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, 53 (12), 5046-54

Characterization of a New Metallo-Beta-Lactamase Gene, bla(NDM-1), and a Novel Erythromycin Esterase Gene Carried on a Unique Genetic Structure in Klebsiella Pneumoniae Sequence Type 14 From India

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Characterization of a New Metallo-Beta-Lactamase Gene, bla(NDM-1), and a Novel Erythromycin Esterase Gene Carried on a Unique Genetic Structure in Klebsiella Pneumoniae Sequence Type 14 From India

Dongeun Yong et al. Antimicrob Agents Chemother.

Abstract

A Swedish patient of Indian origin traveled to New Delhi, India, and acquired a urinary tract infection caused by a carbapenem-resistant Klebsiella pneumoniae strain that typed to the sequence type 14 complex. The isolate, Klebsiella pneumoniae 05-506, was shown to possess a metallo-beta-lactamase (MBL) but was negative for previously known MBL genes. Gene libraries and amplification of class 1 integrons revealed three resistance-conferring regions; the first contained bla(CMY-4) flanked by ISEcP1 and blc. The second region of 4.8 kb contained a complex class 1 integron with the gene cassettes arr-2, a new erythromycin esterase gene; ereC; aadA1; and cmlA7. An intact ISCR1 element was shown to be downstream from the qac/sul genes. The third region consisted of a new MBL gene, designated bla(NDM-1), flanked on one side by K. pneumoniae DNA and a truncated IS26 element on its other side. The last two regions lie adjacent to one another, and all three regions are found on a 180-kb region that is easily transferable to recipient strains and that confers resistance to all antibiotics except fluoroquinolones and colistin. NDM-1 shares very little identity with other MBLs, with the most similar MBLs being VIM-1/VIM-2, with which it has only 32.4% identity. As well as possessing unique residues near the active site, NDM-1 also has an additional insert between positions 162 and 166 not present in other MBLs. NDM-1 has a molecular mass of 28 kDa, is monomeric, and can hydrolyze all beta-lactams except aztreonam. Compared to VIM-2, NDM-1 displays tighter binding to most cephalosporins, in particular, cefuroxime, cefotaxime, and cephalothin (cefalotin), and also to the penicillins. NDM-1 does not bind to the carbapenems as tightly as IMP-1 or VIM-2 and turns over the carbapenems at a rate similar to that of VIM-2. In addition to K. pneumoniae 05-506, bla(NDM-1) was found on a 140-kb plasmid in an Escherichia coli strain isolated from the patient's feces, inferring the possibility of in vivo conjugation. The broad resistance carried on these plasmids is a further worrying development for India, which already has high levels of antibiotic resistance.

Figures

FIG. 1.
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.
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.
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.
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.
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.
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.

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