The FISH procedure using PNA was then adapted to detect Cronobacter
directly in a commercially available PIF, based on the previously work of Mohan Nair and Venkitanarayanan (31
). Initially, we performed an experiment to assess the experimental detection limit of FISH with PNA. For this, Cronobacter muytjensii
ATCC 51329 was resuspended in reconstituted PIF (10% [wt/wt]; NAN 1 Premium; Nestle) at concentrations ranging from 1 × 108
to 1 × 102
CFU/ml. The PIF was reconstituted in water at ca. 60°C, a temperature commonly used for rehydration (13
). One-milliliter aliquots of each dilution were concentrated by centrifugation as described above, and hybridization was performed in suspension or on glass slides. Microscopic visualization showed that this procedure could detect the pathogen at a concentration of 1 × 106
The theoretical detection limit was also calculated based on the assumption that for trustworthy analysis each microscopy field should contain at least five cells. This calculation considered the microscopic field area (0.0158 mm2), the slide well area (200.96 mm2), and the dilution used during the procedure. The value obtained, 2 × 105 cells/ml, was lower than the value determined by laboratory testing. The difference could be explained by the fact that the theoretical value is between the 10-fold dilutions analyzed, 1 × 102 to 1 × 108 CFU/ml. Moreover, during laboratory testing some cells might be lost in the centrifugation steps, decreasing the real cellular concentration.
After determination of the detection limit, we used the FISH procedure with PNA to detect Cronobacter
in PIF samples with different levels of contamination. Serial 10-fold dilutions of Cronobacter
sp. strain ATCC 51329 were prepared using reconstituted formula to obtain final concentrations of Cronobacter
ranging from 1 × 10−4
to 1 × 107
CFU/ml (corresponding to 1 × 10−2
to 1 × 109
CFU/10 g). Given the short lag time and the growth rate of Cronobacter
in PIF, it was expected that a single cell could produce a concentration of more than 1 × 107
CFU/ml in an 8-h enrichment period (19
). For this reason, after 8 h of enrichment at 37°C, 1-ml samples were taken and diluted 1:10, and hybridization was performed in suspension or on glass slides, as described above. A noncontaminated culture was prepared in parallel and exposed to the same conditions. This experiment was performed three times and was repeated with two other Cronobacter
strains (ATCC 29544 and 274). The bacterial concentration in each enriched culture was determined by conventional counting and by FISH with PNA (see Table S1 in the supplemental material). Quantification by FISH with PNA was obtained by epifluorescence microscopy by counting a total of 15 fields. Approximately 57 to 87% of the PNA-FISH-labeled cells were detected by culture methods, and the final concentrations were always higher than the theoretical or experimental detection limit previously determined.
The procedure was able to detect Cronobacter
in PIF samples with an initial concentration of 1 × 10−2
CFU/ml (and even with an initial concentration of 1 × 10−3
CFU/ml for 4 of 27 replicates) after an 8-h preenrichment step. The previously reported levels of Cronobacter
in PIF samples are very low. For instance, Muytjens et al. examined 141 different powdered formulas and isolated Cronobacter
at levels ranging from 0.36 to 66 CFU per 100 g (33
). In other work, Simmons et al. isolated 8 Cronobacter
CFU per 100 g from PIF associated with an outbreak of clinical illness in Tennessee (39
). Based on our findings, our assay is capable of detecting less than 1 CFU per 10 g of PIF, which compares well with previous reports.
Autofluorescence of infant formula proteins remained detectable, but, as the sample was diluted at least 1:10 before hybridization, autofluorescence did not interfere with bacterial detection (Fig. ). Moreover, when hybridization was performed in suspension, autofluorescence was almost undetectable.
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 (more ...)
The experiments with PIF were repeated with the same strains using freeze-dried Cronobacter
cells, as in the experiments performed previously by Iversen and Forsythe (18
). With this approach we intended to investigate if the physiological state of the cells and contamination in powdered or reconstituted infant formula could affect the outcome of FISH with PNA or the concentration determined. The results showed that there was no difference between the two physiological conditions tested.