A Simple and Rapid Cultural Method for Detection of Enterobacter sakazakii in Environmental Samples

Abstract

A method was developed to detect and identify Enterobacter sakazakii in environmental samples. The method is based on selective enrichment at 45+/-0.5 degrees C in lauryl sulfate tryptose broth supplemented with 0.5 M NaCl and 10 mg/liter vancomycin (mLST) for 22 to 24 h followed by streaking on tryptone soy agar with bile salts. When exposed to light during incubation at 37 degrees C, E. sakazakii produces yellow colonies within 24 h; identification was confirmed by testing for alpha-glucosidase activity and by using API 20E strips. All of the E. sakazakii strains tested (n = 99) were able to grow in mLST at 45+/-0.5 degrees C, whereas 35 of 39 strains of potential competitors, all belonging to the Enterobacteriaceae, were suppressed. A survey was carried out with 192 environmental samples from four different milk powder factories. Using this new protocol, E. sakazakii was isolated from almost 40% of the samples, whereas the reference procedure (enrichment in buffered peptone water, isolation on violet red bile glucose agar, and biochemical identification of randomly chosen colonies) only yielded 26% positive results. This selective method can be very useful for the rapid and reliable detection of E. sakazakii in environmental samples.

Comparison of Media for the Isolation of Enterobacter sakazakii

“…All aliquots were additionally inoculated with yellow-pigmented Enterobacteriaceae isolates comprising an ␣-glucosidase-positive strain and an ␣-glucosidase-negative Pantoea strain at 0.4 CFU g Ϫ1 . Four recovery methods were compared: FDA (http://www.cfsan.fda.gov/ ϳcomm/mmesakaz.html), DFI (11; also this study), mLST (6), and AES (http: //www.aeslaboratoire.com/). For convenience, the preenrichment, enrichment, primary isolation, and presumptive identification steps for each of these methods…”

Development of a Novel Screening Method for the Isolation of “ Cronobacter ” spp. ( Enterobacter sakazakii )

“…Assessment of several of these media has shown that they provide comparable sensitivities and specificities (5,12,16). However, it has been established that some isolates of Cronobacter do not grow well in currently proposed enrichment broths, such as modified lauryl sulfate tryptose broth (mLST) and Enterobacteriaceae enrichment broth (6,9,16). Samples containing only such strains could give false-negative results; therefore, the enrichment procedure should be improved.…”

Gesundheitliches Gefährdungspotenzial von Enterobacter sakazakii (Cronobacter spp. nov.) in Säuglingsnahrung

“…Die Produktion eines gelben Pigments wurde zunächst zur Differenzierung verwendet [1,3,5], ist jedoch nicht bei allen Stämmen vorhanden bzw. temperatur-, licht-und medienabhängig ausgeprägt [3,6,7]. Viele E.-sakazakii-Stämme zeigen eine hohe Tenazität gegenüber Hitze [8,9], Trockenheit [10] und osmotischem Stress [11].…”