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Journal of Clinical Microbiology, December 2007, p. 3979-3985, Vol. 45, No. 12
0095-1137/07/$08.00+0     doi:10.1128/JCM.01075-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Genotypic and Phenotypic Analysis of Enterobacter sakazakii Strains from an Outbreak Resulting in Fatalities in a Neonatal Intensive Care Unit in France{triangledown}

J. Caubilla-Barron,1 E. Hurrell,1 S. Townsend,1 P. Cheetham,1 C. Loc-Carrillo,1 O. Fayet,2 M.-F. Prère,2 and S. J. Forsythe1*

School of Biomedical and Natural Sciences, Nottingham Trent University, Clifton Lane, Nottingham, NG11 8NS, United Kingdom,1 Laboratoire de Microbiologie et Génetique Moléculaires, UMR 5100 CNRS et Université Paul Sabatier Toulouse III, 118 route de Narbonne, 31062 Toulouse cedex 9, France2

Received 25 May 2007/ Returned for modification 11 July 2007/ Accepted 25 September 2007


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In 1994, an outbreak of Enterobacter sakazakii infections occurred in a neonatal intensive care unit in France from 5 May to 11 July. During the outbreak, 13 neonates were infected with E. sakazakii, resulting in 3 deaths. In addition, four symptomless neonates were colonized by E. sakazakii. The strains were subjected to 16S rRNA gene sequence analysis, genotyped using pulsed-field gel electrophoresis, and phenotyped for a range of enzyme activities. E. sakazakii was isolated from various anatomical sites, reconstituted formula, and an unopened can of powdered infant formula. A fourth neonate died from septic shock, attributed to E. sakazakii infection, during this period. However, 16S rRNA gene sequence analysis revealed that the organism was Enterobacter cloacae. There were three pulsotypes of E. sakazakii associated with infected neonates, and three neonates were infected by more than one genotype. One genotype matched isolates from unused prepared formula and unfinished formula. However, no pulsotypes matched the E. sakazakii strain recovered from an unopened can of powdered infant formula. One pulsotype was associated with the three fatal cases, and two of these isolates had extended-spectrum β-lactamase activity. It is possible that E. sakazakii strains differ in their pathogenicities, as shown by the range of symptoms associated with each pulsotype.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Enterobacter sakazakii is an opportunistic pathogen associated with the ingestion of reconstituted infant formula and is a rare cause of neonatal meningitis, necrotizing enterocolitis (NEC), and sepsis (9, 10, 11, 23). Such cases often occur among low-birth-weight preterm neonates, who are generally more susceptible to gram-negative bacterial sepsis and endotoxemia associated with NEC (1, 26). The International Commission on Microbiological Specifications for Foods (14) has ranked E. sakazakii as a "severe hazard for restricted populations, life-threatening or substantial chronic sequelae or long duration." A number of reported E. sakazakii outbreaks have been attributed to contaminated reconstituted infant formula (4, 7, 13, 18, 31). Bowen and Braden (4) reviewed 46 cases of invasive E. sakazakii infections and showed a link between symptoms and birth weight but did not consider cases of NEC.

The virulence of E. sakazakii has been studied by Pagotto et al. (23) and Mange et al. (21), who showed the presence of enterotoxins and adhesion to brain cells, respectively. Townsend et al. demonstrated the translocation of E. sakazakii and other intestinal bacteria across the rat intestinal wall in response to the presence of lipopolysaccharide (28). They also demonstrated that E. sakazakii causes chronic-patterned inflammation in the neonatal rat brain, invades capillary endothelial brain cells, is taken up by macrophages, and induces anti-inflammatory cytokine (interleukin-10) expression in vitro and in vivo at various levels according to strain (29). However, these publications did not report the individual case details associated with the isolates under study. Therefore, it is not possible to directly consider correlations between in vitro and in vivo studies.

This study analyzed 31 E. sakazakii strains isolated over a 3-month period in 1994 during a large E. sakazakii outbreak in a neonatal intensive care unit (NICU) in France. This paper primarily considers the genetic and phenotypic diversity of the isolates and is not an epidemiological investigation. However, where appropriate, neonatal details have been included. The strains have been identified using 16S rRNA gene sequence analysis, genotyped using pulsed-field gel electrophoresis (PFGE), and phenotyped for a range of enzyme activities. In addition, antibiograms and determination of extended-spectrum β-lactamase (ESBL) production have been undertaken.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Background. An outbreak of NEC and invasive infections occurred during the period from 5 May to 15 July 1994 in a NICU. An internal hospital investigation, which was not published, determined that the outbreak was due to E. sakazakii. Details of the neonates from whom E. sakazakii was isolated are given in Table 1. All neonates were fed reconstituted powdered infant formula via enteral perfusion. Formula was reconstituted every 24 h and stored in the refrigerator until required. Every 4 to 6 h, syringes were filled from stored formula, and the contents were fed to infants through enteric tubes at ambient temperature.


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  		TABLE 1. Clinical description of neonates

 
NEC case definition. Bell's staging of NEC as modified by Walsh and Kliegman (32) was used by clinicians in the hospital. Infants with stage I disease (NEC I) have suspected NEC, with systemic signs of temperature instability, apnea, bradycardia, and lethargy. Stage II (definite NEC) is characterized by additional symptoms of absence of bowel sounds, radiological signs of intestinal dilation, ileus, and pneumatosis intestinalis. Stage III is advanced NEC, where the neonate is critically ill with metabolic acidosis, neutropenia, signs of generalized peritonitis, and marked tenderness and distension of the abdomen. Neonates without NEC were also tested for E. sakazakii colonization.

Bacterial strains. In 1994, E. sakazakii strains were isolated from various anatomical sites of 17 neonates (Table 2). These included sputum, feces, skin, peritoneal fluid, and conjunctivae. E. sakazakii was also isolated from the remains of prepared formula, unfinished formula collected during the outbreak period, and an unopened can of powdered infant formula that was collected after the end of the outbreak period. The putative E. sakazakii colonies were distinguished on lactose agar plates by their very mucoid aspect. The hospital identified the strains as E. sakazakii using API20E (BioMerieux), with gas production as the confirmatory test. The isolates were kept in long-term storage at –80°C until the studies reported here. One strain (strain 766), originally identified as E. sakazakii, has been reidentified, by using 16S rRNA gene sequence analysis, as Enterobacter cloacae.


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  		TABLE 2. PFGE profiles, biotyping, and plasmid and antibiotic profiles of patient, prepared formula, and powdered infant formula isolates

 
Bacterial identification and 16S rRNA gene cluster group analysis. The recovered bacterial strains were identified using 16S rRNA gene sequence analysis (Accugenix, Newark, DE) as described by Iversen et al. (15). The sequences were compared with E. sakazakii sequences to determine the 16S rRNA gene cluster group.

DNA isolation and PCR. Genomic DNA was prepared using the GenElute bacterial genomic DNA kit (Sigma) and 1.5 ml of overnight culture grown in LB broth according to the manufacturer's instructions. By following methods prescribed by Keyser et al. (19), primers Esak2 (5'-CCCGCATCTCTGCAGGATTCTC-3') and Esak3 (5'-CTAATACCGCATAACGTCTACG-3') were used to amplify an 850-bp PCR product from a region of the E. sakazakii 16S rRNA gene. Lehner et al. (20) used Esakf (5'-GCTYTGCTGACGAGTGGCGG-3') and Esakr (5'-ATCTCTGCAGGATTCTCTGG-3') to amplify a 929-bp PCR product, also from a region of the E. sakazakii 16S rRNA gene. The ompA gene was amplified with primers ESSF (5'-GGATTTAACCGTGAACTTTTCC-3') and ESSR (5'-CGCCAGCGATGTTAGAAGA-3'), resulting in a 469-bp product by using the PCR conditions described by Mohan Nair and Venkitanarayanan (22). The PCR protocols documented above were followed as described in each publication using 2.5 U of GoTaq Flexi DNA polymerase, 5x Green GoTaq Flexi buffer (Promega Corporation, Madison, WI), and a Genius thermocycler (FGEN05TD; Techne Ltd., Cambridge, United Kingdom). E. sakazakii strains NCTC 11467T and ATCC 12868 were used as positive controls. PCR products were visualized on 1% agarose gels stained with 0.5 µg ml–1 ethidium bromide.

PFGE. PFGE of E. sakazakii was performed by following the Pulse Net USA protocol for molecular subtyping of Escherichia coli O157:H7, nontyphoidal Salmonella serotypes, and Shigella sonnei (6). The gel was run at switch times of 5 to 50 s for 20 h at 6 V in a CHEF-DR II system (Bio-Rad, Hercules, CA).

The PFGE patterns were analyzed by Bionumerics software, version 3.5 (Applied Maths, Sint-Martens-Latem, Belgium). The patterns were compared and clustered by the unweighted-pair group method using arithmetic averages (UPGMA) by using the Dice coefficient. The position tolerance was set to 1.5%, and an optimization of 1.5% was applied during the comparison of PFGE fingerprint patterns. PFGE patterns were interpreted according to the criteria of Tenover et al. (27).

Antibiotic sensitivity testing. The susceptibilities of E. sakazakii to antimicrobial agents were determined by the disk diffusion method on Iso-Sensitest agar (catalog no. CM0471; Oxoid Ltd.) according to the British Society for Antimicrobial Chemotherapy protocol (5). The antibiotics tested were amikacin, ampicillin, cefotaxime, cefuroxime, cefpodoxime, ceftazidime, chloramphenicol, ciprofloxacin, amoxicillin-clavulanate, doxycycline, gentamicin, imipenem, piperacillin, and trimethoprim from Oxoid Ltd. UK (Basingstoke, United Kingdom). ESBL production was detected using the combination disc method as described in HPA QSOP 51 (30) using ceftazidime-clavulanic acid, cefotaxime-clavulanic acid, and cefpodoxime-clavulanic acid combination discs in comparison to individual-antibiotic ceftazidime, cefotaxime, and cefpodoxime discs according to the manufacturer's instructions (Mast Diagnostics, Bootle, United Kingdom).

Biotyping. The biotype for each strain was determined according to the Farmer et al. (8) biogrouping scheme as revised by Iversen et al. (17). Standardized biochemical test strips (API20E, ID32, APIZYM) were employed according to the manufacturer's instructions (BioMérieux UK). Additional tests of motility, acid production from sugars, gas production from glucose, and malonate utilization, the methyl red test, the Voges-Proskauer test, and the indole production test were conducted as previously described. Bacterial isolates were subcultured on tryptone soy agar (catalog no. 1.05458; Merck KGaA, Darmstadt, Germany) prior to analysis.

Capsule production. Bacterial cultures were grown overnight at 37°C on milk agar, which was composed of 3 g of agar (catalog no. LP0011; Oxoid Ltd., Basingstoke, United Kingdom) and 0.4 g of ammonium sulfate dissolved into 40 ml of distilled water. After autoclaving at 121°C for 15 min, the mixture was combined with 200 ml of warm (55°C) liquid infant formula (Premium 1, milk-based; Cow & Gate, Trowbridge, United Kingdom) and dispensed into petri dishes. Each strain was evaluated for capsule production by visual comparison with the colony morphology of E. sakazakii strains 1 (noncapsulated) and 2 (capsulated).

Protease activity. Skim milk powder (2%, wt/vol) was added to plate count agar (tryptone glucose yeast agar; catalog no. CM325; Oxoid Ltd.) after autoclaving to make SM-PCA. Plates were inoculated by a simple streak of the bacteria and incubated at 37°C for 72 h. A positive result was indicated by zones of clearing around bacterial growth.

Nucleotide sequence accession numbers. The GenBank accession numbers of the E. sakazakii isolates sequenced in this study are AM778409 to AM778415.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Patients. During the 3-month outbreak period, 18 neonates were infected or asymptomatically colonized, and there were four deaths (Table 1). All infected neonates, except for one (neonate D), were preterm. The average birth weight was 1,461 g, and birth weights ranged from 1,000 to 2,090 g. Nine neonates had severe clinical symptoms: seven cases of NEC (one [neonate F] with abdominal perforation), one case of septicemia (neonate I), and one case of meningitis (neonate H). An autopsy of the latter neonate revealed cerebral lesions. All neonates with NEC had birth weights of <2,000 g. The neonate with meningitis had a birth weight of 1,500 g. The onset of illness was <28 days for the majority (17/18) of neonates. The exception was neonate K, who developed NEC I after 87 days and had a low birth weight (1,180 g). Three neonates who died (F, H, and J) had birth weights of 1,000 g, 1,500 g, and 1,560 g, respectively. The first dates of illness for these neonates were 28, 19, and 15 days after birth. Four other neonates (C, E, O, and Q) were colonized by E. sakazakii without any clinical signs, and two neonates (N and P) had moderate digestive problems.

Identification of bacterial isolates. Thirty-one strains were recovered from the original outbreak collection. No isolates were recovered for neonates I and N. All strains, except isolate 766, were confirmed as E. sakazakii using 16S rRNA gene sequence analysis and were assigned to 16S rRNA gene cluster group 1 (Table 2). The remaining strain (strain 766) was identified as E. cloacae. This strain was associated with the death of neonate R through septic shock during the general E. sakazakii outbreak and is considered in a separate section below.

PFGE typing of bacterial isolates. Three distinguishable E. sakazakii pulsotypes were isolated from neonates and reconstituted formula. A fourth pulsotype was isolated from an unopened can of infant formula (Fig. 1). The pulsotypes were numbered in chronological order.


Figure 1
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  		FIG. 1. Dendrogram generated from PFGE profiles of E. sakazakii isolates (n = 30) by Bionumerics software, version 3.5. Clustering was done with UPGMA by using the Dice coefficient. Pulsotypes are identified on the left. The tolerance in the band was 1.5%, with an optimization of 1.5%.

 
E. sakazakii pulsotype 1 was isolated from 23 March to 19 June and included strains from two cases of NEC and two asymptomatic colonizations (Tables 1 and 2). No clinical records were provided for neonate A, from whom the first isolate of E. sakazakii was obtained. Six strains belonged to pulsotype 1 and were isolated from either the trachea or stool samples.

E. sakazakii pulsotype 2 was isolated from 7 April to 1 July from neonatal peritoneal fluid, sputum, trachea, stools, conjunctivae, and skin. This period overlapped with the period of isolation of pulsotype 1. A total of 16 strains belonged to pulsotype 2. These were obtained from one case of meningitis, seven cases of NEC, two asymptomatic colonizations, and unused prepared formula. Three neonates (F, H, and J) colonized by E. sakazakii pulsotype 2 died from NEC or meningitis. Isolates 701, 767, and 695 from these neonates were obtained from the peritoneal fluid and trachea. E. sakazakii pulsotype 2 strains 705, 706, and 707 were isolated from neonate B on three occasions over 2 months (24 May, 9 June, and 26 June 1994). These strains were from the trachea, stool, and skin. Their identical PFGE profiles formed a subcluster within the pulsotype 2 cluster. Previously, E. sakazakii pulsotype 1 had been isolated from the trachea of this neonate on 25 and 29 April 1994. E. sakazakii pulsotypes 1 and 2 were isolated 3 days apart from neonate C on 9 and 12 May 1994. This neonate was asymptomatically colonized, and the strains were isolated from the trachea on both occasions. E. sakazakii pulsotype 1 was isolated from neonate D on 8 June 1994, and pulsotype 2 was isolated from the same neonate on 13 June and 1 July 1994. On each occasion, the organism was isolated from the stool.

Pulsotype 3 was isolated over a 2-week period from 15 to 27 June. This period overlapped with both pulsotypes 1 and 2. There were five strains in pulsotype 3. Two strains were from the stools of neonates P and Q. Neonate P had a digestive problem, and neonate Q was asymptomatic. Two further strains were obtained 1 week apart (20 and 27 June 1994) from leftover formula, and on the same day (27 June 1994) from unused prepared formula.

An unopened can of powdered infant formula was sampled for bacterial contamination on 11 July. This was after the neonatal pulsotypes had been isolated. Three strains of E. sakazakii (strains 716, 717, and 718) were isolated with PFGE profiles that were more than three bands different from pulsotypes 1 to 3. They were designated pulsotype 4.

Characterization of pulsotypes. Table 2 compares the phenotypic traits of the four pulsotypes. Pulsotypes 1 to 3 corresponded to biotype 13, and pulsotype 4 corresponded to biotype 5 (indole positive). APIZYM profiles did not differ between pulsotypes. All strains had high levels (4 to 5 U) of esterase-lipase and β-galactosidase activities. Moderate (1 to 2 U) levels of {alpha}-glucosidase activity were detected for all strains. No proteolytic activity was detected for pulsotype 4 strains growing on SM-PCA. In addition, pulsotype 4 strains did not produce capsulated colonies on milk agar plates. Some strains in pulsotypes 1 to 3 produced such profuse capsular material that the colonies contacted the inverted petri dish lid.

All E. sakazakii strains were sensitive to ciprofloxacin, amikacin, gentamicin, imipenem, piperacillin, and trimethoprim and resistant to doxycycline. All but two strains (strains 695 and 767) of E. sakazakii were sensitive to ampicillin, cefotaxime, and cefuroxime. Strains 695 and 767 were also resistant to cefpodoxime, ceftazidime, and chloramphenicol. Further analysis using the combination disc method recommended by the HPA (30) demonstrated that these strains possessed ESBLs. These two strains, both pulsotype 2, were isolated in two fatal cases (neonates H and J).

E. cloacae. Strain 766 was isolated as a blood culture from neonate R, who died from septic shock. It was identified in 1994, using API20E, as E. sakazakii. However 16S rRNA gene sequence analysis revealed that it was E. cloacae and had been misidentified. Several methods for the identification of E. sakazakii via PCR have been described and were used in this study (19, 20, 22). PCR products of the indicative size were obtained when genomic DNAs from E. sakazakii strains NCTC 11467T and ATCC 12868 were probed as previously described. However, no corresponding PCR product was obtained for strain 766. This further confirmed that strain 766 was not E. sakazakii. Due to the lack of {alpha}-glucosidase activity, the organism did not produce characteristic blue-green E. sakazakii colonies on the chromogenic E. sakazakii agar DFI (catalog no. CM1055; Oxoid Ltd., Basingstoke, United Kingdom). It also failed to produce any yellow pigmentation on tryptone soy agar after 48 h of incubation at 21°C. The APIZYM profile differed from that of E. sakazakii by the lack of detectable esterase and esterase-lipase activities. Antibiogram profiling showed that the strain was resistant to doxycycline, ampicillin, cefuroxime, and amoxicillin-clavulanate.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This is the largest outbreak of E. sakazakii in a NICU, with the most deaths, that has been reported to date. Unfortunately, because of the long time between the outbreak and this study, a number of clinical details were not available to us. Therefore, this study is not an epidemiological outbreak investigation; instead, it has focused on the diversity of the isolates. Nevertheless, neonatal information regarding gestation age, birth weight, age of onset of symptoms, etc., are reported here (Table 1) and will be of use for future studies on the risk factors for E. sakazakii infections.

This study shows that, over the period from March to July 1994, neonates were infected by three pulsotypes of E. sakazakii for which no definitive sources were identified (Table 2). The first pulsotype was isolated from March to June. It was isolated from five neonates (A to E), including two cases of NEC II, no fatal cases, and two asymptomatic neonates. The two NEC II cases were cocolonized by pulsotype 2. Unfortunately, no records were available for the first case, neonate A. Pulsotype 2 was isolated from April to July. It was associated with six NEC II or NEC III cases, one suspected NEC case, one case of meningitis, and two asymptomatic neonates. There were a total of three deaths. This pulsotype was also isolated from prepared formula but not from an unopened can of powdered infant formula. Pulsotype 3 was isolated over a 2-week period in June from two neonates: neonate O, with a digestive problem, and neonate P, who was asymptomatic. It was also isolated from unused formula and two leftover formulas. Pulsotypes 1 to 3 were not isolated from unopened cans of powdered infant formula analyzed in July, but only from prepared formula on 17 and 27 June 1994 (Table 2). Pulsotype 3 was isolated from unfinished formula on two occasions (20 and 27 June 1994) and from prepared formula on 27 June 1994. No unopened cans of powdered infant formula were tested at this time, and therefore the source of contamination remains uncertain. E. sakazakii was isolated from an unopened can of powdered infant formula (sample date, 11 July 1994). This was 2 weeks after the last case. These strains had a unique pulsotype. Previously, E. sakazakii has been isolated from infant formula preparation equipment, the environment, and the human throat (2, 12, 25). Therefore, given the ubiquity of E. sakazakii, unattributable sources of E. sakazakii could have been caregivers, cross-contamination from other neonates, the environment, and batches of reconstituted infant formula. It is not known if any neonates were fed the batch of powdered infant formula from which E. sakazakii pulsotype 4 was isolated (sample date, 11 July 1994), since there were no neonatal cases in July with that pulsotype.

Multiple E. sakazakii pulsotypes were isolated from neonates B, C, and D. Five isolates from the trachea, stool samples, and skin were obtained from neonate B over a 2-month period (25 April to 26 June 1994) (Table 2). The three pulsotype 2 strains (strains 705, 706, and 707) were from the trachea, stools, and skin, respectively. Due to their identical PFGE profiles, they formed a subcluster within the pulsotype 2 cluster (Fig. 1). This indicates that the neonate was colonized by a specific E. sakazakii strain from 24 May to 26 June. Neonate C was asymptomatic. Pulsotype 1 and 2 strains 708 and 709 were isolated 3 days apart (9 and 12 May 1994), both from the trachea. Neonate D suffered from NEC II. Pulsotype 1 and 2 strains 696 to 698 were isolated from stool samples between 8 June and 1 July 1994. Therefore, multiple isolates from neonates should be pulse typed; otherwise, clinical strains may not be matched with source isolates. Prompt analysis of possible sources is required, especially for substances such as powdered infant formula, since many batches may be in use over a short period of time.

The NICU feeding practices of reconstituting formula every 24 h and administering the formula over a 4- to 6-h period are not currently recommended. After the outbreak, the following internal recommendations were made: to chill the enteral feeding syringe using cryogel and to change the syringe and syringe end every 3 h. These are very similar to the 2-h maximum period between preparation and administration recommended by the FAO-WHO (9).

It should be noted that in 1994 E. sakazakii was not a well-recognized neonatal pathogen and was not associated with contaminated reconstituted infant formula. Therefore, the hospital investigation was not initially focused on the infant formula. The Belgian outbreak that associated E. sakazakii with powdered infant formula, and in which two neonates died, occurred in 1996, and the report of that outbreak was not published until 2001 (31).

All strains were doxycycline resistant. Doxycycline is a tetracycline antibiotic that is not used for this patient group and therefore has no clinical relevance. Cefotaxime and cefuroxime are commonly used first-line treatments for neonates. Therefore, the antibiograms of strains 767 and 695, from fatal cases, are of particular interest. Both these strains had ESBL activity, which may have been acquired by horizontal transfer from other Enterobacteriaceae, since it was absent in the other pulsotype 2 strains. There are more than 30 variants of CTX-M ESBLs, some of which have evolved from the chromosomal β-lactamases of Kluyvera species (3). Cefpirome is stable to the inducible AmpC chromosomal β-lactamases of Enterobacter species, which give a false-positive ESBL result with other cephalosporins that are used. The use of cephalosporins is not recommended for the treatment of Enterobacter species infections, due to the selection of AmpC-derepressed mutants. E. cloacae strain 766 was also from a fatal case and was resistant to ampicillin, cefuroxime, and amoxicillin-clavulanate.

All E. sakazakii strains were in 16S rRNA gene cluster group 1, the largest of the four E. sakazakii genotypes (16). Pulsotype 2 isolates were from eight cases of NEC, one case of septicemia, and one case of meningitis. A total of three deaths were associated with this pulsotype, and therefore it appears to be more virulent than the other pulsotypes. In addition, two pulsotype 2 strains (strains 696 and 767) from neonates H and J, who died, had acquired ESBL activities. Pulsotype 4 strains, isolated from an unopened can of powdered infant formula, differed from the other pulsotypes by the lack of protease activity on SM-PCA and the absence of capsule production. Whether these phenotypes are related to virulence determinants is unknown, but the question is currently under investigation.

Strain 766 was initially identified as E. sakazakii by using API20E and was isolated in a fatal case of septic shock (4 June 1994) during the NICU E. sakazakii outbreak (Table 1). However, 16S rRNA gene sequencing revealed that it did not fall within any of the E. sakazakii cluster groups. Several PCR protocols for the specific amplification of E. sakazakii have been developed that amplify the 16S rRNA gene. More recently, ompA-specific PCR primers were shown to distinguish E. sakazakii from similar organisms (22). Our study used these published protocols and PCR probes in order to obtain a presumptive identification of strain 766 and to further understand at what level the misidentification as E. sakazakii may have occurred. While strains NCTC 11467T and ATCC 12868 were correctly identified by these techniques, no PCR product was obtained from strain 766.

Discrepancies in E. sakazakii identification between commercial biochemical kits have been noted previously (15, 24). Therefore, 16S rRNA gene sequence analysis was used as our "gold standard" for identification. Commercially available specific E. sakazakii chromogenic agars are designed to detect {alpha}-glucosidase activity. Therefore, E. cloacae strain 766 ({alpha}-glucosidase negative) would not have been misidentified by routine use of these agars. Nor would the strain be regarded as a presumptive E. sakazakii strain by use of PCR probes. This further shows the need for rigorous testing and careful consideration of rapid phenotypic tests.

This investigation highlights the need for accurate isolate identification and prompt typing of neonatal and associated isolates. The source of the three pulsotypes could not definitely be identified as contaminated reconstituted infant formula. Nevertheless, the feeding practices of preparation for 24-h periods and prolonged (>2-h) administration could have enabled bacterial growth in the formula and increased the risk of infection.


   ACKNOWLEDGMENTS
 
We thank Stacey Brown for technical assistance, Julie Rainard for translating documents, and Anna Bowen (CDC, Atlanta, GA) for helpful discussions.

We also thank Nottingham Trent University, the Centre National de la Recherche Scientifique, and the Université Paul Sabatier for financial support.


   FOOTNOTES
 
* Corresponding author. Mailing address: School of Biomedical and Natural Sciences, Nottingham Trent University, Clifton Lane, Nottingham, NG11 8NS, United Kingdom. Phone: 115 8483529. Fax: 115 8486636. E-mail: Stephen.forsythe{at}ntu.ac.uk Back

{triangledown} Published ahead of print on 10 October 2007. Back


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Beck-Sague, C., P. Azimi, S. Fonseca, R. Baltimore, D. Powell, L. Bland, M. Arduino, S. McAllister, R. Huberman, R. Sinkowitz, et al. 1994. Bloodstream infections in neonatal intensive care unit patients: results of a multicenter study. Pediatr. Infect. Dis. J. 13:1110-1116.[Medline]
  2. Block, C., O. Peleg, N. Minster, B. Bar-Oz, A. Simhon, I. Arad, and M. Shapiro. 2002. Cluster of neonatal infections in Jerusalem due to unusual biochemical variant of Enterobacter sakazakii. Eur. J. Clin. Microbiol. Infect. Dis. 21:613-616.[CrossRef][Medline]
  3. Bonnet, R. 2004. Growing group of extended-spectrum beta-lactamases: the CTX-M enzymes. Antimicrob. Agents Chemother. 48:1-14.[Free Full Text]
  4. Bowen, A. B., and C. R. Braden. 2006. Invasive Enterobacter sakazakii disease in infants. Emerg. Infect. Dis. 12:1185-1186.[Medline]
  5. British Society for Antimicrobial Chemotherapy. 2006. BSAC methods for antimicrobial susceptibility testing, version 5. http://www.bsac.org.uk/_db/_documents/version_5_.pdf.
  6. CDC. June 2004. Pulse Net USA. One-day (24-48 h) standardized laboratory protocol for molecular subtyping of Escherichia coli O157:H7, non-typhoidal Salmonella serotypes, and Shigella sonnei by pulsed field gel electrophoresis (PFGE). http://www.cdc.gov/pulsenet/protocols/ecoli_salmonella_shigella_protocols.pdf.
  7. Coignard, B., V. Vaillant, J.-P. Vincent, A. Leflèche, P. Mariani-Kurkdjian, C. Bernet, F. L'Hériteau, H. Sénéchal, P. Grimont, E. Bingen, and J.-C. Desenclos. 2006. Infections sévères à Enterobacter sakazakii chez des nouveau-nés ayant consommé une préparation en poudre pour nourrissons, France, octobre-décembre 2004. Bull. Epidémiol. Hebdomadaire 2-3:10-13. http://www.invs.sante.fr/beh/2006/02_03/beh_02_03_2006.pdf.
  8. Farmer, J. J., III, M. A. Asbury, F. W. Hickman, D. J. Brenner, and The Enterobacteriaceae Study Group. 1980. Enterobacter sakazakii: a new species of "Enterobacteriaceae" isolated from clinical specimens. Int. J. Syst. Bacteriol. 30:569-584.
  9. Food and Agriculture Organization-World Health Organization (FAO-WHO). 2004. Enterobacter sakazakii and other microorganisms in powdered infant formula. Meeting report, MRA series 6. World Health Organization, Geneva, Switzerland. http://www.who.int/foodsafety/publications/micro/mra6/en/index.html.
  10. Food and Agriculture Organization-World Health Organization (FAO-WHO). 2006. Enterobacter sakazakii and Salmonella in powdered infant formula. Meeting report, MRA series 10. World Health Organization, Geneva, Switzerland. http://www.who.int/foodsafety/publications/micro/mra10/en/index.html.
  11. Forsythe, S. J. 2005. Enterobacter sakazakii and other bacteria in powdered infant milk formula. Maternal Child Nutr. 1:44-50.[CrossRef]
  12. Gosney, M. A., M. V. Martin, A. E. Wright, and M. Gallagher. 2006. Enterobacter sakazkaii in the mouths of stroke patients and its association with aspiration pneumonia. Eur. J. Intern. Med. 17:185-188.[CrossRef][Medline]
  13. Himelright, I., E. Harris, V. Lorch, and M. Anderson. 2002. Enterobacter sakazakii infections associated with the use of powdered infant formula—Tennessee, 2001. JAMA 287:2204-2205.[Free Full Text]
  14. International Commission on Microbiological Specifications for Foods. 2002. Microorganisms in foods 7. Microbiological testing in food safety management. Kluwer Academic/Plenum Publishers, New York, NY.
  15. Iversen, C., P. Druggan, and S. Forsythe. 2004. A selective differential medium for Enterobacter sakazakii, a preliminary study. Int. J. Food Microbiol. 96:133-139.[CrossRef][Medline]
  16. Iversen, C., M. Waddington, S. L. W. On, and S. Forsythe. 2004. Identification and phylogeny of Enterobacter sakazakii relative to Enterobacter and Citrobacter. J. Clin. Microbiol. 42:5368-5370.[Abstract/Free Full Text]
  17. Iversen, C., M. Waddington, J. J. Farmer III, and S. Forsythe. 2006. The biochemical differentiation of Enterobacter sakazakii genotypes. BMC Microbiol. 6:94.[CrossRef][Medline]
  18. Jarvis, C. 2005. Fatal Enterobacter sakazakii infection associated with powdered infant formula in a neonatal intensive care unit in New Zealand. Am. J. Infect. Control 33:e19.
  19. Keyser, M., R. C. Witthuhn, L. C. Ronquest, and T. J. Britz. 2003. Treatment of winery effluent with upflow anaerobic sludge blanket (UASB)-granular sludges enriched with Enterobacter sakazakii. Biotechnol. Lett. 25:1893-1898.[CrossRef][Medline]
  20. Lehner, A., T. Tasara, and R. Stephan. 2004. 16S rRNA gene based analysis of Enterobacter sakazakii strains from different sources and development of a PCR assay for identification. BMC Microbiol. 4:43.[CrossRef][Medline]
  21. Mange, J. P., R. Stephan, N. Borel, P. Wild, K. S. Kim, A. Pospischil, and A. Lehner. 2006. Adhesive properties of Enterobacter sakazakii to human epithelial and brain microvascular endothelial cells. BMC Microbiol. 6:58.[CrossRef][Medline]
  22. Mohan Nair, M. K., and K. S. Venkitanarayanan. 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.[Abstract/Free Full Text]
  23. Pagotto, F. J., M. Nazarowec-White, S. Bidawid, and J. M. Farber. 2003. Enterobacter sakazakii: infectivity and enterotoxin production in vitro and in vivo. J. Food Prot. 66:370-377.[Medline]
  24. Restaino, L., E. W. Frampton, W. C. Lionberg, and R. J. Becker. 2006. A chromogenic plating medium for the isolation and identification of Enterobacter sakazakii from foods, food ingredients, and environmental sources. J. Food Prot. 69:315-322.[Medline]
  25. Smeets, L. C., A. Voss, H. L. Muytjens, J. F. G. M. Meis, and W. J. G. Melchers. 1998. Genetische karakterisatie van Enterobacter sakazakii—isolaten van Nederlandse patiënten met neonatale meningitis. Ned. Tijdschr. Med. Microbiol. 6:113-115.
  26. Stoll, B. J., N. Hansen, A. A. Fanaroff, and J. A. Lemons. 2004. Enterobacter sakazakii is a rare cause of neonatal septicemia or meningitis in VLBW infants. J. Pediatr. 144:821-823.[Medline]
  27. Tenover, F. C., R. D. Arbeit, R. V. Goering, P. A. Mickelsen, B. E. Murray, D. H. Persing, and B. Swaminathan. 1995. Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing. J. Clin. Microbiol. 33:2233-2239.[Medline]
  28. Townsend, S., J. Caubilla Barron, C. Loc-Carrillo, and S. Forsythe. 2007. The presence of endotoxin in powdered infant formula milk and the influence of endotoxin and Enterobacter sakazakii on bacterial translocation in the infant rat. Food Microbiol. 24:67-74.[CrossRef][Medline]
  29. Townsend, S. M., E. Hurrell, I. Gonzalez-Gomez, J. Lowe, J. G. Frye, S. Forsythe, and J. L. Badger. 2007. Enterobacter sakazakii invades brain capillary endothelial cells, persists in human macrophages influencing cytokine secretion and induces severe brain pathology in the neonatal rat. Microbiology 153:3538-3547.[Abstract/Free Full Text]
  30. United Kingdom Health Protection Agency. 2006. Laboratory detection and reporting of bacteria with extended spectrum β-lactamases. QSOP 51. Health Protection Agency, London, United Kingdom.
  31. van Acker, J., F. De Smet, G. Muyldermans, A. Bougatef, A. Naessens, and S. Lauwers. 2001. Outbreak of necrotizing enterocolitis associated with Enterobacter sakazakii in powdered milk formula. J. Clin. Microbiol. 39:293-297.[Abstract/Free Full Text]
  32. Walsh, M. C., and R. M. Kliegman. 1986. Necrotizing enterocolitis: treatment based on staging criteria. Pediatr. Clin. N. Am. 33:179-201.[Medline]

Journal of Clinical Microbiology, December 2007, p. 3979-3985, Vol. 45, No. 12
0095-1137/07/$08.00+0     doi:10.1128/JCM.01075-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.




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