Evaluation of the motility and capacity of biofilm production by Pseudomonas fluorescens strains in residual milk.

Pseudomonas fluorescens is known to have the ability to adhere and produce biofilm. The formation of biofilms is enhanced by cellular motility, particularly when mediated by flagella. Biofilm formed on surfaces such as those used for food production act as points of contamination, releasing pathogenic or deteriorating microorganisms and compromising the quality of products. We assessed two strains of Pseudomonas fluorescens PL5.4 and PL7.1, sampled from raw, chilled, buffalo milk, which was obtained from a dairy farm. Twitching and swarming motility assays were performed, in addition to the biofilm production evaluations at a temperature of 7 °C. Regarding the motility assays, only the PL5.4 strain scored positive for the swarming assay. On microplates, both strains presented themselves as strong biofilm producers at 7 °C. The PL5.4 strain was also able to form biofilm on a stainless steel structure and maintain this structure for up to 72 hours at refrigeration. The Pseudomonas fluorescens PL5.4 isolate was identified on the basis of a 99% sequence identity with Pseudomonas fluorescens A506, a strain used as a biocontrol in agriculture. Biofilm-forming bacteria, when adapted to low temperatures, become a constant source of contamination, damaging the production, quality, safety and shelf-life of products.

Characterization of the antimicrobial activity produced by Bacillus sp. isolated from wetland sediment.

Bacteria of the genus Bacillus sp. present the potential for inhibiting various pathogens, making them a promising starting point in the search for new antimicrobial substances. In this study, bacteria were isolated from sediment samples from humid areas of a Natural Conservation Unit in the state of Rio Grande do Sul, Brazil. The isolate Bacillus sp. sed 1.4 was selected for production of antimicrobial activity, and was characterized by MALDI-TOF and 16S rDNA sequencing. Phylogenetic analysis showed that Bacillus sed 1.4 was closely related to Bacillus altitudinis and Bacillus pumilus. The cell-free supernatant was partially purified using ammonium sulfate precipitation, gel filtration chromatography (Sephadex G-200) and an ultrafiltration membrane. Partial purification resulted in specific activity of 769.23 AU/mg, with a molecular mass of approximately 148 kDa. This antimicrobial substance showed stability at 100°C for 5 min, and was inactivated by proteolytic enzymes. An antimicrobial effect against Listeria species was observed. Considering the importance of the Listeria genus in the area of food safety, this antimicrobial activity should be further explored, specifically in the field of dairy products and with a focus on food biopreservation studies.

Biological activity of bacteria isolated from wetland sediments collected from a conservation unit in the southern region of Brazil.

Wetlands are ecosystems rich in biodiversity and their ecological importance is recognized worldwide. Sediment samples were subjected to physical-chemical analysis and organic carbon content varied from 3.0% to 4.8%, the clay between 32 and 40%, silt with 41% and 43%, sand coarse varied between 6 and 11% and fine sand between 7 and 16%. The nitrogen values ​​varied from 0.25% to 0.48%, the pH from 5.4 to 7.5 and the humidity ​​varied from 44 to 56%. The selected isolates were evaluated for enzymatic properties and 64% showed positive results for amylase, 16% for gelatinase, 37% for lipase, 91% for protease and 2.7% for inulinase. Six bacterial isolates were selected for the overlapping assay and Bacillus sp. sed 2.2 showed inhibitory activity against Corynebacterium fimi NCTC 7547, and the antimicrobial substance was partially purified. The characterization of the substance was carried and the substance was stable at 100° C for up to 10 minutes and sensitive to the enzymes papain and trypsin. This substance was active against some species of Listeria, including Listeria monocytogenes ATCC 7644. The microorganims obtained from sediment samples were important sources of bioactive compounds, including enzymes and peptides, being a source of bioactive compounds to be studied.

Microbiological and physicochemical characteristics of buffalo milk used for dairy products in southern Brazil.

In Brazil, the buffalo milk market has been growing. However, identity and quality standards have not been established for this raw material, nor have proper distinctions between buffalo milk and bovine milk been defined. Currently, the State of Rio Grande do Sul (RS) has only three producers that supply raw material for officially marketed derivatives. The aim of this study was to determine the identity and quality standards of raw buffalo milk in this region. Samples were obtained biweekly from three farm cooling tanks between June 2017 and August 2018, to reach a total of 69 samples. The averages for the results of the physicochemical parameters fat, protein, lactose, total solids, SNF (solids-not-fat), calcium, density, FP, acidity and SCC were 5.5 g/100 g, 4.06 g/100 g, 5.07 g/100 g, 15.5 g/100 g, 9.96 g/100 g, 0.161 g/100 g, 1.034 g/ml, -0.527°C, 16°D and 95 × 103 cells/ml, respectively. With reference to the microbiological parameters, the mean of the Standard Plate Count (SPC) and thermotolerant coliforms were 9,0 × 104 CFU/ml and 1.6 × 102 MPN/ml, respectively. Regarding coagulase-positive staphylococci, 36 samples tested positive (52% of total). Neither Salmonella spp. nor Listeria monocytogenes, nor antibiotic or antiparasitic residues were detected in any sample. In conclusion, the buffalo milk used as raw material for dairy products in southern Brazil demonstrated satisfactory physicochemical and microbiological characteristics, in accordance with recent scientific literature.

Evaluation of the Physicochemical and Antibacterial Properties of Experimental Adhesives Doped with Lithium Niobate.

The aim of the present study was to formulate dental adhesives with different concentrations of LiNbO3 and to evaluate their physicochemical and antibacterial properties. A dental adhesive was formulated using methacrylate monomers and photoinitiators and used as a control filler-free group. Subsequently, three experimental adhesives doped with LiNbO3 at different concentrations (1 wt.%, 2 wt.%, and 5 wt.%) were also formulated. All the experimental adhesives were assessed to evaluate the degree of conversion (DC), softening in solvent, immediate and long-term microtensile bond-strength (µ-TBS), radiopacity, ultimate tensile strength, and antibacterial activity. The incorporation of 1 wt.% of LiNbO3 had no negative effect on the DC of the adhesive resin compared to the control group (p > 0.05). We observed a decrease in the percentage of softening in solvent in the group LiNbO3 at 1 wt.% (p < 0.05). The addition of LiNbO3 increased the radiopacity at a concentration above 2 wt.%, and there was also an increase in cohesive strength (p < 0.05). The immediate µ-TBS increased for LiNbO3 at 5 wt.% (p < 0.05), and there was no statistical difference for the other groups compared to the control (p > 0.05). After six months, the group with 5 wt.% still presented the highest µ-TBS (p < 0.05). The adhesives showed no antimicrobial activity (p > 0.05). LiNbO3 was successfully incorporated in dental adhesives, increasing the radiopacity and their resistance to degradation. Although LiNbO3 offered no antibacterial properties, the reliability of LiNbO3 incorporation in the adhesive encourages new tests to better investigate the antimicrobial action of LiNbO3 through temperature variation.

Antibacterial activity of bacteriocin-like substance P34 on Listeria monocytogenes in chicken sausage

The antimicrobial activity of the bacteriocin-like substance (BLS) P34 against Listeria monocytogenes was investigated in chicken sausage. The BLS was applied to chicken sausages (256 AU g-1) previously inoculated with a suspension of 10² cfu g-1 of L. monocytogenes. BLS P34 inhibited the indicator microorganism in situ in all incubation times for up to 10 days at 5 °C. The effectiveness of BLS P34 was increased when it was added in combination with nisin. The bacteriocin was also tested in natural eatable natural bovine wrapping (salty semi-dried tripe) against the same indicator microorganism, also showing inhibitory capability in vitro. BLS P34 showed potential to control L. monocytogenes in refrigerated meat products.

Antibacterial activity of bacteriocin-like substance P34 on Listeria monocytogenes in chicken sausage.

The antimicrobial activity of the bacteriocin-like substance (BLS) P34 against Listeria monocytogenes was investigated in chicken sausage. The BLS was applied to chicken sausages (256 AU g(-1)) previously inoculated with a suspension of 10(2) cfu g(-1) of L. monocytogenes. BLS P34 inhibited the indicator microorganism in situ in all incubation times for up to 10 days at 5 °C. The effectiveness of BLS P34 was increased when it was added in combination with nisin. The bacteriocin was also tested in natural eatable natural bovine wrapping (salty semi-dried tripe) against the same indicator microorganism, also showing inhibitory capability in vitro. BLS P34 showed potential to control L. monocytogenes in refrigerated meat products.

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.

Antibacterial activity of a bacteriocin-like substance produced by Bacillus sp. P34 that targets the bacterial cell envelope.

The objective of this study was to investigate the mode of action of BLS P34, a bacteriocin-like substance (BLS) produced by a novel Bacillus sp. strain P34 isolated from the Amazon basin. The effect of the BLS was tested against Listeria monocytogenes, showing a bactericidal effect at 200 AU (activity units) ml(-1), while no inhibition of spore outgrowth of Bacillus cereus was observed with a dose of 1,600 AU ml(-1). Growth of Escherichia coli and Salmonella Enteritidis was inhibited, but only when the chelating agent EDTA was co-added with the BLS. The effect of BLS P34 on L. monocytogenes was also investigated by Fourier transform infrared spectroscopy. Treated cells showed an important frequency increase in 1,452 and 1,397 cm(-1) and decrease in 1,217 and 1,058 cm(-1), corresponding assignments of fatty acids and phospholipids. Transmission electron microscopy showed damaged cell envelope and loss of protoplasmic material. BLS P34 was bactericidal to Gram-positive, and also showed inhibitory effect against Gram-negative bacteria. There is evidence that its mode of action corresponds to that of a membrane-active substance. The knowledge about the mode of action of this BLS is essential to determine its effective application as an antimicrobial agent.

Characterization of a broad range antibacterial substance from a new Bacillus species isolated from Amazon basin.

A Bacillus sp. strain producing a bacteriocin-like substance was characterized by biochemical profiling and 16S rDNA sequencing. The phylogenetic analysis indicated that this strain has low sequence similarity with most Bacillus spp., suggesting a new species was isolated. The antimicrobial activity was detected starting at the exponential growth phase, and maximum activity was observed at stationary phase. The substance was inhibitory to a broad range of indicator strains, including pathogenic and food spoilage bacteria such as Listeria monocytogenes, B. cereus, Aeromonas hydrophila, Erwinia carotovora, Pasteurella haemolytica, Salmonella Gallinarum, among other. The antibacterial substance was stable over a wide pH range, but it was sensitive to pronase E and lipase. The antibacterial substance was bactericidal and bacteriolytic to L. monocytogenes and B. cereus at 160 AU ml(-1). The identification of a broad range bacteriocin-like inhibitory substance active against L. monocytogenes addresses an important aspect of food protection against pathogens and spoilage microorganisms.

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.

Bacteriocin-like substance inhibits potato soft rot caused by Erwinia carotovora.

Soft rot is a major problem encountered in potatoes during postharvest storage. The soft rot bacterium Erwinia carotovora was inhibited by a novel bacteriocin-like substance (BLS) produced by Bacillus licheniformis P40. The BLS caused a bactericidal effect on E. carotovora cells at 30 microg mL(-1). Transmission electron microscopy showed that BLS-treated cells presented wrinkled bacterial surfaces and shrinkage of the whole cell, indicating plasmolysis. Erwinia carotovora cells treated with BLS were analyzed by FTIR showing differences in the 1390 cm(-1) and 1250-1220 cm(-1) bands, corresponding to assignments of membrane lipids. BLS was effective in preventing E. carotovora spoilage on potato tubers, reducing the symptoms of soft rot at 240 microg mL(-1) and higher concentrations. Soft rot development was completely blocked at 3.7 mg mL(-1). This BLS showed potential to protect potato tubers during storage.

Antibacterial activity of cerein 8A, a bacteriocin-like peptide produced by Bacillus cereus.

The mode of action of cerein 8A, a bacteriocin produced by the soil bacterium Bacillus cereus 8A, was investigated. The effect of cerein 8A was tested against Listeria monocytogenes and a bactericidal effect at 400 arbitrary units (AU)/ml was observed. In addition, cerein 8A was bactericidal against Bacillus cereus at 200 AU/ml, and inhibited the growth of Escherichia coli and Salmonella Enteritidis. Stronger inhibition of these gram-negative bacteria was achieved when the chelating agent EDTA was added together with bacteriocin. The effect of cerein 8A on B. cereus and L. monocytogenes was also investigated by Fourier transform infrared spectroscopy (FTIR). Treated cells had an important frequency increase at 2920 cm-1 and a decrease at 1400 cm-1, corresponding to assignments of fatty acids. Transmission electron microscopy showed damaged cell walls and loss of protoplasmic material. These results suggest that the mode of action of cerein 8A is to interfere with cell membranes and the cell wall.

Antibacterial activity of cerein 8A, a bacteriocin-like peptide produced by Bacillus cereus / Actividad antibacteriana de la cereína 8A, un péptido de tipo bacteriocina producido por Bacillus cereus

The mode of action of cerein 8A, a bacteriocin produced by the soil bacterium Bacillus cereus 8A, was investigated. The effect of cerein 8A was tested against Listeria monocytogenes and a bactericidal effect at 400 arbitrary units (AU)/ml was observed. In addition, cerein 8A was bactericidal against Bacillus cereus at 200 AU/ml, and inhibited the growth of Escherichia coli and Salmonella Enteritidis. Stronger inhibition of these gram-negative bacteria was achieved when the chelating agent EDTA was added together with bacteriocin. The effect of cerein 8A on B. cereus and L. monocytogenes was also investigated by Fourier transform infrared spectroscopy (FTIR). Treated cells had an important frequency increase at 2920 cm-1 and a decrease at 1400 cm-1, corresponding to assignments of fatty acids. Transmission electron microscopy showed damaged cell walls and loss of protoplasmic material. These results suggest that the mode of action of cerein 8A is to interfere with cell membranes and the cell wall (AU)

Se investigó el modo de acción de la cereína 8A, una bacteriocina producida por la bacteria del suelo Bacillus cereus 8A. El efecto de la cereína 8A fue probado contra Listeria monocytogenes, obteniendo un efecto bactericida a concentraciones de 400 unidades arbitrarias (AU)/ml. La cereína 8A también tuvo un efecto bactericida contra Bacillus cereus a una concentración de 200 AU/ml. La bacteriocina inhibió el crecimiento de Escherichia coli y Salmonella Enteritidis. Mayor inhibición contra estas bacterias gram-negativas se consiguió cuando a la bacteriocina se le añadió el agente quelante EDTA. El efecto de la cereína 8A sobre B. cereus y L. monocytogenes también fue investigado por espectroscopía de infrarrojos de transformación de Fourier (FTIR). Las células tratadas mostraron un importante crecimiento en frecuencia de 2920 cm-1 y un decrecimiento de 1400 cm-1 de banda, correspondiéndose con la asignación de los ácidos grasos. La microscopía electrónica de transmisión mostró que las células habían padecido daños en la pared celular, con pérdida de material protoplásmico. Los resultados sugieren que el modo de acción de la cereína 8A se produce mediante su intervención en las membranas celulares y en la pared celular (AU)

Screening for antimicrobial activity among bacteria isolated from the Amazon Basin

Bactérias produtoras de atividade antimicrobiana foram identificadas entre 86 isolados de ambientes aquáticos da Bacia Amazônica. Destes, 59 isolados (68.6 per center) apresentaram atividade antimicrobiana contra pelo menos uma bactéria indicadora. A atividade inibitória foi principalmente observada contra bactérias Gram-positivas, como Listeria monocytogenes e Bacillus cereus. As substâncias antimicrobianas produzidas por 19 linhagens que demonstraram maior atividade inibitória foram parcialmente caracterizadas, apresentando resistência térmica até 100ºC e resistência parcial ao tratamento proteolítico. Algumas substâncias foram parcialmente inativadas somente quando tratadas com ácido tricloroacético ou com pronase E na concentração de 2 mg ml-1. A detecção da atividade antimicrobiana em géis de poliacrilamida mostrou que os compostos apresentaram peso molecular inferior à 14 kDa. Várias linhagens apresentaram atividade antibacteriana, que em alguns casos estaria relacionada com peptídios antimicrobianos. O potencial destes microrganismos para produzir substâncias antimicrobianas é grande e merece ser mais explorado.