Lambic sour beers are the products of a spontaneous fermentation that lasts for one to three years before bottling. The present study determined the microbiota involved in the fermentation of lambic beers by sampling two fermentation batches during two years in the most traditional lambic brewery of Belgium, using culture-dependent and culture-independent methods. From 14 samples per fermentation, over 2000 bacterial and yeast isolates were obtained and identified. Although minor variations in the microbiota between casks and batches and a considerable species diversity were found, a characteristic microbial succession was identified. This succession started with a dominance of Enterobacteriaceae in the first month, which were replaced at 2 months by Pediococcus damnosus and Saccharomyces spp., the latter being replaced by Dekkera bruxellensis at 6 months fermentation duration.

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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/SPeDE. IMPORTANCE 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.

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.

Strain NGRI 0510QT , isolated from ryegrass silage, was recently classified as a representative of a novel Pediococcus species, Pediococcus lolii Doi et al. 2009. It was deposited in the DSMZ and JCM culture collections as DSM 19927 T and JCM 15055 T , respectively. A polyphasic taxonomic study, including matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, pheS and 16S rRNA gene sequence analysis, fluorescent amplified fragment length polymorphism and DNA-DNA hybridization, was used to prove that both subcultures of the type, and only, strain of this species are strains of Pediococcus acidilactici.In 2009, Doi and colleagues reported on a novel strain belonging to the genus Pediococcus, NGRI 0510QT , for which they proposed the name Pediococcus lolii (Doi et al., 2009). The description was based on a single strain isolated from ryegrass silage that was deposited in the DSMZ and JCM culture collections as DSM 19927 T and JCM 15055 T , respectively. Their study revealed that the strain exhibited distinct phenotypic characteristics, divergent sequences of the 16S rRNA gene and the 16S-23S rRNA intergenic spacer region, and low rates of DNA-DNA hybridization in comparison with the type strains of Pediococcus acidilactici DSM 20284 T (5LMG 11384 T ) and Pediococcus pentosaceus DSM 20336 T (5LMG 11488 T ).The present study was initiated upon analysis of the P. lolii type strain accessioned from the JCM culture collection, JCM 15055 T (5LMG 25667 T ), by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), which failed to discriminate P. lolii LMG 25667T from P. acidilactici strains. Subsequently, DSM 19927 T (5LMG 27029 T ) was accessioned from the DSMZ culture collection and the polyphasic taxonomic study described below was performed.All strains were grown on MRS agar (Oxoid) at 28 uC in anaerobic atmosphere, except strain P. acidilactici LMG 11384T , which was cultured in an aerobic atmosphere. Prior to MALDI-TOF MS analysis, strains were subcultured twice. Five to ten milligrams of wet cells were suspended in MilliQ-water comprising 75 % pure ethanol. Subsequently, formic acid and acetonitrile were added in a 1 : 1 (v/v) ratio to the bacterial cell pellet. After shaking vigorously, 1 ml supernatant (5the cell extract) was spotted onto a MALDI-TOF MS stainless steel target plate. Spots were overlaid with 1 ml matrix, which consisted of 5 mg acyano-4-hydroxycinnamic acid dissolved in 1 ml acetonitrile/trifluoroacetic acid/MilliQ-solvent (50 : 2 : 48). Prior to analysis, the mass spectrometer was externally calibrated using a peptide mixture of adrenocorticotropic hormone (fragment 18-39) (Sigma-Aldrich), insulin (Sigma-Aldrich), ubiquitin (Sigma-Aldrich), cytochrome C (Sigma-Aldrich) and myoglobin (Sigma-Aldrich). A 4800 Plus MALDI TOF/TOF Analyser (Applied Biosystems) was used in linear mode and covered a mass range of 2-20 kDa. The mass spectrometer used a 200 Hz frequency tripled Nd : YAG laser, operating at a wavelength of 355 nm. Generated ...