RESUMEN
Achyrocline satureioides extracts were tested in vitro against the growth of Paenibacillus larvae. Four different extracts were obtained by liquid-liquid extraction from an aqueous-ethyl alcohol macerate of the aerial parts of the plant. The biological activity was tested by the broth microdilution technique. Hexane extract showed the highest activity (minimum inhibitory concentration = 0.060 ± 0.037 mg/mL). Transmission electron microscopy experiments showed that the main effect exerted by the hexane extract on the cell was at the cellular membrane level. The hexane extract was analyzed by thin-layer chromatography, and the activity of its components was tested by bioautography. Four growth inhibition zones were observed in the bioautographic experiments (using hexane-acetone (7:3) as mobile phase), with that at Rf = 0.57 showing the largest zone of inhibition. High-performance liquid chromatographic experiments, using ultraviolet and electrospray ionization coupled to tandem mass spectrometric detection, showed the presence of one compound with a m/z ratio of 442, which may be related to phloroglucinols α-pyrone compounds recently discovered. The high antibacterial activity of the hexane extract and of the isolated compound determined in this work may be useful for the development of future new alternatives for the treatment of American foulbrood.
Asunto(s)
Paenibacillus/efectos de los fármacos , Extractos Vegetales/farmacología , Pironas/farmacología , Achyrocline/química , Animales , Antibacterianos/aislamiento & purificación , Antibacterianos/farmacología , Abejas/microbiología , Larva/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Paenibacillus/patogenicidad , Extractos Vegetales/química , Pironas/aislamiento & purificaciónRESUMEN
The honeybee disease American foulbrood (AFB) is a serious problem since its causative agent (Paenibacillus larvae) has become increasingly resistant to conventional antibiotics. One of the feasible alternative treatments being used for control of this disease are plants extracts. The aim of the present work was to evaluate the effect of crude extract and fractions of Scutia buxifolia against six Paenibacillus species, including P. larvae, and its potential use for the control of AFB. In vitro activity of S. buxifolia samples against Paenibacillus species were evaluated by the disk diffusion and microdilution methods, and the minimal inhibitory concentration (MIC) were also determined. All Paenibacillus species were sensitive to crude extract and fractions of S. buxifolia. The dichloromethane (DC) fraction showed the better MIC (1.56 mg/mL), followed by ethyl acetate (EtAc) (6.25 mg/mL), n-butanol (BuOH) (25 mg/mL) and Crude extract (CE) (50 mg/mL). Toxic effect of S. buxifolia crude extracts and fractions against bees were also evaluated by the spraying application method of the same concentrations of MICs. The samples tested showed no toxic effects for the bees after 15 days of observation. These results are first time described for this species and showed that S. buxifolia presented a important activity against Paenibacillus species and proved to be a natural alternative for the prevention/control of AFB.
Asunto(s)
Antiinfecciosos/farmacología , Abejas/microbiología , Infecciones por Bacterias Grampositivas/veterinaria , Viabilidad Microbiana/efectos de los fármacos , Paenibacillus/efectos de los fármacos , Extractos Vegetales/farmacología , Rhamnaceae , Animales , Antiinfecciosos/química , Antiinfecciosos/toxicidad , Abejas/efectos de los fármacos , Fraccionamiento Químico , Infecciones por Bacterias Grampositivas/tratamiento farmacológico , Estimación de Kaplan-Meier , Larva/efectos de los fármacos , Larva/microbiología , Longevidad/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Paenibacillus/patogenicidad , Extractos Vegetales/química , Extractos Vegetales/toxicidadRESUMEN
American foulbrood is a bacterial disease of worldwide distribution that affects larvae of the honeybee Apis mellifera. The causative agent is the Gram-positive, spore-forming bacterium Paenibacillus larvae. Several authors have proposed that P. larvae secretes metalloproteases that are involved in the larval degradation that occurs after infection. The aim of the present work was to evaluate the production of a metalloprotease by P. larvae during larval infection. First, the complete gene encoding a metalloprotease was identified in the P. larvae genome and its distribution was evaluated by PCR in a collection of P. larvae isolates from different geographical regions. Then, the complete gene was amplified, cloned and overexpressed, and the recombinant metalloprotease was purified and used to generate anti-metalloprotease antibodies. Metalloprotease production was evaluated by immunofluorescence and fluorescence in situ hybridization. The gene encoding a P. larvae metalloprotease was widely distributed in isolates from different geographical origins in Uruguay and Argentina. Metalloprotease was detected inside P. larvae vegetative cells, on the surface of P. larvae spores and secreted to the external growth medium. Its production was also confirmed in vivo, during the infection of honeybee larvae. This protein was able to hydrolyse milk proteins as described for P. larvae, suggesting that could be involved in larval degradation. This work contributes to the knowledge of the pathogenicity mechanisms of a bacterium of great economic significance and is one step in the characterization of potential P. larvae virulence factors.
Asunto(s)
Proteínas Bacterianas/metabolismo , Abejas/microbiología , Metaloproteasas/metabolismo , Paenibacillus/enzimología , Paenibacillus/fisiología , Animales , Proteínas Bacterianas/genética , Abejas/crecimiento & desarrollo , Abejas/metabolismo , Larva/metabolismo , Larva/microbiología , Metaloproteasas/genética , Paenibacillus/genética , Paenibacillus/patogenicidad , VirulenciaRESUMEN
American Foulbrood is a severe disease affecting larvae of honeybee Apis mellifera, causing significant decrease in the honeybee population, beekeeping industries and agricultural production. In spite of its importance, little is known about the virulence factors secreted by Paenibacillus larvae during larval infection. The aim of the present work was to perform a first approach to the identification and characterization of P. larvae secretome. P. larvae secreted proteins were analyzed by SDS-PAGE and identified by MALDI-TOF. Protein toxicity was evaluated using an experimental model based on feeding of A. mellifera larvae and immunogenicity was evaluated by Western blot, using an antiserum raised against cells and spores of P. larvae. Ten different proteins were identified among P. larvae secreted proteins, including proteins involved in transcription, metabolism, translation, cell envelope, transport, protein folding, degradation of polysaccharides and motility. Although most of these proteins are cytosolic, many of them have been previously detected in the extracellular medium of different Bacillus spp. cultures and have been related to virulence. The secreted proteins resulted highly toxic and immunogenic when larvae were exposed using an experimental model. This is the first description of proteins secreted by the honeybee pathogen P. larvae. This information may be relevant for the elucidation of bacterial pathogenesis mechanisms.
Asunto(s)
Proteínas Bacterianas/metabolismo , Abejas , Paenibacillus/patogenicidad , Animales , Proteínas Bacterianas/toxicidad , Abejas/efectos de los fármacos , Abejas/microbiología , Femenino , Larva/efectos de los fármacos , Ratones , Paenibacillus/enzimología , Paenibacillus/metabolismo , Péptido Hidrolasas/metabolismo , Factores de Virulencia/metabolismoRESUMEN
Paenibacillus larvae is the causative agent of American Foulbrood (AFB), the most severe bacterial disease that affects honeybee larvae. AFB causes a significant decrease in the honeybee population affecting the beekeeping industry and agricultural production. After infection of larvae, P. larvae secretes proteases that could be involved in the pathogenicity. In the present article, we present the secretion of different proteases by P. larvae. Inhibition assays confirmed the presence of metalloproteases. Two different proteases patterns (PP1 and PP2) were identified in a collection of P. larvae isolates from different geographic origin. Forty nine percent of P. larvae isolates showed pattern PP1 while 51% exhibited pattern PP2. Most isolates belonging to genotype ERIC I - BOX A presented PP2, most isolates belonging to ERIC I - BOX C presented PP1 although relations were not significant. Isolates belonging to genotypes ERIC II and ERIC III presented PP2. No correlation was observed between the secreted proteases patterns and geographic distribution, since both patterns are widely distributed in Uruguay. According to exposure bioassays, isolates showing PP2 are more virulent than those showing PP1, suggesting that difference in pathogenicity could be related to the secretion of proteases.