Feeding Honeybee Colonies with Honeybee-Specific Lactic Acid Bacteria (Hbs-LAB) Does Not Affect Colony-Level Hbs-LAB Composition or Paenibacillus larvae Spore Levels, Although American Foulbrood Affected Colonies Harbor a More Diverse Hbs-LAB Community.

The main current methods for controlling American Foulbrood (AFB) in honeybees, caused by the bacterial pathogen Paenibacillus larvae, are enforced incineration or prophylactic antibiotic treatment, neither of which is fully satisfactory. This has led to an increased interest in the natural relationships between the pathogenic and mutualistic microorganisms of the honeybee microbiome, in particular, the antagonistic effects of Honeybee-Specific Lactic Acid Bacteria (hbs-LAB) against P. larvae. We investigated whether supplemental administration of these bacteria affected P. larvae infection at colony level over an entire flowering season. Over the season, the supplements affected neither colony-level hbs-LAB composition nor naturally subclinical or clinical P. larvae spore levels. The composition of hbs-LAB in colonies was, however, more diverse in apiaries with a history of clinical AFB, although this was also unrelated to P. larvae spore levels. During the experiments, we also showed that qPCR could detect a wider range of hbs-LAB, with higher specificity and sensitivity than mass spectrometry. Honeybee colonies are complex super-organisms where social immune defenses, natural homeostatic mechanisms, and microbiome diversity and function play a major role in disease resistance. This means that observations made at the individual bee level cannot be simply extrapolated to infer similar effects at colony level. Although individual laboratory larval assays have clearly demonstrated the antagonistic effects of hbs-LAB on P. larvae infection, the results from the experiments presented here indicate that direct conversion of such practice to colony-level administration of live hbs-LAB is not effective.

Honeybee-Specific Lactic Acid Bacterium Supplements Have No Effect on American Foulbrood-Infected Honeybee Colonies.

Paenibacillus larvae, the causative agent of American foulbrood (AFB), is the primary bacterial pathogen affecting honeybees and beekeeping. The main methods for controlling AFB are incineration of diseased colonies or prophylactic antibiotic treatment (e.g., with tylosin), neither of which is fully satisfactory. The search for superior means for controlling AFB has led to an increased interest in the natural relationships between the honeybee-pathogenic and mutualistic microorganisms and, in particular, the antagonistic effects of honeybee-specific lactic acid bacteria (hbs-LAB) against P. larvae These effects have been demonstrated only on individual larvae in controlled laboratory bioassays. Here we investigated whether supplemental administration of hbs-LAB had a similar beneficial effect on P. larvae infection at colony level. We compared experimentally AFB-infected colonies treated with hbs-LAB supplements to untreated and tylosin-treated colonies and recorded AFB symptoms, bacterial spore levels, and two measures of colony health. To account for the complexity of a bee colony, we focused on (Bayesian) probabilities and magnitudes of effect sizes. Tylosin reduced AFB disease symptoms but also had a negative effect on colony strength. The tylosin treatment did not, however, affect P. larvae spore levels and might therefore "mask" the potential for disease. hbs-LAB tended to reduce brood size in the short term but was unlikely to affect AFB symptoms or spores. These results do not contradict demonstrated antagonistic effects of hbs-LAB against P. larvae at the individual bee level but rather suggest that supplementary administration of hbs-LAB may not be the most effective way to harness these beneficial effects at the colony level.IMPORTANCE The previously demonstrated antagonistic effects of honeybee-derived bacterial microbiota on the infectivity and pathogenicity of P. larvae in laboratory bioassays have identified a possible new approach to AFB control. However, honeybee colonies are complex superorganisms where social immune defenses play a major role in resistance against disease at the colony level. Few studies have investigated the effect of beneficial microorganisms on bee diseases at the colony level. Effects observed at the individual bee level do not necessarily translate into similar effects at the colony level. This study partially fills this gap by showing that, unlike at the individual level, hbs-LAB supplements did not affect AFB symptoms at the colony level. The inference is that the mechanisms regulating the honeybee microbial dynamics within a colony are too strong to manipulate positively through supplemental feeding of live hbs-LAB and that new potential remedies identified through laboratory research have to be tested thoroughly in situ, in colonies.

Improvement of identification methods for honeybee specific Lactic Acid Bacteria; future approaches.

Honeybees face many parasites and pathogens and consequently rely on a diverse set of individual and group-level defenses to prevent disease. The crop microbiota of Apis mellifera, composed of 13 Lactic Acid Bacterial (LAB) species within the genera Lactobacillus and Bifidobacterium, form a beneficial symbiotic relationship with each other and the honeybee to protect their niche and their host. Possibly playing a vital role in honeybee health, it is important that these honeybee specific Lactic Acid Bacterial (hbs-LAB) symbionts can be correctly identified, isolated and cultured, to further investigate their health promoting properties. We have previously reported successful identification to the strain level by culture-dependent methods and we recently sequenced and annotated the genomes of the 13 hbs-LAB. However, the hitherto applied techniques are unfortunately very time consuming, expensive and not ideal when analyzing a vast quantity of samples. In addition, other researchers have constantly failed to identify the 13 hbs-LAB from honeybee samples by using inadequate media and/or molecular techniques based on 16S rRNA gene sequencing with insufficient discriminatory power. The aim of this study was to develop better and more suitable methods for the identification and cultivation of hbs-LAB. We compared currently used bacterial cultivation media and could for the first time demonstrate a significant variation in the hbs-LAB basic requirements for optimal growth. We also present a new bacterial identification approach based on amplicon sequencing of a region of the 16S rRNA gene using the Illumina platform and an error correction software that can be used to successfully differentiate and rapidly identify the 13 hbs-LAB to the strain level.

Effects of a honeybee lactic acid bacterial microbiome on human nasal symptoms, commensals, and biomarkers.

BACKGROUND: Lactic acid bacteria (LAB) can restore commensal microbiomes and prevent infections. Arguably, nasal administrations of LAB may therefore be beneficial in chronic rhinosinusitis (CRS). Previous studies have examined effects of topical/nasal LAB in children with secretory otitis media, but little is as yet known about their effects on the human nasal airway. The aim of this pilot study was to examine effects on nasal symptoms and commensal bacteria in healthy subjects of nasal administration of a honeybee LAB microbiome; ie, a mixture of 9 Lactobacillus spp. and 4 Bifidobacterium spp. obtained from the honeybee Apis mellifera. Furthermore, we aimed to assess whether or not the honeybee LAB produced a local inflammatory response. METHODS: Twenty-two healthy subjects received a single administration of honeybee LAB in a sham-controlled, double-blinded, and crossover design. Using questionnaires, microbiological methods, and nasal lavages, they were assessed regarding symptoms, changes to commensal bacteria, and inflammatory products in nasal lavage fluids. RESULTS: The honeybee LAB did not produce any symptoms or other untoward effects. No changes were observed of commensal bacteria by the honeybee LAB, and no inflammatory response was detected (compared to sham); ie, unaffected nasal lavage fluid levels of monocyte chemoattractant protein-1 (MCP-1), interleukin-8 (IL-8), monokine induced by interferon-γ (MIG), interleukin-15 (IL-15), epidermal growth factor (EGF), eotaxin, interferon gamma-induced protein-10 (IP-10), and interleukin-1 receptor antagonist (IL-1RA). CONCLUSION: A single human nasal administration of a honeybee LAB microbiome is well tolerated. Specifically, it does not affect commensal bacteria and does not produce an inflammatory response.