1) carrying two replicons, classified as Y4 (repA) and FIB36 (repB) by the replicon sequence typing (RST) scheme (32). The backbones of both plasmids are closely related to those of other IncFIIY plasmids carrying blaNDM (Table 1), which have not been well studied but include a conjugation (tra) region and stability (psi, parAB) and maintenance (ccdAB) genes (18, 19).

Both IncA/C2 plasmids carry blaNDM in antibiotic resistance island ARI-A.

In both IncA/C2 plasmids sequenced here, the blaNDM gene is located within an antibiotic resistance island known as ARI-A, which is found in exactly the same location in different type 1 IncA/C2 plasmids, between two tra regions (30, 31). The prototype ARI-A, found in pRMH760, is a complex hybrid transposon structure bounded by 38-bp inverted repeats (IR) interrupted by IS4321 and is inserted upstream of the rhs gene (unknown function) flanked by 5-bp DR (TTGTA) (31, 33). ARI-A in pRMH760 carries a class 1 In/Tn, with IS26-aphA1-IS26 interrupting the Tn402 tni region, and other resistance genes. Islands carrying blaNDM appear to be derived from this structure, with deletions of part of the adjacent rhs gene in some cases (3). In pNDM102337 (Table 1; Fig. 1B), nucleotides 1 to 1,617 of the 3′-conserved segment (3′-CS) of the class 1 integron are followed by a 3,562-bp region carrying a type III restriction-modification system and the rmtC 16S rRNA methylase gene and then 224 bp of the IRR end of ISEcp1. ISEcp1 is truncated by ISKpn14, which is followed by a 198-bp fragment of ISAba14 and then a region found on a number of different plasmids that contains the aac(3)-IId (gentamicin resistance) gene and ISCfr1 (34). The adjacent fragment of the Tn402 tni region has the same boundary with IS26 as in ARI-A of pRMH760, but only 217 bp of IS26 is present. This is followed by an 8,913-bp region matching Acinetobacter plasmids such as pNDM-BJ01, which includes 662 bp of the right end of ISAba14, aphA6, one copy of ISAba125, blaNDM-1, and a fragment of ISCR27.
pNDM10505, pNDM-PstGN576, and pNDM-EcoGN568 (Table 1) have a variant of the pNDM102337 ARI-A with a second ISKpn14 inserted 130 bp upstream of the left end of ISAba125 (Fig. 1B). ISKpn14-mediated deletion may have been responsible for creating the ARI-A variant present in the other closely related type 1 IncA/C2 plasmids pNDM-US, pNDM-US-2, pNDM-KN, and pNDM10469, which lack the aac(3)-IId region (Table 1; Fig. 1B) (3). pKP1-NDM-1, sequenced here, has an almost identical ARI-A, except that only 89 bp of ISAba125 is present adjacent to ISKpn14 upstream of blaNDM. This difference was confirmed by reexamining raw reads and has been seen in other partial sequences (17, 35), and ISKpn14 is ∼89% identical to IS1, which is known to cause adjacent deletions (34). All of these type 1 IncA/C2 plasmids except pNDM-KN have the same cassette array, consisting of a single fused cassette comprised of the first 87 bp of the blaOXA-30 cassette and position 17 to the end of the aacA4 cassette, overlapping by a single A (36). The mechanism(s) responsible for insertion of the blaNDM region into the proposed pNDM102337-like progenitor plasmid is unclear, but it is possible that it involved ISCR27 and/or IS26 and subsequent deletion(s).
The backbone of pEC2-NDM-3 is almost identical to the pNDM102337-like plasmids described above (see Table S1 in the supplemental material), but ISEc23 is inserted 222 bp upstream of ARI-A, flanked by 8-bp DR characteristic of this element. ARI-A of pEC2-NDM-3 includes the same rmtC region as the one described above except that IS3000 is inserted upstream of rmtC, flanked by characteristic 5-bp DR. The region containing blaNDM, however, is different from the one in the other IncA/C2 plasmids and is inserted between ISKpn14 and the aac(3)-IId/ISCfr1/tni402 region. The region matching pNDM-BJ01 encompasses 198 bp of ISAba14, aphA6, one copy of ISAba125, blaNDM, bleMBL, and trpF. ISKpn25, carrying a restriction-modification system, is inserted in ISAba125 upstream of the −35 promoter region, flanked by characteristic 8-bp DR (Fig. 1B). The blaNDM gene has the single nucleotide change giving blaNDM-3 rather than blaNDM-1, and trpF is followed by a truncated blaDHA gene and the associated ampR gene, nucleotides 180 to 1,313 of the 3′-CS and ISCR1. This region is separated from a complete ISAba14 by 934 bp matching the region upstream of ISAba14 in pNDM-BJ01. ARI-A in pEC2-NDM-3 ends with the aac(3)-IId-ISCfr1-tni402 region, but a complete copy of IS26 truncates the rhs gene in the IncA/C2 backbone. The only other known location of the blaNDM-3 variant is on an IncFII plasmid (37) associated with ISCR1 but not with the truncated blaDHA-ampR region present in pEC2-NDM-3.
This context in pEC2-NDM-3 suggests insertion of blaNDM from a circular molecule mediated by ISCR1. ISCR1 is proposed to transpose by a rolling-circle mechanism, similar to the related IS91 family elements (38), in which replication proceeds from the oriIS end, located downstream of rcr (rolling-circle replicase gene), toward the terIS upstream and can continue into and capture an adjacent region. ISCR1 has generally been found associated with class 1 integrons, after position 1,313 of the 3′-CS, suggesting integration of circular molecules by recombination in either the 3′-CS or an existing ISCR1 (38). ISCR1 has previously been suggested to be associated with movement of blaNDM (39) and was recently shown to be responsible for mobilizing a region containing blaNDM and part of the 3′-CS, but without the blaDHAΔ-ampR region, between plasmids (20).
ISCR1 appears to have been responsible for capturing the blaDHAΔ-ampR region from the Morganella morganii chromosome and inserting it into a class 1 integron (40) (Fig. 1C). Generation of a circular molecule by recombination between the two flanking 3′-CS and reintegration at ISCR1 could create the arrangement seen in, e.g., pKP048 (GenBank accession no. NC_014312), with ISCR1 downstream of the blaDHAΔ-ampR region and the 3′-CS, and the usual 3′-CS/ISCR1 boundary (Fig. 1C). ISCR1 may then have mobilized this 3′-CS segment and the blaDHAΔ-ampR region and inserted them downstream of blaNDM, before picking up the blaNDM region as part of a circular molecule (Fig. 1C).
The complete ISAba14 in pEC2-NDM-3 has the same boundary with the aac(3)-IId region as the ISAba14 fragment in pNDM102337, suggesting that homologous recombination between the complete and partial copies of ISAba14 could have been responsible for the insertion of this circular molecule into pEC2-NDM-3 (Fig. 1D). The same circular molecule carrying blaNDM also appears to have inserted in a Proteus mirabilis genomic island to create PGI-PmPEL (39) but in this case by recombination in ISCR1 (Fig. 1D), supporting the proposed mechanism of ISCR1-mediated capture of blaNDM. Regions containing the same ISCR1, 3′-CS, blaDHAΔ-ampR region, but adjacent to shorter fragments of the blaNDM region, are found in the original blaNDM-1 plasmid pKpANDM-1 (FN396876.1) (1) and in plasmids of other Inc types (3) (e.g., the IncL/M plasmid pNDM-HK) (21), suggesting capture of shorter blaNDM regions and/or subsequent deletions.

IncFIIY plasmids carry blaNDM flanked by TIMEs.

Several very closely related IncFIIY plasmids carrying a blaNDM gene have now been identified (Table 1). They all have almost identical backbones with the same insertions of multiple IS elements in the same places, mostly between the replication (repA) and plasmid stability (parA) regions (Fig. 2) and minor sequence differences (see Table S2 in the supplemental material). pKP351 alone appears to have a deletion adjacent to one copy of IS1 (41). In all of these plasmids, blaNDM lies within a 5,945-bp region matching Tn125 that includes 101 bp of ISAba125 and a fragment of ISCR27. This region is flanked by two copies of a 256-bp Tn3-derived inverted-repeat transposable element (TIME), each bounded by 38-bp IRs (42). These TIMEs, previously described as miniature inverted-repeat transposable elements (MITEs), may have been responsible for capturing the blaNDM region from a pNDM-BJ01-like plasmid (18, 19, 43). pEC4-NDM-6 is very closely related to these plasmids (see Table S2 in the supplemental material) but has the single nucleotide change giving blaNDM-6 (44) rather than blaNDM-1, suggesting mutation in this context.
FIG 2
FIG 2 Contexts of blaNDM on IncFIIY plasmids. Features are generally shown as described for Fig. 1. Solid black lines represent the IncFIIY plasmid backbone. Gray-shaded areas indicate matching plasmid backbone regions, with their sizes given. (A) Tn125 in Acinetobacter lwoffii plasmid pNDM-BJ01. (B) Comparison of IncFIIY plasmids. (C) Comparison of rmtC contexts in IncFIIY, plasmids, IncA/C2 ARI-A, and Proteus mirabilis. The sequence shown is the spacer between rmtC and the associated transposable element. The pink triangle indicates the insertion site of the TIME. The sequences used to draw these diagrams are from the GenBank accession numbers listed in Table 1 plus the following: pNDM-BJ01, NC_019268; pNDM-BJ02, NC_019281.1; ISEcp1 transposition unit in P. mirabilis, AB194779.
In most of these IncFIIY plasmids carrying blaNDM, an 11,029-bp region that includes the rmtC gene and an ISCR6-like element separates the TIME upstream of blaNDM-1 from a third copy of this TIME. TIMEs create a 5- to 6-bp DR on transposition like the Tn3 transposons from which they appear to be derived (42). In these plasmids, the 5-bp sequences adjacent to the “inside” of each TIME flanking the rmtC region are identical (TATAA). This configuration could be explained by insertion of a circular molecule, consisting of this region plus one copy of the TIME (flanked by these 5-bp sequences as DR), into the TIME upstream of blaNDM-1 (Fig. 2B). Gain and loss of the rmtC region in this way are supported by the sequences of the IncFIIY plasmids pP10164-NDM and pNDM-EC14653 (Table 1; Fig. 2B), which lack the rmtC region. Removing the TIME and one DR from this circular molecule also gives a region that matches the rmtC region found in ARI-A of IncA/C2 plasmids, also supporting this hypothesis. rmtC was originally identified in a transposition unit flanked by a DR with a complete copy of ISEcp1 that also matches part of this structure (Fig. 2C) (45). The same 30 bp separate rmtC from this complete ISEcp1 and the ISEcp1 fragment in IncA/C2 plasmids, while an additional 10 bp are present between ISCR6 and rmtC. While these contexts are clearly related, without additional examples of rmtC contexts it is difficult to say exactly how each arose.
pECL3-NDM-1 carries the same rmtC region as the other IncFIIY plasmids, but its backbone has a number of confirmed nucleotide differences (see Table S2 in the supplemental material) and a different region carrying blaNDM-1 has been inserted in a different location (Fig. 2B). This region matches pNDM-BJ02, which lacks the copy of ISAba125 downstream of blaNDM (3), rather than pNDM-BJ01, and also includes 1,369 bp of pNDM-BJ02 backbone. An IS903-like element truncates ISAba125, leaving 83 bp upstream of blaNDM-1. This 10,411-bp region replaces a 15,560-bp region present in the other IncFIIY plasmids, and it is possible that the IS903-like element was involved in the insertion of this blaNDM region into pECL3-NDM-1.

Conclusions.

In summary, the analysis presented in this study supplements and complements the catalogue of previously characterized IncA/C2 and IncFIIY plasmids carrying blaNDM. All four plasmids studied here carry segments that align to different parts of the blaNDM regions found on Acinetobacter plasmids. Different mechanisms appear to have been responsible for independently transferring different segments of Tn125 into ARI-A in the same IncA/C2 plasmid backbone (giving pKP1-NDM-1-type plasmids or pEC2-NDM-3). Other less closely related type 1 IncA/C2 plasmids, e.g., pNDM-1_Dok01 from E. coli (46) and pMR0211 from Providencia stuartii (47), also carry segments matching different parts of Tn125 and the adjacent Acinetobacter plasmid backbone within ARI-A, illustrating further variation in the ways in which blaNDM genes appear to have been acquired by similar plasmids. Different mechanisms also appear to have transferred different segments matching blaNDM contexts found in Acinetobacter baumannii to slightly different IncFIIY backbones (giving pEC4-NDM-1-type plasmids or pECL3-NDM-1).
At least theoretically, the transfer of blaNDM segments between Acinetobacter and Enterobacteriaceae plasmids could have occurred either in Acinetobacter or in one or more of the Enterobacteriaceae. Transfer of Acinetobacter plasmids carrying blaNDM into E. coli J53Azr by conjugation has been demonstrated (12, 13), and recently a pNDM-BJ01-like plasmid (p3SP-NDM) was found in an Enterobacter aerogenes isolate (48). IncA/C plasmids have also been reported in a few A. baumannii clinical isolates on the basis of PCR assays (49). While independent transfer from Acinetobacter plasmids to different types of plasmids found in the Enterobacteriaceae is possible, it may be more likely that blaNDM regions have subsequently moved between these plasmids in the Enterobacteriaceae.
The four plasmids in this study were carried by clinical isolates from Australia or New Zealand, from different patients recently returning from India. We have also recently reported partial sequences of blaNDM contexts matching pKP1-NDM-1 (with the 89-bp ISAba125 fragment) in IncA/C plasmids harbored by isolates from a hospital in Pakistan (17) and those matching pECL3-NDM-1 or pEC4-NDM-6 in IncFIIY plasmids in isolates from multiple Australian health care facilities (16). The other related IncA/C2 and IncFIIY plasmids harboring blaNDM genes discussed here were also isolated in several different countries (Table 1). This distribution illustrates the geographical spread of blaNDM genes on these particular plasmid types.
There appears to be an underlying complex network of interactions between blaNDM, different mobile elements, and different plasmids, but without access to the sequences of additional intermediate and progenitor plasmids it is difficult to fully understand the contributions that different factors make to the transmission of blaNDM genes. The different mechanisms observed here to capture relevant genes onto different plasmid types emphasize the capability of Enterobacteriaceae to adapt to their environment, especially when antimicrobial pressure is present.

ACKNOWLEDGMENT

We thank George Jacoby for providing E. coli strain J53Azr.

Supplemental Material

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cover image Antimicrobial Agents and Chemotherapy
Antimicrobial Agents and Chemotherapy
Volume 60Number 7July 2016
Pages: 4082 - 4088
PubMed: 27114281

History

Received: 19 February 2016
Returned for modification: 9 March 2016
Accepted: 22 April 2016
Published online: 20 June 2016

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Authors

Alexander M. Wailan
The University of Queensland, UQ Centre for Clinical Research, Herston, Queensland, Australia
Hanna E. Sidjabat
The University of Queensland, UQ Centre for Clinical Research, Herston, Queensland, Australia
Wan Keat Yam
The University of Queensland, UQ Centre for Clinical Research, Herston, Queensland, Australia
Present address: Wan Keat Yam, Institute of Molecular and Cell Biology, A*STAR, Singapore; Nabil-Fareed Alikhan, Microbiology and Infection Unit, Warwick Medical School, University of Warwick, Coventry, United Kingdom; Nicola K. Petty, The ithree institute, University of Technology Sydney, Sydney, Australia; Anna L. Sartor, Health Protection Branch, Department of Health, Queensland Government, Brisbane, Queensland, Australia; Deborah A. Williamson, Microbiological Diagnostic Unit Public Health Laboratory, Doherty Institute, Melbourne, Australia; Timothy R. Walsh, Department of Medical Microbiology and Infectious Disease, Institute of Infection & Immunity, Heath Park Hospital, Cardiff, United Kingdom, and School of Medicine, Cardiff University, Health Park, Cardiff, United Kingdom.
Nabil-Fareed Alikhan
Australian Infectious Diseases Research Centre, Brisbane, Australia
Present address: Wan Keat Yam, Institute of Molecular and Cell Biology, A*STAR, Singapore; Nabil-Fareed Alikhan, Microbiology and Infection Unit, Warwick Medical School, University of Warwick, Coventry, United Kingdom; Nicola K. Petty, The ithree institute, University of Technology Sydney, Sydney, Australia; Anna L. Sartor, Health Protection Branch, Department of Health, Queensland Government, Brisbane, Queensland, Australia; Deborah A. Williamson, Microbiological Diagnostic Unit Public Health Laboratory, Doherty Institute, Melbourne, Australia; Timothy R. Walsh, Department of Medical Microbiology and Infectious Disease, Institute of Infection & Immunity, Heath Park Hospital, Cardiff, United Kingdom, and School of Medicine, Cardiff University, Health Park, Cardiff, United Kingdom.
Nicola K. Petty
Australian Infectious Diseases Research Centre, Brisbane, Australia
School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, Australia
Present address: Wan Keat Yam, Institute of Molecular and Cell Biology, A*STAR, Singapore; Nabil-Fareed Alikhan, Microbiology and Infection Unit, Warwick Medical School, University of Warwick, Coventry, United Kingdom; Nicola K. Petty, The ithree institute, University of Technology Sydney, Sydney, Australia; Anna L. Sartor, Health Protection Branch, Department of Health, Queensland Government, Brisbane, Queensland, Australia; Deborah A. Williamson, Microbiological Diagnostic Unit Public Health Laboratory, Doherty Institute, Melbourne, Australia; Timothy R. Walsh, Department of Medical Microbiology and Infectious Disease, Institute of Infection & Immunity, Heath Park Hospital, Cardiff, United Kingdom, and School of Medicine, Cardiff University, Health Park, Cardiff, United Kingdom.
Anna L. Sartor
The University of Queensland, UQ Centre for Clinical Research, Herston, Queensland, Australia
Present address: Wan Keat Yam, Institute of Molecular and Cell Biology, A*STAR, Singapore; Nabil-Fareed Alikhan, Microbiology and Infection Unit, Warwick Medical School, University of Warwick, Coventry, United Kingdom; Nicola K. Petty, The ithree institute, University of Technology Sydney, Sydney, Australia; Anna L. Sartor, Health Protection Branch, Department of Health, Queensland Government, Brisbane, Queensland, Australia; Deborah A. Williamson, Microbiological Diagnostic Unit Public Health Laboratory, Doherty Institute, Melbourne, Australia; Timothy R. Walsh, Department of Medical Microbiology and Infectious Disease, Institute of Infection & Immunity, Heath Park Hospital, Cardiff, United Kingdom, and School of Medicine, Cardiff University, Health Park, Cardiff, United Kingdom.
Deborah A. Williamson
Institute of Environmental Science and Research, Wellington, New Zealand
Present address: Wan Keat Yam, Institute of Molecular and Cell Biology, A*STAR, Singapore; Nabil-Fareed Alikhan, Microbiology and Infection Unit, Warwick Medical School, University of Warwick, Coventry, United Kingdom; Nicola K. Petty, The ithree institute, University of Technology Sydney, Sydney, Australia; Anna L. Sartor, Health Protection Branch, Department of Health, Queensland Government, Brisbane, Queensland, Australia; Deborah A. Williamson, Microbiological Diagnostic Unit Public Health Laboratory, Doherty Institute, Melbourne, Australia; Timothy R. Walsh, Department of Medical Microbiology and Infectious Disease, Institute of Infection & Immunity, Heath Park Hospital, Cardiff, United Kingdom, and School of Medicine, Cardiff University, Health Park, Cardiff, United Kingdom.
Brian M. Forde
Australian Infectious Diseases Research Centre, Brisbane, Australia
School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, Australia
Australian Infectious Diseases Research Centre, Brisbane, Australia
School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, Australia
Scott A. Beatson
Australian Infectious Diseases Research Centre, Brisbane, Australia
School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, Australia
David L. Paterson
Australian Infectious Diseases Research Centre, Brisbane, Australia
Infectious Diseases Unit, Royal Brisbane and Women's Hospital, Brisbane, Australia
Pathology Queensland, Brisbane, Australia
Timothy R. Walsh
The University of Queensland, UQ Centre for Clinical Research, Herston, Queensland, Australia
Present address: Wan Keat Yam, Institute of Molecular and Cell Biology, A*STAR, Singapore; Nabil-Fareed Alikhan, Microbiology and Infection Unit, Warwick Medical School, University of Warwick, Coventry, United Kingdom; Nicola K. Petty, The ithree institute, University of Technology Sydney, Sydney, Australia; Anna L. Sartor, Health Protection Branch, Department of Health, Queensland Government, Brisbane, Queensland, Australia; Deborah A. Williamson, Microbiological Diagnostic Unit Public Health Laboratory, Doherty Institute, Melbourne, Australia; Timothy R. Walsh, Department of Medical Microbiology and Infectious Disease, Institute of Infection & Immunity, Heath Park Hospital, Cardiff, United Kingdom, and School of Medicine, Cardiff University, Health Park, Cardiff, United Kingdom.
Sally R. Partridge
Centre for Infectious Diseases and Microbiology, The Westmead Institute for Medical Research, The University of Sydney, Westmead Hospital, New South Wales, Australia

Notes

Address correspondence to Alexander M. Wailan, [email protected], or Sally R. Partridge, [email protected].

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