Next Article in Journal
Spatial Heterogeneity in Soil Microbial Communities Impacts Their Suitability as Bioindicators for Evaluating Productivity in Agricultural Practices
Previous Article in Journal
Exploring the Effect of Enzyme and Protein-Containing Toothpaste on Gum Health: A Systematic Review
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Microorganisms
Volume 13
Issue 5
10.3390/microorganisms13051159
microorganisms-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Selection of Probiotics for Honey Bees: The In Vitro Inhibition of Paenibacillus larvae, Melissococcus plutonius, and Serratia marcescens Strain Sicaria by Host-Specific Lactobacilli and Bifidobacteria

by
Buse Dengiz
1,
Jiří Killer
1,2,*,
Jaroslav Havlík
1,
Pavel Dobeš
3 and
Pavel Hyršl
3
1
Department of Food Science, Czech University of Life Sciences Prague, 165 00 Prague, Czech Republic
2
Laboratory of Anaerobic Microbiology, Institute of Animal Physiology and Genetics, Czech Academy of Sciences, 142 20 Prague, Czech Republic
3
Department of Experimental Biology, Faculty of Science, Masaryk University, 625 00 Brno, Czech Republic
*
Author to whom correspondence should be addressed.
Microorganisms 2025, 13(5), 1159; https://doi.org/10.3390/microorganisms13051159
Submission received: 14 April 2025 / Revised: 15 May 2025 / Accepted: 16 May 2025 / Published: 20 May 2025
(This article belongs to the Special Issue Applied Gut Molecular Microbiology Technology)
Browse Figures
Versions Notes

Abstract

:
Host-specific Lactobacillus and Bifidobacterium species constitute the core microbiota of the honey bee digestive tract and are recognized for their probiotic properties. One of the properties of these bacteria is the inhibition of bacterial pathogens such as Paenibacillus larvae and Melissococcus plutonius, the causative agents of American and European foulbrood, respectively. Additionally, Serratia marcescens has emerged as a relevant opportunistic pathogen. Although several previously published studies have examined the inhibition of selected bacterial pathogens of bees by members of the bee physiological microbiota, none have simultaneously investigated the inhibition of multiple clinical isolates of P. larvae, M. plutonius, and S. marcescens using a wide range of bifidobacterial and lactobacilli strains isolated from various locations within a single country. Thus, this study evaluated the antimicrobial potential of Lactobacillus and Bifidobacterium strains against these pathogens, with a focus on strain-dependent inhibition. A total of 111 bacterial strains (62 Lactobacillus and 49 Bifidobacterium) were isolated from the digestive tracts of honey bees collected from eight sites across the Czech Republic. Using 16S rRNA gene sequencing, the isolates were classified and tested in vitro against four P. larvae isolates, one M. plutonius isolate, and the S. marcescens strain sicaria in modified BHI medium. Twenty-eight strains (~26%) exhibited strong inhibition (≥21 mm) against at least two P. larvae isolates, while 12 strains showed moderate inhibition (16–20 mm) against all four isolates. Inhibition of M. plutonius and S. marcescens was observed in three and twenty strains, respectively. The most effective strains belonged to Bifidobacterium asteroides, B. choladohabitans, B. polysaccharolyticum, Lactobacillus apis, L. helsingborgensis, L. kullabergensis, and L. melliventris. These results underscore the strain-dependent nature of antimicrobial activity and highlight the importance of selecting probiotic strains with broad-spectrum pathogen inhibition to support honey bee health.

1. Introduction

Interest in studying the host-specific microbiota of the digestive tracts of bees and other pollinators has significantly increased over the past decade, during which several bacterial species and genera have been described [1,2,3,4,5,6,7,8,9]. Their presence and activity in the gut are associated with numerous probiotic properties, most of which are attributed to host-specific Lactobacillus species, with some evidence also highlighting the probiotic potential of Bifidobacterium spp. Several species of gut-associated probiotics contribute to honey bee health through two distinct yet complementary mechanisms. First, they are capable of producing a variety of digestive enzymes, such as those that degrade amygdalin, arabinose, xylose, galactose, mannose, lactose, and raffinose, enhancing nutrient absorption and improving the bee’s ability to utilize diverse carbohydrate sources [10,11,12]. These enzymatic activities play a crucial role in breaking down complex polysaccharides, oligosaccharides, and peptides found in the bee diet. Second, some strains have demonstrated the ability to degrade xenobiotics, including harmful insecticides, thus potentially reducing chemical toxicity in the gut environment. This dual functionality, facilitating digestion and detoxification, highlights the potential of certain probiotic strains to enhance honey bee resilience to dietary and environmental stressors [13,14].
Other probiotic functions involve enhancing bee nutrition by synthesizing amino acids and vitamins [15,16], maintaining intestinal homeostasis, stimulating antimicrobial peptide expression [17,18,19], regulating hormone-driven weight gain [20], increasing brood and honey production [21], improving the longevity of queens and overwintering bees [22,23], preserving the nutritional value of bee food [24], mitigating antibiotic-induced dysbiosis [25,26], and even modulating learning [27,28] and memory behavior [29]. However, the most frequently cited probiotic effects are those involving the inhibition of prokaryotic [19,30,31,32,33,34,35] and eukaryotic [17,36,37,38,39] pathogens.
In general, the complex intestinal microbiota of healthy bees plays an irreplaceable role in supporting growth, physiological function, and metabolic processes [40]. It is important to note that not all microorganisms present in this environment can be considered probiotics; true probiotics must meet specific criteria and requirements [41].
Several host-adapted probiotic strains have been shown to inhibit Paenibacillus larvae, the etiological agent of American foulbrood, through distinct antimicrobial mechanisms. For instance, Lactobacillus kunkeei strains isolated from the honey bee crop have demonstrated significant inhibitory effects against P. larvae in vitro, attributed to their production of hydrogen peroxide and organic acids [42]. Likewise, Bifidobacterium asteroides and Lactobacillus apis have been reported to suppress P. larvae growth and to stimulate host immunity in bee larvae, improving survival outcomes during infection [30,43]. Despite these findings, most studies have focused on limited isolate diversity, leaving the inhibition profiles of broader clinical strains largely unexplored. Addressing this gap, the present study investigates the antagonistic potential of 111 Lactobacillus and Bifidobacterium strains, originating from the digestive tracts of honey bees collected across eight regions in the Czech Republic, against four clinical P. larvae isolates, Melissococcus plutonius, and the opportunistic pathogen Serratia marcescens strain sicaria, using a modified BHI co-cultivation approach.

2. Materials and Methods

2.1. Materials

Modified Brain Heart Infusion (mBHI) broth was used as the primary enrichment medium for the isolation of gut-associated bacteria. The composition of the mBHI broth per liter included the following: BHI (Carl Roth, Karlsruhe, Germany) 37 g, glucose 8 g, yeast extract (Oxoid, Hampshire, UK) 3 g, soybean peptone 3 g, meat extract (Carl Roth, Germany) 2 g, KH2PO4 2 g, MgCl2 0.5 g, and 0.5 mL of Tween 80 (Thermo Fisher Scientific, Waltham, MA, USA). The pH was adjusted to 7.1–7.3 using 10 M NaOH. Anaerobic conditions were established by CO2 treatment. For selective bacterial isolation, three types of agar media were used: mBHI agar supplemented with mupirocin (100 mg/L) and acetic acid (1 mL/L) for selective Bifidobacterium isolation [44], Rogosa agar (Oxoid, UK) for Lactobacillus, and MRS agar (Oxoid, UK) to enhance the recovery of other lactic acid bacteria. Anaerobic incubation was achieved using AnaeroGen 3.5 L sachets (Oxoid, UK) in 3.5 L anaerobic jars (Helago-CZ, Prague, Czech Republic). All pure isolates were preserved at −85 °C in 20% (v/v) anaerobic glycerol stocks buffered with 1.2 g/L K2HPO4 and 0.33 g/L KH2PO4, pH: 6.8–7.4.

2.2. Isolation of Bifidobacterium and Lactobacillus Strains

Worker honey bees were sampled from eight different locations in the Czech Republic (Table 1), with 3–5 individuals collected per site. The bees were anesthetized using CO2 and dissected aseptically with sterile forceps. The entire gut contents from each group (weighing 80–200 mg in total) were pooled and transferred into Hungate anaerobic tubes containing 1.8 mL of sterile mBHI broth. Serial dilutions of the gut homogenates were prepared in anaerobic conditions, and aliquots of 0.05–0.2 mL from suitable dilutions were plated on the selective agar media. Plates were incubated at 37 °C for 72 h under anaerobic conditions. Colonies with different morphologies were selected, subcultured in anaerobic mBHI or MRS broth, and preserved for further analysis.

2.3. Isolation of Bacterial Honey Bee Pathogens

Four Paenibacillus larvae strains—PL2, PL41, PL48, and PL52—were provided by the Czech Bee Research Institute (CBRI) in Dol–Máslovice, Czech Republic. Strains PL2, PL48, and PL52 were isolated from larvae exhibiting clinical symptoms of American foulbrood, while strain PL41 was isolated from hive debris. These strains originated from four localities within the Czech Republic: PL2—Brejl, Rakovník (GPS: 50°6′8.248″ N, 13°52′23.312″ E); PL41—Lípa-Paseky (49°11′35.859″ N, 17°46′48.737″ E); PL48—Římovice (49°41′46.842″ N, 14°56′34.802″ E); and PL52—Valtířov (50°40′25.339″ N, 14°7′32.343″ E). Bacterial strains were isolated using the microbiological cultivation technique described previously [45].
The Melissococcus plutonius strain CZ2020, isolated according to the protocol of Thebeau et al. [46], was also supplied by the CBRI for research purposes.
Professor James B. Burritt of the University of Wisconsin–Stout kindly provided the Serratia marcescens strain sicaria, an opportunistic honey bee pathogen [47].

2.4. Classification of Isolates

Overnight cultures (1 mL) grown in mBHI medium were used for chromosomal DNA extraction using the PrepMan Ultra Sample Preparation Reagent (Thermo Fisher Scientific, USA). Diluted DNA samples, ranging from 1:100 to 1:10,000 and reaching concentrations of 20–120 ng/μL, were used for 16S rDNA amplification. PCR mixtures (30 μL) consisted of 1× EliZyme™ FAST Taq MIX Red (Elisabeth Pharmacon, Brno-Zidenice, Czech Republic), 0.5 μM of each primer, PCR-grade ultra-pure H2O, and template DNA at a concentration of 10–100 ng. PCR was performed in a TProfessional Gradient 96 thermocycler (Biometra, Göttingen, Germany) under the following conditions: initial denaturation at 94 °C for 5 min; 36 cycles of denaturation at 94 °C for 1 min, annealing at 49 °C for 100 s, and extension at 72 °C for 2 min; followed by a final extension step at 72 °C for 7 min. PCR amplification was carried out using primer pairs FP1–RP2 [48] or 27F–1542R [49]. Amplicons (~1500 bp) were checked via 1.5% agarose gel electrophoresis and purified using the Monarch PCR & DNA Cleanup Kit (New England Biolabs, Ipswich, MA, USA). The purified products were then bidirectionally sequenced by SEQme (Brno, Czech Republic). Assembled sequences, ranging from 1332 to 1481 nucleotides, were processed in Geneious v7.1.7 (Biomatters Ltd., Auckland, New Zealand) and deposited in the NCBI database using the BankIt submission tool. Taxonomic classification was performed based on 16S rDNA sequence pairwise identity with the closest related taxa using the EzBioCloud database [50]. The same procedure was applied for the classification of the four P. larvae clinical isolates, M. plutonius, and Serratia marcescens strains.

2.5. In Vitro Inhibition of Honey Bee Bacterial Pathogens

A total of one hundred eleven bacterial strains (Table 1) were selected for in vitro assays. All tested strains, including bee (opportunistic) pathogens, were capable of growing in mBHI medium (as described above) under anaerobic conditions. Therefore, this common growth medium, supplemented with bacteriological agar (14 g/L), was used for the inhibition testing. Pathogenic cultures revived in mBHI broth and harvested at the late logarithmic phase were applied in a volume of 0.2 mL (achieving a cell concentration of 6.08–7.4 log CFU—colony-forming units) onto Petri dishes (90 mm in diameter). The inoculated cultures were then overlaid with 20 mL of mBHI agar. After solidification, 0.08 mL of 14–18 h-old bacterial cultures, also in the late logarithmic phase [51] and reaching a concentration of 6.7–7.2 log CFU, was applied to a central well (radius 10 mm). The co-culture samples were incubated under microaerophilic conditions (CampyGen 3.5L; Oxoid, UK) in 3.5 L anaerobic jars (Helago-CZ, Czech Republic) for 120 h at 37 °C. After incubation, the radii of the lytic zones (in mm) were measured. Samples that exhibited inhibition zones were subsequently tested in triplicate. All samples were visually compared with pure cultures of bee pathogens grown under identical conditions.
Selected samples exhibiting inhibition zones were documented by microphotography for publication purposes.

2.6. Phylogenetic Analyses

To evaluate the evolutionary relationships between active and inactive honey bee-associated Bifidobacterium and Lactobacillus strains, phylogenetic trees based on 16S rRNA gene sequences were reconstructed using MEGA version 5.05 and the neighbor-joining method [52]. The Kimura 2-parameter model was applied for the tree construction. All strain types of currently described Bifidobacterium and Lactobacillus species known to exclusively inhabit the honey bee digestive tract were included in the phylogenetic analyses. Their 16S rRNA gene sequences were retrieved from the NCBI nucleotide database (https://www.ncbi.nlm.nih.gov/nuccore/). Particular gene codes are written in parentheses next to specific taxa in the phylogenetic trees.

3. Results

3.1. Classification of Bacterial Strains

Table 1 presents the classification of bacterial strains based on 16S rDNA sequence identity. GenBank accession numbers for the 16S ribosomal RNA genes are also provided. Strains sharing ≥99.4% identity with the closest type strains were classified at the species level. Strains showing ≤98.7% identity [53] were designated by their respective generic names and marked as p.n.sp. (putative new species). Almost all known host-specific species of honey bee-associated Bifidobacterium with the exception of Bifidobacterium apis—were isolated from captured bees [9]. These included the following: B. apousia (2 strains), B. asteroides (12), B. choladohabitans (7), B. mellis (2), B. mizhiense (1), and B. polysaccharolyticum (11). Fourteen additional Bifidobacterium strains may represent potentially novel species. Most host-specific Lactobacillus species (excluding members of the Firm-5 phylotype group, which belongs to the genus Bombilactobacillus) inhabiting the digestive tract of European honey bees were identified as follows: Lactobacillus apis (16 strains), L. helsingborgensis (14), L. kullabergensis (4), L. kimbladii (4), and L. melliventris (20) [8,54]. Two strains are considered potentially novel species within the genus Lactobacillus.
The 16S rRNA gene sequences of clinical isolates PL2, PL41, and PL48 were deposited in the NCBI database under GenBank accession numbers OR050945, OR050947, and OR050948, respectively. These strains, along with PL52, share identical sequences and exhibit 99.86% sequence similarity to Paenibacillus larvae ATCC 9545T. The 16S rRNA gene sequence (1450 bp) of Melissococcus plutonius CZ2020 displayed 100% identity with M. plutonius ATCC 35311T. Similarly, the taxonomic identity of Serratia marcescens strain sicaria (Ss1) was confirmed via comparative analysis [47].

3.2. In Vitro Inhibition of Honey Bee Bacterial Pathogens

Results are presented in Table 1. Inhibitory activity was assessed based on the diameter of inhibition zones and categorized as follows: weak inhibition (16–20 mm), moderate inhibition (21–24 mm), high inhibition (25–30 mm), and strong inhibition (>30 mm). A total of 29 strains (~26%), including 15 Bifidobacterium and 14 Lactobacillus strains, exhibited at least moderate inhibitory activity against two clinical P. larvae isolates. Among these, 12 strains (6 Bifidobacterium and 6 Lactobacillus; ~12%) inhibited all 4 P. larvae clinical strains to some extent. Only 6 strains—Bifidobacterium choladohabitans (2 strains), Bifidobacterium sp. (2), Lactobacillus apis (1), and L. helsingborgensis (1)—demonstrated at least moderate inhibition against 2 or more P. larvae strains as well as S. marcescens strain sicaria. The strongest inhibitory effects against P. larvae were observed in strains belonging to Bifidobacterium asteroides, B. polysaccharolyticum, Bifidobacterium sp., L. helsingborgensis, L. apis, L. kullabergensis, and L. melliventris.
A total of 20 strains (~18%) showed inhibitory activity against the S. marcescens strain sicaria. Of these, 12 strains (8 Bifidobacterium, 4 Lactobacillus) produced at least moderate inhibition. In contrast, M. plutonius CZ2020 was minimally inhibited, with only three strains forming visible inhibition zones. The most active strains overall were affiliated with Bifidobacterium asteroides, B. polysaccharolyticum, B. choladohabitans, Bifidobacterium sp., Lactobacillus apis, and L. helsingborgensis (see Table 1).
Representative microphotographs of inhibition zones against P. larvae and S. marcescens are shown in Figure 1 and Figure 2, respectively.

3.3. Phylogenetic Analyses

Bifidobacterium and Lactobacillus strains exhibiting at least moderate inhibition against at least two P. larvae strains are highlighted in red in Figure 3 and Figure 4, respectively. In both cases, active strains are distributed across multiple species, suggesting that inhibitory activity is strongly strain dependent rather than species specific. In the case of Bifidobacterium (Figure 3), phylogenetic clusters and branches comprising different species are poorly resolved. This likely reflects the high sequence similarity among Bifidobacterium species, as well as the use of a relatively short 16S rRNA gene fragment for tree reconstruction. In contrast, the phylogenetic relationships among Lactobacillus strains are more clearly defined (Figure 4), with distinct clusters corresponding to individual species.

4. Discussion

In this study, we presented the results of a simple technique that reveals the antagonistic activity of key representatives of the honey bee physiological microbiota against the causative agents of the most serious bacterial infections in bees. This technique is based on the co-cultivation of two taxonomically, physiologically, and metabolically distinct groups of bacteria in a modified BHI medium. Paenibacillus larvae strains are characterized as Gram-positive, microaerophilic to facultatively anaerobic, spore-forming, and mostly motile bacteria [55], whereas bifidobacteria and lactobacilli are non-motile, Gram-positive, microaerophilic to anaerobic microorganisms with an obligately fermentative metabolism [56]. Serratia marcescens, phylogenetically belonging to the class Gammaproteobacteria, are Gram-negative, rod- to coccoid-shaped, saprophytic, motile, facultatively anaerobic organisms that can colonize a variety of environments and grow over a wide temperature range (5–40 °C) and rely solely on fermentation for energy production, independent of the presence of oxygen [57]. It is considered an opportunistic pathogen of plants, humans, and some insects, including honey bees [47,58]. Melissococcus plutonius, the causative agent of European foulbrood, is a Gram-positive, anaerobic, lanceolate coccoid bacterium with a fermentative metabolism, classified within the group of lactic acid bacteria [59]. To support the growth of all bacterial strains used in this study, particularly the nutritionally demanding bifidobacteria and lactobacilli, we fortified the BHI medium (ThermoFisher Scientific, Waltham, MA, USA) commonly used for the cultivation of a wide range of bacteria with glucose, yeast extract, and soy peptone. This approach is unique compared to previous studies, which typically applied separate cultivation of bifidobacteria and lactobacilli [30,31] in the initial phase, followed by co-cultivation with P. larvae on different media, mostly MRS broth (agar) and MYPGP agar.
The following taxa exhibited the strongest inhibitory activity against P. larvae strains: Bifidobacterium asteroides, B. polysaccharolyticum, Bifidobacterium sp., Lactobacillus helsingborgensis, L. apis, L. kullabergensis, and L. melliventris (Table 1). Other studies have identified Apilactobacillus kunkeei [30,36,60], Fructobacillus fructosus [35], Lactobacillus apis [4,33], L. panisapium [33], L. melliventris [30,33], L. kullabergensis [30,33], L. kimbladii [30,33], L. mellis [33], and unclassified bifidobacteria from the B. asteroides group [30,33] as active taxa against P. larvae. Compared to these studies, we tested a broader spectrum of bifidobacterial and lactobacilli strains against four different clinical isolates of P. larvae. Only one previous study used four genetically distinct P. larvae strains for similar purposes. Forsgren et al. [30] also observed that only some of the 11 bifidobacterial and lactobacilli strains tested exhibited antagonistic effects on all four clinical isolates. Inhibitory activity against P. larvae was first demonstrated in various Lactobacillus helsingborgensis strains (notably D1/RO1, D1/RO2, P1/MR12, VR5, VSM/RO4; see Table 1), and especially in particular strains of Bifidobacterium asteroides, B. polysaccharolyticum, and Bifidobacterium sp. Notably, the strains designated as Bifidobacterium sp. are likely to represent novel species. The inhibitory effect was strongly strain dependent, as active strains were distributed across different regions of the phylogenetic trees (Figure 3 and Figure 4) and among distinct species clusters. These findings are consistent with the high intra-phylotype diversity observed among lactobacilli and bifidobacteria inhabiting the honey bee gut [54]. The specific bioactive compounds produced by bifidobacteria and lactobacilli responsible for antagonistic effects against P. larvae and other bacterial pathogens remain largely unexplored. Compounds such as heptyl 2-methylbutyrate, di-isobutyl phthalate, heptakis (trimethylsilyl), di-isooctyl phthalate, and hyodeoxycholic acid have been detected in metabolites of Fructobacillus fructosus active against P. larvae [35]. Butler et al. [61] speculated that extracellular proteins of lactic acid bacteria, including bacteriocins and lysozymes, may be the primary antimicrobials in honey bees.
In contrast to previous studies that demonstrated antimicrobial activity of various bifidobacteria [34] and lactobacilli [31,43] against M. plutonius, we observed at most weak inhibition in only three strains (Table 1). This discrepancy may be due to methodological differences and the use of different culture media and also to strain-specific characteristics of the pathogen. Recent genomic analyses have revealed significant heterogeneity among M. plutonius isolates, affecting their susceptibility to antimicrobial agents [62]. Furthermore, environmental conditions such as pH and nutrient availability can influence the production of antimicrobial metabolites by probiotics [63]. For instance, the lack of certain growth-promoting factors under our experimental conditions may have hindered bacteriocin or organic acid production. Finally, increasing reports of antibiotic resistance in M. plutonius, such as Masood et al. [62], suggest that resistance mechanisms may also diminish the efficacy of probiotic interventions. These factors should be considered in future in vitro and in vivo evaluations.
Serratia marcescens is a Gram-negative opportunistic pathogen known to infect a broad spectrum of hosts, including both insects and humans. In honey bees, it has been primarily associated with diseased larvae and only sporadically detected in adult bees, often at very low abundances, which is considered indicative of an imbalanced gut microbiota. Recent studies have reported that certain strains of S. marcescens, such as those isolated from the bee gut or Varroa mites, can increase mortality in adult bees, particularly following exposure to antibiotics or pesticides [58]. Research focusing on the inhibitory effects of the bee gut microbiota against virulent strains of S. marcescens is scarce. One relevant study by Lang et al. [32] reported that Gilliamella apicola and Lactobacillus apis protect honey bees from this opportunistic pathogen. The study by Chen et al. [2] focused on the taxonomic classification and host specificity of B. choladohabitans and did not address its potential probiotic activity against bee pathogens. In our present study, certain B. choladohabitans strains showed remarkable inhibitory effects against important pathogens, especially Paenibacillus larvae. This finding suggests a new functional role for this species and significantly expands the current knowledge about honey bee gut microbiota. In this context, the demonstration of antimicrobial activity of B. choladohabitans highlights its importance as a promising candidate for future probiotic applications. Our study presents the first results on the inhibitory activity of bifidobacteria and lactobacilli against S. marcescens strain sicaria. Since this is a Gram-negative bacterium, active strains, especially B. asteroides, B. polysaccharolyticum, B. choladohabitans, Bifidobacterium sp., and Lactobacillus apis (Table 1), must produce different antimicrobial substances (e.g., H2O2, diacetyl) compared to those effective against Gram-positive pathogens.
It is worth noting that the observed results may be influenced by the methodology. In some cases, the inhibition zones were not sharply defined (e.g., Figure 1: images vi, ix, x, xiv), which may have been caused by the diffusion of antimicrobial compounds throughout the agar medium. Additional factors such as species- or strain-specific growth characteristics, culture medium composition, atmospheric conditions, and temperature could also affect the in vitro inhibitory effects.
The commercial use of probiotics in honey bees is a developing field of research with the aim of improving colony health and productivity and reducing dependence on chemical treatments. Probiotics from the Lactobacillus and Bifidobacterium species are known for their beneficial effects on intestinal health, immune system strengthening, and pathogen suppression properties and have also shown positive results in honey bees [64,65]. Various studies have shown that probiotic strains can modulate the gut microbiota of honey bees and increase their resistance to diseases such as Paenibacillus larvae and Melissococcus plutonius [60,64,66].
In order for probiotics to be used commercially in beekeeping, they must meet certain criteria such as safety, efficacy, and ability to survive in hive conditions. These probiotics must be free of virulence factors that may have adverse effects on honey bee health [67]. They must also be resistant to environmental stressors such as temperature changes and pesticides. Probiotics are expected to effectively colonize bee guts, enhance their antimicrobial properties, and support overall colony health [60,67]. Probiotics may offer a variety of benefits beyond disease prevention. Studies have shown that probiotics can increase the nutrient digestion capacity of honey bees and also increase their resistance to pesticides and malnutrition [65]. These benefits suggest that probiotics may be a sustainable alternative to antibiotics and other chemical treatments, which are often associated with negative ecological consequences such as resistance development.
This study emphasized the concept of strain-dependent inhibition to assess the antimicrobial potential of Lactobacillus and Bifidobacterium strains against major honey bee pathogens, with the broader goal of supporting their potential commercial application as probiotics. The relevance of strain-dependent inhibition lies in its critical role in selecting effective probiotic candidates for honey bee health management. Our results demonstrate that only a fraction of the tested strains exhibited strong and wide-ranging inhibitory effects against multiple pathogenic bacteria. This finding underscores that antimicrobial activity is not consistent at the species level but instead varies significantly between individual strains. Understanding this strain-specific variability is essential for the rational selection of probiotics aimed at enhancing colony resilience against bacterial infections, including foulbrood diseases and opportunistic pathogens like Serratia marcescens. However, more research is needed before probiotics can be widely accepted in beekeeping. Future studies should evaluate the efficacy of probiotics in real-world conditions and examine their long-term effects on hive productivity, colony growth, and survival.
Overall, the antagonistic properties of specific bacterial strains should be experimentally verified in both laboratory and field settings using honey bees infected with relevant pathogens. In future research, the most active strains should also be assessed for additional beneficial effects, such as their potential impact on host immune and metabolic functions. Furthermore, strains with uncertain taxonomic status (e.g., Bifidobacterium sp.) should be taxonomically classified in detail using modern phylogenomic, chemotaxonomic, and biochemical approaches.

5. Conclusions

The results of our study confirmed, as reported in many previous investigations, that the antimicrobial properties of probiotic bacteria are strongly strain dependent. A strong antagonistic effect against P. larvae strains was observed particularly in strains of Bifidobacterium asteroides (~36% of strains showed moderate inhibition of at least two P. larvae strains), B. polysaccharolyticum (~36%), Bifidobacterium sp. (~31%), Lactobacillus helsingborgensis (~43%), L. apis (~19%), L. kullabergensis (~33%), and L. melliventris (20%). Only the strains Bifidobacterium choladohabitans VSM/mBH8 and 2BB/B7; Bifidobacterium sp. 1BB/B1 and 1BB/B2; Lactobacillus apis 1BB/MR8; and L. helsingborgensis VSM/RO7 showed at least moderate inhibition of two or more P. larvae strains and also of S. marcescens.
This is the first study to report on the antimicrobial activity of bifidobacteria and lactobacilli against the Serratia marcescens strain sicaria. The strongest inhibitory effect was observed in strains of Bifidobacterium asteroides, B. polysaccharolyticum, B. choladohabitans, Bifidobacterium sp., Lactobacillus apis, and L. helsingborgensis.
The simple in vitro technique presented here offers a practical and efficient method for screening a broad range of honey bee symbionts against various bacterial pathogens of honey bees. Antagonistic activity against different honey bee pathogens is a crucial probiotic property. In this context, we recommend testing a diverse collection of isolates representing the physiological symbiotic microbiota of the honey bee gut, obtained from different geographic locations, against multiple clinical isolates of P. larvae and other pathogenic or opportunistically pathogenic microorganisms affecting honey bees.
In addition to screening for antimicrobial activity, future studies should also evaluate other probiotic functions such as enzyme production and xenobiotic degradation potential, particularly for the most active strains identified in this study. These properties are important for understanding the broader potential of these probiotics to support honey bee health and promote colony resilience, as they may provide valuable insights into the mechanisms behind the observed antagonistic effects and may help identify strains with additional beneficial traits for honey bee management.

Author Contributions

B.D. and J.K. wrote the manuscript draft, performed the experimental work, and prepared figures and tables. J.K. developed a common culture medium for potentially probiotic bacteria and honey bee bacterial pathogens, conducted the genetic analysis, and prepared figures. J.H. and P.H. obtained funding and provided resources. P.D. critically evaluated the manuscript. All authors reviewed and revised the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the CZECH NATIONAL AGRICULTURAL RESEARCH AGENCY: QK21010088 and METROFOOD-CZ Research Infrastructure (https://metrofood.cz, accessed on 10 February 2025), supported by the Ministry of Education, Youth and Sports of the Czech Republic: Project No. LM2023064.

Institutional Review Board Statement

Not applicable. Ethical approval is specifically required for studies on vertebrates, not insects. There are no restrictions regarding the capture of honey bees in the Czech Republic. However, we followed general ethical recommendations when capturing and killing honey bees.

Informed Consent Statement

Not applicable.

Data Availability Statement

Nearly full-length genes for 16S rRNA of evaluated strains generated by Sanger sequencing are deposited in GenBank under the accession codes PP754609 to PP754719.

Acknowledgments

We gratefully acknowledge professor James B. Burritt from the University of Wisconsin-Stout for kindly providing the Serratia marcescens strain sicaria used in this study.

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. Alberoni, D.; Gaggìa, F.; Baffoni, L.; Modesto, M.M.; Biavati, B.; Di Gioia, D. Bifidobacterium Xylocopae sp. nov. and Bifidobacterium Aemilianum sp. nov., from the Carpenter Bee (Xylocopa Violacea) Digestive Tract. Syst. Appl. Microbiol. 2019, 42, 205–216. [Google Scholar] [CrossRef] [PubMed]
  2. Chen, J.; Wang, J.; Zheng, H. Characterization of Bifidobacterium Apousia sp. nov., Bifidobacterium Choladohabitans sp. nov., and Bifidobacterium Polysaccharolyticum sp. nov., Three Novel Species of the Genus Bifidobacterium from Honeybee Gut. Syst. Appl. Microbiol. 2021, 44, 126247. [Google Scholar] [CrossRef] [PubMed]
  3. Engel, P.; Kwong, W.K.; Moran, N.A. Frischella Perrara gen. nov., sp. nov., a Gammaproteobacterium Isolated from the Gut of the Honeybee, Apis Mellifera. Int. J. Syst. Evol. Microbiol. 2013, 63, 3646–3651. [Google Scholar] [CrossRef] [PubMed]
  4. Killer, J.; Dubná, S.; Sedláček, I.; Švec, P. Lactobacillus Apis sp. nov., from the Stomach of Honeybees (Apis mellifera), Having an in vitro Inhibitory Effect on the Causative Agents of American and European Foulbrood. Int. J. Syst. Evol. Microbiol. 2014, 64, 152–157. [Google Scholar] [CrossRef]
  5. Killer, J.; Kopečný, J.; Mrázek, J.; Havlík, J.; Koppová, I.; Benada, O.; Rada, V.; Kofroňová, O. Bombiscardovia Coagulans gen. nov., sp. nov., a New Member of the Family Bifidobacteriaceae Isolated from the Digestive Tract of Bumblebees. Syst. Appl. Microbiol. 2010, 33, 359–366. [Google Scholar] [CrossRef]
  6. Killer, J.; Votavová, A.; Valterová, I.; Vlková, E.; Rada, V.; Hroncová, Z. Lactobacillus Bombi sp. nov., from the Digestive Tract of Laboratory-Reared Bumblebee Queens (Bombus Terrestris). Int. J. Syst. Evol. Microbiol. 2014, 64, 2611–2617. [Google Scholar] [CrossRef]
  7. Kwong, W.K.; Moran, N.A. Cultivation and Characterization of the Gut Symbionts of Honey Bees and Bumble Bees: Description of Snodgrassella Alvi gen. nov., sp. nov., a Member of the Family Neisseriaceae of the Betaproteobacteria, and Gilliamella Apicola gen. nov., sp. nov., a Member of Orbaceae Fam. nov., Orbales Ord. nov., a Sister Taxon to the Order “Enterobacteriales” of the Gammaproteobacteria. Int. J. Syst. Evol. Microbiol. 2013, 63, 2008–2018. [Google Scholar] [CrossRef]
  8. Olofsson, T.C.; Alsterfjord, M.; Nilson, B.; Butler, È.; Vásquez, A. Lactobacillus Apinorum sp. nov., Lactobacillus Mellifer sp. nov., Lactobacillus Mellis sp. nov., Lactobacillus Melliventris sp. nov., Lactobacillus Kimbladii sp. nov., Lactobacillus Helsingborgensis sp. nov. and Lactobacillus Kullabergensis sp. nov., Isolated from the Honey Stomach of the Honeybee Apis mellifera. Int. J. Syst. Evol. Microbiol. 2014, 64, 3109–3119. [Google Scholar] [CrossRef]
  9. Olofsson, T.C.; Modesto, M.; Pascarelli, S. Bifidobacterium Mellis sp. nov., Isolated from the Honey Stomach of the Honey Bee Apis mellifera. Int. J. Syst. Evol. Microbiol. 2023, 73, 3. [Google Scholar] [CrossRef]
  10. Kešnerová, L.; Mars, R.A.T.; Ellegaard, K.M.; Troilo, M.; Sauer, U.; Engel, P. Disentangling Metabolic Functions of Bacteria in the Honey Bee Gut. PLoS Biol. 2017, 15, e2003467. [Google Scholar] [CrossRef]
  11. Motta, E.V.S.; Gage, A.; Smith, T.E.; Blake, K.J.; Kwong, W.K.; Riddington, I.M.; Moran, N.A. Host-Microbiome Metabolism of a Plant Toxin in Bees. Elife 2022, 11, e82595. [Google Scholar] [CrossRef] [PubMed]
  12. Zheng, H.; Perreau, J.; Elijah Powell, J.; Han, B.; Zhang, Z.; Kwong, W.K.; Tringe, S.G.; Moran, N.A. Division of Labor in Honey Bee Gut Microbiota for Plant Polysaccharide Digestion. Proc. Natl. Acad. Sci. USA 2019, 116, 25909–25916. [Google Scholar] [CrossRef] [PubMed]
  13. Leska, A.; Nowak, A.; Miśkiewicz, K.; Rosicka-Kaczmarek, J. Binding and Detoxification of Insecticides by Potentially Probiotic Lactic Acid Bacteria Isolated from Honeybee (Apis mellifera L.) Environment—An In Vitro Study. Cells 2022, 11, 3743. [Google Scholar] [CrossRef] [PubMed]
  14. Liu, P.; Niu, J.; Zhu, Y.; Li, Z.; Ye, L.; Cao, H.; Shi, T.; Yu, L. Apilactobacillus kunkeei Alleviated Toxicity of Acetamiprid in Honeybee. Insects 2022, 13, 1167. [Google Scholar] [CrossRef]
  15. Nowak, A.; Szczuka, D.; Górczyńska, A.; Motyl, I.; Kręgiel, D. Characterization of Apis Mellifera Gastrointestinal Microbiota and Lactic Acid Bacteria for Honeybee Protection-A Review. Cells 2021, 10, 701. [Google Scholar] [CrossRef]
  16. Smriti; Rana, A.; Singh, G.; Gupta, G. Prospects of Probiotics in Beekeeping: A Review for Sustainable Approach to Boost Honeybee Health. Arch. Microbiol. 2024, 206, 205. [Google Scholar] [CrossRef]
  17. Garrido, P.M.; Porrini, M.P.; Alberoni, D.; Baffoni, L.; Scott, D.; Mifsud, D.; Eguaras, M.J.; Di Gioia, D. Beneficial Bacteria and Plant Extracts Promote Honey Bee Health and Reduce Nosema Ceranae Infection. Probiotics Antimicrob. Proteins 2024, 16, 259–274. [Google Scholar] [CrossRef]
  18. Janashia, I.; Alaux, C. Specific Immune Stimulation by Endogenous Bacteria in Honey Bees (Hymenoptera: Apidae). J. Econ. Entomol. 2016, 109, 1474–1477. [Google Scholar] [CrossRef]
  19. Ye, M.; Li, X.; Yang, F.; Zhou, B. Beneficial Bacteria as Biocontrol Agents for American Foulbrood Disease in Honey Bees (Apis mellifera). J. Insect Sci. 2023, 23, 6. [Google Scholar] [CrossRef]
  20. Zheng, H.; Powell, J.E.; Steele, M.I.; Dietrich, C.; Moran, N.A. Honeybee Gut Microbiota Promotes Host Weight Gain via Bacterial Metabolism and Hormonal Signaling. Proc. Natl. Acad. Sci. USA 2017, 114, 4775–4780. [Google Scholar] [CrossRef]
  21. Alberoni, D.; Baffoni, L.; Gaggìa, F.; Ryan, P.M.; Murphy, K. Impact of Beneficial Bacteria Supplementation on the Gut Microbiota, Colony Development and Productivity of Apis mellifera L. Benef. Microbes 2018, 9, 269–278. [Google Scholar] [CrossRef] [PubMed]
  22. Anderson, K.E.; Ricigliano, V.A.; Mott, B.M.; Copeland, D.C.; Floyd, A.S.; Maes, P. The Queen’s Gut Refines with Age: Longevity Phenotypes in a Social Insect Model. Microbiome 2018, 6, 108. [Google Scholar] [CrossRef] [PubMed]
  23. Wang, H.; Lei, L.; Chen, W.; Chi, X.; Han, K.; Wang, Y.; Ma, L.; Liu, Z.; Xu, B. The Comparison of Antioxidant Performance, Immune Performance, IIS Activity and Gut Microbiota Composition between Queen and Worker Bees Revealed the Mechanism of Different Lifespan of Female Casts in the Honeybee. Insects 2022, 13, 772. [Google Scholar] [CrossRef]
  24. Janashia, I.; Choiset, Y.; Jozefiak, D.; Déniel, F.; Coton, E.; Moosavi-Movahedi, A.A.; Chanishvili, N.; Haertlé, T. Beneficial Protective Role of Endogenous Lactic Acid Bacteria against Mycotic Contamination of Honeybee Beebread. Probiotics Antimicrob. Proteins 2018, 10, 638–646. [Google Scholar] [CrossRef]
  25. Daisley, B.A.; Pitek, A.P.; Chmiel, J.A.; Gibbons, S.; Chernyshova, A.M.; Al, K.F.; Faragalla, K.M.; Burton, J.P.; Thompson, G.J.; Reid, G. Lactobacillus spp. Attenuate Antibiotic-Induced Immune and Microbiota Dysregulation in Honey Bees. Commun. Biol. 2020, 3, 534. [Google Scholar] [CrossRef]
  26. Powell, J.E.; Carver, Z.; Leonard, S.P.; Moran, N.A. Field-Realistic Tylosin Exposure Impacts Honey Bee Microbiota and Pathogen Susceptibility, Which Is Ameliorated by Native Gut Probiotics. Microbiol. Spectr. 2021, 9, e0010321. [Google Scholar] [CrossRef]
  27. Wu, J.; Lang, H.; Mu, X.; Zhang, Z.; Su, Q.; Hu, X.; Zheng, H. Honey Bee Genetics Shape the Strain-Level Structure of Gut Microbiota in Social Transmission. Microbiome 2021, 9, 225. [Google Scholar] [CrossRef]
  28. Zhang, Z.; Mu, X.; Cao, Q.; Shi, Y.; Hu, X.; Zheng, H. Honeybee Gut Lactobacillus Modulates Host Learning and Memory Behaviors via Regulating Tryptophan Metabolism. Nat. Commun. 2022, 13, 2037. [Google Scholar] [CrossRef]
  29. Vernier, C.L.; Nguyen, L.A.; Gernat, T.; Ahmed, A.C.; Chen, Z.; Robinson, G.E. Gut Microbiota Contribute to Variations in Honey Bee Foraging Intensity. ISME J. 2024, 18, wrae030. [Google Scholar] [CrossRef]
  30. Forsgren, E.; Olofsson, T.C.; Vásquez, A.; Fries, I. Novel Lactic Acid Bacteria Inhibiting Paenibacillus Larvae in Honey Bee Larvae Novel Lactic Acid Bacteria Inhibiting Paenibacillus Larvae in Honey Bee Larvae. Apidologie 2010, 41, 99–108. [Google Scholar] [CrossRef]
  31. Iorizzo, M.; Ganassi, S.; Albanese, G.; Letizia, F.; Testa, B.; Tedino, C.; Petrarca, S.; Mutinelli, F.; Mazzeo, A.; De Cristofaro, A. Antimicrobial Activity from Putative Probiotic Lactic Acid Bacteria for the Biological Control of American and European Foulbrood Diseases. Vet. Sci. 2022, 9, 236. [Google Scholar] [CrossRef] [PubMed]
  32. Lang, H.; Duan, H.; Wang, J.; Zhang, W.; Guo, J.; Zhang, X.; Hu, X.; Zheng, H. Specific Strains of Honeybee Gut Lactobacillus Stimulate Host Immune System to Protect against Pathogenic Hafnia Alvei. Microbiol. Spectr. 2022, 23, e0189621. [Google Scholar] [CrossRef]
  33. Truong, A.T.; Kang, J.E.; Yoo, M.S.; Nguyen, T.T.; Youn, S.Y.; Yoon, S.S.; Cho, Y.S. Probiotic Candidates for Controlling Paenibacillus Larvae, a Causative Agent of American Foulbrood Disease in Honey Bee. BMC Microbiol. 2023, 23, 150. [Google Scholar] [CrossRef]
  34. Wu, M.; Sugimura, Y.; Takaya, N.; Takamatsu, D.; Kobayashi, M.; Taylor, D.M.; Yoshiyama, M. Characterization of Bifidobacteria in the Digestive Tract of the Japanese Honeybee, Apis Cerana Japonica. J. Invertebr. Pathol. 2013, 112, 88–93. [Google Scholar] [CrossRef]
  35. Zeid, A.A.A.; Khattaby, A.M.; El-Khair, I.A.A.; Gouda, H.I.A. Detection Bioactive Metabolites of Fructobacillus Fructosus strain HI-1 Isolated from Honey Bee’s Digestive Tract Against Paenibacillus Larvae. Probiotics Antimicrob. Proteins 2022, 14, 476–485. [Google Scholar] [CrossRef]
  36. Arredondo, D.; Castelli, L.; Porrini, M.P.; Garrido, P.M.; Eguaras, M.J.; Zunino, P.; Antúnez, K. Lactobacillus Kunkeei Strains Decreased the Infection by Honey Bee Pathogens Paenibacillus Larvae and Nosema Ceranae. Benef. Microbes 2018, 9, 279–290. [Google Scholar] [CrossRef]
  37. Baffoni, L.; Gaggìa, F.; Alberoni, D.; Cabbri, R.; Nanetti, A.; Biavati, B.; Di Gioia, D. Effect of Dietary Supplementation of Bifidobacterium and Lactobacillus Strains in Apis Mellifera L. Against Nosema ceranae. Benef. Microbes 2016, 7, 45–51. [Google Scholar] [CrossRef]
  38. Iorizzo, M.; Lombardi, S.J.; Ganassi, S.; Testa, B.; Ianiro, M.; Letizia, F.; Succi, M.; Tremonte, P.; Vergalito, F.; Cozzolino, A.; et al. Antagonistic Activity against Ascosphaera apis and Functional Properties of Lactobacillus kunkeei Strains. Antibiotics 2020, 9, 262. [Google Scholar] [CrossRef]
  39. Tejerina, M.R.; Cabana, M.J.; Benitez-Ahrendts, M.R. Strains of Lactobacillus spp. Reduce Chalkbrood in Apis mellifera. J. Invertebr. Pathol. 2021, 178, 107521. [Google Scholar] [CrossRef]
  40. Brar, G.; Ngor, L.; McFrederick, Q.S.; Torson, A.S.; Rajamohan, A.; Rinehart, J.; Singh, P.; Bowsher, J.H. High Abundance of Lactobacilli in the Gut Microbiome of Honey Bees During Winter. Sci. Rep. 2025, 15, 7409. [Google Scholar] [CrossRef]
  41. Reid, G.; Gadir, A.A.; Dhir, R. Probiotics: Reiterating What They Are and What They Are Not. Front. Microbiol. 2019, 10, 424. [Google Scholar] [CrossRef] [PubMed]
  42. Endo, A.; Salminen, S. Honeybees and beehives are rich sources for fructophilic lactic acid bacteria. Syst. Appl. Microbiol. 2013, 36, 444–448. [Google Scholar] [CrossRef] [PubMed]
  43. Vásquez, A.; Olofsson, T.C.; Forsgren, E.; Fries, I.; Paxton, R.J.; Flaberg, E.; Szekely, L.; Odham, G. Symbionts as major modulators of insect health: Lactic acid bacteria and honeybees. PLoS ONE 2012, 7, e33188. [Google Scholar] [CrossRef]
  44. Killer, J.; Kopečný, J.; Mrázek, J.; Rada, V.; Dubná, S.; Marounek, M. Bifidobacteria in the Digestive Tract of Bumblebees. Anaerobe 2010, 16, 165–170. [Google Scholar] [CrossRef]
  45. Matiašovic, J.; Bzdil, J.; Papežíková, I.; Čejková, D.; Vasina, E.; Bizos, J.; Navrátil, S.; Šedivá, M.; Klaudiny, J.; Pikula, J. Genomic Analysis of Paenibacillus larvae Isolates from the Czech Republic and the Neighbouring Regions of Slovakia. Res. Vet. Sci. 2023, 158, 34–40. [Google Scholar] [CrossRef]
  46. Thebeau, J.M.; Liebe, D.; Masood, F.; Kozii, I.V.; Klein, C.D.; Zabrodski, M.W.; Moshynskyy, I.; Sobchishin, L.; Wilson, G.; Guarna, M.M.; et al. Article Investigation of Melissococcus Plutonius Isolates from 3 Outbreaks of European Foulbrood Disease in Commercial Beekeeping Operations in Western Canada. Can. Vet. J. 2022, 63, 935–942. [Google Scholar]
  47. Burritt, N.L.; Foss, N.J.; Neeno-Eckwall, E.C.; Church, J.O.; Hilger, A.M.; Hildebrand, J.A.; Warshauer, D.M.; Perna, N.T.; Burritt, J.B. Sepsis and Hemocyte Loss in Honey Bees (Apis Mellifera) Infected with Serratia Marcescens Strain Sicaria. PLoS ONE 2016, 11, e0167752. [Google Scholar] [CrossRef]
  48. Forster, R.J.; Teather, R.M.; Gong, J.; Deng, S.J. 16S rDNA Analysis of Butyrivibrio Fibrisolvens: Phylogenetic Position and Relation to Butyrate-Producing Anaerobic Bacteria from the Rumen of White-Tailed Deer. Lett. Appl. Microbiol. 1996, 23, 218–222. [Google Scholar] [CrossRef]
  49. Galkiewicz, J.P.; Kellogg, C.A. Cross-Kingdom Amplification Using Bacteria-Specific Primers: Complications for Studies of Coral Microbial Ecology. Appl Environ Microbiol 2008, 74, 7828–7831. [Google Scholar] [CrossRef]
  50. Killer, J.; Mekadim, C.; Pechar, R.; Bunešová, V.; Mrázek, J.; Vlková, E. Gene Encoding the CTP Synthetase as an Appropriate Molecular Tool for Identification and Phylogenetic Study of the Family Bifidobacteriaceae. Microbiologyopen 2018, 7, e00579. [Google Scholar] [CrossRef]
  51. Cheng, L.; Kiewiet, M.B.G.; Logtenberg, M.J.; Groeneveld, A.; Nauta, A.; Schols, H.A.; Walvoort, M.T.C.; Harmsen, H.J.M.; de Vos, P. Effects of Different Human Milk Oligosaccharides on Growth of Bifidobacteria in Monoculture and Co-Culture with Faecalibacterium Prausnitzii. Front. Microbiol. 2020, 11, 569700. [Google Scholar] [CrossRef] [PubMed]
  52. Killer, J.; Havlík, J.; Bunešová, V.; Vlková, E.; Benada, O. Pseudoscardovia radai sp. nov., A Representative of the Family Bifidobacteriaceae Isolated from the Digestive Tract of a Wild Pig (Sus Scrofa Scrofa). Int. J. Syst. Evol. Microbiol. 2014, 64, 2932–2938. [Google Scholar] [CrossRef] [PubMed]
  53. Chun, J.; Oren, A.; Ventosa, A.; Christensen, H.; Arahal, D.R.; da Costa, M.S.; Rooney, A.P.; Yi, H.; Xu, X.W.; De Meyer, S.; et al. Proposed Minimal Standards for the Use of Genome Data for the Taxonomy of Prokaryotes. Int. J. Syst. Evol. Microbiol. 2018, 68, 461–466. [Google Scholar] [CrossRef] [PubMed]
  54. Ellegaard, K.M.; Tamarit, D.; Javelind, E.; Olofsson, T.C.; Andersson, S.G.E.; Vásquez, A. Extensive Intra-Phylotype Diversity in Lactobacilli and Bifidobacteria from the Honeybee Gut. BMC Genom. 2015, 16, 284. [Google Scholar] [CrossRef]
  55. Heyndrickx, M.; Vandemeulebroecke, K.; Hoste, B. Reclassification of Paenibacillus (Formerly Bacillus) Pulvifaciens (Nakamura 1984) Ash et al. 1994, a Later Subjective Synonym of Paenibacillus (Formerly Bacillus) larvae (White 1906) Ash et al. 1994, as a Subspecies of P. Larvae, with Emended Descriptions of P. Larvae as P. Larvae subs P. Larvae and P. Larvae subsp. Pulvifaciens. Int. J. Syst. Bacteriol. 1996, 46, 270–279. [Google Scholar] [CrossRef]
  56. Fijan, S. Microorganisms with Claimed Probiotic Properties: An Overview of Recent Literature. Int. J. Environ. Res. Public Health 2014, 11, 4745–4767. [Google Scholar] [CrossRef]
  57. Hejazi, A.; Falkiner, F.R. Serratia marcescens. J. Med. Microbiol. 1997, 46, 903–912. [Google Scholar] [CrossRef]
  58. Raymann, K.; Coon, K.L.; Shaffer, Z.; Salisbury, S.; Moran, N.A. Pathogenicity of Serratia marcescens Strains in Honey Bees. mBio 2018, 9, e01649-18, Erratum in mBio 2019, 10, e02855-18. [Google Scholar] [CrossRef]
  59. Ansari, M.J.; Al-Ghamdi, A.; Nuru, A.; Ahmed, A.M.; Ayaad, T.H.; Al-Qarni, A.; Alattal, Y.; Al-Waili, N. Survey and Molecular Detection of Melissococcus plutonius, the Causative Agent of European Foulbrood in Honeybees in Saudi Arabia. Saudi J. Biol. Sci. 2017, 24, 1327–1335. [Google Scholar] [CrossRef]
  60. Daisley, B.A.; Pitek, A.P.; Chmiel, J.A.; Al, K.F.; Chernyshova, A.M.; Faragalla, K.M.; Burton, J.P.; Thompson, G.J.; Reid, G. Novel Probiotic Approach to Counter Paenibacillus larvae Infection in Honey Bees. ISME J. 2020, 14, 476–491. [Google Scholar] [CrossRef]
  61. Butler, È.; Alsterfjord, M.; Olofsson, T.C.; Karlsson, C.; Malmström, J.; Vásquez, A. Proteins of Novel Lactic Acid Bacteria from Apis mellifera mellifera: An Insight into the Production of Known Extra-Cellular Proteins during Microbial Stress. BMC Microbiol. 2013, 13, 235. [Google Scholar] [CrossRef] [PubMed]
  62. Masood, F.; Thebeau, J.M.; Cloet, A.; Kozii, I.V.; Zabrodski, M.W.; Biganski, S.; Wood, S.C. Evaluating approved and alternative treatments against an oxytetracycline-resistant bacterium responsible for European foulbrood disease in honey bees. Sci. Rep. 2022, 12, 5906. [Google Scholar] [CrossRef] [PubMed]
  63. Daisley, B.A.; Pitek, A.P.; Mallory, E.; Chernyshova, A.M.; Allen-Vercoe, E.; Reid, G.; Thompson, G.J. Disentangling the microbial ecological factors impacting honey bee susceptibility to Paenibacillus larvae infection. Trends Microbiol. 2023, 31, 521–534. [Google Scholar] [CrossRef]
  64. Iorizzo, M.; Testa, B.; Lombardi, S.J.; Ganassi, S.; Ianiro, M.; Letizia, F.; De Cristofaro, A. Antimicrobial activity against Paenibacillus larvae and functional properties of Lactiplantibacillus plantarum strains: Potential benefits for honeybee health. Antibiotics 2020, 9, 442. [Google Scholar] [CrossRef]
  65. Borges, D.; Guzman-Novoa, E.; Goodwin, P.H. Effects of prebiotics and probiotics on honey bees (Apis mellifera) infected with the microsporidian parasite Nosema ceranae. Microorganisms 2021, 9, 481. [Google Scholar] [CrossRef]
  66. Mallory, E.; Freeze, G.; Daisley, B.A.; Allen-Vercoe, E. Revisiting the role of pathogen diversity and microbial interactions in honeybee susceptibility and treatment of Melissococcus plutonius infection. Front. Vet. Sci. 2024, 11, 1495010. [Google Scholar] [CrossRef]
  67. Toutiaee, S.; Mojgani, N.; Harzandi, N.; Moharrami, M.; Mokhberosafa, L. In vitro probiotic and safety attributes of Bacillus spp. isolated from beebread, honey samples and digestive tract of honeybees Apis mellifera. Lett. Appl. Microbiol. 2022, 74, 656–665. [Google Scholar] [CrossRef]
Microorganisms 13 01159 g001
Figure 1. Examples of inhibition zones observed in co-cultures of selected Lactobacillus and Bifidobacterium strains with Paenibacillus larvae clinical isolates. Shown combinations include: (i) Lactobacillus apis P1/MR8 + PL48; (ii) L. apis P1/MR8 + PL52; (iii) Bifidobacterium choladohabitans VSM/mBH8 + PL48; (iv) B. choladohabitans VSM/mBH8 + PL52; (v) L. melliventris VU/RO9 + PL48; (vi) L. melliventris VU/RO9 + PL52; (vii) L. apis 1BB/MR6 + PL48; (viii) L. apis 1BB/MR6 + PL52; (ix) L. apis 1BB/MR8 + PL2; (x) L. apis 1BB/MR8 + PL41; (xi) L. apis 1BB/MR8 + PL48; (xii) L. apis 1BB/MR8 + PL52; (xiii) Bifidobacterium sp. 1BB/TS7 + PL41; (xiv) Bifidobacterium sp. 1BB/TS7 + PL48; (xv) B. choladohabitans 2BB/B7 + PL48; (xvi) B. choladohabitans 2BB/B7 + PL52; (xvii) L. melliventris 2BB/RO7 + PL48; (xviii) L. melliventris 2BB/RO7 + PL52. Scale bar: 10 mm.
Figure 1. Examples of inhibition zones observed in co-cultures of selected Lactobacillus and Bifidobacterium strains with Paenibacillus larvae clinical isolates. Shown combinations include: (i) Lactobacillus apis P1/MR8 + PL48; (ii) L. apis P1/MR8 + PL52; (iii) Bifidobacterium choladohabitans VSM/mBH8 + PL48; (iv) B. choladohabitans VSM/mBH8 + PL52; (v) L. melliventris VU/RO9 + PL48; (vi) L. melliventris VU/RO9 + PL52; (vii) L. apis 1BB/MR6 + PL48; (viii) L. apis 1BB/MR6 + PL52; (ix) L. apis 1BB/MR8 + PL2; (x) L. apis 1BB/MR8 + PL41; (xi) L. apis 1BB/MR8 + PL48; (xii) L. apis 1BB/MR8 + PL52; (xiii) Bifidobacterium sp. 1BB/TS7 + PL41; (xiv) Bifidobacterium sp. 1BB/TS7 + PL48; (xv) B. choladohabitans 2BB/B7 + PL48; (xvi) B. choladohabitans 2BB/B7 + PL52; (xvii) L. melliventris 2BB/RO7 + PL48; (xviii) L. melliventris 2BB/RO7 + PL52. Scale bar: 10 mm.
Microorganisms 13 01159 g001
Microorganisms 13 01159 g002
Figure 2. Examples of inhibition zones in co-cultures of Serratia marcescens strain sicaria with (a) B. polysaccharolyticum VU/mBH2, (b) B. polysaccharolyticum VSM/mBH5, (c) Bifidobacterium sp. 1BB/B1, (d) Bifidobacterium sp. 1BB/B2, (e) L. apis 1BB/MR8, (f) B. choladohabitans 2BB/B7. Scale bar: 10 mm.
Figure 2. Examples of inhibition zones in co-cultures of Serratia marcescens strain sicaria with (a) B. polysaccharolyticum VU/mBH2, (b) B. polysaccharolyticum VSM/mBH5, (c) Bifidobacterium sp. 1BB/B1, (d) Bifidobacterium sp. 1BB/B2, (e) L. apis 1BB/MR8, (f) B. choladohabitans 2BB/B7. Scale bar: 10 mm.
Microorganisms 13 01159 g002
Microorganisms 13 01159 g003
Figure 3. Phylogenetic tree reconstructed based on the 16S rRNA gene (length: 1236 nt), including bifidobacterial strains from this study and type strains of Bifidobacterium taxa originating exclusively from honey bees. GenBank accession codes for these strains are given in parentheses. Additional GenBank accession numbers are listed in Table 1. Bootstrap values ≥ 70%, based on 1000 replicates, are shown at the nodes. Scale bar: 0.004 substitutions per nucleotide position.
Figure 3. Phylogenetic tree reconstructed based on the 16S rRNA gene (length: 1236 nt), including bifidobacterial strains from this study and type strains of Bifidobacterium taxa originating exclusively from honey bees. GenBank accession codes for these strains are given in parentheses. Additional GenBank accession numbers are listed in Table 1. Bootstrap values ≥ 70%, based on 1000 replicates, are shown at the nodes. Scale bar: 0.004 substitutions per nucleotide position.
Microorganisms 13 01159 g003
Microorganisms 13 01159 g004
Figure 4. Phylogenetic simulation based on the 16S rRNA gene (length: 1330 nt), showing Lactobacillus strains from this study and type strains of Lactobacillus taxa previously isolated exclusively from honey bees. GenBank accession numbers for these strains are given in parentheses; additional accession numbers are listed in Table 1. Bootstrap values ≥ 70%, based on 1000 replicates, are shown at the nodes. Scale bar: 0.007 substitutions per nucleotide position.
Figure 4. Phylogenetic simulation based on the 16S rRNA gene (length: 1330 nt), showing Lactobacillus strains from this study and type strains of Lactobacillus taxa previously isolated exclusively from honey bees. GenBank accession numbers for these strains are given in parentheses; additional accession numbers are listed in Table 1. Bootstrap values ≥ 70%, based on 1000 replicates, are shown at the nodes. Scale bar: 0.007 substitutions per nucleotide position.
Microorganisms 13 01159 g004
Table 1. Inhibition zones (mm) observed in the tested bacterial strains. Standard deviations from triplicate measurements ranged between 2 and 4 mm. A dash (-) indicates no inhibition (negative result). MP—Melissococcus plutonius CZ2020; Ss1—Serratia marcescens strain sicaria. The first letter(s) in the strain designation correspond to the locality of origin: D—Dol (Máslovice), GPS: 50°12′14.327″ N, 14°22′0.412″ E; K—Kralupy nad Vltavou, GPS: 50°14′12.379″ N, 14°18′39.844″ E; P—Postřižín, GPS: 50°14′1.426″ N, 14°23′10.259″ E; V—Větrušice, GPS: 50°11′22.276″ N, 14°22′55.871″ E; VSM—Střezimíř, GPS: 49°31′55.215″ N, 14°36′40.781″ E; VU—Červený Újezd, GPS: 49°33′19.444″ N, 14°36′14.613″ E; BB—Roztoky, GPS: 50°9′24.685″ N, 14°23′24.378″ E; S—Suchdol, GPS: 50°7′48.109″ N, 14°22′25.745″ E.
Table 1. Inhibition zones (mm) observed in the tested bacterial strains. Standard deviations from triplicate measurements ranged between 2 and 4 mm. A dash (-) indicates no inhibition (negative result). MP—Melissococcus plutonius CZ2020; Ss1—Serratia marcescens strain sicaria. The first letter(s) in the strain designation correspond to the locality of origin: D—Dol (Máslovice), GPS: 50°12′14.327″ N, 14°22′0.412″ E; K—Kralupy nad Vltavou, GPS: 50°14′12.379″ N, 14°18′39.844″ E; P—Postřižín, GPS: 50°14′1.426″ N, 14°23′10.259″ E; V—Větrušice, GPS: 50°11′22.276″ N, 14°22′55.871″ E; VSM—Střezimíř, GPS: 49°31′55.215″ N, 14°36′40.781″ E; VU—Červený Újezd, GPS: 49°33′19.444″ N, 14°36′14.613″ E; BB—Roztoky, GPS: 50°9′24.685″ N, 14°23′24.378″ E; S—Suchdol, GPS: 50°7′48.109″ N, 14°22′25.745″ E.
StrainClassificationPL2PL41PL48PL52MPSs1GenBank a.n.
D1/TP2Bifidobacterium apousia-21----PP754609
D1/TP4Bifidobacterium choladohabitans------PP754610
D1/TP5Bifidobacterium sp. (p.n.sp.)------PP754611
D1/TP6Bifidobacterium mellis------PP754612
D1/TP7Bifidobacterium asteroides-203838--PP754613
D1/TP9Bifidobacterium asteroides-1624---PP754614
D1/TP11Bifidobacterium choladohabitans-33----PP754615
D1/TP12Bifidobacterium sp. (p.n.sp.)------PP754616
D1/TP13Bifidobacterium sp. (p.n.sp.)-36----PP754617
D1/MR2Bifidobacterium choladohabitans18162022--PP754618
D1/MR4Bifidobacterium mizhiense------PP754619
D1/MR9.Lactobacillus helsingborgensis---36--PP754620
D1/MR10/B2Bifidobacterium polysaccharolyticum 333442 PP754621
D1/MR10/B4Bifidobacterium choladohabitans19252119--PP754622
D1/MR11Lactobacillus kullabergensis------PP754623
D1/MR12Lactobacillus sp. (p.n.sp.)------PP754624
D1/RO1Lactobacillus helsingborgensis2136-22--PP754625
D1/RO2Lactobacillus helsingborgensis32223234--PP754626
KM1Lactobacillus kimbladii------PP754627
KM10Lactobacillus apis------PP754628
KR1Lactobacillus melliventris--3622--PP754629
KR2Lactobacillus melliventris------PP754630
KR4Lactobacillus melliventris------PP754631
KR5Lactobacillus helsingborgensis2018-2426-PP754632
KR7Lactobacillus melliventris------PP754633
KR8Lactobacillus helsingborgensis--2117--PP754634
KT3Lactobacillus huangpiensis------PP754635
P1/MR1Bifidobacterium asteroides18191818-21PP754636
P1/MR2Lactobacillus melliventris-17-17--PP754637
P1/MR8Lactobacillus apis-241817-18PP754638
P1/MR10Lactobacillus apis-31--17-PP754639
P1/MR11Lactobacillus melliventris------PP754640
P1/MR12Lactobacillus helsingborgensis-443440--PP754641
P1/TP1Bifidobacterium asteroides--21---PP754642
P1/TP2Bifidobacterium sp. (p.n.sp.)------PP754643
P1/TP3Bifidobacterium asteroides171818---PP754644
P1/TP8Bifidobacterium sp. (p.n.sp.)------PP754645
P1/TP9Bifidobacterium apousia---48--PP754646
P1/TP10Bifidobacterium asteroides-46-26--PP754647
P1/TP11Bifidobacterium sp. (p.n.sp.)------PP754648
P1/TP12Bifidobacterium asteroides------PP754649
VM7Lactobacillus apis--18---PP754650
VM9Bifidobacterium polysaccharolyticum19-2520--PP754651
VM10Bifidobacterium polysaccharolyticum20282117--PP754652
VR5Lactobacillus helsingborgensis2222242622-PP754653
VR8Lactobacillus helsingborgensis------PP754654
VR9Lactobacillus melliventris------PP754655
VT5Lactobacillus apis26342624--PP754656
VT6Lactobacillus apis------PP754657
VT9Lactobacillus melliventris------PP754658
VSM/BH8Bifidobacterium asteroides------PP754659
VSM/mBH3Bifidobacterium choladohabitans------PP754660
VSM/mBH4Bifidobacterium mellis---30-20PP754661
VSM/mBH5Bifidobacterium polysaccharolyticum-----24PP754662
VSM/mBH6Bifidobacterium polysaccharolyticum------PP754663
VSM/mBH8Bifidobacterium choladohabitans--2821-21PP754664
VSM/mBH9Bifidobacterium polysaccharolyticum-----23PP754665
VSM/RO2Lactobacillus helsingborgensis------PP754666
VSM/RO4Lactobacillus helsingborgensis483652> 70--PP754667
VSM/RO5Lactobacillus kimbladii------PP754668
VSM/RO6Lactobacillus melliventris18182421--PP754669
VSM/RO7Lactobacillus helsingborgensis--2127-23PP754670
VSM/RO8Lactobacillus helsingborgensis------PP754671
VU/BH3Lactobacillus kimbladii------PP754672
VU/mBH2Bifidobacterium polysaccharolyticum-----21PP754673
VU/mBH4Bifidobacterium sp. (p.n.sp.)-----20PP754674
VU/mBH6Bifidobacterium polysaccharolyticum-----18PP754675
VU/mBH7Bifidobacterium polysaccharolyticum------PP754676
VU/mBH9Bifidobacterium asteroides------PP754677
VU/RO1Lactobacillus melliventris------PP754678
VU/RO3Lactobacillus kimbladii------PP754679
VU/RO6Lactobacillus kullabergensis36263151--PP754680
VU/RO8L. kullabergensis-----18PP754681
VU/RO9L. melliventris--2036--PP754682
1BB/B1Bifidobacterium sp. (p.n.sp.)>703326--25PP754683
1BB/B2Bifidobacterium sp. (p.n.sp.)24-2322-25PP754684
1BB/MF1Bifidobacterium asteroides18-2551-19PP754685
1BB/MF4Bifidobacterium asteroides242418---PP754686
1BB/MR1Bombilactobacillus mellis------PP754687
1BB/MR2Lactobacillus apis--27---PP754688
1BB/MR6Lactobacillus apis--1717-23PP754689
1BB/MR8Lactobacillus apis>703330--22PP754690
1BB/MR10Lactobacillus apis--1616-23PP754691
1BB/RO1Lactobacillus melliventris3927-24--PP754692
1BB/RO3Lactobacillus melliventris>702626---PP754693
1BB/RO7Lactobacillus apis-----18PP754694
1BB/RO8Lactobacillus melliventris------PP754695
1BB/TS1Bifidobacterium sp. (p.n.sp.)---16--PP754696
1BB/TS3Bifidobacterium polysaccharolyticum---20--PP754697
1BB/TS7Bifidobacterium sp. (p.n.sp.)22182919--PP754698
2BB/B1Bifidobacterium sp. (p.n.sp.)2746-16-19PP754699
2BB/B7Bifidobacterium choladohabitans22-2220-25PP754700
2BB/MF2Bifidobacterium polysaccharolyticum--2226--PP754701
2BB/MF3Lactobacillus melliventris---20--PP754702
2BB/MR5Lactobacillus kullabergensis------PP754703
2BB/MR8Lactobacillus apis--2018--PP754704
2BB/RO1Lactobacillus melliventris------PP754705
2BB/RO2Lactobacillus melliventris------PP754706
2BB/RO6Lactobacillus apis--2229--PP754707
2BB/RO7Lactobacillus melliventris--2525--PP754708
SMTP/2Bifidobacterium sp. (p.n.sp.)------PP754709
SMTP/4Bifidobacterium sp. (p.n.sp.)------PP754710
SMTP/6Bifidobacterium asteroides24211718--PP754711
STP/2Lactobacillus helsingborgensis------PP754712
STP/3Lactobacillus apis------PP754713
STP/4Lactobacillus apis------PP754714
STP/5Lactobacillus apis------PP754715
STP/6Lactobacillus melliventris------PP754716
SR1/4Lactobacillus helsingborgensis------PP754717
SR2/2Lactobacillus sp. (p.n.sp.)---16--PP754718
SR2/8Lactobacillus melliventris------PP754719
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Dengiz, B.; Killer, J.; Havlík, J.; Dobeš, P.; Hyršl, P. Selection of Probiotics for Honey Bees: The In Vitro Inhibition of Paenibacillus larvae, Melissococcus plutonius, and Serratia marcescens Strain Sicaria by Host-Specific Lactobacilli and Bifidobacteria. Microorganisms 2025, 13, 1159. https://doi.org/10.3390/microorganisms13051159

AMA Style

Dengiz B, Killer J, Havlík J, Dobeš P, Hyršl P. Selection of Probiotics for Honey Bees: The In Vitro Inhibition of Paenibacillus larvae, Melissococcus plutonius, and Serratia marcescens Strain Sicaria by Host-Specific Lactobacilli and Bifidobacteria. Microorganisms. 2025; 13(5):1159. https://doi.org/10.3390/microorganisms13051159

Chicago/Turabian Style

Dengiz, Buse, Jiří Killer, Jaroslav Havlík, Pavel Dobeš, and Pavel Hyršl. 2025. "Selection of Probiotics for Honey Bees: The In Vitro Inhibition of Paenibacillus larvae, Melissococcus plutonius, and Serratia marcescens Strain Sicaria by Host-Specific Lactobacilli and Bifidobacteria" Microorganisms 13, no. 5: 1159. https://doi.org/10.3390/microorganisms13051159

APA Style

Dengiz, B., Killer, J., Havlík, J., Dobeš, P., & Hyršl, P. (2025). Selection of Probiotics for Honey Bees: The In Vitro Inhibition of Paenibacillus larvae, Melissococcus plutonius, and Serratia marcescens Strain Sicaria by Host-Specific Lactobacilli and Bifidobacteria. Microorganisms, 13(5), 1159. https://doi.org/10.3390/microorganisms13051159

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop