Patient 1 was a 36 year old male Chinese local with lymphocytic meningitis of undetermined cause. He had no recent travel history in the last year. Multi-drug resistant K. pneumoniae 43320 was a clinical isolate from urine during his rehabilitation 3 months after admission. He had a single spike of temperature but was not septic. He recovered without specific antimicrobial treatment. Patient 2 was a 22 year old male foreigner from Vietnam admitted 2 months after Patient 1 to a different ward in the same hospital with T4 hemangioma with cord compression. Multi-drug resistant K. pneumoniae 44951 was a clinical isolate from urine 8 days after admission and 10 days from the isolate from patient 1. As this was a catheter specimen, it was considered as insignificant and no specific antimicrobial treatment was given. Although their hospital stays overlapped, there was no obvious epidemiological link between the 2 patients.
The MICs of 15 antimicrobial agents were determined using the broth microdilution test according to the recommendations of the Clinical and Laboratory Standards Institute.
The 2 carbapenem resistant K. pneumoniae were confirmed to be carrying bla NDM-1 by PCR and subsequent sequencing according to previously published primers for bla NDM-1 . Plasmid conjugation was performed using E. coli J53 azide resistant strain as recipient. Briefly, recipients and bla NDM-1 carrying K. pneumoniae were separately inoculated into brain heart infusion broth (Oxoid Ltd., Basingstoke, England) and incubated at 37 C for 4 h. They were then mixed at a ratio of 1:10 (Donor:Recipient by volume) for overnight incubation at 37 C. A 0.1-ml volume of the overnight broth mixture was spread onto a MacConkey agar plate containing sodium azide (100 micro/mL) and imipenem (2 microg/mL).
Multilocus sequencing typing (MLST) and Pulsed field gel electrophoresis (PFGE) were performed for both bla NDM-1 carrying K. pneumoniae strains. Sequences of seven housekeeping genes for MLST were obtained according to Diancourt et al., Sequences were compared with those on the MLST web site (http://www.pasteur.fr/recherche/genopole/PF8/mlst/Kpneumoniae.html) developed by Diancourt et al., and alleles and sequence types (STs) were assigned accordingly. If there was a difference in two or more alleles the strains were considered to be unrelated.
For PFGE, DNA was prepared as described previously. The restriction enzyme XbaI (New England Biolabs, Beverly, MA, USA) was used at the manufacturer's suggested temperature. Restriction fragments were separated by PFGE in 1% agarose gel (Bio-Rad, Hercules, CA, USA) in 0.5xTBE buffer (45 mM Tris, 45 mM boric acid, 1.0 mM EDTA, pH8.0) for 22 h at 200 V at a temperature of 14 C, with ramped times of 2 to 40 s using the Bio-Rad CHEF MAPPER apparatus (Bio-Rad Laboratories, Richmond, CA, USA). Gels were then stained with ethidium bromide and photographed under ultraviolet light. The resulting genomic DNA profiles, or "fingerprints", were interpreted according to established guidelines. Plasmid replicon typing was performed for transconjugants.
DNA sequencing of the NDM-1-carrying plasmids was performed with a whole genome shotgun approach using 3-kb paired-end libraries. DNA fragments of about 3-kb in length were recovered after hydrodynamic shearing and purified using size exclusion beads (AMPure, Agencourt). The DNA fragments were subsequently linked to adaptors and circularized, then sheared again by nebulization. The resulting nucleotide fragments containing the adaptor were specifically purified, then ligated to oligomers for PCR amplification. The following emulsion-based clonal amplification (emPCR) was performed following standard 454 pyrosequencing protocols. Sequencing was performed using a 454 GS Jr (454 Life Sciences, Branford, CT, USA). The complete nucleotide sequences of plasmid pTR3 and pTR4 have been submitted to GenBank and assigned sequence accession number JQ349086 and JQ349085.
De-novo sequence assembly was performed on a computer workstation using the 454 Newbler, which automatically detects long paired-end reads (Version 2.6, 454 Life Sciences, Branford, CT, USA). The contigs were manually inspected and reassembled using the Phred/Phrap/Consed. Annotation of the plasmid was manually curated after performing automatic annotation on the RAST Server. Insertion sequences and transposons were further annotated using ISfinder (http://www-is.biotoul.fr).