Improved Recovery And Rapid Identification Of Strains, Mixed Strains, Mixed Species, And Various Physiological States Of Foodborne Pathogens Using Infrared Spectroscopy

摘要

Challenges encountered in pathogen identification and detection include the genetic heterogeneity of strains within species of some foodborne pathogens, isolation of injured cells, mixed strains or mixed species contamination of foods, and differentiation between viable and dead cells. The first objective of this research was to evaluate an isolation medium that was based on time-delayed release (5 to 6 h) of selective agents in tablet format to a modified Listeria recovery enrichment broth (mLRB) medium for enhanced and rapid recovery of injured Listeria. The second objective involved the use of Fourier transform infrared (FT-IR) spectroscopy and chemometric analysis for the differentiation of: Listeria monocytogenes epidemic clones (ECs); viable versus heat-killed populations; different mixed strains and mixed species of Listeria; and different injury treatments and repair in Listeria populations. Nitrite- or acid-injured Listeria at approximately 10 CFU/ml were recovered in mLRB medium, and cell populations enumerated at various times (12 to 48 h) of incubation at 37oC. Analysis of variance revealed that acid-injured Listeria populations in mLRBS6 (mLRB plus the selective agents at 6 h) were significantly higher (P u3c 0.05) than those in mLRBS0 (mLRB plus the selective agents at 0 h) at 24 h; however, the differences in populations on these two media were not significant for nitrite-injured Listeria. Cell populations of four strains of Listeria recovered in mLRBTD (mLRB plus the time-delayed release tablets of the selective agents) were significantly higher than when those strains were enriched in the U.S. Food and Drug Administration (FDA), International Organization for Standardization (ISO), and U.S. Department of Agriculture (USDA) broths at 24 h. Comparison between artificially contaminated milk and meat samples with a four-strain cocktail of Listeria resulted in cell populations that were significantly higher (P u3c 0.05) on mLRBTD for contaminated meat than on mLRBTD for contaminated milk at 24 h. FT-IR spectroscopy in the mid-infrared region (4000 to 600 cm-1) and chemometrics was successfully applied to discriminate L. monocytogenes strains belonging to the same EC (ECII or ECIV) (100% accurate spectral classification), intact and heat-killed populations of each EC strain (100% accurate spectral classification), and spectral wavenumbers 1650 to 1390 cm-1 were used to differentiate heat-killed from intact populations. FT-IR spectroscopy and chemometrics in the wavelength region 1800 to 900 cm-1 could successfully discriminate different mixed strains of L. monocytogenes (98.15% accurate spectral classification) and different mixed species of L. monocytogenes and L. innocua (92.06% accurate spectral classification) from individual strains; Wavelength range 1800 to 900 cm-1 was successfully used to discriminate between intact, acid-injured, and heat-injured Listeria, with repaired cells from acid and heat treatments clustering closer to intact cells (93.33% of spectra accurately classified). Delayed-addition of selective agents to broth medium improves recovery of injured Listeria by allowing repair time, could minimize contamination through manual addition of selective agents, and saves analyst time; FT-IR spectroscopy is a highly discriminatory and reproducible technique that can be used for the differentiation of strains and various physiological states of Listeria.