Journal of Antimicrobial Chemotherapy Advance Access published January 20, 2015
J Antimicrob Chemother doi:10.1093/jac/dku576
Characterization of KPC-type carbapenemase-producing Klebsiella pneumoniae strains isolated in the Arabian Peninsula A´gnes Sonnevend1, Akela Ghazawi1, Dania Darwish1, Zulfa AlDeesi2, Ali F. Kadhum2 and Tibor Pa´l1* 1
Department of Microbiology and Immunology, College of Medicine and Health Sciences, United Arab Emirates University, Al Ain, United Arab Emirates; 2Rashid Hospital, Dubai, United Arab Emirates
*Corresponding author. Tel: +971-3-7137480; Fax: +971-3-7671966; E-mail: [email protected]
Keywords: KPC-2, K. pneumoniae, multidrug resistant, MDR
Sir, Since its first description in 1996, variants of KPC have spread throughout the world mostly in the ST258 clone of Klebsiella pneumoniae within isoforms of the Tn4401 transposon located on various plasmids.1 While the Middle East is considered as a reservoir of various carbapenemase-producing bacteria, to date the only Enterobacteriaceae carrying the blaKPC gene reported from the Arabian Peninsula has been an Escherichia coli ST131 strain isolated in Kuwait.2 In a collection of 34 carbapenem-non-susceptible Enterobacteriaceae strains, comprising all clinically relevant, nonrepeat isolates recovered in Rashid Hospital, Dubai, United Arab Emirates (UAE) between March 2012 and February 2014, we identified by PCR3: 3 blaNDM and 1 blaOXA-48-like E. coli; and 3 blaNDM, 6 blaOXA-48-like, 17 blaNDM + blaOXA-48-like and 2 blaKPC K. pneumoniae. Since blaKPC-positive K. pneumoniae has not been described from this region to date, we subjected the two blaKPC-positive K. pneumoniae isolates (ABC220 and ABC224) to detailed investigations. K. pneumoniae ABC220 was isolated in October 2012 from a necrotizing wound of a previously healthy, 40-year-old Filipino male, 25 days after he had been admitted for perforated appendix and sepsis. K. pneumoniae ABC224 was recovered in May 2013 from the sputum of an 80-year-old UAE national with multiple chronic illnesses, continuously hospitalized for .1 year in a different ward for left leg amputation and sepsis. Prior to the isolation of these strains, various samples from both patients had been repeatedly negative for carbapenem-resistant Enterobacteriaceae. As detected by Etest (bioMe´rieux, Marcy-l’E´toile, France) or microdilution, ABC220 and ABC224 exhibited susceptibility to tigecycline (MIC¼0.75 mg/L) and amikacin (MIC¼ 12 mg/L) only. The MICs of all antimicrobials tested are listed in Table S1 (available as Supplementary data at JAC Online). The XbaI-digested
macrorestriction patterns3 of strains exhibited .95% similarity. Both ABC220 and ABC224 belonged to ST14 by MLST.4 Strains were subjected to PCR assays specific for ESBL (blaCTX-M, blaSHV, blaTEM and blaOXA) and AmpC b-lactamase genes, plasmidmediated quinolone and aminoglycoside resistance genes and virulence genes (rmpA, wabG, wcaG, fimH, mrkD, iutA, fyuA, iroN, ireA, kfuBC, traT and clpK).3,5 Sequencing of the amplicons revealed that they both possessed blaTEM-1 , blaSHV-1 , blaCTX-M-15 and aac-6 ′ -1b-cr genes and also virulence-related genes fyuA, kfuBC, fimH, mrkD, traT, clpK, ugeA and wabG. Both strains carried sequences suggestive of a K2 capsule type.6 Outer membrane protein analysis by SDS–PAGE revealed the lack of a band corresponding to OmpK35 from both isolates as compared with the patterns of control strains CSUB10S (Omp36K) and CSUB10S/pSH16K (OmpK35 and OmpK36).7 Plasmid transfer and analysis by electrophoresis, Southern blotting, hybridization, replicon typing and RFLP was performed as described previously. 3 Although blaKPC-carrying plasmids from both strains were conjugally transferred into E. coli J53RAZ, they were not stably maintained in this recipient. Therefore, plasmids were transferred into E. coli GM2163 by transformation. Both ABC220 and ABC224 harboured a 45 kb IncX3 plasmid carrying blaKPC as identified by hybridization. The plasmids exhibited the same RFLP profile when digested with EcoRI and HindIII (data not shown). Sequencing of blaKPC and its flanking regions by PCR mapping and direct sequencing identified the blaKPC-2 allele in both strains located within the Tn4401b transposon inserted into the umuD gene of the IncX3 plasmid (Figure 1) (GenBank accession no. KM983022). At the time of submission of our manuscript, no KPC-producing K. pneumoniae had been reported from the Arabian Peninsula; although, not without causing considerable confusion, the terminology ‘KPC’ was used earlier referring to a strain exhibiting putative carbapenemase activity by modified Hodge test, without any analysis of the bla gene.8 Here, we describe two K. pneumoniae ST14 strains, identical in all features tested, carrying blaKPC-2 on IncX3 plasmids. blaKPC-2-bearing IncX3 plasmids described earlier in ST258 K. pneumoniae in France9 and Hong Kong10 originated from Greece and the USA, respectively, i.e. from KPC-endemic regions. Compared with these strains, the genetic context of blaKPC-2 is different in the UAE isolates: they map within the Tn4401b isoform inserted in the opposite direction into the genetic load region of the IncX3 plasmid disrupting the umuD gene (Figure 1). It is noteworthy that neither of our patients had a history of travel or recent hospitalization prior to their current admission, suggesting that both of them acquired the strains while being treated in different wards. Based on the data available, we cannot exclude or confirm the possibility that the clone was concealed in the hospital environment (including personnel) during the considerable time that elapsed between the isolation of the two strains. At present, it is not possible to predict whether these cases indicate the beginning of a local spread of KPC-positive K. pneumoniae or whether their incidence will remain low. To answer these questions, which may have implications in understanding the global
# The Author 2015. 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 2
Research letter
Funding This work was supported by grant UAEU-NRF 31M085 to A. S. and by grants 50193 from MSD and NP 14-07 from UAEU-CMHS to T. P.
Transparency declarations T. P. was a recipient of a grant from MSD. This fact did not affect the design, analysis or the conclusions of the current study. No reimbursement for preparing the manuscript was received. All other authors: none to declare.
Supplementary data Table S1 is available as Supplementary data at JAC Online (http://jac. oxfordjournals.org/).
References 1 Nordmann P, Poirel L. The difficult-to-control spread of carbapenemase producers in Enterobacteriaceae worldwide. Clin Microbiol Infect 2014; 20: 821–30. 2 Dashti AA, Vali L, Jadaon MM et al. The emergence of carbapenem resistance in ESBL-producing Escherichia coli O25B-ST131 strain from community acquired infection in Kuwait. BMC Proc 2011; 5 Suppl 6: O27. 3 Sonnevend A, Al Baloushi A, Ghazawi A et al. Emergence and spread of NDM-1 producer Enterobacteriaceae with contribution of IncX3 plasmids in the United Arab Emirates. J Med Microbiol 2013; 62: 1044 –50. 4 Diancourt L, Passet V, Verhoef J et al. Multilocus sequence typing of Klebsiella pneumoniae nosocomial isolates. J Clin Microbiol 2005; 43: 4178– 82. Figure 1. Comparison of Tn4401 carrying blaKPC-2 inserted into IncX3-type plasmids. pABC220, current study from UAE (GenBank accession no. KM983022); pKpS90, from a French isolate9 (GenBank accession no. JX461340); and pKPC-NY79, from a Hong Kong isolate10 (GenBank accession no. JX104759). Black arrow, blaKPC-2; grey arrow, other resistance gene; hatched arrow, transposition/insertion-related gene; and white arrow, flanking plasmid gene.
spread of the blaKPC gene and the clones carrying it, systemic surveillance of these isolates in the region is needed.
Acknowledgements We are grateful to Professor Patrice Nordmann for the KPC-positive control strain GR-KPC2, to Professor Jordi Vila for control strains CSUB10S and CSUB10S/pSH16K, to the laboratory personnel of the Microbiology and Infection Control Unit, Rashid Hospital, DHA for collecting the isolates and to Mr Mohammed al Haj for his technical assistance.
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5 Brisse S, Fevre C, Passet V et al. Virulent clones of Klebsiella pneumoniae: identification and evolutionary scenario based on genomic and phenotypic characterization. PLoS One 2009; 4: e4982. 6 Turton JF, Baklan H, Siu LK et al. Evaluation of a multiplex PCR for detection of serotypes K1, K2 and K5 in Klebsiella sp. and comparison of isolates within these serotypes. FEMS Microbiol Lett 2008; 284: 247–52. 7 Dome´nech-Sa´nchez A, Martı´nez-Martı´nez L, Herna´ndez-Alle´s S et al. Role of Klebsiella pneumoniae OmpK35 porin in antimicrobial resistance. Antimicrob Agents Chemother 2003; 47: 3332– 5. 8 Al-Qadheeb NS, Althawadi S, Alkhalaf A et al. Evolution of tigecycline resistance in Klebsiella pneumoniae in a single patient. Ann Saudi Med 2010; 30: 404–7. 9 Kassis-Chikhani N, Frangeul L, Drieux L et al. Complete nucleotide sequence of the first KPC-2- and SHV-12-encoding IncX plasmid, pKpS90, from Klebsiella pneumoniae. Antimicrob Agents Chemother 2013; 57: 618–20. 10 Ho PL, Cheung YY, Lo WU et al. Molecular characterization of an atypical IncX3 plasmid pKPC-NY79 carrying blaKPC-2 in a Klebsiella pneumoniae. Curr Microbiol 2013; 67: 493– 8.
J Antimicrob Chemother doi:10.1093/jac/dku576
Characterization of KPC-type carbapenemase-producing Klebsiella pneumoniae strains isolated in the Arabian Peninsula A´gnes Sonnevend1, Akela Ghazawi1, Dania Darwish1, Zulfa AlDeesi2, Ali F. Kadhum2 and Tibor Pa´l1* 1
Department of Microbiology and Immunology, College of Medicine and Health Sciences, United Arab Emirates University, Al Ain, United Arab Emirates; 2Rashid Hospital, Dubai, United Arab Emirates
*Corresponding author. Tel: +971-3-7137480; Fax: +971-3-7671966; E-mail: [email protected]
Keywords: KPC-2, K. pneumoniae, multidrug resistant, MDR
Sir, Since its first description in 1996, variants of KPC have spread throughout the world mostly in the ST258 clone of Klebsiella pneumoniae within isoforms of the Tn4401 transposon located on various plasmids.1 While the Middle East is considered as a reservoir of various carbapenemase-producing bacteria, to date the only Enterobacteriaceae carrying the blaKPC gene reported from the Arabian Peninsula has been an Escherichia coli ST131 strain isolated in Kuwait.2 In a collection of 34 carbapenem-non-susceptible Enterobacteriaceae strains, comprising all clinically relevant, nonrepeat isolates recovered in Rashid Hospital, Dubai, United Arab Emirates (UAE) between March 2012 and February 2014, we identified by PCR3: 3 blaNDM and 1 blaOXA-48-like E. coli; and 3 blaNDM, 6 blaOXA-48-like, 17 blaNDM + blaOXA-48-like and 2 blaKPC K. pneumoniae. Since blaKPC-positive K. pneumoniae has not been described from this region to date, we subjected the two blaKPC-positive K. pneumoniae isolates (ABC220 and ABC224) to detailed investigations. K. pneumoniae ABC220 was isolated in October 2012 from a necrotizing wound of a previously healthy, 40-year-old Filipino male, 25 days after he had been admitted for perforated appendix and sepsis. K. pneumoniae ABC224 was recovered in May 2013 from the sputum of an 80-year-old UAE national with multiple chronic illnesses, continuously hospitalized for .1 year in a different ward for left leg amputation and sepsis. Prior to the isolation of these strains, various samples from both patients had been repeatedly negative for carbapenem-resistant Enterobacteriaceae. As detected by Etest (bioMe´rieux, Marcy-l’E´toile, France) or microdilution, ABC220 and ABC224 exhibited susceptibility to tigecycline (MIC¼0.75 mg/L) and amikacin (MIC¼ 12 mg/L) only. The MICs of all antimicrobials tested are listed in Table S1 (available as Supplementary data at JAC Online). The XbaI-digested
macrorestriction patterns3 of strains exhibited .95% similarity. Both ABC220 and ABC224 belonged to ST14 by MLST.4 Strains were subjected to PCR assays specific for ESBL (blaCTX-M, blaSHV, blaTEM and blaOXA) and AmpC b-lactamase genes, plasmidmediated quinolone and aminoglycoside resistance genes and virulence genes (rmpA, wabG, wcaG, fimH, mrkD, iutA, fyuA, iroN, ireA, kfuBC, traT and clpK).3,5 Sequencing of the amplicons revealed that they both possessed blaTEM-1 , blaSHV-1 , blaCTX-M-15 and aac-6 ′ -1b-cr genes and also virulence-related genes fyuA, kfuBC, fimH, mrkD, traT, clpK, ugeA and wabG. Both strains carried sequences suggestive of a K2 capsule type.6 Outer membrane protein analysis by SDS–PAGE revealed the lack of a band corresponding to OmpK35 from both isolates as compared with the patterns of control strains CSUB10S (Omp36K) and CSUB10S/pSH16K (OmpK35 and OmpK36).7 Plasmid transfer and analysis by electrophoresis, Southern blotting, hybridization, replicon typing and RFLP was performed as described previously. 3 Although blaKPC-carrying plasmids from both strains were conjugally transferred into E. coli J53RAZ, they were not stably maintained in this recipient. Therefore, plasmids were transferred into E. coli GM2163 by transformation. Both ABC220 and ABC224 harboured a 45 kb IncX3 plasmid carrying blaKPC as identified by hybridization. The plasmids exhibited the same RFLP profile when digested with EcoRI and HindIII (data not shown). Sequencing of blaKPC and its flanking regions by PCR mapping and direct sequencing identified the blaKPC-2 allele in both strains located within the Tn4401b transposon inserted into the umuD gene of the IncX3 plasmid (Figure 1) (GenBank accession no. KM983022). At the time of submission of our manuscript, no KPC-producing K. pneumoniae had been reported from the Arabian Peninsula; although, not without causing considerable confusion, the terminology ‘KPC’ was used earlier referring to a strain exhibiting putative carbapenemase activity by modified Hodge test, without any analysis of the bla gene.8 Here, we describe two K. pneumoniae ST14 strains, identical in all features tested, carrying blaKPC-2 on IncX3 plasmids. blaKPC-2-bearing IncX3 plasmids described earlier in ST258 K. pneumoniae in France9 and Hong Kong10 originated from Greece and the USA, respectively, i.e. from KPC-endemic regions. Compared with these strains, the genetic context of blaKPC-2 is different in the UAE isolates: they map within the Tn4401b isoform inserted in the opposite direction into the genetic load region of the IncX3 plasmid disrupting the umuD gene (Figure 1). It is noteworthy that neither of our patients had a history of travel or recent hospitalization prior to their current admission, suggesting that both of them acquired the strains while being treated in different wards. Based on the data available, we cannot exclude or confirm the possibility that the clone was concealed in the hospital environment (including personnel) during the considerable time that elapsed between the isolation of the two strains. At present, it is not possible to predict whether these cases indicate the beginning of a local spread of KPC-positive K. pneumoniae or whether their incidence will remain low. To answer these questions, which may have implications in understanding the global
# The Author 2015. 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 2
Research letter
Funding This work was supported by grant UAEU-NRF 31M085 to A. S. and by grants 50193 from MSD and NP 14-07 from UAEU-CMHS to T. P.
Transparency declarations T. P. was a recipient of a grant from MSD. This fact did not affect the design, analysis or the conclusions of the current study. No reimbursement for preparing the manuscript was received. All other authors: none to declare.
Supplementary data Table S1 is available as Supplementary data at JAC Online (http://jac. oxfordjournals.org/).
References 1 Nordmann P, Poirel L. The difficult-to-control spread of carbapenemase producers in Enterobacteriaceae worldwide. Clin Microbiol Infect 2014; 20: 821–30. 2 Dashti AA, Vali L, Jadaon MM et al. The emergence of carbapenem resistance in ESBL-producing Escherichia coli O25B-ST131 strain from community acquired infection in Kuwait. BMC Proc 2011; 5 Suppl 6: O27. 3 Sonnevend A, Al Baloushi A, Ghazawi A et al. Emergence and spread of NDM-1 producer Enterobacteriaceae with contribution of IncX3 plasmids in the United Arab Emirates. J Med Microbiol 2013; 62: 1044 –50. 4 Diancourt L, Passet V, Verhoef J et al. Multilocus sequence typing of Klebsiella pneumoniae nosocomial isolates. J Clin Microbiol 2005; 43: 4178– 82. Figure 1. Comparison of Tn4401 carrying blaKPC-2 inserted into IncX3-type plasmids. pABC220, current study from UAE (GenBank accession no. KM983022); pKpS90, from a French isolate9 (GenBank accession no. JX461340); and pKPC-NY79, from a Hong Kong isolate10 (GenBank accession no. JX104759). Black arrow, blaKPC-2; grey arrow, other resistance gene; hatched arrow, transposition/insertion-related gene; and white arrow, flanking plasmid gene.
spread of the blaKPC gene and the clones carrying it, systemic surveillance of these isolates in the region is needed.
Acknowledgements We are grateful to Professor Patrice Nordmann for the KPC-positive control strain GR-KPC2, to Professor Jordi Vila for control strains CSUB10S and CSUB10S/pSH16K, to the laboratory personnel of the Microbiology and Infection Control Unit, Rashid Hospital, DHA for collecting the isolates and to Mr Mohammed al Haj for his technical assistance.
2 of 2
5 Brisse S, Fevre C, Passet V et al. Virulent clones of Klebsiella pneumoniae: identification and evolutionary scenario based on genomic and phenotypic characterization. PLoS One 2009; 4: e4982. 6 Turton JF, Baklan H, Siu LK et al. Evaluation of a multiplex PCR for detection of serotypes K1, K2 and K5 in Klebsiella sp. and comparison of isolates within these serotypes. FEMS Microbiol Lett 2008; 284: 247–52. 7 Dome´nech-Sa´nchez A, Martı´nez-Martı´nez L, Herna´ndez-Alle´s S et al. Role of Klebsiella pneumoniae OmpK35 porin in antimicrobial resistance. Antimicrob Agents Chemother 2003; 47: 3332– 5. 8 Al-Qadheeb NS, Althawadi S, Alkhalaf A et al. Evolution of tigecycline resistance in Klebsiella pneumoniae in a single patient. Ann Saudi Med 2010; 30: 404–7. 9 Kassis-Chikhani N, Frangeul L, Drieux L et al. Complete nucleotide sequence of the first KPC-2- and SHV-12-encoding IncX plasmid, pKpS90, from Klebsiella pneumoniae. Antimicrob Agents Chemother 2013; 57: 618–20. 10 Ho PL, Cheung YY, Lo WU et al. Molecular characterization of an atypical IncX3 plasmid pKPC-NY79 carrying blaKPC-2 in a Klebsiella pneumoniae. Curr Microbiol 2013; 67: 493– 8.
