Arch Microbiol (2012) 194:177–185 DOI 10.1007/s00203-011-0743-4
ORIGINAL PAPER
Antimicrobial factor from Bacillus amyloliquefaciens inhibits Paenibacillus larvae, the causative agent of American foulbrood Lisianne Brittes Benitez • Renata Voltolini Velho Amanda de Souza da Motta • Je´ferson Segalin • Adriano Brandelli
•
Received: 19 January 2011 / Revised: 8 July 2011 / Accepted: 2 August 2011 / Published online: 20 August 2011 Ó Springer-Verlag 2011
Abstract Bacillus amyloliquefaciens LBM 5006 produces an antimicrobial factor active against Paenibacillus larvae, a major honeybee pathogen. The antagonistic effect and the mode of action of the antimicrobial factor were investigated. The antibacterial activity was produced starting at mid-logarithmic growth phase, reaching its maximum during the stationary phase. Exposure of cell suspensions of P. larvae to this antimicrobial resulted in loss of cell viability and reduction in optical density associated with cell lysis. Scanning electron microscopy showed damaged cell envelope and loss of protoplasmic material. The antimicrobial factor was stable for up to 80°C, but it was sensitive to proteinase K and trypsin. Mass spectrometry analysis indicates that the antimicrobial activity is associated with iturin-like peptides. The antimicrobial factor from B. amyloliquefaciens LBM 5006 showed a bactericidal effect against P. larvae cells and spores. This is the first report on iturin activity against P. larvae. This antimicrobial presents potential for use in the control of American foulbrood disease. Keywords Antimicrobial peptide Bacillus American foulbrood disease Iturin Communicated by Erko Stackebrandt. L. B. Benitez A. de Souza da Motta A. Brandelli (&) Laborato´rio de Bioquı´mica e Microbiologia Aplicada, Instituto de Cieˆncia e Tecnologia de Alimentos, Universidade Federal do Rio Grande do Sul, Av. Bento Gonc¸alves 9500, Porto Alegre 91501-970, Brazil e-mail:
[email protected] R. V. Velho J. Segalin Unidade Quı´mica de Proteı´nas e Espectrometria de Massas, Centro de Biotecnologia, UFRGS, Av. Bento Gonc¸alves 9500, Porto Alegre, RS 91501-970, Brazil
Introduction Antimicrobial peptides are widespread produced among bacteria of the genus Bacillus. In particular, different Bacillus amyloliquefaciens strains are producers of antimicrobial substances with potential applications as biocontrol agents to suppress plant pathogens and biological control of spoilage and pathogenic microorganisms in food (Yu et al. 2002; Lee et al. 2007; Caldeira et al. 2008; Huang et al. 2009). Paenibacillus larvae are the causative agent of American foulbrood (AFB), the most severe bacterial disease that affects larvae of the honeybee Apis mellifera. AFB presents a worldwide distribution, causing a significant decrease in honeybee populations and production of honey, pollen, propolis, royal jelly, and beeswax (Genersch et al. 2006). Besides their importance for the beekeeping industry, honey bees play an essential role in the ecology of different environments throughout pollination, being essential for the production of agricultural systems and conservation of natural ecosystems (Antune´z et al. 2009). AFB is a serious bacterial disease of honey bee brood, not only able to kill infected individuals but also potentially lethal to infected colonies (Ashiralieva and Genersch 2006). AFB has unique problems for prevention and control because the spores can remain viable for long periods of time (De Graaf et al. 2006; Hrabak and Martinek 2007) and survive to environmental adversities (Genersch 2010). A control method is burning the diseased colonies (Matheson and Reid 1992; Ratnieks 1992) and is in use in many countries. In Argentina, where disease incidence is high (Alippi et al. 2004), the use of antibiotics appears as an alternative to the burning of infected beehives. Currently, the only antibiotic approved for prevention and control of AFB in honey bee colonies is oxytetracycline;
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however, there is evidence of oxytetracycline-resistant isolates of P. larvae in certain areas of the USA, Canada, and Argentina (Alippi 2000; Evans 2003). The widespread use of antibiotics favors the natural selection of resistant bacterial strains, diminishes the half-life expectation of honey bees, and causes unbalance in the normal microbiota of the beehive with the risks of contamination of honey (Charbonneau et al. 1992). For these reasons, the search for alternative non-contaminating natural biocides for the control of AFB is a great challenge that will improve the honey quality, avoiding the presence of undesirable residues (Gonza´lez and Marioli 2010). B. amyloliquefaciens LBM 5006 produces antimicrobial peptides that show broad antimicrobial spectrum and the antagonistic effect of this strain against phytopathogenic fungi was also recently demonstrated (Benitez et al. 2010). The aim of this work was to investigate the effect and the mode of action of the antimicrobial factor of strain LBM 5006 on P. larvae.
Materials and methods
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cells of P. larvae 1655. Plates were incubated at 37°C for 24 h. The amount of antibacterial activity was determined by the serial two-fold dilution method (Motta and Brandelli 2002). Activity was defined as the reciprocal value of the highest dilution where an inhibition zone was observed and expressed as activity units per milliliter (AU ml-1). Isolation of antimicrobial factor The antimicrobial factor was further purified from the cellfree supernatant by precipitation with ammonium sulfate at 20% (w/v) saturation. After centrifugation at 10,0009g for 15 min, the pellet was suspended in 2 ml of 10 mmol l-1 sodium phosphate buffer pH 6.0 and submitted to gel filtration chromatography using a Sephadex G-100 column (Pharmacia Biotech, Uppsala), eluted with the same buffer. Fractions showing antimicrobial activity were pooled and freeze-dried. This material was dissolved in 10 mmol l-1 phosphate buffer pH 6.0 and then extracted twice with 1-butanol. The organic phases were combined and evaporated under reduced pressure (Benitez et al. 2010). Fractions were suspended in 10 mmol l-1 phosphate buffer pH 6.0 and stored at 4°C.
Bacterial strains and culture conditions The microorganism B. amyloliquefaciens LBM 5006, isolated from the native soil of the Brazilian Atlantic Forest (Lisboa et al. 2006), was used for production of antimicrobial peptides. P. larvae 1655 and P. larvae 165B, isolated from honey bee brood without symptoms of AFB (Schuch et al. 2002), were chosen as the indicator strains to demonstrate and measure antibacterial activity. For longterm storage, the bacteria were kept at -21°C in 20% (v/v) glycerol in BHI broth (Oxoid, Basingstoke, UK). Before the experiments, each bacterial strain was subcultured at least two times for 24 h intervals.
Mass spectrometry
Production of antimicrobial activity by B. amyloliquefaciens LBM 5006
Effect of proteolytic enzymes, heat, and pH on antimicrobial activity
For the production of antimicrobial factor, B. amyloliquefaciens LBM 5006 was grown in 200 ml TSB medium (Oxoid, Basingstoke, UK) at 37°C in a rotary shaker (Cientec, Piracicaba, Brazil) at 125 rpm for 48 h. The cells were harvested by centrifugation at 10,0009g for 15 min at 4°C and the resulting culture supernatant was sterilized by filtration with 0.22 lm membranes (Millipore, Bedford, MA, USA). Bacterial growth (OD 600 nm) and antimicrobial activity were monitored each 4 h during cultivation. The antimicrobial activity against P. larvae was detected by agar disc diffusion assay (Motta and Brandelli 2002). Aliquots of 20 ll were applied onto 6-mm cellulose discs on TSA plates previously inoculated with a suspension of 106
The antimicrobial factor was submitted to stability tests to investigate its peptide nature. The susceptibility to proteolytic enzymes, pH, and heat treatments was evaluated as described elsewhere (Cladera-Olivera et al. 2004). The antimicrobial factor was treated at 37°C for 60 min with 10 mg ml-1 final concentration of trypsin and proteinase K. Samples were boiled for 3 min to inactivate the enzyme. To evaluate pH stability, the antimicrobial substance was incubated at pH 3–10 for 60 min and the pH was adjusted to 7 before testing for antimicrobial activity. To analyze thermal stability, samples were exposed to temperatures ranging 40–100°C for 60 min and 121°C/141 kPa for 15 min. After the treatments, residual antimicrobial
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The antimicrobial factor was applied to C18 cartridges (Vydac, Hesperia, CA, USA) and eluted with 80% acetonitrile 0.046% trifluoroacetic acid. Samples concentrated in a vacuum centrifuge (SpeedVac SC100, Savant, USA) were dissolved in ethanol and analyzed by mass spectrometry in a MALDI-TOF mass spectrometer (Maldi micro MX, Waters Corporation, Milford, MA, USA) operating in reflection mode and using a matrix of a-ciano-4-hydroxycinnamic acid.
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activity against P. larvae by the serial two-fold dilution method as described above. Effect on the growth of P. larvae Overnight cultures of P. larvae 165B and 1655 were obtained by cultivation in BHI at 37°C for 18 h. An aliquot (500 ll) of these cultures containing 106 CFU ml-1 was inoculated in tubes containing 16 ml of BHI and incubated at 37°C. Sterile BHI medium was added to control tubes. The growth was monitored at 2 h intervals by optical density (OD) at 600 nm and by viable cells counts (CFU ml-1). The antimicrobial factor (final concentration 1600 AU ml-1) was added to culture of indicator strain after 4.5 h of cultivation, and the effect of the antimicrobial factor on turbidity and on the number of viable cells was determined at 2 h intervals.
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Effect on P. larvae spores Spore production by P. larvae 165B and P. larvae 1655 was carried out in TSB agar. After incubation at 37°C for 7 days, samples were harvested, suspended in sterile MilliQ water, and treated at 80°C for 10 min to kill vegetative cells). Aliquots were diluted with sterile MilliQ water to the initial concentration of 105–106 spores ml-1. Spores were incubated in BHI for 120 min at 37°C with antimicrobial factor at a concentration of 1,600 AU ml-1 and then plated to determine viable cell counts (Bizani et al. 2005). Plates were incubated at 37°C for up to 5 days before counting. Scanning electron microscopy
A dose response curve was determined using different concentration of antimicrobial factor (between 50 and 3,200 AU ml-1) and an initial inoculum of 105 CFU ml-1 of P. larvae 1655. Viable counts were determined after incubation at 37°C for 120 min. The minimal inhibitory concentration (MIC) values were determined by a standard microbroth dilution method as described previously (Sirtori et al. 2008). Sterile 96-well microplates (Corning, New York, USA) were filled with 100 ll of serial dilutions of LBM 5006 (concentrations ranging from 3,200 to 0 AU ml-1) and then a standardized number of bacteria (100 ll of a 105 CFU ml-1 suspension from P. larvae 165B and 1655) were added into each well. Microplates were incubated at 37°C for 24 h. The MIC was determined by taking into account the higher dilution at which no growth of the test organism was visible. Controls were prepared using sterile 10 mmol l-1 phosphate buffer pH 6.0 instead addition of antimicrobial factor.
The treated and non-treated cells of P. larvae 1655 were prepared by the method described by Kalchayanand et al. (2004) with slight modifications. Cultures of P. larvae 1655 incubated at 37°C for 24 h were centrifuged (3,0009g, 15 min at 20°C). The cells were suspended in BHI broth (controls) or antimicrobial factor (1,600 AU ml-1) and incubated for 120 min at 37°C. To scanning electron microscopy, the cell suspensions were fixed with 2% glutaraldehyde in Na-cacodylate buffer (100 mmol l-1, pH 7.1). Then, the cells were washed to remove glutaraldehyde and suspended in the same buffer. A drop from each suspension was transferred to a poly-L-lysine-treated silicon wafer chips, which were kept for 30 min in a hydrated chamber for cell adhesion. The attached cells were postfixed by immersing the chips in 10 mg ml-1 osmium tetroxide (OsO4) in cacodylate buffer for 30 min, rinsed in the same buffer, and dehydrated in ascending ethanol concentrations (%, v/v) of 50, 70, 95 (2x), and 100 (2x), for 10 min each. The chips were mounted on aluminum stubs and coated with gold in a sputter coater (Emitech K550, Ashford, Kent, England). The chips were viewed at 10 kV accelerating voltage in a scanning electron microscope (JeolÒ JSM-6060).
Measurement of UV-absorbing materials
Toxicity assay
UV-absorbing materials release was measured as an index of cell lysis (Motta et al. 2008). P. larvae 1655 and P. larvae 165B cell suspensions, which corresponded to a 0.5 McFarland turbidity standard solution in 10 mmol l-1 phosphate buffer pH 6.0, were mixed (1:1, v/v) with antimicrobial factor (final concentration 1,600 AU ml-1) and incubated at 37°C. Samples were removed after 120 min and filtered through 0.22 lm membranes. The absorbance of the filtrates was measured at 260 and 280 nm using a spectrophotometer UV-mini 1240 (Shimadzu, Tokyo, Japan). Cultures of the indicator microorganisms without antimicrobial factor were included as controls.
Worker larvae of A. mellifera were obtained in a diseasefree apiary. Larvae were collected and transferred to 96-well microplates (Corning, New York, USA). Larvae were fed ad libitum with a liquid diet consisting of 660 g l-1 royal jelly, 60 g l-1 glucose, 60 g l-1 fructose, and 10 g l-1 yeast extract in sterile distilled water (Evans 2004). Larval food was supplemented with crude and purified fractions of antimicrobial factor at final concentration of 800 AU ml-1. Plates were incubated at 35°C for 48 h. Larvae were considered as dead when they lost their body elasticity or showed a color change to brownish (Evans 2004).
Dose–response curve and MIC determination
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Results Production of antimicrobial factor The culture of the strain B. amyloliquefaciens LBM 5006 was carried out at 37°C in TSB medium. Maximum antimicrobial activity was 6,400 AU ml-1, detected after 32 h of cultivation (Fig. 1), which corresponded to the stationary growth phase. The production of the antimicrobial factor was dependent on the bacterial growth phase. The strain LBM 5006 started to produce antimicrobial activity in TSB medium at 4 h (800 AU ml-1), during the early logarithmic growth phase. The antimicrobial factor was isolated from the culture supernatant, and the mass spectrum revealed a cluster with major peaks at m/z 1,000–1,100 Da (Fig. 2). Another cluster with minor peaks at m/z 1,471, 1,485, and 1,499 was also observed. The antimicrobial factor was sensitive to trypsin and proteinase K at the concentration of 10 mg ml-1 (Table 1), but the activity was maintained when the enzymes were tested at 2 mg ml-1 (data not shown). The antimicrobial factor was stable in the pH range from 3 to 10, remaining 100% its initial activity. The antimicrobial factor was resistant to heating at temperatures up to 80°C (Table 1). Effect on P. larvae The effect of antimicrobial factor (1,600 AU ml-1) on the P. larvae strains 165B and 1655 was examined to establish the mode of action of the antimicrobial factor. This
3
Efflux of UV-absorbing materials To determine whether the antimicrobial factor has an effect on the integrity of cell membranes of P. larvae 1655 and P. larvae 165B, the efflux of cytoplasmic content was measured by optical density (260 and 280 nm) in the culture supernatant of control cells and cells treated for 120 min with the antimicrobial factor. Treatment for P. larvae 1655 and P. larvae 165B cells with 1,600 AU ml-1 of antimicrobial factor caused a leakage of UV-absorbing materials measured at both 260 and 280 nm (Table 2). The release of material absorbing at 280 nm was very similar for both strains, but an increased release of material 260 nm was observed for strain 165B. Dose–response curve and MIC When P. larvae 1655 and 165B cells were incubated with different concentrations of the antimicrobial factor, a dose– response curve was obtained (Fig. 4). The CFU ml-1 were determined initially and after the end of incubation time and compared with the respective control. Reducing the number of viable cells, in both indicators strains, with increasing concentration of antimicrobial factor, reinforces the theory that this antimicrobial has a bactericidal activity. MIC values were determined as 200 AU ml-1 (P. larvae 1655) and 800 AU ml-1 (P. larvae 165B), respectively.
6000 2
4000
1
OD (600 nm)
Antimicrobial activity (AU/mL)
8000
concentration led to marked decrease (about 4 log units) in the number of viable cells, initially 106 cells per ml, during incubation time. This corresponds to a percentile decrease in about 99.99% of the initial population. The decrease in cell counts of P. larvae occurred simultaneously with the decrease in optical density (OD600), indicating a bactericidal effect with simultaneous cell lysis (Fig. 3).
2000
Effect on spore outgrowth The effect of antimicrobial factor on spores of P. larvae 1655 and 165B was investigated. An approximately 2 log10 reduction (from 5.2 to 3.2 and 5.0 to 3.4 log CFU ml-1, respectively) was observed in cell counts when spores were treated with the antimicrobial factor in the tested concentration of 1,600 AU ml-1.
0
0 0
4
8
12
24
32
48
Electron microscopy
Time (h) Fig. 1 Growth (OD 600 nm, filled square) of the strain Bacillus amyloliquefaciens LBM 5006 and kinetics of the production of antimicrobial activity (AU ml-1,). Results are means of three independent experiments
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P. larvae cells were examined by scanning electron microscopy to visualize the changes in morphology following treatment with the antimicrobial factor. Representative photomicrographs at 20,000x magnifications are
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Fig. 2 Mass spectrum of antimicrobial peptides from Bacillus amyloliquefaciens LBM 5006
Table 1 Influence of temperature, pH, and enzymes on the antimicrobial activity
A
8 7
Residual activity (%)
-1
100 0
Trypsin (10 mg ml ) 40°C/60 min
20 100
80°C/60 min
100
100°C/60 min
38
121°C/141 kPa/15 min
100
pH 5.0
100
pH 8.0
100
pH 10.0
4
1 0
0 0
100
Residual activity compared with the antimicrobial activity before the treatment. Data are means of three independent experiments
0,4
3 2
0
pH 3.0
5
OD (600 nm)
Proteinase K (10 mg ml-1)
-1
Control (untreated substance)
0,8 6
log10 CFU mL
Treatment
2
4
6
8
10
12
Time (h) B
9 8
Toxicity Larvae of A. mellifera were fed with a diet containing the antimicrobial factor to evaluate its potential toxicity. The mortality percentage of the control group was 15%. The group that received the antimicrobial factor in the food showed a mortality percentage of 17.3%, although this value increased to 25.5% when the crude antimicrobial was added.
-1
6 5 4
0,4
3
OD (600 nm)
0,8
7
log10 CFU mL
presented in Fig. 5. The control cells (Fig. 5a) showed the characteristic intact rods with normal texture. Exposure to BLS for 120 min at 37°C caused extensive lysis and degradation of the cells, apparently due to changes in the structure of the cell wall and cell membrane (Fig. 5b–c). The disruption of cells in several points has caused the leakage of cellular content. The bactericidal effect of the antimicrobial factor was accompanied by bacterial lysis.
2 1 0
0 0
2
4
6
8
10
12
Time (h) Fig. 3 Effect of antimicrobial peptide of Bacillus amyloliquefaciens LBM 5006 on growth of Paenibacillus larvae 165B (a) and Paenibacillus larvae 1655 (b). Turbidity (open symbols) and viability (black symbols) were monitored in control (squares) and treated (circles) cells with a final concentration of 1,600 AU ml-1. The arrow indicates the time of antimicrobial substance addition. Each point represents the mean of three independent experiments
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Table 2 Release of intracellular UV-absorbing materials of P. larvae 1655 and 165B Nucleic acids (A260
nm)
Proteins (A280
nm)
Control
Treated cells
Control
Treated cells
P. larvae 1655
0.10 ± 0.02
0.31 ± 0.03
0.14 ± 0.04
0.45 ± 0.01
P.larvae 165B
0.09 ± 0.03
0.42 ± 0.09
0.12 ± 0.01
0.44 ± 0.02
log10 CFU mL-¹
3
2
1
0 0
1000
2000
3000 -1
LBM 5006 (AU mL ) Fig. 4 Effect of LBM 5006 concentration on cell viability of P. larvae 1655 (circles) and P. larvae 165B (squares). Viable cell counts were determined after treatment with different concentrations of the antimicrobial substance during 120 min at 37°C. The initial inoculum was 106 CFU ml-1. Each point represents the mean of three independent experiments
Discussion Innocuous bacterial strains that may produce antimicrobial substances are of great interest as natural preservatives to confine or inhibit different pathogens or spoilage microorganisms. The non-toxigenic Bacillus species are considered safe and could be used for agricultural, human, and veterinary purposes (Duc et al. 2004; Motta et al. 2007; Sabate´ et al. 2009). In this context, the inhibitory effect of the antimicrobial factor produced by B. amyloliquefaciens LBM 5006 on P. larvae indicates its potential usefulness to control AFB. The maximum values for antimicrobial activity produced by B. amyloliquefaciens LBM 5006 were coinciding with the stationary growth phase. This agrees with the production kinetic of lipopeptides like surfactin, bacilysin, and iturins, typical secondary metabolites produced by Bacillus spp. (Stein 2005). Other antimicrobial peptides are synthesized by Bacillus during the exponential phase,
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Fig. 5 Scanning electron micrographs of Paenibacillus larvae. Untreated cells (a) and treated cells for 120 min at 37°C with 1,600 AU ml-1 of the bacteriocin (b and c)
including subtilin, TasA, subtilosin, and bacteriocin-like substances (Stein 2005; Barboza-Corona et al. 2007). Bacillus may produce different antimicrobial peptides and their production is under complex regulation (Yao et al. 2003). The modification in the growth conditions such as pH or N source may induce the production of different antimicrobial peptides. During growth in BHI broth, the antifungal activity produced by strain LBM 5006 was associated with iturin A and fengycin A lipopeptides (Benitez et al. 2010). The antimicrobial factor was susceptible to proteolytic enzymes, indicating the peptide nature of the antimicrobial substance. The antimicrobial peptide lichenin A produced by Bacillus licheniformis was completely inactivated by proteinase K treatment but was resistant to trypsin (Pattnaik et al. 2001). This enzyme also eliminates thuricin
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439 activity, a bacteriocin-like peptide produced by Bacillus thuringiensis (Ahern et al. 2003). A broad-range antimicrobial peptide produced by Bacillus sp. P34 was sensitive to trypsin and pronase E (Motta et al. 2007). The maintenance of activity in a broad pH range, relative resistance to heat and proteases resembles the characteristics of cyclic lipopeptides of Bacillus spp. (Stein 2005). The analysis of the mass spectrum showed that strain LBM 5006 produces iturin A in TSB as well. The major peak at m/z 1058 corresponds to iturin A and the adjacent peaks to isoforms containing different fatty acid side chains (Hiradate et al. 2002; Caldeira et al. 2008). Another cluster with minor peaks was also observed at m/z 1485 (only 10% relative abundance in relation to m/z 1058), corresponding to C16-fengycin A [M ? Na]? (Hofemeister et al. 2004). The exact 14 Da difference among the surrounding peaks corresponds to CH2 mass, indicating homologous molecules with different length of fatty acid chain. Although iturin A could not be purified to homogeneity, the results suggest that this peptide is responsible for inhibition of P. larvae. The definitive separation of Bacillus lipopetides is difficult due to the structural variability and co-production of molecules with very similar physico-chemical properties (Caldeira et al. 2008; Chen et al. 2008). Iturin has been typically associated with the inhibition of phytopathogenic fungi (Arrebola et al. 2010) and its antibacterial activity against gram-positive bacteria has been reported (Ongena and Jacques 2008). However, the antagonistic effect of iturins on Paenibacillus spp. has not yet been described. The decrease in the number of viable cells after the addition of the antimicrobial factor suggests that the effect toward P. larvae cells was bactericidal. The reduction in OD readings indicated that cells of indicator strain were lysed. This effect was similar to that observed for the antimicrobial peptide P34 against Bacillus cereus (Motta et al. 2007). The view that the antimicrobial factor was bactericidal and bacteriolytic is consistent with the fact that it damaged the cell envelope (cell wall and membrane) of P. larvae. Indeed, release of UV-absorbing materials was detected after treatment for cells with the antimicrobial factor. It appears from these results that the bacterial cells were injured following treatment with the antimicrobial factor, which was confirmed by scanning electron microscopy. Many antimicrobial peptides exert their bactericidal mode of action by destabilization and permeabilization of cell membranes and may be accompanied by lysis of sensitive cells (Haney et al. 2010). Binding of these peptides to teichoic, lipoteichoic, and teichuronic acids in the cell wall of sensitive bacteria leads to release, and therefore activation, of autolytic enzymes, which under normal conditions are electrostatically bound to these polymers (Cintas et al. 2001). Bacteriocins like pediocin AcH specifically
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destabilize three-dimensional structures of cell walls and cell membranes leading to loss of normal functions including the control of autolysins. This triggers autolysin systems resulting in uncontrolled degradation of the cell wall and eventual cell lysis (Kalchayanand et al. 2004). Antimicrobial peptides from Bacillus like surfactin and fengycin cause lysis by destabilization of membranes, and at higher concentrations, by detergent effect (Deleu et al. 2005). The primary mode of action of iturin is on cytoplasmic membrane, causing osmotic perturbation by the formation of ion-conducting pores (Ongena and Jacques 2008). AFB is a cosmopolitan disease and one of the major threats to beekeeping, since it is highly contagious and able to kill affected colonies (Genersch, et al. 2005). Only honeybee larvae are susceptible to infection and only the spores of P. larvae are infective (Hornitzky 1998). Larvae become infected by ingestion of spore-contaminated larval food (glandular secretions and processed honey). The susceptibility to infection depends on the larval age, the spore dose necessary for successful infection of a larva increases with increasing larval age (Genersch 2010). An interesting finding from this study was the inhibitory activity observed against P. larvae spores treated with the iturin-like peptides. This is particulary relevant considering the P. larvae spores are refractory to harsh environmental conditions (Hrabak and Martinek 2007; Antune´z et al. 2009). Similar results were obtained on Bacillus spores with cerein 8A, a peptide from B. cereus (Bizani et al. 2005) and enterocin EJ97 produced by Enterococcus faecalis (Viedma et al. 2010). The use natural antimicrobial substances have been suggested as promising alternatives to control AFB (Bastos et al. 2008; Gende et al. 2009). Flesar et al. (2010) tested the antibacterial activity of different natural compounds (flavonoids, alkaloids, terpenoids) and crude extracts of plants against P. larvae. Compounds like capsaicin, nordihydroguaiaretic acid, thymoquinone, trans-2-hexanal, and the plant extracts of Humulus lupulus and Myrtus communis showed significant growth inhibition of P. larvae and absence of oral toxicity to worker honey bees. Also, aqueous extracts of the aromatic plants Eucalyptus cinerea and Mintostachys verticillata showed remarkable efficacy for inhibition of P. larvae (Gonza´lez and Marioli 2010). Although terpenes and terpenoids are frequently present in essential oils of aromatic plants, the great variety of chemical composition difficult to determine the exact nature of inhibitory activity. Thus, the absence of side effects on bee and honey quality needs to be evaluated. Biocontrol by antagonistic bacteria is also an interesting alternative to control AFB, and the inhibition of P. larvae by some bacterial strains has been already described (Evans and Armstrong 2005; Olofsson and Vasquez 2008).
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Some Bacillus strains have been reported to inhibit P. larvae but the chemical nature of the metabolites involved in the inhibitory activity was not determined (Alippi and Reynaldi 2006). Only the study of Sabate´ et al. (2009) reported three Bacillus strains isolated from honey samples and bee gut that inhibit P. larvae by surfactin synthesis. This study indicates for the first time that iturin peptides can efficiently inhibit P. larvae, reinforcing the importance of antimicrobial lipopeptides from Bacillus spp. for biological control. Iturin has the additional advantage of presenting low toxicity to mammals and lower toxicity to insects in comparison with surfactin (Klich et al. 1994; Assie´ et al. 2002). Hence, the antimicrobial activity from B. amyloliquefaciens may have a valuable potential as biological control agent, because of its antimicrobial activity against vegetative cells and germinating spores of P. larvae. Despite iturin peptides are often recognized by their low toxicity, there is absence of studies about the effect of such antimicrobial substances on honeybees. Our preliminary results suggest that the antimicrobial factor produced by B. amyloliquefaciens presents low toxicity to bee larvae. However, acute toxicity tests and lethal concentration studies should be conducted before this peptide could be used in field experiments. Acknowledgments Authors thank the technical support of Centro de Microscopia Eletroˆnica (CME-UFRGS). This work was supported by Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq), Brazil.
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