The emergence and dissemination of carbapenemase-producing Enterobacteriaceae is a concerning global threat. The rapid evolution and relatively limited data on the molecular epidemiology of these organisms continue to pose challenges for clinicians and researchers across the world. There are no formal estimates of prevalence for carbapenemase-producing Enterobacteriaceae in Singapore at present. A prospective study offered to all hospitals in Singapore screened 251 Enterobacteriaceae isolates with imipenem or meropenem MIC > 1 mg/L from seven hospitals between September 2010 and October 2012. Of these, carbapenemases were detected by PCR in 122 isolates (48.6%, Table 1). Among carbapenemase producers, blaNDM-type was most prevalent at 68% (83/122), and OXA-48-type producers were the second most common (18/122, 15%; OXA-181 12/18, OXA-48 6/18, Table 1). Notably, two Klebsiella pneumoniae isolates bearing both blaNDM and blaOXA-181 were identified. Here, we describe the molecular characterization of these two unusual isolates.
|Species (number of isolates)||NDM-type||OXA-48-type||KPC-type||IMP-type||NDM-type + OXA-48-type||IMI-A|
|Citrobacter freundii (6)||4||—||—||2||—||—|
|Citrobacter sedlakii (1)||1||—||—||—||—||—|
|Enterobacter cloacae (13)||10||—||2||—||—||1|
|Escherichia coli (36)||32||2||1||1||—||—|
|Klebsiella pneumoniae (63)||35||16||8||2||2||—|
|Morganella morganii (1)||1||—||—||—||—||—|
|Serratia marcescens (1)||—||—||—||1||—||—|
One isolate, KP243, was obtained from an endotracheal aspirate and urine of a 17-year-old Singaporean female admitted for investigation of encephalitis. She required prolonged hospitalization between August and October 2012, including admission to intensive care for 18 days. She had travelled to Malaysia for 5 days in June 2012 but did not have any healthcare contact during this time.
The second isolate, KP1053, was from the urine of a 5-year-old haemodialysis-dependent girl from Bangladesh transferred to Singapore for neurosurgical intervention following admission at two hospitals in Dhaka, Bangladesh for hydrocephalus and hypertensive crisis (Table 2). These patients were admitted to different hospitals in Singapore.
|Isolate||Country of isolation||Country of origin||Hospital||Specimen||Date of Isolation||ST||Resistance determinants||Plasmids||Structures associated with blaNDM-type||Structures associated with blaOXA-181|
|Size (kb)||Replicon(s)||Truncated ISAba125||ble MBL||ISEcp1||ΔlysR-like, ΔereA|
|KP243||Singapore||Singapore||Hospital A||Endotracheal tube aspirate||22/08/2012||29||NDM-1, OXA-181, TEM-1, SHV-11, CTX-M-15, ArmA||~ 340, ~160, ~ 48||A/C||+||+||+||−|
|KP1053||Singapore||Bangladesh||Hospital B||Urine||20/10/2012||231||NDM-5, OXA-181, TEM-1, SHV-11, CTX-M-15, ArmA||~280, ~140, ~48 ~8||FIIA||+||+||+||+|
Species identification of both isolates was confirmed with matrix-assisted laser desorption ionization–time of flight-mass spectrometry (MALDI-ToF-MS; Bruker Daltonik GmbH, Bremen, Germany). Antimicrobial susceptibility testing performed with VITEK-2 and Etest (bioMérieux, Marcy L'Etoile, France) demonstrated high-level resistance to imipenem, meropenem, ertapenem (all MIC >32 mg/L), cefotaxime, ceftazidime, cefepime (all MIC >64 mg/L), amikacin, gentamicin and levofloxacin (all MIC >256 mg/L). Both isolates were susceptible to tigecycline and colistin according to European Committee on Antimicrobial Susceptibility Testing breakpoints using Etest. Synergy was demonstrated with meropenem–dipicolinic acid but not meropenem–boronic acid or meropenem–cloxacillin discs (Rosco Diagnostica A/S, Taastrup, Denmark), suggesting the presence of a metallo-β-lactamase. PCR screening for transmissible β-lactamases including serine carbapenemases (KPC-type) , metallo-β-lactamases (VIM-type, IMP-type, NDM-type) , oxacillinases , extended spectrum β-lactamases (TEM-type, SHV-type, CTX-type) and plasmid-mediated 16S rRNA methylase aminoglycoside resistance determinants (armA, rmtA, rmtB, rmtC, rmtD, npmA)  demonstrated the presence of blaNDM-type and blaOXA-48-type in both isolates. Both isolates were negative for other metallo-β-lactamases or KPC-type β-lactamases but extended spectrum β-lactamase genes (CTX-M-15) and aminoglycoside-resistance determinant armA were observed (Table 2). Full gene sequencing of OXA-48-type amplicons from both isolates revealed 100% identity with blaOXA-181 (GenBank accession no. JN205800.1). Sequencing through the full length of the blaNDM-type amplicons revealed that KP1053 carried blaNDM-5 with 100% identity to GenBank sequence JN104597.1, whereas KP243 carried blaNDM-1 (Table 2).
Multilocus sequence typing (http://www.pasteur.fr/recherche/genopole/PF8/mlst/Kpneumoniae.html) revealed isolates that were different sequence types (ST). KP243 was ST29 whereas KP1053 was ST231, suggesting that they were clonally unrelated (Table 2). Both sequence types have been identified in international surveys as hosts of carbapenemases, in particular NDM-1 [4, 5].
Plasmid analysis by S1 nuclease digestion of whole genomic DNA followed by pulsed-filed gel electrophoresis (S1-PFGE) showed that isolates possessed multiple plasmids of varying sizes (Table 2) . Southern hybridization of S1-PFGE plasmid DNA was performed using DIG DNA Labelling and Detection Kit (Roche, Mannheim, Germany). The DIG-labelled probes of blaNDM and blaOXA-181 were generated and used to localize the bla genes. In KP243, blaOXA-181 was present on a ~160-kb plasmid. However, in KP1053, blaOXA-181 localized to two plasmids of ~280 kb and ~8 kb (data not shown). The blaNDM probe localized to a ~340-kb plasmid in KP243 and to a ~48-kb plasmid in KP1053.
Conjugation experiments using azide-resistant recipient Escherichia coli J53 with selection of transconjugants on LB agar containing sodium azide (50 mg/L) and ampicillin (100 mg/L) generated only transconjugants bearing blaNDM (Table 2). Southern hybridization analysis of transconjugants demonstrated transfer of the ~340-kb plasmid from KP243 and the ~48-kb plasmid from KP1053. The transconjugants were carbapenem resistant (MIC >8 mg/L) but susceptible to non-β-lactam antibiotics. In this study, CTX-M-15 did not co-transfer with plasmids bearing blaNDM.
Using PCR-based replicon typing to identify plasmid incompatibility groups in K. pneumoniae isolates and their transconjugants , one replicon type, IncA/C, was detected in KP243, whereas only FIIA replicons were detected in KP1053 (Table 2). No replicons were detected in the transconjugants indicating that the NDM-bearing plasmids were untypeable by the PCR method of replicon typing. IncA/C digoxigenin-labelled probes when used in S1-PFGE Southern hybridizations localized to the ~160-kb plasmid harbouring blaOXA-181 in KP243. Plasmids encoding OXA-181 of incompatibility group IncA/C were also observed in our previous work characterizing OXA-181 producers . Probing with FIIA revealed localization of the replicon to a ~140-kb plasmid in KP1053, which could possibly carry other drug resistance determinants. Both IncA/C and IncFIIA are conjugative plasmids strongly linked to transmission of multidrug-resistance traits .
The immediate genetic elements around blaOXA-181 and blaNDM were determined by PCR and direct sequencing. Using published primers, the bleomycin-resistance gene, blaMBL, was found downstream while part of the insertion element ISAba125 was found upstream .
Gene mapping using primers (IRF 5′-CCTAGATTCTACGTCAGTAC-3′ and IRR2 5′-CTCTCTAGTCGGACAACACC-3′) designed from GenBank reference isolate KP3, which carries blaOXA-181 within Tn2013 (accession no. JN205800.1) [3, 9], demonstrated the presence of the ISEcp1 element upstream in both of our K. pneumoniae isolates (Table 2). In isolate KP1053, downstream sequences ∆lysR-like (transcriptional regulator) and ∆ereA (erythromycin esterase) were amplified and confirmed by direct amplicon sequencing. Hence, based on plasmid size and genetic features flanking blaOXA-181, we speculate that the ~8-kb plasmid in this isolate is comparable to the 7605-bp ColE2-type blaOXA-181-bearing plasmid . In contrast, ∆lysR-like and ∆ereA sequences could not be amplified from the KP243 isolate, suggesting that blaOXA-181 could be present in a structure different to the previously characterized Tn2013 . These gene mapping results were consistent with our previous observations, that OXA-181 producers derived from local patients with no travel history did not appear to have ΔereA-like, and ΔlysR-like open reading frames .
Here, we have provided evidence of the increasing frequency of transmissible carbapenemase genes among Enterobacteriaceae in Singapore, and the novel finding of isolates carrying both blaOXA-181 and blaNDM genes. Of note, the blaOXA-181 genes were identified in different genetic structures, and the patients from whom they were derived were epidemiologically unrelated with one having no hospital contact overseas in the preceding 12 months. This raises the possibility of emergence within Singapore.