[Skip to Content](https://www.sigmaaldrich.com#main-content) [](https://www.sigmaaldrich.com/CA/en) Products Cart0 CAEN Products [Login / Register](https://www.sigmaaldrich.com/oidc-sign-in) [Order Lookup](https://www.sigmaaldrich.com/CA/en/order-lookup) [Quick Order](https://www.sigmaaldrich.com/CA/en/quick-order) Cart0 [Home](https://www.sigmaaldrich.com/CA/en)[Pathogen & Spoilage Testing](https://www.sigmaaldrich.com/CA/en/applications/microbiological-testing/pathogen-and-spoilage-testing)*Chronobacter* spp. Classic and Detection Methods # *Chronobacter* spp. Classic and Detection Methods ### *Enterobacter sakazakii*, reclassified as *Cronobacter*, is related to neonatal infections *Cronobacter* is a rod-shaped, motile and facultatively-anaerobic bacteria of the family Enterobacteriaceae. Originally *Cronobacter* spp. were listed as yellow-pigmented *Enterobacter cloacae* (__Table 1__). The bacteria was then later on called *Enterobacter sakazakii.* Taxonomic studies have determined that *E. sakazakii* comprises a high genetic heterogeneity and should be reclassified as a novel genus, “*Cronobacter.*”1  __Table 1.__Biochemical reactions from *Cronobacter* spp.  *Cronobacter* spp. are ubiquitous and frequently found on vegetable, meat, fermented bread, diary products and especially in baby food. Consumption of contaminated powdered infant formula milk (IFM) can result in sepsis, infant meningitis and necrotising enterocolitis. In most cases the victims are pre-term infants, low birth weight infants or immunocompromised infants in the first weeks. Mechanisms are not fully understand now but it is assumed that endotoxins are produced. The bacterium can adhere to and penetrate into various types of cells (e.g. endothelial cells). It also survives in macrophages. As *Enterobacteriaceae* are suspectible to heat they do not survive most production processes and it is most likely that contamination happens after the process. The post process contamination can come from the addition of heat sensitive additives such as vitamins or other micronutirents or incorrect handling while reconstitution or storage. It has been shown, that stationary phase *Cronobacter* spp. are remarkably resistant to osmotic and dry stress compared with other species of the Enterobacteriacae group. It is difficult to isolate *Cronobacter* spp. from samples as they are most likely stressed, unevenly distributed throughout the batch and also numbers will probably be fairly low, often lower than 1 CFU per g. For the classical microbiological tests a pre-enrichment is used to recover the stressed cells followed by a selective enrichment step. The FDA method recommends *Enterobacteriaceae* enrichment (EE or Mossel) broth which is then streaked onto VRBG agar and suspect colonies are subcultured onto TSA agar where the yellow pigmented colonies are confirmed by oxidase test and other biochemical tests. See also Table 1 with all kind of biochemical reaction from *Cronobacter* spp. species (not only from FDA). ISO/TS 22964:2008 methods recommends buffered peptone water (BPW) as pre-enrichment medium and modified lauryl sulphate broth with vancomycin (mLST) incubated at 44 °C for the secondary selective enrichment step. The next step is then a chromgenic agar for isolation and identification (see Table 2). | | | | | |----------------|----------------------------------------------------------------|--------------------------------------------|-----------------------------------| | Brand | Product No. | Medium | Description | | Sigma-Aldrich® | [92324](https://www.sigmaaldrich.com/CA/en/product/sial/92324) | HiCrome™ *Cronobacter* spp. Agar | chromogenic media | | Sigma-Aldrich® | [14703](https://www.sigmaaldrich.com/CA/en/product/sial/14703) | HiCrome™ *Cronobacter* spp. Agar, Modified | chromogenic media according ISO | | Sigma-Aldrich® | [69965](https://www.sigmaaldrich.com/CA/en/product/sial/69965) | Mossel Broth (E.E. broth) | for pre-enrichment | | Sigma-Aldrich® | [77187](https://www.sigmaaldrich.com/CA/en/product/sial/77187) | Peptone Water, phosphate-buffered | for pre-enrichment | | Sigma-Aldrich® | [22091](https://www.sigmaaldrich.com/CA/en/product/sial/22091) | Tryptic Soy Agar | for isolation and differentiation | | Sigma-Aldrich® | 79872 | Tryptic Soy Agar | for isolation and differentiation | Table 2.Media for detection of *Cronobacter* spp. There are diverse chromogenic agars available which help to save work and time to result and are more reliable than traditional media. The detection principle is based on the alpha-glucosidase possessed by *Cronobacter* spp. (not by most other *Enterobacteriaceae*) which cleaves the 5-bromo-4-chloro-3-indolyl-a-D-glucopyranoside or similar substrates. The result is a plate with e.g. blue colonies in case of *Cronobacter* spp. (__Figures 2 and 3__) but biochemical confirmation is still required.  __Figure 2.__HiCrome™ *Cronobacter* spp. Agar (92324); *Cronobacter* spp. (blue), *E. aerogenes* (green) *K. pneumoniae* (yellow)  __Figure 3.__HiCrome™ *Cronobacter* spp. Agar, Modified (14703), with *Cronobacter* spp. colonies Today there are also studies that not all *Cronobacter* spp*.* giving yellow pigmented colonies on tryptic soy agar and it was showed that some type of strains did not grow at 44-45 °C. Also some selective media may be too selective to recover all the *Cronobacter* species. There is still some work to do to improve the official methods. | | | | | |----------------|----------------------------------------------------------------|--------------------------------|--------| | Brand | Product No. | Name | Assays | | Sigma-Aldrich® | [12838](https://www.sigmaaldrich.com/CA/en/product/sial/12838) | HybriScan®*D Cronobacter* spp. | 96 | Table 2.Ordering Information ## A Molecular Biology Method for Detecting and Identifying Cronobacter[](https://www.sigmaaldrich.com) Rapid detection and identification of *Cronobacter* species is required, since even low cell numbers have been reported to cause a disease. HybriScan®__*D*__ *Cronobacter* spp. is a rapid molecular test system for detection of bacteria of the genus *Cronobacter* in food, especially in dried infant formula milk and its production environment. The method is based on the detection of rRNA by sandwich hybridisation and so no PCR is needed. It is a 96 well microplate format and the workflow is very similar to an ELISA test. __Figure 4__ shows the specificity of HybriScan®- *Cronobacter* spp. Different cell amounts and related Enterobacteriaceae were tested within a validation study. No signals were observed using 2,3 x 108 *Enterobacter* *cloacae* cells or 7 x 108 *Citrobacter freundii* cells per assay, whereas clear specific signals were detectable using 2,6 x 103, 1,3 x 104, and 2,6 x 104 cells of *Cronobacter* species, respectively. These results demonstrate that the HybriScan® system is highly specific for *Cronobacter* spp.  __Figure 4.__Specificity of HybriScan®-*Cronobacter* spp. Different cell numbers of *Cronobacter* spp. and related Enterobacteriaceae like *E. cloacae* and Citrobacter freundii were tested. Measurement data for HybriScan® analyses represent absorption at 450 nm. A validation study of HybriScan®- *Cronobacter* spp. was performed using two different enrichment procedures: (1) single step enrichment for 24 – 26 hours at 37 °C in ESSB broth (*Enterobacter sakazakii* selective broth) and (2) two step enrichment starting with a pre-enrichment for 18 – 20 hours at 37 °C in buffered peptone water and followed by a selective enrichment for 24 – 26 hours at 45 °C in mLST selective broth. The results of the above mentioned validation study are presented in __Figure 4__. ## Materials Sorry, an unexpected error has occurred Response not successful: Received status code 500 ### References 1\. CAWTHORN D, BOTHA S, WITTHUHN R. 2008. Evaluation of different methods for the detection and identification of Enterobacter sakazakii isolated from South African infant formula milks and the processing environment. International Journal of Food Microbiology. 127(1-2):129-138. [https://doi.org/10.1016/j.ijfoodmicro.2008.06.024](https://doi.org/10.1016/j.ijfoodmicro.2008.06.024) 2\. 2002\. Isolation and Enumeration of Enterobacter sakazakii from Dehydrated Powdered Infant Formula,. \[Internet]. U.S. Food and Drug Administration Center for Food Safety and Applied Nutrition. Available from: [https://wayback.archive-it.org/7993/20170406021728/https://www.fda.gov/Food/FoodScienceResearch/LaboratoryMethods/ucm114665.htm](https://wayback.archive-it.org/7993/20170406021728/https://www.fda.gov/Food/FoodScienceResearch/LaboratoryMethods/ucm114665.htm) 3\. 1997\. Color Atlas and Textbook of Diagnostic Microbiology. 5th Edition. Lippincott Williams &Wilkins. 4\. 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(1):64. [https://doi.org/10.1186/1471-2148-7-64](https://doi.org/10.1186/1471-2148-7-64) 5\. Bergey D, Holt J. 1994. Bergey’s manual of determinative bacteriology. 9th Edition. Lippincott Williams & Wilkins. 6\. Kleiman MB, Allen SD, Neal P, Reynolds J. 1981. Meningoencephalitis and Compartmentalization of the Cerebral Ventricles Caused by Enterobacter sakazakii. 14(3):352-354. [https://doi.org/10.1128/jcm.14.3.352-354.1981](https://doi.org/10.1128/jcm.14.3.352-354.1981) 7\. 2006\. ISO/TS 22964:2006 Milk and milk products detection of Enterobacter Sakazakii. \[Internet]. Available from: [https://www.iso.org/standard/41258.html](https://www.iso.org/standard/41258.html) 8\. GUILLAUME-GENTIL O, SONNARD V, KANDHAI MC, MARUGG JD, JOOSTEN H. 2005. A Simple and Rapid Cultural Method for Detection of Enterobacter sakazakii in Environmental Samples. 68(1):64-69. [https://doi.org/10.4315/0362-028x-68.1.64](https://doi.org/10.4315/0362-028x-68.1.64) 9\. Riedel K, Lehner A. 2007. Identification of proteins involved in osmotic stress response inEnterobacter sakazakii by proteomics. Proteomics. 7(8):1217-1231. [https://doi.org/10.1002/pmic.200600536](https://doi.org/10.1002/pmic.200600536) 10\. PAGOTTO FJ, NAZAROWEC-WHITE M, BIDAWID S, FARBER JM. 2003. Enterobacter sakazakii: Infectivity and Enterotoxin Production In Vitro and In Vivo. 66(3):370-375. [https://doi.org/10.4315/0362-028x-66.3.370](https://doi.org/10.4315/0362-028x-66.3.370) __Related Articles__ - [*Lactobacilli* Basics, Testing & Identification](https://www.sigmaaldrich.com/CA/en/technical-documents/technical-article/clinical-testing-and-diagnostics-manufacturing/bacteriology/lactobacilli) - [Microbial Detection Using Chromogenic and Fluorogenic Culture Media](https://www.sigmaaldrich.com/CA/en/technical-documents/technical-article/clinical-testing-and-diagnostics-manufacturing/bacteriology/overview-chromogenic-media) - [Identification of Microorganisms Based on Color](https://www.sigmaaldrich.com/CA/en/technical-documents/technical-article/clinical-testing-and-diagnostics-manufacturing/cytology-and-microscopy/color-identification-microorganisms) - [Culture Method for Beer Quality Control](https://www.sigmaaldrich.com/CA/en/technical-documents/technical-article/food-and-beverage-testing-and-manufacturing/microbiological-analysis-for-food-and-beverage/culture-method-beer-quality) - [Detection and Differentiation of Staphylococcus Aureus on Baird Parker Agar](https://www.sigmaaldrich.com/CA/en/technical-documents/technical-article/food-and-beverage-testing-and-manufacturing/microbiological-analysis-for-food-and-beverage/detection-and-differentiation) - [Differentiation of Escherichia coli from coliforms](https://www.sigmaaldrich.com/CA/en/technical-documents/technical-article/food-and-beverage-testing-and-manufacturing/microbiological-analysis-for-food-and-beverage/differentiation-of-e-coli-coliforms) - [DiluCult™ Gravimetric Dilutor & Sample Homogenizer](https://www.sigmaaldrich.com/CA/en/technical-documents/technical-article/food-and-beverage-testing-and-manufacturing/microbiological-analysis-for-food-and-beverage/dilucult-gravimetric-dilutor-homogenizer) - [EN ISO 11133 Microbiological Culture Media](https://www.sigmaaldrich.com/CA/en/technical-documents/technical-article/food-and-beverage-testing-and-manufacturing/microbiological-analysis-for-food-and-beverage/en-iso-11133-water-for-the-preparation-and-performance-testing) - [View More](https://www.sigmaaldrich.com/CA/en/search/facet-search?focus=sitecontent&term=facet-search) Top __Sign In To Continue__ To continue reading please sign in or create an account. Sign In__Don't Have An Account?__Register An unknown error has occured. - English - EN - Français - FR [Learn More](https://www.sigmaaldrich.com)