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OXA-181-producing Klebsiella pneumoniae establishing in Singapore

  • Michelle N D Balm1Email author,
  • Grace Ngan1,
  • Roland Jureen1,
  • Raymond T P Lin1 and
  • Jeanette W P Teo1
BMC Infectious DiseasesBMC series ¿ open, inclusive and trusted201313:58

DOI: 10.1186/1471-2334-13-58

Received: 2 November 2012

Accepted: 28 January 2013

Published: 1 February 2013

Abstract

Background

Carbapenemase producing Enterobacteriaceae are becoming a major public health concern globally, however, relatively little is known about the molecular and clinical epidemiology of these organisms in many parts of the world.

Methods

As part of a laboratory surveillance programme, 96 carbapenem non-susceptible Enterobacteriaceae isolates from clinical samples from patients in seven hospitals were referred for investigation for carbapenemases. Using polymerase chain reaction (PCR) to screen for a collection of genes encoding carbapenemases, 33 of 96 (34.5%) isolates were confirmed as carbapenemase producers. NDM-1 producers were the most prevalent at 64% (21/33) whilst OXA-181 was the second most common carbapenemase constituting 24.5% (8/33) of the carbapenemase producing isolates. Seven of these eight OXA-181 positive isolates underwent further characterisation with screening for other transmissible antimicrobial resistance determinants using PCR. Clonal relatedness was explored using Multilocus sequence typing (MLST) and Pulsed Field Gel Electrophoresis (PFGE). Plasmid characterisation was performed including restriction analysis and transfer by conjugation or transformation.

Results

In addition to the OXA-181 gene, all contained other transmissible resistance determinants including extended spectrum β-lactamases, oxacillinases or 16S rRNA methylase genes, but none contained metallo-β-lactamases or serine carbapenemases. All isolates had a multidrug resistant phenotype with two isolates being resistant to every antibiotic tested including colistin. Multilocus sequence typing confirmed five isolates belonged to ST17 and two to ST14, with those belonging to the same sequence type having identical PFGE profiles. The OXA-181 gene was typically carried on large plasmids which were mostly non-conjugative.

Conclusions

OXA-181 carbapenemase appears to be an important and probably under-recognised cause of carbapenem resistance in Enterobacteriaceae in Singapore. Further coordinated research into clinical and molecular epidemiology of carbapenemases is urgently required in Singapore and throughout Asia.

Keywords

Gram negative resistance Carbapenemases Hospital associated infections Beta-lactamases

Background

Acquired resistance to carbapenems in Enterobacteriaceae is a major concern worldwide. This is due both to their importance as human pathogens especially within the hospital setting, and to the highly transmissible nature and propensity for rapid spread shown by these organisms. An increasingly diverse range of enzymes are being recognised as significant, including serine proteases of Ambler Class A, the Class B metallo-β-lactamases and carbapenem-hydrolysing Class D oxacillinases [1]. Originally found in Shewanella species, OXA-48-like carbapenemases have now emerged in Enterobacteriaceae, predominantly Klebsiella pneumoniae. These carbapenem hydrolysing Class D β-lactamases are unusual among carbapenemases in their weak hydrolysis of cephalosporins and resistance to inhibitors such as clavulanate, making them difficult to identify in the laboratory [2]. OXA-181, a variant of OXA-48, was initially reported in India but has been sporadically detected in the United Kingdom, The Netherlands, France, New Zealand, Oman and Singapore [27]. Molecular characterisation of this emerging variant remains limited.

Until recently, the vast majority of carbapenem resistance in this group of organisms was due to extended spectrum β-lactamase (ESBL) or AmpC cephalosporinase production combined with impermeability due to porin mutations [8]. Carbapenemase producing organisms were infrequently reported. Between October 2010 and March 2012, we screened 96 Enterobacteriaceae clinical isolates with MIC ≥ 2 mg/L to meropenem or imipenem for acquired carbapenemases using polymerase chain reaction (PCR). Isolates were referred for investigation from seven of eight (87.5%) hospital or community laboratories in Singapore after initial antimicrobial susceptibility testing showed non-susceptibility to carbapenems. Epidemiological data was not provided by referring laboratories. Carbapenemase genes were detected in 33 (33/96, 34.4%), including eight clinical isolates of OXA-181-producing Klebsiella pneumoniae from patients in three different hospitals. NDM-1 producing isolates dominated this study at 64% (21/33) whilst OXA-181 producers constituted 24.5% (8/33) of carbapenemase producing isolates thus making blaOXA-181 the second most detected carbapenemase gene after blaNDM-1[9]. The remaining were KPC producers (4/33) [10]. Here, we report the molecular characterisation of seven out of the eight of the OXA-181 producing isolates. One isolate was excluded from analysis as consent was not obtained from the referring laboratory.

Methods

All isolates were confirmed as K. pneumoniae using matrix assisted laser desorption ionisation-time of flight-mass spectrometry (MALDI-ToF-MS, Bruker Daltonics GmHB, Bremen, Germany). Antimicrobial susceptibility testing was performed with VITEK-2 instrument and carbapenem MICs confirmed with Etest (bioMérieux, Marcy L’Etoile, France) with susceptibility defined according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints. Presence of carbapenemases were screened phenotypically using disc diffusion assays with meropenem discs supplemented with boronic acid, cloxacillin or dipicolinic acid (Rosco Diagnostica A/S, Taastrup, Denmark) and the modified Hodge test [11].

Isolates underwent screening for transmissible β-lactamase genes including serine carbapenemases (KPC-type), metallo-β-lactamases (MBL; NDM-type, VIM-type, IMP-type), oxacillinases and extended spectrum β-lactamases (ESBL; TEM-type, SHV-type, CTX-M-type) with PCR followed by sequencing of amplicons [9, 10]. Detection of blaOXA was based on a multiplex PCR assay developed by Woodford et al. [12] with addition of blaOXA-48-like primers (OXA-48L 5- GTGGGATGGACAGACGCG-3 and OXA-48 LL 5-CCACACATTATCATCAAGTTC-3) using National Collection of Type Cultures (NTCT) 13442 K. pneumoniae as positive control for blaOXA-48. Plasmid mediated 16S rRNA methylase aminoglycoside resistance determinants (armA, rmtA, rmtB, rmtC, rmtD and npmA) were also analysed by PCR.

Clonal relatedness was investigated using Multilocus sequence typing (MLST, http://​www.​pasteur.​fr/​recherche/​genopole/​PF8/​mlst) and Pulsed Field Gel Electrophoresis (PFGE) of SpeI (New England Biolabs, Ipswich, MA) digested genomic DNA [13].

The number and size of plasmids were analysed by S1 nuclease digestion of whole genomic DNA followed by PFGE (S1-PFGE) [14]. In addition, smaller plasmids were extracted using the QIAprep spin miniprep kit (Qiagen GmbH, Hilden, Germany). Southern hybridisation using a digoxigenin (DIG) labelled blaOXA-181 probe (DIG DNA Labelling and Detection kit, Roche Diagnostics, Mannheim, Germany) was used to localise blaOXA-181. Transformation studies were performed with plasmid DNA extracted using QIAprep Spin Miniprep kit introduced by electroporation into E. coli DH5α cells using Gene Pulser Xcell (Bio-Rad, Hercules, CA) with transformants selected on Luria-Bertani (LB) agar supplemented with imipenem (1 mg/L). Conjugation experiments were performed between K. pneumoniae isolates and azide-resistant recipient E.coli J53 with transconjugants selected on LB agar containing sodium azide (50 mg/L) and imipenem (1 mg/L). PCR-based replicon typing was used to identify plasmid incompatibility groups [15]. To analyse the immediate genetic environment of the blaOXA-181 gene in our isolates, primers (IRF 5-CCTAGATTCTACGTCAGTAC-3 and IRR2 5-CTCTCTAGTCGGACAACACC-3) based on GenBank reference sequence NC_019160.1, designed to walk in from the left and right inverted long repeats (IRL) in the direction of blaOXA-181 were used.

Results and discussion

As part of a surveillance program for carbapenem non-susceptible Enterobacteriaceae in Singapore, 96 isolates from seven hospitals or private laboratories were submitted for investigation. Of these, 33 clinical isolates contained carbapenemase genes as detected by PCR with eight isolates bearing blaOXA-181. Consent from referring laboratories was obtained for seven of these isolates to be subjected to molecular characterisation. The blaOXA-181 positive clinical isolates were confirmed as Klebsiella pneumoniae by MALDI-ToF MS. Most showed high level resistance to carbapenems, and interestingly also to cephalosporins (Table 1), suggesting the presence of other resistance determinants in addition to blaOXA-181. Two of the seven isolates were resistant to all antimicrobial agents tested including colistin. All seven OXA-181 producers were positive by the modified Hodge test. No synergy was detected using disc diffusion assays, the usual pattern seen with oxacillinase presence.
Table 1

Characteristics of OXA-181-producing Klebsiella pneumoniae clinical isolates

Isolate

Hospital

Specimen

Date of Isolation

ST

16S rRNA methylase gene

β-lactamases

Plasmids

MIC (mg/L)

   
 

Size (kb)

Replicon(s)

IMP

MEM

ETP

FOX

CTX

CAZ

AZT

COL

TGC

LEV

GEN

AMK

KPO8

Hospital A

Tracheal aspirate

18/01/2011

17

armA

TEM-116, SHV-11, CTX-M-15, OXA-1

~97, ~170

A/C

>32

>32

>32

>256

>256

64

>256

0.125

2

0.25

>256

>256

KPO9

Hospital A

Sputum

8/02/2011

17

armA

TEM-1, SHV-11, CTX-M-15, OXA-1

~9, ~150, ~170

A/C

>32

>32

>32

>256

>256

64

>256

16

>256

>256

>256

>256

KPO7

Hospital A

Tracheal aspirate

9/12/2010

14

armA

TEM-1, SHV-1, CTX-M-15, OXA-1, OXA-9

~1.5, ~50, ~170, ~194

A/C, F

>32

>32

>32

>256

>256

>256

>256

0.25

12

>256

>256

>256

KPO26

Hospital A

Sputum

14/05/2011

14

ND

TEM-1, SHV-1, CTX-M-15, OXA-1, OXA-9

~1.6, ~194

A/C

>32

>32

>32

>256

>256

>256

>256

32

4

>256

>256

96

KPO6916

Hospital A

Blood

2/03/2012

14

armA

TEM-1, SHV-1, CTX-M-15, OXA-1, OXA-9

~1.6, ~50, ~170, ~194

A/C

8

8

>32

>256

>256

>256

>256

0.25

16

>256

>256

>256

KPO7431

Hospital B

Urine

22/02/2012

14

armA

TEM-1, SHV-11, CTX-M-15, OXA-1, OXA-9

~1.5, ~50, ~170, ~194

A/C, F

4

4

>32

>256

>256

>256

>256

0.25

8

>256

>256

>256

KPO83

Hospital C

Urine

30/12/2011

14

armA

TEM-1, SHV-1, CTX-M-15, OXA-1, OXA-9

~170

A/C

32

8

>32

>256

>256

>256

>256

0.5

16

32

>256

>256

KPO9–T

armA

OXA-181, CTX-M-15

~170

A/C

2

0.5

0.5

2

3

1

0.75

0.125

0.125

0.002

>256

>256

KPO26 –T

ND

OXA-181, OXA-9

~194

A/C

2

0.19

0.25

12

6

2

6

0.125

0.125

0.003

0.125

1

E. coli J53

0.125

0.016

0.04

1

0.064

0.25

0.064

0.125

0.064

0.004

0.125

0.125

Legend:

MIC, minimum inhibitory concentration; IPM, imipenem; MEM, meropenem; ETP, ertapenem; FOX, cefoxitin; CTX, cefotaxime; CAZ, ceftazidime; AZT, aztreonam; COL, colistin; TCG, tigecycline; LEV, levofloxacin; GEN, gentamicin; AMK, amikacin; KPO8, KPO9, KPO7, KPO26, KPO6916, KPO7431, KPO83 carbapenem-resistant K. pneumoniae clinical isolate; KP09-T, KP026-T, K. pneumoniae transconjugant from KP09, KP026; J53, E. coli recipient strain.

Full gene sequencing of amplicons revealed 100% identity with blaOXA-181 [GenBank: JN205800.1]. PCR screening and sequencing showed that all isolates also carried ESBL genes and at least one additional blaOXA-type but none were positive for MLBs or KPC-type β-lactamases (Table 1). armA was detected in six of the seven isolates (Table 1). MLST revealed five isolates of sequence type (ST)14 and two of ST17. Furthermore, isolates belonging to the same sequence type had identical PFGE patterns. OXA-181-producing isolates came from three different hospitals over a 15 month period, and to our knowledge patients did not have an epidemiological link to each other although full data is not accessible.

Analysis of plasmid content using miniprep kit extractions and S1-PFGE revealed the isolates contained multiple small (~1.5kb) and large plasmids (~50 - ~194kb) (Table 1). Southern hybridization studies using a blaOXA-181 probe localised the gene to a large plasmid of ~170kb in six of the seven clinical isolates and to a ~194kb plasmid in isolate KPO26 (Table 1). Transformation studies were not successful in selecting transformants; however, the method would have only extracted small plasmids. This would support localisation of blaOXA-181 to large plasmids. Previously blaOXA-181 has been described in a range of plasmid backbones ranging from a small ColE2-type plasmid of ~7.6kb in K. pneumoniae KP3 to large plasmids of 200-250kb [3, 16]. Transconjugants were obtained for only two isolates (KPO-9, KPO-26) with transfer of a large plasmid carrying blaOXA-181 demonstrated by S1-PFGE and Southern hybridization analysis (data not shown). Transconjugant KPO-26 remained susceptible to non-β-lactam antibiotics whilst for KPO-9, armA was co-transferred with the plasmid (Table 1).

To further characterize the plasmids, PCR- based replicon typing was performed. IncA/C replicons were detected in all isolates including the transconjugants (Table 1), suggesting blaOXA-181 was present on an IncA/C plasmid although Southern hybridisation analysis using IncA/C probes would be needed to confirm this. Plasmids belonging to the IncA/C incompatibility group are of interest as they carry resistance to diverse groups of antimicrobial agents and have a broad host range [17].

blaOXA-181 has been found as part of transposon, Tn2013, with upstream insertion sequence ISEcp1 mediating one-ended transposition of the blaOXA-181 gene [16]. The immediate genetic environment surrounding blaOXA-181 of our isolates was investigated. Gene mapping demonstrated presence of the ISEcp1 element upstream, however, no PCR products were obtained when attempting to amplify downstream sequences (Δlys and Δere). This raises the possibility that blaOXA-181 in our isolates could be present in a structure different to the previously characterized Tn2013[16].

The surveillance project from which these isolates were derived is ongoing. This project has previously identified that NDM-1 containing organisms appear to be well established in different Enterobacteriaceae species, often in patients without travel history within the preceding year and that KPC-2-producing Klebsiella pneumoniae has been introduced to Singapore probably from mainland China [9, 10]. Our work suggests that blaOXA-181 may already have established on plasmids within successful K. pneumoniae strains in Singapore. There is also the likelihood that a much wider reservoir of carbapenemase-producing Enterobacteriaceae already exists here given that this is a voluntary programme and only clinical isolates with obviously elevated carbapenem MICs were referred for investigation. This could be especially true for organisms carrying OXA-48-like carbapenemases which may not exhibit high level resistance to carbapenems or cephalosporins in the absence of other resistance determinants capable of conferring carbapenem non-susceptibility. A major limitation of our study is that clinical data on patients from whom the isolates were obtained is lacking. Although all isolates studied in this collection came from clinical samples, no data is available on whether these represent colonisation or true infection, nor regarding treatment and outcomes. This makes establishing the context of carbapenem resistance in Enterobacteriaceae in Singapore more difficult. Indeed, any data on carbapenemase-producing Enterobacteriaceae is very limited in South East Asia. Given the frequency of travel and economic migration through many parts of the region and the widespread resistance to other antimicrobial classes, carbapenemase production in Enterobacteriaceae is likely to be an under-estimated entity, made more difficult by the lack of availability for diagnostic testing in many areas.

Conclusion

OXA-181 carbapenemase appears to be an emerging and probably under-recognised cause of carbapenem resistance in Enterobacteriaceae in Singapore. This preliminary data, along with the work of others [7], has provided the basis for establishing further coordinated clinical and molecular epidemiological research into carbapenemase-producing Enterobacteriaceae in Singapore aimed at addressing many of the gaps in our current understanding.

Abbreviations

ESBL: 

Extended spectrum β-lactamase

KPC: 

Klebsiella pneumoniae carbapenemase

MALDI-ToF-MS: 

Matrix assisted laser desorption ionisation-time of flight-mass spectrometry

MBL: 

Metallo-β-lactamase

MIC: 

Minimum inhibitory concentration

MLST: 

Multilocus sequence typing

NDM: 

New Delhi metallo-β-lactamase

OXA: 

Oxacillinase

PCR: 

Polymerase chain reaction

PFGE: 

Pulsed field gel electrophoresis

ST: 

Sequence type.

Declarations

Acknowledgements

This work was funded by a Health Service Development Programme Grant provided by the Ministry of Health, Singapore (RL) (Grant # HSDP06/X04) and a National University Health System Clinician Scientist Grant (MB). We thank the referring microbiology laboratories and National Public Health Laboratory for isolate collection.

Authors’ Affiliations

(1)
Department of Laboratory Medicine, National University Health System

References

  1. Nordmann P, Dortet L, Poirel L: Carbapenemase resistance in Enterobacteriaceae: here is the storm!. Trends in Molecular Medicine. 2012, 18: 263-272. 10.1016/j.molmed.2012.03.003.View ArticlePubMed
  2. Poirel L, Potron A, Nordmann P: OXA-48-like carbapenemases: the phantom menace. J Antimicrob Chemother. 2012, 67: 1587-1606.
  3. Castanheira M, Deshpande LM, Mathai D, Bell JM, Jones RN, Mendes RE: Early dissemination of NDM-1- and OXA-181-producing Enterobacteriaceae in Indian hospitals: report from the SENTRY Antimicrobial Surveillance Program, 2006–2007. Antimicrob Agents Chemother. 2011, 55: 1274-1278. 10.1128/AAC.01497-10.PubMed CentralView ArticlePubMed
  4. Dimou V, Dhanji H, Pike R, Livermore DM, Woodford N: Characterization of Enterobacteriaceae producing OXA-48-like carbapenemases in the UK. J Antimicrob Chemother. 2012, 67: 1660-1665. 10.1093/jac/dks124.View ArticlePubMed
  5. Dortet L, Poirel L, Al Yaqoubi F, Nordmann P: NDM-1, OXA-48 and OXA-181 carbapenemase-producing Enterobacteriaceae in Sultanate of Oman. Clin Microbiol Infect. 2011, 18: E144-E148.View Article
  6. Williamson DA, Heffernan H, Sidjabat H, Roberts SA, Paterson DL, Smith M, Freeman JT: Intercontinental transfer of OXA-181-producing Klebsiella pneumoniae into New Zealand. J Antimicrob Chemother. 2011, 66: 2888-2890. 10.1093/jac/dkr396.View ArticlePubMed
  7. Koh TH, Cao DYH, Chan KS, Wijaya L, Low SBG, Lam MS, Ooi EE, Hsu LY: blaOXA-181-positive Klebsiella pneumoniae, Singapore. Emerg Infect Dis. 2012, 18: 1524-1525. 10.3201/eid1809.111727.PubMed CentralView ArticlePubMed
  8. Koh TH: Gram-negative resistance in Singapore: a historical perspective. Ann Acad Med Singapore. 2007, 37: 847-854.
  9. Teo J, Ngan G, Balm M, Jureen R, Krishnan P, Lin R: Molecular characterization of NDM-1 producing Enterobacteriaceae isolates in Singapore hospitals. Western Pacific Surveillance and Response Journal. 2012, 3: 10.5365/wpsar.year.2011.2.4.010.
  10. Balm MND, Ngan G, Jureen R, Lin RTP, Teo J: Molecular characterization of newly emerged blaKPC-2-producing Klebsiella pneumoniae in Singapore. J Clin Microbiol. 2012, 50: 475-476. 10.1128/JCM.05914-11.PubMed CentralView ArticlePubMed
  11. Clinical and Laboratory Standards Institute: Twentieth informational supplement. M100–S20. Performance standards for antimicrobial susceptibility testing. 2010, Wayne (PA): The Institute
  12. Woodford N, Ellington MJ, Coelho JM, Turton JF, Ward ME, Brown S, Amyes SGB, Livermore DL: Multiplex PCR for genes encoding prevalent OXA carbapenemases in Acinetobacter spp. Int J Antimicrob Agents. 2006, 27: 351-353. 10.1016/j.ijantimicag.2006.01.004.View ArticlePubMed
  13. Kaufmann ME: Pulsed-field gel electrophoresis. Molecular Bacteriology: Protocols and Clinical Applications. Edited by: Woodford N, Johnson AP. 1998, Totowa, NJ: Humana Press
  14. Barton BM, Harding GP, Zuccarelli AJ: A general method for detecting and sizing large plasmids. Anal Biochem. 1995, 226: 235-240. 10.1006/abio.1995.1220.View ArticlePubMed
  15. Johnson TJ, Nolan LK: Plasmid replicon typing. Methods Mol Biol. 2009, 551: 27-35. 10.1007/978-1-60327-999-4_3.View ArticlePubMed
  16. Potron A, Nordmann P, Lafeuille E, Al Maskari Z, Al Rashi F, Poirel L: Characterization of OXA-181, a carbapenem-hydrolyzing class D beta-lactamase from Klebsiella pneumoniae. Antimicrob Agents Chemother. 2011, 55: 4896-4899. 10.1128/AAC.00481-11.PubMed CentralView ArticlePubMed
  17. Fernández-Alarcón C, Singer RS, Johnson TJ: Comparative genomics of multidrug resistance-encoding IncA/C plasmids from commensal and pathogenic Escherichia coli from multiple animal sources. PLoS One. 2011, 6: e23415-10.1371/journal.pone.0023415.PubMed CentralView ArticlePubMed
  18. Pre-publication history

    1. The pre-publication history for this paper can be accessed here:http://​www.​biomedcentral.​com/​1471-2334/​13/​58/​prepub

Copyright

© Balm et al.; licensee BioMed Central Ltd. 2013

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://​creativecommons.​org/​licenses/​by/​2.​0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.