Paenibacillus melissococcoides sp. nov., isolated from a honey bee colony affected by European foulbrood disease

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INTRODUCTION
The genus Paenibacillus currently comprises more than 270 species with validly published and correct names [2]. Paenibacillus species are widely distributed in diverse environments, such as soil, plants, earthworms, humans and insects [3]. Among those found in insects, seven species have been associated with honey bees and three of these have been associated with honey bee diseases: Paenibacillus larvae is a well-described pathogen of honey bee brood, causing severe American foulbrood [4]. Paenibacillus alvei and Paenibacillus dendritiformis have been associated with another infectious brood disease, European foulbrood, caused by the bacterium Melissococcus plutonius [5]. Their pathogenicity and role in the course of European foulbrood disease have not been fully established. P. alvei alone caused mortality in honey bee larvae reared under laboratory conditions [6], but it OPEN ACCESS is unknown whether it can cause a disease at the colony level on its own. P. dendritiformis was isolated from diseased colonies, but its pathogenicity in honey bee brood has not yet been tested [7]. For these reasons, these bacteria are described as secondary invaders following M. plutonius infections. Other members of the genus Paenibacillus, Paenibacillus apiarius and Paenibacillus thiaminolyticus, were isolated from healthy honey bees and hive material [8,9], and recently, Paenibacillus apis and Paenibacillus intestini were isolated from honey bee intestines [10].
The novel candidate strains of the genus Paenibacillus described in this report were isolated from a colony of the honey bee Apis mellifera showing symptoms of European foulbrood in Switzerland. They were designated as strains 2.1 T , 1.2 and 3.2. The novel strains are closely related to P. dendritiformis T168 T (=LMG 21716 T ) [11] and P. thiaminolyticus JCM 8360 T (=DSM 7262 T ) [9,12] and are proposed to represent a new species with the name Paenibacillus melissococcoides sp. nov.

ISOLATION AND ECOLOGY
In June 2020, strains 2.1 T , 1.2 and 3.2 were isolated from a European foulbrood diseased colony of Apis mellifera that showed European foulbrood symptoms and tested positive for M. plutonius. The colony was located in the town of Reutigen (46° 41′ N 7° 37′ E, Bern, Switzerland). Droplets of the nutritive jelly given to worker larvae by nurse bees were sampled from several brood cells, diluted into sterile saline buffer, plated on basal medium (BM) agar specifically used for M. plutonius growth [13], and incubated for four days at 36 °C under anoxic conditions. Several colonies not conforming to the morphology of M. plutonius were observed on the plates. Three such colonies were picked and each was transferred into BM broth for growth at 36 °C in oxic conditions. Aliquots of culture were supplemented with 20 % glycerol for long-term storage at −80 °C before phenotypic and phylogenetic-based taxonomic analyses.

16S RNA GENE PHYLOGENY
To perform the phylogenetic studies, the genomic DNA of strains 2.1 T , 1.2 and 3.2 were sequenced using the long-read technology of Pacific Bioscience (PacBio). High molecular weight genomic DNA was recovered using the guanidium thiocyanate method of DNA extraction [14]. DNA was extracted from single colonies grown on solid BM at 36 °C under oxic conditions. Multiplexed SMRTbell libraries were prepared for sequencing on the Sequel apparatus exactly according to the PacBio guidelines (Procedure and Checklist -Preparing Multiplexed Microbial Libraries Using SMRTbell Express Template Prep Kit 2.0; Part Number 101-696-100; version 06; March 2020). Data were assembled de novo using the microbial assembly pipeline (SMRTlink version 9.0) with default parameter settings. The sequences of strains 2.1 T , 1.2 and 3.2 were found to be identical (100 % nucleotide similarity). Eight full-length 16S rRNA gene sequences were assembled for each strain. Seven of the 16S rRNA copies differed by single nucleotide polymorphisms at nine out of 1551 nucleotide sites. For further analysis, the full-length 16S rRNA gene sequence that was present in two identical copies was selected (accession numbers: 2.1 T , OW961637.1; 1.2, OW961636.1; and 3.2, OW961638.1). The full-length 16S rRNA gene sequence of strain 2.1 T was subjected to a blast search against the EzTaxon webserver (www. ezbiocloud.net). The highest pairwise sequence similarity was detected to the 16S rRNA gene of P. dendritiformis (99.7 %), followed by P. thiaminolyticus (98.8%), Paenibacillus popilliae (98.2 %) and Paenibacillus lentimorbus (98.1 %).
To determine the phylogenetic relationships of strains 2.1 T , 1.2 and 3.2 within the genus Paenibacillus, the 16S rRNA gene sequences from each strain were used to generate an alignment (muscle version 3.8.1551 using default parameter settings [15]) with the 236 Paenibacillus strains (235 species with two P. polymyxa strains) that have validly published and correct names [2] and high-quality 16S rRNA gene sequences [16]. Bacillus pumilus was used as an outgroup. The maximum-likelihood tree was calculated using iq-tree (multicore version 1.6.12) with parameter setting '-m TEST -bb 1000 -alrt 1000' on the iq-tree webserver [17][18][19]. The tree (Fig.S1) was visualised using the iTOL webserver [20].
For a better overview of the position of the three strains with the genus, a smaller phylogenetic tree was built based on the same method. The tree comprised honey bee-associated Paenibacillus species as well as the most ancestral species of each cluster ( Fig. 1). In line with the blast search, the closest neighbours of strains 2.1 T , 1.2 and 3.2 were P. dendritiformis, P. thiaminolyticus, P. popilliae and P. lentimorbus. Given that P. dendritiformis and P. thiaminolyticus were previously reported as being associated with honey bees [7,9], they were chosen for further comparative analyses.

GENOME FEATURES
To perform comparative analyses between strains 2.1 T , 1.2, 3.2 and related species, the whole genomes of P. dendritiformis LMG 21716 T and P. thiaminolyticus DSM 7262 T type strains (provided by BCCM LMG and DSMZ GmbH, respectively), were sequenced using the long-read technology of Pacific Biosciences, as previously described. Assembly statistics of strains 2.1 T , 1.2 and 3.2 are listed in Table 1. The levels of pairwise genome-based similarity were evaluated based on average nucleotide identity (ANI) values determined by FastANI [21]. The ANI values between strains 2.1 T , 1.2 and 3.2 were >99.9 % (Table 2), confirming that they belonged to the same species, P. melissococcoides sp. nov. The ANI values of strains 2.1 T , 1.2 and 3.2 versus the closely related species P. dendritiformis LMG 21716 T and P. thiaminolyticus DSM 7262 T were <92.4 % and <91.1 %, respectively ( Table 2). These differences are lower than the cut-off value of 95 % for species-level differentiation [22,23].

PHYSIOLOGY AND CHEMOTAXONOMY
To describe the physiology and chemotaxonomy of strains 2.1 T , 1.2 and 3.2, we studied their growth characteristics and conducted morphological and phenotypic analyses. All quantitative data presented in this section were averages generated from experiments run in duplicate. The growth abilities of the strains were tested using various growth media and compared to those of P. dendritiformis LMG 21716 T and P. thiaminolyticus DSM 7262 T . The following growth media were laboratory-prepared and tested: BM agar, nutrient agar (NA), caso agar (CA) and MYPGP agar commonly used for culture of respectively, M. plutonius [13], P. thiaminolyticus [12], P. dendritiformis [11] and P. larvae [24]. The plates were inoculated with 50 µl sterile saline buffer (0.9 % NaCl) containing 1500 CFU ml −1 , and incubated at 36 °C under oxic conditions. Strains 2.1 T , 1.2, 3.2, LMG 21716 T and DSM 7262 T were all able to grow on all the tested media (Fig. 2). Colonies of the five strains were visible 48 h post-inoculation. As colony morphology on NA and CA was similar, only the results on NA are shown. The colonies of strains 2.1 T , 1.2 and 3.2 were of two  (Fig. S3). All subsequent analyses were performed with bacteria grown on BM agar under optimal conditions, i.e., at 36±1 °C, without NaCl and under oxic conditions.
Gram staining was performed as described in [25]. Strain 2.1 T was grown under optimal conditions for 18 and 36 h, after which single colonies were picked and suspended in 100 µl sterile saline solution (0.9 % NaCl). Ten microlitres of the bacterial suspension were smeared and heat-fixed on microscope slide grids prior to staining. Cell size and spore formation were investigated using a phase-contrast Olympus BX41 microscope. Vegetative cell size was evaluated from 18-h-old cultures. Endospore formation    [27], and on semi-solid BM 0.4 % agar [28]. P. melissococcoides sp. nov. cells stained Gram-positive after 18 h of culture and Gram-negative after 36 h of culture (Fig. S4), suggesting they possess a thick peptidoglycan wall structure, which can be damaged over culture time. Cells were motile (Movie S1, Fig. S5), rod-shaped, 2.2-6.5 µm long and 0.7-1.1 µm wide. They often formed dyads connected by a septum. They formed central or subterminal endospores that were oval-shaped, 0.5-2.0 µm long and 0.2-1.8 µm wide (Fig. 3).
For biochemical and metabolic characterization of strains 2.1 T , 1.2, 3.2, P. dendritiformis LMG 21716 T and P. thiaminolyticus DSM 7262 T , standard assays were performed (Table 3). Catalase activity was tested by the direct addition of a drop of 3 % H 2 O 2 on bacterial cells. The O 2 production and foaming reaction by the strains were interpreted as positive results for the catalase. The presence of cytochrome c oxidase was assessed by spreading bacteria on ashless paper (Schleicher and Schuell) with a drop of 1 % dimethyl-p-phenylenediamine (bioMérieux) and observing whether a blue-violet colouration indicating oxidation by cytochrome c became visible. Strains 2.1 T , 1.2 and 3.2 were positive for catalase and cytochrome c oxidase, whereas LMG 21716 T and DSM 7262 T were only positive for catalase. To determine acid production from carbohydrate analysis, we used API 50 CH strips and API 50 CHE/B Medium (bioMérieux) according to the manufacturer's recommendations. The positive oxidase activity and the absence of acid formation from glycerol and mannose in P. melissococcoides sp. nov. were the major biochemical differences distinguishing the strains from closely related P. dendritiformis LMG 21716 T and P. thiaminolyticus DSM 7262 T (Table 3).
Cellular fatty acid profiles of P. melissococcoides sp. nov. strains 2.1 T , 1.2, 3.2, P. dendritiformis LMG 21716 T and P. thiaminolyticus DSM 7262 T were determined by high-resolution lipid analysis using a gas chromatograph with a flame ionization detector (GC-FID; 6890+, Agilent Technologies) and compared with previously published profiles of two other members of the genus Paenibacillus, P. alvei IFO 3343 T and P. apiarius NRRL NRS-1438 T [9]. The bacteria were grown on BM under optimal conditions. After 48 h culture, bacteria were harvested from the medium using a sterile inoculation loop and lyophilized using a vacuum controller V-855 coupled with a vacuum pump V-700 (Buchi) and a concentrator plus (Eppendorf). Approximately 10 mg dry bacterial material was prepared for analysis by adding 2 µl of the internal standard methyl nonanoate (C9, 0.007 µg µl -1 ) for quantification. Transesterification was initiated by adding 125 µl of KOH/MeOH (2 mol l −1 ) to the samples. After 5 min, 0.3 g of  Table 3. Continued sodium hydrogen sulphate monohydrate was added to stop the reaction. Fatty acid extraction was performed by adding 100 µl of hexane to the samples, which were vortexed and centrifuged. The supernatant (5 µl) containing the fatty acids was injected into the GC-FID column for analysis. The temperature ramp was as follows: 5 min at 60 °C, from 60 to 165 °C at 14 °C per min, 1 min at 165 °C, from 165 to 225 °C at 12 °C per min and 17 min at 225 °C. The chromatograms were integrated using Agilent software (ChemStation). The most abundant cellular fatty acids of strains 2.1 T , 1.2 and 3.2 were anteiso-C 15 : 0 , anteiso-C 17 : 0 , iso-C 15 : 0 , iso-C 17 : 0 and palmitic acid (C 16 : 0 ). The same pattern was observed for P. dendritiformis LMG 21716 T and P. thiaminolyticus DSM 7262 T , except that the proportions shifted towards markedly larger amounts of palmitic acid (C 16 : 0 ) and smaller amounts of anteiso-C 17 : 0 . Previously published cellular fatty acid profiles of members of the genus Paenibacillus showed similar results [9], with anteiso-C 15 : 0 as a major cellular fatty acid (Table 4). Overall, strains 2.1 T , 1.2 and 2.3 could be distinguished from other members of Paenibacillus by larger amounts of anteiso-C 17 : 0 , and lower amount of palmitic acid (C 16 : 0 ).

DESCRIPTION OF PAENIBACILLUS MELISSOCOCCOIDES
The type strain is 2.1 T (=CCOS 2000 T =DSM 113619 T =LMG 32539 T ), isolated from a colony of honey bees, Apis mellifera, presenting symptoms of European foulbrood in Switzerland, canton of Bern. The genomic DNA G+C content of the type strain is 53.3 mol%.

Funding information
This work was partially funded by the Swiss foundation Gottfried and Julia Bangerter-Rhyner to B.D. (PI) and V.D. (co-applicant).