Development and application of a novel peptide nucleic acid probe for the specific detection of Cronobacter genomospecies (Enterobacter sakazakii) in powdered infant formula

Abstract

Here, we report a fluorescence in situ hybridization (FISH) method for rapid detection of Cronobacter strains in powdered infant formula (PIF) using a novel peptide nucleic acid (PNA) probe. Laboratory tests with several Enterobacteriaceae species showed that the specificity and sensitivity of the method were 100%. FISH using PNA could detect as few as 1 CFU per 10 g of Cronobacter in PIF after an 8-h enrichment step, even in a mixed population containing bacterial contaminants.

FIG. 1.

(A) Detection of C. sakazakii 274 on a glass slide using the SakPNA971 probe, a preenriched culture (10% PIF), and an initial concentration of 1 to 3 CFU/100 ml reconstituted PIF. (B) Visualization of the same microscopic field with the green channel (negative control), showing autofluorescence of infant formula proteins and the absence of fluorescent cells.

FIG. 2.

Detection of C. sakazakii ATCC 29544 in suspension with a mixed population using SakPNA971. (A) Reconstituted PIF with C. sakazakii and B. cereus. (B) Reconstituted PIF with C. sakazakii, B. cereus, P. aeruginosa ATCC 10145, and S. enterica serotype Enteritidis ATCC 13076. (C) Reconstituted PIF with C. sakazakii, S. enterica serotype Enteritidis ATCC 13076 (100-fold concentrated), and P. aeruginosa ATCC 10145 (100-fold concentrated). (D) Reconstituted PIF with C. sakazakii and S. enterica serotype Enteritidis ATCC 13076 (100-fold concentrated). (Panels I) Detection of C. sakazakii using the red fluorescent SakPNA971 probe. (Panels II) Counterstaining with DAPI (total population). (Panels III) Visualization of the same microscopic field with the green channel (negative control). The arrows indicate C. sakazakii cells that could easily be visualized in the DAPI channel with the red-labeled SakPNA971 probe. All images were obtained using the same exposure time.