Identification of KPC-2-producing Escherichia coli in a companion animal: a new challenge for veterinary clinicians

Identification of KPC-2-producing Escherichia coli in a companion animal: a new challenge for... Sir, The unbridled dissemination of carbapenemase-producing bacteria in healthcare-associated infections has generated a great threat to public health.1 Since 2001, the occurrence of KPC-producing bacteria has become worrisome, mainly because these bacteria have been frequently related to high rates of morbidity and mortality.2,3 In this regard, the prevalence of KPC has been restricted to humans and anthropogenically impacted environments,3,4 being sporadically reported from broilers in Egypt and mussel samples from a retail market in Tunisia.5,6 In this study, to our knowledge, we report for the first time the occurrence of KPC-2-producing Escherichia coli in an infected dog, raising an alert about the emergence of KPC-2 producers in veterinary medicine. Between June and November 2017, 4228 veterinary clinical samples processed by a large veterinary laboratory in São Paulo, Brazil, were screened by BD Phoenix (BD Diagnostics, Sparks, MD, USA) for potential carbapenemase producers, leading to the identification of one ertapenem-resistant E. coli strain (ECSIC19). The carbapenem-resistant E. coli was isolated from a urine culture of a 13-year-old female mixed-breed dog that succumbed to urinary tract infection. The ECSIC19 strain exhibited an MDR profile to amoxicillin/clavulanic acid (MIC≥16/8 mg/L), ceftiofur (MIC>32 mg/L), ceftazidime (MIC>32 mg/L), cefotaxime (MIC>32 mg/L), cefepime (MIC>32 mg/L), ertapenem (MIC 4 mg/L), imipenem (MIC 4 mg/L), meropenem (MIC 4 mg/L), sulfamethoxazole/trimethoprim (MIC≥4/76 mg/L), enrofloxacin (MIC>32 mg/L), ciprofloxacin (MIC>32 mg/L), gentamicin (MIC≥16 mg/L), levofloxacin (MIC>32 mg/L) nalidixic acid (MIC>32 mg/L) and tetracycline (MIC≥ 64 mg/L), as determined by BD Phoenix (BD Diagnostics) and/or Etest methods.7 ESBL production was confirmed by using a double-disc synergy test, whereas carbapenemase production was detected using an imipenem disc supplemented with 3-aminophenylboronic acid and the modified Hodge test. Additionally, PCR screening and direct sequencing identified blaKPC-2 and blaCTX-M-15 genes. Total DNA was sequencing using Illumina NextSeq 500 paired-end reads (150 bp). De novo assembly was performed using SPAdes v.3.11.1 The resistome, MLST, serotype, plasmidome and virulome were evaluated using bioinformatics tools available from the Center for Genomic Epidemiology (http://genomicepidemiology.org/). E. coli ECSIC19 belonged to serotype O1:H6, MLST ST648 and phylogroup D. ST648 has a pandemic distribution among humans,8 different environments9 and animals,10 being commonly associated with CTX-M-15 enzymes11 and carbapenemase producers.9 Moreover, this E. coli lineage has been globally recognized as a human and animal pathogen.11 The identification of air (enteroaggregative immunoglobulin repeat protein), astA (EAST1 toxin), eilA (Salmonella HilA homologue), iss (increased serum survival) and lpfA (long polar fimbriae) virulence genes revealed a high-virulence profile of E. coli ECSIC19, being defined as an extraintestinal pathogenic E. coli (ExPEC) lineage.11 The resistome of E. coli ECSIC19 confirmed the presence of β-lactam resistance genes (blaKPC-2 and blaCTX-M-15) and other clinically significant resistance determinants conferring resistance to aminoglycosides (aadA5), quinolones (qnrS1), macrolides [mph(A) and erm(B)], sulphonamides (sul1), tetracycline [tet(B)] and trimethoprim (dfrA17). Additionally, point mutations Ser83Leu and Asp87Asn in GyrA and Ser80Ile in ParC were associated with quinolone resistance. E. coli ECSIC19 harboured IncI1, IncFIA, IncFII, IncFII, IncFIB, IncN and p0111 plasmids. The blaKCP-2 gene was found in a hybrid plasmid (pECSIC19) carrying a Tn4401b transposon (Figure 1). The pECSIC19 plasmid (GenBank accession number MG886286) was assembled using de novo contigs and the reference pFCF3SP IncN plasmid (GenBank accession number CP004367) harbouring the blaKPC-2 gene, previously isolated from a human clinical KPC-2-producing Klebsiella pneumoniae identified in Brazil. In this regard, IncN plasmid backbones have dispersed globally within E. coli.12 The preliminary scaffold was refined to solve remaining gaps using an in silico gap closure strategy and automatic/manually annotated by Glimmer 3.2 and ISFinder. The pECSIC19 (52 776 bp) plasmid was confirmed as belonging to the IncN replicon group and ST15, being successfully transferred by conjugation into streptomycin-resistant E. coli strain C600. Figure 1 View largeDownload slide Comparative analysis of IncN-ST15 plasmids carrying the blaKPC-2 gene, isolated from animal (pECSIC19) and human (pFCF3SP and pEC517_KPC) E. coli and K. pneumoniae strains. A high identity (>90% identity) between pECSIC19 (GenBank accession number MG886286), pFCF3SP (GenBank accession number CP004367) and pEC517_KPC (GenBank accession number CP018963) plasmids was observed. This figure appears in colour in the online version of JAC and in black and white in the print version of JAC. Figure 1 View largeDownload slide Comparative analysis of IncN-ST15 plasmids carrying the blaKPC-2 gene, isolated from animal (pECSIC19) and human (pFCF3SP and pEC517_KPC) E. coli and K. pneumoniae strains. A high identity (>90% identity) between pECSIC19 (GenBank accession number MG886286), pFCF3SP (GenBank accession number CP004367) and pEC517_KPC (GenBank accession number CP018963) plasmids was observed. This figure appears in colour in the online version of JAC and in black and white in the print version of JAC. As IncN-ST15 plasmids carrying blaKPC-2::Tn4401b have been previously reported in K. pneumoniae isolates from humans and aquatic environments, in Brazil (GenBank accession numbers CP004367, KC770989 and KC770990), a comparative plasmid analysis was performed using BRIG against pEC517_KPC (GenBank accession number CP018963) and pFCF3SP (GenBank accession number CP004367) plasmids isolated from E. coli and K. pneumoniae of human origin. Comparative analysis revealed >90% nucleotide identity among these plasmids, sharing an identical backbone, whereas only pECSIC19 showed insertion of ISKpn20 (IS3 family) truncating the region of endonuclease EcoRII, similarly to pKp1433 plasmid (JX397875). Additionally, pFCF3SP was different due to absence of type I DNA restriction systems such as ardA, commonly found in conjugative plasmids and assisting in the spread of resistance genes (Figure 1). These results highlight that IncN/ST-15 plasmids are key vectors responsible for the dissemination of the blaKPC-2 gene in Enterobacteriaceae in the human–animal–environment interface in Brazil. In conclusion, to the best of our knowledge, this is the first report of an E. coli co-producing KPC-2, CTX-M-15 and QnrS1 isolated from an animal. These findings suggest that although carbapenems have no legal indications for animals, closer relationships between humans and animals, or the empirical usage of carbapenems to treat ESBL-related infections, could be contributing to the spread of the blaKPC-2 gene and other carbapenemase genes to pets.13 Therefore, the occurrence of MDR bacteria harbouring blaKPC-type genes in animals may represent a new challenge for veterinary medicine and a potential threat to public health. Acknowledgements FAPESP and CNPq research grants are gratefully acknowledged. We thank Cefar Diagnóstica Ltda (Brazil) for kindly supplying antibiotic discs for susceptibility testing. Funding This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP; grant number 2016/08593-9) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq; grant number 462042/2014-6). N. L. is a research fellow of CNPq (grant number: 312249/2017-9). M. R. F. and L. C. are research fellows of FAPESP (grant numbers 2015/13527-2 and 2015/21325-0). Transparency declarations None to declare. References 1 An J , Lai K , Ma Y et al. Emergence of multiple carbapenemase-producing organisms in single patients: an increasing threat to treatment of infection . J Antimicrob Chemother 2018 ; 73 : 544 – 6 . Google Scholar CrossRef Search ADS PubMed 2 Yigit H , Queenan AM , Anderson GJ et al. Novel carbapenem-hydrolyzing β-lactamase, KPC-1, from a carbapenem-resistant strain of Klebsiella pneumoniae . Antimicrob Agents Chemother 2001 ; 45 : 1151 – 61 . Google Scholar CrossRef Search ADS PubMed 3 Wang Q , Zhang Y , Yao X et al. Risk factors and clinical outcomes for carbapenem-resistant Enterobacteriaceae nosocomial infections . Eur J Clin Microbiol Infect Dis 2016 ; 35 : 1679 – 89 . Google Scholar CrossRef Search ADS PubMed 4 Paschoal RP , Campana EH , Corrêa LL et al. Concentration and variety of carbapenemase producers in recreational coastal waters showing distinct levels of pollution . Antimicrob Agents Chemother 2017 ; 61 : e01963–17. Google Scholar CrossRef Search ADS PubMed 5 Hamza E , Dorgham SM , Hamza DA. Carbapenemase-producing Klebsiella pneumoniae in broiler poultry farming in Egypt . J Glob Antimicrob Resist 2016 ; 7 : 8 – 10 . Google Scholar CrossRef Search ADS PubMed 6 Mani Y , Mansour W , Mammeri H et al. KPC-3-producing ST167 Escherichia coli from mussels bought at a retail market in Tunisia . J Antimicrob Chemother 2017 ; 72 : 2403 – 4 . Google Scholar CrossRef Search ADS PubMed 7 Clinical and Laboratory Standards Institute . Performance Standards for Antimicrobial Susceptibility Testing: Twenty-Seventh Informational Supplement M100-S27 . CLSI , Wayne, PA, USA , 2017 . 8 Musicha P , Feasey NA , Cain AK et al. Genomic landscape of extended-spectrum β-lactamase resistance in Escherichia coli from an urban African setting . J Antimicrob Chemother 2017 ; 72 : 1602 – 9 . Google Scholar CrossRef Search ADS PubMed 9 Toleman MA , Bugert JJ , Nizam SA. Extensively drug-resistant New Delhi metallo-β-lactamase-encoding bacteria in the environment, Dhaka, Bangladesh, 2012 . Emerg Infect Dis 2015 ; 21 : 1027 – 30 . Google Scholar CrossRef Search ADS PubMed 10 Bachiri T , Bakour S , Ladjouzi R et al. High rates of CTX-M-15-producing Escherichia coli and Klebsiella pneumoniae in wild boars and Barbary macaques in Algeria . J Glob Antimicrob Resist 2017 ; 8 : 35 – 40 . Google Scholar CrossRef Search ADS PubMed 11 Ewers C , Bethe A , Stamm I et al. CTX-M-15-D-ST648 Escherichia coli from companion animals and horses: another pandemic clone combining multiresistance and extraintestinal virulence? J Antimicrob Chemother 2014 ; 69 : 1224 – 30 . Google Scholar CrossRef Search ADS PubMed 12 Stoesser N , Sheppard AE , Peirano G et al. Genomic epidemiology of global Klebsiella pneumoniae carbapenemase (KPC)-producing Escherichia coli . Sci Rep 2017 ; 7 : 5917 . Google Scholar CrossRef Search ADS PubMed 13 Madec JY , Haenni M , Nordmann P et al. Extended-spectrum β-lactamase/AmpC- and carbapenemase-producing Enterobacteriaceae in animals: a threat for humans? Clin Microbiol Infect 2017 ; 23 : 826 – 33 . Google Scholar CrossRef Search ADS PubMed © The Author(s) 2018. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Antimicrobial Chemotherapy Oxford University Press

Identification of KPC-2-producing Escherichia coli in a companion animal: a new challenge for veterinary clinicians

Journal of Antimicrobial Chemotherapy , Volume Advance Article (8) – May 24, 2018

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Oxford University Press
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© The Author(s) 2018. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com.
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D.O.I.
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Abstract

Sir, The unbridled dissemination of carbapenemase-producing bacteria in healthcare-associated infections has generated a great threat to public health.1 Since 2001, the occurrence of KPC-producing bacteria has become worrisome, mainly because these bacteria have been frequently related to high rates of morbidity and mortality.2,3 In this regard, the prevalence of KPC has been restricted to humans and anthropogenically impacted environments,3,4 being sporadically reported from broilers in Egypt and mussel samples from a retail market in Tunisia.5,6 In this study, to our knowledge, we report for the first time the occurrence of KPC-2-producing Escherichia coli in an infected dog, raising an alert about the emergence of KPC-2 producers in veterinary medicine. Between June and November 2017, 4228 veterinary clinical samples processed by a large veterinary laboratory in São Paulo, Brazil, were screened by BD Phoenix (BD Diagnostics, Sparks, MD, USA) for potential carbapenemase producers, leading to the identification of one ertapenem-resistant E. coli strain (ECSIC19). The carbapenem-resistant E. coli was isolated from a urine culture of a 13-year-old female mixed-breed dog that succumbed to urinary tract infection. The ECSIC19 strain exhibited an MDR profile to amoxicillin/clavulanic acid (MIC≥16/8 mg/L), ceftiofur (MIC>32 mg/L), ceftazidime (MIC>32 mg/L), cefotaxime (MIC>32 mg/L), cefepime (MIC>32 mg/L), ertapenem (MIC 4 mg/L), imipenem (MIC 4 mg/L), meropenem (MIC 4 mg/L), sulfamethoxazole/trimethoprim (MIC≥4/76 mg/L), enrofloxacin (MIC>32 mg/L), ciprofloxacin (MIC>32 mg/L), gentamicin (MIC≥16 mg/L), levofloxacin (MIC>32 mg/L) nalidixic acid (MIC>32 mg/L) and tetracycline (MIC≥ 64 mg/L), as determined by BD Phoenix (BD Diagnostics) and/or Etest methods.7 ESBL production was confirmed by using a double-disc synergy test, whereas carbapenemase production was detected using an imipenem disc supplemented with 3-aminophenylboronic acid and the modified Hodge test. Additionally, PCR screening and direct sequencing identified blaKPC-2 and blaCTX-M-15 genes. Total DNA was sequencing using Illumina NextSeq 500 paired-end reads (150 bp). De novo assembly was performed using SPAdes v.3.11.1 The resistome, MLST, serotype, plasmidome and virulome were evaluated using bioinformatics tools available from the Center for Genomic Epidemiology (http://genomicepidemiology.org/). E. coli ECSIC19 belonged to serotype O1:H6, MLST ST648 and phylogroup D. ST648 has a pandemic distribution among humans,8 different environments9 and animals,10 being commonly associated with CTX-M-15 enzymes11 and carbapenemase producers.9 Moreover, this E. coli lineage has been globally recognized as a human and animal pathogen.11 The identification of air (enteroaggregative immunoglobulin repeat protein), astA (EAST1 toxin), eilA (Salmonella HilA homologue), iss (increased serum survival) and lpfA (long polar fimbriae) virulence genes revealed a high-virulence profile of E. coli ECSIC19, being defined as an extraintestinal pathogenic E. coli (ExPEC) lineage.11 The resistome of E. coli ECSIC19 confirmed the presence of β-lactam resistance genes (blaKPC-2 and blaCTX-M-15) and other clinically significant resistance determinants conferring resistance to aminoglycosides (aadA5), quinolones (qnrS1), macrolides [mph(A) and erm(B)], sulphonamides (sul1), tetracycline [tet(B)] and trimethoprim (dfrA17). Additionally, point mutations Ser83Leu and Asp87Asn in GyrA and Ser80Ile in ParC were associated with quinolone resistance. E. coli ECSIC19 harboured IncI1, IncFIA, IncFII, IncFII, IncFIB, IncN and p0111 plasmids. The blaKCP-2 gene was found in a hybrid plasmid (pECSIC19) carrying a Tn4401b transposon (Figure 1). The pECSIC19 plasmid (GenBank accession number MG886286) was assembled using de novo contigs and the reference pFCF3SP IncN plasmid (GenBank accession number CP004367) harbouring the blaKPC-2 gene, previously isolated from a human clinical KPC-2-producing Klebsiella pneumoniae identified in Brazil. In this regard, IncN plasmid backbones have dispersed globally within E. coli.12 The preliminary scaffold was refined to solve remaining gaps using an in silico gap closure strategy and automatic/manually annotated by Glimmer 3.2 and ISFinder. The pECSIC19 (52 776 bp) plasmid was confirmed as belonging to the IncN replicon group and ST15, being successfully transferred by conjugation into streptomycin-resistant E. coli strain C600. Figure 1 View largeDownload slide Comparative analysis of IncN-ST15 plasmids carrying the blaKPC-2 gene, isolated from animal (pECSIC19) and human (pFCF3SP and pEC517_KPC) E. coli and K. pneumoniae strains. A high identity (>90% identity) between pECSIC19 (GenBank accession number MG886286), pFCF3SP (GenBank accession number CP004367) and pEC517_KPC (GenBank accession number CP018963) plasmids was observed. This figure appears in colour in the online version of JAC and in black and white in the print version of JAC. Figure 1 View largeDownload slide Comparative analysis of IncN-ST15 plasmids carrying the blaKPC-2 gene, isolated from animal (pECSIC19) and human (pFCF3SP and pEC517_KPC) E. coli and K. pneumoniae strains. A high identity (>90% identity) between pECSIC19 (GenBank accession number MG886286), pFCF3SP (GenBank accession number CP004367) and pEC517_KPC (GenBank accession number CP018963) plasmids was observed. This figure appears in colour in the online version of JAC and in black and white in the print version of JAC. As IncN-ST15 plasmids carrying blaKPC-2::Tn4401b have been previously reported in K. pneumoniae isolates from humans and aquatic environments, in Brazil (GenBank accession numbers CP004367, KC770989 and KC770990), a comparative plasmid analysis was performed using BRIG against pEC517_KPC (GenBank accession number CP018963) and pFCF3SP (GenBank accession number CP004367) plasmids isolated from E. coli and K. pneumoniae of human origin. Comparative analysis revealed >90% nucleotide identity among these plasmids, sharing an identical backbone, whereas only pECSIC19 showed insertion of ISKpn20 (IS3 family) truncating the region of endonuclease EcoRII, similarly to pKp1433 plasmid (JX397875). Additionally, pFCF3SP was different due to absence of type I DNA restriction systems such as ardA, commonly found in conjugative plasmids and assisting in the spread of resistance genes (Figure 1). These results highlight that IncN/ST-15 plasmids are key vectors responsible for the dissemination of the blaKPC-2 gene in Enterobacteriaceae in the human–animal–environment interface in Brazil. In conclusion, to the best of our knowledge, this is the first report of an E. coli co-producing KPC-2, CTX-M-15 and QnrS1 isolated from an animal. These findings suggest that although carbapenems have no legal indications for animals, closer relationships between humans and animals, or the empirical usage of carbapenems to treat ESBL-related infections, could be contributing to the spread of the blaKPC-2 gene and other carbapenemase genes to pets.13 Therefore, the occurrence of MDR bacteria harbouring blaKPC-type genes in animals may represent a new challenge for veterinary medicine and a potential threat to public health. Acknowledgements FAPESP and CNPq research grants are gratefully acknowledged. We thank Cefar Diagnóstica Ltda (Brazil) for kindly supplying antibiotic discs for susceptibility testing. Funding This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP; grant number 2016/08593-9) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq; grant number 462042/2014-6). N. L. is a research fellow of CNPq (grant number: 312249/2017-9). M. R. F. and L. C. are research fellows of FAPESP (grant numbers 2015/13527-2 and 2015/21325-0). Transparency declarations None to declare. References 1 An J , Lai K , Ma Y et al. Emergence of multiple carbapenemase-producing organisms in single patients: an increasing threat to treatment of infection . J Antimicrob Chemother 2018 ; 73 : 544 – 6 . Google Scholar CrossRef Search ADS PubMed 2 Yigit H , Queenan AM , Anderson GJ et al. Novel carbapenem-hydrolyzing β-lactamase, KPC-1, from a carbapenem-resistant strain of Klebsiella pneumoniae . Antimicrob Agents Chemother 2001 ; 45 : 1151 – 61 . Google Scholar CrossRef Search ADS PubMed 3 Wang Q , Zhang Y , Yao X et al. Risk factors and clinical outcomes for carbapenem-resistant Enterobacteriaceae nosocomial infections . Eur J Clin Microbiol Infect Dis 2016 ; 35 : 1679 – 89 . Google Scholar CrossRef Search ADS PubMed 4 Paschoal RP , Campana EH , Corrêa LL et al. Concentration and variety of carbapenemase producers in recreational coastal waters showing distinct levels of pollution . Antimicrob Agents Chemother 2017 ; 61 : e01963–17. Google Scholar CrossRef Search ADS PubMed 5 Hamza E , Dorgham SM , Hamza DA. Carbapenemase-producing Klebsiella pneumoniae in broiler poultry farming in Egypt . J Glob Antimicrob Resist 2016 ; 7 : 8 – 10 . Google Scholar CrossRef Search ADS PubMed 6 Mani Y , Mansour W , Mammeri H et al. KPC-3-producing ST167 Escherichia coli from mussels bought at a retail market in Tunisia . J Antimicrob Chemother 2017 ; 72 : 2403 – 4 . Google Scholar CrossRef Search ADS PubMed 7 Clinical and Laboratory Standards Institute . Performance Standards for Antimicrobial Susceptibility Testing: Twenty-Seventh Informational Supplement M100-S27 . CLSI , Wayne, PA, USA , 2017 . 8 Musicha P , Feasey NA , Cain AK et al. Genomic landscape of extended-spectrum β-lactamase resistance in Escherichia coli from an urban African setting . J Antimicrob Chemother 2017 ; 72 : 1602 – 9 . Google Scholar CrossRef Search ADS PubMed 9 Toleman MA , Bugert JJ , Nizam SA. Extensively drug-resistant New Delhi metallo-β-lactamase-encoding bacteria in the environment, Dhaka, Bangladesh, 2012 . Emerg Infect Dis 2015 ; 21 : 1027 – 30 . Google Scholar CrossRef Search ADS PubMed 10 Bachiri T , Bakour S , Ladjouzi R et al. High rates of CTX-M-15-producing Escherichia coli and Klebsiella pneumoniae in wild boars and Barbary macaques in Algeria . J Glob Antimicrob Resist 2017 ; 8 : 35 – 40 . Google Scholar CrossRef Search ADS PubMed 11 Ewers C , Bethe A , Stamm I et al. CTX-M-15-D-ST648 Escherichia coli from companion animals and horses: another pandemic clone combining multiresistance and extraintestinal virulence? J Antimicrob Chemother 2014 ; 69 : 1224 – 30 . Google Scholar CrossRef Search ADS PubMed 12 Stoesser N , Sheppard AE , Peirano G et al. Genomic epidemiology of global Klebsiella pneumoniae carbapenemase (KPC)-producing Escherichia coli . Sci Rep 2017 ; 7 : 5917 . Google Scholar CrossRef Search ADS PubMed 13 Madec JY , Haenni M , Nordmann P et al. Extended-spectrum β-lactamase/AmpC- and carbapenemase-producing Enterobacteriaceae in animals: a threat for humans? Clin Microbiol Infect 2017 ; 23 : 826 – 33 . Google Scholar CrossRef Search ADS PubMed © The Author(s) 2018. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

Journal

Journal of Antimicrobial ChemotherapyOxford University Press

Published: May 24, 2018

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