Amanda de Souza da Motta

Screening for antimicrobial activity among bacteria isolated from the Amazon Basin

Bacteria producing antimicrobial activity were identified among 86 isolates from aquatic environments of Brazilian Amazon Basin. Antimicrobial activity against at least one indicator strain was detected for 59 isolates (68.6%). Inhibitory activity was mostly against Gram-positive bacteria, such as Listeria monocytogenes and Bacillus cereus. The antimicrobial substances produced by 19 strains that showed higher inhibitory activity were partially characterized. These antimicrobial substances showed thermal resistance for up to 100°C and partial resistance to proteolytic treatment. Some antimicrobials were partially resistant to pronase E at 2 mg ml -1 or to treatment with trichloroacetic acid. Detection of antimicrobial activity on polyacrylamide gels revealed that the molecular weight of the compounds was lower than 14 kDa. Several strains showed antibacterial activity, which in some cases appear to be related to antimicrobial peptides. The potential of these microorganisms to produce useful antimicrobial compounds is great and must be better explored.

Essential oil of the leaves of Eugenia uniflora L.: Antioxidant and antimicrobial properties

Essential oil (EO) of the leaves of Eugenia uniflora L. (Brazilian cherry tree) was evaluated for its antioxidant, antibacterial and antifungal properties. The acute toxicity of the EO administered by oral route was also evaluated in mice. The EO exhibited antioxidant activity in the DPPH, ABTS and FRAP assays and reduced lipid peroxidation in the kidney of mice. The EO also showed antimicrobial activity against two important pathogenic bacteria, Staphylococcus aureus and Listeria monocytogenes, and against two fungi of the Candida species, C. lipolytica and C. guilliermondii. Acute administration of the EO by the oral route did not cause lethality or toxicological effects in mice. These findings suggest that the EO of the leaves of E. uniflora may have the potential for use in the pharmaceutical industry.

Evaluation of the in vitro cytotoxicity of the antimicrobial peptide P34

The in vitro cytotoxicity of the antimicrobial peptide P34 was evaluated in different eukaryotic cells. The food‐grade bacteriocin nisin was also analysed for comparison. Vero cells were treated with different concentrations (0.02–2.5 μg·ml−1) of antimicrobial peptide P34 and nisin. Cell viability and plasma membrane integrity were checked by MTT [3‐(4,5‐dimethylthiazole‐2‐yl)‐2,5‐diphenyltetrazolium bromide], NRU (Neutral Red dye uptake) and LDH (lactate dehydrogenase) assays. The EC50 values of the peptide P34 in MTT and NRU assays were 0.60 and 1.25 μg·ml−1 respectively, while values of nisin found were 0.50 and 1.04 μg·ml−1. In the LDH assay, the EC50 values were 0.65 and 0.62 μg·ml−1 for P34 and nisin, respectively. The peptide P34 revealed similar haemolytic activity on human erythrocytes (5.8%) when compared with nisin (4.9%). The effects on viability, motility and acrosomal exocytosis of human sperm were also evaluated. Nisin and P34 showed similar effects on sperm parameters. The evaluation of cytotoxicity of antimicrobial peptides is a critical step to guarantee their safe use.

Purification and Partial Characterization of an Antimicrobial Peptide Produced by a Novel Bacillus sp. Isolated from the Amazon Basin

An antimicrobial peptide produced by a new Bacillus species isolated from the Amazon Basin was purified and characterized. The antimicrobial peptide was purified by ammonium sulfate precipitation, gel filtration, and ion exchange chromatography, and after the final purification step, one active fraction was obtained, designated BLS P34. Direct activity on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was observed. A single band on SDS-PAGE suggested that the peptide was purified to homogeneity and had a molecular mass of about 5 kDa. The molecular weight (MW) was accurately determined by mass spectroscopy as 1456 Da. The purified BLS P34 remained active over a wide temperature range and was susceptible to all proteases tested.

Inhibition of Salmonella Enteritidis by cerein 8A, EDTA and sodium lactate.

The ability of the bacteriocin cerein 8A to inhibit Salmonella Enteritidis in combination with EDTA and sodium lactate was investigated. Salmonella Enteritidis was incubated with combinations of cerein 8A (3200AU/mL) and EDTA (20, 50, 100 mmol/L) or sodium lactate (200 mmol/L). All treatments caused a significant reduction in the OD(600) values of Salmonella Enteritidis cultures. The addition of cerein 8A plus EDTA resulted in higher inhibition in comparison with the bacteriocin alone; the greater the concentration of EDTA, the greater the inhibitory effect. The combination of cerein 8A plus 100 mmol/L EDTA results in a more efficient treatment to reduce the number of viable cells of Salmonella Enteritidis. The combination of cerein 8A plus sodium lactate also showed significant inhibition of the indicator organism. Transmission electron microscopy showed damaged cell walls and loss of protoplasmic material in treated cells. The cells of Salmonella Enteritidis treated with cerein 8A plus EDTA appeared more injured. The bacteriocin cerein 8A may be useful to inhibit Gram-negative bacteria, with enhanced effect in combination with chelating agents. Control of Salmonella Enteritidis, a Gram-negative bacterium constantly linked to food outbreaks, addresses an important aspect of food safety.

Antimicrobial factor from Bacillus amyloliquefaciens inhibits Paenibacillus larvae, the causative agent of American foulbrood

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.

Antiviral activity of a Bacillus sp: P34 peptide against pathogenic viruses of domestic animals

P34 is an antimicrobial peptide produced by a Bacillus sp. strain isolated from the intestinal contents of a fish in the Brazilian Amazon basin with reported antibacterial activity. The aim of this work was to evaluate the peptide P34 for its in vitro antiviral properties against canine adenovirus type 2 (CAV-2), canine coronavirus (CCoV), canine distemper virus (CDV), canine parvovirus type 2 (CPV-2), equine arteritis virus (EAV), equine influenza virus (EIV), feline calicivirus (FCV) and feline herpesvirus type 1 (FHV-1). The results showed that the peptide P34 exhibited antiviral activity against EAV and FHV-1. The peptide P34 inhibited the replication of EAV by 99.9% and FHV-1 by 94.4%. Virucidal activity was detected only against EAV. When P34 and EAV were incubated for 6 h at 37 °C the viral titer reduced from 104.5 TCID50 to 102.75 TCID50, showing a percent of inhibition of 98.6%. In conclusion, our results demonstrated that P34 inhibited EAV and FHV-1 replication in infected cell cultures and it showed virucidal activity against EAV. Since there is documented resistance to the current drugs used against herpesviruses and there is no treatment for equine viral arteritis, it is advisable to search for new antiviral compounds to overcome these infections.

Mode of action of antimicrobial peptide P45 on Listeria monocytogenes.

The mode of action of an antimicrobial peptide produced by Bacillus sp. P45 isolated from the intestine of the Amazonian basin fish Piaractus mesopotamicus was investigated. The antimicrobial peptide was purified from culture supernatants by precipitation with ammonium sulfate and gel filtration chromatography. The peptide has an EC50 of 300 AU (activity units) ml–1 and kills all viable cells of Listeria monocytogenes with a concentration of 800 AU ml–1. A decrease in OD was observed when L. monocytogenes cultures were treated with the peptide, suggesting that cells were lysed. Transmission electron microscopy showed damage of the cell envelope and loss of protoplasmic material. The peptide P45 was bactericidal and bacteriolytic to L. monocytogenes. There is evidence that the mode of action is interefering at cell membranes and the cell wall. The knowledge of the mode of action of antimicrobial peptides is an essential step to consider their utilization in food or clinic. (© 2008 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

Liquid-liquid extraction of antimicrobial peptide P34 by aqueous two-phase and micellar systems.

ABSTRACT Antimicrobial peptide P34 is a promising biopreservative for utilization in the food industry. In this work, aqueous biphasic systems (ABS) and aqueous biphasic micellar systems (ABMS) were studied as prestep for purification of peptide P34. The ABS was prepared with polyethylene glycol (PEG) and inorganic salts and the ABMS with Triton X-114 was chosen as the phase-forming surfactant. Results indicate that peptide P34 partitions preferentially to PEG-rich phase and extraction with ammonium sulfate [(NH4)2SO4], yielding a 75% recovery of the antimicrobial activity, specific activity of 1,530 antimicrobial units per mg of protein, and purification fold of 2.48. Protein partition coefficient and partition coefficient for the biological activity with (NH4)2SO4 system were 0.48 and 64, respectively. Addition of sodium chloride did not affect recovery, but decreased protein amount in the PEG-rich phase, indicating a higher partition of biomolecules. ABMS did not yield good recovery of antimicrobial activity. Purification fold using PEG–(NH4)2SO4 and 1.0 mol l−1 sodium chloride was twice higher than that obtained by conventional protocol, indicating a successful utilization of ABS as a step for purification of peptide P34.

Antimicrobial and antibiofilm activity of Baccharis psiadioides essential oil against antibiotic-resistant Enterococcus faecalis strains

Abstract Context: Baccharis psiadioides (Less.) Joch. Müller (Asteraceae) is considered as a producer of bioactive essential oils and is used in south Brazilian folk medicine for its proprieties as stimulant, antipyretic, anti-inflammatory and as an antidote for snake bites. Objective: To verify the antimicrobial and antibiofilm activities of the essential oil of B. psiadioides (EOBP) against antibiotic-resistant Enterococcus faecalis. Materials and methods: The initial evaluation of EOBP activity was conducted by the agar and microdilution methods against 13 antibiotic-resistant E. faecalis strains. The antibiofilm effect was determined by the application of EOBP in the earlier adherent cells or to the stabilized biofilm for 24 h and was evaluated by crystal violet, viability and scanning electron microscopy (SEM) assays. Chemical composition of EOBP was determined by gas chromatography (GC/FID – GC/MS). Results: The MIC values for EOBP were at least 1.25% and 4–16% for agar and microdilution assays, respectively. The EOBP reduced the microbial adherence and the viability of the cells, but did not cause the complete disruption of biofilms. SEM images indicate that EOBP influences the adherence of cells to a surface. The monoterpene β-pinene was the major constituent identified in EOBP. Conclusion: This research shows the ability of EOBP to control resistant E. faecalis strains and to reduce the biofilm amount attached to abiotic surfaces, indicating its role as a promising new natural antimicrobial agent. Moreover, these results further contribute to the growing number of studies of plant natural products which suggest that these compounds can combat resistant microorganisms.