Rapid identification of Salmonella serovars Enteritidis and Typhimurium using whole cell matrix assisted laser desorption ionization - Time of flight mass spectrometry (MALDI-TOF MS) coupled with multivariate analysis and artificial intelligence.

Salmonella is a common food-borne pathogen with Enteritidis and Typhimurium being among the most important serovars causing numerous outbreaks. A rapid method was investigated to identify these serovars using whole-cell MALDI-TOF MS coupled with multivariate analysis and artificial intelligence and 113 Salmonella strains, including 38 Enteritidis (SE), 38 Typhimurium (ST) and 37 strains from 32 other Salmonella serovars (SG). Datasets of ions (presence/absence) with high discriminative power were created using newly developed criteria and subject to multivariate analyses and eight artificial intelligence (AI) tools. Principal Component Analysis based on 55 or 88 selected ions separated SE, ST and SG without overlap on the first three principal components. Datasets were partitioned using five partitioning methods with 70% of samples for AI model training and 30% for validation. Of the eight AI models evaluated, high performance (HP) SVM and HP Neural were the top performers, identified three serovar groups 97% correctly on average (range 82%-100%) according to the validation results. Selection of serovar specific ions facilitated differentiation of serotypes using unsupervised model PCA and improved the accuracy of classification using AI significantly (p < 0.01). MALDI-TOF MS incorporated with advanced data processing and classification tools is a promising method to allow rapid identification of Salmonella serovars of concern in routine diagnostic laboratories.

Interpretation and Implications of Lognormal Linear Regression Used for Bacterial Enumeration.

BACKGROUND: Bacterial enumeration data are typically log transformed to realize a more normal distribution and stabilize the variance. Unfortunately, statistical results from log transformed data are often misinterpreted as data within the arithmetic domain. OBJECTIVE: To explore the implication of slope and intercept from an unweighted linear regression and compare it to the results of the regression of log transformed data. METHOD: Mathematical formulae inferencing explained using real dataset. RESULTS: For y=Ax+B+ε, where y is the recovery (CFU/g) and x is the target concentration (CFU/g) with error ε homogeneous across x. When B=0, slope A estimates percent recovery R. In the regression of log transformed data, logy=αlogx+ß+εz (equivalent to equation y=Axα·ω), it is the intercept ß=logyx=logA that estimates the percent recovery in logarithm when slope α=1, which means that R doesn't vary over x. Error term ω is multiplicative to x, while εz or log(ω) is additive to log(x). Whether the data should be transformed or not is not a choice, but a decision based on the distribution of the data. Significant difference was not found between the five models (the linear regression of log transformed data, three generalized linear models and a nonlinear model) regarding their predicted percent recovery when applied to our data. An acceptable regression model should result in approximately the best normal distribution of residuals. CONCLUSIONS: Statistical procedures making use of log transformed data should be studied separately and documented as such, not collectively reported and interpreted with results studied in arithmetic domain. HIGHLIGHTS: The way to interpret statistical results developed from arithmetic domain does not apply to that of the log transformed data.

Log Transformation and the Effect on Estimation, Implication, and Interpretation of Mean and Measurement Uncertainty in Microbial Enumeration.

Background: Estimation of measurement uncertainty (MU) has been extensively addressed in documents from standard authorities. In microbiology, bacterial counts are log transformed to get a more normal distribution. Unfortunately, the difference between using original and log-transformed data appears to not have been investigated even in publications focusing on MU estimation. Method: Statistical formulae inferencing and estimation of MU using real bacterial enumeration datasets. Results: Both mean and SD calculated from original data carry the same scale and unit as the original data. However, the mean of log-transformed data becomes a geometric mean in log, and the SD becomes the logarithm of a ratio. Furthermore, calculation of RSD obtained by dividing the SD by the mean is meaningless and misleading for log-transformed data. The ratio, the antilog of the SD of log-transformed data, copes with multiplicative and divisive relationships to geometric mean (without log), instead of the arithmetic mean. The ratio can be converted to an analog ratio, which is similar or almost identical to the RSD of the untransformed data, especially when the within-subject variation is small. When MU is estimated from multiple samples with different measurands, the calculated RSD of original data is independent of the mean and can be pooled; however, for log-transformed data, the SD can be combined to estimate the common uncertainty. Conclusions: Calculation and use of RSD of log-transformed data are meaningless and misleading. Procedures outlining the estimation and interpretation of MU from log-transformed data require re-evaluation.

Evaluation of a Multiplex PCR for Detection of the Top Seven Shiga Toxin-Producing Escherichia coli Serogroups in Ready-to-Eat Meats, Fruits, and Vegetables.

Background: Ready-to-eat (RTE) meats, fruits, and vegetables contaminated by Shiga toxin producing Escherichia coli (STEC) raise serious concerns because they are often consumed directly without further processing. Objective: To evaluate a multiplex PCR for the detection of STEC across food categories. Methods: Samples (25 g) from seven RTE meat and nine fruit and vegetable matrices were inoculated with each of seven STEC (O157:H7, O26, O121, O145, O45, O103, O111) strains targeting 10 CFU/25 g, enriched in 225 mL of modified tryptone soya broth (mTSB), and tested by a multiplex real-time PCR for stx and eae genes, following U.S. Department of Agriculture (USDA) Food Safety and Inspection Service (FSIS) Microbiology Laboratory Guidebook (MLG) 5B, which was originally validated for meat products and environmental sponge. Results: The mTSB was successful at enriching for STEC in RTE meat, fruit, and vegetable matrices, except for sprouts; however, mEHEC resulted in successful enrichment of target organisms in mung bean sprouts. Suppression of eae results by stx in PCR was observed in six fruit and vegetable matrices. Conversely, suppression of stx gene by eae was not observed. PCR solely targeting eae is recommended if a fruit or vegetable sample tested positive for stx and negative for eae. Despite the significant effect from food matrix, strain, and experimental batch, the cycle threshold of PCR was <30 in inoculated samples, and mostly 30-42 and up in uninoculated samples. Conclusions: The multiplex PCR can be adopted for detection of all seven regulated STEC in RTE meat, fruit and vegetable matrices after validation with cut off value selected and justified based on real samples.

51st North American Chemical Residue Workshop.

Manuscripts collected in this 51st North American Chemical Residue Workshop (NACRW) Symposium issue of the Journal of Agricultural and Food Chemistry (JAFC) were originally presented at the 51st NACRW meeting. The 2014 NACRW JAFC symposium collects 14 publications representing the broad range of topics in chemical analyses presented at the 2014 meeting. These include the analysis of chemical residues and contaminants in food, environment, feed, botanical, and bee samples as well as the application of quality control/quality assurance protocols in routine and method development.

Stability of aflatoxins in solution.

The stability of aflatoxins B1, B2, G1, and G2 was studied in solutions containing different concentrations of water, acetonitrile, and/or methanol, and in autosampler vials treated with nitric acid or silanized. When stored at room temperature (20 degrees C) for 24 h, aflatoxins G1 and G2 were stable only in solutions containing 100% organic solvent, whereas aflatoxins B1 and B2 were stable in solutions of methanol-water and acetonitrile-water at greater than 60 and 40% organic content, respectively. At 5 degrees C, aflatoxins G1 and G2 showed a significant decrease in concentration only when kept in less than 20% aqueous organic solvent. Significant loss of aflatoxins was realized in standard, commercially available amber type I borosilicate autosampler vials, but chemical etching of the vials with nitric acid or with silanization prevented aflatoxin degradation. These results indicate that aflatoxins are unstable in aqueous solutions and that this instability can be counteracted by the presence of at least 20% organic solvent and keeping the solutions at 5 degrees C or by the use of treated vials.

Issues in mass spectrometry between bench chemists and regulatory laboratory managers: summary of the roundtable on mass spectrometry held at the 123rd AOAC International Annual Meeting.

At the 123rd AOAC International Annual Meeting in Philadelphia, PA, 45 residue chemists gathered for a roundtable discussion of mass spectrometry (MS) used for regulatory chemical residues analysis. The session was conceived to address current technical and communication issues about MS between "bench chemists and their bosses". The topics covered a range of practical, routine, and recurring issues on capabilities and limitations of MS techniques, and suggestions on how chemists may better communicate their MS results with customers. The customers in this sense include laboratory managers, quality assurance officers, laboratory clients, regulatory officials, policy-makers, lawyers, and others who have interest in the data. The stated goals devised by the roundtable panelists were to provide independent advice, describe limitations, give practical tips, help set realistic expectations, and answer questions from the attendees. The panelists divided the topics into three main themes: practical aspects in routine analysis using MS, choice of MS technique depending on the purpose for analysis, and qualitative identification and confirmation concepts. This report was written to summarize and expand upon the discussion, frame the current issues, and provide advice on handling common situations in MS analysis and reporting of results. Topics included LODs, data quality objectives, quantification and reporting results, matrix effects, calibration, terminology, differences in performance across MS platforms, proficiency testing, qualitative analysis, and laboratory accreditation. Conclusions are presented as a set of questions for structuring a dialog between bench chemists and laboratory managers.

Multiclass, multiresidue drug analysis, including aminoglycosides, in animal tissue using liquid chromatography coupled to tandem mass spectrometry.

A multiresidue, multiclass semiquantitative screening analysis of 39 drug residues covering 8 drug classes, including aminoglycosides in veal muscle, based on a single multiresidue extraction routine and using high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS), is presented. Sample preparation involves extraction of a 5 g diced tissue sample with 10 mL of acetonitrile/ water (86:14), incubated at 60 degrees C for 1 h, and then cooled for 10 min in ice. Formic acid is added to the suspension, then mixed, and centrifuged. The supernatant is retained, and the pellet is extracted with 10 mL of water for aminoglycosides and again centrifuged. Approximately 9.5 mL of each of the supernatants from both extracts is combined and diluted with water to 25 mL. The final solution is then defatted with 20 mL of hexane prior to analysis. Liquid chromatography for the aminoglycosides is carried out with ZIC-HILIC and for the remainder of the compounds with an Atlantis dC18 minicolumn. LC-ESI-MS/MS in positive and negative ionization modes (three injections total) is carried out, and two ion transitions per analyte are monitored. The method provides semiquantitative analysis to identify incurred positive drug classes in a rapid and cost-effective manner. Of particular interest is the detection of numerous compounds in the low nanograms per gram concentration range, which are not typically detected using receptor-based screening methods. All identified drugs were confirmed using internationally recognized regulatory methods, with no apparent false positives.

Ultratrace analysis of nine macrolides, including tulathromycin A (Draxxin), in edible animal tissues with minicolumn liquid chromatography tandem mass spectrometry.

The analysis of nine macrolides is presented, including tulathromycin A (Draxxin), in beef, poultry, and pork muscle with a simple multiresidue extraction and analysis method using high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry. The sample preparation method involves extraction with acetonitrile and defatting with hexane followed by dilution of the extracts for analysis. Separation of the nine macrolides was performed using an Atlantis dC 18, 3 mum, 3.9 mm x 20 mm minicolumn (guard column). Detection was carried out with two multiple reaction monitoring experiments per macrolide. The method detection limits (MDLs) were based on three times standard deviation of eight repeat spikes at 3.0 ng/g of a mix of the nine macrolides in the various tissues. The MDLs and retention times for the macrolides were as follows: lincomycin, 0.19 ng/g (t R = 5.00 min); tulathromycin, 0.46 ng/g (t R = 5.63 min); spiramycin, 0.21 ng/g (t R = 6.06 min); pirlimycin, 0.10 ng/g (t R = 6.04 min); clindamycin, 0.16 ng/g (t R = 6.20 min); tilmicosin, 0.29 ng/g (t R = 6.38 min); erythromycin, 0.19 ng/g (t R = 6.62 min); tylosin, 0.10 ng/g (t R = 6.72 min); and josamycin, 0.09 ng/g (t R = 6.98 min). Precision at 25 ng/g (n = 4) ranged from 2.3 to 9.4% for the compounds from beef muscle. Of interest is the detection of incurred residues of tulathromycin A in edible calf tissue at 0.10-7 mug/g, which is presented here for the first time.

Identification and quantification of eight flavones in root and shoot tissues of the medicinal plant huang-qin (Scutellaria baicalensis Georgi) using high-performance liquid chromatography with diode array and mass spectrometric detection.

A method of analysis of eight flavones using high-performance liquid chromatography (HPLC)-diode array detection (DAD)-mass spectrometry (MS) in root and aerial tissues of the medicinal plant Scutellaria baicalensis was developed. The identity of the analytes was confirmed using retention time, UV-vis and mass spectral comparisons to commercial standards. Both UV-vis and mass spectral patterns were characterized for glycosylated flavones. Two additional flavone glycosides were tentatively identified as chrysin-7-glucuronide and wogonoside, but not quantified. Greenhouse and in vitro-grown tissues were analyzed with flavone concentrations ranges of 0.14-150 and 0.030-1.7 microg/mg for greenhouse root and shoot tissue, respectively, and 0.0068-6.4 and 0.082-1.5 microg/mg for in vitro-grown roots and shoots, respectively.

System for the generation of standard gas mixtures of volatile and semi-volatile organic compounds for calibrations of solid-phase microextraction and other sampling devices.

Standard gases are used for quality control and quality assurance, development of analysis methods and novel air sampling devices. The use of solid-phase microextraction (SPME) and other novel technologies for research in the area of air sampling and analysis requires systems/devices for reliable standard gas generation and sampling. In this paper we describe a new gas standard generating system for volatile organic compounds (VOCs) and semi-VOCs that was designed, built, and tested to facilitate fundamental and applications research with SPME. The system provided for the generation of a wide range of VOC/semi-VOC concentrations and mixing various standard gases, estimation of detection limits, testing the effects of sampling time, air temperature and relative humidity, testing the effects of air velocity and ozone on sampling/extractions. The system can be also used for calibrations of analytical instrumentation, quality control and quality assurance checks, and cross-validations of SPME with/and other sampling techniques.