Antonie van Leeuwenhoek

, Volume 109, Issue 3, pp 405–414 | Cite as

Contribution of the thermotolerance genomic island to increased thermal tolerance in Cronobacter strains

  • Maria Orieskova
  • Michal Kajsik
  • Tomas Szemes
  • Ondrej Holy
  • Stephen Forsythe
  • Jan Turna
  • Hana Drahovska
Original Paper


Cronobacter spp. are opportunistic pathogens associated with serious infections in neonates. Increased stress tolerance, including the thermotolerance of some Cronobacter strains, can promote their survival in production facilities and thus raise the possibility of contamination of dried infant formula which has been identified as a potential source of infection. Some Cronobacter strains contain a genomic island, which might be responsible for increased thermotolerance. By analysis of Cronobacter sequenced genomes this determinant was found to be present in only 49/73 Cronobacter sakazakii strains and in 9/14 Cronobacter malonaticus strains. The island was also found in 16/17 clinical isolates originating from two hospitals. Two configurations of the locus were detected; the first one with the size of 18 kbp containing the thrB-Q genes and a shorter version (6 kbp) harbouring only the thrBCD and thrOP genes. Strains containing the thermotolerance island survived significantly better at 58 °C comparing to a C. sakazakii isogenic mutant lacking the island and strains with the longer version of the island were 2–10 times more tolerant than those with the shortened sequence. The function of the genomic island was further confirmed by its cloning into a low-copy vector and transforming it into the isogenic mutant. Different levels of rpoS, encoding for stress-response sigma factor, expression were also associated with variability in strain thermotolerance.


Cronobacter spp. Heat stress rpoThermotolerance Thermotolerance island 



This publication is the result of the project implementation (ITMS 26240220086) supported by the Research & Development Operational Programme funded by the ERDF and by Slovak Ministry of Education under the Contract No. VEGA 1/0709/12. Project supported by Research Support Foundation, Vaduz - Grant project No. 801100021/39 - Surveillance of Infectious Complications in Hemato-Oncological Patients.

Supplementary material

10482_2016_645_MOESM1_ESM.docx (101 kb)
Supplementary material 1 (DOCX 100 kb)


  1. Alsonosi A, Hariri S, Kajsik M, Orieskova M, Hanulik V, Roderova M, Petrzelova J, Kollarova H, Drahovska H, Forsythe S, Holy O (2015) The speciation and genotyping of Cronobacter isolates from hospitalised patients. Eur J Clin Microbiol Infect Dis 34:1979–1988PubMedCentralCrossRefPubMedGoogle Scholar
  2. Alvarez-Ordonez A, Begley M, Hill C (2012) Polymorphisms in rpoS and stress tolerance heterogeneity in natural isolates of Cronobacter sakazakii. Appl Environ Microbiol 78:3975–3984PubMedCentralCrossRefPubMedGoogle Scholar
  3. Battesti A, Majdalani N, Gottesman S (2011) The RpoS-mediated general stress response in escherichia coli. Annu Rev Microbiol 65:189–213CrossRefPubMedGoogle Scholar
  4. Bojer MS, Struve C, Ingmer H, Hansen DS, Krogfelt KA (2010) Heat resistance mediated by a new plasmid encoded Clp ATPase, ClpK, as a possible novel mechanism for nosocomial persistence of Klebsiella pneumoniae. PLoS ONE 5:e15467PubMedCentralCrossRefPubMedGoogle Scholar
  5. Bojer MS, Struve C, Ingmer H, Krogfelt KA (2013) ClpP-dependent and -independent activities encoded by the polycistronic clpK-encoding locus contribute to heat shock survival in Klebsiella pneumoniae. Res Microbiol 164:205–210CrossRefPubMedGoogle Scholar
  6. Caubilla-Barron J, Forsythe S (2007) Dry stress and survival time of Enterobacter sakazakii and other Enterobacteriaceae in dehydrated powdered infant formula. J Food Prot 70:2111–2117Google Scholar
  7. Caubilla-Barron J, Hurrell E, Townsend S, Cheetham P, Loc-Carrillo C, Fayet O, Prere MF, Forsythe SJ (2007) Genotypic and phenotypic analysis of Enterobacter sakazakii strains from an outbreak resulting in fatalities in a neonatal intensive care unit in France. J Clin Microbiol 45:3979–3985PubMedCentralCrossRefPubMedGoogle Scholar
  8. Dancer GI, Mah JH, Rhee MS, Hwang IG, Kang DH (2009) Resistance of Enterobacter sakazakii (Cronobacter spp.) to environmental stresses. J Appl Microbiol 107:1606–1614CrossRefPubMedGoogle Scholar
  9. Edelson-Mammel SG, Buchanan RL (2004) Thermal inactivation of Enterobacter sakazakii in rehydrated infant formula. J Food Prot 67:60–63PubMedGoogle Scholar
  10. Forsythe S, Dickins B, Jolley KA (2014) Cronobacter, the emergent bacterial pathogen Enterobacter sakazakii comes of age; MLST and whole genome sequence analysis. BMC Genom 15:1121CrossRefGoogle Scholar
  11. Gajdosova J, Benedikovicova K, Kamodyova N, Tothova L, Kaclikova E, Stuchlik S, Turna J, Drahovska H (2011) Analysis of the DNA region mediating increased thermotolerance at 58 °C in Cronobacter sp. and other enterobacterial strains. Antonie Van Leeuwenhoek 100:279–289CrossRefPubMedGoogle Scholar
  12. Gičová A, Oriešková M, Oslanecová L, Drahovská H, Kaclíková E (2014) Identification and characterization of Cronobacter strains isolated from powdered infant foods. Lett Appl Microbiol 58:242–247CrossRefPubMedGoogle Scholar
  13. Holy O, Forsythe S (2014) Cronobacter spp. as emerging causes of healthcare-associated infection. J Hosp Infect 86:169–177CrossRefPubMedGoogle Scholar
  14. Huertas J-P, Álvarez-Ordóñez A, Morrissey R, Ros-Chumillas M, Esteban M-D, Maté J, Palop A, Hill C (2015) Heat resistance of Cronobacter sakazakii DPC 6529 and its behavior in reconstituted powdered infant formula. Food Res Int 69:401–409CrossRefGoogle Scholar
  15. Hunter CJ, Bean JF (2013) Cronobacter: an emerging opportunistic pathogen associated with neonatal meningitis, sepsis and necrotizing enterocolitis. J Perinatol 33:581–585CrossRefPubMedGoogle Scholar
  16. Iversen C, Lehner A, Mullane N, Bidlas E, Cleenwerck I, Marugg J, Fanning S, Stephan R, Joosten H (2007) The taxonomy of Enterobacter sakazakii: proposal of a new genus Cronobacter gen. nov. and descriptions of Cronobacter sakazakii comb. nov. Cronobacter sakazakii subsp. sakazakii, comb. nov., Cronobacter sakazakii subsp. malonaticus subsp. nov., Cronobacter turicensis sp. nov., Cronobacter muytjensii sp. nov., Cronobacter dublinensis sp. nov. and Cronobacter genomospecies 1. BMC Evol Biol 7:64PubMedCentralCrossRefPubMedGoogle Scholar
  17. Iversen C, Mullane N, McCardell B, Tall BD, Lehner A, Fanning S, Stephan R, Joosten H (2008) Cronobacter gen. nov., a new genus to accommodate the biogroups of Enterobacter sakazakii, and proposal of Cronobacter sakazakii gen. nov., comb. nov., Cronobacter malonaticus sp. nov., Cronobacter turicensis sp. nov., Cronobacter muytjensii sp. nov., Cronobacter dublinensis sp. nov., Cronobacter genomospecies 1, and of three subspecies, Cronobacter dublinensis subsp. dublinensis subsp. nov., Cronobacter dublinensis subsp. lausannensis subsp. nov. and Cronobacter dublinensis subsp. lactaridi subsp. nov. Int J Syst Evol Microbiol 58:1442–1447CrossRefPubMedGoogle Scholar
  18. Jaradat ZW, Al Mousa W, Elbetieha A, Al Nabulsi A, Tall BD (2014) Cronobacter spp.—opportunistic food-borne pathogens. A review of their virulence and environmental-adaptive traits. J Med Microbiol 63:1023–1037CrossRefPubMedGoogle Scholar
  19. Jolley KA, Maiden MC (2010) BIGSdb: scalable analysis of bacterial genome variation at the population level. BMC Bioinform 11:595CrossRefGoogle Scholar
  20. Joseph S, Forsythe SJ (2011) Predominance of Cronobacter sakazakii sequence type 4 in neonatal infections. Emerg Infect Dis 17:1713–1715PubMedCentralCrossRefPubMedGoogle Scholar
  21. Joseph S, Cetinkaya E, Drahovska H, Levican A, Figueras MJ, Forsythe SJ (2012a) Cronobacter condimenti sp. nov., isolated from spiced meat, and Cronobacter universalis sp. nov., a species designation for Cronobacter sp. genomospecies 1, recovered from a leg infection, water and food ingredients. Int J Syst Evol Microbiol 62:1277–1283CrossRefPubMedGoogle Scholar
  22. Joseph S, Desai P, Ji Y, Cummings CA, Shih R, Degoricija L, Rico A, Brzoska P, Hamby SE, Masood N, Hariri S, Sonbol H, Chuzhanova N, McClelland M, Furtado MR, Forsythe SJ (2012b) Comparative analysis of genome sequences covering the seven Cronobacter species. PLoS ONE 7:e49455PubMedCentralCrossRefPubMedGoogle Scholar
  23. Masood N, Moore K, Farbos A, Paszkiewicz K, Dickins B, McNally A, Forsythe S (2015) Genomic dissection of the 1994 Cronobacter sakazakii outbreak in a French neonatal intensive care unit. BMC Genom 16:750CrossRefGoogle Scholar
  24. Mercer RG, Zheng J, Garcia-Hernandez R, Ruan L, Ganzle MG, McMullen LM (2015) Genetic determinants of heat resistance in Escherichia coli. Front Microbiol 6:932PubMedCentralCrossRefPubMedGoogle Scholar
  25. Orieskova M, Gajdosova J, Oslanecova L, Ondreickova K, Kaclikova E, Stuchlik S, Turna J, Drahovska H (2013) Function of thermotolerance genomic island in increased stress resistance of Cronobacter sakazakii. J Food Nutr Res 52:37–44Google Scholar
  26. Osaili T, Forsythe S (2009) Desiccation resistance and persistence of Cronobacter species in infant formula. Int J Food Microbiol 136:214–220CrossRefPubMedGoogle Scholar
  27. Patrick ME, Mahon BE, Greene SA, Rounds J, Cronquist A, Wymore K, Boothe E, Lathrop S, Palmer A, Bowen A (2014) Incidence of Cronobacter spp. infections, United States, 2003–2009. Emerg Infect Dis 20:1520–1523PubMedCentralCrossRefPubMedGoogle Scholar
  28. Stephan R, Grim CJ, Gopinath GR, Mammel MK, Sathyamoorthy V, Trach LH, Chase HR, Fanning S, Tall BD (2014) Re-examination of the taxonomic status of Enterobacter helveticus, Enterobacter pulveris and Enterobacter turicensis as members of the genus Cronobacter and their reclassification in the genera Franconibacter gen. nov. and Siccibacter gen. nov. as Franconibacter helveticus comb. nov., Franconibacter pulveris comb. nov. and Siccibacter turicensis comb. nov., respectively. Int J Syst Evol Microbiol 64:3402–3410PubMedCentralCrossRefPubMedGoogle Scholar
  29. Turcovsky I, Kunikova K, Drahovska H, Kaclikova E (2011) Biochemical and molecular characterization of Cronobacter spp. (formerly Enterobacter sakazakii) isolated from foods. Antonie Van Leeuwenhoek 99:257–269CrossRefPubMedGoogle Scholar
  30. Walsh D, Molloy C, Iversen C, Carroll J, Cagney C, Fanning S, Duffy G (2011) Survival characteristics of environmental and clinically derived strains of Cronobacter sakazakii in infant milk formula (IMF) and ingredients. J Appl Microbiol 110:697–703CrossRefPubMedGoogle Scholar
  31. Williams TL, Monday SR, Edelson-Mammel S, Buchanan R, Musser SM (2005) A top-down proteomics approach for differentiating thermal resistant strains of Enterobacter sakazakii. Proteomics 5:4161–4169CrossRefPubMedGoogle Scholar
  32. Yan QQ, Condell O, Power K, Butler F, Tall BD, Fanning S (2012) Cronobacter species (formerly known as Enterobacter sakazakii) in powdered infant formula: a review of our current understanding of the biology of this bacterium. J Appl Microbiol 113:1–15CrossRefPubMedGoogle Scholar

Copyright information

© Springer International Publishing Switzerland 2016

Authors and Affiliations

  • Maria Orieskova
    • 1
  • Michal Kajsik
    • 1
  • Tomas Szemes
    • 1
  • Ondrej Holy
    • 2
  • Stephen Forsythe
    • 3
  • Jan Turna
    • 1
  • Hana Drahovska
    • 1
  1. 1.Department of Molecular Biology, Faculty of Natural SciencesComenius UniversityBratislavaSlovak Republic
  2. 2.Department of Preventive Medicine, Faculty of Medicine and DentistryPalacký University, OlomoucOlomoucCzech Republic
  3. 3.Pathogen Research Group, School of Science and TechnologyNottingham Trent UniversityNottinghamUK

Personalised recommendations