Lactobacillus bombi sp. nov., from the digestive tract of laboratory-reared bumblebee queens (Bombus terrestris)

Abstract

Lactobacillus bombi sp. nov., from the digestive tract of laboratory-reared bumblebee queens (Bombus terrestris) Three bacterial strains belonging to the genus Lactobacillus were isolated from the digestive tracts of laboratory-reared bumblebee queens (Bombus terrestris) using MRS agar under anaerobic conditions. The isolates were identified according to 16S rRNA gene sequence analysis as undescribed members of the genus Lactobacillus, with the highest 16S rRNA gene sequence similarity (96.9 %) to the uncharacterized bacterial strain Lactobacillus sp. Mboho2r2 isolated from the stomach of a European honeybee (Apis mellifera). Lactobacillus tucceti was found to be the closest related species with a validly published name, with 92.9 % 16S rRNA gene sequence similarity to the type strain. However, phylogenetic analyses based on different markers revealed that this species is phylogenetically very distant from the novel strains. The DNA G+C content of the proposed type strain BTLCH M1/2 T is 37.8 mol%. The fatty acids C 19 : 1 v6c and/or C 19 : 0 cyclo v10c/19v6, C 18 : 1 v9c and C 16 : 0 were predominant in all strains. Diphosphatidylglycerol, phosphatidylglycerol, a phospholipid, seven glycolipids and two phosphoglycolipids were detected in the novel strains. Growth was observed at 47 6C. The peptidoglycan type A4a L-Lys-D-Asp was determined for strain BTLCH M1/2 T. Genotypic characteristics and phylogenetic analyses based on the phylogenetic markers hsp60, pheS, rpoA and tuf as well as phenotypic characteristics and the results of chemotaxonomic analyses confirmed that the new isolates belong to a novel species of the genus Lactobacillus, for which the name Lactobacillus bombi sp. nov. is proposed. The type strain is BTLCH M1/2 T (5DSM 26517 T 5CCM 8440 T).

Microorganisms in Honey

“…New methodologies have made it possible to access information about the differences in the profile of this microbiota in different apiculture sources [118][119][120][121], species [53,122] and genetic diversity [116] of honeybees, development stages [53,68,117,[122][123][124][125][126], nutrition [116,127], location inside the gut [49,53,68] and digestive system [120], ontogenetic stage and geographic location [118,122,125], environmental conditions [128], health control [129], and individual [116,125].…”

“…This access has been carried out mainly by sequencing the coding region of the 16S subunit of the bacterial ribosome [53,121,130], both from genomic DNA from microorganisms growing on selective media as Man-Rogosa-Sharpe agar, Sabouraud dextrose agar, and Candida agar [117,120,131,132], such as process-independent culture as specific PCR [68], denaturing gradient gel electrophoresis [124,125], mixed and deep 16S sequencing [49,128], pyrosequencing [53,116,121], and clone library [115,118,120,122]. While culturedependent methods are ideal for quantification of microorganisms and phenotypic testing, culture-independent methods generally have greater coverage in relation to the amount of different species accessed and are ideal for fingerprinting studies, and the identification of these species may be performed by real-time PCR analysis [49,68,125,128].…”

“…Several microorganisms present in the honey and in the gut of honeybees have antagonistic effects on honeybees and human pathogens, especially of Bacillus genus [123,134], lactic acid bacteria as Lactobacillus [71,121,124,[130][131][132]135], Enterococcus [130], Bifidobacteria [116,132,[136][137][138], and Acetobacteraceae [117,121,133]. These same microorganisms can be accessed for other purposes, such as its potential as fermenters [116,130,133] or probiotics [116].…”

“…However, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry profiling was used for acetobacterium identification from bumble bee crop [133] and clustering of lactic acid bacteria of a bumble bee gut microbiota [139]. Several studies have shown the effectiveness of these microorganisms to inhibit human pathogens such as Staphylococcus aureus, Escherichia coli O157: H7, Salmonella, and Listeria [130,140] or pathogens of honeybees as Melissococcus plutonius [124,138], the causative agent of European foulbrood and Paenibacillus larvae [123,124,130,134], and the causative agent of American foulbrood, among others. This effectiveness is generally associated with the production of acid, bacteriocins [130], and other antimicrobial molecules [140].…”

Genomics of the honey bee microbiome

“…These have been called F-4, F-5 (or Firm4 and Firm5) and “Bifido” [3–5]. Within the two “Firm” clusters, strains have been given multiple species names within Lactobacillus [19]; likewise “Bifido” corresponds to a species cluster that includes Bifidobacterium asteroides from honey bee [20] and several species described from bumble bees [21–23] (Table 1). Two other species clusters are from distantly related clusters of Alphaproteobacteria, initially called Alpha1 and Alpha2 [4].…”