Isolation and Molecular Identification of Cronobacter spp. from Non-dairy Foods in Indonesia

Introduction: Cronobacter spp. (formerly Enterobacter sakazakii) is a group of emerging pathogens that have been implicated as the causative agents of meningitis and necrotizing enterocolitis in certain groups of infants. In Indonesia several isolates have been obtained mostly from powder infant formula and weaning foods. 

Purpose: This research aimed to isolate Cronobacter spp from non-dairy foods, i.e., flour, starch and spice powder and evaluate the biochemical as well as molecular characteristics using rapid biochemical test and PCR, respectively. 

Methods: The biochemical assay was conducted using API 20E test while PCR was done on  the 16S rRNA gene using 2 primer pairs, i.e., 16 SUNI-L/Saka 2b (Segment 1) and ESA1/16 SUNI-R (Segment 2). Products of the DNA amplification were sequenced and then analyzed for their relatedness to reference isolates of C. sakazakii from GenBank using BLAST program. 

Results: In this study, 36 samples of flour, starch and spices were screened for the presence of Cronobacter spp.  Twelve typical colonies on DFI were obtained and the biochemical test suggested that 11 were Enterobacter sakazakii while one was identified as E. cloacae.  The PCR suggested that the 12 isolates including the one identified as E. cloacae were confirmed as C. sakazakii and C. muytjensii. Relationships between strains for segment 1 depicted in a dendrogram using the neighbor-joining method show that the twelve isolates can be differentiated into two groups.  Eleven isolates had 97-99% homology with C. sakazakii in the GenBank, while one had high homology with C. muytjensii ATCC 51329 (99%).

Significance: This study signifies the importance of non-dairy foods as the source of Cronobacter spp.