mRNA biomarkers selection based on Partial Least Square algorithm in order to further predict Bacillus weihenstephanensis acid resistance.

In order to integrate omics data to quantitative microbiological risk assessment in foods, gene expressions may serve as bacterial behaviour biomarkers. In this study an integrative approach encompassing predictive modelling and mRNAs quantifications, was followed to select molecular biomarkers to further predict the acid resistance of Bacillus weihenstephanensis. A multivariate analysis was performed to correlate the acid bacterial resistance and the gene expression of vegetative cells with or without exposure to stressing conditions. This mathematical method provides the advantage to take gene expressions and their interactions into account. The use of the Partial Least Squares algorithm allowed the selection of nine genes as acid resistance biomarkers among thirty targeted genes. According to their involvement in the general acid stress response of Bacillus, these genes were assigned to three different biological modules namely, metabolic rearrangements, general stress response and oxidative stress response. The oxidative stress response appeared as the major activated biological module in B. weihenstephanensis cells submitted to acid stress conditions. Furthermore, as a firstly described model, the developed concept showed promising results to further be used to predict bacterial resistance using gene expression. Thus, this study underlines the possibility to integrate the bacterial physiology state, using omics biomarkers, into bacterial behaviour modelling and provide mechanistic understanding in acid bacterial resistance mechanisms.

Validation of the Thermo Scientific™ SureTect™ Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus PCR Assay for the Detection of Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus in Seafood Matrixes: AOAC Performance Tested MethodsSM 022301.

BACKGROUND: The Thermo Scientific™ SureTect™ Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus PCR Assay method is a real-time PCR method for the multiplex detection of Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus in seafood. OBJECTIVE: The Thermo Scientific SureTect Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus Assay was evaluated for AOAC Performance Tested MethodsSM certification. METHOD: Inclusivity/exclusivity, matrix, product consistency/stability, and robustness studies were conducted to assess the method's performance. For the matrix study, the method was validated using the Applied Biosystems™ QuantStudio™ 5 Real-Time PCR Food Safety Instrument and the Applied Biosystems™ 7500 Fast Real-Time PCR Food Safety Instrument against the U.S. Food and Drug Administration Bacteriological Analytical Manual, Chapter 9 (2004), Vibrio and ISO 21872-1:2017 Microbiology of the food chain-Horizontal method for the determination of Vibrio spp.-Part 1: Detection of potentially enteropathogenic Vibrio parahaemolyticus, Vibrio cholerae, and Vibrio vulnificus reference methods. RESULTS: Matrix studies showed equivalent or superior performance of the candidate method compared to the reference method and, overall, no difference between presumptive and confirmed results, except for one matrix due to high background flora. The inclusivity/exclusivity study correctly identified/excluded all strains analyzed. Robustness testing showed no statistically significant differences in assay performance under varied test conditions. Product consistency and stability studies demonstrated no statistically significant differences between assay lots with different expiration dates. CONCLUSIONS: The data presented show that the assay constitutes a rapid and reliable workflow for the detection of V. cholerae, V. parahaemolyticus, and V. vulnificus in seafood matrixes. HIGHLIGHTS: The SureTect PCR Assay method allows for fast, reliable detection of stipulated strains in seafood matrixes with results obtained in as little as 80 min post-enrichment.

An integrative approach to identify Bacillus weihenstephanensis resistance biomarkers using gene expression quantification throughout acid inactivation.

The aim of this study was to define an integrative approach to identify resistance biomarkers using gene expression quantification and mathematical modelling. Mid-exponentially growing cells were transferred into acid conditions (BHI, pH 4.6) to obtain inactivation kinetics, performed in triplicate. The inactivation curve was fitted with a mixed Weibull model. This model allowed to differentiate two subpopulations with various acid resistances among the initial population. In parallel, differential gene expression was quantified by RT-qPCR. While narL was down-regulated throughout acid inactivation, sigB and katA were up-regulated. sigB expression up-regulation peak was correlated to the less resistant subpopulation when katA up-regulation, was correlated to the more resistant subpopulation. Moreover, differences in population structure were highlighted between each replicate. The higher proportion of the more resistant subpopulation was linked to a higher katA gene expression. These results suggest that sigB and katA might be used as different types of biomarkers, for instance to track moderate and high acid-resistance, respectively. The use of this approach combining RT-qPCR and predictive modelling to track cellular biomarker variations appears as an interesting tool to take into account physiological cell responses into mathematical modelling, allowing an accurate prediction of microbial behaviour.

Polyphasic approach for quantitative analysis of obligately heterofermentative Lactobacillus species in cheese.

Obligately heterofermentative lactobacilli (OHL) present in cheese during ripening can influence the flavour and texture of the final product. In order to better evaluate, follow and control this population, there is a current need for easy-to-use tools. In this study, a culture-dependent quantitative method (ABEV medium) was set up for direct and selective enumeration of total OHL from cheese, and a culture-independent method based on specific real time PCR (qPCR) assays was developed to target Lactobacillus fermentum and Lactobacillus parabuchneri individual species. These tools were applied for OHL quantification in manufactured Emmental and Tomme cheeses. The ABEV medium was well adapted for specific enumeration and isolation of OHL species present in milk-derived samples, even in the presence of background microbiota. qPCR assays showed 100% specificity and could accurately quantify the targeted species in various types of cheese. Culture-dependent and -independent techniques evaluated in manufactured cheese samples generated similar bacterial counts. The behaviour of L. fermentum and L. parabuchneri was characterized from milk samples to the end of ripening. In addition, PCR-TTGE was used to confirm the presence of inoculated species and to globally analyze the composition of naturally present species. This polyphasic approach illustrates the complementarity of the different methods.

Reverse transcription quantitative PCR revealed persistency of thermophilic lactic acid bacteria metabolic activity until the end of the ripening of Emmental cheese.

For Emmental manufacture two kinds of adjunct culture are added: (i) thermophilic lactic acid bacteria (starters) such as Lactobacillus helveticus (LH), and Streptococcus thermophilus (ST) growing the first day of the manufacture and (ii) ripening culture. ST and LH have a key role in curd acidification and proteolysis at the beginning of the manufacture but are considered to be lyzed for a great part of them at the ripening step. The aim of this work was to assess the metabolic activity of these bacteria throughout manufacture and ripening. During Emmental cheesemaking, LH and ST were subjected to i) population quantification by numerations and by quantitative PCR (qPCR) ii) reverse transcription (RT) Temporal Temperature Gel Electrophoresis (TTGE) iii) transcript quantification by RT-qPCR targeting 16S rRNA, tuf and groL mRNAs to evaluate bacterial metabolic activity. During ripening, ST and LH numerations showed a 2.5 log(10) loss of culturability whereas qPCR on pelleted cells revealed only one log(10) of decrease for both of these species. 10(9) ST and 10(8) LH cells/g of cheese still remained. They contained a stable number of 16S transcript and at least 10(6) copies of mRNAs per 10(9) cells until the end of ripening. These results prove the unexpected persistency of thermophilic lactic acid bacteria starters (ST and LH) metabolic activity until the end of ripening and open new perspectives in term of their involvement in the quality of cheeses during ripening.

Validation of the Thermo Scientific SureTect™ Escherichia coli O157:H7 and STEC Screening PCR Assay and SureTect™ Escherichia coli STEC Identification PCR Assay for the Detection of Escherichia coli O157:H7 and the Escherichia coli STEC Serotypes (O26, O45, O103, O111, O121, O145) from Fresh Raw Spinach, Fresh Baby Leaves, Fresh Cut Tomatoes, Frozen Raw Beef, Raw Beef Trim, and Beef Carcass Sponges: AOAC Performance Tested MethodSM 012102.

BACKGROUND: The Thermo Scientific SureTect™ Escherichia coli O157:H7 and STEC Screening PCR Assay and SureTect Escherichia coli STEC Identification PCR Assay are real-time PCR kits for the rapid detection of E. coli O157:H7 and non-E. coli O157 Shiga toxin-producing E. coli (STEC) serotypes (O26, O45, O103, O111, O121, O145) from fresh raw spinach, fresh baby leaves, fresh cut tomatoes, frozen raw beef, raw beef trim, and beef carcass sponges. OBJECTIVE: Both assays were evaluated for AOAC®Performance Tested MethodsSM certification. METHODS: Detection and confirmation inclusivity/exclusivity, matrix, product consistency and stability, and robustness studies were conducted. In the matrix studies, the candidate method was validated against United States and international reference methods for STEC serotypes. RESULTS: Matrix studies showed no statistically significant differences between the candidate and reference method results when analyzed by probability of detection. For each inclusivity/exclusivity study, all inclusivity strains and no exclusivity strains were detected by either kit. Robustness testing demonstrated that the identification assay performed reliably despite method deviations; however, although not statistically significant, the screening assay performance was impacted. Product consistency and stability testing demonstrated no statistically significant differences between kit lots and storage time points. CONCLUSION: The data presented show that both assays constitute a rapid and reliable workflow for the detection and confirmation of E. coli O157:H7 and stipulated non-E. coli O157:H7 STEC serotypes from the tested matrixes. HIGHLIGHTS: Results are obtained in 80 min post-enrichment with both assays run simultaneously, allowing for the detection and confirmation of STEC within a single workflow.

Validation of the Thermo Scientific™ SureTect™Staphylococcus aureus PCR Assay for the Detection of Staphylococcus aureus in Dairy Matrixes: AOAC Performance Tested MethodSM 052101.

BACKGROUND: The Thermo Scientific™ SureTect™Staphylococcus aureus PCR Assay is a real-time PCR assay for the detection of Staphylococcus aureus in dairy samples. OBJECTIVE: The Thermo Scientific SureTect Staphylococcus aureus PCR Assay was evaluated for AOAC Performance Tested MethodSM certification. METHODS: Inclusivity/exclusivity, matrix studies, product consistency and stability, and robustness testing were conducted to assess the method's performance. For the matrix study, the method was validated on the Applied Biosystems™ QuantStudio™ 5 Real-Time PCR instrument and the Applied Biosystems 7500 Fast Real-Time PCR instrument against the ISO 6888-3:2003 Microbiology of food and animal feeding stuffs-Horizontal method for the enumeration of coagulase-positive staphylococci (Staphylococcus aureus and other species)-Part 3: Detection and MPN technique for low numbers, and the U.S. Food and Drug Administration (FDA) Bacteriological Analytical Manual (BAM) Ch. 12, Staphylococcus aureus, 2016, reference methods. RESULTS: Matrix studies showed no statistically significant differences between the candidate and reference methods or between presumptive and confirmed results. The inclusivity/exclusivity study correctly identified/excluded all strains analyzed. Robustness testing showed no statistically significant difference in assay performance after set method parameter deviations, and product consistency and stability studies demonstrated no statistically significant differences in performance between kit lots at different expiration points. CONCLUSION: The data presented show that the assay is a rapid and reliable workflow for the detection of S. aureus from dairy matrixes. HIGHLIGHTS: The PCR assay allows for fast, reliable detection of S. aureus in dairy matrixes with results obtained in as little as 80 min post enrichment.

Validation of the Thermo Scientific SureTect™ Campylobacter jejuni, C. coli, and C. lari PCR Kit for the Detection of Campylobacter jejuni, C. coli, and C. lari in Raw Poultry and Ready-to-Cook Poultry Products: AOAC Performance Tested MethodSM 012101.

BACKGROUND: The Thermo Scientific SureTect™ Campylobacter jejuni, C. coli, and C. lari PCR Kit is a real-time PCR assay for the detection and differentiation of C. jejuni, C. coli, and C. lari from raw poultry, ready-to-cook poultry products, and environmental samples. OBJECTIVE: The Thermo Scientific SureTect Campylobacter jejuni, C. coli, and C. lari PCR Kit was evaluated for AOAC®Performance Tested MethodsSM certification. METHODS: Inclusivity/exclusivity, matrix studies, product consistency and stability, and robustness testing were conducted to assess the method's performance. In the matrix studies, the method was validated against United States and international reference methods for Campylobacter detection. RESULTS: There were no statistically significant differences found in the matrix studies between the candidate and reference methods when analyzed by probability of detection. All 52 inclusivity strains and none of the 51 exclusivity strains tested were detected by the assay. Robustness testing demonstrated that the assay gave reliable performance with specific method deviations outside of the recommended parameters, and the real-time stability testing demonstrated that there were no statistically significant differences between kit lots, validating the stated shelf life of the kit. CONCLUSION: The data presented support the product claims that the Thermo Scientific SureTect Campylobacter jejuni, C. coli, and C. lari PCR assay is suitable for the detection and differentiation of C. jejuni, C. coli, and C. lari from raw poultry, ready-to-cook poultry products, and environmental samples. HIGHLIGHTS: Presumptive results can be obtained in as little as 23 h. Microaerophilic incubators are not required for enrichment.

Evaluation of the Thermo Scientific SureTect Salmonella Species PCR Assay in a Broad Range of Foods and Select Environmental Surfaces: Pre-Collaborative and Collaborative Study: First Action 2021.02.

BACKGROUND: The Thermo Scientific™ SureTect™ Salmonella species PCR Assay utilizes Solaris™ reagents for performing PCR for the rapid and specific detection of Salmonella species in a broad range of foods and select environmental surfaces. OBJECTIVE: The aims were to demonstrate the reproducibility of the Thermo Scientific SureTect Salmonella species PCR Assay in a collaborative study using a challenging matrix, cocoa powder, and to extend the scope of the method. METHOD: In the collaborative study, the candidate method was compared to the US Food and Drug Administration (FDA) Bacteriological Analytical Manual (BAM) Chapter 5 Salmonella reference method. The candidate method used two PCR thermocyclers, the Applied Biosystems™ QuantStudio™ 5 Real-Time PCR instrument (QS5) and the Applied Biosystems 7500 Fast Real-Time PCR instrument (7500 Fast). Fourteen participants from nine laboratories located within the United States and Europe were solicited for the collaborative study, with 12 participants submitting valid data. Three levels of contamination were evaluated for each matrix. Statistical analysis was conducted according to the probability of detection statistical model. In addition, 11 matrix studies were performed comparing the candidate method to the FDA/BAM Chapter 5 or US Department of Agriculture, Food Safety and Inspection Service, Microbiology Laboratory Guidebook 4.10 Isolation and Identification of Salmonella from Meat, Poultry, Pasteurized Egg, and Siluriformes (Fish) Products and Carcass and Environmental Sponges reference method. Nine of these matrices were also compared to the EN ISO 6579-1:2017/Amd.1:2020(E) Microbiology of the food chain-Horizontal method for the detection, enumeration and serotyping of Salmonella-Part 1: Detection of Salmonella spp.-AMENDMENT 1: Broader range of incubation temperatures, amendment to the status of Annex D, and correction of the composition of MSRV and SC reference method. RESULTS: In the collaborative study, the difference in laboratory results indicates equivalence between the candidate method and reference method for the matrix evaluated, and the method demonstrated acceptable interlaboratory reproducibility as determined in the collaborative evaluation. False-positive and false-negative rates were determined for the matrix and produced values of <2%. The two PCR thermocyclers (QS5, 7500 Fast) performed equivalently. There were no result differences between candidate method confirmations and reference method confirmations. In the pre-collaborative matrix extension, the results from the matrix studies showed a comparable performance between the candidate method and the tested reference methods. CONCLUSIONS: Based on the data generated, the method demonstrated acceptable interlaboratory reproducibility data and statistical analysis. HIGHLIGHTS: Due to the COVID-19 pandemic, some participants had to be trained remotely. Additionally, 375 g cocoa powder is known to be a challenging matrix for PCR methods. No unusual cross-contamination or false-positive/negative was reported, highlighting the ease of use, reproducibility, and robustness of the method.

Evaluation of the Thermo Scientific SureTectTM Listeria Species PCR Assay in a Broad Range of Foods and Selected Environmental Surfaces: Pre-Collaborative and Collaborative Study, First Action 2021.06.

BACKGROUND: The Thermo Scientific SureTect™ Listeria species PCR assay utilizes SolarisTM reagents for performing PCR for the rapid and specific detection of Listeria species in a broad range of foods and selected environmental surfaces. OBJECTIVE: To demonstrate reproducibility of the Thermo Scientific SureTect Listeria species PCR assay in a collaborative study using a challenging matrix, full-fat cottage cheese (25 g), to extend the scope of the method. METHODS: In the collaborative study, the candidate method was compared to the US Food and Drug Administration/Bacteriological Analytical Manual (FDA/BAM) Ch. 10 Listeria reference method. The candidate method used two PCR thermocyclers, the Applied Biosystems QuantStudio™ 5 Real-Time PCR instrument (QS5) and the Applied Biosystems 7500 Fast Real-Time PCR instrument (7500 Fast). The candidate method included its own confirmation procedure. Eighteen participants from 10 laboratories located within the United States and Europe were solicited for the collaborative study, with 12 participants submitting valid data. Statistical analysis was conducted according to the probability of detection (POD) statistical model. In addition, in order to extend the scope of the method, seven matrix studies were performed comparing the candidate method to the FDA/BAM reference method. One of these matrixes was also compared to the ISO 11290-1:2017 Microbiology of the food chain-Horizontal method for the detection and enumeration of Listeria monocytogenes and of Listeria spp.-Part 1: Detection method reference method. RESULTS: In the collaborative study, the difference in laboratory results indicates equivalence between the candidate method and reference method for the matrix evaluated and the method demonstrated acceptable inter-laboratory reproducibility as determined in the collaborative evaluation. The two PCR instruments used in the study performed equivalently. All presumptive positives were confirmed via the alternative confirmation procedure. In the pre-collaborative studies, the results showed comparable performances between the candidate method and the reference method for all matrixes tested. CONCLUSION: Based on the data generated, the method demonstrated acceptable inter-laboratory reproducibility data and statistical analysis. HIGHLIGHTS: Due to the COVID-19 pandemic, some participants had to be trained remotely. Additionally, 25 g full-fat cottage cheese is known to be a challenging matrix to test. No unusual cross-contamination, or false-positive/negative data was reported, highlighting the ease of use, reproducibility, and robustness of the candidate method.

Evaluation of the Thermo ScientificTM SureTectTMListeria monocytogenes PCR Assay in a Broad Range of Foods and Selected Environmental Surfaces: Pre-Collaborative and Collaborative Study, First Action 2021.05.

BACKGROUND: The Thermo Scientific™ SureTect™ Listeria monocytogenes PCR Assay uses Solaris reagents for performing PCR for the rapid and specific detection of Listeria monocytogenes in a broad range of foods and selected environmental surfaces. OBJECTIVE: To demonstrate reproducibility of the SureTect Listeria monocytogenes PCR Assay in a collaborative study using a challenging matrix, full-fat cottage cheese (25 g). To extend the scope of the method. METHOD: In the collaborative study, the candidate method was compared to the United States Food and Drug Administration/Bacteriological Analytical Manual (FDA/BAM) Chapter 10 Listeria reference method. The candidate method used two PCR thermocyclers, the Applied Biosystems™ QuantStudio™ 5 Real-Time PCR instrument (QS5) and the Applied Biosystems 7500 Fast Real-Time PCR instrument (7500 Fast). Eighteen participants from 10 laboratories located within the United States and Europe were solicited for the collaborative study, with 12 participants submitting valid data. Three levels of contamination were evaluated for each matrix. Statistical analysis was conducted according to the probability of detection (POD) statistical model. In addition, to extend the scope, six matrix studies were performed comparing the candidate method to the FDA/BAM reference method. One of these matrixes was also compared to the ISO 11290-1:2017 Microbiology of the Food Chain-Horizontal Method for the Detection and Enumeration of Listeria monocytogenes and of Listeria spp.-Part 1: Detection Method Reference Method. RESULTS: In the collaborative study, the difference in laboratory results indicates equivalence between the candidate method and reference method for the matrix evaluated, and the method demonstrated acceptable inter-laboratory reproducibility as determined in the collaborative evaluation. The two PCR instruments used in the study performed equivalently. All presumptive positives were confirmed via the alternative confirmation procedure. In the pre-collaborative studies, the results showed comparable performances between the candidate method and the reference method for all matrixes tested. CONCLUSIONS: Based on the data generated, the method demonstrated acceptable inter-laboratory reproducibility data and statistical analysis. HIGHLIGHTS: Due to the COVID-19 pandemic, some participants had to be trained remotely. Additionally, 25 g full-fat cottage cheese is known to be a challenging matrix to test. No unusual cross-contamination or false positive/negative data were reported, highlighting the ease of use, reproducibility, and robustness of the method.

Recent advances in quantitative PCR (qPCR) applications in food microbiology.

Molecular methods are being increasingly applied to detect, quantify and study microbial populations in food or during food processes. Among these methods, PCR-based techniques have been the subject of considerable focus and ISO guidelines have been established for the detection of food-borne pathogens. More particularly, real-time quantitative PCR (qPCR) is considered as a method of choice for the detection and quantification of microorganisms. One of its major advantages is to be faster than conventional culture-based methods. It is also highly sensitive, specific and enables simultaneous detection of different microorganisms. Application of reverse-transcription-qPCR (RT-qPCR) to study population dynamics and activities through quantification of gene expression in food, by contrast with the use of qPCR, is just beginning. Provided that appropriate controls are included in the analyses, qPCR and RT-qPCR appear to be highly accurate and reliable for quantification of genes and gene expression. This review addresses some important technical aspects to be considered when using these techniques. Recent applications of qPCR and RT-qPCR in food microbiology are given. Some interesting applications such as risk analysis or studying the influence of industrial processes on gene expression and microbial activity are reported.

Validation of the Thermo Scientific™ SARS-CoV-2 RT-PCR Detection Workflow for the Detection of SARS-CoV-2 from Stainless-Steel Environmental Surface Swabs: AOAC Performance Tested MethodSM 012103.

BACKGROUND: The Thermo Scientific™ SARS-CoV-2 reverse transcription-polymerase chain reaction (RT-PCR) Detection Workflow, packaged with Applied Biosystems™ TaqMan™ 2019-nCoV Assay Kit v1 targets three different SARS-CoV-2 genomic regions in a single RT-PCR reaction. OBJECTIVE: To validate the Thermo Scientific SARS-CoV-2 RT-PCR Workflow, for the detection of SARS-CoV-2 virus on stainless-steel surfaces as part of the AOAC Performance Tested MethodSM Emergency Response Validation program. METHOD: The Applied Biosystems TaqMan 2019-nCoV Assay Kit v1, as part of the Thermo Scientific SARS-CoV-2 RT-PCR Workflow, was evaluated for specificity using in silico analysis of 15 764 SARS-CoV-2 sequences and 65 exclusivity organisms. The Thermo Scientific SARS-CoV-2 RT-PCR Workflow was evaluated in an unpaired study for one environmental surface (stainless steel) and compared to the U.S. Centers for Disease Control and Prevention 2019-Novel Coronavirus RT-PCR Diagnostic Panel, Instructions for Use (Revision 4, Effective 6/12/2020). RESULTS: In silico analysis showed that, of the 15 756 target SARS-CoV-2 genomes analyzed, 99% of the strains/isolates are perfectly matched to at least two of the three assays, and more than 90% have 100% homology to all three assays (ORF1ab, N-gene, S-gene) in the SARS-CoV-2 Kit. None of the 65 non-target strain genomes analyzed showed matching sequences. In the matrix study, the Thermo Scientific SARS-CoV-2 workflow showed comparable detection to the centers of disease control and prevention (CDC) method. CONCLUSIONS: The Thermo Scientific SARS-CoV-2 RT-PCR Workflow is an effective procedure for detection of RNA from SARS-CoV-2 virus from stainless steel. HIGHLIGHTS: The workflow provides equivalent performance results with the two tested RNA extraction platforms and the two tested RT-PCR instruments.

Validation of an alternative method for counting Enterobacteriaceae in foods based on accuracy profile.

Several years ago, microbiologists developed a harmonized standard for the validation of alternative methods against the reference method, namely International Organization for Standardization (ISO) 16140:2003. Since then a new and better statistical approach known as the accuracy profile concept has been proposed for interpretation of data collected during a validation study. This approach defines acceptability limits that are used to verify that an alternative analytical method can produce results acceptable for a defined analytical objective. This paper demonstrates how the accuracy profile approach can be efficiently applied to any data set collected according to ISO guidelines and how to interpret the final calculations to decide if a method is valid or not. The procedure was applied to data obtained in the validation of a commercial kit for the enumeration of Enterobacteriaceae in foods. A comparison of several statistical outputs derived from application of the accuracy profile demonstrated that this simple numerical method can be applied despite the non-normal distribution of bacterial colony counts.

Confirmation and Identification of Salmonella spp., Cronobacter spp., and Other Gram-Negative Organisms by the Bruker MALDI Biotyper Method: Collaborative Study Method Extension to Include Campylobacter Species, Revised First Action 2017.09.

Background: The Bruker MALDI Biotyper® method utilizes matrix-assisted laser desorption/ionization time-of-flight MS for the rapid and accurate identification and confirmation of Gram-negative bacteria from select media types. The alternative method was evaluated in a method extension study of AOAC INTERNATIONAL First Action Official MethodSM 2017.09 using nonselective and selective agars to identify Cronobacter spp., Salmonella spp., Campylobacter spp., and select Gram-negative bacteria. Results obtained by the Bruker MALDI Biotyper were compared to the traditional biochemical methods as prescribed in the appropriate reference methods. Methods: Two collaborative studies were organized, one in the United States focusing on Cronobacter spp. and other Gram-negative bacteria and one in Europe focusing on Salmonella spp. and other Gram-negative bacteria. Fourteen collaborators from seven laboratories located within the United States participated in the first collaborative study for Cronobacter spp. Fifteen collaborators from 15 service laboratories located within Europe participated in the second collaborative study for Salmonella spp. For each target organism (either Salmonella spp. or Cronobacter spp.), a total of 24 blind-coded isolates were evaluated. In each set of 24 organisms, there were 16 inclusivity organisms (Cronobacter spp. or Salmonella spp.) and 8 exclusivity organisms (non-Cronobacter spp. and non-Salmonella spp. closely related Gram-negative organisms). For the Campylobacter spp. method extension, 17 collaborators from eight laboratories located within the United States (seven laboratories) and Canada (one laboratory) participated in the collaborative study. A total of 24 blind-coded isolates were evaluated. In each set of 24 organisms, there were 16 inclusivity organisms (Campylobacter spp.) and 8 exclusivity organisms (non-Campylobacter spp. closely related Gram-negative organisms). Results: After testing was completed, the total percentage of correct identifications from each agar type for each strain was determined at a percentage of 100.0% to the genus level for the Cronobacter study and a percentage of 100.0% to the genus level for the Salmonella study. For the Campylobacter method extension, a correct identification and confirmation rate of 100.0% was obtained for the Campylobacter organisms at the species level. For non-Cronobacter, non-Salmonella, and non-Campylobacter organisms, 100.0% were correctly identified. Conclusions: The results indicated that the alternative method produced equivalent results when compared to the confirmatory procedures specified by each reference method. Highlights: The method extension can be modified to include the identification and confirmation of Campylobacter jejuni, Campylobacter coli, and Campylobacter lari.

Confirmation and Identification of Salmonella spp., Cronobacter spp., and Other Gram-Negative Organisms by the Bruker MALDI Biotyper Method: Collaborative Study, First Action 2017.09.

The Bruker MALDI Biotyper® method utilizes matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS for the rapid and accurate identification and confirmation of Gram-negative bacteria from select media types. The alternative method was evaluated using nonselective and selective agars to identify Cronobacter spp., Salmonella spp., and select Gram-negative bacteria. Results obtained by the Bruker MALDI Biotyper were compared to the traditional biochemical methods as prescribed in the appropriate reference methods. Two collaborative studies were organized, one in the United States focusing on Cronobacter spp. and other Gram-negative bacteria, and one in Europe focusing on Salmonella spp. and other Gram-negative bacteria. Fourteen collaborators from seven laboratories located within the United States participated in the first collaborative study for Cronobacter spp. Fifteen collaborators from 15 service laboratories located within Europe participated in the second collaborative study for Salmonella spp. For each target organism (either Salmonella spp. or Cronobacter spp.), a total of 24 blind-coded isolates were evaluated. In each set of 24 organisms, there were 16 inclusivity organisms (Cronobacter spp. or Salmonella spp.) and 8 exclusivity organisms (closely related non-Cronobacter spp. and non-Salmonella spp. Gram-negative organisms). After testing was completed, the total percentage of correct identifications from each agar type for each strain was determined at a percentage of 100.0% to the genus level for the Cronobacter study and a percentage of 100.0% to the genus level for the Salmonella study. For both non-Cronobacter and non-Salmonella organisms, a percentage of 100.0% was correctly identified. The results indicated that the alternative method produced equivalent results when compared to the confirmatory procedures specified by each reference method.

Confirmation and Identification of Listeria monocytogenes, Listeria spp. and Other Gram-Positive Organisms by the Bruker MALDI Biotyper Method: Collaborative Study, First Action 2017.10.

The Bruker MALDI Biotyper® method utilizes matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS for the rapid and accurate confirmation and identification of Gram-positive bacteria from select media types. This alternative method was evaluated using nonselective and selective agar plates to identify and confirm Listeria monocytogenes, Listeria species, and select Gram-positive bacteria. Results obtained by the Bruker MALDI Biotyper were compared with the traditional biochemical methods as prescribed in the appropriate reference method standards. Sixteen collaborators from 16 different laboratories located within the European Union participated in the collaborative study. A total of 36 blind-coded isolates were evaluated by each collaborator. In each set of 36 organisms, there were 16 L. monocytogenes strains, 12 non-monocytogenes Listeria species strains, and 8 additional Gram-positive exclusivity strains. After testing was completed, the total percentage of correct identifications (to both genus and species level) and confirmation from each agar type for each strain was determined at a percentage of 99.9% to the genus level and 98.8% to the species level. The results indicated that the alternative method produced equivalent results when compared with the confirmatory procedures specified by each reference method.

Easy DNA extraction method and optimisation of PCR-Temporal Temperature Gel Electrophoresis to identify the predominant high and low GC-content bacteria from dairy products.

Molecular fingerprinting of bacterial ecosystems has recently increased in food microbiology. The aim of this work was to develop a rapid and easy method to extract DNA from various cheeses, and to optimize the separation of low and high GC-content bacteria by PCR-Temporal Temperature Gel Electrophoresis (PCR-TTGE). Seventy six strains belonging to 50 of the most frequently encountered bacterial species in dairy products were used to construct a database. Specific PCR-TTGE ladders containing 17 species forming a regular scale were created. Amplicons of these species were sequenced and the GC-content plotted against the migration distance: the correlation coefficients obtained were r(2)=0.97 and r(2)=0.99, respectively for high and low GC-contents. The extraction method developed did not use any harmful solvent such as phenol/chloroform. The concentrations of DNA extracted from hard cooked and pressed cheeses, quantified by picogreen molecular probes, were between 0.7 and 6 microg/g for core samples and 8 to 30 microg/g for rind samples. Experimental as well as commercial dairy products were analysed using the developed method and the reproducibility of the profiles was 89%. The method appears to be particularly efficient in the characterization of the ecosystem of cheese rinds.

Yeast ecology in French cider and black olive natural fermentations.

In this study, rDNA ITS restriction analysis was used to identify yeasts from two naturally fermented products: French ciders and black olives. In cider musts and bottled ciders, the PCR-RFLP method generated 15 different ITS/RFLP profiles for a total of 208 isolates. The predominant yeasts corresponded to Saccharomyces bayanus, Saccharomyces cerevisiae, Lachancea cidri and Dekkera anomala. Three identified species: Candida sake, Candida tropicalis and Kluyveromyces marxianus had never been described before in ciders. For the black olive fermentation, the method allowed for identification of 11 profiles for a total of 137 isolates. A sequential apparition of yeasts was observed with Pichia anomala, Candida boidinii and Debaryomyces etchellsii being the predominant species. Four isolates did not correspond to any known species based on the sequencing of the D1/D2 region of the 26S rRNA gene. By using the rDNA ITS method, valuable information on yeast population biodiversity and dynamics in the naturally fermented food products studied was obtained.

Modelling the influence of single acid and mixture on bacterial growth.

Depending on environmental factors, the prediction of bacterial growth is made difficult by the complexity of foodstuff. Although the influence of temperature, pH, and water activity are usually taken into account, models have to be completed with the influence of acid mixture. Nine strains of Listeria spp., four Salmonella spp., one Staphylococcus aureus, one Escherichia coli, and Listeria innocua ATCC 33090 were used for this study to extend model proposed by [Le Marc, Y., Huchet, V., Bourgeois, C., Guyonnet, J., Mafart, P., Thuault, D., 2002. Modelling the growth kinetics of Listeria as a function of temperature, pH and organic acid concentration. International Journal of Food Microbiology 73, 219-237]. Derived from data of [Houtsma, P.C., Kusters, B.J., De Wit, J.C., Rombouts, F.M., Zwietering, M.H., 1994. Modelling growth rates of Listeria innocua as a function of lactate concentration. International Journal of Food Microbiology 24, 113-123] and our own data, the extended model described accurately different effects of addition of acid salts in the medium (decrease of water activity and pH, variation of undissociated weak acid form, and variation of synergetic effect between environmental factors). This previous model was implemented to describe the observed variability of behaviour of the different studied strains. alpha reflected the general behaviour of species (sensitiveness to low or high undissociated acid concentration), and MIC(U) reflected the various resistances of strains. From this simple model, a new model was built for describing the effects of concentrations of several mixed acids on bacterial growth rates. Simulations of growth were carried out from three acids mixtures by inputting parameter estimates previously obtained. Despite a very variable effect of investigated acids on growth, the new model yielded fair predictions.