Antonie van Leeuwenhoek

, Volume 109, Issue 7, pp 1029–1040 | Cite as

Comparative analyses of phenotypic methods and 16S rRNA, khe, rpoB genes sequencing for identification of clinical isolates of Klebsiella pneumoniae

  • Yanxia He
  • Xianguang Guo
  • Shifei Xiang
  • Jiao Li
  • Xiaoqin Li
  • Hui Xiang
  • Jinlei He
  • Dali Chen
  • Jianping Chen
Original Paper

Abstract

The present work aimed to evaluate 16S rRNA, khe and rpoB gene sequencing for the identification of Klebsiella pneumoniae in comparison with phenotypic methods. Fifteen clinical isolates were examined, which were initially identified as K. pneumoniae subsp. pneumoniae using the automated VITEK 32 system in two hospitals in Enshi City, China. Their identity was further supported by conventional phenotypic methods on the basis of morphological and biochemical characteristics. Using Bayesian phylogenetic analyses and haplotypes network reconstruction, 13 isolates were identified as K. pneumoniae, whereas the other two isolates (K19, K24) were classified as Shigella sp. and Enterobacter sp., respectively. Of the three genes, 16S rRNA and khe gene could discriminate the clinical isolates at the genus level, whereas rpoB could discriminate Klebsiella at the species and even subspecies level. Overall, the gene tree based on rpoB is more compatible with the currently accepted classification of Klebsiella than those based on 16S rRNA and khe genes, showing that rpoB can be a powerful tool for identification of K. pneumoniae isolates. Above all, our study challenges the utility of khe as a species-specific marker for identification of K. pneumoniae.

Keywords

16S rRNA khe rpoB Bayesian phylogenetic analysis Species identification Klebsiella pneumoniae 

Notes

Acknowledgments

This work was supported by the National Natural Science Foundation of China (31572240, 81171607, and J1103604), and the fund of collaboration between Sichuan University and Luzhou City (2013CDLZ-S16). Xianguang Guo was supported by the National Natural Science Foundation of China (31272281).

Supplementary material

10482_2016_702_MOESM1_ESM.docx (29 kb)
Supplementary material 1 (DOCX 30 kb)
10482_2016_702_MOESM2_ESM.docx (24 kb)
Supplementary material 2 (DOCX 24 kb)

References

  1. Adékambi T, Drancourt M, Raoult D (2009) The rpoB gene as a tool for clinical microbiologists. Trends Microbiol 17:37–45CrossRefPubMedGoogle Scholar
  2. Albuquerque P, Mendes MV, Santos CL (2009) DNA signature-based approaches for bacterial detection and identification. Sci Total Environ 407:3641–3651CrossRefPubMedGoogle Scholar
  3. Baraniak A, Izdebski R, Fiett J et al (2016) NDM-producing Enterobacteriaceae in Poland, 2012–14: inter-regional outbreak of Klebsiella pneumoniae ST11 and sporadic cases. J Antimicrob Chemother 71:85–91CrossRefPubMedGoogle Scholar
  4. Borer A, Saidel-Odes L, Riesenberg K et al (2009) Attributable mortality rate for carbapenem-resistant Klebsiella pneumoniae bacteremia. Infect Control Hosp Epidemiol 30:972–976CrossRefPubMedGoogle Scholar
  5. Boye K, Hansen DS (2003) Sequencing of 16S rDNA of Klebsiella: taxonomic relations within the genus and to other Enterobacteriaceae. Int J Med Microbiol 292:495–503CrossRefPubMedGoogle Scholar
  6. Brisse S, Duijkeren E (2005) Identification and antimicrobial susceptibility of 100 Klebsiella animal clinical isolates. Vet Microbiol 105:307–312CrossRefPubMedGoogle Scholar
  7. Brisse S, Verhoef J (2001) Phylogenetic diversity of Klebsiella pneumoniae and Klebsiella oxytoca clinical isolates revealed by randomly amplified polymorphic DNA, gyrA and parC genes sequencing and automated ribotyping. Int J Syst Evol Microbiol 51:915–924CrossRefPubMedGoogle Scholar
  8. Brisse S, van Himbergen T, Kusters K, Verhoef J (2004) Development of a rapid identification method for Klebsiella pneumoniae phylogenetic groups and analysis of 420 clinical isolates. Clin Microbiol Infect 10:942–945CrossRefPubMedGoogle Scholar
  9. Brisse S, Passet V, Grimont PA (2014) Description of Klebsiella quasipneumoniae sp. nov., isolated from human infections, with two subspecies, Klebsiella quasipneumoniae subsp. quasipneumoniae subsp. nov. and Klebsiella quasipneumoniae subsp. similipneumoniae subsp. nov., and demonstration that Klebsiella singaporensis is a junior heterotypic synonym of Klebsiella variicola. Int J Syst Evol Microbiol 64:3146–3152CrossRefPubMedGoogle Scholar
  10. Carter JS, Bowden FJ, Bastian I (1999) Phylogenetic evidence for reclassification of Calymmatobacterium granulomatis as Klebsiella granulomatis comb. nov. Int J Syst Bacteriol 49:1695–1700CrossRefPubMedGoogle Scholar
  11. Chen Z, Liu M, Cui Y et al (2014) A novel PCR-based genotyping scheme for clinical Klebsiella pneumoniae. Future Microbiol 9:21–32CrossRefPubMedGoogle Scholar
  12. Chuang YC, Su JH, Lin CN (2002) Cloning of a gene encoding a unique hemolysin from Klebsiella pneumoniae and its potential use as a species-specific gene probe. Microb Pathog 33:1–6CrossRefGoogle Scholar
  13. Clement M, Posada D, Crandall K (2000) TCS: a computer program to estimate gene genealogies. Mol Ecol 9:1657–1660CrossRefPubMedGoogle Scholar
  14. Diancourt L, Passet V, Verhoef J et al (2005) Multilocus sequence typing of Klebsiella pneumoniae nosocomial isolates. J Clin Microbiol 43:4178–4182CrossRefPubMedPubMedCentralGoogle Scholar
  15. Drancourt M, Bollet C, Carta A (2001) Phylogenetic analyses of Klebsiella species delineate Klebsiella and Raoultella gen. nov., with description of Raoultella ornithinolytica comb. nov., Raoultella terrigena comb. nov., Raoultella planticola comb. nov. Int J Syst Evol Microbiol 51:925–932CrossRefPubMedGoogle Scholar
  16. Dunwell JM (1998) Sequence analysis of the cupin gene family in Synechocystis PCC6803. Microb Comp Genomics 3:141–148CrossRefPubMedGoogle Scholar
  17. Escobar Pérez JA, Olarte Escobar NM, Castro-Cardozo B et al (2013) Outbreak of NDM-1-producing Klebsiella pneumoniae in a neonatal unit in Colombia. Antimicrob Agents Chemother 57:1957–1960CrossRefPubMedPubMedCentralGoogle Scholar
  18. Fevre C, Passet V, Deletoile A et al (2011) PCR-based identification of Klebsiella pneumoniae subsp. rhinoscleromatis, the agent of rhinoscleroma. PLoS Negl Trop Dis 5:e1052CrossRefPubMedPubMedCentralGoogle Scholar
  19. Fontana C, Favaro M, Pelliccioni M (2005) Use of the MicroSeq 500 16S rRNA gene-based sequencing for identification of bacterial isolates that commercial automated systems failed to identify correctly. J Clin Microbiol 43:615–619CrossRefPubMedPubMedCentralGoogle Scholar
  20. Garcia-Garrote F, Cercenado E, Bouza E (2000) Evaluation of a new system, VITEK 2, for identification and antimicrobial susceptibility testing of enterococci. J Clin Microbiol 38:2108–2111PubMedPubMedCentralGoogle Scholar
  21. Gouy M, Guindon S, Gascuel O (2010) SeaView version 4: a multiplatform graphical user interface for sequence alignment and phylogenetic tree building. Mol Biol Evol 27:221–224CrossRefPubMedGoogle Scholar
  22. Hansen DS, Aucken HM, Abiola T et al (2004) Recommended test panel for differentiation of Klebsiella species on the basis of a trilateral interlaboratory evaluation of 18 biochemical tests. J Clin Microbiol 42:3665–3669CrossRefPubMedPubMedCentralGoogle Scholar
  23. Harris KA, Hartley JC (2003) Development of broad-range 16S rDNA PCR for use in the routine diagnostic clinical microbiology service. J Med Microbiol 52:685–691CrossRefPubMedGoogle Scholar
  24. Hashimoto JG, Stevenson BS, Schmidt TM (2003) Rates and consequences of recombination between rRNA operons. J Bacteriol 185:966–972CrossRefPubMedPubMedCentralGoogle Scholar
  25. Hawkey PM (2006) Identification of Enterobacteriaceae. In: Gillespie SH, Hawkey PM (eds) Principles and Practice of Clinical Bacteriology, 2nd edn. Wiley & Sons, England, pp 341–345CrossRefGoogle Scholar
  26. He JY, Qu JF, Liu MY et al (2012) Polymerase chain reaction analysis for detecting highly virulent serotypes of Klebsiella pneumoniae. Letters in Biotechnology 23:554–557 (in Chinese) Google Scholar
  27. Jerassy Z, Yinnon AM, Mazouz-Cohen S et al (2006) Prospective hospital-wide studies of 505 patients with nosocomial bacteraemia in 1997 and 2002. J Hosp Infect 62:230–236CrossRefPubMedGoogle Scholar
  28. Johnson M, Zaretskaya I, Raytselis Y (2008) NCBI BLAST: a better web interface. Nucleic Acids Res 36:W5–W9CrossRefPubMedPubMedCentralGoogle Scholar
  29. Jonas D, Spitzmüller B, Daschner FD (2004) Discrimination of Klebsiella pneumoniae and Klebsiella oxytoca phylogenetic groups and other Klebsiella species by use of amplified fragment length polymorphism. Res Microbiol 155:17–23CrossRefPubMedGoogle Scholar
  30. Kovtunovych G, Lytvynenko T, Negrutska V (2003) Identification of Klebsiella oxytoca using a specific PCR assay targeting the polygalacturonase pehX gene. Res Microbiol 154:587–592CrossRefPubMedGoogle Scholar
  31. Kumar V, Sun P, Vamathevan J et al (2011) Comparative genomics of Klebsiella pneumoniae strains with different antibiotic resistance profiles. Antimicrob Agents Chemother 55:4267–4276CrossRefPubMedPubMedCentralGoogle Scholar
  32. Lane DJ (1991) 16S and 23S rRNA sequencing. In: Stackebrandt E, Goodfellow M (eds) Nucleic acids techniques in bacterial systematics. John Wiley & Sons, Chichester, pp 115–175Google Scholar
  33. Martínez J, Martínez L, Rosenblueth M (2004) How are gene sequence analyses modifying bacterial taxonomy? The case of Klebsiella. Int Microbiol 7:261–268PubMedGoogle Scholar
  34. Martínez-Murcia AJ, Antón AI, Rodríguez-Valera F (1999) Patterns of sequence variation in two regions of the 16S rRNA multigene family of Escherichia coli. Int J Syst Bacteriol 49:601–610CrossRefPubMedGoogle Scholar
  35. Mollet C, Drancourt M, Raoult D (1997) rpoB sequence analysis as a novel basis for bacterial identification. Mol Microbiol 26:1005–1011CrossRefPubMedGoogle Scholar
  36. Noller HF, Woese CR (1981) Secondary structure of 16S ribosomal RNA. Science 212:403–411CrossRefPubMedGoogle Scholar
  37. Pena C, Pujol M, Ardanuy C et al (2001) An outbreak of hospital-acquired Klebsiella pneumoniae bacteraemia, including strains producing extended-spectrum beta-lactamase. J Hosp Infect 47:53–59CrossRefPubMedGoogle Scholar
  38. Podschun R, Ullmann U (1998) Klebsiella spp. as nosocomial pathogens: epidemiology, taxonomy, typing methods, and pathogenicity factors. Clin Microbiol Rev 11:589–603PubMedPubMedCentralGoogle Scholar
  39. Podschun R, Pietsch S, Höller C, Ullmann U (2001) Incidence of Klebsiella species in surface waters and their expression of virulence factors. Appl Environ Microbiol 67:3325–3327CrossRefPubMedPubMedCentralGoogle Scholar
  40. Posada D, Crandall KA (1998) MODLTEST: testing the model of DNA substitution. Bioinformatics 14:817–818CrossRefPubMedGoogle Scholar
  41. Posada D, Crandall KA (2001) Intraspecific gene genealogies: trees grafting into networks. Trends Ecol Evol 16:37–45CrossRefPubMedGoogle Scholar
  42. Ronquist F, Teslenko M, van der Mark P et al (2012) MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Syst Biol 61:539–542CrossRefPubMedPubMedCentralGoogle Scholar
  43. Rosenblueth M, Martínez L, Silva J (2004) Klebsiella variicola, a novel species with clinical and plant-associated isolates. Syst Appl Microbiol 27:27–35CrossRefPubMedGoogle Scholar
  44. Thompson JD, Gibson TJ, Plewniak F (1997) The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25:4876–4882CrossRefPubMedPubMedCentralGoogle Scholar
  45. Ueda K, Seki T, Kudo T (1999) Two distinct mechanisms cause heterogeneity of 16S rRNA. J Bacteriol 181:78–82PubMedPubMedCentralGoogle Scholar
  46. Weaver TM, Smith JA, Hocking JM (2009) Structural and functional studies of truncated hemolysin A from Proteus mirabilis. J Biol Chem 284:22297–22309CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer International Publishing Switzerland 2016

Authors and Affiliations

  • Yanxia He
    • 1
    • 2
  • Xianguang Guo
    • 4
  • Shifei Xiang
    • 5
  • Jiao Li
    • 1
  • Xiaoqin Li
    • 6
  • Hui Xiang
    • 5
  • Jinlei He
    • 1
  • Dali Chen
    • 1
  • Jianping Chen
    • 1
    • 3
  1. 1.Department of Parasitology, West China School of Preclinical and Forensic MedicineSichuan UniversityChengduChina
  2. 2.Department of Pathogen Biology and Immunology, Medical CollegeHubei University for NationalitiesEnshiChina
  3. 3.Animal Disease Prevention and Food Safety Key Laboratory of Sichuan ProvinceSichuan UniversityChengduChina
  4. 4.Chengdu Institute of BiologyChinese Academy of SciencesChengduChina
  5. 5.Affiliated Minda HospitalHubei University for NationalitiesEnshiChina
  6. 6.Central Hospital of Enshi Autonomous PrefectureEnshiChina

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