Evaluation of Fourier transform infrared (FT-IR) spectroscopy and chemometrics as a rapid approach for sub-typing Escherichia coli O157:H7 isolates.

The importance of tracking outbreaks of foodborne illness and the emergence of new virulent subtypes of foodborne pathogens have created the need for rapid and reliable sub-typing methods for Escherichia coli O157:H7. Fourier transform infrared (FT-IR) spectroscopy coupled with multivariate statistical analyses was used for sub-typing 30 strains of E. coli O157:H7 that had previously been typed by multilocus variable number tandem repeat analysis (MLVA) and pulsed field gel electrophoresis (PFGE). Hierarchical cluster analysis (HCA) and canonical variate analysis (CVA) of the FT-IR spectra resulted in the clustering of the same or similar MLVA types and separation of different MLVA types of E. coli O157:H7. The developed FT-IR method showed better discriminatory power than PFGE in sub-typing E. coli O157:H7. Results also indicated the spectral relatedness between different outbreak strains. However, the grouping of some strains was not in complete agreement with the clustering based on PFGE and MLVA. Additionally, HCA of the spectra differentiated the strains into 30 sub-clusters, indicating the high specificity and suitability of the method for strain level identification. Strains were also classified (97% correct) based on the type of Shiga toxin present using CVA of the spectra. This study demonstrated that FT-IR spectroscopy is suitable for rapid (≤16 h) and economical sub-typing of E. coli O157:H7 with comparable accuracy to MLVA typing. This is the first report of using an FT-IR-based method for sub-typing E. coli O157:H7.

Differentiation of live, dead and treated cells of Escherichia coli O157:H7 using FT-IR spectroscopy.

AIMS: To apply specific collection techniques and spectroscopy to differentiate between live and dead Escherichia coli O157:H7 cells, as well as cells subjected to various inactivation treatments, including heat, salt, UV, antibiotics and alcohol. METHODS AND RESULTS: Fourier transform-infrared (FT-IR) spectroscopy was used to analyse E. coli O157:H7 cells, after filtration or immunomagnetic collection. Partial least squares analysis of the spectra quantified live E. coli O157:H7 in the presence of dead cells with an R(2) > 0·996. Canonical variate analysis (CVA) not only differentiated between spectra of 100% dead and 100% live cells but also between 1% live : 99% dead and 100% dead. CVA using principal components also differentiated between the spectra of the differentially treated cells at a 95% confidence level, and Cooman plots showed clear separation between clusters of spectra of bacteria exposed to the different inactivation treatments. Mahalanobis distances (MD) corroborated the results of CVA. CONCLUSIONS: These results demonstrated the effectiveness of rapid cell collection and FT-IR spectroscopy techniques to differentiate between live and dead E. coli O157:H7 cells. SIGNIFICANCE AND IMPACT OF THE STUDY: This technique has potential applications for use with foods subjected to various inactivation treatments.

Detection and differentiation of live and heat-treated Salmonella enterica serovars inoculated onto chicken breast using Fourier transform infrared (FT-IR) spectroscopy.

AIMS: To evaluate Fourier transform infrared (FT-IR) techniques for detecting, quantifying, and differentiating viable and heat-treated cells of Salmonella enterica serovars from chicken breast. METHODS AND RESULTS: Salmonella enterica serovars were captured from inoculated chicken breast by filtration and immunomagnetic separation (IMS) prior to spectral collection using an FT-IR spectrometer and IR microscopy. The detection limits, based on amide II peak area (1589 to 1493 cm(-1) ), for the Filtration-FT-IR and IMS-FT-IR methods were 10(6) and 10(4) CFU g(-1) , respectively. The bacteria were detectable after 6 h of culture enrichment during a sensitivity experiment with lower initial inoculum of 10(1) CFU g(-1) . Canonical variate analysis differentiated experimental from control spectra at a level of 10(3) CFU g(-1) . Partial least squares models were established for the quantification of Salm. enterica from chicken breast using Filtration-FT-IR (R(2) ≥ 0·95, RMSEC ≤ 0·62) and IMS-FT-IR (R(2) ≥ 0·80, RMSEC ≤ 1·61) methods. Filtration-FT-IR was also used to detect and quantify live Salm. enterica in the presence of heat-treated cells with R(2) = 0·996, and this approach was comparable to the results of a commercial stain (BacLight™; R(2) = 0·998). Discriminant and canonical variate analyses of the spectra differentiated live and dead cells of different serovars of Salm. enterica. CONCLUSIONS: FT-IR analysis coupled with separation methods is useful for the rapid detection and differentiation of Salm. enterica separated from chicken. SIGNIFICANCE AND IMPACT OF THE STUDY: FT-IR-based methods are faster than traditional microbiological methods and can be used for the detection of live and dead bacteria from complex foods.

A bioluminescence-based assay for enumeration of lytic bacteriophage.

A bioluminescence-based assay for enumeration of lytic bacteriophage was developed. The assay consists of a bioluminescent Escherichia coli as the host bacterium, the lytic bacteriophage T4 and an automated luminometer measuring luminescence over time. The assay is based on the decrease in luminescence as the bioluminescent host cells are lysed by T4. The T4 concentration, bioluminescent E. coli concentration, phage suspension medium, and temperature (25 degrees C and 37 degrees C) were varied. There was a strong negative correlation between bioluminescence intensities and T4 phage concentrations at both room temperature (R(2)=0.993) and 37 degrees C (R(2)=0.970). Phage was detected more rapidly at 37 degrees C than at 25 degrees C. The detection limit was also lower when the assay was performed at 37 degrees C with a minimum detection level of 2.4 log CFU/ml compared to 3.4 log CFU/ml for 25 degrees C. The assay was used to determine thermal inactivation using T4 phages heated at 70 degrees C for 0 to 30 min, and phage concentrations were determined using the bioluminescence assay and a standard plaque assay. There was no significant difference between the two enumeration methods (P>0.01). This study suggests the bioluminescence-based assay can be used as an alternative for quantitatively monitoring phage infectivity, instead of conventional standard plaque assays.

Interaction of environmental moisture with powdered green tea formulations: effect on catechin chemical stability.

Green tea and tea catechins must be stable in finished products to deliver health benefits; however, they may be adversely affected by tea processing/storage conditions and the presence of other components. The objective of this study was to determine the effects of storage relative humidity (RH) and addition of other ingredients on catechin stability in simulated dry beverage mixtures. Samples of green tea powder alone and mixed with sucrose, citric acid, and/or ascorbic acid were prepared and stored in desiccators at 22 degrees C and 0-85% RH for up to 3 months. Epicatechin, epigallocatechin, epicatechin gallate, and epigallocatechin gallate were determined by high-performance liquid chromatography (HPLC). Formulation and the interaction of formulation and RH significantly promoted catechin degradation ( P < 0.0001). The chemical degradation of total and individual catechins in green tea powder formulations was significantly increased ( P < 0.0001) by exposure to increasing RH, and the degradation was exacerbated at > or = 58% RH by the presence of powdered citric acid and at > or = 75% RH by the presence of ascorbic acid. Catechins degraded the most in formulations containing both acids. Although catechin chemical stability was maintained at < or = 43% RH in all samples stored at 22 degrees C for 3 months, caking was observed in samples at these relative humidities. These results are the first to demonstrate that addition of other dry components to tea powders may affect catechin stability in finished dry blends and highlight the importance of considering the complex interplay between a multicomponent system and its environment for developing stable products.

Effect of heat on the distribution of fluorescently labeled plasminogen and plasminogen activators in bovine skim milk.

Differentially fluorescently labeled bovine plasminogen (PG-594) and human tissue- and urokinase-type plasminogen activators (tPA-647 and uPA-546) were added to bovine skim milk to track the effect of heat on the location and concentration of these plasmin system components following acid precipitation or ultracentrifugation. In unheated milk, the majority (71.7% to 89.0%) of the added PG and PAs associated with casein micelles or acid curd, and PG-594 in the serum fraction was partially due to associations with nonsedimentable caseins. Heat treatment (85 degrees C for 16 s) significantly (P < 0.05) affected distribution of PG-594, tPA-647, and uPA-546, resulting in reduced concentrations of PG and PAs in the serum fractions and reciprocal increases in their levels in the nonsedimentable casein fractions. Overall, almost all of the added PG and PAs (95.9% to 97.5%) became associated with caseins following heat treatment. This is the 1st study to successfully use fluorescent labeling to quantify effects of heat on the location of plasmin components in skim milk.

Detection of Escherichia coli O157:H7 and Salmonella typhimurium using filtration followed by Fourier-transform infrared spectroscopy.

Fourier-transform infrared spectroscopy has been successfully used as a nondestructive method for identifying, distinguishing, and classifying pathogens. In this study, a less time-consuming Fourier-transform infrared procedure was developed to identify Escherichia coli O157:H7 and Salmonella Typhimurium. Samples containing 10(9) CFU/ml were prepared in tryptic soy broth and then serially diluted (up to eight times) to obtain bacterial solutions of 10(9) to 10 CFU/ml. These dilutions were incubated at 37 degrees C for 6 h, samples were filtered through a Metricel filter hourly (for 0 to 6 h), and spectra were obtained using a ZnSe contact attenuated total reflectance accessory on a Continu mum infrared microscope. Midinfrared spectra (4,000 to 700 cm(-1)) of Salmonella Typhimurium and E. coli O157:H7 were generated, and peak areas in the region of 1,589 to 1,493 cm(-1) were used to detect the pathogens. Initially, detection limits were between 10(6) and 10(7) CFU/ml without preenrichment, and samples starting with 500 CFU/ml were detectable following incubation for 6 h, when counts reached at least 10(6) CFU/ ml. Compared with results of previously published studies in which Fourier-transform infrared spectroscopy was used to identify select pathogens, this method is more rapid and less expensive for practical large-scale sample analysis.

Differentiation of outer membrane proteins from Salmonellaenterica serotypes using Fourier transform infrared spectroscopy and chemometrics.

AIMS: To differentiate between outer membrane proteins (OMPs) from six Salmonellaenterica serotypes using a Fourier transform infrared (FTIR) spectroscopy method and chemometrics. METHODS AND RESULTS: The OMPs from Salmonella serotypes (Typhimurium, Enteritidis, Thomasville, Hadar, Seftenberg and Brandenburg) were isolated using a sarcosyl extraction method. OMP profiles on SDS-PAGE exhibited two or three bands between 48 and 54 kDa. Spectra of 10 microl of OMP preparations (5 mg ml(-1)) dried on a gold reflective slide were collected using 128 scans at 4 cm(-1) resolution and units of log (1/R) and analyzed using canonical variate analysis (CVA) and linear discriminant analysis (LDA). The CVA of Salmonella OMP spectra in the 1800-1500 cm(-1) region separated the serotypes and LDA provided a 100% correct classification. CONCLUSIONS: The use of a FTIR method combined with chemometrics provided better differentiation of Salmonella OMPs than the OMP pattern analysis by SDS-PAGE. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study to demonstrate that spectra of OMP extracts from Salmonella serotypes can be used for 100% correct classification of the serotypes studied.

Fluorescent labeling study of plasminogen concentration and location in simulated bovine milk systems.

A fluorescent labeling method was developed to study plasminogen (PG) concentration and location in simulated bovine milk. Activity and stability of PG labeled with Alexa Fluor 594 (PG-594) were comparable to those of native PG. The fluorescent signal of PG-594 exhibited pH, temperature, and storage stability, and remained stable throughout typical sample treatments (stirring, heating, and ultracentrifugation). These characteristics indicate broad applicability of the fluorescent labeling technique for milk protease characterization. In an example application, PG-594 was added to simulated milk samples to study effects of heat and beta-lactoglobulin (beta-LG) on the distribution of PG. Before heating, about one-third of the PG-594 remained soluble in the whey fraction (supernatant) whereas the rest became associated with the casein micelle. Addition of beta-LG to the system slightly shifted PG-594 distribution toward the whey fraction. Heat-induced PG-594 binding to micelles in whey-protein-free systems was evidenced by a decrease of PG-594 from 31 to 15% in the whey fraction accompanied by an increase of PG-594 from 69 to 85% in casein micelle fractions. When beta-LG was present during heating, more than 95% of PG-594 became associated with the micelle. A comparison with the distribution pattern of PG-derived activities revealed that heat-induced PG binding to micelles accompanies heat-induced PG inactivation in the micelle fraction. Incubation of the casein micelles with the reducing agent beta-mercaptoethanol revealed that disulfide bonds formed between PG and casein or between PG and casein-bound beta-LG are the mechanisms for heat-induced PG binding to casein micelles. Western blotting and zymography results correlated well with fluorescent labeling studies and activity studies, respectively. Theoretically important findings are: 1) when heated, serum PG is capable of covalently binding to micellar casein or complexing with beta-LG in whey and then coadhering to micelles, and 2) PG that associated with micellar casein through lysine binding sites before heating is capable of developing heat-induced disulfide bonds with casein. The overall results are PG covalently binding to micelles and inactivation thereafter. Our results suggest that, instead of thermal denaturation through irreversible unfolding, covalent bond formation between PG and other milk proteins is the mechanism of PG inhibition during thermal processing.

Use of Fourier transform infrared spectra of crude bacterial lipopolysaccharides and chemometrics for differentiation of Salmonella enterica serotypes.

AIMS: To evaluate Fourier transform infrared spectroscopy (FTIR) and chemometrics for differentiating intact cells and crude lipopolysaccharide (LPS) extracts from Salmonella serotypes. METHODS AND RESULTS: Intact cells and crude LPS extracts from six different Salmonella enterica serotypes (Typhimurium, Enteritidis, Thomasville, Brandenburg, Hadar and Seftenberg) were used. The crude Salmonella LPS extracts were visualized using deoxycholic acid-polyacrylamide gel electrophoresis (DOC-PAGE) and appeared heterogeneous on the gel with two exceptions: S. Enteritidis and S. Brandenburg, and S. Thomasville and S. Seftenberg. Canonical variate analysis (CVA) of spectra of crude LPS extracts provided 100% correct classification. CVA of spectra of intact cells was not useful for classifying the Salmonella serotypes, having only 47 and 50% correct classifications in the 1200-900 and 4000-700 cm(-1) regions respectively. These data were confirmed by greater Mahalanobis distances between crude LPS spectra than intact cell spectra. CONCLUSIONS: CVA of FTIR spectra of crude LPS extracts from Salmonella serotypes provided a 100% correct serotype classification. SIGNIFICANCE AND IMPACT OF THE STUDY: This study suggests that the FTIR analytical procedure provides chemical detail as well as a better separation of Salmonella serotypes using spectra of crude LPS extracts than analysis using DOC-PAGE.