Isolation and identification of Enterobacter sakazakii from fresh vegetables and fruits samples from Bogor Indonesia

Abstract

E.sakazakii is classified as a pathogenic bacterium that causes illness mostly to infants and immunocompromised individuals. This bacterium belongs to the group of Gram negative Enterobacteriaceae species, which was re-classified as Cronobacter spp. based on DNA hybridization and phenotyping. E. sakazakii has been widely implicated in a number of foodborne diseases, which are characterized by illnesses like meningitis and enteritis especially in pre-termed neonates and or with low birth weight infants. The bacterium has been isolated from various foods including fresh vegetables and fruits and also survives refrigeration temperatures. This research aim was to isolate E.sakazakii from a number of fresh vegetables and fruits samples available in local market surrounding Darmaga area, Bogor, Indonesia. The isolation followed FDA (2002) method modified by Iversen and Forsythe (2004). Rapid biochemical kit, API 20E, was used for biochemical characterization and identification. Molecular confirmation consisted of isolation of DNA and PCR to amplify the gene encoding for 16S rRNA using two different pair of primers, ESA-1/16SUNI-(R) and 16SUNI-L/Saka 2b. A total number of 19 samples tested positive to pre-enrichment and enrichment broth, in BPW (buffered peptone water) and EE (Enterobacteriaceae enrichment) broth, respectively. Isolation in VRBG (Violet Red Bile Glucose) agar yielded all positive results for all samples. However test on DFI (Druggan–Forsythe–Iversen) agar showed only four isolates producing typical blue/green colony, i.e. those isolated from cabbage, chaisin, apple “fuji” and apple “manalagi”. The biochemical test kit API 20E (Analytical Profile Index; bioMe´rieux) showed that none of the samples were positive for E.sakazakii. The PCR results showed that only one isolate i.e. from cabbage, successfully produced an amplicon of the 16S rRNA. This study suggests that isolation rate of E.sakazakii in vegetable is low at 5.2 %.