NDM-1 encoded by a pNDM-BJ01-like plasmid p3SP-NDM in clinical Enterobacter aerogenes

A carbapenem-nonsusceptible Enterobacter aerogenes strain named 3-SP was isolated from a human case of pneumonia in a Chinese teaching hospital. NDM-1 carbapenemase is produced by a pNDM-BJ01-like conjugative plasmid designated p3SP-NDM to account for carbapenem resistance of 3-SP. p3SP-NDM was fully sequenced and compared with all publically available pNDM-BJ01-like plasmids. The genetic differences between p3SP-NDM and pNDM-BJ01 include only 18 single nucleotide polymorphisms, a 1 bp deletion and a 706 bp deletion. p3SP-NDM and pNDM-BJ01 harbor an identical Tn125 element organized as ISAba125, blaNDM−1, bleMBL, ΔtrpF, dsbC, cutA, ΔgroES, groEL, ISCR27, and ISAba125. The blaNDM−1 surrounding regions in these pNDM-BJ01-like plasmids have a conserved linear organization ISAba14-aphA6-Tn125-unknown IS, with considerable genetic differences identified within or immediately downstream of Tn125. All reported pNDM-BJ01-like plasmids are exclusively found in Acinetobacter, whereas this is the first report of identification of a pNDM-BJ01-like plasmid in Enterobacteriaceae.

Enterobacter aerogenes is a Gram-negative bacterium widely found in the human gastrointestinal tract and in the environment, and generally non-pathogenic to healthy humans. Since 1990s, E. aerogenes has become an important opportunistic pathogen commonly affecting those with weakened immune systems to cause hospital-acquired infections such as pneumonia, bacteremia, urinary tract infection, surgical site infection, and meningitis (Georghiou et al., 1995;Davin-Regli et al., 1996;De Gheldre et al., 1997;Jalaluddin et al., 1998;Ronveaux et al., 1999).
The 47.27 Kb plasmid pNDM-BJ01 is isolated from a clinical A. lwoffii strain in China in 2010 (Hu et al., 2012) and it cannot be assigned into any known incompatibility group. In this study, phenotypic and biochemical experiments combined with plasmid sequencing and comparative genomics analyses disclose that production of NDM-1 by a pNDM-BJ01-like conjugative plasmid p3SP-NDM accounts for carbapenem resistance of a clinical E. aerogenes isolate recovered from a human case of pneumonia in a Chinese teaching hospital.

Bacterial Strains and Identification
The use of human specimens and all related experimental protocols were approved by the Committee on Human Research of indicated institutions and carried out in accordance with the approved guidelines, and moreover the informed consent was obtained from indicated patient. All the bacterial strains in this study were subjective for species identification by BioMérieux VITEK 2, Bruker MALDI Biotyper, and 16s rRNA gene sequencing. For determination of 16S rRNA gene sequence, the almost complete coding region of 16S rRNA gene was amplified by PCR with the universal primers 27f (AGAGTTTGATC-CTGGCTCAG) and 1492r (TACCTTGTTACGACTT) (Frank et al., 2008). The major carbapenemase and ESBL genes as listed in Table S1 were subjected to PCR detection. All PCR amplicons were sequenced on ABI 3730 Sequencer with the same primers for PCR.

Plasmid Transfer
Plasmid conjugal transfer experiments were carried out with the rifampin-resistant E. coli EC600 being used as recipient and the bla NDM -positive strain 3-SP as donor. 3 ml of overnight culture of each of donor and recipient bacteria were mixed together, harvested, and resuspended in 80 µl of Brain Heart Infusion (BHI) broth (BD Biosciences). The mixture was spotted on a 1 cm 2 filter membrane that was placed on BHI agar (BD Biosciences) plate, and then incubated for mating at 37 • C for 12-18 h. Bacteria were washed from filter membrane and spotted on Muller-Hinton (MH) agar (BD Biosciences) plate containing 750 µg/ml rifampin and 200 µg/ml ampicillin for selection of bla NDM -positive E. coli transconjugants.
To prepare competent cells for plasmid electroporation, 200 ml of overnight culture of E. coli DH10B in Super Optimal Broth (SOB) at an optical density (OD 600 ) of 0.4-0.6 was washed three times with electroporation buffer (0.5 M mannitol and 10% glycerol) and concentrated into a final volume of 2 ml. 1 µg of plasmid DNA, which was isolated from 3-SP with QIAGEN Plasmid Midi Kit, were mixed with 100 µl of competent cells for electroporation at 25 µF, 200 , and 2.5 Kv. The resulting cells were suspended in 500 µl of SOB, and an appropriate aliquot was spotted on SOB agar plate containing 200 µg/ml ampicillin for selection of bla NDM -positive E. coli electroporants.

S1-PFGE and Southern Blot
Bacterial genomic DNA was prepared in agarose plugs and digested with S1 nuclease (Takara). The linearized plasmids and partially digested genomic DNA were separated through the CHEF-Mapper XA PFGE system (Bio-Rad). The DNA fragments were stained with ethidium bromide (EtBr), transferred to a Hybond N + membrane (GE Amersham Biosciences) and hybridized with a DIG-labeled probe specific to bla NDM (Rasheed et al., 2013). Probe labeling and signal detection were carried out with DIG high primer DNA labeling and detection starter kit II according to the manufacturer's instructions (Roche Diagnostics).

Determination of Minimum Inhibitory Concentration (MIC)
The MIC values of indicated bacterial strains were tested by using VITEK 2 according to manufacturer's instructions, and antimicrobial susceptibility was judged by Clinical and Laboratory Standards Institute (CLSI) standard.

Determination of Plasmid DNA Sequence
The chromosome DNA-free plasmid DNA was isolated from the cell cultures of indicated E. coli transconjugant using a Qiagen large construct kit, and then sequenced by using wholegenome shotgun strategy in combination with Illumina HiSeq 2500 sequencing technology. The contigs were assembled with Velvet, and the gaps were filled through combinatorial PCR and Sanger Sequencing on ABI 3730 Sequencer. The genes were predicted with GeneMarkS and further annotated by BLASTP against UniPort and NR databases.

Nucleotide Sequence Accession Numbers
The complete sequence of plasmid p3SP-NDM was submitted to GenBank under accession number KP900015.

Carbapenem-nonsusceptible E. aerogenes 3-SP
In June 2012, an 86-year-old male with cough and fever visited a teaching hospital in Xi'an city of China. The patient had underlying sequelae of cerebral hemorrhage, and complained of recurrent pulmonary infection. The patient received oral administration with cefradine for a week, but his symptoms did not improve. The patient was subsequently hospitalized, and chest X-ray examination confirmed presence of bilateral pulmonary infection and he was accordingly diagnosed to have pneumonia. The sputum specimens were sampled on the same day of admission. On the next day, round bacterial colonies were observed after cultivation of sputum on MH agar, and the bacterial isolate designated 3-SP was identified as E. aerogenes by VITEK 2, Bruker MALDI Biotyper, and 16s rRNA gene sequencing. The antimicrobial susceptibility test using VITEK 2 indicated 3-SP was resistant to multiple β-lactam antibiotics including imipenem and meropenem but remained susceptible to fluoroquinolones. The patient accordingly received intravenous administration with levofloxacin, and his symptoms associated with pulmonary infection disappeared and he was discharged after 10 days of antimicrobial treatment.

Comparative Genomics of pNDM-BJ01-like Plasmids
p3SP-NDM is highly similar to pNDM-BJ01 with genetic differences including only 18 single nucleotide polymorphisms and an 1 bp deletion (Table S2) and a 706 bp deletion (Figure S3, see also below).
The above eight plasmids contain a highly conserved backbone composed of two separate regions of plasmid replication/transfer and one region of type VI section system, with only one structural polymorphism that a 706 bp fragment (nucleotide position 43,861-44,566 in pNDM-BJ01; located within the plasmid replication/transfer region and contains only one annotated gene encoding hypothetical protein) is deleted from p3SP-NDM and pNDM-Iz4b relative to all the other plasmids (Figure 2).  As for accessory modules (Figure 2), each of these eight plasmids contains a bla NDM−1 gene cluster located around nucleotide position 5685; in addition, pNDM-AB harbors an additional 3.5 Kb accessory region, which is located around nucleotide position 5570 and composed of an ISAba14 element and a gene encoding type I restriction-modification system methyl transferase subunit.
The bla NDM−1 gene clusters from the above eight plasmids show a conserved linear organization ISAba14-aphA6-Tn125unknown IS, and the ISAba14-aphA6 and unknown IS fragments are essentially identical structurally in these plasmids while structural differences occur within or immediately downstream of the composite transposon Tn125 (Figure 3). pNDM-BJ01 and p3SP-NDM contain the prototype Tn125, which is sequentially organized as ISAba125, bla NDM−1 , ble MBL (bleomycin resistance), trpF, dsbC, cutA, groES, groEL, ISCR27, and ISAba125 (Figure 3); Tn125 is inserted into a site downstream of aphA6 (aminoglycoside resistance), which is evidenced by presence of GTT direct repeats at both ends, and the two copies of ISAba125 likely target bla NDM−1 surrounding sequences to promote formation and transposition of Tn125 .

Discussion
NDM, initially identified in Klebsiella pneumoniae in 2009, is a metallo-β-lactamase (MBL) capable of hydrolyzing almost all clinically used β-lactams (Tiwari and Moganty, 2013), and the bla NDM genes have been found in a large collection of Gramnegative bacteria of clinical, environmental and animal origins, especially including Acinetobacter, Enterobacteriaceae, and Pseudomonas Johnson and Woodford, 2013;Dortet et al., 2014). Fourteen NDM variants have been described, differing by several amino acid changes, and a few of them have been tested for their enzymatic kinetics, which denotes that amino acid substitution is a major source of MBL activity extension Tada et al., 2013). Nevertheless, a systematic characterization of enzymatic kinetics of all the identified NDM variants is needed.
At the time of writing this paper, there are at least eight additional pNDM-BJ01-like plasmids have been deposited in GenBank. All the above plasmids are exclusively found in Acinetobacter species including A. lwoffii, A. baumannii, A. ereziniae, A. pittii, and an unidentified Acinetobacter species from China, India, and Pakistan. This is the first report of identification of a pNDM-BJ01-like plasmid in Enterobacteriaceae, indicating spread of pNDM-BJ01-like plasmids from Acinetobacter to Enterobacteriaceae.
There is only one preliminary report describing detection of bla NDM in E. aerogenes, and this strain harbors a ∼50 Kb bla NDM−1 -encoding plasmid and is recovered from the stool sample of a 1-year-old infant with cough and intermittent fever in Hunan Province of China (Ho et al., 2012). This work presents extended evidence that NDM-1 is produced by a conjugative 46.57 Kb plasmid p3SP-NDM, and accounts for carbapenem resistance of clinical E. aerogenes; phenotypic and biochemical experiments combined with plasmid sequencing and comparative genomics analyses give a deeper understanding of antibiotic resistance mechanism of this NDM-1-producing E. aerogenes strain.
Figure S1 | PCR detection of bla genes. The major ESBL and carbapenemase genes (Table S1) were screened by PCR with strains 3-SP, 3-SP-NDM-EC600, 3-SP-NDM-DH10B, ATCC BAA-2146 (a NDM-1-producing reference strain of K. pneumoniae, Rasheed et al., 2013), EC600, and DH10B. Of all the bla genes detected, only bla NDM was shown to be present in each of 3-SP, 3-SP-NDM-EC600, and 3-SP-NDM-DH10B. Figure S2 | S1-PFGE/Southern blot. The S1-digested genomic DNA samples was analyzed on an EtBr-stained PFGE gel (A), and then subjected to Southern blot hybridization with a DIG-labeled probe specific to bla NDM−1 (B). Figure S3 | Detection of carbapenemase activity. In the presence of any carbapenemase, relevant carbapenems are hydrolyzed and transformed into its carboxylic form, thus leading to a pH decrease which is detected by a color change of phenol red solution (red to yellow-orange). Ambler class A carbapenemases are, at least partially, inhibited by tazobactam, whereas class B carbapenemases (metallo-ß-lactamases) are inhibited by divalent cation chelators such as EDTA. There is no available chemical inhibitor for class D carbapenemases. In this study, the bla NDM -psitive strains 3-SP, 3-SP-NDM-EC600, 3-SP-NDM-DH10B, and ATCC BAA-2146 had class B carbapenemase activity. As expected, E. coli EC600 and DH10B had no carbapenemase activity.