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. 2019 Jul 2;25:104206. doi: 10.1016/j.dib.2019.104206

Data associated with the characterization and presumptive identification of Bacillus and related species isolated from honey samples by using HiCrome Bacillus agar

Adriana M Alippi 1
PMCID: PMC6626980  PMID: 31338401

Abstract

The dataset described in this paper provides information on the morphological features of 24 different species of the genera Bacillus, Paenibacillus, Brevibacillus, Lysinibacillus, and Rummeliibacilluswhen growing in HiCrome Bacillus agar. The species studied are common contaminants of honey. In support to the recent publication entitled “HiCrome Bacillus agar for presumptive identification of Bacillus and related species isolated from honey samples” (2), a collection of 197 bacterial isolates belonging to 24 different species of aerobic spore-forming bacteria have been screened for their colony appearance and color and any substrate color change of HiCrome Bacillus agar at 24 and 48 h of incubation. Two simple flowcharts utilizing a combination of colony and media characteristics in the chromogenic medium and a set of simple biochemical and morphological tests were developed for quick presumptive identification.

Keywords: HiCrome bacillus agar, Honey, Bacillus, Brevibacillus, Lysinibacillus, Paenibacillus, Rummeliibacillus, Aerobic spore-forming bacteria, Chromogenic media

Specifications table

Subject area Microbiology, Food Microbiology
More specific subject area Microbiological Methods, Bioinformatics
Type of data Tables, Figures, Flowcharts
How data was acquired Digital camera, PCR, purification, sequencing, and Phylogenetic analysis
Data format Analyzed
Experimental factors Isolation of spore-forming bacteria
Genomic DNA from pure bacterial cultures
Experimental features Isolation and cultivation of bacteria, 16S rRNA sequencing, microbiological tests, colony morphology, and microscopy
Data source location Bacteria were isolated from samples from different geographical areas
The analysis was performed at CIDEFI - Facultad de Ciencias Agrarias y Forestales, Universidad Nacional de La Plata, Calles 60 y 119 S/N, 1900 La Plata, Buenos Aires, Argentina
Data accessibility Data are available with this article.
16S rRNA sequences of selected bacterial strains (n = 56) isolated from honey or honeybee larvae have been deposited in GenBank (https://www.ncbi.nlm.nih.gov/genbank/) under accession numbers summarized in Table 1.
Related research article HiCrome Bacillus agar for presumptive identification of Bacillus and related species isolated from honey samples by Alippi and Abrahamovich (International Journal of Food Microbiology, 2019, DOI: 10.1016/j.ijfoodmicro.2019.108245)
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Value of the data
  • •

    Data presented to describe the colony appearance on HiCrome Bacillus agar of several Bacillus and related species commonly found in honey.

  • •

    Data generated serve as a point of reference for further research in microbial diversity, microbial ecology, and microbial taxonomy.

  • •

    The information presented could be beneficial for other researchers that are interested in the microbiology of honey and is potentially also of benefit for research on other food products.

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1. Data

The dataset described in this paper provides information on the morphological features of 24 different species of the genera Bacillus, Paenibacillus, Brevibacillus, Lysinibacillus, and Rummeliibacillus when growing in HiCrome Bacillus agar. The species studied here have been previously reported in honey [1], [2], [3], [5], [7], [10], [12], [13], [14], [15].

A collection of 197 bacterial isolates belonging to the 24 species tested have been screened for their colony appearance and color and any substrate color change of HiCrome Bacillus agar at 24 and 48 h of incubation (Fig. 1 and Table 3). Colors of colonies and substrate observed were compared with a Pantone international chart and identified with a PMS number (http://www.cal-print.com/InkColorChart.htm).

Fig. 1.

Fig. 1

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Comparison of colony appearance on HiCrome Bacillus agar of several Bacillus and related species commonly found in honey: A. Bacillus cereus m87, B. Bacillus megaterium m344, C. Paenibacillus alvei mv82, D. Bacillus amyloliquefaciens m39, E. Bacillus subtilis ATCC 7061, F. Bacillus subtilis m191, G. Lysinibacillus sphaericus m533, H. Bacillus licheniformis mv68, I. Bacillus pumilus mv49b, J. Complex sample of naturally contaminated honey containing (Clockwise from upper left) B. cereus, B. licheniformis, B. pumilus B. amyloliquefaciens, and B. megaterium.

Table 3.

Colony appearance and growth of selected strains tested in HiCrome Bacillus agar.

Species Strain/Isolate designation Colonies in HiCrome Bacillus Agar Ecometric Code
HiCrome Control Bacillus medium
Bacillus amyloliquefaciens m39 Image 1 5 5
Bacillus badius ATTC 14574 Image 2 3.8 3.4
Bacillus cereus ATCC 11778 Image 3 5 5
Bacillus cereus m388 Image 4 5 5
Bacillus circulans ATCC 4515 Image 5 4 5
Bacillus clausii FR231 Image 6 1.6 5
Bacillus coagulans NRRL NRS 609 Image 7 5 5
Bacillus firmus ATCC 8247 Image 8 5 5
Bacillus licheniformis NRRL B-1001 Image 9 5 5
Bacillus megaterium NRRL B-939 Image 10 4.6 5
Bacillus mycoides ATCC 10206 Image 11 4 5
Bacillus pumilus ATCC 7061 Image 12 5 5
Bacillus subtilis m191 Image 13 5 5
NRRL B-543 Image 14 4 5
Bacillus thuringiensis ATCC 10792 Image 15 5 5
Brevibacillus borstelensis RC Image 16 4.2 5
Brevibacillus brevis ATCC 8246 Image 17 4.4 5
Brevibacillus laterosporus CCT 0031 Image 18 2.2 5
Lysinibacillus fusiformis mv119 Image 19 3.8 5
Lysinibacillus sphaericus ATCC 245 Image 20 1.4 5
Paenibacillus alvei NRRL B-383 Image 21 1 5
Paenibacillus amylolyticus NRRL B-14940 Image 22 4 5
Paenibacillus apiarius ATCC 29575 Image 23 4 5
Paenibacillus larvae ERIC I ATCC 9545 Image 24 2 5
ERIC IV ATCC 13537 Image 25 2 5
ERIC II SAG 290 Image 26 3.8 5
Paenibacillus polymyxa NRRL B-510 Image 27 5 5
Rummeliibacillus stabekisii mv111 Image 28 5 5
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The Ecometric technique was used for comparative evaluation of HiCrome Bacillus agar and the control medium (Fig. 2, Fig. 3 and Table 3). E-values (Table 3) were obtained for 28 selected isolates tested by using previously published methods [2], [8].

Fig. 2.

Fig. 2

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Bacillus cereus ATCC 11778 growing on A: BHIT and B. HiCrome Bacillus agar showing luxuriant growth (+++) and E value = 5.

Fig. 3.

Fig. 3

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Rummeliibacillus stabekisii mv111 growing on A: MYPGP and B. HiCrome Bacillus agar showing good growth (++) and E value = 5.

Two Flowcharts were prepared by a combination of colony and media characteristics in HiCrome Bacillus agar and a set of selected biochemical and morphological tests that are used routinely in Microbiological laboratories. The first chart (Fig. 4) permits the identification of the aerobic spore-forming bacteria reported in honey by a few simple tests. The more simplified flowchart presented in Fig. 5 allows differentiating typical strains of aerobic spore-forming species by direct isolation from honey.

Fig. 4.

Fig. 4

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Flowchart - Main steps for the identification of common strains of Bacillus and related species from honey by using a combination of selected morphological and biochemical tests and HiCrome Bacillus agar.

Fig. 5.

Fig. 5

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Flowchart - Simplified steps for the identification of common strains of Bacillus and related species from honey by using isolation in HiCrome Bacillus agar and selected morphological and biochemical tests.

The bacterial identity of selected strains isolated from honey or honeybee larvae (n = 56) (Table 1) were confirmed by sequencing the 16S rDNA. Sequences were deposited in the DDBJ/EMBL/Genbank under the Accession Numbers listed in Table 1. For comparisons, 16S rRNA sequences from type cultures (n = 32 plus 1 outlier) were used and are listed in Table 2.

Table 1.

Source and accession numbers of bacterial strains and isolates used in this study.

Species Strain/Isolate designation Source and geographical origin Accesion number
Bacillus amyloliquefaciens xx Honeybee larvae-Argentina KP177517.1
Bacillus amyloliquefaciens mv35 Honey- Argentina MG004186.1
Bacillus amyloliquefaciens m39 Honey- Argentina MG004187.1
Bacillus amyloliquefaciens m163b Honey- Argentina MG004188.1
Bacillus amyloliquefaciens m164b Honey- Argentina MG004193.1
Bacillus amyloliquefaciens m287b Honey- Argentina MG004189.1
Bacillus amyloliquefaciens m291b Honey- Argentina MG004190.1
Bacillus badius CCT 0196 CCT N/A
Bacillus cereus ATCC 11778 ATCC AF290546.1
Bacillus cereus cm4 Honey- Argentina N/A
Bacillus cereus m6c Honey- Argentina KP005456.1
Bacillus cereus cm7 Honey- Argentina N/A
Bacillus cereus cm8 Honey- Argentina N/A
Bacillus cereus m9a Honey- Argentina N/A
Bacillus cereus m10a Honey- Argentina N/A
Bacillus cereus m10b Honey- Argentina N/A
Bacillus cereus m12 Honey- Argentina N/A
Bacillus cereus m19 Honey- Argentina N/A
Bacillus cereus m21 Honey- Argentina N/A
Bacillus cereus m28 Honey - Argentina N/A
Bacillus cereus m31 Honey- Argentina N/A
Bacillus cereus mv33 Honey- Argentina KU230015.1
Bacillus cereus cm37 Honey- Argentina N/A
Bacillus cereus mv39b Honey- Argentina N/A
Bacillus cereus mv41x Honey- Argentina N/A
Bacillus cereus mv54 Honey- Argentina N/A
Bacillus cereus m54 Honey- Argentina N/A
Bacillus cereus mv67 Honey- Argentina N/A
Bacillus cereus m73 Honey- Argentina N/A
Bacillus cereus mv73 Honey- Argentina N/A
Bacillus cereus mv75 Honey- Argentina N/A
Bacillus cereus mv76 Honey- Argentina N/A
Bacillus cereus mv77 Honey- Argentina N/A
Bacillus cereus mv78 Honey- Argentina N/A
Bacillus cereus mv79 Honey- Argentina N/A
Bacillus cereus mv80 Honey- Argentina N/A
Bacillus cereus m84 Honey- Argentina N/A
Bacillus cereus m85 Honey- Argentina N/A
Bacillus cereus mv86 Honey- Argentina N/A
Bacillus cereus mv87 Honey- Argentina N/A
Bacillus cereus m90 Honey- Argentina N/A
Bacillus cereus m91 Honey- Argentina N/A
Bacillus cereus m97 Honey- Argentina N/A
Bacillus cereus m105 Honey- Argentina N/A
Bacillus cereus mv114 Honey- Argentina N/A
Bacillus cereus mv117 Honey- Argentina N/A
Bacillus cereus cm117 Honey- Argentina N/A
Bacillus cereus cm118 Honey- Argentina N/A
Bacillus cereus m134 Honey- Argentina N/A
Bacillus cereus m139b Honey- Argentina N/A
Bacillus cereus m143b Honey- Argentina N/A
Bacillus cereus m143c Honey- Argentina N/A
Bacillus cereus m157 Honey- Italy N/A
Bacillus cereus m158 Honey- Argentina N/A
Bacillus cereus m163a Honey- Argentina N/A
Bacillus cereus m167 Honey- Argentina N/A
Bacillus cereus m189 Honey- Argentina N/A
Bacillus cereus m193 Honey- Argentina N/A
Bacillus cereus m225a Honey- Argentina N/A
Bacillus cereus m228 Honey- Argentina N/A
Bacillus cereus m243 Honey- Argentina N/A
Bacillus cereus m244 Honey- Argentina N/A
Bacillus cereus m248 Honey- Argentina N/A
Bacillus cereus m262 Honey- Argentina N/A
Bacillus cereus m267 Honey- Argentina N/A
Bacillus cereus cm281 Honey- Argentina N/A
Bacillus cereus m282a Honey- Argentina N/A
Bacillus cereus m287a Honey- Argentina N/A
Bacillus cereus m292 Honey- Argentina N/A
Bacillus cereus m296 Honey- Argentina N/A
Bacillus cereus m298 Honey- Argentina N/A
Bacillus cereus m305 Honey- Argentina N/A
Bacillus cereus m308 Honey- Argentina N/A
Bacillus cereus m309 Honey- Argentina N/A
Bacillus cereus m316 Honey- Argentina N/A
Bacillus cereus m365 Honey- Argentina N/A
Bacillus cereus m370 Honey- Argentina N/A
Bacillus cereus m383 Honey- Argentina N/A
Bacillus cereus m385 Honey- Argentina N/A
Bacillus cereus m387 Honey- Argentina KP005455.1
Bacillus cereus m388 Honey- Argentina N/A
Bacillus cereus m434 Honey- Argentina KU230027.1
Bacillus cereus m436 Honey- Argentina N/A
Bacillus cereus m437b Honey- Argentina N/A
Bacillus cereus m438 Honey- Argentina N/A
Bacillus cereus m439 Honey- Argentina N/A
Bacillus cereus m444 Honey- Argentina N/A
Bacillus cereus m445b Honey - Argentina N/A
Bacillus cereus LPcer1 Honeybee larvae- Argentina KX431225.1
Bacillus cereus MexB Honey- Mexico KU230012.1
Bacillus cereus MexC Honey- Mexico KU230013.1
Bacillus circulans ATCC 4515 ATCC N/A
Bacillus clausii Fr231 Honey- France KU230014.1
Bacillus clausii m448b Honey- Brazil KX685159.1
Bacillus coagulans NRRL NRS 609 NRRL N/A
Bacillus firmus ATCC 8247 ATCC N/A
Bacillus licheniformis mv55 Honey-Argentina KU230018.1
Bacillus licheniformis mv68 Honey-Argentina MF187633.1
Bacillus licheniformis mv72 Honey- Argentina N/A
Bacillus licheniformis m112 Honey- Argentina N/A
Bacillus licheniformis NRRL B-1001 NRRL N/A
Bacillus megaterium m280 Honey- Argentina N/A
Bacillus megaterium m327 Honey- Argentina MF187637.1
Bacillus megaterium m344 Honey- Argentina N/A
Bacillus megaterium m373 Honey- Argentina N/A
Bacillus megaterium m435 Honey- Mexico KU230028.1
Bacillus megaterium m441 Honey- Argentina N/A
Bacillus megaterium m458 Honey- Brazil N/A
Bacillus megaterium NRRL B-939 NRRL N/A
Bacillus mycoides m336 Honey- Argentina MF187638.1
Bacillus mycoides m425 Honey- Argentina N/A
Bacillus mycoides ATCC 10206 ATCC N/A
Bacillus pumilus mv41aA Honey- Argentina MG366818.1
Bacillus pumilus mv49b Honey- Argentina KU230016.1
Bacillus pumilus mv74 Honey- Argentina MF972935.1
Bacillus pumilus mv81 Honey- Argentina KU230019.1
Bacillus pumilus m108 Honey- Argentina N/A
Bacillus pumilus m116 Honey- Argentina KU230020.1
Bacillus pumilus m157 Honey- Italy N/A
Bacillus pumilus m187 Honey- Argentina N/A
Bacillus pumilus m225b Honey- Argentina N/A
Bacillus pumilus m288 Honey- Argentina MF187635.1
Bacillus pumilus m330 Honey- Argentina MF187646.1
Bacillus pumilus m335 Honey- Argentina N/A
Bacillus pumilus m339 Honey- Argentina MG366884.1
Bacillus pumilus m350 Honey- Argentina KU230023.1
Bacillus pumilus m354 Honey- Argentina N/A
Bacillus pumilus m357 Honey- Argentina MF187634.1
Bacillus pumilus m358 Honey- Argentina MG345110.1
Bacillus pumilus m360 Honey- Argentina MF187636.1
Bacillus pumilus m363 Honey- Argentina KU230024.1
Bacillus pumilus m414 Honey- Argentina KU230026.1
Bacillus pumilus ATCC 7061T ATCC AY876289.1
Bacillus subtilis m11 Honey- Argentina N/A
Bacillus subtilis m13 Honey - Argentina MF187645.1
Bacillus subtilis m45 Honey- Argentina N/A
Bacillus subtilis cm45 Honey- Argentina MF187639.1
Bacillus subtilis mv49a Honey- Argentina N/A
Bacillus subtilis mv51 Honey- Argentina N/A
Bacillus subtilis mv53b Honey- Argentina N/A
Bacillus subtilis mv63 Honey- Argentina N/A
Bacillus subtilis mv64 Honey- Argentina N/A
Bacillus subtilis mv65 Honey- Argentina N/A
Bacillus subtilis mv66 Honey- Argentina N/A
Bacillus subtilis mv70 Honey- Argentina N/A
Bacillus subtilis mv71 Honey- Argentina N/A
Bacillus subtilis m107 Honey- Argentina N/A
Bacillus subtilis m117 Honey- Argentina N/A
Bacillus subtilis m119 Honey- Argentina N/A
Bacillus subtilis m191 Honey- Argentina MF187644.1
Bacillus subtilis m192 Honey- Argentina N/A
Bacillus subtilis m197 Honey- Argentina N/A
Bacillus subtilis m291b Honey- Argentina N/A
Bacillus subtilis m329 Honey- Argentina KU230021.1
Bacillus subtilis m334 Honey- Argentina KU230022.1
Bacillus subtilis m347 Honey- Argentina KP177515.1
Bacillus subtilis m351 Honey- Argentina KP177516.1
Bacillus subtilis m384 Honey- Argentina N/A
Bacillus subtilis m386 Honey- Argentina N/A
Bacillus subtilis m389 Honey- Argentina N/A
Bacillus subtilis m392 Honey- Argentina MF187640.1
Bacillus subtilis m412 Honey- Argentina N/A
Bacillus subtilis NRRL B-543 NRRL N/A
Bacillus thuringiensis ATCC 10792T ATCC D16281.1
Bacillus thuringiensis m5 Honey - Argentina N/A
Bacillus thuringiensis mv50b Honey - Argentina KU230017.1
Bacillus thuringiensis m391 Honey - Argentina N/A
Bacillus thuringiensis m395 Honey- Argentina KU230025.1
Bacillus thuringiensis m401 Honey- Argentina N/A
Brevibacillus borstelensis m348 Honey- Argentina MF187641.1
Brevibacillus borstelensis RC Honey- Argentina KP177514.1
Brevibacillus brevis ATCC 8246 ATCC N/A
Brevibacillus laterosporus CCT 0031 CCT N/A
Brevibacillus laterosporus BLAT169 Honeybee larvae - Argentina KX102627.1
Brevibacillus laterosporus BLAT170 Honeybee larvae - Argentina KX431223.1
Brevibacillus laterosporus BLAT171 Honeybee larvae - Argentina KX431224.1
Lysinibacillus fusiformis mv119 Honey- Argentina MG004185.1
Lysinibacillus sphaericus ATCC 245 ATCC N/A
Lysinibacillus sphaericus m533 Honey- Argentina MG001492.1
Lysinibacillus sphaericus LMDZA Honeybee larvae - Argentina MG004191.1
Paenibacillus alvei NRRL B-383 NRRL N/A
Paenibacillus alvei mv82 Honey- Argentina MF187643.1
Paenibacillus alvei m291a Honey- Argentina MF187632.1
Paenibacillus alvei m420 Honey- Argentina MF187642.1
Paenibacillus amylolyticus NRRL B-14940 NRRL N/A
Paenibacillus apiarius ATCC 29575 ATCC N/A
Paenibacillus larvae ERIC I ATCC 9545T ATCC NR_118956.1
Paenibacillus larvae ERIC IV ATCC 13537T ATCC KT363749.1
Paenibacillus larvae ERIC I PL38 Honeybee larvae-Argentina N/A
Paenibacillus larvae ERIC I PL45 Honeybee larvae- France N/A
Paenibacillus larvae ERIC I PL58 Honeybee larvae- Sweden N/A
Paenibacillus larvae ERIC II SAG 290 Honey - Unknown N/A
Paenibacillus larvae ERIC II SAG 10367 Honey- Unknown CP020557
Paenibacillus larvae ERIC II SAG 10754 Honey- Unknown N/A
Paenibacillus polymyxa NRRL B-510 NRRL N/A
Rummeliibacillus stabekisii mv111 Honey- Argentina MF972934.1
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ATCC: American Type Culture Collection, USA; CCT: Colleçao de Culturas Tropical, Brazil; NRRL: Northern Utilization Research and Development Division, USA; SAG: Servicio Agrícola Ganadero, Chile.

N/A: Not applicable.

Table 2.

Accession numbers of 16 S rRNA sequences from Type cultures used for sequence analysis.

Species Strain Accession number
Bacillus amyloliquefaciens NBRC 15535 NR_112685.1
Bacillus badius ATCC 14574 X77790.1
Bacillus cereus ATCC 11778 NR_074540.1
Bacillus circulans ATCC 4513 AY724690.1
Bacillus clausii DSM 8716 X76440.1
Bacillus coagulans ATCC 7050 DQ297928.1
Bacillus firmus NBRC 15306 NR_112635.1
Bacillus flexus IFO15715 NR_024691.1
Bacillus licheniformis ATCC 14580 NR_074923.1
Bacillus megaterium IAM 13418 D16273.1
Bacillus mycoides ATCC 6462 NR_115993.1
Bacillus niabensis 4T19 AY998119.2
Bacillus pumilus ATCC 7061 AY876289.1
Bacillus simplex DSM 1321 AJ439078
Bacillus subtilis DSM 10 JQ424889.1
Bacillus thuringiensis IAM 12077 D16281.1
Bacillus xiamenensis MCCC 1A00008 NR_148244.1
Brevibacillus borstelensis DSM 6347 AB112721
Brevibacillus brevis NBRC 15304 NR_041524.1
Brevibacillus centrosporus NRRL NRS-664 NR_043414.1
Brevibacillus formosus DSM 9885 AB112712.1
Brevibacillus laterosporus IAM 12465 D16271
Lysinibacillus fusiformis DSM 2898 AJ310083.1
Lysinibacillus sphaericus ATCC 14577 NR_115724.1
Paenibacillus alvei DSM 29 AJ320491
Paenibacillus amylolyticus NRRL NRS-290 D85396.2
Paenibacillus apiarius NRRL NRS-1438 NR_118834.1
Paenibacillus larvae subsp. larvae ATCC 9545 NR_118956.1
Paenibacillus larvae subsp. pulvifaciens ATCC 13537 KT363749.1
Paenibacillus macerans IAM 12467 NR_040886
Paenibacillus polymyxa DSM 36 AJ320493.1
Rummeliibacillus stabekisii NBRC 104870 NR_114270
Micrococcus luteus (outlier) DSM 20030 AJ536198.1
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2. Experimental design, materials, and methods

A collection of 197 bacterial isolates of Bacillus, Brevibacillus, Lysinibacillus, Paenibacillus, and Rummeliibacillus belonging to different species that have been reported in honey [1], [2], [3], [5], [7], [10], [12], [13], [14], [15] were screened for their abilities to grow and colony appearance and color, and any substrate color change by using HiCrome Bacillus agar. The collection includes 167 isolates from honey samples from different geographical areas including Argentina, Brazil, France, Italy, and Mexico; 9 isolates from honeybee larvae from different geographical areas including Argentina, France and Sweden; and 21 strains from Culture Collections used for comparison and quality control. Bacteria were maintained as stock cultures at −80 °C in the correspondent broth medium, Müller-Hinton Broth, yeast extract, potassium phosphate, glucose, and pyruvate (MYPGP) [4] or Brain heart infusion (BHI) plus 20% glycerol (v/v). For short-term storage, the strains were kept at 4 °C in screw-capped vials containing MYPGP or BHI semi-solid (0.4% agar).

Bacterial smears stained by Schaeffer-Fulton technique were examined for the presence and location of spores within cells, as well as for the size and shape of vegetative cells [9], [11]. Also, the presence of unstained globules in the cytoplasm [6], [9], [11] was examined by phase contrast microscopy (Leica, ICC50).

Bacterial cultures were also tested by catalase reaction, anaerobic growth, nitrate reduction, Voges-Proskauer reaction (VP), pH in VP broth, indol and urease production, mannitol, L-arabinose, and citrate utilization, starch and gelatin hydrolysis, decomposition of tyrosine, growth in 7% and 10% of NaCl and at different temperatures (30-37-50 and 55 °C) according to standard protocols [6], [9], [11].

The Ecometric technique was used for comparative evaluation of HiCrome Bacillus agar and the correspondent control media (BHI or MYPGP). Overnight cultures were adjusted to 0.5 Mc Farland in sterile distilled water. One loop of 10 μl of each suspension was sequentially diluted from streak to streak onto each medium by inoculating 21 streaks (5 per quadrant and 1 in the center). Growth on the plates was recorded as a score. Readings were presented as absolute growth indices with possible values of 0–5, where 0 is an absence of growth in any streak and 5 was the maximum score obtained when all of the streaks in the four quadrants and also the last streak showed visible bacterial growth [2], [8]. Twenty-eight bacterial strains with different colony types (Table 2) were used for the evaluation. Plates were inoculated and incubated in duplicate for 24–48 h at 37 °C. Scores for HiCrome and control plates were compared to estimate the degree of inhibition due to the chromogenic mixture (Table 3, Fig. 2, Fig. 3).

The identity of selected strains (n = 56) was confirmed by sequencing the 16S rDNA. Universal eubacterial primers used for 16S rDNA sequence analysis were 27f (5′-AGAGTTTGATCMTGGCTCAG - 3′) and 1492r (5′-TACGGYTACCTTGTTACGACTT- 3′).

For purification of PCR products the following enzymatic procedure was used: The mixture contained 0.5 μl Antarctic phosphatase buffer (NEB, Migliore Lacaustra, Argentina), 0.6 μl Antarctic phosphatase (NEB, Migliore Lacaustra, Argentina), 0.6 μl Exonuclease I (NEB, Migliore Lacaustra, Argentina), 4 μl unpurified PCR product and 3.3 μl double distilled sterile deionized water. The Thermal cycler protocol consisted of one step of 37 °C for 20 min and the second of 80 °C for 20 min.

The quality and quantity of PCR products were assessed by gel electrophoresis (1 μl/1.6% agarose/molecular weight marker QuantiMarker, Promega, Argentina) and DNA concentration was estimated by using a Genova Nano spectrophotometer (JenWay). The purified PCR products of approximately 1,400 bp were sequenced by the dideoxy termination method by the commercial services of Macrogen Inc. (Seoul, Korea) or Unidad de Genómica, Instituto de Biotecnología, CICVYA - INTA (Hurlingham, Argentina). Sequence assembly and contig editing were performed by using CodonCode Aligner software (Codon Code Corporation, MA, USA). The partial sequences obtained were subjected to both Blast-N (http://www.ncbi.nlm.nih.gov), and EZBiocloud (http://www.ezbiocloud.n) search to identify sequences with the highest similarity by comparison only with sequences obtained from Type Cultures [2].

Acknowledgments

This work was financially supported by the Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT) [PICT 2012/0189 and PICT 2017/2014] and the Comisión de Investigaciones Científicas de la Provincia de Buenos Aires (CICBA) [Grants 1194/14 and 274/16]. AMA is a member of the Scientific Research Career of CICBA. I thank Dr. Ivo Siegrist for his helpful advice on HiCrome Bacillus agar and Dr. Eliana Abrahamovich for helping with the artwork and photographs.

Conflict of interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

References

  • 1.Alippi A.M. Detection of Bacillus larvae spores in Argentinian honeys by using a semi-selective medium. Microbiología (Madrid) 1995;11:343–350. [PubMed] [Google Scholar]
  • 2.Alippi A.M., Abrahamovich E. HiCrome Bacillus agar for presumptive identification of Bacillus and related species isolated from honey samples. Int. J. Food Microbiol. 2019 doi: 10.1016/j.ijfoodmicro.2019.108245. [DOI] [PubMed] [Google Scholar]
  • 3.Alippi A.M., Reynaldi F.J., López A.C., De Giusti M.R., Aguilar O.M. Molecular epidemiology of Paenibacillus larvae larvae and incidence of American foulbrood in argentinean honeys from Buenos Aires Province. J. Apic. Res. 2004;43:135–143. [Google Scholar]
  • 4.Dingman D.W., Stahly D.P. Medium promoting sporulation of Bacillus larvae and metabolism of medium components. Appl. Environ. Microbiol. 1983;46:860–869. doi: 10.1128/aem.46.4.860-869.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Gilliam M. Microbiology of pollen and bee bread: the genus Bacillus. Apidologie. 1979;10:269–274. [Google Scholar]
  • 6.Gordon R.E., Haynes W.C., Pang C.H.-N. Agricultural Research Service, USDA; Washington, D.C.: 1973. The Genus Bacillus: Agriculture Handbook No. 427. [Google Scholar]
  • 7.Iurlina M.O., Fritz R. Characterization of microorganisms in Argentinean honeys from different sources. Int. J. Food Microbiol. 2005;105:297–304. doi: 10.1016/j.ijfoodmicro.2005.03.017. [DOI] [PubMed] [Google Scholar]
  • 8.Kornacki J.L., Gurtler J.B., Yan Z., Cooper C.H. Evaluation of several modifications of the ecometric technique for assessment of media performance. J. Food Prot. 2003;66:1727–1732. doi: 10.4315/0362-028x-66.9.1727. [DOI] [PubMed] [Google Scholar]
  • 9.Parry J.M., Turnbull P.C.B., Gibson J.R. Wolfe Medical Publications Ltd.; Ipswich, England: 1983. A Colour Atlas of Bacillus Species. [Google Scholar]
  • 10.Piccini C., Antúnez K., Zunino P. An approach to the characterization of the honey bee hive bacterial flora. J. Apic. Res. 2004;43:101–104. [Google Scholar]
  • 11.Priest F.G., Goodfellow M., Todd C. A numerical classification of the genus Bacillus. J. Gen. Microbiol. 1988;143:1847–1882. doi: 10.1099/00221287-134-7-1847. [DOI] [PubMed] [Google Scholar]
  • 12.Silva M.S., Rabadzhiev Y., Eller M.R., Iliev I., Ivanova I., Santana W.C. Chapter 11: Microorganisms in honey. In: de Toledo V.A., editor. Honey Analysis. 2017. pp. 233–234. [Google Scholar]
  • 13.Sinacori M., Francesca N., Alfonzo A., Cruciata M., Sannino C., Settanni L., Moschetti G. Cultivable microorganisms associated with honeys of different geographical and botanical origin. Food Microbiol. 2014;38:284–294. doi: 10.1016/j.fm.2013.07.013. [DOI] [PubMed] [Google Scholar]
  • 14.Snowdon J.A., Cliver D.O. Microorganisms in honey. Int. J. Food Microbiol. 1996;31:1–26. doi: 10.1016/0168-1605(96)00970-1. [DOI] [PubMed] [Google Scholar]
  • 15.Wen Y., Wang L., Yue J., Zhang J., Su L., Zhang X., Zhou J., Li Y. The microbial community dynamics during the vitex honey ripening process in the honeycomb. Front. Microbiol. 2017;8:1649. doi: 10.3389/fmicb.2017.01649. [DOI] [PMC free article] [PubMed] [Google Scholar]

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