The gram-positive bacterium
Paenibacillus larvae
is the etiological agent of American Foulbrood of honey bees, a notifiable disease in many countries. Hence,
P. larvae
can be considered as an ...entomopathogen of considerable relevance in veterinary medicine.
P. larvae
is a highly specialized pathogen with only one established host, the honey bee larva. No other natural environment supporting germination and proliferation of
P. larvae
is known. Over the last decade, tremendous progress in the understanding of
P. larvae
and its interactions with honey bee larvae at a molecular level has been made. In this review, we will present the recent highlights and developments in
P. larvae
research and discuss the impact of some of the findings in a broader context to demonstrate what we can learn from studying “exotic” pathogens.
(
) is a bacterial pathogen causing American foulbrood (AFB), the most serious disease of honeybee larvae. The food of young larvae could play an important role in the resistance of larvae against ...AFB. It contains antibacterial substances produced by honeybees that may inhibit the propagation of the pathogen in larval midguts. In this study, we identified and investigated the antibacterial effects of one of these substances,
-10-hydroxy-2-decenoic acid (10-HDA), against
strains including all Enterobacterial Repetitive Intergenic Consensus (ERIC) genotypes. Its inhibitory activities were studied by determining the minimum inhibitory concentrations (MICs). It was found that 10-HDA efficacy increases substantially with decreasing pH; up to 12-fold differences in efficacy were observed between pH = 5.5 and pH = 7.2.
strains showed different susceptibility to 10-HDA; up to 2.97-fold differences existed among various strains with environmentally important ERIC I and ERIC II genotypes. Germinating spores of the pathogen were generally more susceptible to 10-HDA than vegetative cells. Our findings suggest that 10-HDA could play significant role in conferring antipathogenic activity to larval food in the midguts of young larvae and contribute to the resistance of individual larvae to
.
American foulbrood (AFB), caused by the highly virulent, spore-forming bacterium
, poses a significant threat to honey bee brood. The widespread use of antibiotics not only fails to effectively ...combat the disease but also raises concerns regarding honey safety. The current computational study was attempted to identify a novel therapeutic drug target against
, a causative agent of American foulbrood disease in honey bee.
We investigated effective novel drug targets through a comprehensive
pan-proteome and hierarchal subtractive sequence analysis. In total, 14 strains of
genomes were used to identify core genes. Subsequently, the core proteome was systematically narrowed down to a single protein predicted as the potential drug target. Alphafold software was then employed to predict the 3D structure of the potential drug target. Structural docking was carried out between a library of phytochemicals derived from traditional Chinese flora (
> 36,000) and the potential receptor using Autodock tool 1.5.6. Finally, molecular dynamics (MD) simulation study was conducted using GROMACS to assess the stability of the best-docked ligand.
Proteome mining led to the identification of Ketoacyl-ACP synthase III as a highly promising therapeutic target, making it a prime candidate for inhibitor screening. The subsequent virtual screening and MD simulation analyses further affirmed the selection of ZINC95910054 as a potent inhibitor, with the lowest binding energy. This finding presents significant promise in the battle against
Computer aided drug design provides a novel approach for managing American foulbrood in honey bee populations, potentially mitigating its detrimental effects on both bee colonies and the honey industry.
American Foulbrood is a worldwide distributed, fatal disease of the brood of the Western honey bee (Apis mellifera). The causative agent of this fatal brood disease is the Gram-positive, ...spore-forming bacterium Paenibacillus larvae, which can be classified into four different genotypes (ERIC I-IV), with ERIC I and II being the ones isolated from contemporary AFB outbreaks. P. larvae is a peritrichously flagellated bacterium and, hence, we hypothesized that P. larvae is capable of coordinated and cooperative multicellular behaviors like swarming motility and biofilm formation. In order to analyze these behaviors of P. larvae, we firstly established appropriate functional assays. Using these assays we demonstrated that P. larvae ERIC II, but not P. larvae ERIC I, was capable of swarming. Swarming motility was hampered in a P. larvae ERIC II-mutant lacking production of paenilarvin, an iturin-like lipopeptide exclusively expressed by this genotype. Both genotypes were able to form free floating biofilm aggregates loosely attached to the walls of the culture wells. Visualizing the biofilms by Congo red and thioflavin S staining suggested structural differences between the biofilms formed. Biofilm formation was shown to be independent from paenilarvin production because the paenilarvin deficient mutant was comparably able to form a biofilm.
American foulbrood (AFB) is a highly virulent disease afflicting honey bees (Apis mellifera). The causative organism, Paenibacillus larvae, attacks honey bee brood and renders entire hives ...dysfunctional during active disease states, but more commonly resides in hives asymptomatically as inactive spores that elude even vigilant beekeepers. The mechanism of this pathogenic transition is not fully understood, and no cure exists for AFB. Here, we evaluated how hive supplementation with probiotic lactobacilli (delivered through a nutrient patty; BioPatty) affected colony resistance towards a naturally occurring AFB outbreak. Results demonstrated a significantly lower pathogen load and proteolytic activity of honey bee larvae from BioPatty-treated hives. Interestingly, a distinctive shift in the microbiota composition of adult nurse bees occurred irrespective of treatment group during the monitoring period, but only vehicle-supplemented nurse bees exhibited higher P. larvae loads. In vitro experiments utilizing laboratory-reared honey bee larvae showed Lactobacillus plantarum Lp39, Lactobacillus rhamnosus GR-1, and Lactobacillus kunkeei BR-1 (contained in the BioPatty) could reduce pathogen load, upregulate expression of key immune genes, and improve survival during P. larvae infection. These findings suggest the usage of a lactobacilli-containing hive supplement, which is practical and affordable for beekeepers, may be effective for reducing enzootic pathogen-related hive losses.
, the causative agent of American foulbrood (AFB), is the primary bacterial pathogen affecting honeybees and beekeeping. The main methods for controlling AFB are incineration of diseased colonies or ...prophylactic antibiotic treatment (e.g., with tylosin), neither of which is fully satisfactory. The search for superior means for controlling AFB has led to an increased interest in the natural relationships between the honeybee-pathogenic and mutualistic microorganisms and, in particular, the antagonistic effects of honeybee-specific lactic acid bacteria (hbs-LAB) against
These effects have been demonstrated only on individual larvae in controlled laboratory bioassays. Here we investigated whether supplemental administration of hbs-LAB had a similar beneficial effect on
infection at colony level. We compared experimentally AFB-infected colonies treated with hbs-LAB supplements to untreated and tylosin-treated colonies and recorded AFB symptoms, bacterial spore levels, and two measures of colony health. To account for the complexity of a bee colony, we focused on (Bayesian) probabilities and magnitudes of effect sizes. Tylosin reduced AFB disease symptoms but also had a negative effect on colony strength. The tylosin treatment did not, however, affect
spore levels and might therefore "mask" the potential for disease. hbs-LAB tended to reduce brood size in the short term but was unlikely to affect AFB symptoms or spores. These results do not contradict demonstrated antagonistic effects of hbs-LAB against
at the individual bee level but rather suggest that supplementary administration of hbs-LAB may not be the most effective way to harness these beneficial effects at the colony level.
The previously demonstrated antagonistic effects of honeybee-derived bacterial microbiota on the infectivity and pathogenicity of
in laboratory bioassays have identified a possible new approach to AFB control. However, honeybee colonies are complex superorganisms where social immune defenses play a major role in resistance against disease at the colony level. Few studies have investigated the effect of beneficial microorganisms on bee diseases at the colony level. Effects observed at the individual bee level do not necessarily translate into similar effects at the colony level. This study partially fills this gap by showing that, unlike at the individual level, hbs-LAB supplements did not affect AFB symptoms at the colony level. The inference is that the mechanisms regulating the honeybee microbial dynamics within a colony are too strong to manipulate positively through supplemental feeding of live hbs-LAB and that new potential remedies identified through laboratory research have to be tested thoroughly
, in colonies.
and
are two bacteria that are members of the
family. Both are commonly found in beehives and have historically been difficult to distinguish from each other due to related genetic and phenotypic ...characteristics and a shared ecological niche. Here, we discuss the likely mischaracterization of three 16S rRNA sequences previously published as
and provide the phylogenetic evidence that supported the GenBank reassignment of the sequences as
We explore the issues that arise by using only 16S rRNA or other single-gene analyses to distinguish between these bacteria. We also present three sets of molecular markers, two sets that distinguish
from
and other closely related species within the
genus and a third set that distinguishes
from
and other closely related species within the
genus. These molecular markers provide a tool for proper identification of these oft-mistaken species.
16S rRNA gene sequencing in bacteria has long been held as the gold standard for typing bacteria and, for the most part, is an excellent method of taxonomically identifying different bacterial species. However, the high level of 16S rRNA sequence similarity of some published strains of
and
, as well as possible horizontal gene transfer events within their shared ecological niche, complicates the use of 16S rRNA sequence as an effective molecular marker for differentiating these two species. Additionally, shared characteristics of these bacteria limit the effectiveness of using traditional phenotypic identification assays, such as the catalase test. The results from this study provide PCR methods to quickly differentiate between these two genera and will be useful when studying
,
, and other disease-relevant bacteria commonly found in beehives.
The American Foulbrood Disease (AFB) is a fatal larval bee infection. The etiologic agent is the bacterium Paenibacillus larvae. The treatment involves incineration of all contaminated materials, ...leading to high losses. The Glycerol Monolaurate (GML) is a known antimicrobial potential compound, however its use is reduced due to its low solubility in water and high melting point. The nanoencapsulation of some drugs offers several advantages like improved stability and solubility in water. The present study aimed to evaluate the antimicrobial activity against P. larvae and the toxicity in bees of GML nanoparticles. The nanocapsules were produced and presented mean diameter of 210 nm, polydispersity index of 0.044, and zeta potential of −23.4 mV demonstrating the acceptable values to predict a stable system. The microdilution assay showed that it is necessary 142 and 285 μg/mL of GML nanocapsules to obtain a bacteriostatic and bactericidal effect respectively. The time-kill curve showed the controlled release of compound, exterminating the microorganism after 24 h. The GML nanocapsules were able to kill the spore form of Paenibacillus larvae while the GML do not cause any effect. The assay in bees showed that the GML has a high toxicity while the GML nanoparticles showed a decrease on toxic effects. Concluding, the formulation shows positive results in the action to combat AFB besides not causing damage to bees.
•The nanoparticle was effective against the Paenibacillus larvae.•The nanocapsules was able to kill Paenibacillus larvae spores.•The nanoparticle was not toxic to the bees.•GML Nanoparticle was able to combat infection agent without adverse effects.
American Foulbrood (AFB) is a deadly bacterial disease affecting pupal and larval honey bees. AFB is caused by the endospore-forming bacterium
(PL). Propolis, which contains a variety of organic ...compounds, is a product of bee foraging and is a resinous substance derived from botanical substances found primarily in trees. Several compounds from the class of caffeic acid esters, which are commonly found in propolis, have been shown to have antibacterial activity against PL. In this study, six different caffeic acid esters were synthesized, purified, spectroscopically analyzed, and tested for their activity against PL to determine the minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs). Caffeic acid isopropenyl ester (CAIE), caffeic acid benzyl ester (CABE), and caffeic acid phenethyl ester (CAPE) were the most effective in inhibiting PL growth and killing PL cell with MICs and MBCs of 125 µg/mL when used individually, and a MIC and MBC of 31.25 µg/mL for each compound alone when CAIE, CABE, and CAPE are used in combination against PL. These compounds inhibited bacterial growth through a bactericidal effect, which revealed cell killing but no lysis of PL cells after 18 h. Incubation with CAIE, CABE, and CAPE at their MICs significantly increased reactive oxygen species levels and significantly changed glutathione levels within PL cells. Caffeic acid esters are potent bactericidal compounds against PL and eliminate bacterial growth through an oxidative stress mechanism.
Aims
To explore alternative nonchemical control measures against two honeybee pathogens, Paenibacillus larvae and Ascosphaera apis, 37 plant species were screened for antimicrobial activity.
Methods ...and Results
The activity of selected plant extracts was screened using an in vitro disc diffusion assay and the minimal inhibitory concentration (MIC) was determined by the broth microdilution method. The results showed that 36 plant extracts had some antibacterial activity on P. larvae by disc diffusion assay. Chromolaena odorata showed the greatest antibacterial activity against P. larvae (MIC 16–64 μg ml−1). Of the 37 tested plants, only seven species, Amomum krervanh, Allium sativum, Cinnamomum sp., Piper betle, Piper ribesioides, Piper sarmentosum and Syzygium aromaticum had inhibitory effects on A. apis (MICs of 32–64 μg ml−1). The results demonstrated that promising plant extracts were not toxic to adult bees at the concentrations used in this study.
Conclusions
The results demonstrate the potential antimicrobial activity of natural products against honeybee diseases caused by P. larvae and A. apis. Chromolaena odorata in particular showed high bioactivity against P. larvae. Further study is recommended to develop these nonchemical treatments against American foulbrood and chalkbrood in honeybees.
Significance and Impact of the Study
This work proposes new natural products for the control of American foulbrood and chalkbrood in honeybees.
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