BRILLIANCE ENTEROBACTER SAKAZAKII AGAR (DFI formulation)

Code: CM1055

Brilliance™ Enterobacter sakazakii Agar (formerly Chromogenic Enterobacter sakazakii Agar) is for the differentiation and enumeration of Enterobacter sakazakii from infant formula and other food samples.

Typical Formula *	gm/litre
Tryptone	15.0
Soya peptone	5.0
Sodium chloride	5.0
Ferric ammonium citrate	1.0
Sodium desoxycholate	1.0
Sodium thiosulphate	1.0
Chromogen	0.1
Agar	15.0
Final pH 7.3 ± 0.2 @ 25°C

 * Adjusted as required to meet performance standards

Directions
Suspend 43.1g of Brilliance Enterobacter sakazakii Agar in 1 litre of distilled water. Mix well and bring to the boil to dissolve completely. Sterilize by autoclaving at 121°C for 15 minutes. Cool the medium to 50°C. Mix well and pour into sterile Petri dishes.

Description
Brilliance Enterobacter sakazakii Agar (DFI formulation¹) is based on the a-glucosidase reaction, which is detected by incorporating the chromogenic substrate 5-bromo-4-chloro-3-indolyl-a-D-glucopyranoside in the medium. The enzyme a-glucosidase, present in Enterobacter sakazakii, hydrolyses the substrate producing blue-green colonies on this pale yellow medium. Proteus vulgaris is also weakly a-glucosidase positive and could grow to give colonies of a similar colour to E. sakazakii. However, on this medium, Proteus spp. grow as grey colonies: they produce hydrogen sulphide in the presence of ferric ions forming ferrous sulphide. Desoxycholate inhibits the growth of most Gram-positive organisms.

Enterobacter sakazakii is a Gram-negative rod-shaped bacterium that rarely causes disease in healthy adults. It has been implicated in outbreaks of disease in premature infants (neonates). Research suggests that neonates, or those infants who have other medical conditions, are more susceptible to this infection. Most reported cases of infection are severe, including sepsis (bacteria in the blood), meningitis, or necrotizing enterocolitis (severe intestinal infection). Neurological damage may be permanent, and the death rate is reported to be as high as 40-80%².

Enterobacter sakazakii is an opportunistic pathogen which has been isolated at low levels from powdered infant formulas. The organisms’ high tolerance to desiccation provides a competitive advantage for Enterobacter sakazakii in dry environments, as found in milk powder factories, and thereby increases the risk of post-pasteurisation contamination of the finished product³.

The current FDA method³ for the detection of Enterobacter sakazakii is based on yellow pigment production and originated from pioneering work of Muytjens et al.⁴. Samples are incubated in water overnight then enriched in EE Broth (CM0317), followed by plating on VBRGA (CM0485) to isolate Enterobacteriaceae. Five colonies are selected and streaked on Tryptone Soya Agar (CM0131), incubated for up to 3 days and observed for yellow colonies, typical of Enterobacter sakazakii. However, this method does not select for Enterobacter sakazakii and the combined use of EE broth and VRBGA could allow other Enterobacteria to outgrow Enterobacter sakazakii and give false negative results. It is not possible to select for Enterobacter sakazakii colonies from VRBGA plates on the basis of colony morphology as they will appear the same as other Enterobacteria.

Technique
Please note that the following is only a suggested method of use. Refer to current guidelines and recommendations.

Oxoid Brilliance Enterobacter sakazakii Agar has been shown to increase the recovery of Enterobacter sakazakii from powdered infant formula and other food samples when it is used to replace the two plating media in the FDA method 2002¹.

1. Using an inoculating loop remove 10µl from the incubated EE broth and streak or spread onto the surface of a Brilliance Enterobacter sakazakii Agar plate
2. Incubate the plate at 35-37°C for 24 hours, and observe for blue-green colonies
3. Confirm presumptive blue-green colonies as Enterobacter sakazakii, biochemically. This may be carried out using systems such as Microbact^TM 24E MB1131A or MB1074A.

Storage conditions and Shelf life
The dehydrated medium should be stored at 10-30°C and used before the expiry date on the label.
Prepared medium may be stored for up to 2 weeks at 2-8°C.

Appearance
Dehydrated powder: a straw coloured, free-flowing powder
Prepared medium: a pale yellow gel