Inhibition of Paenibacillus larvae and Ascosphaera apis by Bacillus subtilis isolated from honeybee gut and honey samples (original) (raw)
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Revista Argentina De Microbiologia, 2004
The fungus Ascosphaera apis, the causative agent of chalkbrood disease in honeybee larvae, occurs throughout the world and is found in many beekeeping areas of Argentina. The potential as biocontrol agents of 249 aerobic spore-forming bacterial antagonists isolated from honey samples was evaluated. Each isolate was screened against A. apis by a central disk test assay. Ten bacterial strains that showed the best antagonistic effect to A. apis were selected for further study and identified as Bacillus cereus (m363, mv86, mv81, mv75), Bacillus circulans (Fr231, m448b), Bacillus megaterium (m435), Bacillus pumilus (m354), Bacillus subtilis (m329), and Paenibacillus alvei (m321). For testing the efficiency of the selected strains, a paired culture test was used with 5 replicates of each combination bacterial antagonist / A. apis strain, and 5 replications for each control on 4 different culture media. The analysis of variance and posterior comparison of means according to LSD method showed that the best antagonists when using YGPSA medium were B. subtilis (m329) and B. megaterium (m435), and in the case of MYPGP medium the most efficient were B. circulans strains Fr 231 and m448b.
Revista Argentina de microbiología
The fungus Ascosphaera apis, the causative agent of chalkbrood disease in honeybee larvae, occurs throughout the world and is found in many beekeeping areas of Argentina. The potential as biocontrol agents of 249 aerobic spore-forming bacterial antagonists isolated from honey samples was evaluated. Each isolate was screened against A. apis by a central disk test assay. Ten bacterial strains that showed the best antagonistic effect to A. apis were selected for further study and identified as Bacillus cereus (m363, mv86, mv81, mv75), Bacillus circulans (Fr231, m448b), Bacillus megaterium (m435), Bacillus pumilus (m354), Bacillus subtilis (m329), and Paenibacillus alvei (m321). For testing the efficiency of the selected strains, a paired culture test was used with 5 replicates of each combination bacterial antagonist/A. apis strain, and 5 replications for each control on 4 different culture media. The analysis of variance and posterior comparison of means according to LSD method showed...
Microbiological Research, 2019
The inhibitory action that a Brevibacillus laterosporus strain isolated from the honeybee body causes against the American Foulbrood (AFB) etiological agent Paenibacillus larvae was studied by in-vitro experiments. A protein fraction isolated from B. laterosporus culture supernatant was involved in the observed inhibition of P. larvae vegetative growth and spore germination. As a result of LC-MS/MS proteomic analyses, the bacteriocin laterosporulin was found to be the major component of this fraction, followed by other antimicrobial proteins and substances including lectins, chaperonins, various enzymes and a number of putative uncharacterized proteins. The results obtained in this study highlight the potential of B. laterosporus as a biological control agent for preserving and improving honeybee health.
Journal of Invertebrate Pathology, 2006
The bacterium Paenibacillus larvae, the causative agent of American foulbrood disease of honeybee larvae, occurs throughout the world and is found in many beekeeping areas of Argentina. The potential as biocontrol agents of antagonic aerobic spore-forming bacteria isolated from honey samples and other apiarian sources were evaluated. Each isolate was screened against one strain of Paenibacillus larvae (ATCC 9545) by using a perpendicular streak technique. Ten randomly selected bacterial strains from the group that showed the best antagonistic eVect to P. larvae ATCC 9545 were selected for further study. These were identiWed as Bacillus subtilis , B. pumilus (m350), B. licheniformis (m347), B. cereus (mv33), B. cereus (m387), B. cereus (m6c), B. megaterium (m404), Brevibacillus laterosporus (BLAT169), B. laterosporus (BLAT170), and B. laterosporus (BLAT171). The antagonistic strains were tested against 17 P. larvae strains from diVerent geographical origins by means of a spot test in wells. The analysis of variance and posterior comparison of means by Tukey method (P < 0.01) showed that the best antagonists were B. megaterium (m404), B. licheniformis (m347), B. cereus (m6c), B. cereus (mv33), and B. cereus (m387).
PLoS ONE, 2014
The Gram-positive bacterium Paenibacillus larvae is the etiological agent of American Foulbrood. This bacterial infection of honey bee brood is a notifiable epizootic posing a serious threat to global honey bee health because not only individual larvae but also entire colonies succumb to the disease. In the recent past considerable progress has been made in elucidating molecular aspects of host pathogen interactions during pathogenesis of P. larvae infections. Especially the sequencing and annotation of the complete genome of P. larvae was a major step forward and revealed the existence of several giant gene clusters coding for non-ribosomal peptide synthetases which might act as putative virulence factors. We here present the detailed analysis of one of these clusters which we demonstrated to be responsible for the biosynthesis of bacillibactin, a P. larvae siderophore. We first established culture conditions allowing the growth of P. larvae under ironlimited conditions and triggering siderophore production by P. larvae. Using a gene disruption strategy we linked siderophore production to the expression of an uninterrupted bacillibactin gene cluster. In silico analysis predicted the structure of a trimeric trithreonyl lactone (DHB-Gly-Thr) 3 similar to the structure of bacillibactin produced by several Bacillus species. Mass spectrometric analysis unambiguously confirmed that the siderophore produced by P. larvae is identical to bacillibactin. Exposure bioassays demonstrated that P. larvae bacillibactin is not required for full virulence of P. larvae in laboratory exposure bioassays. This observation is consistent with results obtained for bacillibactin in other pathogenic bacteria.
Biocontrol Science and Technology, 2020
An in vitro study was conducted to screen for the inhibitory potential of lactic acid bacteria (LAB), isolated from fermented food matrices, against the honeybee American Foulbrood agent Paenibacillus larvae. Various strains of Lactobacillus plantarum showed significant antagonism associated with their cell-free culture supernatant (CFS). The characterisation of this inhibition was based on assays and analyses involving different fraction and extracts. A significant effect was associated with media acidification resulting from LAB growth. However, this antimicrobial activity was found to be heat-stable and specific experiments suggested the implication of small antimicrobial peptides and/or antibiotics. Accordingly, a possible role of plantaricins released by L. plantarum in the culture supernatant was confirmed by the expression of different plantaricin-related genes. The results obtained in this study support further investigations on LAB strains as biological control agents for application in the apiary to preserve and improve honeybee health.
antagonistic potential of gut bacteria, 2020
Paenibacillus larvae is one of the major bacterial pathogens of honey bee broods and the causative agent of American foul-brood (AFB) disease. The factors responsible for the pathogenesis of AFB disease are still not fully understood, and the increasing resistance of P. larvae to commonly used antibiotics requires a search for new agents to control this disease. An in vitro screen was carried out to determine the antagonistic activity of gut bacteria isolated from indigenous honey bees, Apis mellifera jemenitica of Saudi Arabia against P. larvae. The gut bacterial isolates were evaluated individually against P. larvae ATCC9545 strain by the disc diffusion method. Seven of the 100 evaluated gut bacterial isolates, Fructobacillus fructosus (KY027123); Proteus mirabilis (KY027132); Bacillus licheniformis (KY027142); Lactobacillus kunkeei (KY027158); Bacillus subtilis (KY027169); Enterobacter kobei (KY027178); and Morganella morganii (KY027186) showed strong inhibitory effects. To our knowledge, this the first demonstration of antagonistic activity of F. fructosus, P. mirabilis, E. kobei, and M. morganii isolated from honey bee gut against P. larvae. The tested gut isolates exhibited significant antimicrobial activities against P. larvae, and they could play an important role in the treatment or prevention of AFB disease.
Novel lactic acid bacteria inhibiting Paenibacillus larvae in honey bee larvae
Apidologie, 2009
We evaluated the antagonistic effects of newly identified lactic acid bacteria (LAB) in the genera Lactobacillus and Bifidobacterium, originating from the honey stomach, on the honey bee pathogen, Paenibacillus larvae. We used inhibition assays on agar plates and honey bee larval bioassays to investigate the effects of honey bee LAB on P. larvae growth in vitro and on AFB infection in vivo. The individual LAB phylotypes showed different inhibition properties against P. larvae growth on agar plates, whereas a combination of all eleven LAB phylotypes resulted in a total inhibition (no visible growth) of P. larvae. Adding the LAB mixture to the larval food significantly reduced the number of AFB infected larvae in exposure bioassays. The results demonstrate that honey bee specific LAB possess beneficial properties for honey bee health. Possible benefits to honey bee health by enhancing growth of LAB or by applying LAB to honey bee colonies should be further investigated.
Journal of Apicultural Research, 2005
While honey bees (Apis mellifera) support a diverse microbial community, the impacts of most of their associated microbes on honey bee health remain unresolved. Here, pairwise inhibition assays were used to identify honey bee bacterial symbionts that inhibit a primary pathogen, the Gram-positive bacterium Paenibacillus larvae larvae. Four bacterial taxa isolated from bee larvae appeared especially promising with respect to inhibition of P. l. larvae and, in fact, completely inhibited P. l. larvae growth in pairwise plate assays. These isolates were identified by 16S ribosomal RNA sequencing as Stenotrophomonas maltophilia, Acinetobacter sp., Brevibacillus formosus and Bacillus fusiformis. A PCR-based survey confirmed that these bacterial isolates are present in bee larvae, at frequencies ranging from 2% (1/48) for B. formosus to 79% (39/48) for Acinetobacter sp. An understanding of the distributions of these co-occurring bacteria could elucidate variation across colonies in susceptibility to American foulbrood disease. In addition, supplementation of colonies with these naturally occurring bacteria or their antagonistic products can provide a novel way of controlling foulbrood disease.
Antibiotics, 2020
Paenibacillus larvae is the causative agent of American foulbrood (AFB), a severe bacterial disease that affects larvae of honeybees. The present study evaluated, in vitro, antimicrobial activity of sixty-one Lactiplantibacillus plantarum strains, against P. larvae ATCC 9545. Five strains (P8, P25, P86, P95 and P100) that showed the greatest antagonism against P. larvae ATCC 9545 were selected for further physiological and biochemical characterizations. In particular, the hydrophobicity, auto-aggregation, exopolysaccharides production, osmotic tolerance, enzymatic activity and carbohydrate assimilation patterns were evaluated. The five L. plantarum selected strains showed suitable physical and biochemical properties for their use as probiotics in the honeybee diet. The selection and availability of new selected bacteria with good functional characteristics and with antagonistic activity against P. larvae opens up interesting perspectives for new biocontrol strategies of diseases suc...