Large-scale cultivation of the bumblebee gut microbiota reveals an underestimated bacterial species diversity capable of pathogen inhibition.

A total of 1940 isolates from gut samples of 60 bumblebees representing Bombus pascuorum, Bombus terrestris, Bombus lucorum and Bombus lapidarius was collected and identified through state-of the-art taxonomic methods. The bacterial species diversity in these Bombus species exceeded that suggested by phylotype analysis through 16S rRNA amplicon sequencing, and revealed that B. pascuorum and B. terrestris had a unique microbiota composition, each. Representatives of most phylotypes reported earlier and detected in the present study were effectively isolated, and included several novel bacterial taxa and species reported for the first time in the bumblebee gut. Isolates were screened in pectin degradation assays and growth inhibition assays against the honeybee pathogens Paenibacillus larvae, Melissococcus plutonius and Ascosphaera apis and the bumblebee parasite Crithidia bombi. While inhibitory activity against each of these pathogens was observed, only one single culture was able to degrade pectin and polygalacturonic acid in vitro. The availability of accurately identified microbial isolates will facilitate future evaluation of the functional potential of the bumblebee gut microbiota.

Apibacter mensalis sp. nov.: a rare member of the bumblebee gut microbiota.

Isolates LMG 28357T (=R-53146T) and LMG 28623 were obtained from gut samples of Bombus lapidarius bumblebees caught in Ghent, Belgium. They had identical 16S rRNA gene sequences which were 95.7 % identical to that of Apibacter adventoris wkB301T, a member of the family Flavobacteriaceae. Both isolates had highly similar matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS and randomly amplified polymorphic DNA (RAPD) profiles. A draft genome sequence was obtained for strain LMG 28357T (Gold ID Gp0108260); its DNA G+C content was 30.4%, which is within the range reported for members of the family Flavobacteriaceae (27 to 56 mol%) and which is similar to that of the type strain of A. adventoris (29.0 mol%). Whole-cell fatty acid methyl ester analysis of strain LMG 28357T revealed many branched-chain fatty acids, a typical characteristic of bacteria of the family Flavobacteriaceae and a profile that was similar to that reported for A. adventoris wkB301T. MK6 was the major respiratory quinone, again conforming to bacteria of the family Flavobacteriaceae. The isolates LMG 28357T and LMG 28623 could be distinguished from A. adventoris strains through their oxidase activity. On the basis of phylogenetic, genotypic and phenotypic data, we propose to classify both isolates as representatives of a novel species of the genus Apibacter, Apibacter mensalis sp. nov., with LMG 28357T (=DSM 100903T=R-53146T) as the type strain.

Bombella intestini gen. nov., sp. nov., an acetic acid bacterium isolated from bumble bee crop.

In the frame of a bumble bee gut microbiota study, acetic acid bacteria (AAB) were isolated using a combination of direct isolation methods and enrichment procedures. MALDI-TOF MS profiling of the isolates and a comparison of these profiles with profiles of established AAB species identified most isolates as Asaia astilbis or as 'Commensalibacter intestini', except for two isolates (R-52486 and LMG 28161(T)) that showed an identical profile. A nearly complete 16S rRNA gene sequence of strain LMG 28161(T) was determined and showed the highest pairwise similarity to Saccharibacter floricola S-877(T) (96.5%), which corresponded with genus level divergence in the family Acetobacteraceae. Isolate LMG 28161(T) was subjected to whole-genome shotgun sequencing; a 16S-23S rRNA internal transcribed spacer (ITS) sequence as well as partial sequences of the housekeeping genes dnaK, groEL and rpoB were extracted for phylogenetic analyses. The obtained data confirmed that this isolate is best classified into a new genus in the family Acetobacteraceae. The DNA G+C content of strain LMG 28161(T) was 54.9 mol%. The fatty acid compositions of isolates R-52486 and LMG 28161(T) were similar to those of established AAB species [with C18:1ω7c (43.1%) as the major component], but the amounts of fatty acids such as C19:0 cyclo ω8c, C14:0 and C14:0 2-OH enabled to differentiate them. The major ubiquinone was Q-10. Both isolates could also be differentiated from the known genera of AAB by means of biochemical characteristics, such as their inability to oxidize ethanol to acetic acid, negligible acid production from melibiose, and notable acid production from d-fructose, sucrose and d-mannitol. In addition, they produced 2-keto-d-gluconate, but not 5-keto-d-gluconate from d-glucose. Therefore, the name Bombella intestini gen nov., sp. nov. is proposed for this new taxon, with LMG 28161(T) ( =DSM 28636(T) =R-52487(T)) as the type strain of the type species.

Bifidobacterium commune sp. nov. isolated from the bumble bee gut.

Bifidobacteria were isolated from the gut of Bombus lapidarius, Bombus terrestris and Bombus hypnorum bumble bees by direct isolation on modified trypticase phytone yeast extract agar. The MALDI-TOF MS profiles of four isolates (LMG 28292(T), R-53560, R-53124, LMG 28626) were found to be identical and did not cluster with the profiles of established Bifidobacterium species. Analysis of the 16S rRNA gene sequence of strain LMG 28292(T) revealed that LMG 28292(T) is most closely related to the Bifidobacterium bohemicum type strain (96.8%), which was also isolated from bumble bee gut specimens. The hsp60 gene of strain LMG 28292(T) shows 85.8% sequence similarity to that of the B. bohemicum type strain. The (GTG)5-PCR profiles and the hsp60 sequences of all four isolates were indistinguishable; however, three different phenotypes were observed among the four isolates by means of the API 50CHL microtest system. Based on the phylogenetic, genotypic and phenotypic data, we propose to classify the four isolates within the novel species Bifidobacterium commune sp. nov., with LMG 28292(T) (= DSM 28792(T)) as the type strain.

Novel lactic acid bacteria isolated from the bumble bee gut: Convivina intestini gen. nov., sp. nov., Lactobacillus bombicola sp. nov., and Weissella bombi sp. nov.

Twelve isolates of lactic acid bacteria (LAB) were obtained in the course of a bumble bee gut microbiota study and grouped into four matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry clusters. Comparative 16S rRNA gene sequence analysis revealed that cluster 1 isolates, represented by strain LMG 28288(T), are most closely related to Lactobacillus apis (97.0% sequence similarity to that of L. apis LMG 26964(T)). Cluster 2 isolates represented by strain LMG 28290(T) are most closely related to Weissella hellenica (99.6% sequence similarity to that of W. hellenica LMG 15125(T)). The single cluster 3 and 4 isolates had identical 16S rRNA gene sequences which were 94.8% similar to that of Leuconostoc mesenteroides subsp. mesenteroides LMG 6893(T), their nearest phylogenetic neighbour. A polyphasic taxonomic study additionally including comparative pheS sequence analysis, DNA-DNA hybridization experiments, DNA G+C content analysis, (GTG)5-PCR fingerprinting and a biochemical characterization, demonstrated that cluster 1 isolates represent a novel Lactobacillus species for which we propose the name Lactobacillus bombicola sp. nov. with LMG 28288(T) (= DSM 28793(T)) as the type strain; and that cluster 2 isolates represent a novel Weissella species for which we propose the name Weissella bombi sp. nov. with LMG 28290(T) (= DSM 28794(T)) as the type strain. Cluster 3 and 4 isolates, in contrast, represented a very distinct, novel taxon that could be distinguished from members of the genera Leuconostoc and Fructobacillus, its nearest phylogenetic neighbours, by its cellular morphology, non-fructophilic metabolism and DNA G+C content. We therefore classify both isolates into a novel species representing a novel LAB genus for which the name Convivina intestini gen. nov., sp. nov. is proposed with LMG 28291(T) (= DSM 28795(T)) as the type strain.

Enterococcus bulliens sp. nov., a novel lactic acid bacterium isolated from camel milk.

Four lactic acid bacteria isolates obtained from fresh dromedary camel milk produced in Dakhla, a city in southern Morocco, were characterised in order to determine their taxonomic position. The four isolates had highly similar MALDI-TOF MS and RAPD fingerprints and identical 16S rRNA gene sequences. Comparative sequence analysis revealed that the 16S rRNA gene sequence of the four isolates was most similar to that of Enterococcus sulfureus ATCC 49903(T) and Enterococcus italicus DSM 15952(T) (99.33 and 98.59% similarity, respectively). However, sequence analysis of the phenylalanyl-tRNA synthase (pheS), RNA polymerase (rpoA) and ATP synthase (atpA) genes revealed that the taxon represented by strain LMG 28766(T) was well separated from E. sulfureus LMG 13084(T) and E. italicus LMG 22039(T), which was further confirmed by DNA-DNA hybridization values that were clearly below the species demarcation threshold. The novel taxon was easily differentiated from its nearest neighbour species through sequence analysis of protein encoding genes, MALDI-TOF mass spectrometry and multiple biochemical tests, but had a similar percentage G+C content of about 39%. We therefore propose to formally classify these isolates as Enterococcus bulliens sp. nov., with LMG 28766(T) (=CCMM B1177(T)) 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.

Phylogenomic Analyses of Snodgrassella Isolates from Honeybees and Bumblebees Reveal Taxonomic and Functional Diversity.

Snodgrassella is a genus of Betaproteobacteria that lives in the gut of honeybees (Apis spp.) and bumblebees (Bombus spp). It is part of a conserved microbiome that is composed of a few core phylotypes and is essential for bee health and metabolism. Phylogenomic analyses using whole-genome sequences of 75 Snodgrassella strains from 4 species of honeybees and 14 species of bumblebees showed that these strains formed a monophyletic lineage within the Neisseriaceae family, that Snodgrassella isolates from Asian honeybees diverged early from the other species in their evolution, that isolates from honeybees and bumblebees were well separated, and that this genus consists of at least seven species. We propose to formally name two new Snodgrassella species that were isolated from bumblebees: i.e., Snodgrassella gandavensis sp. nov. and Snodgrassella communis sp. nov. Possible evolutionary scenarios for 107 species- or group-specific genes revealed very limited evidence for horizontal gene transfer. Functional analyses revealed the importance of small proteins, defense mechanisms, amino acid transport and metabolism, inorganic ion transport and metabolism and carbohydrate transport and metabolism among these 107 specific genes. IMPORTANCE The microbiome of honeybees (Apis spp.) and bumblebees (Bombus spp.) is highly conserved and represented by few phylotypes. This simplicity in taxon composition makes the bee's microbiome an emergent model organism for the study of gut microbial communities. Since the description of the Snodgrassella genus, which was isolated from the gut of honeybees and bumblebees in 2013, a single species (i.e., Snodgrassella alvi), has been named. Here, we demonstrate that this genus is actually composed of at least seven species, two of which (Snodgrassella gandavensis sp. nov. and Snodgrassella communis sp. nov.) are formally described and named in the present publication. We also report the presence of 107 genes specific to Snodgrassella species, showing notably the importance of small proteins and defense mechanisms in this genus.

Healthcare-Associated COVID-19 across Five Pandemic Waves: Prediction Models and Genomic Analyses.

BACKGROUND: Healthcare-associated SARS-CoV-2 infections need to be explored further. Our study is an analysis of hospital-acquired infections (HAIs) and ambulatory healthcare workers (aHCWs) with SARS-CoV-2 across the pandemic in a Belgian university hospital. METHODS: We compared HAIs with community-associated infections (CAIs) to identify the factors associated with having an HAI. We then performed a genomic cluster analysis of HAIs and aHCWs. We used this alongside the European Centre for Disease Control (ECDC) case source classifications of an HAI. RESULTS: Between March 2020 and March 2022, 269 patients had an HAI. A lower BMI, a worse frailty index, lower C-reactive protein (CRP), and a higher thrombocyte count as well as death and length of stay were significantly associated with having an HAI. Using those variables to predict HAIs versus CAIs, we obtained a positive predictive value (PPV) of 83.6% and a negative predictive value (NPV) of 82.2%; the area under the ROC was 0.89. Genomic cluster analyses and representations on epicurves and minimal spanning trees delivered further insights into HAI dynamics across different pandemic waves. The genomic data were also compared with the clinical ECDC definitions for HAIs; we found that 90.0% of the 'definite', 87.8% of the 'probable', and 70.3% of the 'indeterminate' HAIs belonged to one of the twenty-two COVID-19 genomic clusters we identified. CONCLUSIONS: We propose a novel prediction model for HAIs. In addition, we show that the management of nosocomial outbreaks will benefit from genome sequencing analyses.

Gilliamella intestini sp. nov., Gilliamella bombicola sp. nov., Gilliamella bombi sp. nov. and Gilliamella mensalis sp. nov.: Four novel Gilliamella species isolated from the bumblebee gut.

Spectra of five isolates (LMG 28358T, LMG 29879T, LMG 29880T, LMG 28359T and R-53705) obtained from gut samples of wild bumblebees of Bombus pascuorum, Bombus lapidarius and Bombus terrestris were grouped into four MALDI-TOF MS clusters. RAPD analysis revealed an identical DNA fingerprint for LMG 28359T and R-53705 which also grouped in the same MALDI-TOF MS cluster, while different DNA fingerprints were obtained for the other isolates. Comparative 16S rRNA gene sequence analysis of the four different strains identified Gilliamella apicola NCIMB 14804T as nearest neighbour species. Average nucleotide identity values of draft genome sequences of the four isolates and of G. apicola NCIMB 14804T were below the 96% threshold value for species delineation and all four strains and G. apicola NCIMB 14804T were phenotypically distinct. Together, the draft genome sequences and phylogenetic and phenotypic data indicate that the four strains represent four novel Gilliamella species for which we propose the names Gilliamella intestini sp. nov., with LMG 28358T as the type strain, Gilliamella bombicola sp. nov., with LMG 28359T as the type strain, Gilliamella bombi sp. nov., with LMG 29879T as the type strain and Gilliamella mensalis sp. nov., with LMG 29880T as the type strain.

Extensive cultivation of soil and water samples yields various pathogens in patients with cystic fibrosis but not Burkholderia multivorans.

BACKGROUND: While the epidemiology of Burkholderia cepacia complex (Bcc) bacteria in cystic fibrosis (CF) patients suggests that Burkholderia multivorans is acquired from environmental sources, this species has rarely been isolated from soil and water samples. METHODS: Multiple isolation strategies were applied to water and soil samples that were previously shown to be B. multivorans PCR positive. These included direct plating and liquid enrichment procedures and the use of selective media, acclimatizing recovery and co-cultivation with CF sputum. MALDI-TOF mass spectrometry and sequence analysis of 16S rRNA and housekeeping genes were used to identify all isolates. RESULTS: None of the approaches yielded B. multivorans isolates. Other Burkholderia species, several Gram-negative non-fermenting bacteria (including Cupriavidus, Inquilinus, Pandoraea, Pseudomonas and Stenotrophomonas) and rapidly growing mycobacteria (including Mycobacterium chelonae) were all isolated from water and soil samples. CONCLUSIONS: The use of Bcc isolation media yielded a surprisingly wide array of rare but often clinically relevant CF pathogens, confirming that soil and water are reservoirs of these infectious agents.