|Year : 2016 | Volume
| Issue : 1 | Page : 121-123
Presence of a novel variant NDM-10, of the New Delhi metallo-beta-lactamase in a Klebsiella pneumoniae isolate
Atul Khajuria1, Ashok Kumar Praharaj2, Mahadevan Kumar1, Naveen Grover1
1 Department of Microbiology, Armed Forces Medical College, Pune, Maharashtra, India
2 Department of Microbiology, AIIMS, Bhubaneswar, Odisha - 751019, India
|Date of Submission||26-May-2014|
|Date of Acceptance||20-Jul-2015|
|Date of Web Publication||15-Jan-2016|
Department of Microbiology, Armed Forces Medical College, Pune, Maharashtra
Source of Support: None, Conflict of Interest: None
|How to cite this article:|
Khajuria A, Praharaj AK, Kumar M, Grover N. Presence of a novel variant NDM-10, of the New Delhi metallo-beta-lactamase in a Klebsiella pneumoniae isolate. Indian J Med Microbiol 2016;34:121-3
|How to cite this URL:|
Khajuria A, Praharaj AK, Kumar M, Grover N. Presence of a novel variant NDM-10, of the New Delhi metallo-beta-lactamase in a Klebsiella pneumoniae isolate. Indian J Med Microbiol [serial online] 2016 [cited 2020 Jan 22];34:121-3. Available from: http://www.ijmm.org/text.asp?2016/34/1/121/174101
Emerging carbapenem resistance in Enterobacteriaceae represents a significant threat to public health because it reduces the effectiveness of antimicrobial treatment. Broncheo-alveolar lavage fluid from a 65 years old patient grew Gram-negative, non-motile bacilli in pure culture. The organism was identified as Klebsiella pneumoniae (Labelled as KPN 78) by Vitek-2 system using VITEK-GNI cards (bioMérieux, Marcy l'Etoile, France). Antibiotic sensitivity test was performed by standard Kirby-Bauer disc diffusion technique as per the guidelines of the Clinical Laboratory Standards Institute (CLSI) with commercially available discs (Hi-Media, Mumbai, India) on Mueller Hinton agar plates. The isolate was resistant to all beta-lactams and their minimum inhibitory concentrations (MICs) of are shown in [Table 1], whereas MICs of colistin and tigecycline are 0.75 µg/ml and 1 µg/ml respectively as determined by VITEK-2 and E-test as per CLSI breakpoints. Modified Hodge's Test and MBL (IP/IPI) E-test (bioMérieux, Marcy l'Etoile, France) for MBL production was positive. Bacterial DNA was extracted using the spin column method (QIAGEN; GmbH, Hilden, Germany) as per manufacturer's instructions. Polymerase chain reaction (PCR)-based analysis for beta-lactamase genes (blaCTXM, blaOXA, blaSHV and blaTEM), Ambler class B MBLs (blaIMP, blaVIM, blaSPM, blaGIM, blaSIM, blaNDM-1), Ambler classD (blaOXA-23, blaOXA-24, blaOXA48), serine carbapenemases (blaKPC, blaGES and blaNMC) and for 16S rRNA methylase gene was carried out in a Gene Amp 9700 PCR System (Applied Biosystems, Singapore). PCR primers used as described earlier  were procured from Sigma-Aldrich, India. The isolates were positive for blaNDM and harbored 16S rRNA armA methylase gene, encoding high-level resistance to all aminoglycosides. The amplicons were purified using QIAquick PCR purification kit (QIAGEN; GmbH, Hilden, Germany) and sequenced with the ABI 3730XL capillary sequencer (Applied Biosystems, Foster City, CA, USA). The chromatogram was analysed, consensus sequences were aligned using BioEdit software and showed five mutations corresponding to the five amino acid substitutions compared with blaNDM-1. Sequencing revealed a novel blaNDM type sequence, showing 99% similarity to the previous known sequences as in NCBI database that was designated as blaNDM-10 by the curators of the Lahey database of beta-lactamases (http://www.lahey.org/Studies/) and was deposited under GenBank accession number KF361506. Analysis of the predicted amino acid sequence revealed five substitutions at positions 32 (Arg→Ser), 36 (Gly→Asp), 69 (Gly→Ser), 74 (Ala→Thr) and 200 (Gly→Arg). In addition to blaNDM-10, isolate was positive for extended-spectrum beta-lactamase genes and sequencing showed the presence of blaCTX-M-15, blaTEM-1 and blaSHV-28. The point mutations at position 94 (C→A), 107 (G→A), 205 (G→A), 220 (G→A) and 598 (G→C) responsible for the amino acid substitutions were confirmed by reamplification and sequencing of gene from fresh DNA preparation. The NDM-10 gene was localised on a plasmid of ca. 130 kb in size as demonstrated by Southern hybridization with a blaNDM-specific probe. Liqid broth mating-out assays was carried out at 37°C using K. pneumoniae isolate (Parental strain) as donos and an azide-resistant Escherichia More Details coli J53 as the recipient strain in 1:10 ratio. Selection of the transconjugants was based on growth on MacConkey agar supplemented with sodium azide (100 μg/ml), cefoxitin (10 μg/ml) and ceftazidime (30 μg/ml). Mating-out assays followed by PCR of transconjugant [Table 1] showed blaNDM-10 gene on a 130-kb plasmid and blaCTX-M-15, blaTEM-1 and blaSHV-28 on 70 kb plasmid. Using the PCR-based replicon typing method as described previously. BlaNDM-10 gene was located on a plasmid typed as IncFII-type (Amplicon size of 270 bp). Plasmid carrying blaCTX-M-15, blaTEM-1 and blaSHV-28 showed association with multiple replicons (IncFIB and IncFIA). A ca. 130-kb plasmid was successfully transferred to E. coli DH10B by electroporation and transformant displayed similar beta-lactam resistance expression pattern as that of NDM-10 [Table 1]. Here, we report a novel NDM-10 in K. pneumoniae KPN-78 isolate and due to these mutations, isolate is showing higher MICs values against beta-lactam antibiotics as compared to NDM-1 [Table 1]. Due to higher MICs the patient is clinically resistant to all beta-lactam group of antibiotics, fluoroquinolones, aminoglycosides, beta-lactam and beta-lactam inhibitor combinations and beta-lactam and an aminiglycoside combination. Colistin is the mainstay of therapy.
|Table 1: MIC of beta-lactams for KPN-78 clinical isolate, the transformant E. coli DH10B harbouring natural plasmid from KPN-78, azide resistant J53 transconjugant, E. coli DH10B, E. coli azide resistant J53 and NDM-1 positive KPN|
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