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WO2023230736A1 - Composition pour le traitement et la prévention de la loque américaine (la) - Google Patents

Composition pour le traitement et la prévention de la loque américaine (la) Download PDF

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Publication number
WO2023230736A1
WO2023230736A1 PCT/CL2022/050059 CL2022050059W WO2023230736A1 WO 2023230736 A1 WO2023230736 A1 WO 2023230736A1 CL 2022050059 W CL2022050059 W CL 2022050059W WO 2023230736 A1 WO2023230736 A1 WO 2023230736A1
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WO
WIPO (PCT)
Prior art keywords
composition
composition according
larvae
treatment
consortium
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Ceased
Application number
PCT/CL2022/050059
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English (en)
Spanish (es)
Inventor
Alejandro OLMOS PAREDES
Camilo BRAVO QUIROGA
Stephanie SLEBOS VERGARA
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Beetechnology SpA
Fundacion Copec - Universidad Catolica
Universidad Mayor
Original Assignee
Beetechnology SpA
Fundacion Copec - Universidad Catolica
Universidad Mayor
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Publication date
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Priority to PCT/CL2022/050059 priority Critical patent/WO2023230736A1/fr
Priority to ARP230101369A priority patent/AR129477A1/es
Publication of WO2023230736A1 publication Critical patent/WO2023230736A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
    • A01N63/22—Bacillus
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K50/00—Feeding-stuffs specially adapted for particular animals
    • A23K50/90—Feeding-stuffs specially adapted for particular animals for insects, e.g. bees or silkworms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/12—Unicellular algae; Culture media therefor

Definitions

  • the present invention is related to the beekeeping industry. Specifically, the invention relates to a composition for the prevention and treatment of American Foulbrood caused by Paenibacillus larvae in honey bees. The invention also relates to a manufacturing process and the use of said compositions.
  • honey bees are subject to many diseases and pests, which are the main obstacle in beekeeping.
  • the disease American Foulbrood, LA (American Foulbrood, AFB), is one of the main diseases that attack bees, caused by the bacteria Paenibacillus larvae that affects the brood, developing in their intestine and subsequently causing the death of the bees. larva.
  • the disease is difficult to control because P. larvae forms spores that are extremely resistant to the environment, cold and heat, humidity, chemical disinfectants and UV radiation, being able to survive up to 40 years in a natural environment, although they experience decrease in its viability in that period. Therefore, the most effective form of treatment is the total incineration of the hive and work material, being the most used worldwide.
  • American foulbrood is particularly lethal in the first 48 hours of life of the larvae, so curative actions are not effective and preventive control measures must be taken to prevent the disease from spreading in the hive.
  • an efficient but costly disease control method considers frequent inspection of hives by qualified apiary inspectors, accompanied by a search and destroy strategy, to minimize damage to apiaries, where the entire population of Infected bees are removed using poison gas and materials from the affected colony are disinfected with sodium hydroxide or burned, burying the dead bees and removed material to prevent worker bees from healthy colonies from being exposed to contaminated material.
  • Chemical methods to control AFB involve the administration of antibiotics, such as lincomycin or tylosin, or sodium sulfathiazole, in various formulations, mixed with powdered sugar or syrup. Since antibiotics and sulfonamides prevent the multiplication of the pathogenic bacteria, but do not kill the spores, the multiplication of P. larvae may begin again soon after the treatment is given, so the chemical treatment must be repeated periodically at intervals each time. shorter periods and higher concentrations, due to the generation of resistant strains, together with the simultaneous disinfection of the hives, with the risk of contaminating the honey with residues of antibiotics or sodium sulfathiazole.
  • antibiotics such as lincomycin or tylosin
  • Chemical treatment to control AFB can be carried out only in countries where it is permitted (USA and Canada, for example), under the supervision of a veterinarian; However, in Europe this method is prohibited.
  • oxytetracycline and sodium sulfathiazole are used, although in the United States the use of sulfathiazole has stopped due to the high residuality it has in honey, even more than a year, and due to the appearance of resistant bacterial strains.
  • tylosin tartrate it is still considered an effective alternative, because it is not toxic to bees, its residuality in honey is low (although it is recommended to apply it before the honey production season) and the disease does not recur. up to one year after treatment carried out.
  • the treatment does not eliminate the spores, multiple markets, including the European one, reject this type of treatment and imports with traces of chemical antibiotics, because they are a potential threat of entry of spores.
  • honey from an affected apiary could be contaminated with P. larvae spores, so care must be taken with honey processing and waste disposal areas.
  • Figure 1 Separated bacteria. Using spectrophotometric techniques, the growth of P. larvae is measured; the increase in turbidity is considered effective growth. Optical density is measured at 600 nm of the culture medium (BHI supplemented with thiamine), growth control of P. larvae, proteins against P. larvae from B. laterosporus, E. faecalis and the microbial consortium (proteins A, B and C, respectively), and the pathogen presented to the different treatments A, B and C. The average ⁇ the standard deviation is graphed. Significantly different from the control: ****, p ⁇ 0.0001, ANOVA analysis and Tukey test.
  • FIG. 1 Spores. Germination and development of P. larvae from its spores in liquid medium (BHI+t) with consortium proteins (concentration 1,029.84 pg/mL). Optical density is measured at 600 nm of the sterile culture medium ("Culture Medium”); consortium proteins ("C proteins”); spore growth and germination control ("Spore Germination”); spores on BH I medium treated with proteins C ("Treated spores”); and spores that were pretreated with protein C, washed and resuspended in BHI medium free of treatment (“Resuspended spores”). The average ⁇ standard deviation is plotted. Significantly different from the control: ****, p ⁇ 0.0001, ANOVA analysis and Tukey test.
  • FIG. 3 Bactericide. Turbidity measurement, related to P. larvae growth. The optical density is measured at 600 nm in sterile BHI medium; normal growth control of P. larvae ATCC 9545; P. larvae treated with consortium proteins (1,029.84 pg/mL); and the pathogen washed with PBS and resuspended in BHI medium after being treated with proteins of the consortium (1,029.84 pg/mL). The average ⁇ standard deviation, ANOVA and multiple comparison by Tukey (p ⁇ 0.05) are plotted; values with the same letter do not have significant differences.
  • FIG. 4A Growth of P. larvae faced with various v/v concentrations of the biocontroller (from 10 to 100%) against the SAG environmental strain (a) and ATCC 9545 strain (b). The measurement of the optical density at 600 nm is observed versus the controls and the different concentrations of the v/v biocontroller. The average ⁇ standard deviation, ANOVA and multiple comparison by Tukey (p ⁇ 0.05) are plotted; values with the same letter do not have significant differences.
  • Figure 4B Minimum Dose. Growth of P. larvae faced with various dilutions of the biocontroller against the ATCC 9545 strain. The measurement of the optical density at 600 nm is observed versus the controls (Culture medium and P. larvae Growth), and the different concentrations of the proteins of the consortium (205.96 pg/mL, 411.93 pg/mL, 617.9 pg/mL and 2,059.68 pg/mL). The average ⁇ standard deviation, ANOVA and multiple comparison by Tukey (p ⁇ 0.05) are plotted; values with the same letter do not have significant differences.
  • the result after the 8 days of the experiment is graphed with the average ⁇ standard deviation, ANOVA and multiple comparison by Tukey (p ⁇ 0.05), values with the same letter do not have significant differences.
  • Figure 7 Growth of P. larvae from its spores in liquid medium with the biocontroller at 50% v/v. The measurement of the optical density at 600 nm is observed versus the controls (BH I medium, SAG strain and ATCC strain) and the different treatments (biocontroller + SAG strain and ATCC strain. The average ⁇ the standard deviation is plotted. Significantly different from the control: ****, p ⁇ 0.0001, ANOVA analysis and Tukey test.
  • Figure 8 Growth of Lactobacillus kunkeei. Turbidity measurement, related to the growth of L kunkeei. The optical density at 600 nm is measured in the sterile MRS medium, normal growth control of L. kunkeei, the consortium proteins (Proteins C) and L. kunkeei treated with the proteins (37.37 pg/mL). The average ⁇ standard deviation, ANOVA and multiple comparison by Tukey (p ⁇ 0.05) are plotted; values with the same letter do not have significant differences.
  • the present invention corresponds to a composition that comprises the protein fraction of the culture of a bacterial consortium that includes strains of Brevibacillus laterosporus, Bacillus licheniformis, Enterococcus faecalis and Clostridium perfringens.
  • This consortium is deposited with the ATCC® on February 14, 2022 under the number PTA-127132.
  • the composition of the invention efficiently inhibits the growth of Paenibacillus larvae, which causes the American foulbrood disease, even eliminating the spores of the bacteria.
  • a protein composition it is a safer format for application compared to synthetic chemical compositions or antibiotics.
  • a protein composition is advantageous over the use of live bacterial probiotics directly, since the handling of a protein composition is more convenient and also does not alter the bee microbiota.
  • composition can be administered as a nutritional supplement, in mixture with bee food or in syrup.
  • a set of proteins produced by a bacterial consortium is obtained, where the composition that contains different proteins produced by the bacteria of the consortium are capable of inhibiting the growth of Paenibacillus larvae.
  • FIG. 1 shows a comparison between treatments with proteins from B. laterosporus (treatment A), E. faecalis (treatment B) and the microbial consortium, demonstrating that the use of the consortium (treatment C) is what reduces effective growth. much better than the use of B. laterosporus and E. faecalis separately, which is consistent with a synergistic effect.
  • consortium has demonstrated a bactericidal effect, which is verified when evaluating growth in normal culture medium, when treating it with proteins from the consortium and when cultivating after treatment with said proteins, in both treatments with a concentration of 1,029.84 pg /mL, equivalent to a concentration of about 50%, of consortium proteins (see Figure 3).
  • the protein production process by the bacterial consortium includes the following stages:
  • BHI Brain Heart Infusion Broth
  • Merck supplemented with 1 - 5 mg/mL of thiamine hydrochloride is preferably used.
  • Filter with a pore size of 0.2 pm preferably using a membrane with low protein affinity, in order to avoid decreasing the concentration of proteins in the eluate.
  • a membrane filter made of polyethersulfone (PES), regenerated cellulose (RC), cellulose acetate (CA) or polyvinylidene fluoride (PVDF).
  • the concentration of consortium proteins (active ingredient) obtained can be diluted according to the desired final concentration.
  • This sterile protein supernatant constitutes the composition of the invention, and can be administered directly to the hive by direct application to the brood frames or mixed with fructose or dextrose, or other sugars, as a syrup.
  • Fructose or dextrose or other sugars are used in a concentration between 20 to 50% w/v.
  • composition and/or proteins obtained from the consortium of the present invention can be administered in a mixture with other vehicles, such as water, culture medium, nutritional supplement, food, wax or other solid substrates.
  • any food grade ingredient to be incorporated into the hive such as Potassium sorbate: between 3 and 8 mg/L; sodium propionate: between 3 and 8 mg/L, citric acid: between 18 and 26 mg/L, calcium propionate: between 3 and 8 mg/L, soy flour: between 200 to 300 g/L, dehydrated egg: between 200 and 300 g/L, fructose or dextrose: between 300 and 700 g/L, among others.
  • Potassium sorbate between 3 and 8 mg/L
  • sodium propionate between 3 and 8 mg/L
  • citric acid between 18 and 26 mg/L
  • calcium propionate between 3 and 8 mg/L
  • soy flour between 200 to 300 g/L
  • dehydrated egg between 200 and 300 g/L
  • fructose or dextrose between 300 and 700 g/L, among others.
  • hives For direct application to hives, without mixing with sugars, it can be applied to adult bees, larvae and to any apiary instrument that may be in direct contact with the bee hive: to brood frames, boards and hive covers. , in the stamping and/or in the hive feeder by mechanical application.
  • the composition can be administered by spraying, dipping, impregnation or combinations thereof, as well as being administered through the hive feeder.
  • the recommended dose is 500 ml per hive in a final concentration range from 205 pg/ml to 2 mg/mL of consortium proteins.
  • the composition obtained directly (100%) in one embodiment of the invention, is diluted to concentrations equivalent to a protein concentration of 409.4 pg/mL or higher. And in any case, the protein concentration of the consortium will always be applied in concentrations greater than 37 pg/ml.
  • Figure 4A shows that even from 15% dilutions inhibition of the growth of P. larvae could be achieved, so an evaluation of the minimum dose was carried out, using concentrations of 205.96 pg/mL, 411, 93 pg/mL, 617.9 pg/mL and 2,059.68 pg/mL (Figure 4B); From this it is obtained that concentrations above 617.9 pg/mL achieve the maximum biocontrol effect. Therefore, the biocontrol effect is preferably observed in a concentration range from 205.96 pg/mL to 2,060 pg/mL, where dilutions can be made with water, culture medium or the vehicle used for application in the hive.
  • the indicated ranges are derived from experimental results where the supernatant of the isolated microbial consortium is capable of inhibiting the growth and germination of Paenibacillus larvae spores at concentrations greater than 20% and 50% v/v, respectively.
  • the composition has a protein concentration of approximately 1,500 to 2,500 pg/mL for each production batch, preferably 2,040 ⁇ 10 pg/mL, with a shelf life of 32 and 40 weeks stored at room temperature. and 4°C, respectively, maintaining its inhibitory characteristics against P. larvae.
  • the PierceTM Coomassie (Bradford) Protein Assay kit from Thermo-Fisher was used to determine the protein concentration of the supernatant. After filtering and pH neutralization, the protein concentration was measured against the Bradford reagent in the NanoQuant Tecan Infinite m200 PRO equipment at OD 595nm, with dilution factor 1/4. The absorbance results are extrapolated to a BSA calibration curve of the kit between the ranges of 25pg/mL to 1,500 pg/mL to determine the protein concentration of the biocontroller. Notwithstanding the foregoing, the protein concentration may be determined according to any method available in the state of the art, without changing the scope of the present invention.
  • the cell lines were exposed to the supernatant according to the protocol of the CytoTox 96® Non-Radioactive Cytotoxicity Assay kit from Promega®.
  • Medium with cells was placed in a 96-well plate and the supernatant, as well as a control for maximum release of lactate dehydrogenase (LDH), were incubated at 37°C for the CaCo-2 cell line and at 27°C for Sf9 for a 3 hour period. Subsequently, it was left at room temperature in the dark for 30 minutes and then the Stop solution was added and absorbance was measured at 490nm at the time of adding the Stop solution. The results were calculated according to Equation 1.
  • Equation 1 Calculation of percentage of cytotoxicity of the CytoTox 96® Non-Radioactive Cytotoxicity Assay kit from Promega®.
  • Percent cytotoxicity 100 x - - - - - - - - - - - - - - - - —
  • Oral and contact toxicity tests on Apis mellifera determined an LD5O greater than 100 pg/bee for both tests, which means low lethality associated with the biocontroller.
  • composition is characterized by not producing toxicity by ingestion, contact or inhalation in mammals.
  • composition of the invention has been shown to inhibit the growth of the Paenibacillus larvae bacteria and by using the composition against P. larvae spores, its development and growth is prevented.
  • the composition is capable of eliminating only the P. Larvae bacteria, without damaging the beneficial microbiota of the bees.
  • the bacteria Lactobacillus kunkeei which has been reported as a predominant bacteria in the bee intestine, is not affected when using the composition according to the invention.
  • composition of the present invention has bacteriolytic, bactericidal and bacteriostatic potential, which allows us to affirm that once the disease is controlled, it will prevent its recurrence in the hive.
  • composition according to the present invention is of natural origin and does not leave residues in the products that could affect their commercialization.
  • bacteria instead of bacteria in the microbial consortium makes it a more attractive option for disease control in bees.
  • the use of bacteria as biocontrollers can alter the normal microbiota of bees, sugars or food could be fermented, or the conditions may not exist for the bacteria to
  • Example 1 Obtaining cell-free supernatant
  • the microbial consortium composed of bacteria of the genera Brevibacillus laterosporus, Bacillus licheniformis, Enterococcus faecalis and Clostridium perfringens (deposit number PTA-127132) was grown in BHI medium with 3 mg/mL thiamine for 6 days at 37°C, centrifuged at 10,000 rcf for 30 minutes at room temperature, collecting 0.5 L of the supernatant.
  • the pH was adjusted by adding 1M NaOH until reaching pH 7.2.
  • Example 2 Administration of the composition through food
  • Solid cakes or bars were used to feed the bees, these were placed on the frames and under the top lid of the hive.
  • Foods made with the composition of the invention comprising the cell-free supernatant may optionally contain sugar syrup as indicated below.
  • the amount of protein composition was determined so that the final dose of the supernatant is greater than 205.96 ug/ml and less than 2 mg/ml, as recommended.
  • Syrup is a sugar-based product that is typically administered through the feeders of each hive.
  • granulated sugar diluted in water was used to reach a viscous consistency. For every kilo of granulated sugar, 0.5 liters of water were used. The water was hot at approximately 60°C, the sugar was added slowly while stirring so that all the sugar was dissolved and a homogeneous syrup was formed without crystals in suspension. It was allowed to cool to 35°C and the same amount of cell-free supernatant was incorporated for each liter of water in the syrup, that is, 0.5 liters of cell-free supernatant. The amount of protein composition allowed the final recommended dose of the supernatant to be greater than 37 ug/ml and less than 2 mg/ml.
  • a syrup could be made where the sugar is diluted directly in the cell-free supernatant, where for each kilo of granulated sugar 1 liter of cell-free supernatant is used.
  • concentrations from 10% to 100% v/v of the cell-free supernatant of the consortium were used against P. larvae in liquid culture medium. For example, to obtain 10 mL at a concentration of 30% v/v, 3 mL of cell-free supernatant and 7 mL of culture medium are used. For this, the optical density (OD) was measured by observing its growth curve over time in the NanoQuant Tecan Infinite m200 PRO equipment at OD 600nm. The ATCC 9545 strain was used as a control without the addition of supernatant, under the same conditions.
  • Table 3 OD at 660nm for different treatments in P. larvae ATCC 9545
  • Example 5 Evaluation of inhibitory capacity against spore germination.
  • the procedure to promote sporulation in P. larvae consisted of culturing on BHIT plates at 37°C for 6 to 7 days, microscopically monitoring cell growth and sporulation. After the incubation time, the spores were removed by washing three times with sterile water and subsequent centrifugation at 12,000 x g for 15 minutes at 4°C to concentrate the spores. The pellet was resuspended in 30 mL of cold sterile water, centrifuged and washed four more times, discarding supernatant. The final resuspension was made in 5 mL of cold sterile water and stored at 4°C.
  • a concentration of 800 to 1,000 spores was estimated in BHI media supplemented with thiamine and 50% biocontroller.
  • the concentration of BHIT was maintained at IX, and it was incubated for 48 hours at 37°C.
  • Example 6 Evaluation of the toxicity of the biocontroller by acute oral administration in adult bees (Apis mellifera).
  • Dimethoate which was evaluated in 5 doses (0.06, 0.13, 0.25, 0.50 and 1.00 pg/bee).
  • Example 7 Evaluation of the acute toxicity of the biocontroller by contact in adult bees (Apis mellifera).

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Abstract

Composition pour le traitement et la prévention de la loque américaine (LA), pour la protection des ruchers d'abeilles miélifères, procédé pour l'élaboration de cette composition et son utilisation dans la prévention et le traitement de maladies microbiennes, notamment causées par la bactérie Paenibacillus larvae. La composition de l'invention comprend des protéines obtenues à partir d'une communauté microbienne déposée auprès de l'ATCC sous la référence PTA-127132 qui comprend les bactéries Brevibacillus laterosporus, Bacillus licheniformis, Enterococcus faecalis et Clostridium perfringens.
PCT/CL2022/050059 2022-05-30 2022-05-30 Composition pour le traitement et la prévention de la loque américaine (la) Ceased WO2023230736A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
PCT/CL2022/050059 WO2023230736A1 (fr) 2022-05-30 2022-05-30 Composition pour le traitement et la prévention de la loque américaine (la)
ARP230101369A AR129477A1 (es) 2022-05-30 2023-05-30 Composición para el tratamiento y prevención de loque americana (la)

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Application Number Priority Date Filing Date Title
PCT/CL2022/050059 WO2023230736A1 (fr) 2022-05-30 2022-05-30 Composition pour le traitement et la prévention de la loque américaine (la)

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WO2023230736A1 true WO2023230736A1 (fr) 2023-12-07

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Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
ALIPPI, A. M. ET AL.: "Inhibition of the growth of Paenibacillus larvae, the causal agent of American foulbrood of honeybees, by selected strains of aerobic spore-forming bacteria isolated from apiarian sources", JOURNAL OF INVERTEBRATE PATHOLOGY, vol. 91, no. 3, 2006, pages 141 - 146, XP024948652, DOI: 10.1016/j.jip. 2005.12.00 2 *
BARTEL LAURA CECILIA, ABRAHAMOVICH ELIANA, MORI CONSUELO, LÓPEZ ANA CLAUDIA, ALIPPI ADRIANA MÓNICA: "Bacillus and Brevibacillus strains as potential antagonists of Paenibacillus larvae and Ascosphaera apis", JOURNAL OF APICULTURAL RESEARCH, INTERNATIONAL BEE RESEARCH ASSOCIATION, CARDIFF, GB, vol. 58, no. 1, 1 January 2019 (2019-01-01), GB , pages 117 - 132, XP093121679, ISSN: 0021-8839, DOI: 10.1080/00218839.2018.1495439 *
KAČÁNIOVÁ MIROSLAVA, TERENTJEVA MARGARITA, ŽIAROVSKÁ JANA, KOWALCZEWSKI PRZEMYSŁAW ŁUKASZ: "In Vitro Antagonistic Effect of Gut Bacteriota Isolated from Indigenous Honey Bees and Essential Oils against Paenibacillus Larvae", INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES, MOLECULAR DIVERSITY PRESERVATION INTERNATIONAL (MDPI), BASEL, CH, vol. 21, no. 18, Basel, CH , pages 6736, XP093121684, ISSN: 1422-0067, DOI: 10.3390/ijms21186736 *
MARCHE, M. G. ET AL.: "Inhibition of Paenibacillus larvae by an extracellular protein fraction from a honeybee-borne Brevibacillus laterosporus strain", MICROBIOLOGICAL RESEARCH, vol. 227, 2019, pages 126303, XP085774189, DOI: 10.1016/j. micros. 2019.126303 *

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