Article Figures & Data
Figures
- FIG. 1.
Alignment of novel erythromycin esterase EreC (this study) with EreA and EreA2. Amino acid changes between EreC and EreA are indicated with asterisks. The single amino acid difference between EreA and EreA2 is highlighted in gray.
- FIG. 2.
Three characterized antibiotic resistance-conferring regions from K. pneumoniae 05-506. (A) The 4.3-kb region is linked to the 4.8-kb complex class 1 integron region. The genes encoding the efflux pump and lactate dehydrogenase (gray diagonal lines) are of Klebsiella origin. blaNDM-1 (dark gray) is flanked between the pathogenicity island (vertical black lines) and IS26/Tn3 (black small squares). This region lies downstream of the 4.8-kb complex class 1 integron containing Int (checkered area), arr-2, ere2A, aadA1, and cmlA7 as gene cassettes and qacΔ1 (white boxes). Downstream is an intact copy of ISCR1 (black and white diagonal lines). Arrows, direction of transcription; black ellipses, 59-base elements; Δ, genes that are truncated. (B) blaCMY-4 (gray) is located between ISEcP1 (black) and blc (white), as reported previously (16).
- FIG. 3.
Alignment of the amino acid sequences of NDM-1 with the amino acid sequences of IMP-1, IMP-2, IMP-8, VIM-1, VIM-2, GIM-1, SPM-1, SIM-1, and KHM-1. Conserved residues coordinating zinc ions are denoted with asterisks. Key amino acids are also numbered according to the BBL scheme (12). Additional amino acids unique to NDM-1 are enclosed in a light gray box. Amino acid substitutions near the active site are highlighted in plain boxes (13).
- FIG. 4.
Phylogenetic tree obtained for MBL subgroup B1. The numbers for the substitutions should be multiplied by 100. The alignment used was prepared with the CLUSTALW program. The GenBank accession numbers for the sequences are as follows: IMP-1, S71932; IMP-2, AJ243491; IMP-8, AF322577; VIM-1, Y18050; VIM-2, AF191564; GIM-1, AJ620678; SPM-1, AJ492820; SIM-1, AY887066; KHM-1, AB364006.
- FIG. 5.
PFGE of plasmids from K. pneumoniae 05-506, conjugant E. coli J53(pNDM-1), and E. coli NF-NDM-1 (from the patient's normal flora). The bands with white arrows showed positive signals by Southern blot hybridization with the NDM-1 probe (data nor shown). K. pneumoniae 05-506 carries blaNDM-1 on a 180-kb plasmid, whereas E. coli NF-NDM-1 carries blaNDM-1 on a plasmid of approximately 140 kb.
- FIG. 6.
PFGE findings (A and C) and Southern blot hybridization (B and D) with blaNDM-1 (A and B) and an ISCR1 probe (C and D). The genomic DNA of the index strain (K. pneumoniae 05-506) was undigested (lane 1) or was digested with S1 nuclease (lane 2), XbaI (lane 4), SpeI (lane 5), or I-CeuI (lane 6). Lane 3, DNA size marker.
Tables
- TABLE 1.
Antimicrobial susceptibility patterns of K. pneumoniae 05-560, E. coli NF-NDM-1, transformants, and conjugants
Antibiotic MIC (μg/ml) K. pneumoniae 05-560 E. coli TOP10 E. coli TOP10(NDM-1) E. coli J53 E. coli J53(pNDM-1) E. coli NF-NDM-1 Ampicillin >256 12 >256 4 >256 >256 Piperacillin >256 0.5 NT 1 >256 >256 Cephalothin >256 8 >256 4 >256 >256 Cefoxitin >256 4 >256 2 >256 >256 Cefotaxime >256 0.125 >256 0.064 >256 >256 Cefuroxime >256 8 >256 8 >256 >256 Ceftazidime >256 1 >256 0.125 >256 >256 Aztreonam >256 0.094 0.25 0.064 24 24 Cefepime >256 0.032 8 0.064 24 24 Ertapenem >32 0.125 24 0.5 >32 >32 Imipenem >32 0.094 12 0.25 16 >32 Meropenem >32 0.064 12 0.064 >32 >32 Ciprofloxacin >32 0.004 0.006 0.032 0.047 4 Colistin 0.75 0.38 0.38 0.25 0.25 0.25 - TABLE 2.
Steady-state kinetic constants of NDM-1, IMP-1, and VIM-2
Compound NDM-1 IMP-1a VIM-2b Km (μM) kcat (s−1) kcat/Km (s−1/μM) Km (μM) kcat (s−1) kcat/Km (s−1/μM) Km (μM) kcat (s−1) kcat/Km (s−1/μM) Penicillin G 16 11 0.68 520 320 0.62 49 56 1.14 Ampicillin 22 15 0.66 200 950 4.8 DNA Piperacillin 12 14 1.17 NDc ND ND 72 33 0.45 Cephalothin 10 4 0.40 21 48 2.4 44 57 1.28 Cefoxitin 49 1 0.02 8 16 2 24 3 0.12 Cefotaxime 10 6 0.58 4 1.3 0.35 32 28 0.86 Cefuroxime 8 5 0.61 37 8 0.22 22 12 0.55 Ceftazidime 181 5 0.03 44 8 0.18 98 89 0.90 Aztreonam ND >1,000 >0.01 <0.0001 ND <0.5 ND Cefepime 77 13 0.17 11 7 0.66 184 5 0.03 Imipenem 94 20 0.21 39 46 1.2 10 10 0.99 Meropenem 49 12 0.25 10 50 0.12 5 1 0.28 Clavulanic acid ND NRd NR
