A RAPID POLYMERASE CHAIN REACTION ASSAY FOR ENTEROBACTER SAKAZAKII DETECTION IN INFANT MILK FORMULAS

Abstract

ABSTRACTEnterobacter sakazakii is a cause of invasive infection with high mortality rates in neonates. In the present study, a polymerase chain reaction (PCR) method for the rapid detection of E. sakazakii in infant milk formulas was developed, using a set of primers designed from a region of the 16S ribosomal RNA gene. The PCR detection limit was established using infant formulas artificially inoculated with serial dilutions of E. sakazakii. In addition, 50 commercial samples were analyzed by PCR and were compared with a conventional culture method. In order to determine statistical significance between the comparison, McNemar's test (a chi‐square [χ2]) was used. Experiments to determine the sensitivity of PCR indicated that it could detect as few as 105 cfu/mL of E. sakazakii in infant formulas directly and 100 cfu/mL after an 8‐h enrichment step. Of 50 samples analyzed, 39 were positive by PCR, whereas 31 were confirmed as E. sakazakii by the microbiological method. The chi‐square obtained was 6.125, which showed that the difference (P < 0.05) between the tests was significant.PRACTICAL APPLICATIONSAt the present, the Mexican food industry must confirm the absence of Enterobacter sakazakii in order to export ingredients used in infant milk formulas to the U.S.A. The E. sakazakii detection and isolation method adopted in Mexico is the U.S. Food and Drug Administration 2002 procedure that is time‐consuming and labor intensive. The polymerase chain reaction (PCR) assay developed in this study, which takes only 14 h, saves up to 5 days for analysis and eliminates the need for plating samples on selective or diagnostic agar as well as the biochemical confirmation steps needed in the reference method. Thus, this PCR method can speed up the detection of E. sakazakii and may be used to rapidly screen infant formula samples and their ingredients. In addition, it would greatly benefit the Mexican food industry by avoiding the rejection and seizure of tainted products in the export market, as well as by facilitating the monitoring of this pathogen in Mexican health facilities, thereby greatly reducing the incidence of E. sakazakii‐related infant diseases.