Characterisation of Cronobacter strains isolated from hospitalised adult patients

Abstract

Bacteria belonging to the genus Cronobacter are opportunistic pathogens known for causing rare but serious infections in neonates, including meningitis, necrotising enterocolitis and sepsis. Cronobacter infections occur also in adult populations, however, they generally have milder manifestations and their prevalence is uncertain. In this study, the presence of Cronobacter strains from adult patients in the University Hospital in Bratislava was investigated and overall 18 confirmed isolates from 321 patients (5.3%) were recovered. No Cronobacter positive sample was detected in 215 sputum samples from outpatients. The highest occurrence of Cronobacter strains was observed from stroke patients and this may be associated with an abnormal swallowing ability. The isolated strains belonged to the species Cronobacter sakazakii and Cronobacter malonaticus. In silico genotyping (MLST, CRISPR-cas array profiling) of whole genome sequences assigned the strains to three different MLST clones. The majority (12/18) of the isolated strains were sequence type ST513 or single locus variants ST514 and ST515, thereby being members of C. sakazakii pathovar clonal complex CC4. However, according to core genome MLST analysis the ST513-ST515 strains created a unique cluster substantially different from other CC4 strains. The isolated strains were susceptible to 18 tested antibiotics. All strains possess a genomic island encoding for increased thermal tolerance. As Cronobacter strains are frequently present in dried foods of plant origin, spread of a specific clone within a hospital may be caused by food transmission and may be facilitated by its tolerance to environmental stresses such as desiccation and temperature.

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References

  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 34:1979–1988

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  2. Aziz RK, Bartels D, Best AA, DeJongh M, Disz T, Edwards RA, Formsma K, Gerdes S, Glass EM, Kubal M, Meyer F, Olsen GJ, Olson R, Osterman AL, Overbeek RA, McNeil LK, Paarmann D, Paczian T, Parrello B, Pusch GD, Reich C, Stevens R, Vassieva O, Vonstein V, Wilke A, Zagnitko O (2008) The RAST server: rapid annotations using subsystems technology. BMC Genom 9:75. https://doi.org/10.1186/1471-2164-9-75

    Article  CAS  Google Scholar 

  3. Baltimore RS, Duncan RL, Shapiro ED, Edberg SC (1989) Epidemiology of pharyngeal colonization of infants with aerobic Gram-negative rod bacteria. J Clin Microbiol 27:91–95

    PubMed  PubMed Central  CAS  Google Scholar 

  4. Bankevich A, Nurk S, Antipov D, Gurevich AA, Dvorkin M, Kulikov AS, Lesin VM, Nikolenko SI, Pham S, Prjibelski AD, Pyshkin AV, Sirotkin AV, Vyahhi N, Tesler G, Alekseyev MA, Pevzner PA (2012) SPAdes: a new genome assembly algorithm and its applications to single-cell se-quencing. J Comput Biol 19:455–477

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  5. Blazkova M, Javurkova B, Vlach J, Goselova S, Karamonova L, Ogrodzki P, Forsythe S, Fukal L (2015) Diversity of O antigens within the genus Cronobacter: from disorder to order. Appl Environ Microbiol 81:5574–5582

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  6. 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 164:205–210

    Article  PubMed  CAS  Google Scholar 

  7. Boll EJ, Marti R, Hasman H, Overballe-Petersen S, Stegger M, Ng K, Knochel S, Krogfelt KA, Hummerjohann J, Struve C (2017) Turn up the heat—food and clinical Escherichia coli isolates feature two transferrable loci of heat resistance. Front Microbiol 8:579

    Article  PubMed  PubMed Central  Google Scholar 

  8. 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 70:2111–2117

    Article  Google Scholar 

  9. Chase HR, Gopinath GR, Eshwar AK, Stoller A, Fricker-Feer C, Gangiredla J, Patel IR, Cinar HN, Jeong H, Lee C, Negrete F, Finkelstein S, Stephan R, Tall BD, Lehner A (2017) Comparative genomic characterization of the highly persistent and potentially virulent Cronobacter sakazakii ST83, CC65 strain H322 and other ST83 strains. Front Microbiol 8:1136

    Article  PubMed  PubMed Central  Google Scholar 

  10. Cui JH, Yu B, Xiang Y, Zhang Z, Zhang T, Zeng YC, Cui ZG, Huo XX (2017) Two cases of multi-antibiotic resistant Cronobacter spp. infections of infants in China. Biomed Environ Sci 30:601–605

    PubMed  Google Scholar 

  11. Forsythe S, Dickins B, Jolley KA (2014) Cronobacter, the emergent bacterial pathogen Enterobacter sakazakii comes of age; MLST and whole genome sequence analysis. BMC Genomics 15:1121

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  12. Franco AA, Hu L, Grim CJ, Gopinath G, Sathyamoorthy V, Jarvis KG, Lee C, Sadowski J, Kim J, Kothary MH, McCardell BA, Tall BD (2011) Characterization of putative virulence genes on the related RepFIB plasmids harbored by Cronobacter spp. Appl Environ Microbiol 77:3255–3267

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  13. 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 ºC in Cronobacter sp. and other enterobacterial strains. Antonie Van Leeuwenhoek 100:279–289

    Article  PubMed  CAS  Google Scholar 

  14. Gattringer R, Nikš M, Ostertág R, Schwarza K, Medvedovica H, Graninger W, Georgopoulos A (2002) Evaluation of MIDITECH automated colorimetric MIC reading for antimicrobial susceptibility testing. J Antimicrob Chemother 49:651–659

    Article  PubMed  CAS  Google Scholar 

  15. Gosney M, Martin MV, Wright AE (2006a) The role of selective decontamination of the digestive tract in acute stroke. Age Ageing 35:42–47

    Article  PubMed  CAS  Google Scholar 

  16. Gosney MA, Martin MV, Wright AE, Gallagher M (2006b) Enterobacter sakazakii in the mouths of stroke patients and its association with aspiration pneumonia. Eur J Intern Med 17:185–188

    Article  PubMed  Google Scholar 

  17. Gupta SK, Padmanabhan BR, Diene SM, Lopez-Rojas R, Kempf M, Landraud L, Rolain JM (2014) ARG-annot, a new bioinformatic tool to discover antibiotic resistance genes in bacterial genomes. Antimicrob Agents Chemother 58:212–220

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  18. Holy O, Forsythe S (2014) Cronobacter spp. as emerging causes of healthcare-associated infection. J Hosp Infect 86:169–177

    Article  PubMed  CAS  Google Scholar 

  19. Holy O, Petrzelova J, Hanulik V, Chroma M, Matouskova I, Forsythe SJ (2013) Epidemiology of Cronobacter spp. isolates from patients admitted to the Olomouc University Hospital (Czech Republic). Epidemiol Mikrobiol Imunol 63:69–72

    Google Scholar 

  20. Hunter CJ, Petrosyan M, Ford HR, Prasadarao NV (2008) Enterobacter sakazakii: an emerging pathogen in infants and neonates. Surg Infect (Larchmt) 9:533–539

    Article  Google Scholar 

  21. 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 7:64

    Article  PubMed  PubMed Central  Google Scholar 

  22. 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 62:1277–1283

    Article  PubMed  CAS  Google Scholar 

  23. Joseph S, Sonbol H, Hariri S, Desai P, McClelland M, Forsythe SJ (2012b) Diversity of the Cronobacter genus as revealed by multilocus sequence typing. J Clin Microbiol 50:3031–3039

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  24. Kandhai MC, Heuvelink AE, Reij MW, Beumer RR, Dijk R, van Tilburg JJHC, van Schothorst M, Gorris LGM (2010) A study into the occurrence of Cronobacter spp. in The Netherlands between 2001 and 2005. Food Control 21:1127–1136

    Article  Google Scholar 

  25. Kucerova E, Clifton SW, Xia XQ, Long F, Porwollik S, Fulton L, Fronick C, Minx P, Kyung K, Warren W, Fulton R, Feng D, Wollam A, Shah N, Bhonagiri V, Nash WE, Hallsworth-Pepin K, Wilson RK, McClelland M, Forsythe SJ (2010) Genome sequence of Cronobacter sakazakii BAA-894 and comparative genomic hybridization analysis with other Cronobacter species. PLoS ONE 5:e9556

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  26. Lai KK (2001) Enterobacter sakazakii infections among neonates, infants, children, and adults. Case reports and a review of the literature. Medicine 80:113–122

    Article  PubMed  CAS  Google Scholar 

  27. Lehner A, Nitzsche S, Breeuwer P, Diep B, Thelen K, Stephan R (2006) Comparison of two chromogenic media and evaluation of two molecular based identification systems for Enterobacter sakazakii detection. BMC Microbiol 6:15

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  28. Liu H, Cui JH, Cui ZG, Hu GC, Yang YL, Li J, Shi YW (2013) Cronobacter carriage in neonate and adult intestinal tracts. Biomed Environm Sci 26:861–864

    Google Scholar 

  29. Maiden MC, Jansen van Rensburg MJ, Bray JE, Earle SG, Ford SA, Jolley KA, McCarthy ND (2013) MLST revisited: the gene-by-gene approach to bacterial genomics. Nat Rev Microbiol 11:728–736

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  30. Marti R, Muniesa M, Schmid M, Ahrens CH, Naskova J, Hummerjohann J (2016) Short communication: heat-resistant Escherichia coli as potential persistent reservoir of extended-spectrum beta-lactamases and Shiga toxin-encoding phages in dairy. J Dairy Sci 99:8622–8632

    Article  PubMed  CAS  Google Scholar 

  31. Masood N, Moore K, Farbos A, Hariri S, Paszkiewicz K, Dickins B, McNally A, Forsythe S (2013) Draft genome sequence of the earliest Cronobacter sakazakii sequence type 4 strain, NCIMB 8272. Genome Announc 1:e00782-13

    PubMed  PubMed Central  Article  Google Scholar 

  32. 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 Genomics 16:750

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  33. McArthur AG, Waglechner N, Nizam F, Yan A, Azad MA, Baylay AJ, Bhullar K, Canova MJ, De Pascale G, Ejim L, Kalan L, King AM, Koteva K, Morar M, Mulvey MR, O’Brien JS, Pawlowski AC, Piddock LJ, Spanogiannopoulos P, Sutherland AD, Tang I, Taylor PL, Thaker M, Wang W, Yan M, Yu T, Wright GD (2013) The comprehensive antibiotic resistance database. Antimicrob Agents Chemother 57:3348–3357

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  34. Mercer RG, Zheng J, Garcia-Hernandez R, Ruan L, Ganzle MG, McMullen LM (2015) Genetic determinants of heat resistance in Escherichia coli. Front Microbiol 6:932

    Article  PubMed  PubMed Central  Google Scholar 

  35. Mercer RG, Walker BD, Yang X, McMullen LM, Ganzle MG (2017) The locus of heat resistance (LHR) mediates heat resistance in Salmonella enterica, Escherichia coli and Enterobacter cloacae. Food Microbiol 64:96–103

    Article  PubMed  CAS  Google Scholar 

  36. Mohan Nair MK, Venkitanarayanan KS (2006) Cloning and sequencing of the ompA gene of Enterobacter sakazakii and development of an ompA-targeted PCR for rapid detection of Enterobacter sakazakii in infant formula. Appl Environ Microbiol 72:2539–2546

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  37. Moine D, Kassam M, Baert L, Tang Y, Barretto C, Bru CN, Klijn A, Descombesa P (2016) Fully closed genome sequences of five type strains of the genus Cronobacter and one Cronobacter sakazakii strain. Genome Announc 4:e00142-16

    Article  PubMed  PubMed Central  Google Scholar 

  38. Nguyen SV, Harhay GP, Bono JL, Smith TP, Harhay DM (2017) Genome sequence of the thermotolerant foodborne pathogen Salmonella enterica serovar Senftenberg ATCC 43845 and phylogenetic analysis of loci encoding increased protein quality control mechanisms. mSystems 2:e00190–e001916

  39. Ogrodzki P, Forsythe SJ (2016) CRISPR-cas loci profiling of Cronobacter sakazakii pathovars. Future Microbiol 11:1507–1519

    Article  PubMed  CAS  Google Scholar 

  40. Ogrodzki P, Forsythe SJ (2017) DNA-sequence based typing of the Cronobacter genus using MLST, CRISPR-cas array and capsular profiling. Front Microbiol 8:1875

    Article  PubMed  PubMed Central  Google Scholar 

  41. 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 52:37–44

    CAS  Google Scholar 

  42. Orieskova M, Kajsik M, Szemes T, Holy O, Forsythe S, Turna J, Drahovska H (2016) Contribution of the thermotolerance genomic island to increased thermal tolerance in Cronobacter strains. Antonie Van Leeuwenhoek 109:405–414

    Article  PubMed  CAS  Google Scholar 

  43. 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 20:1520–1523

    Article  PubMed  PubMed Central  Google Scholar 

  44. Schmid MC, Iversen Gontia I, Stephan R, Hofmann A, Hartmann A, Jha B, Eberl L, Riedel K, Lehner A (2009) Evidence for a plant-associated natural habitat for Cronobacter spp. Res Microbiol 160:608–614

    Article  PubMed  Google Scholar 

  45. 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 64:3402–3410

    Article  PubMed  PubMed Central  Google Scholar 

  46. Sun Y, Wang M, Wang Q, Cao B, He X, Li K, Feng L, Wang L (2012) Genetic analysis of the Cronobacter sakazakii O4 to O7 O-antigen gene clusters and development of a PCR assay for identification of all C. sakazakii O serotypes. Appl Environ Microbiol 78:3966–3974

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  47. 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 99:257–269

    Article  PubMed  Google Scholar 

  48. Vojkovska H, Karpiskova R, Orieskova M, Drahovska H (2016) Characterization of Cronobacter spp. isolated from food of plant origin and environmental samples collected from farms and from supermarkets in the Czech Republic. Int J Food Microbiol 217:130–136

    Article  PubMed  CAS  Google Scholar 

  49. Yan Q, Power KA, Cooney S, Fox E, Gopinath GR, Grim CJ, Tall BD, McCusker MP, Fanning S (2013) Complete genome sequence and phenotype microarray analysis of Cronobacter sakazakii SP291: a persistent isolate cultured from a powdered infant formula production facility. Front Microbiol 4:256

    Article  PubMed  PubMed Central  Google Scholar 

  50. Zankari E, Hasman H, Cosentino S, Vestergaard M, Rasmussen S, Lund O, Aarestrup FM, Larsen MV (2012) Identification of acquired antimicrobial resistance genes. J Antimicrob Chemother 67:2640–2644

    Article  PubMed  PubMed Central  CAS  Google Scholar 

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Acknowledgements

This publication is the outcome of BIOREKPROT project (Grant No. ITMS 26240220048) supported by the Research and Development Operational Programme funded by the ERDF.

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Correspondence to Hana Drahovska.

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Kadlicekova, V., Kajsik, M., Soltys, K. et al. Characterisation of Cronobacter strains isolated from hospitalised adult patients. Antonie van Leeuwenhoek 111, 1073–1085 (2018). https://doi.org/10.1007/s10482-017-1008-2

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Keywords

  • Cronobacter spp.
  • Hospital Infection
  • Genotyping
  • Thermotolerance island