Effect of Lactobacillus spp. cell-free supernatant against planktonic growth and biofilm formation of foodborne Escherichia coli isolates.

The aim of this work was to evaluate the anti-Escherichia coli effect of cell-free supernatant (CFS) of Lactobacillus spp. against planktonic and biofilm forms of foodborne isolates. Escherichiacoli strains (P12, P25, P35 and P36), previously isolated from fresh filets of fish, were subjected to antimicrobial susceptibility determination by the disc-diffusion agar method. Subsequently, the antagonistic effect between probiotic and pathogenic strains was determined by spot overlay assay. Finally, the CFS activity against pre-established (12 h) biofilms was demonstrated through biomass quantification by crystal violet staining and scanning electron microscopy (SEM). All isolates presented some pattern of resistance, primarily to ampicillin and tetracycline. Probiotic strains presented high antagonistic effects against all E. coli strains, presenting inhibition zones (R) ranging from 15.60 to 20.67 mm. Additionally, the residual biomass of pre-established (12 h) biofilm was drastically reduced about 50% after CFS treatment (P < 0.01). What can be noted by SEM images, which show less surface-attached cells of CFS-treated biofilms of E. coli (P12). Thus, cell-free preparations produced from Lactobacillus spp. may represent a tool in the battle against planktonic cells and biofilm forms of antibiotic-resistant E. coli.

Comparative study of the chemical composition, antibacterial activity and synergic effects of the essential oils of Croton tetradenius baill. And C. pulegiodorus baill. Against Staphylococcus aureus isolates.

The aim of this work was to evaluate the chemical composition and antibacterial activity of Croton tetradenius Baill. (CTEO) and C. pulegiodorus Baill. (CPEO) essential oils against Staphylococcus aureus, and their synergism with antibiotics. The essential oils (EOs) were extracted by hydrodistillation and chemically characterized by gas chromatography-mass spectrometry (CG-MS) and gas chromatography with flame ionization detection (CG-FID). The antimicrobial action of the EOs was tested against two standard strains and four clinical isolates of S. aureus using the disk-diffusion agar method and the microdilution assay. The bacterial kinetic growth was also determined. The synergistic effect between EOs and antimicrobials was analyzed by the checkerboard test. CTEO and CPEO yielded 0.47 and 0.37% w/w and the most common components were p-cymene (28.24%), camphor (17.76%) and α-phellandrene (8.98%), and trans-chrysanthenyl acetate (27.05%), α-terpinene (19.21%) and p-cymene (12.27%), respectively. The disk-diffusion test showed that the bacteria are sensitive to the agents tested. The MIC in the presence of the CTEO it was 4000 µg/mL, while for the CPEO it was 8000 µg/mL, except for clinical isolate 4B. The MBC for strains treated with CTEO were 8000 µg/mL, with the exception of isolates 8B and 0 A 4000 µg/mL. For the CPEO, all strains showed a concentration above 8000 µg/mL. The growth curve showed that CTEO and CPEO altered growth kinetics, delaying the lag phase and reducing the log phase. In combination with antibiotics, both essential oils showed synergisms effect with oxacillin and ampicillin, and additive effect with benzylpenicillin. CTEO and CPEO showed antibacterial action against S. aureus strains, showing as a promise natural alternative in clinical therapy.

Anti-Staphylococcal Activity of Cinnamomum zeylanicum Essential Oil against Planktonic and Biofilm Cells Isolated from Canine Otological Infections.

The aim of this study was to evaluate the phytochemical profile of Cinnamomum zeylanicum essential oil (CZEO) and their antimicrobial and antibiofilm activity against Staphylococcus strains isolated from canine otitis. First, the CZEO chemical composition was determined by gas chromatography-mass spectrometry (CG-MS). External otitis samples collected from dogs were submitted to staphylococcal isolation, followed by MALDI-TOF mass spectrometry identification. The antimicrobial action was tested against the isolates using the disk-diffusion and microdilution methods. The antibiofilm activity was evaluated by CZEO-based concentrations, subMIC for biofilm formation and supraMIC against preformed biofilm, quantified by crystal violet (CV) staining and CFU counting. The chemical analysis revealed that (E)-cinnamaldehyde, eugenol and (E)-cinnamyl acetate were the main compounds in the CZEO, representing 77.42, 8.17 and 4.50%, respectively. Two strains of three different species, S. saprophyticus, S. schleiferi and S. pseudintermedius, were identified. The disk-diffusion test showed an inhibitory zone diameter, ranging from 34.0 to 49.5 mm, while the MIC and MBC values were around 500 and 1000 µg/mL. SubMIC demonstrated an inhibition on biofilm formation against 4 out the 6 strains tested. On mature biofilm, the CZEO-based supraMIC groups had slightly change on biomass, however, the biofilm cell viability decreased the CFU in 3 magnitude orders.

Antimicrobial and Antibiofilm Activity of Lys-[Trp6]hy-a1 Combined with Ciprofloxacin Against Gram-Negative Bacteria.

BACKGROUND: Ciprofloxacin (Cip) is the most commonly used quinolone in clinical practice; however large-scale use has favored the increase of multiresistant pathogenic microorganisms. Antimicrobial peptides (AMPs) appear to be a promising alternative in potentiating these conventional drugs. OBJECTIVE: The aim of this study was to evaluate the effect of the peptide Lys-[Trp6]hy-a1 (lys-a1) on the antimicrobial and antibiofilm activity of ciprofloxacin against clinically relevant gram-negative bacteria. METHODS: The antimicrobial effects of Cip and lys-a1 were assessed by determining the minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs). The synergistic action of Cip and lys-a1 was determined by checkerboard assay. The time-kill curve was constructed for the Cip/lys-a1 combination against Pseudomonas aeruginosa ATCC 9027. The antibiofilm activity of this combination was analyzed by crystal violet, colony-forming unit count and atomic force microscopy (AFM). RESULTS: The data demonstrated that lys-a1 was able to inhibit planktonic growth of strains of P. aeruginosa and Klebsiella pneumoniae both at 125 µg/mL. The fractional inhibitory concentration index (FICi) showed a synergistic effect between Cip and lys-a1 against P. aeruginosa, decreasing the MICs of the individual antimicrobial agents by 4- and 8-fold, respectively. This effect was also observed for the death kinetics and antibiofilm activity. Analysis of the early biofilms (6 h) as well as isolated cells by AFM images evidenced the cell perturbation caused by Cip/lys-a1 treatment. CONCLUSION: These data suggest that lys-a1 has biotechnological potential as a therapeutic tool for the treatment of infections caused by clinically relevant microorganisms, especially P. aeruginosa.

Evaluation of the proteomic profiles of ejaculated spermatozoa from Saanen bucks ( Capra hircus ).

The Saanen goat breed has been widely explored in breeding programmes; however, there are few reports about the breed's genetic and molecular composition. Thus, this study aimed to characterize the proteomic profile of spermatozoa from Saanen breeding goats. Five breeding animals with proven fertility were selected, the spermatozoa were collected, and the protein was extracted. Subsequently, the proteins were separated and analysed by two-dimensional electrophoresis and mass spectrometry; the proteins were then identified with the SwissProt database. A total of 31 proteins involved in reproduction were identified, including binding proteins on spermatozoa for fusion with the egg, acrosomal membrane proteins, metabolic enzymes, heat shock proteins, cytoskeletal proteins and spermatozoa motility proteins. The characterization of such proteins clarifies the molecular mechanisms of spermatogenesis and the modifications that ensure the success of fertilization.

Diversity of ejaculated sperm proteins in Moxotó bucks (Capra hircus ) evaluated by multiple extraction methods.

This study aimed to develop protocols for the extraction of sperm proteins from Moxotó goats (Capra hircus) and to compare the resulting proteomic maps. The sperm proteins were isolated using an extraction buffer containing 7 M urea and 2 M thiourea, 20 mM DTT, and one of the following detergents: 1% or 4% CHAPS; 1% or 4% SDS; 1% or 4% Triton X-100; or a combination of CHAPS and SDS. The 1-DE and 2-DE profiles of the isolated proteins revealed that the various isolation methods were efficient. Qualitative and quantitative differences in the 1-DE and 2-DE profiles were observed. 2-DE maps indicated that the amount and diversity of proteins visualized depended on the detergent that was used. Furthermore, this work revealed that the combination of detergents increased the resolution of some spots and retained the characteristics of the individual detergents, depending on their concentrations.

Purification, Biochemical Characterization, and Amino Acid Sequence of a Novel Type of Lectin from Aplysia dactylomela Eggs with Antibacterial/Antibiofilm Potential.

A new lectin from Aplysia dactylomela eggs (ADEL) was isolated by affinity chromatography on HCl-activated Sepharose™ media. Hemagglutination caused by ADEL was inhibited by several galactosides, mainly galacturonic acid (Ka = 6.05 × 106 M-1). The primary structure of ADEL consists of 217 residues, including 11 half-cystines involved in five intrachain and one interchain disulfide bond, resulting in a molecular mass of 57,228 ± 2 Da, as determined by matrix-assisted laser desorption/ionization time of flight mass spectrometry. ADEL showed high similarity with lectins isolated from Aplysia eggs, but not with other known lectins, indicating that these lectins could be grouped into a new family of animal lectins. Three glycosylation sites were found in its polypeptide backbone. Data from peptide-N-glycosidase F digestion and MS suggest that all oligosaccharides attached to ADEL are high in mannose. The secondary structure of ADEL is predominantly ß-sheet, and its tertiary structure is sensitive to the presence of ligands, as observed by CD. A 3D structure model of ADEL was created and shows two domains connected by a short loop. Domain A is composed of a flat three-stranded and a curved five-stranded ß-sheet, while domain B presents a flat three-stranded and a curved four-stranded ß-sheet. Molecular docking revealed favorable binding energies for interactions between lectin and galacturonic acid, lactose, galactosamine, and galactose. Moreover, ADEL was able to agglutinate and inhibit biofilm formation of Staphylococcus aureus, suggesting that this lectin may be a potential alternative to conventional use of antimicrobial agents in the treatment of infections caused by Staphylococcal biofilms.