Biotyping of Local Isolates Entrobacter sakazakii (Cronobacter spp.) and Genetic Variability among Several Isolates based on infB Gene.

Abstract

E. sakazakii was initially described as E. cloacae with yellow pigment, and later were classified based on their biochemical reactions, antibiotics susceprability and DNA-DNA hybridization. In 2007, E. sakazakii was reclassified into Cronobacter spp. consisting of C. sakazakii, C. malonaticus, C. muytjensii, C. dublinensis and C. turicensis based on f-AFLP and production of acid from dulcitol, production of indole, malonate utilization and production of acid from methyl-α-D-glucoside. All local isolates obtained from powder infant formula, weaning foods, starch, flours and spices in Indonesia have been identified as Cronobacter spp. and were reported to have close genetic relatedness based on its partial gene sequence encoding for 16S rRNA. However, they have been reported to have different thermal resistance (Seftiono 2012). This study aims to identify and classify local isolates of C. sakazakii based on their biochemical properties and genetic relatedness based on infB genes which known thermal kinetics. Identification of the 19 local isolates using RapID onE suggested that 9 isolates were identified as E. sakazakii, 9 others were E. cloacae and 1 was E. cancerogenus. When the results were compared to the identification using API 20E (Gitapratiwi 2011; Hamdani 2012), 8 of the 19 isolates were identified as E. sakazakii by both kits. Based on classification using 4 biochemical reaction, 15 isolates can be identified as Cronobacter spp. whereas 4 isolates can not be identified. By evaluating biochemical reaction in the RapID onE test kit, it can be conclude that reaction of pyrohydonyl-β-naphthylamide can be used as the additional testing to classify the previously unclassified local isolates into C. sakazakii. Ten of the 19 biochemical tests in RapID onE were selected for the biotyping of Cronobacter spp local isolates since they give different result for all isolates studied. Based on the ten biochemical profiles, the 19 local isolates of Cronobacter spp. can be classified into 16 biotypes. When the results of biotyping with rapid kit RapID onE was compared to result with the four biochemical reaction by Iversen, biotype I-VIII is C. sakazakii; while biotype IX, X, XV and XVI is C. malonaticus; biotype XIII and XIV is C. muytjensii; and biotype XI, XII and XVI is C. turicensis. v Genotypic characterization of infB gene among local isolates showed that all tested isolates where closely related. Based on the BLAST program, it was revealed that the contig sequence of the infB genes was as a translation initiation factor IF2. Based on the infB gene the local isolates can be classified into 2 groups. Isolat DES b7a, DES b10, DES c13, YR t3a and E6 belong to C. sakazakii, while isolate YR c3a is C. malonaticus and C. dublinensis.