Validation of a clinical assay for botulinum neurotoxins through mass spectrometric detection.

Botulism is a paralytic disease due to the inhibition of acetylcholine exocytosis at the neuromuscular junction, which can be lethal if left untreated. Botulinum neurotoxins (BoNTs) are produced by some spore-forming Clostridium bacteria. The current confirmatory assay to test for BoNTs in clinical specimens is the gold-standard mouse bioassay. However, an Endopep-MS assay method has been developed to detect BoNTs in clinical samples using benchtop mass spectrometric detection. This work demonstrates the validation of the Endopep-MS method for clinical specimens with the intent of method distribution in public health laboratories. The Endopep-MS assay was validated by assessing the sensitivity, robustness, selectivity, specificity, and reproducibility. The limit of detection was found to be equivalent to or more sensitive than the mouse bioassay. Specificity studies determined no cross-reactivity between the different serotypes and no false positives from an exclusivity panel of culture supernatants of enteric disease organisms and non-toxigenic strains of Clostridium. Inter-serotype specificity testing with 19 BoNT subtypes was 100% concordant with the expected results, accurately determining the presence of the correct serotype and the absence of incorrect serotypes. Additionally, a panel of potential interfering substances was used to test selectivity. Finally, clinical studies included clinical specimen stability and reproducibility, which was found to be 99.9% from a multicenter evaluation study. The multicenter validation study also included a clinical validation study, which yielded a 99.4% correct determination rate. Use of the Endopep-MS method will improve the capacity and response time for laboratory confirmation of botulism in public health laboratories.

Comprehensive characterization of toxins during progression of inhalation anthrax in a non-human primate model.

Inhalation anthrax has three clinical stages: early-prodromal, intermediate-progressive, and late-fulminant. We report the comprehensive characterization of anthrax toxins, including total protective antigen (PA), total lethal factor (LF), total edema factor (EF), and their toxin complexes, lethal toxin and edema toxin in plasma, during the course of inhalation anthrax in 23 cynomolgus macaques. The toxin kinetics were predominantly triphasic with an early rise (phase-1), a plateau/decline (phase-2), and a final rapid rise (phase-3). Eleven animals had shorter survival times, mean±standard deviation of 58.7±7.6 hours (fast progression), 11 animals had longer survival times, 113±34.4 hours (slow progression), and one animal survived. Median (lower-upper quartile) LF levels at the end-of-phase-1 were significantly higher in animals with fast progression [138 (54.9-326) ng/mL], than in those with slow progression [23.8 (15.6-26.3) ng/mL] (p = 0.0002), and the survivor (11.1 ng/mL). The differences were also observed for other toxins and bacteremia. Animals with slow progression had an extended phase-2 plateau, with low variability of LF levels across all time points and animals. Characterization of phase-2 toxin levels defined upper thresholds; critical levels for exiting phase-2 and entering the critical phase-3, 342 ng/mL (PA), 35.8 ng/mL (LF), and 1.10 ng/mL (EF). The thresholds were exceeded earlier in animals with fast progression (38.5±7.4 hours) and later in animals with slow progression (78.7±15.2 hours). Once the threshold was passed, toxin levels rose rapidly in both groups to the terminal stage. The time from threshold to terminal was rapid and similar; 20.8±7.4 hours for fast and 19.9±7.5 hours for slow progression. The three toxemic phases were aligned with the three clinical stages of anthrax for fast and slow progression which showed that anthrax progression is toxin- rather than time-dependent. This first comprehensive evaluation of anthrax toxins provides new insights into disease progression.

Inaccurately Reported Statin Use Affects the Assessing of Lipid Profile Measures and Their Association with Coronary Artery Disease Risk.

BACKGROUND: Lipid profiling is central for coronary artery disease (CAD) risk assessment. Nonadherence or unreported use of lipid-lowering drugs, particularly statins, can significantly complicate the association between lipid profile measures and CAD clinical outcomes. By combining medication history evaluation with statin analysis in plasma, we determined the effects of inaccurately reported statin use on lipid profile measures and their association with CAD risk. METHODS: We compared medication history of statin use with statin concentration measurements, by liquid chromatography-tandem mass spectrometry, in 690 participants undergoing coronary angiography (63 ± 11 years of age). Nominal logistic regression was employed to model CAD diagnosis with statin measurements, phenotypic, and lipid profile characteristics. RESULTS: Medication history of statin use was confirmed by statin assay for 81% of the patients. Surprisingly, statins were detected in 46% of patients without statin use records. Nonreported statin use was disproportionately higher among older participants. Stratifying samples by statin history resulted in underestimated LDL-lipid measures. Apolipoprotein B concentrations had a significant inverse CAD association, which became nonsignificant upon re-stratification using the statin assay data. CONCLUSIONS: Our study uncovered prominent discrepancies between medication records and actual statin use measured by mass spectrometry. We showed that inaccurate statin use assessments may lead to overestimation and underestimation of LDL levels in statin user and nonuser categories, exaggerating the reverse epidemiology association between LDL levels and CAD diagnosis. Combining medication history and quantitative statin assay data can significantly improve the design, analysis, and interpretation of clinical and epidemiological studies.

Mass Spectrometric Detection and Differentiation of Enzymatically Active Abrin and Ricin Combined with a Novel Affinity Enrichment Technique.

Abrin and ricin are toxic proteins produced by plants. Both proteins are composed of two subunits, an A-chain and a B-chain. The A-chain is responsible for the enzymatic activity, which causes toxicity. The B-chain binds to glycoproteins on the cell surface to direct the A-chain to its target. Both toxins depurinate 28S rRNA, making it impossible to differentiate these toxins based on only their enzymatic activity. We developed an analytical workflow for both ricin and abrin using a single method and sample. We have developed a novel affinity enrichment technique based on the ability of the B-chain to bind a glycoprotein, asialofetuin. After the toxin is extracted with asialofetuin-coated magnetic beads, an RNA substrate is added. Then, depurination is detected by a benchtop matrix-assisted laser desorption/ionization time-of-flight (MALDI TOF) mass spectrometer to determine the presence or absence of an active toxin. Next, the beads are subjected to tryptic digest. Toxin fingerprinting is done on a benchtop MALDI-TOF MS. We validated the assay through sensitivity and specificity studies and determined the limit of detection for each toxin as nanogram level for enzymatic activity and µg level for toxin fingerprinting. We examined potential cross-reactivity from proteins that are near neighbors of the toxins and examined potential false results in the presence of white powders.

Inclusion of deuterated glycopeptides provides increased sequence coverage in hydrogen/deuterium exchange mass spectrometry analysis of SARS-CoV-2 spike glycoprotein.

RATIONALE: Hydrogen/deuterium exchange mass spectrometry (HDX-MS) can provide precise analysis of a protein's conformational dynamics across varied states, such as heat-denatured versus native protein structures, localizing regions that are specifically affected by such conditional changes. Maximizing protein sequence coverage provides high confidence that regions of interest were located by HDX-MS, but one challenge for complete sequence coverage is N-glycosylation sites. The deuteration of peptides post-translationally modified by asparagine-bound glycans (glycopeptides) has not always been identified in previous reports of HDX-MS analyses, causing significant sequence coverage gaps in heavily glycosylated proteins and uncertainty in structural dynamics in many regions throughout a glycoprotein. METHODS: We detected deuterated glycopeptides with a Tribrid Orbitrap Eclipse mass spectrometer performing data-dependent acquisition. An MS scan was used to identify precursor ions; if high-energy collision-induced dissociation MS/MS of the precursor indicated oxonium ions diagnostic for complex glycans, then electron transfer low-energy collision-induced dissociation MS/MS scans of the precursor identified the modified asparagine residue and the glycan's mass. As in traditional HDX-MS, the identified glycopeptides were then analyzed at the MS level in samples labeled with D2 O. RESULTS: We report HDX-MS analysis of the SARS-CoV-2 spike protein ectodomain in its trimeric prefusion form, which has 22 predicted N-glycosylation sites per monomer, with and without heat treatment. We identified glycopeptides and calculated their average isotopic mass shifts from deuteration. Inclusion of the deuterated glycopeptides increased sequence coverage of spike ectodomain from 76% to 84%, demonstrated that glycopeptides had been deuterated, and improved confidence in results localizing structural rearrangements. CONCLUSION: Inclusion of deuterated glycopeptides improves the analysis of the conformational dynamics of glycoproteins such as viral surface antigens and cellular receptors.

An antibody-free evaluation of an mRNA COVID-19 vaccine.

This manuscript describes the use of an analytical assay that combines transfection of mammalian cells and isotope dilution mass spectrometry (IDMS) for accurate quantification of antigen expression. Expired mRNA COVID-19 vaccine material was stored at 4 °C, room temperature (∼25 °C), and 56 °C over a period of 5 weeks. The same vaccine was also exposed to 5 freeze-thaw cycles. Every week, the spike protein antigenic expression in mammalian (BHK-21) cells was evaluated. Housekeeping proteins, ß-actin and GAPDH, were simultaneously quantified to account for the variation in cell counts that occurs during maintenance and growth of cell cultures. Data show that vaccine stored at elevated temperatures results in reduced spike protein expression. Also, maintaining the vaccine in ultracold conditions or exposing the vaccine to freeze-thaw cycles had less effect on the vaccine's ability to produce the antigen in mammalian cells. We describe the use of IDMS as an antibody-free means to accurately quantify expressed protein from mammalian cells transfected with mRNA vaccine.

Vitamin E Acetate in Bronchoalveolar-Lavage Fluid Associated with EVALI.

BACKGROUND: The causative agents for the current national outbreak of electronic-cigarette, or vaping, product use-associated lung injury (EVALI) have not been established. Detection of toxicants in bronchoalveolar-lavage (BAL) fluid from patients with EVALI can provide direct information on exposure within the lung. METHODS: BAL fluids were collected from 51 patients with EVALI in 16 states and from 99 healthy participants who were part of an ongoing study of smoking involving nonsmokers, exclusive users of e-cigarettes or vaping products, and exclusive cigarette smokers that was initiated in 2015. Using the BAL fluid, we performed isotope dilution mass spectrometry to measure several priority toxicants: vitamin E acetate, plant oils, medium-chain triglyceride oil, coconut oil, petroleum distillates, and diluent terpenes. RESULTS: State and local health departments assigned EVALI case status as confirmed for 25 patients and as probable for 26 patients. Vitamin E acetate was identified in BAL fluid obtained from 48 of 51 case patients (94%) in 16 states but not in such fluid obtained from the healthy comparator group. No other priority toxicants were found in BAL fluid from the case patients or the comparator group, except for coconut oil and limonene, which were found in 1 patient each. Among the case patients for whom laboratory or epidemiologic data were available, 47 of 50 (94%) had detectable tetrahydrocannabinol (THC) or its metabolites in BAL fluid or had reported vaping THC products in the 90 days before the onset of illness. Nicotine or its metabolites were detected in 30 of 47 of the case patients (64%). CONCLUSIONS: Vitamin E acetate was associated with EVALI in a convenience sample of 51 patients in 16 states across the United States. (Funded by the National Cancer Institute and others.).

Analysis of the N-glycosylation profiles of the spike proteins from the Alpha, Beta, Gamma, and Delta variants of SARS-CoV-2.

N-Glycosylation plays an important role in the structure and function of membrane and secreted proteins. Viral proteins used in cell entry are often extensively glycosylated to assist in protein folding, provide stability, and shield the virus from immune recognition by its host (described as a "glycan shield"). The SARS-CoV-2 spike protein (S) is a prime example, having 22 potential sites of N-glycosylation per protein protomer, as predicted from the primary sequence. In this report, we conducted mass spectrometric analysis of the N-glycosylation profiles of recombinant spike proteins derived from four common SARS-CoV-2 variants classified as Variant of Concern, including Alpha, Beta, Gamma, and Delta along with D614G variant spike as a control. Our data reveal that the amino acid substitutions and deletions between variants impact the abundance and type of glycans on glycosylation sites of the spike protein. Some of the N-glycosylation sequons in S show differences between SARS-CoV-2 variants in the distribution of glycan forms. In comparison with our previously reported site-specific glycan analysis on the S-D614G and its ancestral protein, glycan types on later variants showed high similarity on the site-specific glycan content to S-D614G. Additionally, we applied multiple digestion methods on each sample, and confirmed the results for individual glycosylation sites from different experiment conditions to improve the identification and quantification of glycopeptides. Detailed site-specific glycan analysis of a wide variety of SARS-CoV-2 variants provides useful information toward the understanding of the role of protein glycosylation on viral protein structure and function and development of effective vaccines and therapeutics.

The small HDL particle hypothesis of Alzheimer's disease.

We propose the hypothesis that small high-density lipoprotein (HDL) particles reduce the risk of Alzheimer's disease (AD) by virtue of their capacity to exchange lipids, affecting neuronal membrane composition and vascular and synaptic functions. Concentrations of small HDLs in cerebrospinal fluid (CSF) and plasma were measured in 180 individuals ≥60 years of age using ion mobility methodology. Small HDL concentrations in CSF were positively associated with performance in three domains of cognitive function independent of apolipoprotein E (APOE) ε4 status, age, sex, and years of education. Moreover, there was a significant correlation between levels of small HDLs in CSF and plasma. Further studies will be aimed at determining whether specific components of small HDL exchange across the blood, brain, and CSF barriers, and developing approaches to exploit small HDLs for therapeutic purposes.

Confirmation of Statin and Fibrate Use from Small-Volume Archived Plasma Samples by High-Throughput LC-MS/MS Method.

Designing studies for lipid-metabolism-related biomarker discovery is challenging because of the high prevalence of various statin and fibrate usage for lipid-lowering therapies. When the statin and fibrate use is determined based on self-reports, patient adherence to the prescribed statin dose regimen remains unknown. A potentially more accurate way to verify a patient's medication adherence is by direct analytical measurements. Current analytical methods are prohibitive because of the limited panel of drugs per test and large sample volume requirement that is not available from archived samples. A 4-min-long method was developed for the detection of seven statins and three fibrates using 10 µL of plasma analyzed via reverse-phase liquid chromatography and tandem mass spectrometry. The method was applied to the analysis of 941 archived plasma samples collected from patients before cardiac catheterization. When statin use was self-reported, statins were detected in 78.6% of the samples. In the case of self-reported atorvastatin use, the agreement with detection was 90.2%. However, when no statin use was reported, 42.4% of the samples had detectable levels of statins, with a similar range of concentrations as the samples from the self-reported statin users. The method is highly applicable in population studies designed for biomarker discovery or diet and lifestyle intervention studies, where the accuracy of statin or fibrate use may strongly affect the statistical evaluation of the biomarker data.

Welder's Anthrax: A Tale of 2 Cases.

Bacillus anthracis has traditionally been considered the etiologic agent of anthrax. However, anthrax-like illness has been documented in welders and other metal workers infected with Bacillus cereus group spp. harboring pXO1 virulence genes that produce anthrax toxins. We present 2 recent cases of severe pneumonia in welders with B. cereus group infections and discuss potential risk factors for infection and treatment options, including antitoxin.

Human Innate Immune Cells Respond Differentially to Poly-γ-Glutamic Acid Polymers from Bacillus anthracis and Nonpathogenic Bacillus Species.

The poly-γ-glutamic acid (PGA) capsule produced by Bacillus anthracis is composed entirely of d-isomer glutamic acid, whereas nonpathogenic Bacillus species produce mixed d-, l-isomer PGAs. To determine if B. anthracis PGA confers a pathogenic advantage over other PGAs, we compared the responses of human innate immune cells to B. anthracis PGA and PGAs from nonpathogenic B. subtilis subsp. chungkookjang and B. licheniformis Monocytes and immature dendritic cells (iDCs) responded differentially to the PGAs, with B. anthracis PGA being least stimulatory and B. licheniformis PGA most stimulatory. All three elicited IL-8 and IL-6 from monocytes, but B. subtilis PGA also elicited IL-10 and TNF-α, whereas B. licheniformis PGA elicited all those plus IL-1ß. Similarly, all three PGAs elicited IL-8 from iDCs, but B. subtilis PGA also elicited IL-6, and B. licheniformis PGA elicited those plus IL-12p70, IL-10, IL-1ß, and TNF-α. Only B. licheniformis PGA induced dendritic cell maturation. TLR assays also yielded differential results. B. subtilis PGA and B. licheniformis PGA both elicited more TLR2 signal than B. anthracis PGA, but only responses to B. subtilis PGA were affected by a TLR6 neutralizing Ab. B. licheniformis PGA elicited more TLR4 signal than B. anthracis PGA, whereas B. subtilis PGA elicited none. B. anthracis PGA persisted longer in high m.w. form in monocyte and iDC cultures than the other PGAs. Reducing the m.w. of B. anthracis PGA reduced monocytes' cytokine responses. We conclude that B. anthracis PGA is recognized less effectively by innate immune cells than PGAs from nonpathogenic Bacillus species, resulting in failure to induce a robust host response, which may contribute to anthrax pathogenesis.

Clindamycin Protects Nonhuman Primates Against Inhalational Anthrax But Does Not Enhance Reduction of Circulating Toxin Levels When Combined With Ciprofloxacin.

BACKGROUND: Inhalational anthrax is rare and clinical experience limited. Expert guidelines recommend treatment with combination antibiotics including protein synthesis-inhibitors to decrease toxin production and increase survival, although evidence is lacking. METHODS: Rhesus macaques exposed to an aerosol of Bacillus anthracis spores were treated with ciprofloxacin, clindamycin, or ciprofloxacin + clindamycin after becoming bacteremic. Circulating anthrax lethal factor and protective antigen were quantitated pretreatment and 1.5 and 12 hours after beginning antibiotics. RESULTS: In the clindamycin group, 8 of 11 (73%) survived demonstrating its efficacy for the first time in inhalational anthrax, compared to 9 of 9 (100%) with ciprofloxacin, and 8 of 11 (73%) with ciprofloxacin + clindamycin. These differences were not statistically significant. There were no significant differences between groups in lethal factor or protective antigen levels from pretreatment to 12 hours after starting antibiotics. Animals that died after clindamycin had a greater incidence of meningitis compared to those given ciprofloxacin or ciprofloxacin + clindamycin, but numbers of animals were very low and no definitive conclusion could be reached. CONCLUSION: Treatment of inhalational anthrax with clindamycin was as effective as ciprofloxacin in the nonhuman primate. Addition of clindamycin to ciprofloxacin did not enhance reduction of circulating toxin levels.

Detection of ricin activity and structure by using novel galactose-terminated magnetic bead extraction coupled with mass spectrometric detection.

Ricin is a toxic protein derived from the castor bean plant (Ricinus communis) and has potential for bioterrorism or criminal use. Therefore, sensitive and rapid analytical methods are needed for its confirmatory detection in environmental samples. Our laboratory previously reported on the development of a confirmatory method to detect ricin involving antibody capture of ricin followed by mass spectrometric detection of ricin's enzymatic activity and of tryptic fragments unique to ricin. Here, we describe a novel ricin capture method of magnetic beads coated with 4-aminophenyl-1-thiol-ß-galactopyranoside, using ricin's lectin characteristics. The assay has been adapted for use on a simple, benchtop MALDI-TOF MS mass spectrometer common in clinical microbiology laboratories. Validation of the novel assay includes establishment of a limit of detection, and an examination of assay selectivity. The limit of detection of the enzymatic activity method is 8 ng/mL and 500 ng/mL for the confirmatory tryptic fragment assay. The assay is highly selective with no cross-reactivity from near neighbors and highly specific with a panel of 19 cultivars all testing positive. Additionally, there were no interferences found during testing of a panel of white powders. This allows for a confirmatory detection method for ricin in laboratories lacking expensive, sophisticated mass spectrometers.

Comprehensive Analysis of the Glycan Complement of SARS-CoV-2 Spike Proteins Using Signature Ions-Triggered Electron-Transfer/Higher-Energy Collisional Dissociation (EThcD) Mass Spectrometry.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to a global pandemic of coronavirus disease 2019 (COVID-19). The spike protein expressed on the surface of this virus is highly glycosylated and plays an essential role during the process of infection. We conducted a comprehensive mass spectrometric analysis of the N-glycosylation profiles of the SARS-CoV-2 spike proteins using signature ions-triggered electron-transfer/higher-energy collision dissociation (EThcD) mass spectrometry. The patterns of N-glycosylation within the recombinant ectodomain and S1 subunit of the SARS-CoV-2 spike protein were characterized using this approach. Significant variations were observed in the distribution of glycan types as well as the specific individual glycans on the modification sites of the ectodomain and subunit proteins. The relative abundance of sialylated glycans in the S1 subunit compared to the full-length protein could indicate differences in the global structure and function of these two species. In addition, we compared N-glycan profiles of the recombinant spike proteins produced from different expression systems, including human embryonic kidney (HEK 293) cells and Spodoptera frugiperda (SF9) insect cells. These results provide useful information for the study of the interactions of SARS-CoV-2 viral proteins and for the development of effective vaccines and therapeutics.

Limited Tryptic Digestion-Isotope Dilution Mass Spectrometry (LTD-IDMS): A Reagent-Free Analytical Assay To Quantify Hemagglutinin of A(H5N1) Vaccine Material.

Avian influenza viruses, such as A(H5N1) and A(H7N9), are primary public health concerns due to their pandemic potential. Influenza vaccines represent the most effective response to this threat especially with timely provision. The current pandemic response timelines require a substantial period for strain-specific reference antigen and sera preparation for use with single-radial immunodiffusion (SRID), the accepted vaccine potency assay. To address this time lag, the isotope dilution mass spectrometry (IDMS) method was developed to quantify the absolute hemagglutinin (HA, the main influenza antigen) amount in the vaccine without the need for purified, inactivated, and calibrated virus reference antigens. However, an additional challenge in determining potency is to differentiate between vaccine antigens in their most potent form from other less potent, stressed antigen forms. The limited trypsin digestion (LTD) method has been developed and does not require strain-specific full-length reference antigens or antibodies; instead, stressed HA is selectively degraded, leaving the more potent form to be measured. LTD, followed by precipitation and IDMS, allows for efficient differentiation between potent and significantly less potent HA for vaccine release and potency testing across the vaccine's shelf life. In this study, we tested the LTD-IDMS assay on A(H5N1) vaccine material that had been stressed by low pH, heat, and multiple freeze-thaw cycles. The results showed that the LTD-IDMS method effectively quantified the potent HA in A(H5N1) vaccine material with results comparable to SRID. As such, it shows great promise to complement and potentially replace SRID in a pandemic when strain-specific reagents may not be readily available.

Intracellular changes of a swine tracheal cell line infected with a Mycoplasma hyopneumoniae pathogenic strain.

Mycoplasma hyopneumoniae is the etiological agent of enzootic pneumonia (EP), a widespread disease that causes major economic losses to the pig industry. The swine host response plays an important role in the outcome of M. hyopneumoniae infections. The whole proteome of newborn pig trachea (NPTr) epithelial cells infected with the M. hyopneumoniae pathogenic strain 7448 was analyzed using an LC-MS/MS approach to shed light on intracellular processes triggered in response to the pathogen. Overall, 853 swine protein species were identified, 156 of which were differentially represented in response to M. hyopneumoniae 7448 infection in comparison with non-infected control cells. These differentially represented proteins were categorized by function. Fifty-seven of them were assigned to the immune system and/or response to stimulus functional subcategories. Comparative expression analysis of these immune-related proteins in NPTr cells infected with attenuated or non-pathogenic mycoplasmas (M. hyopneumoniae J strain and M. flocculare, respectively) revealed proteins whose abundance was altered only in response to the pathogenic M. hyopneumoniae 7448 strain. Among these proteins, calcium homeostasis and endoplasmic reticulum stress-related biomarkers were detected, providing evidence of molecular mechanisms that might lead to swine cell apoptosis.

Evaluation of Bronchoalveolar Lavage Fluid from Patients in an Outbreak of E-cigarette, or Vaping, Product Use-Associated Lung Injury - 10 States, August-October 2019.

CDC, the Food and Drug Administration (FDA), state and local health departments, and multiple public health and clinical partners are investigating a national outbreak of e-cigarette, or vaping, product use-associated lung injury (EVALI). Based on data collected as of October 15, 2019, 86% of 867 EVALI patients reported using tetrahydrocannabinol (THC)-containing products in the 3 months preceding symptom onset (1). Analyses of THC-containing product samples by FDA and state public health laboratories have identified potentially harmful constituents in these products, such as vitamin E acetate, medium chain triglyceride oil (MCT oil), and other lipids (2,3) (personal communication, D.T. Heitkemper, FDA Forensic Chemistry Center, November 2019). Vitamin E acetate, in particular, might be used as an additive in the production of e-cigarette, or vaping, products; it also can be used as a thickening agent in THC products (4). Inhalation of vitamin E acetate might impair lung function (5-7).

Vagococcus bubulae sp. nov., isolated from ground beef, and Vagococcus vulneris sp. nov., isolated from a human foot wound.

Two unusual catalase-negative, Gram-stain-positive, Vagococcus-like isolates that were referred to the CDC Streptococcus Laboratory for identification are described. Strain SS1994T was isolated from ground beef and strain SS1995T was isolated from a human foot wound. Comparative 16S rRNA gene sequence analysis of isolates SS1994T and SS1995T against Vagococcus type strain sequences supported their inclusion in the genus Vagococcus. Strain SS1994T showed high sequence similarity (>97.0 %) to the two most recently proposed species, Vagococcus martis (99.2 %) and Vagococcus teuberi (99.0 %) followed by Vagococcus penaei (98.8 %), strain SS1995T (98.6 %), Vagococcus carniphilus (98.0 %), Vagococcus acidifermentans (98.0 %) and Vagococcus fluvialis (97.9 %). The 16S rRNA gene sequence of strain SS1995T was most similar to V. penaei (99.1 %), followed by SS1994T (98.6 %), V. martis (98.4 %), V. teuberi (98.1 %), V. acidifermentans (97.8 %), and both V. carniphilus and V. fluvialis (97.5 %). A polyphasic taxonomic study using conventional biochemical and the rapid ID 32 STREP system, MALDI-TOF MS, cell fatty acid analysis, pairwise sequence comparisons of the 16S rRNA, rpoA, rpoB, pheS and groL genes, and comparative core and whole genome sequence analyses revealed that strains SS1994T and SS1995T were two novel Vagococcus species. The novel taxonomic status of the two isolates was confirmed with core genome phylogeny, average nucleotide identity <84 % and in silico DNA-DNA hybridization <28 % to any other Vagococcus species. The names Vagococcusbubulae SS1994T=(CCUG 70831T=LMG 30164T) and Vagococcusvulneris SS1995T=(CCUG 70832T=LMG 30165T) are proposed.