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Journal of Clinical Microbiology, June 2007, p. 2048-2050, Vol. 45, No. 6
0095-1137/07/$08.00+0     doi:10.1128/JCM.00961-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Comparison of the Phenotyping Methods ID 32E and VITEK 2 Compact GN with 16S rRNA Gene Sequencing for the Identification of Enterobacter sakazakii{triangledown}

Nadège Fanjat,1 Alexandre Leclercq,2 Han Joosten,3 and Denis Robichon1*

bioMérieux, Research and Development Microbiology, La Balme Les Grottes, France,1 Institut Pasteur, Microbiology Department, 28 rue du Dr Roux, 75724 Paris cedex 15, France,2 Nestle Research Centre, Quality and Safety Department, CH-1000 Lausanne 26, Switzerland3

Received 8 May 2006/ Returned for modification 23 June 2006/ Accepted 30 March 2007


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ABSTRACT
 
A total of 34 isolates (28 Enterobacter sakazakii and 6 Enterobacteriaceae) from infant formulae, milk powder, and the production environment of milk powder factories were identified using ID 32E and VITEK 2 compact GN systems (bioMérieux, France). The ID 32E version 3.0 and VITEK 2 compact GN version 01.01b correctly identified 100% (28) of the Enterobacter sakazakii isolates tested, whereas the previous software version 2.0 for ID 32E showed only 71.4% correct results. None of the non-E. sakazakii isolates tested were misidentified as E. sakazakii with either of the identification systems used.


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TEXT
 
Enterobacter sakazakii, initially referred to as "yellow-pigmented Enterobacter cloacae," is an opportunistic pathogen implicated in food-borne outbreaks or sporadic cases worldwide (5, 15, 21). This bacterium, a member of the Enterobacteriaceae family, is the causative agent of meningitis, septicemia, and necrotizing colitis in infants, particularly neonates and infants with underlying chronic health conditions. At least 76 cases of E. sakazakii infections with 19 deaths in infants and children have been reported to date (2, 5-7, 15, 19). Fatality rates in neonates are approximately 20 to 50% (13), and survivors often suffer from severe neurological sequelae. Epidemiological investigations suggest milk-based powdered infant formulae as the major vehicle for human infection (1, 5). Contamination of infant formulae seems to be associated with the production environment rather than originating from utensils or equipment in hospital settings, but it is assumed that improper storage conditions of the reconstituted products in the hospital have also played a key role in causing the outbreaks. Until recently there were no specific legal requirements with respect to E. sakazakii in infant formulae, but this opportunistic pathogen was covered by the general criteria for coliforms. However, in 2005 new regulations were implemented in Europe requiring the absence of E. sakazakii in 30 samples of 10 g each (http://eurlex.europa.eu/LexUriServ/site/en/oj/2005/l_338/l_33820051222en00010026.pdf), and it is expected that other countries will develop similar legislation. To ensure legal compliance, sensitive detection and accurate identification methods for E. sakazakii have to be available.

Detection procedures for E. sakazakii are often based on standardized methods for enumeration and detection of Enterobacteriaceae followed by identification of colonies (http://www.cfsan.fda.gov/~comm/mmesakaz.html). Recently, selective enrichment procedures and chromogenic agar media have been developed for E. sakazakii (4, 9, 20). However, confirmation of colonies on these media is still necessary to verify the identity of the isolates. Utilization of commercially available biochemical galleries or PCR-based assays for routine food and clinical microbiology laboratories can be used to rapidly and specifically identify bacteria from a wide variety of sources (16, 18). The most utilized biochemical gallery is the API 20E (bioMérieux, France) (5), but Iversen et al. (8, 9) reported that the API 20E and ID 32E (bioMérieux, France) could give false-negative and false-positive results. On the other hand, Drudy et al. (3) tested 57 isolates and found 98% correct for ID 32E. We therefore decided to evaluate the performance of the ID 32E system and the VITEK 2 compact GN (bioMérieux, France) for the identification of Enterobacteriaceae and nonfastidious gram-negative bacteria on a number of Enterobacter sakazakii and non-Enterobacter sakazakii isolates.

The study was carried out on 28 E. sakazakii strains and 6 other Enterobacteriaceae that were initially misidentified as E. sakazakii by routine food microbiology laboratories (Table 1). These strains were selected based on typical appearance of their colonies (blue/green, indicative of {alpha}-glucosidase activity) on Druggan Forsyth Iversen agar (9) which presumed that they were E. sakazakii.


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  		TABLE 1. Strains used in this study

The isolates were subcultured on Trypticase soy agar (bioMérieux, France) and incubated at 36°C for 18 to 24 h. Each strain was identified to the species level by 16S rRNA gene sequencing, which is considered the reference method. Briefly, DNA was extracted using an enzymatic method, and PCR amplification of the 16S rRNA gene was performed with three primer pairs (universal forward and reverse) (14). The same primers were used for sequencing. Comparative analyses against the 16S rRNA gene sequence for the E. sakazakii type strain (ATCC 29544) were done by BLAST 2 sequences (version BLASTN 2.2.13; http://www.ncbi.nlm.nih.gov/BLAST/).

All isolates were also tested with each of the two miniaturized biochemical identification systems for Enterobacteriaceae and other nonfastidious gram-negative bacteria, according to the manufacturer's instructions. Data obtained with the ID 32E system were read automatically and interpreted with the database version 2.0 of the ATB Plus software. The data were also interpreted with an improved version 3.0 (available since the end of 2006). The VITEK 2 compact GN colorimetric card was read and interpreted automatically with the VITEK 2 compact system, version 01.01b.

Results obtained by 16S rRNA gene sequencing were compared to those obtained using the miniaturized biochemical identification systems. Biochemical identifications were classified into five categories: correct identification to one taxon (excellent, very good, good, or acceptable identification), correct identification including more than one taxon (excellent, very good, good, acceptable genus, or low discrimination), doubtful profile, unidentified (not reliable identification), and incorrect. The first two categories were assigned to the correct identification.

Identification by 16S rRNA sequencing, percentage of similarity with type strain, and clustering for each E. sakazakii isolate are presented in Table 1. Among the 34 isolates, 28 sharing 98 to 100% similarity with the type strain were classified as E. sakazakii. According to Iversen and her colleagues, E. sakazakii can be divided into four DNA cluster groups (10). The large majority of clinical and food isolates belong to DNA cluster group 1 (11, 12), and all of the 28 E. sakazakii strains that were used for our study were also assigned to this DNA cluster group. The six other strains were less than 96% similar to the type strain, which suggested that these isolates should not be identified as E. sakazakii.

Regarding identification rates obtained for E. sakazakii isolates with the ID 32E, 20 isolates (71.4%) were correctly identified as E. sakazakii using the current database version 2.0. For the other eight isolates (28.6%), the profile was doubtful because the L-arabinose and/or {alpha}-maltosidase tests were negative, whereas these tests were expected to be 100% positive for E. sakazakii according to the database. Taking these new results into account, the ID 32E database was modified accordingly, which decreased the corresponding positive percentages for both tests (L-arabinose and {alpha}-maltosidase) to 99%. Using this improved database (version 3.0), a correct species identification for all isolates (100%) was obtained, which represents a significant improvement (P < 0.05 by binomial test) over that obtained with version 2.0.

For the VITEK 2 compact GN, an excellent identification rate was obtained directly, since 28 (100%) of the isolates were correctly identified to the species level.

Concerning the six other Enterobacteriaceae isolates tested in this study (Table 1), one was identified to the genus Enterobacter (strain number 05 01 120) with both bioMérieux systems and by 16S sequencing according to 96% similarity with the type strain. For the remaining five isolates, 16S rRNA gene sequence analysis did not allow identification of the strains at the genus level, and inconsistent results were obtained with ID 32E and VITEK 2 compact GN kits. Three of these strains (v429, v444, and v450) correspond with "unknown DNA cluster group 6" (12) and fruit powder isolates described by Lehner et al. (17). Among these six strains, none were incorrectly identified as E. sakazakii by any of the methods used.

In conclusion, these results show that identifications of the tested strains as E. sakazakii species by ID 32E and VITEK 2 compact GN were fully comparable with results obtained by the 16S rRNA sequencing reference method. The ID 32E version 3.0 and VITEK 2 compact GN version 01.01.b are well suited for E. sakazakii, since 100% of the studied isolates were correctly identified and did not need supplemental tests. Our study showed that neither of the systems gave false identifications.

Nevertheless, this study did not evaluate a large number of isolates, and only strains belonging to DNA cluster group 1 were included. The reliability of the systems for the identification of larger numbers of strains belonging to the four DNA cluster groups of E. sakazakii should be established.


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ACKNOWLEDGMENTS
 
We thank Isabelle Desforges for contributions in manuscript writing and for helpful discussion, Nicole Garcia for technical support, Sonia Chatellier for providing 16S sequencing results, and Dave Pincus for helpful comments and critical discussions.


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FOOTNOTES
 
* Corresponding author. Mailing address: bioMerieux, Research and Development Microbiology, 3 Route de Port Michaud, 38 390 La Balme Les Grottes, France. Phone: 33 (0)4 74 95 26 22. Fax: 33 (0)4 74 95 26 32. E-mail: denis.robichon{at}eu.biomerieux.com Back

{triangledown} Published ahead of print on 11 April 2007. Back


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Journal of Clinical Microbiology, June 2007, p. 2048-2050, Vol. 45, No. 6
0095-1137/07/$08.00+0     doi:10.1128/JCM.00961-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Iversen, C., Lehner, A., Mullane, N., Marugg, J., Fanning, S., Stephan, R., Joosten, H. (2007). Identification of "Cronobacter" spp. (Enterobacter sakazakii). J. Clin. Microbiol. 45: 3814-3816 [Abstract] [Full Text]  

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