Emergence of a New Delhi metallo-β-lactamase-1-producing Pseudomonas aeruginosa in Singapore

Dear Editor,

Among the clinically significant carbapenemases, the New Delhi metallo-β-lactamase (NDM) is one of the most formidable. NDM efficiently hydrolyses β-lactams and last-resort carbapenems. Hence, therapeutic options for infections by NDM-producers are restricted to a handful of antibiotics, such as colistin, tigecycline, and fosfomycin.¹ NDM is predominantly associated with Enterobacteriaceae. This carbapenemase has also been described in Acinetobacter spp. but has been much less frequently detected in Pseudomonas aeruginosa.¹

The first report of NDM-1 production in P. aeruginosa came from Serbia in 2011.² It is now acknowledged that the NDM-1 gene is endemic to the Balkan states.^3,4 NDM-producing P. aeruginosa has since been isolated from other European countries,^4,5,6,7 as well as in India⁸ and Egypt.⁹ Local hospital laboratory surveillance suggests that approximately 12% of P. aeruginosa isolates are not susceptible to carbapenems.¹⁰ Previously, only producers of the metallo-β-lactamases (MBLs) VIM and IMP had been sporadically detected.¹¹ Here, we describe the first observed case of NDM-1-producing P. aeruginosa in Southeast Asia.

P. aeruginosa was cultured from the endotracheal aspirate of a 90-year-old female patient with colon cancer in March 2015. The isolate exhibited multidrug resistance to carbapenems (meropenem, imipenem, minimum inhibitory concentrations (MICs) > 32 mg/L), cephalosporin (ceftazidime, cefepime, MICs > 256 mg/L), aminoglycosides (gentamicin, amikacin, MICs > 256 mg/L), and fluoroquinolones (ciprofloxacin, levofloxacin, MICs > 32 mg/L). The isolate was partially resistant to aztreonam (MIC 16 mg/L). Colistin susceptibility was observed at an MIC of 1 mg/L. Phenotypic testing for carbapenemases using the KPC/MBL Confirm Kit (Rosco Diagnostica A/S, Taastrup, Denmark) indicated the presence of an MBL.

Comprehensive polymerase chain reaction (PCR) screening for β-lactamase genes was performed.¹² The isolate was positive for bla_NDM and determined to be bla_NDM-1 by full-length gene sequencing. The isolate was negative for other MBLs (IMP, VIM, SPM, DIM, AIM) and for genes encoding class A carbapenemases (KPC, GES). TEM and CTX-M extended spectrum β-lactamases (ESBLs) were found to be present.

Plasmid analysis using S1 nuclease pulsed-field gel electrophoresis¹³ and spin column plasmid extractions (QIAprep Spin Miniprep Kit, QIAGEN, Valencia, CA, USA) did not the reveal the presence of plasmids. Southern blot analysis with a bla_NDM-1 probe of S1 nuclease-treated DNA agarose plugs indicated that the probe hybridized to high molecular weight chromosomal DNA, suggesting that bla_NDM-1 was situated on the chromosome (data not shown). Furthermore, solid media conjugation assays were performed to assess the transferability of bla_NDM-1 from the clinical isolate to the azide-resistant recipient Escherichia coli J53. No transconjugants were obtained, suggesting the non-transmissibility of bla_NDM-1 (at least to E. coli), again suggesting a chromosomal position. Because the isolate was highly resistant to most antibiotics, including rifampicin, this excluded the use of the rifampicin-resistant laboratory strain of P. aeruginosa for conjugation. Hence, we were unable to assess the intra-species transmissibility of bla_NDM-1. P. aeruginosa bla_NDM-1 may be present chromosomally^2,5,6 or on a plasmid; in the latter context, bla_NDM-1 is transmissible.⁸

The nucleotide sequences immediately flanking bla_NDM-1 were determined by an inverse PCR and primer walking approach. Two copies of bla_NDM-1 were detected and separated by an Insertion Sequence Common Region (ISCR) element (Figure 1). Sequencing of this ISCR element revealed a 97% nucleotide homology to ISCR24. ISCR24 has been identified in the genetic environment of a novel bla_PME-1 (Pseudomonas aeruginosa ESBL 1) and implicated in the acquisition of the ESBL by P. aeruginosa.¹⁵ This is not surprising because ISCR elements mediate the mobilization of almost every class of antibiotic resistance genes, including those encoding ESBLs and carbapenemases.¹⁶ ISCR elements, such as ISCR1, have been found to be associated with NDM-1 from P. aeruginosa.^3,5,7 Jovcic et al.³ reported two copies of bla_NDM-1 in P. aeruginosa (Figure 1), where it is presumed that the ISCR1 element, as part of its rolling-circling mechanism of transposition, duplicates adjacent genetic segments.¹⁶ Because ISCR elements are known to construct extended clusters of antibiotic resistance genes on plasmids as well as on chromosomes,¹⁶ it would be interesting to investigate the presence of other resistance determinants surrounding bla_NDM-1 and the ISCR24-like region that may contribute to its multidrug resistance.

Figure 1.

Comparative schematic diagrams of the diverse genetic organizations of bla_NDM-1 in P. aeruginosa. The selected bla_NDM-1 sequences are chromosomally located and associated with ISCR elements and class I integrons.^3,5 Typical common genetic features surrounding bla_NDM-1 are the insertion sequence ISAba125 and the bleomycin resistance gene (ble_MBL).¹⁴

After this isolate was identified, two other NDM-1-producing P. aeruginosa isolates with antibiograms identical to the initial isolate were cultured from the sputum samples of two other patients in April and May 2015. One of the isolates was from a 58-year-old male with intracranial bleeding from a ruptured aneurysm, and the other was from an 84-year-old female with a femur fracture, complicated by pancreatitis and small intestine perforation. The genetic relatedness of the three NDM-1 P. aeruginosa isolates was investigated by DiversiLab rep-PCR fingerprinting (bioMérieux, Marcy l'Etoile, France), which revealed that the rep-PCR profiles were indistinguishable, suggesting that the three isolates were clonal in nature. PCR mapping and sequencing of the two latter isolates reveal a bla_NDM genetic context identical to that of the first isolate. Because all three patients stayed in the same surgical intensive care unit, the detection of indistinguishable NDM-1-positive P. aeruginosa suggested a transmission event.

In summary, this is the first report of the emergence of NDM-1 in P. aeruginosa in Southeast Asia in an unusual genetic context. The apparent intra-ward transmission of this extremely drug-resistant isolate highlights the gravity of this escalating public health issue.

Nucleotide sequence accession number

The bla_NDM-1 sequence from the initial P. aeruginosa isolate has been deposited into Genbank under the accession number KT364224.