Bacterial Agents for Biocontrol of American Foulbrood (AFB) of Larvae Honey Bee
1
Division of Infectious Diseases and Veterinary Administration, Department of Epizootiology with Exotic Animal and Bird Clinic, Wrocław University of Environmental and Life Sciences, 50-375 Wrocław, Poland
2
Beneficial Insect Diseases Laboratory, Department of Epizootiology with Exotic Animal and Bird Clinic, Wrocław University of Environmental and Life Sciences, 50-375 Wrocław, Poland
3
Division of Apiculture, Institute of Animal Husbandry and Breeding, Wrocław University of Environmental and Life Sciences, 50-375 Wrocław, Poland
4
ProBiotics Polska Wytwórnia Probiotyków, 62-720 Bratuszyn, Poland
*
Authors to whom correspondence should be addressed.
Microbiol. Res. 2024, 15(4), 2394-2413; https://doi.org/10.3390/microbiolres15040161
Submission received: 27 September 2024
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Revised: 16 November 2024
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Accepted: 20 November 2024
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Published: 24 November 2024
Abstract
:Bee colonies are constantly threatened by a bacterial larvae disease called American foulbrood, caused by the Gram-positive bacterium Paenibacillus larvae. This is a highly specialized pathogen with only one established host, the honey bee larvae. The current state of legislation throughout the European Union prevents the inclusion of pharmacotherapy treatment, and the only control is based on the physical elimination of infected colonies upon veterinary orders. The purpose of this study was to investigate the antimicrobial activity of selected bacteria with proven probiotic potential with typical characteristics meeting the definition of a probiotic that could reduce the American foulbrood pressure by promoting the development of the larvae microbiome that competes with and blocks the excessive proliferation and production of P. larvae endospores. The antimicrobial activity of inoculants of the following selected strains was studied: Bacillus pumilus, Bacillus licheniformis, Streptomyces narbonensis, Lysinibacillus fusiformis, Levilactobacillus brevis, Bacillus megaterium against Paenibacillus larvae ATCC 9545 (formerly Paenibacillus larvae sub sp. larvae), and Paenibacillus larvae CCUG 48973. Analyses were performed by the well diffusion method according to EUCAST standards (clinical breakpoints and dosing of antibiotics) with modifications due to the specificity of the bacteria used. The largest zone of growth inhibition of P. larvae was confirmed with S. narbonensis, B. licheniformis, and B. megaterium, and medium activity was observed with L. brevis and B. pumilus. Negligible activity was shown by L. fusiformis. Differences were noticed in the resistance of indicator strains of P. larvae and between the media and carriers used.
Keywords:
AFB; Paenibacillus larvae; bees; probiotics; biocontrol; biosecurity; beneficial; microorganisms; welfare1. Introduction
The work conducted by honey bee workers for the benefit of humans and the ecosystem is invaluable. EFSA reports that the annual pollination by bees has been valued at around 22 billion Euros, and in Poland alone, this value exceeds 964 million Euros [1,2]. Worldwide, the economic value of pollination by honey bee workers has been estimated at 265 billion Euros per year [3,4]. Researchers have been searching for many years for factors that reduce losses in apiary management and increase the security of the honey bee as the main pollinator in the food production chain [5,6,7]. Bee colonies are constantly threatened by brood and adult bee diseases, susceptibility to xenobiotics (including pesticides), an impoverished forage base, and lack of diverse protein food results in massive worker malnutrition (nutritional stress), environmental stress, and newer pests arriving from different parts of the world (populations of the small hive beetle or Asian hornet) [8,9]. The honey bee is also accompanied by a microflora whose biodiversity is modified by many environmental factors, including stress factors such as pesticides, forage shortages, pressure from the pathogenic microbiome, and immune impairment [10,11,12]. According to various literature sources, there are between 6000 and 8000 different microbial strains in the microbiome of bee colonies [13].
One microbial threat to the balance of the colony microbiome is Paenibacillus larvae, a Gram-positive, peridomestic, mobile, virulent, flagellated, spore-forming bacterium highly adapted to its only known host; honey bee larvae that cause a brood disease are called American foulbrood [14]. The bacteria Paenibacillus larvae, which produces approximately one billion spores per infected larva, is the main vector of this disease [15].
Increasingly, the so-called orphan bacteria, i.e., bacteria that are highly adapted to a specific host or extreme environment, are the object of scientific interest for many scientists. Such bacteria include the P. larvae, which attack bee larvae that become infected through the activity of feeder bees and hub bees, which take care of the brood and provide food, often already contaminated with P. larvae endospores. Due to ongoing proteolytic processes, the larvae are broken down into a purulent infectious mass. After drying, this mass continues to exhibit infectious properties. When most of the larvae are infected and die, the whole colony is sealed and collapses. The pathogenic bacteria become a natural opponent for the probiotic bacteria that are in the larvae’s native gut microbiome and can completely sterilize the microflora and dominate this environment through the expansion of P. larvae. The metabolism of these bacteria shows that they can produce peptide antibiotics either through genes encoding for the synthesis of ribosomal peptides or through giant gene clusters encoding for enzyme complexes that non-ribosomally synthesize antimicrobial active peptides (NRP) or polyketides (PK). Gene clusters are responsible for producing low molecular weight compounds, such as bacillibactin, were identified. They act as an iron chelator, meaning that they bind iron in the environment, making it difficult for other microorganisms to access this element. This gives bacteria that produce bacillobactin an advantage in the competition for resources in a given ecosystem. The destructive effect of cytotoxins such as paenilarvins, in which antifungal properties have been confirmed against yeasts and filamentous fungi, or sevadicins or paenilamicins with bactericidal and fungicidal effects, is also suspected. The bacteriocins and antimicrobial compounds secreted by P. larvae first eliminate the microbes in the intestine of the larvae, thus ensuring that the infection is only potentiated by the P. larvae. During the colonization of the midgut lumen, P. larvae peptide antibiotics (e.g., paenilamicin and iturin) eliminate bacterial and fungal microorganisms ensuring that P. larvae are the only bacterium. In addition, P. larvae secrete enzymes (lytic polysaccharide monooxygenases) that break down chitin, which protects epithelial cells. Specific toxins secreted by P. larvae Eric II, i.e., Plx1, Plx2, or cytotoxic secondary metabolites secreted by both genotypes (paenilarvin by Eric II and paenilamicin secreted by Eric I/II) attack essential cellular functions and the bacteria are attached to exposed epithelial cells through the S-layer. Two important mechanisms should be considered: the degradation of chitin and the degradation of the midgut of larvae, which are estimated to be key steps in the pathogenesis of P. larvae infection [16].
Spores can survive for up to 50 years under favorable conditions and exhibit infectious properties for 35 years. A temperature of 100 °C destroys spores after 5 days, while at 140–170 °C they die after only 2 h. Spores are also killed in 5–10% formalin within 6 h, while soda lye kills spores at a concentration of 2% within 4 min. It has been found that 5% sodium hypochlorite is also effective. Vegetative forms die at 60 °C [16,17,18]. An infected and disease-dead larva can contain up to 2.5 billion endospores, but only 10 to 35 spores are needed to infect another larva, indicating high virulence and a high rate of disease spread [19,20]. Spores can reside in honey, wax, royal jelly, propolis, pollen, and bee feathers. Once in the digestive tract of the larvae, they germinate after 24 h, leading to bacterial superinfection manifested by rapid growth of vegetative forms. The vegetative cells damage the epithelium and intestinal walls and later attack all internal organs leading to cytolysis and histolysis [21,22,23]. Nine days after infection, spores begin to form. The maggots die after 2–3 days after the cell is sealed [24]. The dead larvae become flaccid, changing color first to yellow, then to a brown, sticky, and malleable mass. Five serotypes of American foulbrood are currently recognized worldwide: Eric I, Eric II, Eric III, Eric IV, and Eric V [25,26].
The epizootiological agent for all varieties is the same bacterial species, Paenibacillus larvae. Sick bee colonies are physically eradicated on the recommendation of veterinarians, legislation in the EU does not allow the use of any form of pharmacotherapy, and in Poland, American foulbrood is eradicated ex officio. In the past, antibiotics and sulfonamides were used. Currently, only administrative procedures are available for American foulbrood control [27]. Once American foulbrood is confirmed, the district veterinarian may decide to eradicate the colonies or, in justified cases, order a double relocation procedure to a new or decontaminated hive on frames with hoses [28]. Most cases with confirmed cases of foulbrood are classified as a high-risk group and end up with the burning of infected colonies. Prevention consists primarily of maintaining appropriate sanitary standards involving, for example, frequent changes in combs, and disinfection of hives and tools (e.g., with Virkon S produced by Bayer, active substance: pentapotassium bis(sulfate) bis(peroxymonosulfate)). Other prevention methods include descriptions of the use of essential oils: cinnamon, rosemary, thyme, lemon, or aniseed. The effect of extracts of various herbal species on P. larvae cells is also known from in vitro laboratory studies [29,30]. Solutions described as effective in reducing P. larvae abundance include products to be applied as food additives (syrup or cake) and products to be applied as sprays or sprinkled on the inter-frame streets [31].
In Poland, there are no specific legal restrictions on the movement of migratory apiaries, which exacerbates the epidemiological situation.
The phenomenon of competition between microorganisms is well-known. Bacterial metabolites, also known as metabiotics, have become an object of scientific interest in recent years. Micro-organisms isolated from the digestive tracts of bees with in vitro antimicrobial activity against P. larvae are described in the literature [32,33]. Such activity may be called bioassurance. The definition given by Zygmunt Pejsak includes specific indications to be implemented to reduce or eliminate the risk of pathogen transmission into the flock. The aim of bioassurance is, among other things, to reduce the risk of introducing an infectious agent into the herd [34]. In the definition of another researcher Thomas Gillespie, bioassurance must reduce the risk of pathogen intrusion and must consist of bioexclusion, which will control the transmission of pathogens. This element of bioassurance has been referred to as biocontainment [35]. Bioassurance is the so-called biological protection consisting of protecting the animals through preventive and sanitary measures using biocontainment on the farm as well as in the immediate surroundings [36,37]. Biotisation as a tool for the introduction of bioassurance may be a new biotechnological approach to animal husbandry. It consists of inoculating the animal environment with beneficial micro-organisms such as fungi or bacteria to increase their dominance in the environmental microbiome by increasing the tolerance of animals to biotic and abiotic stresses (in practice this is spraying or fogging) [38,39,40]. Beneficial micro-organisms, also known as beneficial or effective microorganisms (EM), have an important function in restoring the microbial balance in the native microbiome of animals and also supply animals with valuable metabolites and protect them from pathogens [40,41].
The lack of effective product solutions on the market to prevent the development of superinfection, the unstable administrative and legal status of American foulbrood, the risky practices of beekeepers and growers exacerbating the American foulbrood problem, the increased interest on the market in natural products like targeted microbial biopreparations with increased metabolite content, the untapped potential of biotechnology and microbiology in the control and prevention of livestock diseases and animal welfare are just some elements of why this topic of work was undertaken.
The complexity of the research problem concerns is as follows: (1) the reduction in American foulbrood pressure in bee colonies by supporting the development of the physiological microbiome of honey bee larvae, competing with and blocking the excessive proliferation and production of P. larvae endospores; and (2) testing the sensitivity of P. larvae vegetative cells to biologically active substances, constituted by biotechnological processes aimed at obtaining a high content of bioactive compounds in the final product using bacterial probiotic components.
The aim of the study was (1) to test the biological activity of selected bacterial components with confirmed probiotic potential that could constitute the composition of a pilot pre-formulation of future metabolic preparation, and (2) to compare the antimicrobial activity of inoculants and post-culture (post-fermentation) fluids of selected, functional bacterial strains against Paenibacillus larvae Eric I ATCC 9545 (Labiol) and Eric II CCUG 48973 (Labiol).
The overall scope of the work involves making inoculants of functional bacterial strains by revitalizing the bacterial strains by hydration of lyophilizes according to in-house procedures, then carrying out the actual multiplication (fermentation) in glass bioreactors and obtaining the first half pre-formulations on a laboratory scale. This consisted of transferring the functional bacteria cultures to selected media and multiplying by a technological process aimed at obtaining bioactive metabolites. The final step was to determine the antimicrobial activity of the culture fluids by establishing the inhibition zones of the indicator strain. This was performed by inoculating bloody agar with a suspension obtained from the selective multiplication of biological material on selective nutrient slants for P. larvae. Results were presented after 48–72 h of incubation at 37 °C.
2. Materials and Methods
2.1. Bacterial Strains
2.1.1. Beneficial Microorganisms
The working material consisted of the functional bacterial strains which were used in an experiment to check their antimicrobial activity: Bacillus pumilus R5 strain, Bacillus licheniformis M/542/M/18 strain, Streptomyces narbonensis R71 strain, Lysinibacillus fusiformis E23 strain, Levilactobacillus brevis M/495/M/17 strain, and Bacillus megaterium R7 strain. The names of the strains have internal numbering and coding. The strains were deposited at the Institute of Microbiological Technology in Turek, Poland. The test strains had confirmed probiotic potential, meeting the FAO definition and the essential characteristics of a probiotic.
2.1.2. Pathogens
The biological material consisted of the bacterial, pathogenic, indicator strains belonging to Paenibacillus larvae ATCC 9545 (Labiol) and Paenibacillus larvae CCUG 48973 (Labiol). The strains were deposited at the Institute of Microbiological Technology in Turek, Poland.
2.2. Cultivation of Microbial Cultures—Bacterial Inocula
Banked bacterial strains were revived. The strains belonging to Bacillus pumilus R5, Bacillus licheniformis M/542/M/18, Lysinibacillus fusiformis E23, and Bacillus megaterium R7 were seeded on nutrient agar (Argenta, Poznań, Poland) and incubation took place at 37 °C under aerobic conditions. A strain belonging to Streptomyces narbonensis R71 was seeded on Streptomyces medium (Sigma-Aldrich, Saint Louis, MO, USA) and incubation took place at 28 °C under aerobic conditions. The strain belonging to Levilactobacillus brevis M/495/M/17 was seeded on MRS agar (Argenta) and incubation took place at 30 °C under anaerobic conditions. At the end of the incubation period, so-called microbial washes were made from the 24 h cultures of pure bacterial cultures and inocula with an optical density of OD = 0.5° McFarland Standards (approximately 1.0 × 108 CFU/cm3 (densitometer DEN-1B, Biosan, Poland) were obtained.
2.3. Preparation of Microorganism Cultures in Bioreactors
The prepared inoculants were used to inoculate in the bioreactors (Donserv, LPP Equipment, Warszawa, Poland) an industrial medium (autoclaved 4% aqueous solution of organic cane molasses on distilled water) at 4% by volume and a second, liquid tryptone peptone with glucose and yeast extract medium (BTL) also at 4%. Cultures were prepared in 18-L glass bioreactors at 30–38 °C for 10 days under anaerobic or aerobic conditions depending on the specific bacteria used. Basic biotechnological parameters were observed, such as kinetics of pH value changes, ORP-Redox conductivity [µs/cm], sugar content [°Brix], an increase in culture density [CFU/cm3], general contamination aerobic microorganisms, and fungi Clostridium perfringens. If a particular strain starts to multiply on the medium then the pH should normally decrease, ORP-Redox should increase, and optical density should also increase. The absence of these changes may indicate that the multiplication process is not going well, for example, due to infections. The MARTA (Bionet F1, 2021) software supplied to the bioreactors by LPP Equipment allows internal measurement of biotechnology parameters. These coincide with external measurements of collected samples using basic laboratory equipment. All experimental conditions are consistent and the same for all experimental systems. There are 920 bacteria in the strain bank. Only selected bacteria from the genera Bacillus, Streptomyces, and Lactobacillus were included in the study. This action is motivated by a review of the scientific literature, which indicates the high antimicrobial activity of the bacterial species selected in the experiment (biological agents for American foulbrood biocontrol) against Paenibacillus larvae, genotypes: Eric I and Eric II [42,43].
2.4. Working Methodology
Agar Diffusion Assay
For the determination of antimicrobial activity, the well diffusion method was used according to EUCAST standards with modifications due to the specificity of the bacteria used. For this purpose, the P. larvae Eric I and Eric II strains were revived by transplanting colonies onto blood agar medium (Argenta) and incubated at 37 °C for 24–48 h in a CO2 atmosphere (incubator BE400, Memmert, ZALMED, Warszawa, Poland). A so-called microbial wash was then performed with an optical density of OD = 0.5° McFarland Standards (approximately 1.0 × 108 CFU/cm3, densitometer DEN-1B, Biosan, Józefów-Otwock, Poland). Each of the functional inoculants was combined, respectively, with the media used in beekeeping to feed the bee colonies (sterile water and sterile sucrose solutions of 50% and 60%, Merck, Darmstadt, Germany), and mixtures of 3, 4, 5, and 10% v/v were obtained. The culture fluid in undiluted form was treated to a concentration of 100%. Media without bacteria were used as control groups, i.e., sterile water and sterile sucrose solutions of 50% and 60% (Merck). All Petri dishes (Thermo Scientific, Warszawa, Poland) were incubated for 48–72 h at 37 °C in a CO2 atmosphere (incubator BE400, Memmert, ZALMED, Poland). After incubation, the ‘halo’ zones were measured, which are the sole criterion for eligibility of a strain for further work.
2.5. Statistical Analysis
Statistical analysis of the results obtained was performed using one-way ANOVA (Tukey’s parametric post hoc test) at a significance level of p-value < 0.05. The statistical analysis included results from the evaluation of antimicrobial potential (v/v concentrations of the inoculant—4 groups versus the carriers: water, sucrose 50%, and sucrose 60%—3 groups).
3. Results
3.1. Parameters of Microorganism Cultures in Bioreactors
Strains belonging to the species B. pumilus, B. licheniformis, S. narbonensis, L. fusiformis, L. brevis, and B. megaterium were successfully multiplied in a volume of 18 L on target media. The cultivation lasted for 10 days. The release parameters met the final specifications of the process.
The strain belonging to the species Bacillus pumilus successfully multiplied in a volume of 18 L on target media. The culture lasted for 10 days. The final culture density was 4.5 × 105 CFU/cm3 in a cane molasses medium to 4.7 × 105 CFU/cm3 in a tryptone peptone medium. There is a marked decrease in pH values, a slight decrease in ORP-Redox, and a slight consumption of sugars manifested by a weak decrease in Brix values. No infections with foreign microflora are observed (Table 1).
Strain belonging to the species Bacillus licheniformis successfully multiplied in a volume of 18 L on target media. The culture lasted for 11 days. The final culture density was 3.5 × 107 CFU/cm3 in a cane molasses medium to 3.7 × 107 CFU/cm3 in a tryptone peptone medium. There is a marked decrease in pH values, a significant increase in ORP-Redox, and little consumption of sugars is manifested by a weak decrease in Brix values. No infections with foreign microflora are observed (Table 2).
Strain belonging to the species Streptomyces narbonensis successfully multiplied in a volume of 18 L on target media. The culture lasted for 12 days. The final culture density was 4.5 × 106 CFU/cm3 in a reed molasses medium to 4.7 × 106 CFU/cm3 in a tryptone peptone medium. There is a marked decrease in pH values, a significant increase in ORP-Redox, and little consumption of sugars is manifested by a weak decrease in Brix values. No infections with foreign microflora are observed (Table 3).
Strain belonging to the species Lysinibacillus fusiformis successfully multiplied in a volume of 18 L on target media. The culture lasted for 10 days. The final culture density was 3.5 × 107 CFU/cm3 in a cane molasses medium to 3.7 × 107 CFU/cm3 in a tryptone peptone medium. There is a marked decrease in pH values, a significant increase in ORP-Redox, and little consumption of sugars is manifested by a weak decrease in Brix values. No infections with foreign microflora are observed (Table 4).
Strain belonging to the species Levilactobacillus brevis successfully multiplied in a volume of 18 liters on target media. The culture lasted for 8 days. The final culture density was 4.5 × 108 CFU/cm3 in cane molasses medium to 3.4 × 105 CFU/cm3 in a tryptone peptone medium. There is a marked decrease in pH values, a significant increase in ORP-Redox, and a significant consumption of sugars manifested by a significant decrease in Brix values. No infections with foreign microflora are observed (Table 5).
Strain belonging to the species Bacillus megaterium successfully multiplied in a volume of 18 liters on target media. The culture lasted for 10 days. The final culture density was 2.7 × 108 CFU/cm3 in a cane molasses medium to 2.6 × 108 CFU/cm3 in a tryptone peptone medium. There is a marked decrease in pH values, a slight increase in ORP-Redox for the cane molasses medium, and a decrease in ORP-Redox for the tryptone peptone medium, as well as a slight consumption of sugars manifested by a slight decrease in Brix values. No infections with foreign microflora are observed (Table 6).
3.2. Results Based on Agar Diffusion Assay
3.2.1. Antimicrobial Activity of Bacterial Suspensions Against P. larvae ATCC 9545 (Eric I Genotype)
An attempt to inhibit the growth of strain Paenibacillus larvae belonging to Eric I genotype using inoculants of selected bacteria with probiotic potential under in vitro conditions was carried out successfully. Comparative analysis of the antimicrobial activity of culture fluids of selected bacterial species against Paenibacillus larvae (Eric I genotype) under in vitro conditions showed differences between the inoculants tested and thus, between the bacterial strains used, as well as between the media and culture media used. Under in vitro conditions, 9 out of 12 single inoculants tested showed suppressive effects against indicator strains belonging to the P. larvae (Eric I genotype) species and additionally bacterial consortia labeled as Mix (Table 7).
The greatest zone of growth inhibition of P. larvae was confirmed for culture fluids of bacterial species such as B. pumilus (suspended in water, molasses culture), Mix (suspended in 60% sucrose, molasses culture), L. brevis (suspended in 50% sucrose, molasses culture), and S. narbonensis (suspended in 60% sucrose, molasses culture) qualifying these microorganisms into the group with the greatest antimicrobial activity.
The group with high antimicrobial activity was assigned to strains belonging to the species B. megaterium (suspended in 60% sucrose, molasses culture), L. brevis (suspended in 60% sucrose and 60% sucrose, molasses culture), L. brevis (suspended in 50% sucrose and 60% sucrose, tryptone peptone culture), L. fusiformis (suspended in 60% sucrose, tryptone peptone culture), S. narbonensis (suspended in 50% sucrose, molasses culture), B. licheniformis (suspended in 50% sucrose, molasses culture) and B. pumilus (suspended in 50% sucrose and 60% sucrose, molasses culture).
The group with lower antimicrobial activity was assigned to strains belonging to the species B. licheniformis (suspended in water, molasses culture), S. narbonensis (suspended in 50% sucrose, tryptone peptone culture), S. narbonensis (suspended in water, molasses culture), L. fusiformis (suspended in 50% sucrose, tryptone-peptone culture), L. brevis (suspended in water, molasses culture), B. megaterium (suspended in all carriers, tryptone-peptone culture), B. megaterium (suspended in water and 50% sucrose, molasses culture), Mix (suspended in water, tryptone peptone culture), and Mix (suspended in water, molasses culture).
The group with negligible antimicrobial activity included a strain belonging to the species B. pumilus (suspended in all carriers, tryptone peptone culture), B. licheniformis (suspended in all carriers, tryptone peptone culture), S. narbonensis (suspended in water and 60% sucrose, tryptone peptone culture), L. fusiformis (suspended in water, tryptone peptone culture), L. fusiformis (suspended in all carriers, molasses culture), and L. brevis (suspended in water, tryptone peptone culture) (Table 7 and Figure 1).
The study also showed the superiority of the ‘Mix’ bacterial consortium over single inoculants for biocontrol of the development of P. larvae infection (Table 7).
For some samples, bacterial overgrowth or infection of the plates marked with ‘P’ and ‘Z’ was noticed so those samples were removed from statistical analyses. The results of the interaction of the control samples (with water, 50% sucrose, and 60% sucrose) always indicated a level of 0 mm. This confirms that the sucrose solution alone does not have an antimicrobial effect (Table 7). Statistical analysis of the results obtained was performed using one-way ANOVA (Tukey’s parametric post hoc test) at a significance level of p-value < 0.05. It has been proven that there is a correlation and a statistically significant correlation between the microorganisms used multiplied in the biotechnological fermentation process and the subsequent antimicrobial activity of the metabolites and bacteria remaining in the culture fluid. In some cases (experimental systems), there is a correlation and a statistically significant correlation between the inoculant concentration used and its subsequent antimicrobial activity. The analysis of the antimicrobial activity showed statistically significant differences between the inoculants tested and, therefore, between the bacterial strains used and between the media, media, and indicator strains used. A one-way ANOVA analysis indicated a superior efficacy of the Mix against the pathogen analyzed. Statistical analysis (Tukey’s parametric post hoc test) indicates increased resistance of the Paenibacillus larvae strain ATCC 9545 (Eric I genotype) to the tested agents for American foulbrood biocontrol.
3.2.2. Antimicrobial Activity of Bacterial Suspensions Against P. larvae CCUG 48973 (Eric II Genotype)
Comparative analysis of the antimicrobial activity of culture fluids of selected bacterial species against strain belonging to Paenibacillus larvae (Eric II genotype) under in vitro conditions showed differences between the inoculants tested and thus, between the bacterial strains used, as well as between the media and culture media used. Under in vitro conditions, 9 out of 12 single inoculants tested showed suppressive effects against indicator strains belonging to the P. larvae (Eric II genotype) species and additionally, bacterial consortia labeled as Mix (Table 8 and Figure 1).
The greatest zone of growth inhibition of P. larvae was confirmed for culture fluids of bacterial species such as B. licheniformis (suspended in water, molasses culture), Mix (suspended in water, 60% sucrose, and 50% sucrose, tryptone peptone culture), B. megaterium (suspended in water, tryptone peptone culture), S. narbonensis (suspended in water, tryptone peptone culture) qualifying these microorganisms into the group with the greatest antimicrobial activity.
The group with high antimicrobial activity was assigned to strains belonging to the species L. brevis (suspended in 50% sucrose and 60% sucrose, tryptone peptone culture and suspended in water, 50% sucrose, 60% sucrose, molasses culture), B. licheniformis (suspended in 50% sucrose and 60% sucrose, molasses culture), S. narbonensis (suspended in 50% sucrose and 60% sucrose, tryptone peptone culture), S. narbonensis (suspended in water, molasses culture), B. megaterium (suspended in 50% sucrose, tryptone peptone culture), B. megaterium (suspended in all carriers, molasses culture) and B. pumilus (suspended in water, 50% sucrose and 60% sucrose, molasses culture).
The group with lower antimicrobial activity was assigned to strains belonging to the species S. narbonensis (suspended in 50% sucrose and 60% sucrose, molasses culture), L. fusiformis (suspended in all carriers, tryptone peptone culture), L. fusiformis (suspended in 60% sucrose, molasses culture), B. megaterium (suspended in 60% sucrose, tryptone peptone culture), and Mix (suspended in all carriers, molasses culture).
The group with negligible antimicrobial activity included a strain belonging to the species L. fusiformis (suspended in water and 50% sucrose, molasses culture), L. brevis (suspended in water, tryptone peptone culture), B. licheniformis (suspended in all carriers, tryptone peptone culture), and B. pumilus (suspended in all carriers, tryptone peptone culture) (Table 8 and Figure 1).
The study also showed the superiority of the ‘Mix’ bacterial consortium over single inoculants for biocontrol of the development of P. larvae ser. Eric II infection (Table 8).
For some samples, bacterial overgrowth or infection of the plates marked with ‘P’ and ‘Z’ was noticed, so those samples were removed from statistical analyses. The results of the interaction of the control samples (with water, 50% sucrose, and 60% sucrose) always indicated a level of 0 mm. This confirms that the sucrose solution alone does not have an antimicrobial effect (Table 8). Statistical analysis of the results obtained was performed using one-way ANOVA (Tukey’s parametric post hoc test) at a significance level of p-value < 0.05. It has been proven that there is a correlation and a statistically significant correlation between the microorganisms used multiplied in the biotechnological fermentation process and the subsequent antimicrobial activity of the metabolites and bacteria remaining in the culture fluid. In some cases (experimental systems), there is a correlation and a statistically significant correlation between the inoculant concentration used and its subsequent antimicrobial activity. The analysis of antimicrobial activity showed statistically significant differences between the inoculants tested and, therefore, between the bacterial strains used and between the media, media, and indicator strains used. A one-way ANOVA analysis indicated a superior efficacy of the Mix against the pathogen analyzed. There is no increased resistance of Paenibacillus larvae CCUG 48973 (Eric II genotype) to the inoculants used.
4. Discussion
The environmental microbiota is constantly disrupted due to various environmental factors, so the constant supplementation with probiotics (agriculture, veterinary, and medicine) seems justified. In line with the provisions of the European Field-to-Table Strategy and the Green Deal, a reduction in the use of antibiotics in the ecosystem cycle is recommended. This is also indirectly related to increasing antibiotic resistance [44,45].
Also, in the environment of the honey bee population, there is a continuous reduction in beneficial microbes. Despite prohibitions, beekeepers often use prohibited antimicrobial substances, including antibiotics, which then easily pass into bee products and become a real threat to consumers in the food safety chain. To overcome these problems, steps should be taken to develop a leak-proof prevention system and to implement natural product solutions, which could contain proven functional microorganisms, metabolites, or plant extracts to displace and reduce the pressure of pathogens from the bee colonies’ physiological microbiome. The research undertaken in this study has achieved TRL Technology Readiness Level III and will certainly continue to develop an effective composition to reduce bacterial superinfections caused by Paenibacillus larvae.
Researchers searching for microbial antagonistic agents for American foulbrood biocontrol are most often limited to testing strains from the genera Lactobacillus and Bifidobacterium. Studies in which bacteria isolated from healthy bee colonies have been also encountered. These are specific microorganisms derived from the microbiome of bee colonies and usually involve the genera Bacillus sp., Streptococcus, Candida sp., Saccharomyces, Pseudobacterium, Fructobacillus, Parasaccharibacter, Ochrabactum, Acinetobacter, Stenotrophomonas, Gilliamella, Snodgrassella, Actinomadura, Streptomyces, Apilactobacillus, Bombilactobacillus, and many others. As there are between 6000 and 8000 different microbial strains in the bee microbiome, it seems nothing new or harmful to administer specific probiotics and supplement the microflora. In human medicine, the effect of so-called probiotic interventions and the administration of specific strains to support, for example, diabetes, cancer, or obesity, modifying the diseased microbiome, is increasingly being studied. Diagnostic tests based on the presence of specific microorganisms as biomarkers are also being developed [46,47,48,49].
The results from the comparative analysis of the antimicrobial activity of culture fluids of selected probiotic bacterial species against Paenibacillus larvae belonging to Eric I and II genotypes under in vitro conditions are consistent with literature data and tests evaluated by E. Forsgren, S. Lamei, and T. Olofsson [50,51,52]
High efficacy in antimicrobial activity was also achieved in a 2006 study conducted by Alippi and Reynaldi, confirming high antimicrobial activity for Bacillus pumilus, Bacillus licheniformis, and Bacillus megaterium against P. larvae ATCC 9545 [53].
Audisio et al. described the effective combination of a Bacillus subtilis strain with a plant extract against P. larvae highlighting the high utility of Bacillus spp. [54].
Also, Bartel and Alippi in 2018 identified natural antagonists of P. larvae by selecting strains of Bacillus species and related genera producing a broad range of antimicrobial compounds, with activity against bacteria and fungi that include peptides, lipopeptides, bacteriocins, and bacteriocin-like inhibitory substances. By using biological tools, they evaluated the antagonistic activity of 34 bacterial strains against Paenibacillus larvae and Ascosphaera apis, the causal agents of American Foulbrood and Chalkbrood diseases of honey bee larvae, respectively. In the work in question, a strong bactericidal effect attributed to a strain of Bacillus megaterium and Bacillus licheniformis was demonstrated. Similarly, in studies by other researchers, B. megaterium showed strong antimicrobial activity due to the production of megacins by Bacillus megaterium and lichenin and lichenicidin produced by Bacillus licheniformis [55].
Studies by other authors have documented that Bacillus licheniformis and another strain tested in the experiment reduced the mortality of American foulbrood-infected larvae. Treatments showed positive effects and reduced mortality [56].
Many studies by other authors confirm the efficacy of the bacterial species analyzed in reducing American foulbrood. This demonstrates the pertinent and successful selection of bacterial strains that can find application in the prevention of this disease. In other studies, B. licheniformis and B. subtilis also showed antimicrobial activity against P. larvae isolated from the bee environment in Saudi Arabia [57].
There are no sources that report what antagonistic effect Streptomyces narbonensis species may have. Korean researchers demonstrated that isolates extracted from forest soil, also identified as human pathogens, containing mainly strains of the genus Streptomyces showed strong pressure and antimicrobial activity against Paenibacillus larvae isolates supporting the theory that the source of the search for microbial agents need not only be the healthy bee microbiome but also other sources and natural resources [58].
Bacterial metabolites are reported in detail by Adrian A. Pinto-Tomas and co-workers, who isolated Actinobacteria in the genus Streptomyces from foraging bees, and especially common in pollen stores. One strain, isolated from pollen stores, exhibited pronounced inhibitory activity against Paenibacillus larvae [59].
Q. Jamal, on the other hand, describes the widespread use of bacteria of the genus Lysinibacillus. Moreover, some Lysinibacillus species have antimicrobial potential due to bacteriocins, peptide antibiotics, and other therapeutic molecules.
This shows a well-chosen vector of search and selection of strains for the work in question. Good probiotic properties, high survival rates, and origin from the hive microbiome are reported by Polish researchers in the context of B. pumilus and B. licheniformis which again shows the correct selection in microbial agents for biocontrol of American foulbrood. Therefore, it is confirmed that B. pumilus and B. licheniformis species also occur in the hive environment [60].
Levilactobacillus brevis also shows the ability to reduce the biofilm formation of P. larvae isolated from honey bee guts or fresh pollen samples, as indicated by the mechanism of action of this species [61].
In a study by Polish scientists, Levilactobacillus brevis B50 increased the expression of pattern recognition receptors and genes encoding antimicrobial peptides (defensin-1 and abaecin [62]. The LAB isolated from the honey bee gut has been demonstrated to be helpful for the inhibition of P. larvae. The antibiotic action of L. brevis is also based on its secretion of organic acids [63,64].
By testing bacterial growth under bioreactor conditions on a semi-industrial scale, it was shown that the most promising bacteria with the highest antimicrobial activity can be cultured on a large scale in the future. The research presented in this paper is distinguished by the fact that the antimicrobial properties of the functional strains were tested not on laboratory media as is the case with most available studies and publications, but scaled up to a larger production scale, and only then were these antimicrobial properties reproduced and confirmed on industrial media. Therefore, this is an added value to the present work. It should also be ensured in further stages that these results are validated under in vivo conditions by checking that the antimicrobial properties are also confirmed on living organisms (larvae and bees).
5. Conclusions
Using beneficial microorganisms as an alternative to chemical treatments is a promising novel technique for tackling honey bee diseases and improving their immunity. Factors capable of reducing American foulbrood pressure in bee colonies by promoting the development of a physiological colony microbiome that might compete with P. larvae and can block excessive proliferation and production of P. larvae endospores were found, and strains were selected for further selection, biocontrol of American foulbrood, and development of a bioactive composition. An attempt to inhibit the growth of strains belonging to Paenibacillus larvae Eric I and Eric II genotypes using inoculants of selected bacteria with probiotic potential under in vitro conditions was carried out successfully. Results from a comparative analysis of the antimicrobial activity of culture fluids of selected probiotic bacterial species against Paenibacillus larvae Eric I and Eric II genotypes under in vitro conditions are consistent with literature data (for example, with the evaluation by E. Forsgren and T. Olofsson). Analysis of antimicrobial activity showed differences between the inoculants tested and thus between the bacterial strains used and between the media, media, and indicator strains used. Under in vitro conditions, 9 out of 12 single inoculants tested showed suppressive activity against indicator strains. There is a correlation and a statistically significant correlation between the microorganisms used in the biotechnological fermentation process and the subsequent antimicrobial activity of the metabolites remaining in the culture fluid. In some cases (experimental systems) there is a correlation and a statistically significant correlation between the inoculant concentration used and their subsequent antimicrobial activity. The greatest zone of growth inhibition of P. larvae was confirmed for culture fluids using S. narbonensis, B. megaterium, and B. licheniformis classifying these microorganisms into the group with the highest antimicrobial activity. Strains belonging to species were assigned to the group with lower antimicrobial activity: L. brevis and B. pumilus. The group with negligible antimicrobial activity included a strain belonging to the species L. fusiformis. The study also showed the superiority of the bacterial consortium ‘Mix’ over single inoculants for biocontrol of the development of P. larvae infection.
Author Contributions
Writing—original draft preparation, P.N.; conceptualization, P.N. and B.G.; methodology, P.N., P.C., P.M. and B.G.; investigation, P.N., P.C., P.M. and B.G.; validation, P.C., P.M. and B.G.; supervision and investigation. All authors contributed to writing the original draft, reviewing, and editing. All authors have read and agreed to the published version of the manuscript.
Funding
This research was funded by the Polish Ministry of Science and Higher Education, grant number N0CBR000.7117.UWD.6/CBR/2021.
Institutional Review Board Statement
Not applicable.
Informed Consent Statement
Not applicable.
Data Availability Statement
All data generated or analyzed during this study are included in this published article and are available from the authors upon reasonable request.
Acknowledgments
We thank the laboratory specialists at the Institute of Microbiological Technologies in Turek for excellent technical assistance.
Conflicts of Interest
Author Bogusław Górski was employed by the company ProBiotics Polska Wytwórnia Probiotyków. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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Figure 1.
Observed ‘halo’ effects with a visible zone of growth inhibition against strains belonging to the P. larvae species. (a–f) Tests were performed using the well diffusion method according to EUCAST guidelines with modifications due to the specific bacteria used. (a,c,d,f) Antimicrobial mechanisms are of interest, showing antagonism between the microorganisms used, which may include, for example, the secretion of metabiotics, i.e., metabolites and bacteriocins, called antimicrobial substances or competition for food and growth conditions. (b,e) Impressive bacterial overgrowth of beneficial microorganisms is also observed, which takes away the growth capacity of pathogenic cells and even leads to lysis of pathogenic cells.
Figure 1.
Observed ‘halo’ effects with a visible zone of growth inhibition against strains belonging to the P. larvae species. (a–f) Tests were performed using the well diffusion method according to EUCAST guidelines with modifications due to the specific bacteria used. (a,c,d,f) Antimicrobial mechanisms are of interest, showing antagonism between the microorganisms used, which may include, for example, the secretion of metabiotics, i.e., metabolites and bacteriocins, called antimicrobial substances or competition for food and growth conditions. (b,e) Impressive bacterial overgrowth of beneficial microorganisms is also observed, which takes away the growth capacity of pathogenic cells and even leads to lysis of pathogenic cells.
Table 1.
Reactor cultivation parameters for Bacillus pumilus R5 strain using BPW (buffered peptone water), PCA (plate count agar), DRBC (dichloran rose bengal agar), MRS (De Man–Rogosa–Sharpe), ISA (iron sulfite agar), where symbol ‘G’ means growth and ‘NG’ means no growth.
Table 1.
Reactor cultivation parameters for Bacillus pumilus R5 strain using BPW (buffered peptone water), PCA (plate count agar), DRBC (dichloran rose bengal agar), MRS (De Man–Rogosa–Sharpe), ISA (iron sulfite agar), where symbol ‘G’ means growth and ‘NG’ means no growth.
| Strain: Bacillus pumilus R5 | |||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| Culture Stage | Parameter | Test Conditions and Media | Incubation | Direct Culture | Non-Selective Multiplication | Device Reading | External Measurement | ||||
| CM | TP | CM | TP | CM | TP | CM | TP | ||||
| After filling the reactor with a 4% sterilized solution of cane molasses (CM) or tryptone-pepton (TP) in distilled water [about 50 µs/cm], 18 L | pH | - | - | - | - | - | - | 5.31 | 7.10 | 5.22 | 7.30 |
| Conductivity [µs/cm] | - | - | - | - | - | - | 2800 | 2788 | 2810 | 2600 | |
| Brix | - | - | - | - | - | - | 3.7 | 3.7 | 3.6 | 3.4 | |
| General contamination | BPW | Aerobic, 37 °C, 24 h | NG | NG | NG | NG | - | - | - | - | |
| Aerobic microorganisms | PCA only/with BPW | Aerobic, 30 °C, 72 h | NG | NG | NG | NG | - | - | - | - | |
| Fungi | DRBC only/with | Aerobic, 25 °C, 72 h | NG | NG | NG | NG | - | - | - | - | |
| LAB | MRS only/with BPW | Anaerobic, 37 °C, 72 h | NG | NG | NG | NG | - | - | - | - | |
| Clostridium sp. | ISA only/with BPW | Anaerobic, 37 °C, 48 h | NG | NG | NG | NG | - | - | - | - | |
| After inoculation and completion of culture | pH | - | - | - | - | - | - | 4.87 | 6.50 | 4.67 | 6.70 |
| Conductivity [µs/cm] | - | - | - | - | - | - | 2607 | 2500 | 2540 | 2700 | |
| Brix | - | - | - | - | - | - | 3.6 | 3.4 | 3.5 | 3.5 | |
| General contamination | BPW | Aerobic, 37 °C, 24 h | - | - | G | G | - | - | 3.3 × 105 CFU/cm3 | 3.5 × 105 CFU/cm3 | |
| Aerobic microorganisms | PCA only/with BPW | Aerobic, 30 °C, 72 h | G | G | G | G | - | - | 4.5 × 105 CFU/cm3 | 4.7 × 105 CFU/cm3 | |
| Fungi | DRBC only/with | Aerobic, 25 °C, 72 h | NG | NG | NG | NG | - | - | <1.0 × 100 CFU/cm3 | <1.0 × 100 CFU/cm3 | |
| LAB | MRS only/with BPW | Anaerobic, 37 °C, 72 h | NG | NG | NG | NG | - | - | <1.0 × 100 CFU/cm3 | <1.0 × 100 CFU/cm3 | |
| Clostridium sp. | ISA only/with BPW | Anaerobic, 37 °C, 48 h | NG | NG | NG | NG | - | - | <1.0 × 100 CFU/cm3 | <1.0 × 100 CFU/cm3 | |
Table 2.
Reactor cultivation parameters for Bacillus licheniformis M/542/M/18 strain using BPW (buffered peptone water), PCA (plate count agar), DRBC (dichloran rose bengal agar), MRS (De Man–Rogosa–Sharpe), ISA (iron sulfite agar), where symbol ‘G’ means growth and ‘NG’ means no growth.
Table 2.
Reactor cultivation parameters for Bacillus licheniformis M/542/M/18 strain using BPW (buffered peptone water), PCA (plate count agar), DRBC (dichloran rose bengal agar), MRS (De Man–Rogosa–Sharpe), ISA (iron sulfite agar), where symbol ‘G’ means growth and ‘NG’ means no growth.
| Strain: Bacillus licheniformis M/542/M/18 | |||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| Culture Stage | Parameter | Test Conditions and Media | Incubation | Direct Culture | Non-Selective Multiplication | Device Reading | External Measurement | ||||
| CM | TP | CM | TP | CM | TP | CM | TP | ||||
| After filling the reactor with a 4% sterilized solution of cane molasses (CM) or tryptone-pepton (TP) in distilled water [about 50 µs/cm], 18 L | pH | - | - | - | - | - | - | 5.00 | 7.12 | 5.18 | 7.20 |
| Conductivity [µs/cm] | - | - | - | - | - | - | 2200 | 2400 | 2220 | 2410 | |
| Brix | - | - | - | - | - | - | 3.7 | 3.7 | 3.8 | 3.8 | |
| General contamination | BPW | Aerobic, 37 °C, 24 h | NG | NG | NG | NG | - | - | - | - | |
| Aerobic microorganisms | PCA only/with BPW | Aerobic, 30 °C, 72 h | NG | NG | NG | NG | - | - | - | - | |
| Fungi | DRBC only/with | Aerobic, 25 °C, 72 h | NG | NG | NG | NG | - | - | - | - | |
| LAB | MRS only/with BPW | Anaerobic, 37 °C, 72 h | NG | NG | NG | NG | - | - | - | - | |
| Clostridium sp. | ISA only/with BPW | Anaerobic, 37 °C, 48 h | NG | NG | NG | NG | - | - | - | - | |
| After inoculation and completion of culture | pH | - | - | - | - | - | - | 4.37 | 6.40 | 4.47 | 6.60 |
| Conductivity [µs/cm] | - | - | - | - | - | - | 3030 | 2900 | 2940 | 2800 | |
| Brix | - | - | - | - | - | - | 3.6 | 3.5 | 3.7 | 3.5 | |
| General contamination | BPW | Aerobic, 37 °C, 24 h | - | - | G | G | - | - | 3.1 × 107 CFU/cm3 | 3.6 × 107 CFU/cm3 | |
| Aerobic microorganisms | PCA only/with BPW | Aerobic, 30 °C, 72 h | G | G | G | G | - | - | 3.5 × 107 CFU/cm3 | 3.7 × 107 CFU/cm3 | |
| Fungi | DRBC only/with | Aerobic, 25 °C, 72 h | NG | NG | NG | NG | - | - | <1.0 × 100 CFU/cm3 | <1.0 × 100 CFU/cm3 | |
| LAB | MRS only/with BPW | Anaerobic, 37 °C, 72 h | NG | NG | NG | NG | - | - | <1.0 × 100 CFU/cm3 | <1.0 × 100 CFU/cm3 | |
| Clostridium sp. | ISA only/with BPW | Anaerobic, 37 °C, 48 h | NG | NG | NG | NG | - | - | <1.0 × 100 CFU/cm3 | <1.0 × 100 CFU/cm3 | |
Table 3.
Reactor cultivation parameters for Streptomyces narbonensis R71 strain using BPW (buffered peptone water), PCA (plate count agar), DRBC (dichloran rose bengal agar), MRS (De Man–Rogosa–Sharpe), ISA (iron sulfite agar), where symbol ‘G’ means growth and ‘NG’ means no growth.
Table 3.
Reactor cultivation parameters for Streptomyces narbonensis R71 strain using BPW (buffered peptone water), PCA (plate count agar), DRBC (dichloran rose bengal agar), MRS (De Man–Rogosa–Sharpe), ISA (iron sulfite agar), where symbol ‘G’ means growth and ‘NG’ means no growth.
| Strain: Streptomyces narbonensis R71 | |||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| Culture Stage | Parameter | Test Conditions and Media | Incubation | Direct Culture | Non-Selective Multiplication | Device Reading | External Measurement | ||||
| CM | TP | CM | TP | CM | TP | CM | TP | ||||
| After filling the reactor with a 4% sterilized solution of cane molasses (CM) or tryptone-pepton (TP) in distilled water [about 50 µs/cm], 18 L | pH | - | - | - | - | - | - | 5.12 | 7.50 | 5.22 | 7.70 |
| Conductivity [µs/cm] | - | - | - | - | - | - | 2300 | 2350 | 2380 | 2370 | |
| Brix | - | - | - | - | - | - | 3.6 | 3.6 | 3.7 | 3.7 | |
| General contamination | BPW | Aerobic, 37 °C, 24 h | NG | NG | NG | NG | - | - | - | - | |
| Aerobic microorganisms | PCA only/with BPW | Aerobic, 30 °C, 72 h | NG | NG | NG | NG | - | - | - | - | |
| Fungi | DRBC only/with | Aerobic, 25 °C, 72 h | NG | NG | NG | NG | - | - | - | - | |
| LAB | MRS only/with BPW | Anaerobic, 37 °C, 72 h | NG | NG | NG | NG | - | - | - | - | |
| Clostridium sp. | ISA only/with BPW | Anaerobic, 37 °C, 48 h | NG | NG | NG | NG | - | - | - | - | |
| After inoculation and completion of culture | pH | - | - | - | - | - | - | 4.77 | 7.00 | 4.87 | 7.40 |
| Conductivity [µs/cm] | - | - | - | - | - | - | 2800 | 2880 | 2840 | 2800 | |
| Brix | - | - | - | - | - | - | 3.6 | 3.5 | 3.6 | 3.5 | |
| General contamination | BPW | Aerobic, 37 °C, 24 h | - | - | G | G | - | - | 4.2 × 106 CFU/cm3 | 3.9 × 106 CFU/cm3 | |
| Aerobic microorganisms | PCA only/with BPW | Aerobic, 30 °C, 72 h | G | G | G | G | - | - | 4.5 × 106 CFU/cm3 | 4.7 × 106 CFU/cm3 | |
| Fungi | DRBC only/with | Aerobic, 25 °C, 72 h | NG | NG | NG | NG | - | - | <1.0 × 100 CFU/cm3 | <1.0 × 100 CFU/cm3 | |
| LAB | MRS only/with BPW | Anaerobic, 37 °C, 72 h | NG | NG | NG | NG | - | - | <1.0 × 100 CFU/cm3 | <1.0 × 100 CFU/cm3 | |
| Clostridium sp. | ISA only/with BPW | Anaerobic, 37 °C, 48 h | NG | NG | NG | NG | - | - | <1.0 × 100 CFU/cm3 | <1.0 × 100 CFU/cm3 | |
Table 4.
Reactor cultivation parameters for Lysinibacillus fusiformis E23 strain using BPW (buffered peptone water), PCA (plate count agar), DRBC (dichloran rose bengal agar), MRS (De Man–Rogosa–Sharpe), ISA (iron sulfite agar), where symbol ‘G’ means growth and ‘NG’ means no growth.
Table 4.
Reactor cultivation parameters for Lysinibacillus fusiformis E23 strain using BPW (buffered peptone water), PCA (plate count agar), DRBC (dichloran rose bengal agar), MRS (De Man–Rogosa–Sharpe), ISA (iron sulfite agar), where symbol ‘G’ means growth and ‘NG’ means no growth.
| Strain: Lysinibacillus fusiformis E23 | |||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| Culture Stage | Parameter | Test Conditions and Media | Incubation | Direct Culture | Non-Selective Multiplication | Device Reading | External Measurement | ||||
| CM | TP | CM | TP | CM | TP | CM | TP | ||||
| After filling the reactor with a 4% sterilized solution of cane molasses (CM) or tryptone-pepton (TP) in distilled water [about 50 µs/cm], 18 L | pH | - | - | - | - | - | - | 5.65 | 7.30 | 5.52 | 7.40 |
| Conductivity [µs/cm] | - | - | - | - | - | - | 2400 | 2450 | 2480 | 2470 | |
| Brix | - | - | - | - | - | - | 3.8 | 3.8 | 3.9 | 3.8 | |
| General contamination | BPW | Aerobic, 37 °C, 24 h | NG | NG | NG | NG | - | - | - | - | |
| Aerobic microorganisms | PCA only/with BPW | Aerobic, 30 °C, 72 h | NG | NG | NG | NG | - | - | - | - | |
| Fungi | DRBC only/with | Aerobic, 25 °C, 72 h | NG | NG | NG | NG | - | - | - | - | |
| LAB | MRS only/with BPW | Anaerobic, 37 °C, 72 h | NG | NG | NG | NG | - | - | - | - | |
| Clostridium sp. | ISA only/with BPW | Anaerobic, 37 °C, 48 h | NG | NG | NG | NG | - | - | - | - | |
| After inoculation and completion of culture | pH | - | - | - | - | - | - | 4.88 | 6.80 | 4.80 | 6.60 |
| Conductivity [µs/cm] | - | - | - | - | - | - | 2800 | 2880 | 2840 | 2800 | |
| Brix | - | - | - | - | - | - | 3.6 | 3.6 | 3.7 | 3.7 | |
| General contamination | BPW | Aerobic, 37 °C, 24 h | - | - | G | G | - | - | 3.2 × 107 CFU/cm3 | 3.5 × 107 CFU/cm3 | |
| Aerobic microorganisms | PCA only/with BPW | Aerobic, 30 °C, 72 h | G | G | G | G | - | - | 3.5 × 107 CFU/cm3 | 3.7 × 107 CFU/cm3 | |
| Fungi | DRBC only/with | Aerobic, 25 °C, 72 h | NG | NG | NG | NG | - | - | <1.0 × 100 CFU/cm3 | <1.0 × 100 CFU/cm3 | |
| LAB | MRS only/with BPW | Anaerobic, 37 °C, 72 h | NG | NG | NG | NG | - | - | <1.0 × 100 CFU/cm3 | <1.0 × 100 CFU/cm3 | |
| Clostridium sp. | ISA only/with BPW | Anaerobic, 37 °C, 48 h | NG | NG | NG | NG | - | - | <1.0 × 100 CFU/cm3 | <1.0 × 100 CFU/cm3 | |
Table 5.
Reactor cultivation parameters for Levilactobacillus brevis M/495/M/17 strain using BPW (buffered peptone water), PCA (plate count agar), DRBC (dichloran rose bengal agar), MRS (De Man–Rogosa–Sharpe), ISA (iron sulfite agar), where symbol ‘G’ means growth and ‘NG’ means no growth.
Table 5.
Reactor cultivation parameters for Levilactobacillus brevis M/495/M/17 strain using BPW (buffered peptone water), PCA (plate count agar), DRBC (dichloran rose bengal agar), MRS (De Man–Rogosa–Sharpe), ISA (iron sulfite agar), where symbol ‘G’ means growth and ‘NG’ means no growth.
| Strain: Levilactobacillus brevis M/495/M/17 | |||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| Culture Stage | Parameter | Test Conditions and Media | Incubation | Direct Culture | Non-Selective Multiplication | Device Reading | External Measurement | ||||
| CM | TP | CM | TP | CM | TP | CM | TP | ||||
| After filling the reactor with a 4% sterilized solution of cane molasses (CM) or tryptone-pepton (TP) in distilled water [about 50 µs/cm], 18 L | pH | - | - | - | - | - | - | 5.45 | 7.60 | 5.56 | 7.55 |
| Conductivity [µs/cm] | - | - | - | - | - | - | 2000 | 2150 | 2080 | 2170 | |
| Brix | - | - | - | - | - | - | 3.9 | 3.9 | 3.9 | 3.9 | |
| General contamination | BPW | Aerobic, 37 °C, 24 h | NG | NG | NG | NG | - | - | - | - | |
| Aerobic microorganisms | PCA only/with BPW | Aerobic, 30 °C, 72 h | NG | NG | NG | NG | - | - | - | - | |
| Fungi | DRBC only/with | Aerobic, 25 °C, 72 h | NG | NG | NG | NG | - | - | - | - | |
| LAB | MRS only/with BPW | Anaerobic, 37 °C, 72 h | NG | NG | NG | NG | - | - | - | - | |
| Clostridium sp. | ISA only/with BPW | Anaerobic, 37 °C, 48 h | NG | NG | NG | NG | - | - | - | - | |
| After inoculation and completion of culture | pH | - | - | - | - | - | - | 4.15 | 6.70 | 4.20 | 6.65 |
| Conductivity [µs/cm] | - | - | - | - | - | - | 2900 | 2980 | 2940 | 2960 | |
| Brix | - | - | - | - | - | - | 3.3 | 3.1 | 3.2 | 3.1 | |
| General contamination | BPW | Aerobic, 37 °C, 24 h | - | - | G | G | - | - | 1.8 × 105 CFU/cm3 | 1.1 × 104 CFU/cm3 | |
| Aerobic microorganisms | PCA only/with BPW | Aerobic, 30 °C, 72 h | G | G | G | G | - | - | 1.7 × 108 CFU/cm3 | 1.6 × 104 CFU/cm3 | |
| Fungi | DRBC only/with | Aerobic, 25 °C, 72 h | NG | NG | NG | NG | - | - | <1.0 × 100 CFU/cm3 | <1.0 × 100 CFU/cm3 | |
| LAB | MRS only/with BPW | Anaerobic, 37 °C, 72 h | G | G | G | G | - | - | 4.5 × 108 CFU/cm3 | 3.4 × 105 CFU/cm3 | |
| Clostridium sp. | ISA only/with BPW | Anaerobic, 37 °C, 48 h | NG | NG | NG | NG | - | - | <1.0 × 100 CFU/cm3 | <1.0 × 100 CFU/cm3 | |
Table 6.
Reactor cultivation parameters for Bacillus megaterium R7 strain using BPW (buffered peptone water), PCA (plate count agar), DRBC (dichloran rose bengal agar), MRS (De Man–Rogosa–Sharpe), ISA (iron sulfite agar), where symbol ‘G’ means growth and ‘NG’ means no growth.
Table 6.
Reactor cultivation parameters for Bacillus megaterium R7 strain using BPW (buffered peptone water), PCA (plate count agar), DRBC (dichloran rose bengal agar), MRS (De Man–Rogosa–Sharpe), ISA (iron sulfite agar), where symbol ‘G’ means growth and ‘NG’ means no growth.
| Strain: Bacillus megaterium R7 | |||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| Culture Stage | Parameter | Test Conditions and Media | Incubation | Direct Culture | Non-Selective Multiplication | Device Reading | External Measurement | ||||
| CM | TP | CM | TP | CM | TP | CM | TP | ||||
| After filling the reactor with a 4% sterilized solution of cane molasses (CM) or tryptone-pepton (TP) in distilled water [about 50 µs/cm], 18 L | pH | - | - | - | - | - | - | 5.50 | 7.45 | 5.56 | 7.50 |
| Conductivity [µs/cm] | - | - | - | - | - | - | 2300 | 2450 | 2350 | 2500 | |
| Brix | - | - | - | - | - | - | 3.7 | 3.7 | 3.8 | 3.8 | |
| General contamination | BPW | Aerobic, 37 °C, 24 h | NG | NG | NG | NG | - | - | - | - | |
| Aerobic microorganisms | PCA only/with BPW | Aerobic, 30 °C, 72 h | NG | NG | NG | NG | - | - | - | - | |
| Fungi | DRBC only/with | Aerobic, 25 °C, 72 h | NG | NG | NG | NG | - | - | - | - | |
| LAB | MRS only/with BPW | Anaerobic, 37 °C, 72 h | NG | NG | NG | NG | - | - | - | - | |
| Clostridium sp. | ISA only/with BPW | Anaerobic, 37 °C, 48 h | NG | NG | NG | NG | - | - | - | - | |
| After inoculation and completion of culture | pH | - | - | - | - | - | - | 5.15 | 7.00 | 4.20 | 6.65 |
| Conductivity [µs/cm] | - | - | - | - | - | - | 2400 | 2080 | 2480 | 2300 | |
| Brix | - | - | - | - | - | - | 3.5 | 3.5 | 3.6 | 3.7 | |
| General contamination | BPW | Aerobic, 37 °C, 24 h | - | - | G | G | - | - | 2.6 × 106 CFU/cm3 | 2.1 × 107 CFU/cm3 | |
| Aerobic microorganisms | PCA only/with BPW | Aerobic, 30 °C, 72 h | G | G | G | G | - | - | 2.7 × 108 CFU/cm3 | 2.6 × 108 CFU/cm3 | |
| Fungi | DRBC only/with | Aerobic, 25 °C, 72 h | NG | NG | NG | NG | - | - | <1.0 × 100 CFU/cm3 | <1.0 × 100 CFU/cm3 | |
| LAB | MRS only/with BPW | Anaerobic, 37 °C, 72 h | G | G | G | G | - | - | <1.0 × 100 CFU/cm3 | <1.0 × 100 CFU/cm3 | |
| Clostridium sp. | ISA only/with BPW | Anaerobic, 37 °C, 48 h | NG | NG | NG | NG | - | - | <1.0 × 100 CFU/cm3 | <1.0 × 100 CFU/cm3 | |
Table 7.
Data show antimicrobial activity against Paenibacillus larvae ATCC 9545 (Eric I genotype), using the well diffusion method according to EUCAST standards [mm] with heat map effect (red color means the lowest antimicrobial activity or lack of antimicrobial properties, orange color means lower antimicrobial activity, yellow color means medium-higher antimicrobial activity, green color means high activity so from lowest to highest antimicrobial activity).
Table 7.
Data show antimicrobial activity against Paenibacillus larvae ATCC 9545 (Eric I genotype), using the well diffusion method according to EUCAST standards [mm] with heat map effect (red color means the lowest antimicrobial activity or lack of antimicrobial properties, orange color means lower antimicrobial activity, yellow color means medium-higher antimicrobial activity, green color means high activity so from lowest to highest antimicrobial activity).
| Antimicrobial Activity against Paenibacillus larvae ATCC 9545, Using Well-Diffusion Method according EUCAST Standards [mm] | Bacillus pumilus | Bacillus pumilus | Bacillus licheniformis | Bacillus licheniformis | Streptomyces narbonensis | Streptomyces narbonensis | Lysinibacillus fusiformis | Lysinibacillus fusiformis | Levilactobacillus brevis | Levilactobacillus brevis | Bacillus megaterium | Bacillus megaterium | Mix | Mix | ||||||||||||||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| (Tryptone Peptone Broth) | (Molasses) | (Tryptone Peptone Broth) | (Molasses) | (Tryptone Peptone Broth) | (Molasses) | (Tryptone Peptone Broth) | (Molasses) | (Tryptone Peptone Broth) | (Molasses) | (Tryptone Peptone Broth) | (Molasses) | (Tryptone Peptone Broth) | (Molasses) | |||||||||||||||||||||||||||||
| Concentration [%] | water | 50% sucrose | 60% sucrose | water | 50% sucrose | 60% sucrose | water | 50% sucrose | 60% sucrose | water | 50% sucrose | 60% sucrose | water | 50% sucrose | 60% sucrose | water | 50% sucrose | 60% sucrose | water | 50% sucrose | 60% sucrose | water | 50% sucrose | 60% sucrose | water | 50% sucrose | 60% sucrose | water | 50% sucrose | 60% sucrose | water | 50% sucrose | 60% sucrose | water | 50% sucrose | 60% sucrose | water | 50% sucrose | 60% sucrose | water | 50% sucrose | 60% sucrose |
| C | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | |
| 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | |
| Average | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| 3 | 1 | 0 | 0 | 9 | 7 | 7.3 | 0 | 1 | 1 | 4 | 7 | 6 | 0 | 5 | 0 | 5 | 6.7 | 7 | 0 | 5 | 7.3 | 0 | 0 | 0 | 0 | 7 | 6 | 5 | 8 | 5.8 | 4.3 | 4 | 4.8 | 4.8 | 5 | 7 | 2.3 | 5 | 7 | 5 | 8 | 9.1 |
| 1 | 1 | 0 | 9 | 7 | 7.3 | 0.5 | 1 | 1 | 5 | 7 | 6 | 0 | 5 | 0.5 | 5 | 7 | 8.4 | 0 | 5.1 | 7 | 0 | 0 | 0 | 0 | 7.4 | 6.2 | 5.4 | 9 | 6 | 4.5 | 3 | 4.8 | 4.9 | 4.9 | 6.8 | 2.7 | 5 | 7.4 | 6 | 8 | 9 | |
| 1 | 0 | 0 | 9 | 7.5 | 7 | 0 | 1.5 | 1.5 | 5 | 7.5 | 6.5 | 0 | 6 | 0 | 5.4 | 6.7 | 8.8 | 0.5 | 5.4 | 7.4 | 0 | 0 | 0 | 0 | 7.3 | 6.1 | 5.5 | 9.1 | 5.8 | 4.5 | 4 | 4.4 | 5.1 | 4.9 | 6.6 | 2.5 | 5 | 7.5 | 5.4 | 8.3 | 9.1 | |
| Average | 1 | 0.33 | 0 | 9 | 7.17 | 7.2 | 0.17 | 1.17 | 1.17 | 4.67 | 7.17 | 6.17 | 0 | 5.33 | 0.17 | 5.13 | 6.8 | 8.07 | 0.17 | 5.17 | 7.23 | 0 | 0 | 0 | 0 | 7.23 | 6.1 | 5.3 | 8.7 | 5.87 | 4.43 | 3.67 | 4.67 | 4.93 | 4.93 | 6.8 | 2.5 | 5 | 7.3 | 5.47 | 8.1 | 9.07 |
| 4 | 1 | 1 | 1 | 9.5 | 7 | 6.5 | 0 | 1 | 1 | 5 | 7 | 6 | 0 | 5 | 0 | 4.3 | 6.5 | 7 | 0 | 5 | 7.3 | 0 | 0 | 0 | 0 | 6.8 | 6 | 5.3 | 8 | 6 | 4 | 4.3 | 5 | 4 | 5.5 | 6.3 | 3 | 7 | 7 | 5.3 | 7 | 5.7 |
| 1 | 1 | 0 | 9 | 7 | 6 | 0 | 2 | 1 | 5 | 7.5 | 6 | 0 | 5 | 0 | 4.5 | 6.8 | 7.2 | 0 | 6.7 | 7.4 | 0 | 0 | 0 | 0 | 7 | 6.2 | 5.3 | 8.4 | 6.2 | 5 | 4.5 | 5.3 | 3.9 | 5.6 | 6.5 | 4 | 6.8 | 7 | 5.4 | 7.2 | 5.7 | |
| 1 | 0 | 0 | 9 | 7.5 | 6.5 | 0.5 | 1 | 1 | 5.5 | 7 | 6 | 0 | 4 | 0 | 4.5 | 6.3 | 7.2 | 0 | 5.9 | 7.2 | 0 | 0 | 0 | 0 | 7 | 6.2 | 5.4 | 8.4 | 6.2 | 5.6 | 4.3 | 5 | 3.8 | 5.5 | 6.5 | 4 | 6.8 | 7 | 5.4 | 7.2 | 5.6 | |
| Average | 1 | 0.67 | 0.33 | 9.17 | 7.17 | 6.33 | 0.17 | 1.33 | 1 | 5.17 | 7.17 | 6 | 0 | 4.67 | 0 | 4.43 | 6.53 | 7.13 | 0 | 5.87 | 7.3 | 0 | 0 | 0 | 0 | 6.93 | 6.13 | 5.33 | 8.27 | 6.13 | 4.87 | 4.37 | 5.1 | 3.9 | 5.53 | 6.43 | 3.67 | 6.87 | 7 | 5.37 | 7.13 | 5.67 |
| 5 | 2 | 2 | 1 | P | 5 | 7 | 0 | P | P | 5 | 7 | 6 | 0 | 4.8 | 0 | 5 | 6 | 7 | 0 | 5 | 7.5 | 0 | 0 | 0 | 0 | 7 | 7 | 5 | 5.5 | 6 | 5 | 5 | 5 | 5 | 5.5 | 6 | 3 | 7 | 6 | 5 | 7 | 6 |
| 1 | 2 | 1 | P | 5 | 6.5 | 0 | P | P | 5 | 8 | 5 | 0 | 5 | 0 | 5.4 | 7 | 7.4 | 0 | 5.7 | 7.5 | 0 | 0 | 0 | 0 | 7 | 7 | 5 | 5.3 | 6.7 | 5 | 5.1 | 5.2 | 4.8 | 6 | 7 | 3.1 | 7.4 | 5.8 | 5.3 | 7.9 | 5.9 | |
| 2 | 1 | 1 | P | 6 | 7 | 0 | P | P | 6 | 7 | 6 | 0 | 4 | 0 | 5.2 | 7.1 | 7.5 | 0 | 5.7 | 7.8 | 0 | 0 | 0 | 0 | 7 | 7 | 5.1 | 5.6 | 6.5 | 4.8 | 3.7 | 5.2 | 5.1 | 5.8 | 6.4 | 3.6 | 7.4 | 5.8 | 5.3 | 7.9 | 5.9 | |
| Average | 1.67 | 1.67 | 1 | 0 | 5.33 | 6.83 | 0 | 0 | 0 | 5.33 | 7.33 | 5.67 | 0 | 4.6 | 0 | 5.2 | 6.7 | 7.3 | 0 | 5.47 | 7.6 | 0 | 0 | 0 | 0 | 7 | 7 | 5.03 | 5.47 | 6.4 | 4.93 | 4.6 | 5.13 | 4.97 | 5.77 | 6.47 | 3.23 | 7.27 | 5.87 | 5.2 | 7.6 | 5.93 |
| 10 | 2 | 2 | 2 | P | 6 | 7 | 0 | P | P | 5 | 7.5 | 6 | 0 | 5 | 0 | 5 | 7 | 7 | 0 | 5.9 | 0 | 0 | 0 | 0 | 0 | 7 | 7 | 5 | 6 | 6.3 | 4 | 5 | 4.8 | 4.3 | 5.8 | P | 2.8 | 7 | 7.3 | 5 | 7 | P |
| 3 | 2 | 1 | P | 5 | 7 | 0 | P | P | 6 | 8 | 6 | 0 | 6 | 0 | 6.5 | 6.8 | 7.2 | 0 | 5.9 | 0 | 0 | 0 | 0 | 0 | 7 | 7.1 | 4.9 | 6.2 | 6.5 | 4.3 | 4.7 | 5.1 | 4.4 | 5.8 | P | 1.9 | 8 | 7.5 | 5.2 | 7 | P | |
| 2 | 2 | 2 | P | 6.5 | 6 | 0 | P | P | 5.5 | 8 | 6.5 | 0 | 7 | 0 | 6 | 7.1 | 6.9 | 0 | 5 | 0 | 0 | 0 | 0 | 0 | 7.3 | 7 | 4.9 | 6.2 | 6.5 | 4.3 | 5 | 5.1 | 4.3 | 5.7 | p | 2.5 | 8.1 | 7.5 | 5.2 | 7 | P | |
| Average | 2.33 | 2 | 1.67 | 0 | 5.83 | 6.67 | 0 | 0 | 0 | 5.5 | 7.83 | 6.17 | 0 | 6 | 0 | 5.83 | 6.97 | 7.03 | 0 | 5.6 | 0 | 0 | 0 | 0 | 0 | 7.1 | 7.03 | 4.93 | 6.13 | 6.43 | 4.2 | 4.9 | 5 | 4.33 | 5.77 | 0 | 2.4 | 7.7 | 7.43 | 5.13 | 7 | 0 |
Table 8.
Data show antimicrobial activity against Paenibacillus larvae CCUG 48973 (Eric II genotype), using the well diffusion method according to EUCAST standards [mm] with heat map effect (red color means the lowest antimicrobial activity or lack of antimicrobial properties, orange color means lower antimicrobial activity, yellow color means medium-higher antimicrobial activity, green color means high activity so from lowest to highest antimicrobial activity).
Table 8.
Data show antimicrobial activity against Paenibacillus larvae CCUG 48973 (Eric II genotype), using the well diffusion method according to EUCAST standards [mm] with heat map effect (red color means the lowest antimicrobial activity or lack of antimicrobial properties, orange color means lower antimicrobial activity, yellow color means medium-higher antimicrobial activity, green color means high activity so from lowest to highest antimicrobial activity).
| Antimicrobial Activity against Paenibacillus larvae CCUG 48973 Using Well-Diffusion Method according EUCAST Standards [mm] | Bacillus pumilus | Bacillus pumilus | Bacillus licheniformis | Bacillus licheniformis | Streptomyces narbonensis | Streptomyces narbonensis | Lysinibacillus fusiformis | Lysinibacillus fusiformis | Levilactobacillus brevis | Levilactobacillus brevis | Bacillus megaterium | Bacillus megaterium | Mix | Mix | ||||||||||||||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| (Tryptone Peptone Broth) | (Molasses) | (Tryptone Peptone Broth) | (Molasses) | (Tryptone Peptone Broth) | (Molasses) | (Tryptone Peptone Broth) | (Molasses) | (Tryptone Peptone Broth) | (Molasses) | (Tryptone Peptone Broth) | (Molasses) | (Tryptone Peptone Broth) | (Molasses) | |||||||||||||||||||||||||||||
| Concentration [%] | water | 50% sucrose | 60% sucrose | water | 50% sucrose | 60% sucrose | water | 50% sucrose | 60% sucrose | water | 50% sucrose | 60% sucrose | water | 50% sucrose | 60% sucrose | water | 50% sucrose | 60% sucrose | water | 50% sucrose | 60% sucrose | water | 50% sucrose | 60% sucrose | water | 50% sucrose | 60% sucrose | water | 50% sucrose | 60% sucrose | water | 50% sucrose | 60% sucrose | water | 50% sucrose | 60% sucrose | water | 50% sucrose | 60% sucrose | water | 50% sucrose | 60% sucrose |
| C | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | |
| 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | |
| Average | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| 3 | 0 | 0 | 0 | 5.3 | 6 | 5.33 | 0 | 0 | 0 | 9 | 6 | 6 | 9 | 5 | 6 | 5.3 | Z | 5 | 4 | 4 | Z | 0 | 0 | 4.5 | 0 | 8 | 5 | 6.25 | 6 | 5.75 | 9 | 5.25 | 5 | 6 | 6 | 4 | 8.3 | 9 | 10 | 6 | P | 4 |
| 0 | 0 | 0 | 5.3 | 6 | 5.4 | 0 | 0 | 0 | 9.3 | 6.1 | 6 | 9.3 | 5 | 6.1 | 5.3 | 5 | 5 | P | 4.4 | 4 | 0 | 0 | 4.5 | 0 | 8.6 | 5 | 6.5 | 6.5 | 5.8 | 9.35 | 5.25 | 5 | 6.1 | 6 | 4.5 | 8.5 | 9.4 | 10 | 6 | 5 | 4 | |
| 0 | 0 | 0 | 5.4 | 6.1 | 5.3 | 0 | 0 | 0 | 9.3 | 5.9 | 6.3 | 9.3 | 5.1 | 6.1 | 5 | 4.8 | 5 | P | P | 4.1 | 0 | 0 | 4.5 | 0 | 8.5 | 5 | 6.25 | 6.5 | 5.55 | 9.5 | 5.2 | 5.15 | 6 | 6.1 | 4.3 | 8.3 | 9.3 | 10.1 | 6 | 5.2 | 4.1 | |
| Average | 0 | 0 | 0 | 5.33 | 6.03 | 5.34 | 0 | 0 | 0 | 9.2 | 6 | 6.1 | 9.2 | 5.03 | 6.07 | 5.2 | 3.27 | 5 | 1.33 | 2.8 | 2.7 | 0 | 0 | 4.5 | 0 | 8.37 | 5 | 6.33 | 3 | 5.7 | 9.28 | 5.23 | 5.05 | 6.03 | 6.03 | 4.27 | 8.37 | 9.23 | 10.03 | 6 | 3.4 | 4.03 |
| 4 | 0 | 0 | 0 | 6.3 | 6.3 | 6 | 0 | 0 | 0 | 10 | 5 | 5.3 | 9 | 5 | 6 | 5 | 5 | 5 | 5 | 5 | 5 | 0 | 0 | 5 | 0 | 7.3 | 5 | 6 | 6.3 | 6 | 9 | 5 | 5 | 6 | 6 | 5 | 8.75 | 9 | 10 | 7 | 5 | 4 |
| 0 | 0 | 0 | 6.4 | 6.3 | 5.9 | 0 | 0 | 0 | 10.5 | 5.1 | 5.4 | 9 | 5 | 6 | 5 | 5.1 | 5 | 5.1 | 5.2 | 5.1 | 0 | 0 | 5.1 | 0 | 7.1 | 5 | 5.8 | 6.4 | 6.1 | 9 | 5 | 5.15 | 6.2 | 6.1 | 5.1 | 9 | 9.1 | 10 | 7.1 | 5.1 | 4.2 | |
| 0 | 0 | 0 | 6.3 | 6.3 | 6 | 0 | 0 | 0 | 10.4 | 5 | 5.4 | 9 | 5 | 6 | 5 | 5 | 5 | 5.1 | 5.2 | 5.1 | 0 | 0 | 5.1 | 0 | 7.3 | 5 | 5.9 | 6.5 | 6 | 9.35 | 5.15 | 5.45 | 6.1 | 6.2 | 5.1 | 9.1 | 9.2 | 11 | 7 | 5 | 4.1 | |
| Average | 0 | 0 | 0 | 6.33 | 6.3 | 5.97 | 0 | 0 | 0 | 10.3 | 5.03 | 5.37 | 9 | 5 | 6 | 5 | 5.03 | 5 | 5.07 | 5.13 | 5.07 | 0 | 0 | 5.07 | 0 | 7.23 | 5 | 5.9 | 6.4 | 6.03 | 9.12 | 5.05 | 5.2 | 6.1 | 6.1 | 5.07 | 8.95 | 9.1 | 10.33 | 7.03 | 5.03 | 4.1 |
| 5 | 0 | 0 | 0 | 6 | 6 | 6 | 0 | 0 | 0 | 10 | 6 | 6 | 9 | 5 | 5.8 | 6 | 5 | 4 | 5 | 5 | 5 | 0 | 0 | 4.25 | 0 | 6 | 5.75 | 6.5 | 6.3 | 5 | 9 | 5.25 | 4.75 | 5.7 | 6 | 5 | 9 | 9 | 10 | 6 | P | 4 |
| 0 | 0 | 0 | 7.2 | 6 | 7 | 0 | 0 | 0 | 10.5 | 6.3 | 6 | 8.9 | 5 | 5.9 | 6.1 | 5 | 3.9 | 5.1 | 5.2 | 5.1 | 0 | 0 | 4.28 | 0 | 6.2 | 5.5 | 6.8 | 6.3 | 5.4 | 9 | 5.35 | 4.5 | 5.7 | 6 | 5.45 | 9.2 | 9.1 | 10 | 5.9 | 4 | 4.2 | |
| 0 | 0 | 0 | 6.7 | 6.2 | 7.1 | 0 | 0 | 0 | 10.5 | 6.2 | 6 | 9 | 5 | 5.8 | 6.1 | 5.3 | 3.9 | 5.1 | 5.2 | 5.1 | 0 | 0 | 4.3 | 0 | 6.11 | 5.67 | 6.5 | 6.3 | 5.95 | 9.2 | 5.25 | 4.7 | 5.6 | 6 | 5 | 9.2 | 9 | 10.4 | 6 | 4.1 | 4 | |
| Average | 0 | 0 | 0 | 6.63 | 6.07 | 6.7 | 0 | 0 | 0 | 10.33 | 6.17 | 6 | 8.97 | 5 | 5.83 | 6.07 | 5.1 | 3.93 | 5.07 | 5.13 | 5.07 | 0 | 0 | 4.28 | 0 | 6.1 | 5.64 | 6.6 | 6.3 | 5.45 | 9.07 | 5.28 | 4.65 | 5.67 | 6 | 5.15 | 9.13 | 9.03 | 10.13 | 5.97 | 2.7 | 4.07 |
| 10 | 0 | 0 | 0 | 5.6 | 6 | 6 | 0 | 0 | 0 | 10 | 6 | 6 | 8 | 5 | 5.3 | 6.3 | 7.1 | P | P | 5.4 | 5 | 0 | 0 | 4.5 | 0 | 5 | 5.25 | 6 | 6 | 5 | 9.25 | 5 | 5 | 5.7 | 6 | 5 | 9 | 10 | 10 | 6 | P | 4 |
| 0 | 0 | 0 | 5.9 | 6 | 6.1 | 0 | 0 | 0 | 11 | 7 | 6.4 | 9 | 5.1 | 5.2 | 6.3 | P | P | 5 | 5.2 | 5 | 0 | 0 | 4.5 | 0 | 5 | 5.35 | 6.1 | 5.9 | 5.9 | 9.8 | 5 | 5 | 5.65 | 6.1 | 5.25 | 10 | 10.6 | 10 | 6 | 4 | 4 | |
| 0 | 0 | 0 | 5.9 | 6 | 6 | 0 | 0 | 0 | 11.2 | 7.3 | 6.4 | 8.8 | 5.3 | 5.2 | 6.3 | P | P | 5.1 | P | 5 | 0 | 0 | 4.2 | 0 | 5.5 | 5.3 | 6 | 5.9 | 5.75 | 10 | 5 | P | 5 | 6 | 5.25 | 10 | 10.5 | 10 | 6 | 4 | 4 | |
| Average | 0 | 0 | 0 | 5.8 | 6 | 6.03 | 0 | 0 | 0 | 10.73 | 6.77 | 6.27 | 8.6 | 5.13 | 5.23 | 6.3 | 2.37 | 0 | 3.37 | 3.53 | 5 | 0 | 0 | 4.4 | 0 | 5.17 | 5.3 | 6.03 | 5.93 | 5.55 | 6.35 | 5 | 3.33 | 5.45 | 6.03 | 5.17 | 9.67 | 10.37 | 10 | 6 | 2.67 | 4 |
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Nowotnik, P.; Chorbiński, P.; Migdał, P.; Górski, B. Bacterial Agents for Biocontrol of American Foulbrood (AFB) of Larvae Honey Bee. Microbiol. Res. 2024, 15, 2394-2413. https://doi.org/10.3390/microbiolres15040161
AMA Style
Nowotnik P, Chorbiński P, Migdał P, Górski B. Bacterial Agents for Biocontrol of American Foulbrood (AFB) of Larvae Honey Bee. Microbiology Research. 2024; 15(4):2394-2413. https://doi.org/10.3390/microbiolres15040161
Chicago/Turabian StyleNowotnik, Piotr, Paweł Chorbiński, Paweł Migdał, and Bogusław Górski. 2024. "Bacterial Agents for Biocontrol of American Foulbrood (AFB) of Larvae Honey Bee" Microbiology Research 15, no. 4: 2394-2413. https://doi.org/10.3390/microbiolres15040161
APA StyleNowotnik, P., Chorbiński, P., Migdał, P., & Górski, B. (2024). Bacterial Agents for Biocontrol of American Foulbrood (AFB) of Larvae Honey Bee. Microbiology Research, 15(4), 2394-2413. https://doi.org/10.3390/microbiolres15040161
