Eupransor demetentiae gen. nov., sp. nov., a novel fructophilic lactic acid bacterium from bumble bees.

Strain LMG 33000T was isolated from a Bombus lapidarius gut sample. It shared the highest percentage 16S rRNA sequence identity, average amino acid identity, and amino acid identity of conserved genes with Convivina intestini LMG 28291T (95.86 %, 69.9 and 76.2 %, respectively), and the highest percentage OrthoANIu value with Fructobacillus fructosus DSM 20349T (71.4 %). Phylogenomic analyses by means of 107 or 120 conserved genes consistently revealed Convivina as nearest neighbour genus. The draft genome of strain LMG 33000T was 1.44 Mbp in size and had a DNA G+C content of 46.1 mol%. Genomic and physiological analyses revealed that strain LMG 33000T was a typical obligately fructophilic lactic acid bacterium that lacked the adhE and aldh genes and that did not produce ethanol during glucose or fructose metabolism. In contrast, Convivina species have the adhE and aldh genes in their genomes and produced ethanol from glucose and fructose metabolism, which is typical for heterofermentative lactic acid bacteria. Moreover, strain LMG 33000T exhibited catalase activity, an unusual characteristic among lactic acid bacteria, that is not shared with Convivina species. Given its position in the phylogenomic trees, and the difference in genomic percentage G+C content and in physiological and metabolic characteristics between strain LMG 33000T and Convivina species, we considered it most appropriate to classify strain LMG 33000T into a novel genus and species within the Lactobacillaceae family for which we propose the name Eupransor demetentiae gen. nov., sp. nov., with LMG 33000T (=CECT 30958T) as the type strain.

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.

Microbial diversity and antimicrobial susceptibility in endotracheal tube biofilms recovered from mechanically ventilated COVID-19 patients.

In patients with acute respiratory failure, mechanical ventilation through an endotracheal tube (ET) may be required to correct hypoxemia and hypercarbia. However, biofilm formation on these ETs is a risk factor for infections in intubated patients, as the ET can act as a reservoir of microorganisms that can cause infections in the lungs. As severely ill COVID-19 patients often need to be intubated, a better knowledge of the composition of ET biofilms in this population is important. In Spring 2020, during the first wave of the COVID-19 pandemic in Europe, 31 ETs were obtained from COVID-19 patients at Ghent University Hospital (Ghent, Belgium). Biofilms were collected from the ET and the biofilm composition was determined using culture-dependent (MALDI-TOF mass spectrometry and biochemical tests) and culture-independent (16S and ITS1 rRNA amplicon sequencing) approaches. In addition, antimicrobial resistance was assessed for isolates collected via the culture-dependent approach using disc diffusion for 11 antimicrobials commonly used to treat lower respiratory tract infections. The most common microorganisms identified by the culture-dependent approach were those typically found during lung infections and included both presumed commensal and potentially pathogenic microorganisms like Staphylococcus epidermidis, Enterococcus faecalis, Pseudomonas aeruginosa and Candida albicans. More unusual organisms, such as Paracoccus yeei, were also identified, but each only in a few patients. The culture-independent approach revealed a wide variety of microbes present in the ET biofilms and showed large variation in biofilm composition between patients. Some biofilms contained a diverse set of bacteria of which many are generally considered as non-pathogenic commensals, whereas others were dominated by a single or a few pathogens. Antimicrobial resistance was widespread in the isolates, e.g. 68% and 53% of all isolates tested were resistant against meropenem and gentamicin, respectively. Different isolates from the same species recovered from the same ET biofilm often showed differences in antibiotic susceptibility. Our data suggest that ET biofilms are a potential risk factor for secondary infections in intubated COVID-19 patients, as is the case in mechanically-ventilated non-COVID-19 patients.

Paraburkholderia gardini sp. nov. and Paraburkholderia saeva sp. nov.: Novel aromatic compound degrading bacteria isolated from garden and forest soil samples.

Three forest and four botanical garden top soil isolates with unique MALDI-TOF mass spectra were identified as Paraburkholderia strains closely related to Paraburkholderia sartisoli through recA gene sequence analysis. OrthoANIu, digital DNA-DNA hybridization analyses and phylogenomic analyses demonstrated that the five strains represented two new Paraburkholderia species closely related to P. sartisoli. The genome of strain LMG 31841T had a cumulative size of 6.3 Mb and a G + C content of 62.64 mol%; strain LMG 32171T had a genome size of 5.8 Mb and a G + C content of 62.91 mol%. Hemolysis on horse blood agar, beta-galactosidase and phosphoamidase activity, and assimilation of adipic acid and trisodium citrate allowed phenotypic differentiation of strains LMG 31841T, LMG 32171T and P. sartisoli LMG 24000T. An analysis of the genomic potential for aromatic compound degradation yielded additional differences among strains representing these three species, but also highlighted some discrepancies between the presence of genes and pathways, and the phenotype revealed through growth experiments using a mineral salts medium supplemented with single aromatic compounds as carbon sources. We propose to classify all isolates from the present study into two novel Paraburkholderia species, for which we propose the names Paraburkholderia gardini with LMG 32171T (=CECT 30344T) as the type strain, and Paraburkholderia saeva with LMG 31841T (=CECT 30338T) as the type strain.

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).

Network Analysis Based on Unique Spectral Features Enables an Efficient Selection of Genomically Diverse Operational Isolation Units.

Culturomics-based bacterial diversity studies benefit from the implementation of MALDI-TOF MS to remove genomically redundant isolates from isolate collections. We previously introduced SPeDE, a novel tool designed to dereplicate spectral datasets at an infraspecific level into operational isolation units (OIUs) based on unique spectral features. However, biological and technical variation may result in methodology-induced differences in MALDI-TOF mass spectra and hence provoke the detection of genomically redundant OIUs. In the present study, we used three datasets to analyze to which extent hierarchical clustering and network analysis allowed to eliminate redundant OIUs obtained through biological and technical sample variation and to describe the diversity within a set of spectra obtained from 134 unknown soil isolates. Overall, network analysis based on unique spectral features in MALDI-TOF mass spectra enabled a superior selection of genomically diverse OIUs compared to hierarchical clustering analysis and provided a better understanding of the inter-OIU relationships.

Genomics of an endemic cystic fibrosis Burkholderia multivorans strain reveals low within-patient evolution but high between-patient diversity.

Burkholderia multivorans is a member of the Burkholderia cepacia complex (Bcc), notorious for its pathogenicity in persons with cystic fibrosis. Epidemiological surveillance suggests that patients predominantly acquire B. multivorans from environmental sources, with rare cases of patient-to-patient transmission. Here we report on the genomic analysis of thirteen isolates from an endemic B. multivorans strain infecting four cystic fibrosis patients treated in different pediatric cystic fibrosis centers in Belgium, with no evidence of cross-infection. All isolates share an identical sequence type (ST-742) but whole genome analysis shows that they exhibit peculiar patterns of genomic diversity between patients. By combining short and long reads sequencing technologies, we highlight key differences in terms of small nucleotide polymorphisms indicative of low rates of adaptive evolution within patient, and well-defined, hundred kbps-long segments of high enrichment in mutations between patients. In addition, we observed large structural genomic variations amongst the isolates which revealed different plasmid contents, active roles for transposase IS3 and IS5 in the deactivation of genes, and mobile prophage elements. Our study shows limited within-patient B. multivorans evolution and high between-patient strain diversity, indicating that an environmental microdiverse reservoir must be present for this endemic strain, in which active diversification is taking place. Furthermore, our analysis also reveals a set of 30 parallel adaptations across multiple patients, indicating that the specific genomic background of a given strain may dictate the route of adaptation within the cystic fibrosis lung.

Burkholderia Bacteria Produce Multiple Potentially Novel Molecules that Inhibit Carbapenem-Resistant Gram-Negative Bacterial Pathogens.

Antimicrobial resistance in Gram-negative pathogens represents a global threat to human health. This study determines the antimicrobial potential of a taxonomically and geographically diverse collection of 263 Burkholderia (sensu lato) isolates and applies natural product dereplication strategies to identify potentially novel molecules. Antimicrobial activity is almost exclusively present in Burkholderia sensu stricto bacteria and rarely observed in the novel genera Paraburkholderia, Caballeronia, Robbsia, Trinickia, and Mycetohabitans. Fourteen isolates show a unique spectrum of antimicrobial activity and inhibited carbapenem-resistant Gram-negative bacterial pathogens. Dereplication of the molecules present in crude spent agar extracts identifies 42 specialized metabolites, 19 of which represented potentially novel molecules. The known identified Burkholderia metabolites include toxoflavin, reumycin, pyrrolnitrin, enacyloxin, bactobolin, cepacidin, ditropolonyl sulfide, and antibiotics BN-227-F and SF 2420B, as well as the siderophores ornibactin, pyochelin, and cepabactin. Following semipreparative fractionation and activity testing, a total of five potentially novel molecules are detected in active fractions. Given the molecular formula and UV spectrum, two of those putative novel molecules are likely related to bactobolins, and another is likely related to enacyloxins. The results from this study confirm and extend the observation that Burkholderia bacteria present exciting opportunities for the discovery of potentially novel bioactive molecules.

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.

Achromobacter veterisilvae sp. nov., from a mixed hydrogen-oxidizing bacteria enrichment reactor for microbial protein production.

Strain LMG 30378T was isolated from a hydrogen-oxidizing bacteria enrichment reactor inoculated with forest soil. Phylogenetic analysis based on the 16S rRNA gene sequence indicated that this strain belonged to the genus Achromobacter. Multilocus sequence analysis combined with sequence analysis of a 765 bp nrd A gene fragment both showed Achromobacter agilis LMG 3411T and Achromobacter denitrificans LMG 1231T to be the closest-related neighbours to strain LMG 30378T. Genome sequence analysis revealed a draft genome of 6.81 Mb with a G+C content of 67.2 mol%. In silico DNA-DNA hybridization with A. denitrificans LMG 1231T and A. agilis LMG 3411T showed 42.7 and 42.5% similarity, respectively, confirming that strain LMG 30378T represented a novel Achromobacter species. Phenotypic and metabolic characterization revealed acid phosphatase activity and the absence of phosphoamidase activity as distinctive features. The draft genome composes all necessary metabolic components to fix carbon dioxide and to oxidize molecular hydrogen, suggesting that strain LMG 30378T is a key organism in the enrichment reactor. Together, these data demonstrate that strain LMG 30378T represents a novel species of the genus Achromobacter, for which the name Achromobacter veterisilvae sp. nov. is proposed. The type strain is LMG 30378T (=CCUG 71558T).

Comparative Genomics of Pandoraea, a Genus Enriched in Xenobiotic Biodegradation and Metabolism.

Comparative analysis of partial gyrB, recA, and gltB gene sequences of 84 Pandoraea reference strains and field isolates revealed several clusters that included no taxonomic reference strains. The gyrB, recA, and gltB phylogenetic trees were used to select 27 strains for whole-genome sequence analysis and for a comparative genomics study that also included 41 publicly available Pandoraea genome sequences. The phylogenomic analyses included a Genome BLAST Distance Phylogeny approach to calculate pairwise digital DNA-DNA hybridization values and their confidence intervals, average nucleotide identity analyses using the OrthoANIu algorithm, and a whole-genome phylogeny reconstruction based on 107 single-copy core genes using bcgTree. These analyses, along with subsequent chemotaxonomic and traditional phenotypic analyses, revealed the presence of 17 novel Pandoraea species among the strains analyzed, and allowed the identification of several unclassified Pandoraea strains reported in the literature. The genus Pandoraea has an open pan genome that includes many orthogroups in the 'Xenobiotics biodegradation and metabolism' KEGG pathway, which likely explains the enrichment of these species in polluted soils and participation in the biodegradation of complex organic substances. We propose to formally classify the 17 novel Pandoraea species as P. anapnoica sp. nov. (type strain LMG 31117T = CCUG 73385T), P. anhela sp. nov. (type strain LMG 31108T = CCUG 73386T), P. aquatica sp. nov. (type strain LMG 31011T = CCUG 73384T), P. bronchicola sp. nov. (type strain LMG 20603T = ATCC BAA-110T), P. capi sp. nov. (type strain LMG 20602T = ATCC BAA-109T), P. captiosa sp. nov. (type strain LMG 31118T = CCUG 73387T), P. cepalis sp. nov. (type strain LMG 31106T = CCUG 39680T), P. commovens sp. nov. (type strain LMG 31010T = CCUG 73378T), P. communis sp. nov. (type strain LMG 31110T = CCUG 73383T), P. eparura sp. nov. (type strain LMG 31012T = CCUG 73380T), P. horticolens sp. nov. (type strain LMG 31112T = CCUG 73379T), P. iniqua sp. nov. (type strain LMG 31009T = CCUG 73377T), P. morbifera sp. nov. (type strain LMG 31116T = CCUG 73389T), P. nosoerga sp. nov. (type strain LMG 31109T = CCUG 73390T), P. pneumonica sp. nov. (type strain LMG 31114T = CCUG 73388T), P. soli sp. nov. (type strain LMG 31014T = CCUG 73382T), and P. terrigena sp. nov. (type strain LMG 31013T = CCUG 73381T).

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.

The BCESM genomic region contains a regulator involved in quorum sensing and persistence in Burkholderia cenocepacia J2315.

AIM: In this study, we investigated the function of BCAM0257-8-9 located in the BCESM region of the Burkholderia cenocepacia J2315 genome. MATERIALS & METHODS: Differential RNA sequencing was used to determine transcription start sites. The phenotype of overexpression mutants was studied and a transcriptome analysis of the BCAM0258 overexpression mutant was performed. RESULTS: BCAM0257 and BCAM0258 were identified as belonging to an operon, positively regulated by BCAM0259. We found that this operon is involved in persistence and that BCAM0258 functions as a regulator influencing quorum sensing and activating pathways related to iron acquisition and biofilm formation. Overexpression of BCAM0257 increased virulence. CONCLUSION: The BCESM genomic region contains an operon that contributes to quorum sensing and is involved in persistence, biofilm formation and virulence. BCAM0257-8-9 is found in all sequenced B. cenocepacia ET12 genomes and these results may help explain why infections with strains of the B. cenocepacia ET12 lineage are difficult to treat.

Genomewide screening for genes involved in biofilm formation and miconazole susceptibility in Saccharomyces cerevisiae.

Infections related to fungal biofilms are difficult to treat due to the reduced susceptibility of sessile cells to most antifungal agents. Previous research has shown that 1-10% of sessile Candida cells survive treatment with high doses of miconazole (a fungicidal imidazole). The aim of this study was to identify genes involved in fungal biofilm formation and to unravel the mechanisms of resistance of these biofilms to miconazole. To this end, a screening of a Saccharomyces cerevisiae deletion mutant bank was carried out. Our results revealed that genes involved in peroxisomal transport and the biogenesis of the respiratory chain complex IV play an essential role in biofilm formation. On the other hand, genes involved in transcription and peroxisomal and mitochondrial organization seem to highly influence the susceptibility to miconazole of yeast biofilms. Additionally, our data confirm previous findings on genes involved in biofilm formation and in general stress responses. Our data suggest the involvement of peroxisomes in biofilm formation and miconazole resistance in fungal biofilms.

Antibacterial activity of a lectin-like Burkholderia cenocepacia protein.

Bacteriocins of the LlpA family have previously been characterized in the γ-proteobacteria Pseudomonas and Xanthomonas. These proteins are composed of two MMBL (monocot mannose-binding lectin) domains, a module predominantly and abundantly found in lectins from monocot plants. Genes encoding four different types of LlpA-like proteins were identified in genomes from strains belonging to the Burkholderia cepacia complex (Bcc) and the Burkholderia pseudomallei group. A selected recombinant LlpA-like protein from the human isolate Burkholderia cenocepacia AU1054 displayed narrow-spectrum genus-specific antibacterial activity, thus representing the first functionally characterized bacteriocin within this ß-proteobacterial genus. Strain-specific killing was confined to other members of the Bcc, with mostly Burkholderia ambifaria strains being susceptible. In addition to killing planktonic cells, this bacteriocin also acted as an antibiofilm agent.