Identification of synergistic interactions among microorganisms in biofilms by digital image analysis

Digital image analysis showed that reductions in biofilm plating efficiency were due to the loss of protection provided by two benzoate-degrading strains of Pseudomonas fluorescens. This loss in protection was due to the spatial separation of the protective organisms from benzoate-sensitive organisms during the dilution process. Communities were cultivated in flow cells irrigated with trypticase soy broth. When the effluent from these flow cells was plated on 0.15% benzoic acid, satellite colonies formed only in the vicinity of primary colonies. A digital image analysis procedure was developed to measure the size and spatial distribution of these satellites as a function of distance from the primary colony. The size of satellites served as a measure of growth, and the number per unit area served as a measure of survival. At the three dilutions tested, the size and concentration of satellite colonies varied inversely with distance from the primary colonies. When these measurements were plotted, the slopes were used to quantify the effect of bacterial association on the growth and survivability of the satellites. In the absence of the primary colonies, satellites grew in axenic culture only at low benzoate concentrations. Thus benzoate-degrading organisms are capable of creating a protective microenvironment for other members of biofilm communities

Effect of Lactobacillus plantarum Fermentation on the Surface and Functional Properties of Pea Protein-Enriched Flour

Istražen je utjecaj fermentacije s pomoću Lactobacillus plantarum na funkcionalna i fizikalno-kemijska svojstva brašna graška obogaćenog proteinima. Tijekom fermentacije povećavao se stupanj hidrolize do maksimuma od 13,5 % nakon 11 h. Prije mjerenja površinske hidrofobnosti i naboja te ispitivanja funkcionalnih svojstava podešena je pH-vrijednost fermentiranog brašna na pH=4 ili 7. Pri pH=4 površinski naboj, mjeren pomoću zeta potencijala, povećao se s +14 na +27 mV nakon 1 h fermentacije, a zatim smanjio na +10 mV nakon 11 h, dok se pri pH=7 naboj postepeno povećavao s -37 na -27 mV tijekom fermentacije. Površinska hidrofobnost znatno se smanjila tijekom fermentacije pri pH=4, dok se pri pH=7 neznatno smanjila. Kapacitet pijenjenja bio je najveći u emulziji brašna fermentiranog tijekom 5 h pri pH=4, dok je stabilnost pjene bila niska pri obje pH-vrijednosti u svim uzorcima. Emulgirajuća svojstva naglo su se smanjila nakon 5 h fermentacije pri pH=4, a stabilnost emulzija poboljšala se nakon 5 h fermentacije pri pH=7, u usporedbi s kontrolom. Sposobnost vezivanja ulja povećala se s 1,8 g/g pri 0 h na 3,5 g/g nakon 11 h fermentacije, a vode smanjila nakon 5 h, te zatim povećala nakon 9 h fermentacije. Rezultati pokazuju da se fermentacijom brašna graška obogaćenog proteinima mogu mijenjati njegova svojstva te na taj način proizvesti novi funkcionalni sastojci.The effect of Lactobacillus plantarum fermentation on the functional and physicochemical properties of pea protein-enriched flour (PPF) was investigated. Over the course of the fermentation the extent of hydrolysis increased continuously until reaching a maximum degree of hydrolysis of 13.5 % after 11 h. The resulting fermented flour was then adjusted to either pH=4 or 7 prior to measuring the surface and functional attributes as a function of fermentation time. At pH=4 surface charge, as measured by zeta potential, initially increased from +14 to +27 mV after 1 h of fermentation, and then decreased to +10 mV after 11 h; whereas at pH=7 the charge gradually increased from –37 to –27 mV over the entire fermentation time. Surface hydrophobicity significantly increased at pH=4 as a function of fermentation time, whereas at pH=7 fermentation induced only a slight decrease in PPF surface hydrophobicity. Foam capacity was highest at pH=4 using PPF fermented for 5 h whereas foam stability was low at both pH values for all samples. Emulsifying activity sharply decreased after 5 h of fermentation at pH=4. Emulsion stability improved at pH=7 after 5 h of fermentation as compared to the control. Oil-holding capacity improved from 1.8 g/g at time 0 to 3.5 g/g by the end of 11 h of fermentation, whereas water hydration capacity decreased after 5 h, then increased after 9 h of fermentation. These results indicate that the fermentation of PPF can modify its properties, which can lead towards its utilization as a functional food ingredient

Effect of Fermentation on the Protein Digestibility and Levels of Non-Nutritive Compounds of Pea Protein Concentrate

Radi utvrđivanja utjecaja fermentacije na kakvoću proteina u koncentratu proteina graška ispitani su sljedeći parametri: udjel ukupnih fenola i tanina, aktivnost inhibitora proteaze, sastav aminokiselina i probavljivost proteina in vitro nakon 11 sati fermentacije s pomoću bakterije Lactobacillus plantarum. Maseni se udjel fenola u koncentratu proteina graška, izražen kao ekvivalent katehina, povećao na bazi suhe tvari s 2,5 pri 0 h na 4,9 mg/g nakon 11 sati fermentacije. Udjel tanina se povećao s 0,14 pri 0 h na maksimalnih 0,96 mg/g koncentrata nakon 5 h fermentacije, a zatim se smanjio na 0,79 mg/g nakon 11 h fermentacije. Nakon 9 h fermentacije smanjila se aktivnost inhibitora tripsina, međutim, pri svim ostalim vremenima fermentacije dobivene su vrijednosti slične onima pri 0 h. Aktivnost se inhibitora kimotripsina smanjila s 3,7 na 1,1 jedinicu inhibicije kimotripsina po mg nakon 11 sati fermentacije. Probavljivost je proteina dosegla maksimalnu vrijednost od 87,4 % nakon 5 sati fermentacije, međutim vrijednost aminokiselina koje sadržavaju sumpor smanjila se s 0,84 pri 0 h na 0,66 nakon 11 h fermentacije. Smanjenjem udjela sumpora promijenila se vrijednost aminokiselina korigirana probavljivošću proteina in vitro s 67,0 pri 0 h na 54,6 % nakon 11 h fermentacije. Dobiveni podaci potvrđuju da je, iako je fermentacija valjana metoda za smanjenje udjela nekih nenutritivnih sastojaka u koncentratu proteina graška, potrebno odabrati odgovarajuće bakterije koje nemaju izraženu sposobnost razgradnje aminokiselina što sadržavaju sumpor.In order to determine the impact of fermentation on protein quality, pea protein concentrate (PPC) was fermented with Lactobacillus plantarum for 11 h and total phenol and tannin contents, protease inhibitor activity, amino acid composition and in vitro protein digestibility were analyzed. Phenol levels, expressed as catechin equivalents (CE), increased on dry mass basis from 2.5 at 0 h to 4.9 mg CE per 1 g of PPC at 11 h. Tannin content rose from 0.14 at 0 h to a maximum of 0.96 mg CE per 1 g of PPC after 5 h, and thereafter declined to 0.79 mg/g after 11 h. After 9 h of fermentation trypsin inhibitor activity decreased, however, at all other fermentation times similar levels to the PPC at time 0 h were produced. Chymotrypsin inhibitor activity decreased from 3.7 to 1.1 chymotrypsin inhibitory units (CIU) per mg following 11 h of fermentation. Protein digestibility reached a maximum (87.4 %) after 5 h of fermentation, however, the sulfur amino acid score was reduced from 0.84 at 0 h to 0.66 at 11 h. This reduction in sulfur content altered the in vitro protein digestibility-corrected amino acid score from 67.0 % at 0 h to 54.6 % at 11 h. These data suggest that while fermentation is a viable method of reducing certain non-nutritive compounds in pea protein concentrate, selection of an alternative bacterium which metabolises sulfur amino acids to a lesser extent than L. plantarum should be considered

Origin and Epidemiological History of HIV-1 CRF14_BG

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Users must also make clear the license terms under which the work was published. CC BY Licence: http://creativecommons.org/licenses/by/4.0/Background: CRF14_BG isolates, originally found in Spain, are characterized by CXCR4 tropism and rapid disease progression. This study aimed to identify the origin of CRF14_BG and reconstruct its epidemiological history based on new isolates from Portugal.Methodology/Principal Findings: C2V3C3 env gene sequences were obtained from 62 samples collected in 1993–1998 from Portuguese HIV-1 patients. Full-length genomic sequences were obtained from three patients. Viral subtypes, diversity, divergence rate and positive selection were investigated by phylogenetic analysis. The molecular structure of the genomes was determined by bootscanning. A relaxed molecular clock model was used to date the origin of CRF14_BG. Geno2pheno was used to predict viral tropism. Subtype B was the most prevalent subtype (45 sequences; 73%) followed by CRF14_BG (8; 13%), G (4; 6%), F1 (2; 3%), C (2; 3%) and CRF02_AG (1; 2%). Three CRF14_BG sequences were derived from 1993 samples. Near full-length genomic sequences were strongly related to the CRF14_BG isolates from Spain. Genetic diversity of the Portuguese isolates was significantly higher than the Spanish isolates (0.044 vs 0.014, P,0.0001). The mean date of origin of the CRF14_BG cluster was estimated to be 1992 (range, 1989 and 1996) based on the subtype G genomic region and 1989 (range, 1984–1993) based on the subtype B genomic region. Most CRF14_BG strains (78.9%) were predicted to be CXCR4. Finally, up to five amino acids were under selective pressure in subtype B V3 loop whereas only one was found in the CRF14_BG cluster.Conclusions: CRF14_BG emerged in Portugal in the early 1990 s soon after the beginning of the HIV-1 epidemics, spread to Spain in late 1990 s as a consequence of IVDUs migration and then to the rest of Europe. CXCR4 tropism is a general characteristic of this CRF that may have been selected for by escape from neutralizing antibody response

Prevalence of Escherichia coli O157 in Saskatchewan Cattle: Characterization of Isolates by Using Random Amplified Polymorphic DNA PCR, Antibiotic Resistance Profiles, and Pathogenicity Determinants

The prevalence of Escherichia coli O157 associated with feedlot cattle in Saskatchewan was determined in a 10-month longitudinal study (3 feedlots) and a point prevalence study (20 feedlots). The prevalence of E. coli O157 at the three different sites in the horizontal study varied from 2.5 to 45%. The point prevalence of E. coli O157 among Saskatchewan cattle from 20 different feedlots ranged from 0% to a high of 57%. A statistically significant (P = 0.003) positive correlation was determined to exist between the density of cattle and the E. coli O157 prevalence rate. A significant correlation (P = 0.006) was also found between the E. coli O157 percent prevalence and the number of cattle housed/capacity ratio. All 194 E. coli O157 isolates obtained were highly virulent, and random amplified polymorphic DNA PCR analysis revealed that the isolates grouped into 39 different E. coli O157 subtypes, most of which were indigenous to specific feedlots. Two of the most predominant subtypes were detected in 11 different feedlots and formed distinct clusters in two geographic regions in the province. Antimicrobial susceptibility testing of the E. coli O157 isolates revealed that 10 were multidrug resistant and that 73 and 5 were resistant to sulfisoxazole and tetracycline, respectively

Adaptive Resistance and Differential Protein Expression of Salmonella enterica Serovar Enteritidis Biofilms Exposed to Benzalkonium Chloride

The development of adaptive resistance of Salmonella enterica serovar Enteritidis ATCC 4931 biofilms following exposure to benzalkonium chloride (BC) either continuously (1 μg ml(−1)) or intermittently (10 μg ml(−1) for 10 min daily) was examined. Biofilms adapted to BC over a 144-h period could survive a normally lethal BC challenge (500 μg ml(−1) for 10 min) and then regrow, as determined by increases in biofilm thickness, total biomass, and the ratio of the viable biomass to the nonviable biomass. Exposure of untreated control biofilms to the lethal BC challenge resulted in biofilm erosion and cell death. Proteins found to be up-regulated following BC adaptation were those involved in energy metabolism (TpiA and Eno), amino acid and protein biosynthesis (WrbA, TrxA, RplL, Tsf, Tuf, DsbA, and RpoZ), nutrient binding (FruB), adaptation (CspA), detoxification (Tpx, SodB, and a probable peroxidase), and degradation of 1,2-propanediol (PduJ and PduA). A putative universal stress protein (YnaF) was also found to be up-regulated. Proteins involved in proteolysis (DegQ), cell envelope formation (RfbH), adaptation (UspA), heat shock response (DnaK), and broad regulatory functions (Hns) were found to be down-regulated following adaptation. An overall increase in cellular protein biosynthesis was deduced from the significant up-regulation of ribosomal subunit proteins, translation elongation factors, and amino acid biosynthesis protein and down-regulation of serine endoprotease. The cold shock response, stress response, and detoxification are suggested to play roles in the adaptive resistance of Salmonella serovar Enteritidis biofilms to BC

Treatment of Salmonella enterica Serovar Enteritidis with a Sublethal Concentration of Trisodium Phosphate or Alkaline pH Induces Thermotolerance

The responses of Salmonella enterica serovar Enteritidis to a sublethal dose of trisodium phosphate (TSP) and its equivalent alkaline pH made with NaOH were examined. Pretreatment of S. enterica serovar Enteritidis cells with 1.5% TSP or pH 10.0 solutions resulted in a significant increase in thermotolerance, resistance to 2.5% TSP, resistance to high pH, and sensitivity to acid and H(2)O(2). Protein inhibition studies with chloramphenicol revealed that thermotolerance, unlike resistance to high pH, was dependent on de novo protein synthesis. Two-dimensional polyacrylamide gel electrophoresis (PAGE) of total cellular proteins from untreated control cells resolved as many as 232 proteins, of which 22 and 15% were absent in TSP- or alkaline pH-pretreated cells, respectively. More than 50% of the proteins that were either up- or down-regulated by TSP pretreatment were also up- or down-regulated by alkaline pH pretreatment. Sodium dodecyl sulfate-PAGE analysis of detergent-insoluble outer membrane proteins revealed the up-regulation of at least four proteins. Mass spectrometric analysis showed the up-regulated proteins to include those involved in the transport of small hydrophilic molecules across the cytoplasmic membrane and those that act as chaperones and aid in the export of newly synthesized proteins by keeping them in open conformation. Other up-regulated proteins included common housekeeping proteins like those involved in amino acid biosynthesis, nucleotide metabolism, and aminoacyl-tRNA biosynthesis. In addition to the differential expression of proteins following TSP or alkaline pH treatment, changes in membrane fatty acid composition were also observed. Alkaline pH- or TSP-pretreated cells showed a higher saturated and cyclic to unsaturated fatty acid ratio than did the untreated control cells. These results suggest that the cytoplasmic membrane could play a significant role in the induction of thermotolerance and resistance to other stresses following TSP or alkaline pH treatment

High pH during Trisodium Phosphate Treatment Causes Membrane Damage and Destruction of Salmonella enterica Serovar Enteritidis

Trisodium phosphate (TSP) is now widely used during the processing of poultry and red meats, but the mechanism whereby it inactivates gram-negative bacteria such Salmonella spp. remains unclear. Thus, Salmonella enterica serovar Enteritidis (ATCC 4931) cells were treated with different concentrations of TSP (1.5, 2.0, and 2.5% [wt/vol]) and compared with (i) cells treated with the same pH as the TSP treatments (pH 10.0, 10.5, and 11.0, respectively) and (ii) cells treated with different concentrations of TSP (1.5, 2.0, and 2.5% [wt/vol]) adjusted to a pH of 7.0 ± 0.2 (mean ± standard deviation). Cell viability, loss of membrane integrity, cellular leakage, release of lipopolysaccharides, and cell morphology were accordingly examined and quantified under the above treatment conditions. Exposure of serovar Enteritidis cells to TSP or equivalent alkaline pH resulted in the loss of cell viability and membrane integrity in a TSP concentration- or alkaline pH-dependent manner. In contrast, cells treated with different concentrations of TSP whose pH was adjusted to 7.0 did not show any loss of cell viability or membrane integrity. A 30-min pretreatment with 1.0 mM EDTA significantly enhanced the loss of membrane integrity only when followed by TSP or alkaline pH treatments. Measuring the absorbance at 260 nm, agarose gel electrophoresis, Bradford assay, and Tricine-sodium dodecyl sulfate gel electrophoresis of filtrates of treated cell suspensions revealed considerable release of DNA, proteins, and lipopolysaccharides compared to controls and pH 7.0 TSP treatments. Electron microscopic examination of TSP- or alkaline pH-treated cells showed disfigured cell surface topology and wrinkled appearance and showed evidence of a TSP concentration- and pH-dependent disruption of the cytoplasmic and outer membranes. These results demonstrate that TSP treatment permeabilizes and disrupts the cytoplasmic and outer membranes of serovar Enteritidis cells because of the alkaline pH, which in turn leads to release of intracellular contents and eventual cell death