Identification of a Novel NDM Variant, blaNDM-3, From a Multidrug-Resistant Acinetobacter baumannii

To the Editor—Acinetobacter baumannii is a rapidly emerging pathogen in the healthcare setting. Acinetobacter infections usually occur in severely ill patients in the intensive care unit, and the associated mortality rate is high, ranging from 26% to 68%. ¹ Acinetobacter species have a tendency to rapidly develop antimicrobial resistance to many classes of antibiotics. Increasing antimicrobial resistance leaves few therapeutic options to treat Acinetobacter infections. In the present study, A. baumanii was isolated from a patient who suffered serious burn injuries. The susceptibility of A. baumanii was tested against antimicrobial agents according to the Clinical and Laboratory Standards Institute (CLSI) broth microdilution procedure and interpretation criteria. A. baumanii showed resistance to the following antibiotics: aztreonem (≥32 mg/L), ceftazidime/clavulanic acid (≥128 mg/L), cefepime (≥64 mg/L), tobramycin (≥128 mg/L), ceftriaxone (≥32 mg/L), gentamicin (≥16 mg/L), amikacin (≥64 mg/L), imipenem (≥32 mg/L), meropenem (≥32 mg/L), colistin (≥16 mg/L) and tigecycline (≥16 mg/L).

Multiplex PCR approaches were used to search for Ambler class B (blaIMP, blaVIM, blaSIM, blaSPM, and blaNDM-1) and carbapenem-hydrolyzing class D β-lactamase genes (blaOXA-51, blaOXA-23, blaOXA-24, and blaOXA-58) as previously described. ^{2 , 3} Multiplex PCRs for class D β-lactamases and MBL genes were positive only for blaOXA-51 and blaNDM-1 genes in A. baumanii. The sequence analysis of the PCR product showed 99% identity with the blaNDM-2 previously reported in A. baumannii from Egypt. ⁴ The sequence of the blaNDM gene detected in this study showed 4 substitutions at the protein level, compared with blaNDM-1. The first change resulted in an amino acid substitution from P (proline) to A (alanine) at position 28, as was previously described and named blaNDM-2. ⁴ The other 3 amino acid substitutions revealed in this study have not been reported previously: (1) from A to T (threonine) at position 99, (2) from P to L (leucine) at position 150, and (3) from L to P at position 221. This new variant was designated as blaNDM-3 (GenBank accession no. KU220611).

Plasmid identification experiments were unsuccessful. ⁵ Genetic transformation of electrocompetent Escherichia coli DH5α with cell-free extract of A. baumannii also failed, which suggests that the blaNDM-3 gene was chromosomally encoded in A. baumannii. Detection of blaNDM-1 gene on the chromosome has also been reported previously. ⁴ To determine the genetic structure surrounding the blaNDM-3 gene, inverse PCR was performed. An analysis of the genetic surroundings showed that the blaNDM-2 gene was similar to that described for plasmid pNDM-HK ⁶ with ble (bleomycin resistance) and trpF (N-[5′-phosphoribosyl] anthranilate isomerase) genes downstream and an insertion sequence (ISAba125) upstream close to the promoter region (Figure 1). Thus, blaNDM-3 flanked by an insertion sequence can be shuttled between plasmids and the chromosome.

FIGURE 1 Genetic surroundings of the bla _NDM-3 in A. baumannii. IS, insertion sequence; ble, bleomycin resistance gene; trpF, N-(5′-phosphoribosyl) anthranilate isomerase.

In conclusion, this study reports for the first time a new variant of blaNDM-1 producing a multidrug-resistant A. baumannii isolate from India. The presence of an insertion sequence around the blaNDM-3 gene may contribute to the dissemination of the blaNDM-like genes with a likelihood of the emergence of more variants. Epidemiological control and identification of new mechanisms of resistance will contribute to better applications of therapeutic measures and to the prevention of an increase in resistance.