The power, potential, benefits, and challenges of implementing high-throughput sequencing in food safety systems.

The development and application of modern sequencing technologies have led to many new improvements in food safety and public health. With unprecedented resolution and big data, high-throughput sequencing (HTS) has enabled food safety specialists to sequence marker genes, whole genomes, and transcriptomes of microorganisms almost in real-time. These data reveal not only the identity of a pathogen or an organism of interest in the food supply but its virulence potential and functional characteristics. HTS of amplicons, allow better characterization of the microbial communities associated with food and the environment. New and powerful bioinformatics tools, algorithms, and machine learning allow for development of new models to predict and tackle important events such as foodborne disease outbreaks. Despite its potential, the integration of HTS into current food safety systems is far from complete. Government agencies have embraced this new technology, and use it for disease diagnostics, food safety inspections, and outbreak investigations. However, adoption and application of HTS by the food industry have been comparatively slow, sporadic, and fragmented. Incorporation of HTS by food manufacturers in their food safety programs could reinforce the design and verification of effectiveness of control measures by providing greater insight into the characteristics, origin, relatedness, and evolution of microorganisms in our foods and environment. Here, we discuss this new technology, its power, and potential. A brief history of implementation by public health agencies is presented, as are the benefits and challenges for the food industry, and its future in the context of food safety.

Escherichia coli O157:H7 Cluster Associated With Deer Harvested at a Single Wildlife Hunting Area, Oregon, 2017.

The Oregon Health Authority routinely investigates clusters of reportable enteric diseases identified by whole-genome sequencing. While investigating 2 cases of Escherichia coli O157:H7 in 2019, in which both patients were exposed to the same home-processed "jerky" and clinical isolates matched within 2 single nucleotide polymorphisms (SNPs), we discovered, by searching the National Library of Medicine's National Center for Biotechnology Information website, 3 other cases of E coli O157:H7 from 3 Oregon counties-Tillamook, Umatilla, and Douglas-whose clinical isolates were within 9 SNPs of the 2 initial matched cases. We analyzed interview data for 3 case patients and followed up with additional hypothesis-generating questions. Onset of illness for the Tillamook, Umatilla, and Douglas county cases were October 7, 2017, October 27, 2017, and April 30, 2018, respectively. The median age of the 5 case patients was 16 years. Parents of 2 of the 5 case patients, each from a different county, had harvested deer approximately 20 miles from each other in the same Douglas County wildlife hunting unit in late September 2017. The case from Umatilla County was lost to follow-up. Although it is well documented that deer are a viable and substantial reservoir of E coli O157:H7, to our knowledge, this is the first time that venison from a common wildlife hunting unit was found to be associated with a cluster of illnesses. This finding suggests a geographic nidus for E coli O157:H7. We recommend routinely asking about wildlife hunting units when developing exposure hypotheses involving potential venison-associated clusters.

Acquisition of the L452R Mutation in the ACE2-Binding Interface of Spike Protein Triggers Recent Massive Expansion of SARS-CoV-2 Variants.

We report that there is a recent global expansion of numerous independent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with mutation L452R in the receptor-binding domain (RBD) of the spike protein. The massive emergence of L452R variants was first linked to lineage B.1.427/B.1.429 (clade 21C) that has been spreading in California since November and December 2020, originally named CAL.20C and currently variant of interest epsilon. By PCR amplification and Sanger sequencing of a 541-base fragment coding for amino acids 414 to 583 of the RBD from a collection of clinical specimens, we identified a separate L452R variant that also recently emerged in California but derives from the lineage B.1.232, clade 20A (named CAL.20A). Notably, CAL.20A caused an infection in gorillas in the San Diego Zoo, reported in January 2021. Unlike the epsilon variant that carries two additional mutations in the N-terminal domain of spike protein, L452R is the only mutation found in the spike proteins of CAL.20A. Based on genome-wide phylogenetic analysis, emergence of both viral variants was specifically triggered by acquisition of L452R, suggesting a strong positive selection for this mutation. Global analysis revealed that L452R is nearly omnipresent in a dozen independently emerged lineages, including the most recent variants of concern/interest delta, kappa, epsilon and iota, with the lambda variant carrying L452Q. L452 is in immediate proximity to the angiotensin-converting enzyme 2 (ACE2) interaction interface of RBD. It was reported that the L452R mutation is associated with immune escape and could result in a stronger cell attachment of the virus, with both factors likely increasing viral transmissibility, infectivity, and pathogenicity.

Validation of the AtlasTMCampylobacter Detection Assay: AOAC Performance Tested MethodSM 032101.

BACKGROUND: Campylobacter spp. are a major causal agent for diarrheal illness in humans. Detection of Campylobacter spp. in food is critical to reduce foodborne illness, and to provide safe foods. OBJECTIVE: The aim was to evaluate the Atlas Campylobacter Detection Assay for AOAC Performance Tested MethodsSM certification for detecting C. jejuni, C. coli, and C. lari in foods after 12 h enrichment. METHOD: The Atlas Campylobacter Detection Assay was compared to the ISO 10272-1:2017 reference culture method for chicken carcass rinse, turkey carcass sponge, raw ground poultry, raw ground pork, and ready-to-eat (RTE) meats. Inclusivity, exclusivity, product consistency, product stability, and robustness studies were also performed. An independent laboratory evaluated the performance of the Atlas Campylobacter Detection Assay method on chicken carcass rinse. RESULTS: No significant differences were observed between the Atlas Campylobacter Detection Assay and the reference ISO method in spiked food matrixes. The Atlas Campylobacter Detection Assay detected all 50 inclusive organisms and none of the 30 exclusive organisms. Product consistency and stability studies showed no statistical differences between lots or over the term of the shelf-life using accelerated method study. Finally, the robustness study showed no statistical difference between different sample volumes, enrichment times, and storage time after sample transfer. CONCLUSIONS: The results of this study indicate that the Atlas Campylobacter Detection Assay is comparable to ISO 10272-1:2017 for detecting Campylobacter in chicken carcass rinse, turkey carcass sponge, raw ground poultry, raw ground pork, and RTE meats. HIGHLIGHTS: The Atlas Campylobacter Detection Assay is a rapid, accurate molecular method able to detect C. jejuni, C. coli, and C. lari in in chicken carcass rinse, turkey carcass sponge, raw ground poultry, raw ground pork, and RTE meats within 12-18 h.

Acquisition of the L452R mutation in the ACE2-binding interface of Spike protein triggers recent massive expansion of SARS-Cov-2 variants.

The recent rise in mutational variants of SARS-CoV-2, especially with changes in the Spike protein, is of significant concern due to the potential ability for these mutations to increase viral infectivity, virulence and/or ability to escape protective antibodies. Here, we investigated genetic variations in a 414-583 amino acid region of the Spike protein, partially encompassing the ACE2 receptor-binding domain (RBD), across a subset of 570 nasopharyngeal samples isolated between April 2020 and February 2021, from Washington, California, Arizona, Colorado, Minnesota and Illinois. We found that samples isolated since November have an increased number of amino acid mutations in the region, with L452R being the dominant mutation. This mutation is associated with a recently discovered CAL.20C viral variant from clade 20C, lineage B.1.429, that since November-December 2020 is associated with multiple outbreaks and is undergoing massive expansion across California. In some samples, however, we found a distinct L452R-carrying variant of the virus that, upon detailed analysis of the GISAID database genomes, is also circulating primarily in California, but emerged even more recently. The newly identified variant derives from the clade 20A (lineage B.1.232) and is named CAL.20A. We also found that the SARS-CoV-2 strain that caused the only recorded case of infection in an ape - gorillas in the San Diego Zoo, reported in January 2021 - is CAL.20A. In contrast to CAL.20C that carries two additional to L452R mutations in the Spike protein, L452R is the only mutation found in CAL.20A. According to the phylogenetic analysis, however, emergence of CAL.20C was also specifically triggered by acquisition of the L452R mutation. Further analysis of GISAID-deposited genomes revealed that several independent L452R-carrying lineages have recently emerged across the globe, with over 90% of the isolates reported between December 2020 - February 2021. Taken together, these results indicate that the L452R mutation alone is of significant adaptive value to SARS-CoV-2 and, apparently, the positive selection for this mutation became particularly strong only recently, possibly reflecting viral adaptation to the containment measures or increasing population immunity. While the functional impact of L452R has not yet been extensively evaluated, leucine-452 is positioned in the receptor-binding motif of RBD, in the interface of direct contact with the ACE2 receptor. Its replacement with arginine is predicted to result in both a much stronger binding to the receptor and escape from neutralizing antibodies. If true, this in turn might lead to significantly increased infectivity of the L452R variants, warranting their close surveillance and in-depth functional studies.

Prevalence of SARS-CoV-2 Contamination on Food Plant Surfaces as Determined by Environmental Monitoring.

ABSTRACT: The SARS-CoV-2 pandemic has presented new challenges to food manufacturers. During the early phase of the pandemic, several large outbreaks of coronavirus disease 2019 (COVID-19) occurred in food manufacturing plants resulting in deaths and economic loss, with approximately 15% of personnel diagnosed as asymptomatic for COVID-19. Spread by asymptomatic and presymptomatic individuals has been implicated in large outbreaks of COVID-19. In March 2020, we assisted in implementation of environmental monitoring programs for SARS-CoV-2 in zones 3 and 4 of 116 food production facilities. All participating facilities had already implemented measures to prevent symptomatic personnel from coming to work. During the study period, from 17 March to 3 September 2020, 1.23% of the 22,643 environmental samples tested positive for SARS-CoV-2, suggesting that infected individuals were actively shedding virus. Virus contamination was commonly found on frequently touched surfaces such as doorknobs, handles, table surfaces, and sanitizer dispensers. Most processing plants managed to control their environmental contamination when they became aware of the positive findings. Comparisons of positive test results for plant personnel and environmental surfaces in one plant revealed a close correlation. Our work illustrates that environmental monitoring for SARS-CoV-2 can be used as a surrogate for identifying the presence of asymptomatic and presymptomatic personnel in workplaces and may aid in controlling infection spread.

Microbiologique Microfilm™ EBEc - a Rapid Method for the Simultaneous Enumeration of Enterobacteriaceae and Escherichia coli.

BACKGROUND: The Enterobacteriaceae and generic Escherichia coli are routinely enumerated in foods as part of product release criteria, or in the case of swabs, for environmental monitoring. OBJECTIVE: Microbiologique Microfilm™ EBEc is intended to provide a rapid and easy-to-use method for simultaneous enumeration of Enterobacteriaceae and E. coli on foods and environmental surfaces. Methods: This study evaluated the performance of Microfilm™ EBEc against ISO methods (ISO 21528-2:2017 for Enterobacteriaceae and ISO 16649-2: 2001 for E. coli) in 20 food matrixes and two environmental surfaces. Inclusivity, exclusivity, lot-to-lot reproducibility, ruggedness and stability studies were also performed on Microfilm™ EBEc. RESULTS: No significant differences and high correlation coefficients (R2) were observed between the Microfilm™ EBEc and the corresponding ISO methods in spiked food matrixes and environmental samples. Inclusivity studies showed expected results for all the E. coli and Enterobacteriaceae strains tested. In terms of exclusivity testing, all the strains tested failed to grow on Microfilm™ EBEc. A lot-to-lot study showed no significant differences in mean difference (log10) counts among the three lots of the Microfilm™ EBEc. Ruggedness studies showed no significant differences in mean difference (log10) counts at varying incubation temperatures and times. Stability studies on the Microfilm™ EBEc showed that it is stable at 2-25°C for 12 months, and at 45°C for 6 weeks. CONCLUSIONS: The results of this study indicate that the Microfilm™ EBEc is equivalent to the corresponding ISO methods for enumeration of Enterobacteriaceae and E. coli. Highlights: Microfilm™ EBEc offers a convenient and relatively fast test method for simultaneous enumeration of Enterobacteriaceae and E. coli in 24 h and has an advantage over the corresponding ISO methods that require two assays on the same sample for enumeration of Enterobacteriaceae and E. coli Gram-negative indicator groups.

Case Report: Allergic Reactivity to Mahaleb (Prunus mahaleb) Spice in a Subject With Almond and Other Tree Nut Allergies.

BACKGROUND: Mahaleb is an aromatic spice prepared from the fruit stone of the St. Lucie Cherry that is used as a flavoring agent in traditional Turkish and Middle Eastern baking. Immunodiagnostic kits for almond, which are based on polyclonal almond-specific IgG antibodies, have been shown to demonstrate considerable cross-reactivity with mahaleb as was incidentally discovered during a cluster of allergen-related food recalls in 2015. OBJECTIVE: Though acute allergy to almond is somewhat common, allergies to mahaleb have not been previously documented. However, based on antigenic similarity observed with almond-specific IgG, it is predicted that mahaleb nut proteins would exhibit some level of cross-reactivity with almond-specific IgE and may therefore potentiate acute allergic symptoms in individuals with food allergy to almond.Case Presentation: Herein, we report on a 40-year old Caucasian female with longitudinal history of multiple tree nut allergies including allergy to almond, presenting with moderate pruritus and oropharyngeal swelling shortly following ingestion of mahaleb seed kernels. METHODS AND RESULTS: Skin-prick testing using extracts compounded from pistachio, almond, and mahaleb revealed positive wheals measuring 8, 3, and 7 mm respectfully. Indirect enzyme-linked immunosorbent assay (ELISA) using plate-bound antigens prepared from pistachio, almond, and mahaleb revealed IgG positive responses to all three targets. ELISA and Western blot analysis performed using goat anti-almond polyclonal IgG demonstrated significant cross-reactivity between almond and mahaleb, but not to pistachio. CONCLUSION: This is the first documented case of acute allergy to mahaleb, co-occurring in the context of plural tree nut allergies, providing novel evidence that mahaleb may pose a risk to nut-allergic individuals and indicating a need for awareness of spice contamination with nut and mahaleb residues.

Unification of Abyssicoccus albus Jiang et al. 2016 and Auricoccus indicus Prakash et al. 2017 and the status of the genus Auricoccus Prakash et al. 2017.

Based on the high phylogenetic relatedness of Auricoccus indicus Prakash et al. 2017 and Abyssicoccus albus Jiang et al. 2016, it is proposed to unite them with retaining the latter name as having nomenclatural priority. As the result of the species unification, the genus Auricoccus name is proposed to consider as illegitimate in the boundaries determined by Rule 51a of the International Code of Nomenclature of Prokaryotes.

Application of Sample6 DETECT™ HT/L Kit for the Detection of Listeria in Mixed Leafy Greens.

BACKGROUND: Early and accurate detection of Listeria in foods is vital. Current methods require 24 h enrichment for detection. OBJECTIVE: This study aimed to demonstrate enhanced early detection of Listeria in mixed leafy greens using Sample6 DETECT™ HT/L, a phage-based detection system. METHODS: A method comparison between the reference method (U.S. Food and Drug Administration Bacteriological Analytical Manual Chapter 10) for the detection of Listeria spp. and the Sample6 DETECT HT/L using a new proprietary R2 Medium was performed in mixed leafy greens. RESULTS: Using the R2 Medium enrichment with Sample6 DETECT HT/L, detection of L. innocua was possible at 12 h (with a centrifugation step), and L. monocytogenes was detected by 18 h, with equivalent performance as the 24 h enrichments using the reference method detection. The Sample6 DETECT HT/L gave an equivalent performance for L. innocua at 15 h as the reference method at 24 h. Detection was accomplished by the addition of reagents in the kit, following the package insert, and reading results in a detection chamber using a 96-well plate reader that detects a fluorescent signal. CONCLUSIONS: Results indicate the new R2 Medium and Sample6 DETECT HT/L allowed for earlier detection of Listeria spp. in mixed leafy greens. HIGHLIGHTS: Sample6 DETECT HT/L with the new R2 Medium allowed the early detection of Listeria spp. and may be applied in other food matrices and environmental samples.

Extension of xMAP Food Allergen Detection Assay To Include Sesame.

An estimated 0.1 to 0.2% of the North American population is allergic to sesame, and deaths due to anaphylactic shock have been reported. Detecting and quantifying sesame in various food samples is critical to safeguard the allergic population by ensuring accurate ingredient labeling. Because of the modular nature of the xMAP Food Allergen Detection Assay (FADA), it was possible through method extension to add sesame as a validated additional analyte. Because raw and toasted sesame are both commonly used and the two display significantly different antigenicity, three antibodies, one monoclonal and two polyclonal, were conjugated to bead sets to ensure reliable detection. The modified xMAP FADA successfully detected sesame incurred or spiked in baked muffins, spice mix, canola oil, and in both raw and toasted sesame oils with limit of quantitation values ≤ 1.3 ppm of sesame. Canola oil, sesame oil, toasted sesame oil, and olive oil inhibited sesame detection, as did the detection of sesame incurred in foods containing oil (e.g., hummus). Despite this inhibition, the xMAP FADA was still able to reliably detect sesame at levels throughout the dynamic range of the assay (22 to 750 ng of protein per mL) in all the foods examined. Further, the high signal-to-noise ratio of the lowest calibration standard and preliminary studies conjugating the antibodies at higher concentrations indicate an ability to increase the sensitivity of the assay should the need arise.

Reclassification of Burkholderia insecticola as Caballeronia insecticola comb. nov. and reliability of conserved signature indels as molecular synapomorphies.

In the last 4 years, most of the species previously classified as members of the genus Burkholderia have been transferred to the novel genera Paraburkholderia, Caballeronia, Robbsia, Mycetohabitans and Trinickia. However, there have been objections to splitting the genus Burkholderiasensu lato, and based on this taxonomic opinion, strain RPE64T, which has the 16S rRNA gene sequence identical to that of Caballeronia peredens LMG 29314T, has recently been proposed as the type strain of Burkholderia insecticolasp. nov. The arguments against the split were analysed in this study and found to be not convincing enough to revise the taxonomic positions of members of the novel genera. Therefore, based on the results of phylogenetic analyses, including comparisons of 16S rRNA gene sequences and those of concatenated proteins, as well as on the fact that strain RPE64T had all molecular signatures included as Caballeronia-specific markers in the genus description, we propose to reclassify B. insecticola as Caballeronia insecticola comb. nov. The results of this study also showed that 'Burkholderia novacaledonica' and 'Burkholderia ultramafica' should be transferred to the genera Caballeronia and Paraburkholderia, respectively.

Occurrence and Levels of Salmonella, Enterohemorrhagic Escherichia coli, and Listeria in Raw Wheat.

HIGHLIGHTS: Prevalence of Salmonella and E. coli in raw wheat emphasizes the need to cook wheat products. 3,891 grain samples were tested for E. coli and Salmonella; 1,285 were tested for Listeria. Of wheat berries sampled, 0.44% were positive for E. coli and 1.23% were positive for Salmonella. Salmonella diversity was high, indicating various animal sources that are difficult to prevent. Cooking wheat products is the best preventative measure against foodborne illness from wheat.

Application of an Environmental Phage-Based Assay (Sample6 Detect HT/L) for the Detection of Listeria spp. in Ice Cream.

Background: Dairy products are common sources of Listeria outbreaks, and early detection of the pathogen is critical to prevent outbreaks of illnesses and financial losses for dairy producers. Objective: This study aimed to evaluate Sample6 Detect HT/L for effective detection of Listeria monocytogenes and L. innocua in ice cream. Methods: Performance of the Sample6 DETECT HT/L was compared with U.S. Food and Drug Administration (FDA) Bacteriological Analytical Manual (BAM) Chapter 10 method for detection of Listeria spp. in ice cream using an unpaired study design. Results: R2-enriched samples tested with Sample6 Detect HT/L performed as well as the reference method at all time points tested from 15 to 24 h. R2 is a proprietary blend for use with the test kit that helps with early detection. All the dPODC values (Sample6 Detect HT/L presumptive and confirmed results) equaled zero, indicating 100% concordance between the methods. Both Sample6 Detect HT/L and FDA BAM results showed low dPODC values, with confidence intervals indicating no significant differences between Sample6 Detect HT/L and reference method results. Conclusions: Sample6 Detect HT/L is suitable to detect Listeria spp. in ice cream, even with a 12 h enrichment. Sample6 Detect HT/L demonstrated equivalent detection of L. monocytogenes and L. innocua from R2-enriched samples as expected with 15 and 18 h enrichment when compared with the 24 h FDA BAM method for L. monocytogenes. Highlights: These results indicate that Sample6 Detect HT/L, primarily developed for environmental samples, can be used to detect Listeria spp. in ice cream with less incubation time, resulting in faster detection.

Simultaneous Detection of Multiple Wine-Spoilage Organisms Using a PCR-Based DNA Dipstick Assay.

Background: The presence of microbial contaminants such as Brettanomyces in wine can lead to undesirable wine. Therefore, monitoring for the presence of these spoilage organisms is critical for winemakers to ensure the quality of their end product. Objective: To address this problem, Molecular Epidemiology, Inc. (MEI, Seattle, WA) has developed a wine-spoilage organism detection kit consisting of a multiplex PCR DNA dipstick that simultaneously detects these organisms. Methods: Wine samples obtained from local wineries that tested negative by routine microbiological culture were spiked with the target microorganisms, while samples that were designated as spoiled by the wineries were used as-is without spiking for assessing the performance characteristics of the DNA dipstick assay. Microbial enumeration was performed following standard microbiological plating methods. Samples spiked with low cell numbers (<5 cells per 100 mL) were enriched using wine enrichment media (WSE; optional component of the kit) prior to analysis using the DNA dipstick assay. Suitability of WSE medium to support the growth of wine-spoilage microorganisms was compared with standard microbiological media. Results: Testing of 92 diverse bacterial and yeast strains commonly found in winery and food operations and 50 various strains of spoilage organisms isolated from wineries indicated that the dipstick assay can exclusively detect the target wine-spoilage microorganisms. All target spoilage organisms in samples containing low cell numbers (<5 cells per 100 mL) were detected by dipstick assay 48 h postenrichment in WSE, except for a few strains of Brettanomyces bruxellensis that required longer incubation times. Conclusions: The wine-spoilage organism detection kit has a detection limit of 10 cells/mL. Highlights: The kit can be used at different stages of the wine-making process to detect multiple spoilage-causing microorganisms in a single assay, thus offering a convenient test system for winemakers interested in monitoring the quality of their product.

Quantitative Detection of Chicken and Turkey Contamination in Cooked Meat Products by ELISA.

Background: Concerns about the contamination of meat products with undeclared meats and new regulations for the declaration of meat adulterants have established the need for a rapid test to detect chicken and turkey adulteration. Objective: To address this need, Microbiologique, Inc. has developed an ELISA that can quantify the presence of chicken and turkey down to 0.1% (w/w) in cooked pork, horse, beef, goat, and lamb meats. Results: This chicken/turkey authentication ELISA has an analytical sensitivity of 0.000037% and 0.000048% (w/v) for cooked and autoclaved chicken, respectively, and an analytical range of quantitation of 0.025-2% (w/v), in the absence of other meats. The assay cross-reacts with cooked duck and pheasant but does not demonstrate any cross-reactivity with cooked pork, horse, beef, goat, and lamb meats, egg, or common food matrixes. Conclusions: The assay is rapid, can be completed in 70 min, and can detect a 0.1% level of meat adulteration. Highlights: The Microbiologique Cooked Chicken/Turkey ELISA can quantitate cooked chicken/turkey in the presence of pork, horse, chicken, goat, or sheep meat to 0.1% (w/w) and is not affected by common food matrixes.

Quantitative Detection of Beef Contamination in Cooked Meat Products by ELISA.

Background: Concerns about the contamination of meat products with undeclared meats, and new regulations for the declaration of meat adulterants have established the need for a rapid test to detect beef adulteration to 0.1% sensitivity. Objective: To address this need, Microbiologique, Inc. has developed an ELISA that can quantify the presence of beef down to 0.1% (w/w) in cooked pork, horse, chicken, goat, and sheep meat. Results: The beef-authentication ELISA has an analytical sensitivity of 0.00022 and 0.00012% (w/v) for cooked and autoclaved beef, respectively, and an analytical range of quantitation of 0.025 to 2% (w/v), in the absence of other meats. Moreover, the assay is specific for cooked beef and does not cross react with common food matrixes. Conclusions: The assay is rapid, can be completed in 70 min, and can detect a 0.1% level of meat adulteration. The assay is an improvement over a previous U.S. Department of Agriculture's tested assay, which is sensitive to 1% adulteration and takes 2.5-3 h to complete. Highlights: The Microbiologique Cooked Beef ELISA can quantitate cooked beef in the presence of pork, horse, chicken, goat, and sheep meat to 0.1% (w/w) and is not affected by common food matrixes.

Reclassification of Eubacterium combesii and discrepancies in the nomenclature of botulinum neurotoxin-producing clostridia: Challenging Opinion 69. Request for an Opinion.

To clarify the taxonomic position of Eubacterium combesii, the whole genome of its type strain, DSM 20696T, was sequenced. Comparison of this sequence with known sequences of other bacteria confirmed that E. combesii represented a member of the Clostridium sporogenes/Clostridium botulinum Group I clade. However, the results of phylogenetic analysis also demonstrated that the latter two species did not form the same genetic entity and that E. combesii was in the C. botulinum Group I subclade. Meanwhile, we showed that E. combesii DSM 20696T did not produce botulinum neurotoxins (BoNTs) and thus should be identified as a strain of C. sporogenes in accordance with the current nomenclature of BoNT-producing clostridia, which is based, in particular, on Opinion 69 issued by the Judicial Commission of the ICSB. However, review of the corresponding Request for an Opinion revealed that it had been based on an erroneous statement. Therefore, we request reconsideration of Opinion 69 and propose to reclassify Eubacterium combesii as a later synonym of Clostridium botulinum. The results of phylogenetic analysis of the other five groups of BoNT-producing clostridia indicated that all the groups were far distant from each other. However, the members of Groups IV-VI are classified as strains of different species, while all members of Groups I-III are designated C. botulinum. Meanwhile, similarly to Group I, Groups II and III are also polyphyletic and appear to consist of two and four species, respectively. These results demonstrate, once again, discrepancies in the nomenclature of BoNT-producing bacteria and corroborate our request for reconsideration of Opinion 69.