Emerg Infect DisEmerging Infect. DisEIDEmerging Infectious Diseases1080-60401080-6059Centers for Disease Control and Prevention21888801332208711-026010.3201/eid1709.110260DispatchPredominance of Cronobacter sakazakii Sequence Type 4 in Neonatal InfectionsRunning Head: Cronobacter sakazakii ST4JosephSusanForsytheStephen J.Author affiliation: Nottingham Trent University, Nottingham, UKAddress for correspondence: Stephen Forsythe, School of Science and Technology, Nottingham Trent University, Clifton Lane, Nottingham NG11 8NS, UK; email: stephen.forsythe@ntu.ac.uk9201117917131715

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.

Keywords: bacteriaCronobacter sakazakiibacteriamultilocus sequence typing (MLST)neonatal meningitisUnited Kingdomdispatch

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.

Strains used in study of <italic>Cronobacter sakazakii</italic> genotypes and disease severity and clinical details derived from original case histories*
StrainPatient type/age (EGA)†Clinical signs/outcomeIsolation siteYearCountrySTReference
553Neonate/1 dUNKUNK1977Netherlands4 (15)
557Neonate/5 dUNKUNK1979Netherlands4 (15)
693Neonate/13 d (41 wk)AsymptomaticFeces1994France13 (3)
695Neonate/15 d (32 wk)Fatal NEC IITrachea1994France4 (3,6)
701Neonate/28 d (28 wk)Fatal NEC IIIPeritoneal fluid1994France4 (3,6)
709Neonate/18 d (29 wk)SepticemiaTrachea1994France4 (3,6)
767Neonate/19 d (31 wk)Fatal meningitisTrachea1994France4 (3,6)
721Neonate/2 wkMeningitisCSF2003USA4
978Neonate/<1 wkUNKEnteral feeding tube2007UK3 (12)
696Neonate/17 d (32 wk)NEC IIFeces1994France12 (3,6)
984Neonate/3–4 wkUNKEnteral feeding tube2007UK3 (12)
690Neonate/27 d (31 wk)AsymptomaticFeces1994France12 (3)
1218Neonate/<1 mo (30 wk)Fatal meningitisCSF2001USA1
1219Neonate/<1 mo (36 wk)Fatal meningitisCSF2002USA4
1221Neonate/<1 moMeningitis, adverse neurologic outcomeCSF2003USA4
1225Neonate/<1 mo (35 wk)Fatal meningitisBlood2007USA4
1231Neonate (33 wk)Fatal neurologic damageFeces2004New Zealand4 (2)
HPB 3290Neonate (33 wk)MeningitisCSF2001USA1 (1)
1249NeonateFatal infectionUNK2009UK31
1220Infant/6 wk (37 wk)Brain abscess, nonfatalCSF2003USA4
1223Infant/6 wk (31 wk)UNK, in ICUBlood2004USA4
1240Infant/7 wkFatal meningitisCSF2008USA4 (11)
1242Infant/7 wkFatal meningitisBrain2008USA4 (11)
1241Infant/7 moSudden infant death syndromeBlood2008USA1 (11)
1222Infant/8 moFever, recoveredBlood2003USA4
1224Infant/10 moFever, severe combined immunodeficiencyBlood2004USA4
HPB 2856Child/6 yUNKUNK2002Canada15 (10)
ATCC 29544ChildUNKThroat1980USA8 (13)
20Child/6 yUNKFeces2004Czech Rep4
12Adult/74 yUNKFeces2004Czech Rep1
CDC 0743–75UNKFoot woundWound1975USA41 (13)
CDC 407–77UNKUNKSputum1977USA8 (13)
CDC 996–77UNKUNKSpinal fluid1977USA8 (13)
NCTC 9238UNKUNKAbdomen pus1953UK18 (15)
HPB 2852UNKUNKUNK1990Canada8 (10)
HPB 2853UNKUNKUNK1990Canada4 (10)
511UNKUNKUNK1983Czech Rep8 (14)
513UNKUNKUNK1983Czech Rep8 (14)
520UNKUNKUNK1983Czech Rep12 (14)
526UNKUNKUNK1983Czech Rep8 (14)
558UNKUNKUNK1983Netherlands4 (15)

*EGA, estimated gestational age; ST, sequence type; UNK, unknown; NEC, necrotizing enterocolitis; CSF, cerebrospinal fluid; ICU, intensive care unit; Czech Rep, Czech Republic; CDC, Centers for Disease Control and Prevention.
†Values <37 weeks are considered premature.

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.

Summary of <italic>Cronobacter sakazakii</italic> sequence types and source details from study of <italic>Cronobacter sakazakii</italic> genotypes and disease severity*
STNo. infectionsPatient details
Clinical signs
Neonate†Infant‡ChildAdultUNKMeningitisBacteremiaNECInfectionAsymptomaticUNK
14211211
3222
4201071291226
871616
12321111
13111
15111
18111
31111
41
1




1




1


Total41188311112235217

*ST, sequence type; UNK, unknown; NEC, necrotizing enterocolitis.
†Age <28 d.
‡Age 28–364 d.

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.

Suggested citation for this article: Joseph S, Forsythe SJ. Predominance of Cronobacter sakazakii sequence type 4 in neonatal infections. Emerg Infect Dis [serial on the Internet]. 2011 Sep [date cited]. http://dx.doi.org/10.3201/eid1709.110260

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.

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.

ReferencesHimelright I, Harris E, Lorch V, Anderson M Enterobacter sakazakii infections associated with the use of powdered infant formula—Tennessee, 2001. JAMA. 2002;287:22045 10.1001/jama.287.17.220411987295Jarvis 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 10.1016/j.ajic.2005.04.012Caubilla-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 10.1128/JCM.01075-0717928419Bowen AB, Braden CR Clinical characteristics and outcomes of infants with invasive Enterobacter sakazakii disease. Emerg Infect Dis. 2006;12:1185916965695Bowen 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.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 10.1186/1471-2180-8-6418423002Kucerova 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 10.1371/journal.pone.000955620221447Kucerova E, Joseph S, Forsythe S The Cronobacter genus: ubiquity and diversity. Quality Assurance and Safety of Crops and Foods. 2011 In press 10.1111/j.1757-837X.2011.00104.xBaldwin 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 10.1186/1471-2180-9-22319852808Pagotto FJ, Nazarowec-White M, Bidawid S, Farber JM Enterobacter sakazakii: infectivity and enterotoxin production in vitro and in vivo. J Food Prot. 2003;66:370512636287Centers for Disease Control and Prevention Cronobacter species isolation in two infants—New Mexico, 2008. MMWR Morb Mortal Wkly Rep. 2009;58:117983 .10.1097/INF.0b013e3181cb86c919875980Hurrell 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 10.1186/1471-2334-9-14619723318Farmer 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 10.1099/00207713-30-3-569Aldová E, Hausne O, Postupa R Tween esterase activity in Enterobacter sakazakii. Zentralbl Bakteriol Mikrobiol Hyg [A]. 1983;256:10386659742Muytjens 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;18:115206885983