Refermentation and maturation of lambic beer in bottles: a necessary step for gueuze production.

The production of gueuze beers through refermentation and maturation of blends of lambic beer in bottles is a way for lambic brewers to cope with the variability among different lambic beer batches. The resulting gueuze beers are more carbonated than lambic beers and are supposed to possess a unique flavor profile that varies over time. To map this refermentation and maturation process for gueuze production, a blend of lambic beers was made and bottled, whereby one of them was produced with the old wheat landrace Zeeuwse Witte. Through the use of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and high-throughput sequencing of bacterial and fungal amplicons, in combination with metabolite target analysis, new insights into gueuze production were obtained. During the initial stages of refermentation, the conditions in the bottles were similar to those encountered during the maturation phase of lambic beer productions in wooden barrels, which was also reflected microbiologically (presence of Brettanomyces species, Pediococcus damnosus, and Acetobacter lambici) and biochemically (ethanol, higher alcohols, lactic acid, acetic acid, volatile phenolic compounds, and ethyl esters). However, after a few weeks of maturation, a switch from a favorable environment to one with nutrient and dissolved oxygen depletion resulted in several changes. Concerning the microbiology, a sequential prevalence of three lactic acid bacterial species occurred, namely, P. damnosus, Lentilactobacillus buchneri, and Lactobacillus acetotolerans, while the diversity of the yeasts decreased. Concerning the metabolites produced, mainly those of the Brettanomyces yeasts determined the metabolic profiles encountered during later stages of the gueuze production.IMPORTANCEGueuze beers are the result of a refermentation and maturation process of a blend of lambic beers carried out in bottles. These gueuze beers are known to have a long shelf life, and their quality typically varies over time. However, knowledge about gueuze production in bottles is scarce. The present study provided more insights into the varying microbial and metabolite composition of gueuze beers during the first 2 years of this refermentation and maturation process. This will allow gueuze producers to gain more information about the influence of the refermentation and maturation time on their beers. These insights can also be used by gueuze producers to better inform their customers about the quality of young and old gueuze beers.

Isolation and Identification of a Bacillus sp. from Freshwater Sediment Displaying Potent Activity Against Bacteria and Phytopathogen Fungi.

A bacterium strain isolated from freshwater sediment of San Pablo river of Santiago de Cuba, Cuba was identified as a Bacillus sp. by Matrix-Assisted Laser Desorption/Ionization Time Of Flight Mass Spectrometry. A 16S rRNA gene analysis showed that the isolate A3 belongs to the operational group Bacillus amyloliquefaciens, while the phylogenetic analysis of the gyrA gene sequence grouped it within B. amyloliquefaciens subsp. plantarum cluster, referred now as Bacillus velezensis. In vitro antibacterial studies demonstrated the capacity of the isolate A3 to produce bioactive metabolites against Bacillus subtilis ATCC 11,778, Bacillus cereus ATCC 6633, and Staphylococcus aureus ATCC 25,923 by cross-streak, overlay, and microdilution methods. The strain also showed a high potential against the multidrug-resistant Staphylococcus aureus ATCC 700,699, ATCC 29,213, and ATCC 6538. At pH 8 and 96 h in the medium 2 of A3 culture conditions, the produced metabolites with antibacterial potential were enhanced. Some alterations in the morphology of the phytopathogens Aspergillus niger ATCC 9642, Alternaria alternata CECT 2662, and Fusarium solani CCEBI 3094 were induced by the cell-free supernatant of B. velezensis A3. A preliminary study of the nature of the bioactive compounds produced by the strain A3 showed the presence of both lipids and peptides in the culture. Those results highlight B. velezensis A3 as a promissory bacterium capable to produce bioactive metabolites with antibacterial and antifungal properties against pathogens.

Gluconacetobacter dulcium sp. nov., a novel Gluconacetobacter species from sugar-rich environments.

A phylogenomic analysis based on 107 single-copy core genes revealed that three strains from sugar-rich environments, i.e. LMG 1728T, LMG 1731 and LMG 22058, represented a single, novel Gluconacetobacter lineage with Gluconacetobacter liquefaciens as nearest validly named neighbour. OrthoANIu and digital DNA-DNA hybridization analyses among these strains and Gluconacetobacter type strains confirmed that the three strains represented a novel Gluconacetobacter species. Biochemical characteristics and MALDI-TOF mass spectra allowed differentiation of this novel species from the type strains of G. liquefaciens and other closely related Gluconacetobacter species. We therefore propose to classify strains LMG 1728T, LMG 1731 and LMG 22058 in the novel species Gluconacetobacter dulcium sp. nov., with LMG 1728T (=CECT 30142T) as the type strain.

Genomic Aromatic Compound Degradation Potential of Novel Paraburkholderia Species: Paraburkholderia domus sp. nov., Paraburkholderia haematera sp. nov. and Paraburkholderia nemoris sp. nov.

We performed a taxonomic and comparative genomics analysis of 67 novel Paraburkholderia isolates from forest soil. Phylogenetic analysis of the recA gene revealed that these isolates formed a coherent lineage within the genus Paraburkholderia that also included Paraburkholderiaaspalathi, Paraburkholderiamadseniana, Paraburkholderiasediminicola, Paraburkholderiacaffeinilytica, Paraburkholderiasolitsugae and Paraburkholderiaelongata and four unidentified soil isolates from earlier studies. A phylogenomic analysis, along with orthoANIu and digital DNA-DNA hybridization calculations revealed that they represented four different species including three novel species and P. aspalathi. Functional genome annotation of the strains revealed several pathways for aromatic compound degradation and the presence of mono- and dioxygenases involved in the degradation of the lignin-derived compounds ferulic acid and p-coumaric acid. This co-occurrence of multiple Paraburkholderia strains and species with the capacity to degrade aromatic compounds in pristine forest soil is likely caused by the abundant presence of aromatic compounds in decomposing plant litter and may highlight a diversity in micro-habitats or be indicative of synergistic relationships. We propose to classify the isolates representing novel species as Paraburkholderia domus with LMG 31832T (=CECT 30334) as the type strain, Paraburkholderia nemoris with LMG 31836T (=CECT 30335) as the type strain and Paraburkholderia haematera with LMG 31837T (=CECT 30336) as the type strain and provide an emended description of Paraburkholderia sediminicola Lim et al. 2008.

Novel acetic acid bacteria from cider fermentations: Acetobacter conturbans sp. nov. and Acetobacter fallax sp. nov.

Strains LMG 1627T, LMG 1636T and LMG 1637 were all isolated from cider fermentations in the 1940s and 1950s. A recent study based on MALDI-TOF MS and dnaK gene sequence analyses suggested they represented novel Acetobacter species. In the present study, we determined the whole-genome sequences of these strains and analysed their phenotypic and chemotaxonomic characteristics. A phylogenomic analysis based on 107 single-copy core genes revealed that they represented a single Acetobacter lineage with Acetobacter aceti, Acetobacter sicerae, Acetobacter musti and Acetobacter oeni, Acetobacter estunensis and with Acetobacter nitrogenifigens as an outgroup to this cluster. OrthoANIu value and dDDH analyses among these and other Acetobacter type strains confirmed that these three strains represented two novel Acetobacter species, which could be differentiated from other closely related type strains of Acetobacter by different phenotypic tests, such as ketogenesis from glycerol. We therefore propose to classify strain LMG 1627T in the novel species Acetobacter conturbans sp. nov., with LMG 1627T (=NCIMB 8945T) as the type strain, and to classify strains LMG 1636T and LMG 1637 in the novel species Acetobacter fallax sp. nov., with LMG 1636T (=NCIMB 8956T) as the type strain.

Micromonospora fluminis sp. nov., isolated from mountain river sediment.

During a bioprospection of bacteria with antimicrobial activity, the actinomycete strain A38T was isolated from a sediment sample of the Carpintero river located in the Gran Piedra Mountains, Santiago de Cuba province (Cuba). This strain was identified as a member of the genus Micromonospora by means of a polyphasic taxonomy study. Strain A38T was an aerobic Gram-positive filamentous bacterium that produced single spores in a well-developed vegetative mycelium. An aerial mycelium was absent. The cell wall contained meso-diaminopimelic acid and the whole-cell sugars were glucose, mannose, ribose and xylose. The major cellular fatty acids were isoC15:0, 10 methyl C17:0, anteiso-C17:0 and iso-C17:0. The predominant menaquinones were MK-10(H4) and MK-10(H6). Phylogenetic analysis of 16S rRNA gene sequences revealed that this strain was closely related to Micromonospora tulbaghiae DSM 45142T (99.5 %), Micromonospora citrea DSM 43903T (99.4 %), Micromonospora marina DSM 45555T (99.4 %), Micromonospora maritima DSM 45782T (99.3 %), Micromonospora sediminicola DSM 45794T (99.3 %), Micromonospora aurantiaca DSM 43813T (99.2 %) and Micromonospora chaiyaphumensis DSM 45246T (99.2 %). The results of OrthoANIu analysis showed the highest similarity to Micromonospora chalcea DSM 43026T (96.4 %). However, the 16S rRNA and gyrB gene sequence-based phylogeny and phenotypic characteristics provided support to distinguish strain A38T as a novel species. On the basis of the results presented here, we propose to classify strain A38T (=LMG 30467T=CECT 30034T) as the type strain of the novel species Micromonospora fluminis sp. nov.

Nocardiopsis dassonvillei subsp. crassaminis subsp. nov., isolated from freshwater sediment, and reappraisal of Nocardiopsis alborubida Grund and Kroppenstedt 1990 emend. Nouioui et al. 2018.

An actinomycete, strain D1T, was isolated from a freshwater sediment sample collected from the San Pablo river in the La Risueña community, Santiago de Cuba province, Cuba. The strain was identified as a member of the genus Nocardiopsis by means of a polyphasic taxonomic study. It produced a light yellow non-fragmented substrate mycelium, a white well-developed aerial mycelium and straight to flexuous hyphae. No specific spore chains were observed. Strain D1T contained meso-diaminopimelic acid, no diagnostic sugars, and MK-10(H2), MK-10(H4), MK-10 and MK-10(H6) as predominant menaquinones, but not phosphatidylcholine as diagnostic polar lipid of the genus Nocardiopsis. The predominant fatty acids were iso-C16 : 0, 10-methyl-C18 : 0 and anteiso-C17 : 0. Strain D1T showed the highest degree of 16S rRNA gene sequence similarity to Nocardiopsis synnematoformans DSM 44143T (99.8 %), Nocardiopsis dassonvillei subsp. albirubida NBRC 13392T (99.8 %) and Nocardiopsis dassonvillei subsp. dassonvillei DSM 43111T (99.6 %). A genomic OrthoANIu value between D1T and N. dassonvillei subsp. dassonvillei DSM 43111T of 97.63 % and a dDDH value of 78.9 % indicated that strain D1T should be classified in N. dassonvillei. However, phenotypic characteristics distinguished strain D1T from its nearest neighbour taxon. On basis of these results we propose to classify strain D1T (=LMG 30468T=CECT 30033T) as a representative of a novel subspecies of the genus Nocardiopsis, for which the name Nocardiopsis dassonvillei subsp. crassaminis subsp. nov. is proposed. In addition, the genomic distance between N. dassonvillei subsp. albirubida NBRC 13392T and N. dassonvillei subsp. dassonvillei DSM 43111T as determined through OrthoANIu (93.64 %) and dDDH (53.40 %), along with considerable phenotypic and chemotaxonomic differences reported in earlier studies, indicated that the classification of this taxon as Nocardiopsis alborubida Grund and Kroppenstedt 1990 is to be preferred over its classification as N. dassonvillei subsp. albirubida Evtushenko et al. 2000.

Actinomadura roseirufa sp. nov., producer of semduramicin, a polyether ionophore.

The taxonomic position of 'Actinomadura roseorufa' LMG 30035T, a semduramicin-producing mutant of strain ATCC 53666P, which was isolated from a soil sample collected in Yamae Village, Kamamoto, Japan, was clarified in the present study using a polyphasic approach. This Gram-positive, aerobic actinomycete formed a well-developed, extensively branched, non-fragmenting substrate and aerial mycelia which differentiated into single, smooth-appearing spores. Based on analysis of nearly complete 16S rRNA gene sequence, strain LMG 30035T was found to be closely related to the type strains of Actinomadura fibrosa ATCC 49459T (98.88 %) and Actinomadura formosensis JCM 7474T (98.82 %) (pairwise similarity values in parentheses). Digital DNA-DNA hybridisation experiments revealed unambiguously that strain LMG 30035T represents a novel Actinomadura species (OrthoANIu values less than 83.1 %; dDDH values less than 27.2 % with type strains of validly named Actinomadura species). Analysis of the cell wall revealed the presence of meso-diaminopimelic acid in the peptidoglycan. The whole-cell sugars were glucose, madurose, galactose, ribose and rhamnose. The major polar lipids included phosphatidylinositol and diphosphatidylglycerol. The predominant menaquinones were MK-9(H6), MK-9(H8), MK-9(H4) and MK-9(H2). The major fatty acids were C16 : 00, 10-methyl C18 : 0, C18 : 1 ω9c and C18 : 00. The DNA G+C content of its genome was 72.5 mol%. In summary, these characteristics distinguish strain LMG 30035T from validly named species of the genus Actinomadura, and therefore, we propose to classify this strain formally as the novel species Actinomadura roseirufa sp. nov. with LMG 30035T (=CECT 9808T,=ATCC 53664T) as the type strain.

Characterization of novel Gluconobacter species from fruits and fermented food products: Gluconobacter cadivus sp. nov., Gluconobacter vitians sp. nov. and Gluconobacter potus sp. nov.

Strains LMG 1744T, LMG 1745, LMG 31484T, LMG 1764T and R-71646 were isolated from rotting fruits and fermented food products. A phylogenomic analysis based on 107 single-copy core genes revealed that they grouped in a Gluconobacter lineage comprising Gluconobacter oxydans, Gluconobacter roseus, Gluconobacter sphaericus, Gluconobacter kanchanaburiensis, Gluconobacter albidus, Gluconobacter cerevisiae, Gluconobacter kondonii and Gluconobacter aidae. OrthoANIu and digital DNA hybridization analyses demonstrated that these five strains represented three novel Gluconobacter species, which could be differentiated from the type strains of closely related Gluconobacter species by multiple phenotypic characteristics. We therefore propose to classify strains LMG 1744T and LMG 1745 in the novel species Gluconobacter cadivus sp. nov., with LMG 1744T (=CECT 30141T) as the type strain; to classify strain LMG 31484T as the novel species Gluconobacter vitians sp. nov., with LMG 31484T (=CECT 30132T) as the type strain; and to classify strains LMG 1764T and R-71646 in the novel species Gluconobacter potus sp. nov., with LMG 1764T (=CECT 30140T) as the type strain.

The microbial diversity of an industrially produced lambic beer shares members of a traditionally produced one and reveals a core microbiota for lambic beer fermentation.

The microbiota involved in lambic beer fermentations in an industrial brewery in West-Flanders, Belgium, was determined through culture-dependent and culture-independent techniques. More than 1300 bacterial and yeast isolates from 13 samples collected during a one-year fermentation process were identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry followed by sequence analysis of rRNA and various protein-encoding genes. The bacterial and yeast communities of the same samples were further analyzed using denaturing gradient gel electrophoresis of PCR-amplified V3 regions of the 16S rRNA genes and D1/D2 regions of the 26S rRNA genes, respectively. In contrast to traditional lambic beer fermentations, there was no Enterobacteriaceae phase and a larger variety of acetic acid bacteria were found in industrial lambic beer fermentations. Like in traditional lambic beer fermentations, Saccharomyces cerevisiae, Saccharomyces pastorianus, Dekkera bruxellensis and Pediococcus damnosus were the microorganisms responsible for the main fermentation and maturation phases. These microorganisms originated most probably from the wood of the casks and were considered as the core microbiota of lambic beer fermentations.

Microbiota and metabolites of aged bottled gueuze beers converge to the same composition.

Gueuze beers are prepared by mixing young and old lambic beers and are bottle-refermented spontaneously for aging. The present study analyzed the microbiota and metabolites present in gueuze beers that were aged between a few months and up to 17 years. Yeasts were cultivated from all beers sampled, but bacteria could not be grown from beers older than 5 years. Lactic acid and ethyl lactate concentrations increased steadily during aging, whereas ethanol concentrations remained constant. The concentrations of isoamyl acetate and ethyl decanoate decreased during the aging process. Hence, ethyl lactate and ethyl decanoate can be considered as positive and negative gueuze beer-aging metabolite biomarkers, respectively. Nevertheless, considerable bottle-to-bottle variation in the metabolite profiles was found, which hindered the generalization of the effects seen during the aging of the gueuze beers examined, but which illustrated the unique character of the lambic beers. The present results further indicate that gueuze beers are preferably aged for less than 10 years.

Effects of growth medium on matrix-assisted laser desorption-ionization time of flight mass spectra: a case study of acetic acid bacteria.

The effect of the growth medium used on the matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectra generated and its consequences for species and strain level differentiation of acetic acid bacteria (AAB) were determined by using a set of 25 strains. The strains were grown on five different culture media that yielded a total of more than 600 mass spectra, including technical and biological replicates. The results demonstrate that the culture medium can have a profound effect on the mass spectra of AAB as observed in the presence and varying signal intensities of peak classes, in particular when culture media do not sustain optimal growth. The observed growth medium effects do not disturb species level differentiation but strongly affect the potential for strain level differentiation. The data prove that a well-constructed and robust MALDI-TOF mass spectrometry identification database should comprise mass spectra of multiple reference strains per species grown on different culture media to facilitate species and strain level differentiation.

Various cold storage-backslopping cycles show the robustness of Limosilactobacillus fermentum IMDO 130101 as starter culture for Type 3 sourdough production.

Type 3 sourdoughs, which are starter culture-initiated and subsequently backslopped, are less studied than other sourdough types. Yet, they can serve as a model to assess how competitive starter culture strains for sourdough production are and how the microbial composition of such sourdoughs may evolve over time. In the present study, Limosilactobacillus fermentum IMDO 130101 was used to produce Type 3 sourdoughs, prepared from wheat and wholemeal wheat flours. Therefore, an initial fermentation of the flour-water mixture was performed at 30 °C for 48 h. This was followed by cold storage-backslopping cycles, consisting of refreshments (50 %, v/v), fermentation steps of 16 h, and storage at 4 °C each week, every three weeks, and every six weeks. The microbial dynamics (culture-dependent and -independent approaches) and metabolite dynamics were measured. In all sourdoughs produced, starter culture strain monitoring, following an amplicon sequence variant approach, showed that Liml. fermentum IMDO 130101 prevailed during one month when the sourdoughs were refreshed each week, during 24 weeks when the sourdoughs were refreshed every three weeks, and during 12 weeks when the sourdoughs were refreshed every six weeks. This suggested the competitiveness and robustness of Liml. fermentum IMDO 130101 for a considerable duration but also showed that the strain is prone to microbial interference. For instance, Levilactobacillus brevis and Pediococcus spp. prevailed upon further cold storage and backslopping. Also, although no yeasts were inoculated into the flour-water mixtures, Kazachstania unispora, Torulaspora delbrueckii, and Wickerhamomyces anomalus were the main yeast species found. They appeared after several weeks of storage and backslopping, which however indicated the importance of an interplay between LAB and yeast species in sourdoughs. The main differences among the mature sourdoughs obtained could be explained by the different flours used, the refreshment conditions applied, and the sampling time (before and after backslopping). Finally, the metabolite quantifications revealed continued metabolite production during the cold storage periods, which may impact the sourdough properties and those of the breads made thereof.

Novel Ralstonia species from human infections: improved matrix-assisted laser desorption/ionization time-of-flight mass spectrometry-based identification and analysis of antimicrobial resistance patterns.

A collection of 161 Ralstonia isolates, including 90 isolates from persons with cystic fibrosis, 27 isolates from other human clinical samples, 8 isolates from the hospital environment, 7 isolates from industrial samples, and 19 environmental isolates, was subjected to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) identification and yielded confident species level identification scores for only 62 (39%) of the isolates, including four that proved misidentified subsequently. Whole-genome sequence analysis of 32 representative isolates for which no confident MALDI-TOF MS species level identification was obtained revealed the presence of seven novel Ralstonia species, including three and four that were isolated from cystic fibrosis or other human clinical samples, respectively, and provided the basis for updating an in-house MALDI-TOF MS database. A reanalysis of all mass spectra with the updated MALDI-TOF MS database increased the percentage of isolates with confident species level identification up to 77%. The antimicrobial susceptibility of 30 isolates mainly representing novel human clinical and environmental Ralstonia species was tested toward 17 antimicrobial agents and demonstrated that the novel Ralstonia species were generally multi-resistant, yet susceptible to trimethoprim/sulfamethoxazole, ciprofloxacin, and tigecycline. An analysis of genomic antimicrobial resistance genes in 32 novel and publicly available genome sequences revealed broadly distributed beta-lactam resistance determinants.IMPORTANCEThe present study demonstrated that a commercial matrix-assisted laser desorption/ionization time-of-flight mass spectrometry identification database can be tailored to improve the identification of Ralstonia species. It also revealed the presence of seven novel Ralstonia species, including three and four that were isolated from cystic fibrosis or other human clinical samples, respectively. An analysis of minimum inhibitory concentration values demonstrated that the novel Ralstonia species were generally multi-resistant but susceptible to trimethoprim/sulfamethoxazole, ciprofloxacin, and tigecycline.

A comparative genomic analysis of Fructobacillus evanidus sp. nov. from bumble bees.

The increase in studies on bee microbiomes is prompted by concerns over global pollinator declines. Bumble bees host core and non-core microbiota which may contribute to increased lifetime fitness. The presence of Fructobacillus in the gut microbiomes of bumble bee workers, or the replacement of core symbionts with Fructobacillus bacteria, has been considered a marker of dysbiosis. A phylogenomic analysis and functional genomic characterization of the genomes of 21 Fructobacillus isolates from bumble bees demonstrated that they represented four species, i.e. Fructobacillus cardui, Fructobacillus fructosus, Fructobacillus tropaeoli, and the novel species Fructobacillus evanidus sp. nov. Our results confirmed and substantiated the presence of two phylogenetically and functionally distinct Fructobacillus species clades that differ in genome size, percentage G + C content, the number of coding DNA sequences and metabolic characteristics. Clade 1 and clade 2 species differed in amino acid and, to a lesser extent, in carbohydrate metabolism, with F. evanidus and F. tropaeoli genomes featuring a higher number of complete metabolic pathways. While Fructobacillus genomes encoded genes that allow adhesion, biofilm formation, antibacterial activity and detoxification, other bacteria isolated from the bumble bee gut appeared better equipped to co-exist with the bumble bee host. The isolation and identification of multiple Fructobacillus species from several bumble bee gut samples in the present study also argued against a specific partnership between Fructobacillus species and their bumble bee hosts.

Brytella acorum gen. nov., sp. nov., a novel acetic acid bacterium from sour beverages.

Polyphasic taxonomic and comparative genomic analyses revealed that a series of lambic beer isolates including strain LMG 32668T and the kombucha isolate LMG 32879 represent a novel species among the acetic acid bacteria, with Acidomonas methanolica as the nearest phylogenomic neighbor with a valid name. Overall genomic relatedness indices and phylogenomic and physiological analyses revealed that this novel species was best classified in a novel genus for which we propose the name Brytella acorum gen. nov., sp. nov., with LMG 32668T (=CECT 30723T) as the type strain. The B. acorum genomes encode a complete but modified tricarboxylic acid cycle, and complete pentose phosphate, pyruvate oxidation and gluconeogenesis pathways. The absence of 6-phosphofructokinase which rendered the glycolysis pathway non-functional, and an energy metabolism that included both aerobic respiration and oxidative fermentation are typical metabolic characteristics of acetic acid bacteria. Neither genome encodes nitrogen fixation or nitrate reduction genes, but both genomes encode genes for the biosynthesis of a broad range of amino acids. Antibiotic resistance genes or virulence factors are absent.

Bacterial species identification using MALDI-TOF mass spectrometry and machine learning techniques: A large-scale benchmarking study.

Today machine learning methods are commonly deployed for bacterial species identification using MALDI-TOF mass spectrometry data. However, most of the studies reported in literature only consider very traditional machine learning methods on small datasets that contain a limited number of species. In this paper we present benchmarking results on an unprecedented scale for a wide range of machine learning methods, using datasets that contain almost 100,000 spectra and more than 1000 different species. The size and the diversity of the data allow to compare three important identification scenarios that are often not distinguished in literature, i.e., identification for novel biological replicates, novel strains and novel species that are not present in the training data. The results demonstrate that in all three scenarios acceptable identification rates are obtained, but the numbers are typically lower than those reported in studies with a more limited analysis. Using hierarchical classification methods, we also demonstrate that taxonomic information is in general not well preserved in MALDI-TOF mass spectrometry data. For the novel species scenario, we apply for the first time neural networks with Monte Carlo dropout, which have shown to be successful in other domains, such as computer vision, for the detection of novel species.

Burkholderia perseverans sp. nov., a bacterium isolated from the Restinga ecosystem, is a producer of volatile and diffusible compounds that inhibit plant pathogens.

Gram-negative, aerobic, rod-shaped, non-spore-forming, motile bacteria, designated CBAS 719 T, CBAS 732 and CBAS 720 were isolated from leaf litter samples, collected in Espírito Santo State, Brazil, in 2008. Sequences of the 16S rRNA, gyrB, lepA and recA genes showed that these strains grouped with Burkholderia plantarii LMG 9035 T, Burkholderia gladioli LMG 2216 T and Burkholderia glumae LMG 2196 T in a clade of phytopathogenic Burkholderia species. Digital DNA-DNA hybridization experiments and ANI analyses demonstrated that strain CBAS 719 T represents a novel species in this lineage that is very closely related with B. plantarii. The genome sequence of the type strain is 7.57 Mbp and its G + C content is 69.01 mol%. The absence of growth on TSA medium supplemented with 3% (w/v) NaCl, citrate assimilation, ß-galactosidase (PNPG) activity, and of lipase C14 activity differentiated strain CBAS 719 T from B. plantarii LMG 9035 T, its nearest phylogenetic neighbor. Its predominant fatty acid components were C16:0, C18:1 ω7c, cyclo-C17:0 and summed feature 3 (C16:1 ω7c and/or C15:0 iso 2-OH). Based on these genotypic and phenotypic characteristics, the strains CBAS 719 T, CBAS 732 and CBAS 720 are classified in a novel Burkholderia species, for which the name Burkholderia perseverans sp. nov. is proposed. The type strain is CBAS 719 T (= LMG 31557 T = INN12T).

Burkholderia cepacia Complex Taxon K: Where to Split?

The objective of the present study was to provide an updated classification for Burkholderia cepacia complex (Bcc) taxon K isolates. A representative set of 39 taxon K isolates were analyzed through multilocus sequence typing (MLST) and phylogenomic analyses. MLST analysis revealed the presence of at least six clusters of sequence types (STs) within taxon K, two of which contain the type strains of Burkholderia contaminans (ST-102) and Burkholderia lata (ST-101), and four corresponding to the previously defined taxa Other Bcc groups C, G, H and M. This clustering was largely supported by a phylogenomic tree which revealed three main clades. Isolates of B. contaminans and of Other Bcc groups C, G, and H represented a first clade which generally shared average nucleotide identity (ANI) and average digital DNA-DNA hybridization (dDDH) values at or above the 95-96% ANI and 70% dDDH thresholds for species delineation. A second clade consisted of Other Bcc group M bacteria and of four B. lata isolates and was supported by average ANI and dDDH values of 97.2 and 76.1% within this clade and average ANI and dDDH values of 94.5 and 57.2% toward the remaining B. lata isolates (including the type strain), which represented a third clade. We therefore concluded that isolates known as Other Bcc groups C, G, and H should be classified as B. contaminans, and propose a novel species, Burkholderia aenigmatica sp. nov., to accommodate Other Bcc M and B. lata ST-98, ST-103, and ST-119 isolates. Optimized MALDI-TOF MS databases for the identification of clinical Burkholderia isolates may provide correct species-level identification for some of these bacteria but would identify most of them as B. cepacia complex. MLST facilitates species-level identification of many taxon K strains but some may require comparative genomics for accurate species-level assignment. Finally, the inclusion of Other Bcc groups C, G, and H into B. contaminans affects the phenotype of this species minimally and the proposal to classify Other Bcc group M and B. lata ST-98, ST-103, and ST-119 strains as a novel Burkholderia species is supported by a distinctive phenotype, i.e., growth at 42°C and lysine decarboxylase activity.

Introducing SPeDE: High-Throughput Dereplication and Accurate Determination of Microbial Diversity from Matrix-Assisted Laser Desorption-Ionization Time of Flight Mass Spectrometry Data.

The isolation of microorganisms from microbial community samples often yields a large number of conspecific isolates. Increasing the diversity covered by an isolate collection entails the implementation of methods and protocols to minimize the number of redundant isolates. Matrix-assisted laser desorption-ionization time-of-flight (MALDI-TOF) mass spectrometry methods are ideally suited to this dereplication problem because of their low cost and high throughput. However, the available software tools are cumbersome and rely either on the prior development of reference databases or on global similarity analyses, which are inconvenient and offer low taxonomic resolution. We introduce SPeDE, a user-friendly spectral data analysis tool for the dereplication of MALDI-TOF mass spectra. Rather than relying on global similarity approaches to classify spectra, SPeDE determines the number of unique spectral features by a mix of global and local peak comparisons. This approach allows the identification of a set of nonredundant spectra linked to operational isolation units. We evaluated SPeDE on a data set of 5,228 spectra representing 167 bacterial strains belonging to 132 genera across six phyla and on a data set of 312 spectra of 78 strains measured before and after lyophilization and subculturing. SPeDE was able to dereplicate with high efficiency by identifying redundant spectra while retrieving reference spectra for all strains in a sample. SPeDE can identify distinguishing features between spectra, and its performance exceeds that of established methods in speed and precision. SPeDE is open source under the MIT license and is available from https://github.com/LM-UGent/SPeDEIMPORTANCE Estimation of the operational isolation units present in a MALDI-TOF mass spectral data set involves an essential dereplication step to identify redundant spectra in a rapid manner and without sacrificing biological resolution. We describe SPeDE, a new algorithm which facilitates culture-dependent clinical or environmental studies. SPeDE enables the rapid analysis and dereplication of isolates, a critical feature when long-term storage of cultures is limited or not feasible. We show that SPeDE can efficiently identify sets of similar spectra at the level of the species or strain, exceeding the taxonomic resolution of other methods. The high-throughput capacity, speed, and low cost of MALDI-TOF mass spectrometry and SPeDE dereplication over traditional gene marker-based sequencing approaches should facilitate adoption of the culturomics approach to bacterial isolation campaigns.