Skip directly to site content Skip directly to page options Skip directly to A-Z link Skip directly to A-Z link Skip directly to A-Z link
Volume 17, Number 9—September 2011
Dispatch

Predominance of Cronobacter sakazakii Sequence Type 4 in Neonatal Infections

Tables
Article Metrics
139
citations of this article
EID Journal Metrics on Scopus
Author affiliations: Author affiliation: Nottingham Trent University, Nottingham, UK

Cite This Article

Abstract

A 7-loci (3,036 nt) multilocus sequence typing scheme was applied to 41 clinical isolates of Cronobacter sakazakii. Half (20/41) of the C. sakazakii strains were sequence type (ST) 4, and 9/12 meningitis isolates were ST4. C. sakazakii ST4 appears to be a highly stable clone with a high propensity for neonatal meningitis.

Cronobacter is a genus within the family Enterobacteriaceae and was previously known as Enterobacter sakazakii. It is closely related to the genera Enterobacter and Citrobacter. Cronobacter spp. have been frequently isolated from the environment, plant material (wheat, rice, herbs, and spices), and various food products, including powdered infant formula (PIF). Cronobacter spp. have come to prominence because of their association with severe neonatal infections, which can be fatal (13). Our current knowledge of the virulence and epidemiology of this organism is limited. However, because neonates are frequently fed reconstituted PIF, this product has been the focus of attention for reducing infection risk to neonates because the number of exposure routes is limited (1,2).

Infections with Cronobacter spp. occur across all age groups, and most infections, albeit less severe, are in the adult population. However, neonates, particularly those of low birthweight, are the major identified group at risk, because the organism can cause meningitis, necrotizing enterocolitis (NEC), and sepsis in patients in neonatal intensive care units and has a high mortality rate (13). Bowen and Braden (4) reviewed 46 cases of invasive (non-NEC) infant Cronobacter infections to define risk factors and provide guidance for prevention and treatment. Although these infections have been associated with intrinsically and extrinsically contaminated PIF, other environmental sources are possible and several non–infant formula–associated cases have been reported (5). Cronobacter spp. have been shown to invade human intestinal cells, replicate in macrophages, and invade the blood–brain barrier (6). Kucerova et al. (7,8) used comparative genomic hybridization-based analysis to describe a range of virulence traits in Cronobacter spp., including iron acquisition mechanisms, fimbriae, and macrophage survival.

Recently, Baldwin et al. (9) constructed a comprehensive multilocus sequence typing (MLST) scheme for Cronobacter spp. based on 7 housekeeping genes (atpD, fusA, glnS, gltB, gyrB, infB, ppsA; 3,036 nt concatenated length). The MLST scheme currently has 66 defined sequence types covering all Cronobacter spp. (www.pubMLST.org/cronobacter). However, the scheme has not been applied for any epidemiologic purposes. Therefore, we investigated whether severity of infection by Cronobacter spp. is associated with particular genotype(s) by compiling patient details, isolation site, and clinical signs for clinical C. sakazakii isolates and comparing these with the sequence type (ST) profile of the isolates.

The Study

Error! Hyperlink reference not valid.Forty-one clinical C. sakazakii strains were included in the study. These strains were from 7 countries and had been isolated during 1953–2008. The strains included those of recent (13,1012) and those of more historic interest (>25 years 1315; ). Strains used in this study, along with patient details and clinical signs, are shown in Table 1. Details of clinical signs were collated from information in the associated publication or supplied by the strain provider (Centers for Disease Control and Prevention, Atlanta, GA, USA). Primers and conditions for amplification and sequencing of the 7 MLST genes atpD (390 bp), fusA (438 bp), glnS (363 bp), gltB (507 bp), gyrB (402 bp), infB (441 bp) and ppsA (495 bp) were as described (9). All sequences are available for download and independent analysis through open access at www.pubMLST.org/cronobacter.

Comparative analysis with the online Cronobacter MLST database (covering isolates from all sources) showed that the clinical isolates were in 10 of 30 STs defined for C. sakazakii spp. However, the clinical strains were not evenly distributed across the STs. Of particular interest was that half (20/41) of the strains were ST4 (Table 2). The remaining strains were ST8 (7), ST1 (4), ST12 (3), ST3 (2), ST13, ST15, ST18, ST31, and ST41 (1 each). Of the 20 ST4 strains, 10 were from neonates, 7 from infants, and 1 from a child; 2 had no patient details. Similarly, most (9/12) isolates from meningitis cases were ST4 strains; 7 were isolated from cerebrospinal fluid and the others from blood and the trachea. The remaining ST4 strains were from bacteremia cases (1), NEC (2), and undefined infection (1), with 6 from unknown sources. ST4 was the main ST associated with neonates (10/18); this ST has been reported by Baldwin et al. (9) for the high incidence of PIF isolates.

The ST4 clinical strains were from 6 countries (the Netherlands, France, United States, New Zealand, Czech Republic, and Canada) and had been isolated during 1977–2008 (Table 1). Of the 30 strains with known patient details, only 1 isolate (ST1) was from an adult patient. To date, all other isolates from adults have been identified as C. malonaticus (S. Joseph, unpub. data).

Conclusions

The 7 housekeeping genes for MLST analysis are not virulence related, but a large proportion of severe neonatal infections were caused by a single sequence type. Whether this is caused by survival characteristics increasing persistence under desiccated conditions, and hence neonatal exposure or particular virulence capabilities, is uncertain. It is plausible that different age groups are exposed to different genotypes of C. sakazakii according to their diet and lifestyle. C. sakazakii ST4 appears to be a stable clone because strains have been isolated from 7 countries for >50 years. The earliest (1951) nonclinical isolate was from a can of dried milk (13). Whether this clonal nature occurs in other Cronobacter spp. awaits future investigation.

Ms Joseph is a research student at Nottingham Trent University. She is currently investigating the genomic diversity of Cronobacter spp.

Dr Forsythe is professor of microbiology at Nottingham Trent University. His research interests are foodborne pathogens, especially emergent bacterial pathogens and the origin of their virulence.

Top

Acknowledgments

We thank Nadia Chuzhanova for providing statistical advice and those who provided cultures, particularly Maria-Francoise Preré, Harry Muytjens, Ivo Safarík, Jeff Farber, and Matthew Arduino.

This study was supported by Nottingham Trent University.

Top

References

  1. Himelright  I, Harris  E, Lorch  V, Anderson  M. Enterobacter sakazakii infections associated with the use of powdered infant formula—Tennessee, 2001. JAMA. 2002;287:22045. DOIPubMedGoogle Scholar
  2. Jarvis  C. Fatal Enterobacter sakazakii infection associated with powdered infant formula in a neonatal intensive care unit in New Zealand. Am J Infect Control. 2005;33:e19. DOIGoogle Scholar
  3. Caubilla-Barron  J, Hurrell  E, Townsend  S, Cheetham  P, Loc-Carrillo  C, Fayet  O, 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. 2007;45:397985. DOIPubMedGoogle Scholar
  4. Bowen  AB, Braden  CR. Clinical characteristics and outcomes of infants with invasive Enterobacter sakazakii disease. Emerg Infect Dis. 2006;12:11859.PubMedGoogle Scholar
  5. Bowen  AB, Braden  CR. Enterobacter sakazakii disease and epidemiology. In: Farber JM, Forsythe SJ, editors. Emerging issues in food safety: Enterobacter sakazakii. Washington: American Society for Microbiology Press; 2008;4:101–25.
  6. Townsend  S, Hurrell  E, Forsythe  SJ. Virulence studies of Enterobacter sakazakii isolates associated with a neonatal intensive care unit outbreak. BMC Microbiol. 2008;8:64. DOIPubMedGoogle Scholar
  7. Kucerova  E, Clifton  SW, Xia  X-Q, Long  F, Porwollik  S, Fulton  L, Genome sequence of Cronobacter sakazakii BAA-894 and comparative genomic hybridization analysis with other Cronobacter species. PLoS ONE. 2010;5:e9556. DOIPubMedGoogle Scholar
  8. Kucerova  E, Joseph  S, Forsythe  S. The Cronobacter genus: ubiquity and diversity. Quality Assurance and Safety of Crops and Foods. 2011. In press. DOIGoogle Scholar
  9. Baldwin  A, Loughlin  M, Caubilla-Barron  J, Kucerova  E, Manning  G, Dowson  C, Multilocus sequence typing of Cronobacter sakazakii and Cronobacter malonaticus reveals stable clonal structures with clinical significance, which do not correlate with biotypes. BMC Microbiol. 2009;9:223. DOIPubMedGoogle Scholar
  10. Pagotto  FJ, Nazarowec-White  M, Bidawid  S, Farber  JM. Enterobacter sakazakii: infectivity and enterotoxin production in vitro and in vivo. J Food Prot. 2003;66:3705.PubMedGoogle Scholar
  11. Centers for Disease Control and Prevention. Cronobacter species isolation in two infants—New Mexico, 2008. MMWR Morb Mortal Wkly Rep. 2009;58:117983 .DOIPubMedGoogle Scholar
  12. Hurrell  E, Kucerova  E, Loughlin  M, Caubilla-Barron  J, Hilton  A, Armstrong  R, Neonatal enteral feeding tubes as loci for colonisation by members of the Enterobacteriaceae. BMC Infect Dis. 2009;9:146. DOIPubMedGoogle Scholar
  13. Farmer  JJ III, Asbury  MA, Hickman  FW, Brenner  DJ. The Enterobacteriaceae study group. Enterobacter sakazakii: a new species of “Enterobacteriaceae” isolated from clinical specimens. Int J Syst Bacteriol. 1980;30:56984. DOIGoogle Scholar
  14. Aldová  E, Hausne  O, Postupa  R. Tween esterase activity in Enterobacter sakazakii. Zentralbl Bakteriol Mikrobiol Hyg [A]. 1983;256:1038.PubMedGoogle Scholar
  15. Muytjens  HL, Zanen  HC, Sonderkamp  HJ, Kollée  LA, Washsmuth  K, Farmer  JJ. Analysis of eight cases of neonatal meningitis and sepsis due to Enterobacter sakazakii. J Clin Microbiol. 1983;18:11520.PubMedGoogle Scholar

Top

Tables

Top

Cite This Article

DOI: 10.3201/eid1709.110260

Table of Contents – Volume 17, Number 9—September 2011

EID Search Options
presentation_01 Advanced Article Search – Search articles by author and/or keyword.
presentation_01 Articles by Country Search – Search articles by the topic country.
presentation_01 Article Type Search – Search articles by article type and issue.

Top

Comments

Please use the form below to submit correspondence to the authors or contact them at the following address:

Stephen Forsythe, School of Science and Technology, Nottingham Trent University, Clifton Lane, Nottingham NG11 8NS, UK

Send To

10000 character(s) remaining.

Top

Page created: September 06, 2011
Page updated: September 06, 2011
Page reviewed: September 06, 2011
The conclusions, findings, and opinions expressed by authors contributing to this journal do not necessarily reflect the official position of the U.S. Department of Health and Human Services, the Public Health Service, the Centers for Disease Control and Prevention, or the authors' affiliated institutions. Use of trade names is for identification only and does not imply endorsement by any of the groups named above.
file_external