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

Abstract

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.

Clinical efficacy of a topical lactic acid bacterial microbiome in chronic rhinosinusitis: A randomized controlled trial

“…With regard to the microbiological identification part of this study, The MALDI‐TOF method was chosen over 16S rRNA gene sequencing as it gives sufficient information on the entire microbiome at the strain level . No change to the nasal microbiota was observed for LAB compared to sham, neither for commensals nor CRS pathogens.…”

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 composition and total amount of bacteria in the hbs-LAB supplement to be used at colony level (10 6 colony forming units (CFU)/colony) was extrapolated from previous doseresponse research at individual level, in laboratory bioassays, through multiplying the optimum per-larva dose (2500 CFU/ larva) [18] [20,21], were incubated individually and anaerobically at 35°C in Man, Rogosa & Sharpe (MRS; Oxoid, England) broth, supplemented with 2% fructose (Merck, Sollentuna, Sweden) and 0.1% L-cysteine (Sigma-Aldrich, Stockholm, Sweden) [35]. A 30-mL mixture of all 13 hbs-LAB was prepared from equal volumes of each individual bacterial strain at 10 7 CFU/mL, and was subsequently freeze-dried in ten aliquots.…”

“…DNA was extracted from 100 μL of honeycrop homogenate using the DNeasy Blood and Tissue kit (Qiagen, Germany), with minor modifications of the manufacturer's protocol as described previously [35]. The DNA of the different hbs-LAB was quantified by qPCR using a modified version of Nadkarni et al [40], using proprietary primers developed by ConCellae AB (Lund, Sweden) based on selective gene sequences to allow the specific detection of each of the hbs-LAB species and strains investigated here.…”

“…Bacteria in the honey crop homogenates were cultured in tenfold serial dilutions on MRS agar supplemented with 2% fructose (Merck, Sollentuna, Sweden) and 0.1% L-cysteine (Sigma-Aldrich, Sweden) [31,35]. Fifteen individual bacterial colonies from each sample were transferred to a 96-well steel target plate (Bruker Daltonics, Sweden) and covered with 1 μL 70% formic acid and 1 μL of 10 mg/mL α-cyano 4hydroxycinnamic acid (HCCA) in a matrix solution (Sigma-Aldrich), as described previously [29].…”

The secretome of honey bee-specific lactic acid bacteria inhibits Paenibacillus larvae growth

“…The 13 Hma3N and Bifidobacterium coryneforme Bma6N) were cultured individually in supplemented MRS medium (sMRS) containing 2% fructose (Merck, Sollentuna, Sweden) and 0.1% L-cysteine (Sigma-Aldrich, Stockholm, Sweden) as previously described (Lamei et al, 2017). The hbs-LAB mixture used for experimentation was prepared by mixing the 13 species, each at a starting cell density of OD 600 ¼0.02, followed by incubation of the mixture in sMRS broth for 18 h at 35 C.…”