Journal of Antimicrobial Chemotherapy Advance Access published January 26, 2016
J Antimicrob Chemother doi:10.1093/jac/dkv471
Detection and characterization of two NDM-1-producing Klebsiella pneumoniae strains from Bulgaria
1
Department of Medical Microbiology, University of Antwerp, Antwerp, Belgium; 2Vaccine & Infectious Disease Institute, University of Antwerp, Antwerp, Belgium; 3Department of Medical Microbiology, Faculty of Medicine, Medical University of Sofia, Sofia, Bulgaria; 4Central Laboratory of Microbiology, Alexandrovska University Hospital, Sofia, Bulgaria *Corresponding author. Department of Medical Microbiology, University of Antwerp, Antwerp, Belgium. Tel: +32-3-2652550; E-mail: [email protected]
Sir, Carbapenemase-producing Enterobacteriaceae (CPE) have proven themselves as a substantial threat to human health, spreading efficiently in many countries where numerous cases and outbreaks have been reported.1 Since its first discovery in 2008 in Klebsiella pneumoniae, New Delhi metallo-b-lactamase 1 (NDM-1) has appeared in many countries around the world.2 One of the main reservoirs of NDM, apart from the Indian subcontinent and the Middle East, has been the Balkan region.3 NDM-1-harbouring K. pneumoniae strains were detected in Croatia and Turkey in 2012, and an outbreak was reported in Greece in 2014.4 There is also evidence of single cases of NDM-1-producing Enterobacteriaceae found in Kosovo, Montenegro and Serbia, as well as exported cases described in other countries in Europe.5,6 An NDM-1 Escherichia coli outbreak was reported in 2014 in a hospital in Bulgaria.7 In March and August 2014, we recovered two carbapenem-resistant K. pneumoniae, which were later identified to be NDM-harbouring strains, from patients at the Alexandrovska University Hospital in Sofia, Bulgaria. The aim of this study was to describe the first two clinical cases of NDM-1-producing K. pneumoniae in Bulgaria and to characterize the genetic context of these blaNDM. The first isolate, Kpn240, was recovered in March 2014 from a urine sample of a female outpatient in her early thirties with clinical symptoms of a urinary tract infection. The patient had been admitted previously to the University Hospital for Active Treatment and Emergency Medicine “N. I. Pirogov” in Sofia within 1 month preceding the recovery of the strain. However, she had not travelled or stayed abroad during the prior 6 months. There was no information about antibiotic treatment at the time of urine sampling. The second isolate, Kpn235, was obtained from
# The Author 2016. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: [email protected]
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T. Kostyanev1,2*, T. Strateva3, B. B. Xavier1,2, Y. Marteva-Proevska4, C. Lammens1,2, B. Markova4, H. Goossens1,2 and S. Malhotra-Kumar1,2
a tracheal aspirate of a man in his late seventies who was admitted to the ICU at the Alexandrovska University Hospital in August 2014. He had been admitted for a routine surgical intervention, but developed left-sided pneumonia and was transferred to the ICU, where, 2 weeks after the initial admission, Kpn235 was recovered. The patient had been admitted to another hospital in Southern Bulgaria 2 months prior to the recovery of Kpn235. He had no history of stays abroad during the previous 6 months. The isolates were stored and later sent to the University of Antwerp for further testing. Strain identification was (re)confirmed with MALDI-TOF MS (Bruker Daltonics), and antibiotic susceptibility testing was performed using Etest (bioMe´rieux). Results were interpreted according to EUCAST guidelines (http:// www.eucast.org/clinical_breakpoints/). The MIC values of imipenem were 32 mg/L for both strains, confirming the antibioticsusceptibility testing results obtained in Alexandrovska University Hospital. Meropenem MICs were 4 mg/L (Kpn240) and 8 mg/L (Kpn235). Both strains showed similar MIC values of most of the antibiotics tested: .32 mg/L of cefotaxime, ceftazidime, ciprofloxacin, levofloxacin and trimethoprim/sulfamethoxazole; 64 mg/L of cefepime; .256 mg/L of piperacillin/tazobactam; and 0.064 mg/L of colistin. Kpn240 showed MIC values of amikacin and gentamicin .256 mg/L, while Kpn235 showed lower MICs of these two aminoglycosides, i.e. 32 mg/L. The presence of ESBL and carbapenemase genes was tested by PCR and Sanger sequencing.8 Both strains were found to harbour blaCTX-M-15, blaSHV-11 and blaNDM-1. Kpn240 also harboured blaTEM-1. MLST identified both K. pneumoniae strains as ST11 (allelic profile: 3-3-1-1-1-1-4), a single-locus variant of the pandemic clone ST258. Genomic DNA from both strains was extracted using a MasterPureTM DNA purification kit (Epicentre Technologies). WGS of both isolates was performed via 2×150 bp paired-end sequencing (Nextera XT sample preparation kit and MiSeq, Illumina) and assembled using SPAdes v. 3.5.1 (de novo assembly). De novo contigs were screened for plasmid origin using Plasmid Finder (https:// cge.cbs.dtu.dk//services/PlasmidFinder/). Reference mapping was done using plasmid contigs as template (CLC Genomics Workbench v. 7.5.1, CLC bio, Denmark). Mapped reads were extracted and de novo assembled and annotated using the online server Bacterial Annotation System (BASys).9 The NDM-1-carrying plasmids in both strains were highly similar (99%) to each other at nucleotide level and in size (pkpn-240-BG at 76 980 bp harbouring 85 protein-coding genes and pkpn-235-BG at 76 365 bp harbouring 85 protein-coding genes) (Figure 1a). The average read coverage for each plasmid was .200-fold. Also, the 76 kb pkpn-240-BG and pkpn-235-BG plasmids showed 99% sequence similarity to the previously sequenced 97 kb NDM-1-carrying plasmid (pB-300cz, accession number KJ958926) from the Czech Republic.10 Analysis of the pB-300cz sequence showed that the 21 kb fragment that was missing from pkpn-240-BG and pkpn-235-BG comprised a transposable element harbouring multiple antibiotic resistance genes, including aadA1a and aacA4-T329 flanked by ISKpn14 and Tn3 (Figure 1b). Both plasmids belonged to the incompatibility type IncFII. As also reported previously, blaNDM-1 co-occurred with bleMBL downstream of the ISAba125 element.7
Research letter
(a)
Figure 1. (a) Linear map of the NDM-1-coding pkpn-240-BG isolated from Kpn240. (b) Comparison of NDM-1-harbouring plasmids showing high similarity levels by the National Center for Biotechnology Information Basic Local Alignment Search Tool (NCBI BLAST) with pkpn-240-BG. Comparators are the pB-300cz and pS-300cz co-isolated from a Czech strain, K. pneumoniae plasmid pKPX-1 from Taiwan (93% similarity) and Enterobacter hormaechei plasmid pEh1A from Brazil (78% similarity). Similar sequences are shown as blocks of the same colour.
In silico whole-genome mapping comparison with AflII using unweighted pair group method with arithmetic mean (UPGMA) clustering analysis further showed that Kpn240 and Kpn235 were 97.2% similar and that there were 16 unmatched fragments between the two strains (Figure S1, available as Supplementary data at JAC Online). To our knowledge, these are the first two documented cases of NDM-1-producing K. pneumoniae in Bulgaria. Both patients had a history of previous admissions to healthcare institutions in different parts of the country. However, there was no evidence of travel to NDM-endemic regions or contact with proximal countries with recently reported outbreaks or cases of ST11 NDM-1 K. pneumoniae.4,10 It is thus highly likely that the two described cases are unrelated events with different origins, which underscores the probability of several as yet undetected foci of CPE
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in Bulgaria and the need for consistent and widespread epidemiologic surveillance to facilitate their detection.
Plasmid sequences The plasmid sequences were deposited in GenBank (accession numbers: pkpn-240-BG, KT852335; and pkpn-235-BG, KT852336).
Funding T. K. and the work on the genetic analysis of the Kpn240 and Kpn235 strains were supported by funding from the Innovative Medicines Initiative project COMBACTE (Combatting Bacterial Resistance in Europe) (IMI Grant Agreement No. 115523) and COMBACTE-CARE (IMI
Downloaded from http://jac.oxfordjournals.org/ at Orta Dogu Teknik University Library (ODTU) on February 1, 2016
(b)
Research letter
Grant Agreement No. 115620). The isolation of the Kpn240 strain is part of the project ‘European Survey on Carbapenemase-Producing Enterobacteriaceae in Europe (EuSCAPE)’ (framework contract ECDC/ 2012/055). B. B. X. is supported by University of Antwerp Research Funds (BOF-DOCPRO 2012-27450).
4 Voulgari E, Gartzonika C, Vrioni G et al. The Balkan region: NDM-1producing Klebsiella pneumoniae ST11 clonal strain causing outbreaks in Greece. J Antimicrob Chemother 2014; 69: 2091– 7.
Transparency declarations
5 Glasner C, Albiger B, Buist G et al. Carbapenemase-producing Enterobacteriaceae in Europe: a survey among national experts from 39 countries, February 2013. Euro Surveill 2013; 18: pii¼20525.
3 Meletis G, Oustas E, Bagkeri M. Carbapenemase reports from the Balkans: a systematic review. Infez Med 2014; 2: 85– 106.
6 Baraniak A, Izdebski R, Fiett J et al. NDM-producing Enterobacteriaceae in Poland, 2012 –14: inter-regional outbreak of Klebsiella pneumoniae ST11 and sporadic cases. J Antimicrob Chemother 2016; 71: 85–91.
Supplementary data Figure S1 is available as Supplementary data at JAC Online (http://jac. oxfordjournals.org/).
7 Poirel L, Savov E, Nazli A et al. Outbreak caused by NDM-1- and RmtB-producing Escherichia coli in Bulgaria. Antimicrob Agents Chemother 2014; 58: 2472– 4.
References
8 Poirel L, Walsh TR, Cuvillier V et al. Multiplex PCR for detection of acquired carbapenemase genes. Diagn Microbiol Infect Dis 2011; 70: 119–23.
1 Mathers AJ, Peirano G, Pitout JD. The role of epidemic resistance plasmids and international high-risk clones in the spread of multidrug-resistant Enterobacteriaceae. Clin Microbiol Rev 2015; 28: 565 – 91. 2 Johnson AP, Woodford N. Global spread of antibiotic resistance: the example of New Delhi metallo-b-lactamase (NDM)-mediated carbapenem resistance. J Med Microbiol 2013; 62: 499–513.
9 Van Domselaar GH, Stothard P, Shrivastava S et al. BASys: a web server for automated bacterial genome annotation. Nucleic Acids Res 2005; 33 (Web Server issue): W455– 9. 10 Studentova V, Dobiasova H, Hedlova et al. Complete nucleotide sequences of two NDM-1-encoding plasmids from the same sequence type 11 Klebsiella pneumoniae strain. Antimicrob Agents Chemother 2015; 59: 1325– 8.
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Downloaded from http://jac.oxfordjournals.org/ at Orta Dogu Teknik University Library (ODTU) on February 1, 2016
None to declare.
J Antimicrob Chemother doi:10.1093/jac/dkv471
Detection and characterization of two NDM-1-producing Klebsiella pneumoniae strains from Bulgaria
1
Department of Medical Microbiology, University of Antwerp, Antwerp, Belgium; 2Vaccine & Infectious Disease Institute, University of Antwerp, Antwerp, Belgium; 3Department of Medical Microbiology, Faculty of Medicine, Medical University of Sofia, Sofia, Bulgaria; 4Central Laboratory of Microbiology, Alexandrovska University Hospital, Sofia, Bulgaria *Corresponding author. Department of Medical Microbiology, University of Antwerp, Antwerp, Belgium. Tel: +32-3-2652550; E-mail: [email protected]
Sir, Carbapenemase-producing Enterobacteriaceae (CPE) have proven themselves as a substantial threat to human health, spreading efficiently in many countries where numerous cases and outbreaks have been reported.1 Since its first discovery in 2008 in Klebsiella pneumoniae, New Delhi metallo-b-lactamase 1 (NDM-1) has appeared in many countries around the world.2 One of the main reservoirs of NDM, apart from the Indian subcontinent and the Middle East, has been the Balkan region.3 NDM-1-harbouring K. pneumoniae strains were detected in Croatia and Turkey in 2012, and an outbreak was reported in Greece in 2014.4 There is also evidence of single cases of NDM-1-producing Enterobacteriaceae found in Kosovo, Montenegro and Serbia, as well as exported cases described in other countries in Europe.5,6 An NDM-1 Escherichia coli outbreak was reported in 2014 in a hospital in Bulgaria.7 In March and August 2014, we recovered two carbapenem-resistant K. pneumoniae, which were later identified to be NDM-harbouring strains, from patients at the Alexandrovska University Hospital in Sofia, Bulgaria. The aim of this study was to describe the first two clinical cases of NDM-1-producing K. pneumoniae in Bulgaria and to characterize the genetic context of these blaNDM. The first isolate, Kpn240, was recovered in March 2014 from a urine sample of a female outpatient in her early thirties with clinical symptoms of a urinary tract infection. The patient had been admitted previously to the University Hospital for Active Treatment and Emergency Medicine “N. I. Pirogov” in Sofia within 1 month preceding the recovery of the strain. However, she had not travelled or stayed abroad during the prior 6 months. There was no information about antibiotic treatment at the time of urine sampling. The second isolate, Kpn235, was obtained from
# The Author 2016. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: [email protected]
1 of 3
Downloaded from http://jac.oxfordjournals.org/ at Orta Dogu Teknik University Library (ODTU) on February 1, 2016
T. Kostyanev1,2*, T. Strateva3, B. B. Xavier1,2, Y. Marteva-Proevska4, C. Lammens1,2, B. Markova4, H. Goossens1,2 and S. Malhotra-Kumar1,2
a tracheal aspirate of a man in his late seventies who was admitted to the ICU at the Alexandrovska University Hospital in August 2014. He had been admitted for a routine surgical intervention, but developed left-sided pneumonia and was transferred to the ICU, where, 2 weeks after the initial admission, Kpn235 was recovered. The patient had been admitted to another hospital in Southern Bulgaria 2 months prior to the recovery of Kpn235. He had no history of stays abroad during the previous 6 months. The isolates were stored and later sent to the University of Antwerp for further testing. Strain identification was (re)confirmed with MALDI-TOF MS (Bruker Daltonics), and antibiotic susceptibility testing was performed using Etest (bioMe´rieux). Results were interpreted according to EUCAST guidelines (http:// www.eucast.org/clinical_breakpoints/). The MIC values of imipenem were 32 mg/L for both strains, confirming the antibioticsusceptibility testing results obtained in Alexandrovska University Hospital. Meropenem MICs were 4 mg/L (Kpn240) and 8 mg/L (Kpn235). Both strains showed similar MIC values of most of the antibiotics tested: .32 mg/L of cefotaxime, ceftazidime, ciprofloxacin, levofloxacin and trimethoprim/sulfamethoxazole; 64 mg/L of cefepime; .256 mg/L of piperacillin/tazobactam; and 0.064 mg/L of colistin. Kpn240 showed MIC values of amikacin and gentamicin .256 mg/L, while Kpn235 showed lower MICs of these two aminoglycosides, i.e. 32 mg/L. The presence of ESBL and carbapenemase genes was tested by PCR and Sanger sequencing.8 Both strains were found to harbour blaCTX-M-15, blaSHV-11 and blaNDM-1. Kpn240 also harboured blaTEM-1. MLST identified both K. pneumoniae strains as ST11 (allelic profile: 3-3-1-1-1-1-4), a single-locus variant of the pandemic clone ST258. Genomic DNA from both strains was extracted using a MasterPureTM DNA purification kit (Epicentre Technologies). WGS of both isolates was performed via 2×150 bp paired-end sequencing (Nextera XT sample preparation kit and MiSeq, Illumina) and assembled using SPAdes v. 3.5.1 (de novo assembly). De novo contigs were screened for plasmid origin using Plasmid Finder (https:// cge.cbs.dtu.dk//services/PlasmidFinder/). Reference mapping was done using plasmid contigs as template (CLC Genomics Workbench v. 7.5.1, CLC bio, Denmark). Mapped reads were extracted and de novo assembled and annotated using the online server Bacterial Annotation System (BASys).9 The NDM-1-carrying plasmids in both strains were highly similar (99%) to each other at nucleotide level and in size (pkpn-240-BG at 76 980 bp harbouring 85 protein-coding genes and pkpn-235-BG at 76 365 bp harbouring 85 protein-coding genes) (Figure 1a). The average read coverage for each plasmid was .200-fold. Also, the 76 kb pkpn-240-BG and pkpn-235-BG plasmids showed 99% sequence similarity to the previously sequenced 97 kb NDM-1-carrying plasmid (pB-300cz, accession number KJ958926) from the Czech Republic.10 Analysis of the pB-300cz sequence showed that the 21 kb fragment that was missing from pkpn-240-BG and pkpn-235-BG comprised a transposable element harbouring multiple antibiotic resistance genes, including aadA1a and aacA4-T329 flanked by ISKpn14 and Tn3 (Figure 1b). Both plasmids belonged to the incompatibility type IncFII. As also reported previously, blaNDM-1 co-occurred with bleMBL downstream of the ISAba125 element.7
Research letter
(a)
Figure 1. (a) Linear map of the NDM-1-coding pkpn-240-BG isolated from Kpn240. (b) Comparison of NDM-1-harbouring plasmids showing high similarity levels by the National Center for Biotechnology Information Basic Local Alignment Search Tool (NCBI BLAST) with pkpn-240-BG. Comparators are the pB-300cz and pS-300cz co-isolated from a Czech strain, K. pneumoniae plasmid pKPX-1 from Taiwan (93% similarity) and Enterobacter hormaechei plasmid pEh1A from Brazil (78% similarity). Similar sequences are shown as blocks of the same colour.
In silico whole-genome mapping comparison with AflII using unweighted pair group method with arithmetic mean (UPGMA) clustering analysis further showed that Kpn240 and Kpn235 were 97.2% similar and that there were 16 unmatched fragments between the two strains (Figure S1, available as Supplementary data at JAC Online). To our knowledge, these are the first two documented cases of NDM-1-producing K. pneumoniae in Bulgaria. Both patients had a history of previous admissions to healthcare institutions in different parts of the country. However, there was no evidence of travel to NDM-endemic regions or contact with proximal countries with recently reported outbreaks or cases of ST11 NDM-1 K. pneumoniae.4,10 It is thus highly likely that the two described cases are unrelated events with different origins, which underscores the probability of several as yet undetected foci of CPE
2 of 3
in Bulgaria and the need for consistent and widespread epidemiologic surveillance to facilitate their detection.
Plasmid sequences The plasmid sequences were deposited in GenBank (accession numbers: pkpn-240-BG, KT852335; and pkpn-235-BG, KT852336).
Funding T. K. and the work on the genetic analysis of the Kpn240 and Kpn235 strains were supported by funding from the Innovative Medicines Initiative project COMBACTE (Combatting Bacterial Resistance in Europe) (IMI Grant Agreement No. 115523) and COMBACTE-CARE (IMI
Downloaded from http://jac.oxfordjournals.org/ at Orta Dogu Teknik University Library (ODTU) on February 1, 2016
(b)
Research letter
Grant Agreement No. 115620). The isolation of the Kpn240 strain is part of the project ‘European Survey on Carbapenemase-Producing Enterobacteriaceae in Europe (EuSCAPE)’ (framework contract ECDC/ 2012/055). B. B. X. is supported by University of Antwerp Research Funds (BOF-DOCPRO 2012-27450).
4 Voulgari E, Gartzonika C, Vrioni G et al. The Balkan region: NDM-1producing Klebsiella pneumoniae ST11 clonal strain causing outbreaks in Greece. J Antimicrob Chemother 2014; 69: 2091– 7.
Transparency declarations
5 Glasner C, Albiger B, Buist G et al. Carbapenemase-producing Enterobacteriaceae in Europe: a survey among national experts from 39 countries, February 2013. Euro Surveill 2013; 18: pii¼20525.
3 Meletis G, Oustas E, Bagkeri M. Carbapenemase reports from the Balkans: a systematic review. Infez Med 2014; 2: 85– 106.
6 Baraniak A, Izdebski R, Fiett J et al. NDM-producing Enterobacteriaceae in Poland, 2012 –14: inter-regional outbreak of Klebsiella pneumoniae ST11 and sporadic cases. J Antimicrob Chemother 2016; 71: 85–91.
Supplementary data Figure S1 is available as Supplementary data at JAC Online (http://jac. oxfordjournals.org/).
7 Poirel L, Savov E, Nazli A et al. Outbreak caused by NDM-1- and RmtB-producing Escherichia coli in Bulgaria. Antimicrob Agents Chemother 2014; 58: 2472– 4.
References
8 Poirel L, Walsh TR, Cuvillier V et al. Multiplex PCR for detection of acquired carbapenemase genes. Diagn Microbiol Infect Dis 2011; 70: 119–23.
1 Mathers AJ, Peirano G, Pitout JD. The role of epidemic resistance plasmids and international high-risk clones in the spread of multidrug-resistant Enterobacteriaceae. Clin Microbiol Rev 2015; 28: 565 – 91. 2 Johnson AP, Woodford N. Global spread of antibiotic resistance: the example of New Delhi metallo-b-lactamase (NDM)-mediated carbapenem resistance. J Med Microbiol 2013; 62: 499–513.
9 Van Domselaar GH, Stothard P, Shrivastava S et al. BASys: a web server for automated bacterial genome annotation. Nucleic Acids Res 2005; 33 (Web Server issue): W455– 9. 10 Studentova V, Dobiasova H, Hedlova et al. Complete nucleotide sequences of two NDM-1-encoding plasmids from the same sequence type 11 Klebsiella pneumoniae strain. Antimicrob Agents Chemother 2015; 59: 1325– 8.
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Downloaded from http://jac.oxfordjournals.org/ at Orta Dogu Teknik University Library (ODTU) on February 1, 2016
None to declare.
