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We investigated the feasibility of visible and near-infrared (VNIR) hyperspectral imaging for rapid presumptive-positive
screening of six representative non-O157 Shiga-toxin producing Escherichia coli (STEC) serogroups (O26, O45, O103,
O111, O121, and O145) on spread plates of mixed cultures. Although the traditional culture method is still the "gold
standard" for presumptive-positive pathogen screening, it is time-consuming, labor-intensive, not effective in testing
large amount of food samples, and cannot completely prevent unwanted background microflora from growing together
with target microorganisms on agar media. A previous study was performed using the data obtained from pure cultures
individually inoculated on spot and/or spread plates in order to develop multivariate classification models differentiating
each colony of the six non-O157 STEC serogroups and to optimize the models in terms of parameters. This study dealt
with the validation of the trained and optimized models with a test set of new independent samples obtained from
colonies on spread plates of mixed cultures. A new validation protocol appropriate to a hyperspectral imaging study for
mixed cultures was developed. One imaging experiment with colonies obtained from two serial dilutions was performed.
A total of six agar plates were prepared, where O45, O111 and O121 serogroups were inoculated into all six plates and
each of O45, O103 and O145 serogroups was added into the mixture of the three common bacterial cultures. The number
of colonies grown after 24-h incubation was 331 and the number of pixels associated with the grown colonies was
16,379. The best model found from this validation study was based on pre-processing with standard normal variate and
detrending (SNVD), first derivative, spectral smoothing, and k-nearest neighbor classification (kNN, k=3) of scores in
the principal component subspace spanned by 6 principal components. The independent testing results showed 95%
overall detection accuracy at pixel level and 97% at colony level. The developed model was proven to be still valid even
for the independent samples although the size of a test set was small and only one experiment was performed. This study
was an important first step in validating and updating multivariate classification models for rapid screening of ground
beef samples contaminated by non-O157 STEC pathogens using hyperspectral imaging.
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