Tobias Olofsson

Symbionts as major modulators of insect health: Lactic Acid Bacteria and honeybees

Lactic acid bacteria (LAB) are well recognized beneficial host-associated members of the microbiota of humans and animals. Yet LAB-associations of invertebrates have been poorly characterized and their functions remain obscure. Here we show that honeybees possess an abundant, diverse and ancient LAB microbiota in their honey crop with beneficial effects for bee health, defending them against microbial threats. Our studies of LAB in all extant honeybee species plus related apid bees reveal one of the largest collections of novel species from the genera Lactobacillus and Bifidobacterium ever discovered within a single insect and suggest a long (>80 mya) history of association. Bee associated microbiotas highlight Lactobacillus kunkeei as the dominant LAB member. Those showing potent antimicrobial properties are acquired by callow honey bee workers from nestmates and maintained within the crop in biofilms, though beekeeping management practices can negatively impact this microbiota. Prophylactic practices that enhance LAB, or supplementary feeding of LAB, may serve in integrated approaches to sustainable pollinator service provision. We anticipate this microbiota will become central to studies on honeybee health, including colony collapse disorder, and act as an exemplar case of insect-microbe symbiosis.

Detection and Identification of a Novel Lactic Acid Bacterial Flora Within the Honey Stomach of the Honeybee Apis mellifera.

This investigation concerned the question of whether honeybees collect bacteria that are beneficial for humans from the flowers that contribute to formation of their honey. Bacteria originating from the types of flowers involved, and found in different anatomic parts of the bees, in larvae, and in honey of different types, were sampled during a 2-year period. 16S rRNA sequencing of isolates and clones was employed. A novel bacterial flora composed of lactic acid bacteria (LAB) of the genera Lactobacillus and Bifidobacterium, which originated in the honey stomach of the honeybee, was discovered. It varied with the sources of nectar and the presence of other bacterial genera within the honeybee and ended up eventually in the honey. It appeared that honeybees and the novel LAB flora may have evolved in mutual dependence on one another. It was suggested that honey be considered a fermented food product because of the LAB involved in honey production. The findings are seen as having clear implications for future research in the area, as providing a better understanding the health of honeybees and of their production and storage of honey, and as having clear relevance for future honeybee and human probiotics.

Novel lactic acid bacteria inhibiting Paenibacillus larvae in honey bee larvae

We evaluated the antagonistic effects of newly identified lactic acid bacteria (LAB) in the genera Lactobacillus and Bifidobacterium, originating from the honey stomach, on the honey bee pathogen, Paenibacillus larvae. We used inhibition assays on agar plates and honey bee larval bioassays to investigate the effects of honey bee LAB on P. larvae growth in vitro and on AFB infection in vivo. The individual LAB phylotypes showed different inhibition properties against P. larvae growth on agar plates, whereas a combination of all eleven LAB phylotypes resulted in a total inhibition (no visible growth) of P. larvae. Adding the LAB mixture to the larval food significantly reduced the number of AFB infected larvae in exposure bioassays. The results demonstrate that honey bee specific LAB possess beneficial properties for honey bee health. Possible benefits to honey bee health by enhancing growth of LAB or by applying LAB to honey bee colonies should be further investigated.ZusammenfassungDie Amerikanische Faulbrut (AFB) ist eine Krankheit, die junge Honigbienenlarven befällt. Sie ist eine der schädlichsten Bienenkrankheiten und hat große ökonomische Bedeutung für die Imkerei weltweit. Der Erreger der AFB ist das sporenbildende Bakterium Paenibacillus larvae, das den Mitteldarm junger Larven durch kontaminiertes Futter befällt. Die Besiedelung des larvalen Mitteldarms stellt einen der Schlüsselfaktoren für die Pathogenese von P. larvae dar und bestimmte Zusammensetzungen der Mikroflora des Darmes könnten das Wachstum des Krankheitserregers unterdrücken. Kürzlich wurden eine neue Flora von Milchsäurebakterien (LAB) der Gattungen Lactobacillus und Bifidobacterium aus dem Honigmagen der Bienen beschrieben. LAB sind zwar bekannt für die Produktion von antimikrobiellen Substanzen, jedoch gibt es Variationen bezüglich der nutzbringenden Eigenschaften zwischen Arten und Gattungen. In dieser Untersuchung wurde der antagonistische Effekt der Honigbienen-LAB auf P. larvae beurteilt. Wir verwendeten Hemmtests auf Agarplatten und Biotests mit Honigbienenlarven, um diese Effekte zu untersuchen. Die individuellen LAB-Phylotypen zeigten unterschiedliche Hemmeigenschaften gegenüber auf Agarplatten wachsenden P. larvae, während eine Kombination aller 11 LAB-Phylotypen sogar eine totale Hemmung (kein sichtbares Wachstum mehr) von P. larvae bewirkte. Eine Zugabe des LAB-Mix zum Larvenfutter reduzierte signifikant die Anzahl an AFB-infizierten Larven im Biotest.Die Ergebnisse zeigen, dass die für Honigbienen spezifischen LAB nutzbringende Eigenschaften für die Bienengesundheit besitzen. Der mögliche Nutzen einer Applikation von LAB in Bienenvölkern sollte untersucht werden.

The lactic acid bacteria involved in the production of bee pollen and bee bread

Summary Recently a large flora of lactic acid bacteria (LAB) was identified in the honey stomach of the honey bee Apis mellifera. In this study, the presence of this flora in bee pollen and bee bread was investigated. Pollen was collected from the legs of honey bees, and both two week old and two month old bee bread were also obtained for the study. Bacterial isolates cultivated from these bee products were identified using 16S rRNA gene analyzes. The majority of the honey stomach LAB flora was recovered in a viable state from both the pollen and the two week old bee bread, but not from the two month old bee bread. It is demonstrated for the first time that bee bread is probably fermented by the honey stomach LAB flora that has been added to the pollen via regurgitated nectar from the honey stomach. This discovery helps to explain how honey bees standardize the production of bee bread and how it is stored. The presence of the honey stomach LAB and its antimicrobial substances in bee bread also suggests a possible role in the defence against honey bee diseases since the bee bread is consumed by both the larvae and the adult bees.

Lactobacillus apinorum sp. nov., Lactobacillus mellifer sp. nov., Lactobacillus mellis sp. nov., Lactobacillus melliventris sp. nov., Lactobacillus kimbladii sp. nov., Lactobacillus helsingborgensis sp. nov. and Lactobacillus kullabergensis sp. nov., isolated from the honey stomach of the honeybee Apis mellifera

We previously discovered a symbiotic lactic acid bacterial (LAB) microbiota in the honey stomach of the honeybee Apis mellifera. The microbiota was composed of several phylotypes of Bifidobacterium and Lactobacillus. 16S rRNA gene sequence analyses and phenotypic and genetic characteristics revealed that the phylotypes isolated represent seven novel species. One grouped with Lactobacillus kunkeei and the others belong to the Lactobacillus buchneri and Lactobacillus delbrueckiisubgroups of Lactobacillus. We propose the names Lactobacillus apinorum sp. nov., Lactobacillus mellifer sp. nov., Lactobacillus mellis sp. nov., Lactobacillus melliventris sp. nov., Lactobacillus kimbladii sp. nov., Lactobacillus helsingborgensis sp. nov. and Lactobacillus kullabergensis sp. nov. for these novel species, with the respective type strains being Fhon13NT ( = DSM 26257T = CCUG 63287T), Bin4NT ( = DSM 26254T = CCUG 63291T), Hon2NT ( = DSM 26255T = CCUG 63289T), Hma8NT ( = DSM 26256T = CCUG 63629T), Hma2NT ( = DSM 26263T = CCUG 63633T), Bma5NT ( = DSM 26265T = CCUG 63301T) and Biut2NT ( = DSM 26262T = CCUG 63631T).

The bacterial flora of vacuum-packed cold-smoked salmon stored at 7 degrees C, identified by direct 16S rRNA gene analysis and pure culture technique.

Aims:  The indigenous flora of freshly chilled cold‐smoked salmon just after the vacuum packaging, and the spoilage flora after storage, in vacuum package at 7°C for 19 days, were to be investigated with two different sampling strategies.

Impact of malignant stem cell burden on therapy outcome in newly diagnosed chronic myeloid leukemia patients

Chronic myeloid leukemia (CML) stem cells appear resistant to tyrosine kinase inhibitors (TKIs) in vitro, but their impact and drug sensitivity in vivo has not been systematically assessed. We prospectively analyzed the proportion of Philadelphia chromosome-positive leukemic stem cells (LSCs, Ph+CD34+CD38−) and progenitor cells (LPCs, Ph+CD34+CD38+) from 46 newly diagnosed CML patients both at the diagnosis and during imatinib or dasatinib therapy (ClinicalTrials.gov NCT00852566). At diagnosis, the proportion of LSCs varied markedly (1–100%) between individual patients with a significantly lower median value as compared with LPCs (79% vs 96%, respectively, P=0.0001). The LSC burden correlated with leukocyte count, spleen size, hemoglobin and blast percentage. A low initial LSC percentage was associated with less therapy-related hematological toxicity and superior cytogenetic and molecular responses. After initiation of TKI therapy, the LPCs and LSCs rapidly decreased in both therapy groups, but at 3 months time point the median LPC level was significantly lower in dasatinib group compared with imatinib patients (0.05% vs 0.68%, P=0.032). These data detail for the first time the prognostic significance of the LSC burden at diagnosis and show that in contrast to in vitro data, TKI therapy rapidly eradicates the majority of LSCs in patients.

A scientific note on the lactic acid bacterial flora in honeybees in the USA - A comparison with bees from Sweden

Recently, it was discovered by Olofsson and Vasquez (2008) that a novel flora composed of lactic acid bacteria (LAB) of the genera Lactobacillus and Bifidobacterium (Fig. 1), exists in the honey stomach of the honeybee Apis mellifera. The ten different flora members varied numerically with the sources of nectar and the presence of other bacterial genera within the honeybee. Closely related phylotypes of some of the lactic acid bacteria have been encountered before in previous work (Scardovi and Trovatelli, 1969; Jeyaprakash et al., 2003; Babendreier et al., 2007) but only in the intestines of honeybees. It appeared that honeybees and the novel LAB flora evolved in mutual dependence on one another, the LAB obtaining a niche in which nutrients were available, the honeybees and their honey in turn being protected by the LAB from harmful microorganisms. In addition, four novel bacterial phylotypes belonging to the family Pasteurellaceae, were discovered (Fig. 1). In other studies, (Jeyaprakash et al., 2003; Babendreier et al., 2007) clones with high sequence similarities have been found (Fig. 1), but again only in the intestines of honeybees. By comparison, honey stomachs of honeybees (Apis mellifera and A. m. scutellata) collected in the USA were sampled. During spring, honeybees were foraging on flowers that were in bloom (see Tab. I)

Proteins of novel lactic acid bacteria from Apis mellifera mellifera: an insight into the production of known extra-cellular proteins during microbial stress

BackgroundLactic acid bacteria (LAB) has been considered a beneficial bacterial group, found as part of the microbiota of diverse hosts, including humans and various animals. However, the mechanisms of how hosts and LAB interact are still poorly understood. Previous work demonstrates that 13 species of Lactobacillus and Bifidobacterium from the honey crop in bees function symbiotically with the honeybee. They protect each other, their hosts, and the surrounding environment against severe bee pathogens, bacteria, and yeasts. Therefore, we hypothesized that these LAB under stress, i.e. in their natural niche in the honey crop, are likely to produce bioactive substances with antimicrobial activity.ResultsThe genomic analysis of the LAB demonstrated varying genome sizes ranging from 1.5 to 2.2 mega-base pairs (Mbps) which points out a clear difference within the protein gene content, as well as specialized functions in the honeybee microbiota and their adaptation to their host. We demonstrate a clear variation between the secreted proteins of the symbiotic LAB when subjected to microbial stressors. We have identified that 10 of the 13 LAB produced extra-cellular proteins of known or unknown function in which some are arranged in interesting putative operons that may be involved in antimicrobial action, host interaction, or biofilm formation. The most common known extra-cellular proteins secreted were enzymes, DNA chaperones, S-layer proteins, bacteriocins, and lysozymes. A new bacteriocin may have been identified in one of the LAB symbionts while many proteins with unknown functions were produced which must be investigated further.ConclusionsThe 13 LAB symbionts likely play different roles in their natural environment defending their niche and their host and participating in the honeybee’s food production. These roles are partly played through producing extracellular proteins on exposure to microbial stressors widely found in natural occurring flowers. Many of these secreted proteins may have a putative antimicrobial function. In the future, understanding these processes in this complicated environment may lead to novel applications of honey crop LAB proteins.

Extensive intra-phylotype diversity in lactobacilli and bifidobacteria from the honeybee gut

BackgroundIn the honeybee Apis mellifera, the bacterial gut community is consistently colonized by eight distinct phylotypes of bacteria. Managed bee colonies are of considerable economic interest and it is therefore important to elucidate the diversity and role of this microbiota in the honeybee. In this study, we have sequenced the genomes of eleven strains of lactobacilli and bifidobacteria isolated from the honey crop of the honeybee A. mellifera.ResultsSingle gene phylogenies confirmed that the isolated strains represent the diversity of lactobacilli and bifidobacteria in the gut, as previously identified by 16S rRNA gene sequencing. Core genome phylogenies of the lactobacilli and bifidobacteria further indicated extensive divergence between strains classified as the same phylotype. Phylotype-specific protein families included unique surface proteins. Within phylotypes, we found a remarkably high level of gene content diversity. Carbohydrate metabolism and transport functions contributed up to 45% of the accessory genes, with some genomes having a higher content of genes encoding phosphotransferase systems for the uptake of carbohydrates than any previously sequenced genome. These genes were often located in highly variable genomic segments that also contained genes for enzymes involved in the degradation and modification of sugar residues. Strain-specific gene clusters for the biosynthesis of exopolysaccharides were identified in two phylotypes. The dynamics of these segments contrasted with low recombination frequencies and conserved gene order structures for the core genes. Hits for CRISPR spacers were almost exclusively found within phylotypes, suggesting that the phylotypes are associated with distinct phage populations.ConclusionsThe honeybee gut microbiota has been described as consisting of a modest number of phylotypes; however, the genomes sequenced in the current study demonstrated a very high level of gene content diversity within all three described phylotypes of lactobacilli and bifidobacteria, particularly in terms of metabolic functions and surface structures, where many features were strain-specific. Together, these results indicate niche differentiation within phylotypes, suggesting that the honeybee gut microbiota is more complex than previously thought.