A 38-year-old Taiwanese man was repatriated to Taiwan after undergoing an operation in a medical center in New Delhi, India. This patient had stayed in an Indian intensive care unit for 8 days. K. pneumoniae carrying bla NDM-1 (designated KPX) was isolated from two rectal swab samples of this patient as part of his routine clinical treatment. The identification and the antimicrobial profile of these isolates were fully characterized (Table 1). About one month after the first isolation of the NDM-1 positive K. pneumoniae, the Taiwan CDC announced that the bacterium had not been detected in three consecutive samples from rectal swabs.
The cultured samples were inoculated onto MacConkey agar plates (BBL, Becton Dickinson Microbiology Systems, Cockeysville, MD) containing imipenem (1 microg/ml). Bacteria that grew on these plates were tested for the presence of carbapenemases by the Modified Hodge test (MHT). Bacteria that gave a positive result for the MHT were screened for the presence of bla NDM-1 by PCR using primers targeting the bla NDM-1 gene (GenBank accession number FN396876.1). Identification of KPX was performed by conventional biochemical methods and by 16S rRNA sequence analysis. Multilocus sequence typing (MLST) was used to identify the clonality of the isolate. Minimal inhibitory concentrations of various antimicrobials were determined by the reference broth microdilution method using custom-designed panels (Sensititre, Trek Diagnostics, West Essex, England) following the guidelines of the Clinical and Laboratory Standards Institute and by the E-test following the manufacturer's recommendations.
To identify the genetic determinates of antimicrobial resistance we performed shotgun sequencing of the KPX plasmids. The plasmids were extracted using a Qiagen large-construct kit (Qiagen, Valencia, CA, USA). Shotgun sequencing was performed to a 160-fold coverage using a Genome Sequencer Junior instrument (Roche) using a library consisting of 800-bp shotgun fragments. Additional small-size (3~4-kb and 5~8-kb inserts) shotgun libraries were constructed from the plasmid DNA and then sequenced to 2.3-fold coverage of the targets using ABI3730xl automated capillary electrophoresis sequencers (Applied Biosystems, Foster City, CA, USA). Taking the linking information of paired sequences, contigs were inspected manually and reassembled, using the Consed visualization and editing system. Two circular DNA sequences (pKPX-1 and pKPX-2) were eventually assembled; and the orientation and position of the constituent contigs were confirmed by matrix PCR and by sequencing selected cloned BamHI and EcoRI fragments.
Open reading frames (ORF) were predicted based on the start and termination codons. Sequence homology was searched against the GenBank database using the BLAST program (http://blast.ncbi.nlm.nih.gov/). Manual annotation was carried out throughout the sequence; and any discrepancies were curated.
KPX was cultured in 200-ml Luria-Bertani (LB) broth for overnight at 37 C. Bacterial cells were harvested by centrifugation and plasmids were extracted as for DNA sequencing. To separate different plasmids, 0.6 microg plasmid DNA from KPX was electroporated into ElectroMAX  DH10B  T1 Phage-Resistant electrocompetent E. coli cells (Invitrogen, Carlsbad, CA, USA) following the manufacturer's instructions. Two transformants selected from the Luria agar plate containing either 4 microg/ml imipenem (ECX-1) or 100 microg/ml ampicillin (ECX-2) were confirmed for the presence of the NDM-1 gene by antimicrobial susceptibility test and PCR.
To verify the DNA sequence assembly, plasmid DNA from KPX, ECX-1 and ECX-2 digested with different restriction enzymes was separated by either conventional agarose gel electrophoresis or pulsed field gel electrophoresis (PFGE) using a CHEF-DEII system (Bio-Rad Laboratories, Hercules, CA, USA). The PGFE was carried out using a 1% agarose gel and 0.5X TBE buffer at 6 volts per centimeter and 14 C for 16 hours, with a switch time of 1-25 seconds and a rotated angle of 120 degrees. To investigate the genetic organization of the bla NDM-1 locus, Southern blotting was performed using DIG-labeled probes (Roche Diagnostics, Mannheim, Germany).
To test the conjugal ability of KPX plasmids, the original K. pneumoniae strain, KPX, was mated to an E. coli recipient, J53, which is resistant to sodium azide, at 25 C, at 30 C, and at 37 C using overnight culture. The mating mixture was then resuspended in LB broth and spread onto plates containing both 100 microg/ml sodium azide and 100 microg/ml ampicillin. After overnight culture, the grown colonies were tested for their resistance to either imipenem or gentamicin.
In order to identify the genetic determinants for amikacin or chloramphenicol resistance, two shotgun libraries with average insert sizes of 3.5-kb and 6-kb were constructed. In total, 1920 colonies were tested for growth in the presence of amikacin or chloramphenicol medium at different concentrations of antibiotic (16, 32, and 64 microg/ml). Colonies that grew on the individual selection medium were picked. Thirty clones conferring amikacin resistance and twenty clones conferring chloramphenicol resistance were sequenced from both ends of their inserts.
Bacterial KPX was spread onto LB agar plates with different concentrations of imipenem. After overnight incubation, multiple single colonies from each plate were inoculated into LB broth that contained the same concentration of imipenem and grown at 37 C for 4-7 hours. Genomic DNA derived from each colony was extracted by Wizard DNA purification kit (Promega, Madison, WI, USA). To estimate the relative copy number of the NDM-1 gene, two sequences that could be amplified from pKPX-1 - namely pcoB (pcoB-F: TGCCATGCAGATGCCAGCAGAT; pcoB-R: ATCCGACCGTTGCCATTCCAG) and bla NDM-1 (NDM_qPCR_F: TGCCCAATATTATGCACCCGG; NDM_qPCR_R: CGAAACCCGGCATGTCGAGA) - were designed. Quantitative PCR reactions were carried out using 5 microl KAPA SYBR  FAST ABI Prism  2X qPCR Master Mix (Kapa Biosystems, Woburn, MA, USA), 2 ng genomic DNA as template, and 2 pmole of each primer in a 10 microl reaction. The Ct values of each sample was measured under appropriate PCR conditions (preheat at 50 C for 2 min; denature at 95 C for 20 sec; 40 amplified cycles at 95 C for 3 sec and 60 C for 20 sec) on an ABI PRISM 7900HT. The relative fold change of bla NDM-1 for each sample was normalized against a housekeeping gene (pcoB) by using the comparative Ct method.
This research did not involve human subjects. The bacterial samples used in this study were previously gathered as part of routine clinical work. The clinical information presented in this paper on the patient has been published previously. We consulted NHRI's Institutional Review Board regarding whether this study would require review and none was required.