Molecular Characterization of Ndm-1 Producing Enterobacteriaceae Isolates in Singapore Hospitals

Objective: In this study, we molecularly characterized 12 NDM-1 producing clinical Enterobacteriaceae (Klebsiella pneumoniae, Escherichia coli, Enterobacter cloacae) isolates that were part of a collection of non-carbapenem susceptible isolates obtained during a one-year period. These isolates were obtained from four local general hospitals in Singapore. Methods: Polymerase chain reaction (PCR) assays and sequencing was used to determine the presence of β-lactamase encoding genes (bla) including bla NDM-1 and plasmid-mediated quinolone and aminoglycoside resistance determinants. Conjugation experiments were performed to determine the transferability of bla NDM-1. Isolate relatedness was determined by multilocus sequence typing (MLST). Results: The isolates were completely resistant to the second-and third-generation cephalosporins tested as well as carbapenems. Susceptibility profiling of the isolates indicated that 100% retained susceptibility to tigecycline while 11/12 (91.7%) were susceptible to colistin. The bla NDM-1 gene was encoded on plasmids that were easily transferable. None of the patients had a travel history to countries where NDM-1 has been reported. The isolates appear clonally unrelated with MLST, revealing a diversity of clonal types among the K. pneumoniae and E. coli isolates. Conclusion: The ease of NDM-1 plasmid transmissibility may help their dissemination among the Enterobacteriaceae. Although it appears that the isolates are clonally unrelated, epidemiological links cannot be fully excluded without further research. T he discovery of a novel carbapenemase, the New Delhi metallo--lactamase-1 (NDM-1), generated much global alarm. These NDM-1 producing isolates gained media notoriety being labelled as superbugs which had the reputation of being impossible to treat. The first carbapenem-resistant NDM-1 isolates characterized in 2009 were Klebsiella pneumoniae and Escherichia coli isolated from a Swedish patient who had sought medical care in New Delhi, India. The strains were resistant to all antibiotics tested except colistin. The ease of β-lactamase encoding genes (bla NDM-1) dissemination has become apparent with the worldwide detection of NDM-1 producers. In this study, we provide the molecular characterization and epidemiology for 12 NDM-1 positive clinical isolates. These isolates were obtained as part of a hospital surveillance programme for carbapenem non-susceptible Enterobacteriaceae. 52 non-duplicate carbapenem non-susceptible clinical isolates from local hospitals were analysed. The isolates were submitted from four hospitals that represented 40% of the general hospitals in Singapore. The 52 isolates comprised the following species: 31 K. pneumoniae, 13 E. coli, seven E. cloacae and one Enterobacter aerogenes. Two of these isolates (594 and 693) were obtained from the same patient but from different collection sites (Table 1). …

T he discovery of a novel carbapenemase, the New Delhi metallo--lactamase-1 (NDM-1), generated much global alarm.These NDM-1 producing isolates gained media notoriety being labelled as superbugs which had the reputation of being impossible to treat.The first carbapenem-resistant NDM-1 isolates characterized in 2009 were Klebsiella pneumoniae and Escherichia coli isolated from a Swedish patient who had sought medical care in New Delhi, India.The strains were resistant to all antibiotics tested except colistin. 1[4][5][6] In this study, we provide the molecular characterization and epidemiology for 12 NDM-1 positive clinical isolates.These isolates were obtained as part of a hospital surveillance programme for carbapenem nonsusceptible Enterobacteriaceae.

Antimicrobial susceptibility testing
Carbapenem resistance and resistance to other classes of antibiotics were confirmed by the Etest (bioMérieux) method with minimum inhibitory concentrations (MICs) determined after a 24-hour incubation at 37°C.Susceptibility was defined according to the breakpoints of the European Committee on Antimicrobial Susceptibility Testing.E. coli ATCC 25922 was used as the quality control strain for antimicrobial susceptibility testing.

Plasmid analysis
Plasmids were extracted using the Plasmid Mini Kit (Qiagen, Hilden, Germany).Plasmids were separated on 0.7% Megabase Agarose (Bio-Rad, Hercules, CA, USA) and their sizes estimated using BAC-Tracker TM Supercoiled DNA Ladder (Epicentre, Madison, WI, USA) as a reference.Southern hybridization analysis was performed using DIG DNA Labelling and Detection Kit (Roche Diagnostics, Mannheim, Germany) and digoxigenin-labelled 291 bp bla NDM-1 was used as the probe.

The NDM-1 producers are genotypically unrelated
Five different sequence types were obtained for the six K. pneumoniae isolates.Two isolates with identical sequence type (ST 437) were isolated one month apart in separate wards.Isolate 547 and 594 derived from the same patient had dissimilar MLSTs.All the E. coli isolates had differing sequence types.Hence, NDM-1 producing isolates comprised a variety of sequence types and were therefore genetically different (Table 1).

bla NDM-1 is plasmid borne and transferable
Conjugation experiments indicated that bla NDM-1 was transferable and likely via a plasmid-mediated event.

Conjugation assays
Conjugation experiments were performed between the clinical donor isolates and azide resistant E. coli J53 as a recipient.Transconjugants were recovered from Luria-Bertani agar plates containing sodium azide (100 mg/L) and imipenem (5 mg/L) with PCR confirming the presence of bla NDM-1 .

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A typical agarose gel electrophorectic profile of the plasmids analysed is shown in Figure 1.Plasmid content from clinical donor strains and transconjugants revealed that clinical NDM-1 isolates and their respective transconjugants carried a common band of covalently closed circular DNA larger than 28 kb in size (Figure 1).Southern hybridization analysis demonstrated localization of the NDM-1 gene to these plasmids (data not shown).
We were unable to size the plasmids more accurately due to limitations by conventional gel electrophoresis.

PCR screening for bla and other drug resistance determinants
All the isolates were PCR negative for MBLs and serine carbapenemase genes (OXA-48, KPC-1, GES-1, 2, 3, 4, 5 and 7).PCR results showed that it was not uncommon for the clinical NDM-1 producers to carry bla genes for more than one type of extended spectrum -lactamases as well as plasmid-encoded AmpC -lactamases (Table 1).These bla genes were transferable and conferred high levels of resistance to second-and third-generation cephalosporins and carbapenems to their recipient strain (Table 3).
Plasmid-mediated quinolone (qnr genes) and aminoglycoside resistance determinants (armA, rmtA, rmtB, rmtC, rmtD and npmA) were analysed by PCR.E. cloacae 459 was the only isolate found to be positive for qnrB.This determinant was not transferred to the transconjugant (Table 1).16S rRNA methylase genes were detected in only one isolate, E. cloacae 241.In this isolate, armA was co-detected with rmtC (Table 1).

DISCUSSION
In this study, we observed differences from an initial report from Singapore regarding NDM-1 producers in which the NDM-1 positive isolates were likely to have been imported from India and Bangladesh. 2 In contrast, the patients in our study had no recent travel history to countries where NDM-1 producers have been reported.The investigated isolates were obtained from four local hospitals and did not include the hospital of the initial report.Isolates from Tan Tock Seng Hospital were isolated over a four-month period, while the isolates at the National University Hospital were isolated over five months.No patient information was available for the isolates from the private hospital.Since the isolates originated from patients in differing wards and hospitals with their emergence being detected over a period of several months, we believe they were unlikely to have an epidemiological link.Epidemiological investigations done so far do not suggest nosocomial transmission of NDM-1 producing Enterobacteriaceae in these hospitals.However, we cannot completely rule out nosocomial transmission and further research is required to determine whether NDM-1 producing Enterobacteriaceae are endemic in Singapore.
The diversity of MLST strain types also indicates that the clones were genetically unrelated.Clonal diversity appears to be a characteristic of NDM-1 producers. 12his was reflected in a study looking at isolates of global origin, which revealed a large variety of strain types. 12he ease of dissemination of plasmid-bearing bla NDM-1 was apparent by the ability to obtain transconjugants for all the clinical donor strains. Due to the limited research capacity at our laboratory, we could perform only basic plasmid characterization.However, we acknowledge that PCR-based replicon typing 14 is an important tool for epidemiologically tracing these bla NDM-1 plasmids.