Effects of dietary supplementation with fenugreek seeds, alone or in combination with probiotics, on gilthead seabream (Sparus aurata L.) skin mucosal immunity.

Despite increasing interest in modulating the immune response of fish, providing a combination of probiotics and herbal immunostimulants in aquafeed has rarely has been studied. The effects on gilthead seabream (Sparus aurata L.) of the dietary administration of fenugreek (Trigonella foenum graecum) seeds alone (FE), or combined with one of the following probiotic strains: Bacillus licheniformis (FEBL), Lactobacillus plantarum (FELP) or Bacillus subtilis (FEBS) were evaluated. Fish were fed a control or one of the supplemented diets for 3 weeks. After 2 and 3 weeks of the feeding trial, the abundance of terminal carbohydrates, IgM levels, enzymatic activities (proteases, alkaline phosphatase, esterase and ceruloplasmin) and bactericidal activity were determined in skin mucus. Our results demonstrated that the dietary administration of FE in combination with L. plantarum, particularly, increased carbohydrate abundance, the activity of certain enzymes such as ceruloplasmin, and bactericidal activity against the pathogenic bacterium Photobacterium damselae and the non-pathogenic bacterium B. subtilis in skin mucus at the end of the trial. The carbohydrates most affected by the FELP diet were mannose/glucose, N-acetyl-d-galactosamine and N-acetyl-ß-d-glucosamine. Interestingly, IgM levels were significantly higher in fish fed the FELP and FEBS diets whilst protease activity generally increased in all supplemented diets, which could suggests that the main effect in this activity was to the result of FE supplementation although that fact cannot be confirmed because the effects of probiotics addition alone were not studied. These results suggest that the combined dietary administration of fenugreek and L. plantarum will best enhance the skin mucosal immunity response of gilthead seabream.

Quality and antioxidant response of gilthead seabream (Sparus aurata L.) to dietary supplements of fenugreek (Trigonella foenum graecum) alone or combined with probiotic strains.

The present study was conducted to determine the potential effect of the dietary intake of fenugreek (Trigonella foenum graecum) seeds alone or in combination with Bacillus licheniformis, Lactobacillus plantarum or B. subtilis on gilthead seabream quality and antioxidant response after 2 and 3 weeks of experimental feeding. The results showed that the supplements did not affect the percentage of the fatty acid profiles of muscle, demonstrating that all the additives tested can be administrated without any negative effect on biochemical composition and quality of gilthead seabream. The quantification of thiobarbituric acid reactive substances in muscle demonstrated the significant beneficial effect of the experimental diets compared with the control one. Besides, an increase in superoxide dismutase and catalase in liver was recorded after 3 weeks of administration of experimental diets. Furthermore, real time qPCR revealed that dietary supplementation with FEBS significantly enhances the expression of scavenging enzymes, such as cat and gr genes in the liver after 3 weeks. The findings suggest that the administration of fenugreek supplement alone or combined with probiotic strains could be considered as a good source of natural antioxidants and as a functional aquafeed ingredient for gilthead seabream.

Effects of dietary administration of fenugreek seeds, alone or in combination with probiotics, on growth performance parameters, humoral immune response and gene expression of gilthead seabream (Sparus aurata L.).

The use of immunostimulants is considered a promising preventive practice that may help to maintain animal welfare and a healthy environment, while increasing production and providing higher profits. The purpose of this study was to evaluate the effects on gilthead seabream (Sparus aurata L.) of the dietary administration of fenugreek (Trigonella foenum graecum) seeds, alone or combined with one of the following probiotic strains: Bacillus licheniformis (TSB27), Lactobacillus plantarum or Bacillus subtilis (B46). Gilthead seabream were fed a control or one of the supplemented diets for 3 weeks. The effects of these supplemented diets on growth performance parameters and the humoral immune response (natural haemolytic complement, peroxidase, total IgM levels, proteases and antiproteases activities) were evaluated after 2 and 3 weeks of feeding. Simultaneously, the expression levels of some immune-relevant genes (igm, tcr-ß, csfr1 and bd) were measured in the head-kidney. Interestingly, all probiotic supplemented diets increased seabream growth rates, especially the B. licheniformis supplemented diet. Generally, humoral immune parameters were enhanced by the dietary supplementation at the different time points measured. The results showed a significant increases in the immune parameters, principally in fish fed only fenugreek or fenugreek combined with B. subtilis. Furthermore, real time qPCR revealed that dietary supplementation significantly enhances the expression of immune-associated genes in the head-kidney, particularly igm gene expression. These results suggest that fenugreek alone or combined with one of the probiotic strains mentioned enhances the immune response of gilthead seabream, a species with one of the highest rates of production in marine aquaculture.

Acid stress suggests different determinants for polystyrene and HeLa cell adhesion in Lactobacillus casei.

Adhesion has been regarded as one of the basic features of probiotics. The aim of this study was to investigate the influence of acid stress on the functional properties, such as hydrophobicity, adhesion to HeLa cells, and composition of membrane fatty acids, of Lactobacillus probiotics strains. Two strains of Lactobacillus casei were used. Adhesion on polystyrene, hydrophobicity, epithelial cells adhesion, and fatty acids analysis were evaluated. Our results showed that the membrane properties such as hydrophobicity and fatty acid composition of stressed strains were significantly changed with different pH values. However, we found that acid stress caused a change in the proportions of unsaturated and saturated fatty acid. The ratio of saturated fatty acid to unsaturated fatty acids observed in acid-stressed Lactobacillus casei cells was significantly higher than the ration in control cells. In addition, we observed a significant decrease in the adhesion ability of these strains to HeLa cells and to a polystyrene surface at low pH. The present finding could first add new insight about the acid stress adaptation and, thus, enable new strategies to be developed aimed at improving the industrial performance of this species under acid stress. Second, no relationship was observed between changes in membrane composition and fluidity induced by acid treatment and adhesion to biotic and abiotic surfaces. In fact, the decrease of cell surface hydrophobicity and the adhesion ability to abiotic surface and the increase of the capacity of adhesion to biotic surface demonstrate that adhesive characteristics will have little relevance in probiotic strain-screening procedures.

Effect of dietary supplementation of probiotics and palm fruits extracts on the antioxidant enzyme gene expression in the mucosae of gilthead seabream (Sparus aurata L.).

Antioxidant activity is particularly important, since oxidation is an unavoidable reaction in all living bodies. At present, natural antioxidants to be used on food as an alternative to synthetic ones are being sought. Gilthead seabream (Sparus aurata L.) specimens were fed for 4 weeks with diets enriched with bacterial probiotics (Shewanella putrefaciens Pdp11 and Bacillus sp), single or in combination with Tunisian dates palm fruit extracts. The expression of the main antioxidant enzyme genes (superoxide dismutase, catalase and glutathione reductase) in the mucosae (gut, skin and gill) was evaluated after 2 and 4 weeks. Previously, free radical scavenging and several antioxidant assays were developed to know the antioxidant properties present on the palm fruits extracts. The results demonstrated that experimental diets alter the expression of the studied antioxidant genes, primarily in the gill and skin. Furthermore, the tested probiotics and mainly, the aqueous date palm fruits extracts had significant antioxidant properties based on their protective effect against the levels of intracellular reactive oxygen species, especially when administering during 4 weeks. For this reason, probiotics and date palm fruit extracts may serve as good natural antioxidants and could potentially be considered as a functional food ingredient for fish in farms.

Preliminary characterization of the probiotic properties of Candida famata and Geobacillus thermoleovorans.

BACKGROUND AND OBJECTIVE: Probiotics are live microbial feed supplements which beneficially affect the host animal by improving its intestinal microbial balance, producing metabolites which inhibit the colonization or growth of other microorganisms or by competing with them for resources such as nutrients or space. The aim of this study was to investigate the probiotic properties of Candida famata and Geobacillus thermoleovorans. MATERIAL AND METHODS: In this study, yeast and bacterial strains isolated from pure oil waste were identified using Api 50 CHB and Api Candida Systems and their probiotic properties were studied through antimicrobial activity, biofilm production, adherence assay and enzymatic characterization. RESULTS AND CONCLUSION: According to biochemical analyses, these strains corresponded to Geobacillus thermoleovorans and Candida famata. Antagonism assay results showed that the tested strains have an inhibitory effect against tested pathogenic bacteria. The yeast Candida famata was unable to produce biofilm on Congo Red Agar (CRA), while the bacterial strain was a slime producer. Adherence assays to abiotic surfaces revealed that the investigated strains were fairly adhesive to polystyrene with values ranging from 0.18 to 0.34 at 595 nm. The enzymatic characterization revealed that the tested strains expressed enzymes such as phosphatase alkaline, esterase lipase (C8), amylase, lipase, lecitenase and caseinase. The obtained results may allow the isolated strains to be considered as having the potential to be candidate probiotics.

Antifungal properties of Salvadora persica and Juglans regia L. extracts against oral Candida strains.

We report in this work, and for the first time, the potent antifungal activities of Salvadora persica and Juglans regia L. on different Candida species. Methanol, ethyl acetate, and diluted acetone extracts of S. persica (fresh and dry plant) and J. regia L. were screened for in vitro activity against some Candida species. These plants were selected due to their traditional use for the treatment of oral infections. Plant preparations were screened for antifungal activity using a standard agar disc diffusion assay. Following study of the antifungal activity of plant extracts, their minimal inhibitory concentration (MIC) and minimal fungicidal concentration (MFC) values were determined using broth microdilution assay. Among S. persica and J. regia L. extracts, ethyl acetate J. regia L. extract had potent antifungal activity against all Candida strains. The MIC values of the J. regia L. against Candida strains ranged from 0.006 to 0.195 mg/ml. Two C. albicans strains showed a high MIC value (3.125 mg/ml). These results indicate that extracts can contain compounds with therapeutic potential against Candida strains and, hence, their possible use as therapeutic agents.

Molecular typing of clinical Candida strains using random amplified polymorphic DNA and contour-clamped homogenous electric fields electrophoresis.

AIMS: This report describes an investigation into the genetic profiles of 38 Candida albicans and 19 Candida glabrata strains collected from a dental hospital of Monastir (Tunisia) and the Laboratory of Parasitology, Farhat Hached Hospital of Sousse (Tunisia), using two typing methods: random amplified polymorphic DNA (RAPD) and contour-clamped homogenous electric fields (CHEF). METHODS AND RESULTS: The two methods (RAPD and CHEF electrophoresis) were able to identify clonal-related isolates from different patients. RAPD method using two primers (CA1 and CA2) exhibited the highest discriminatory power by discriminating 22 genotypes for C. albicans with CA1 oligonucleotides and 19 genotypes with CA2 primer. For C. glabrata, 17 genotypes were obtained when both primers CA1 and CA2 were combined. The CHEF karyotyping of C. albicans has discriminated only 17 different karyotypes. CONCLUSION: The genotype of each isolate and genotypic difference among C. albicans and C. glabrata isolates were patient specific and not associated with the site of infection, geographic origin or date of isolation. SIGNIFICANCE AND IMPACT OF THE STUDY: Identification of relatedness between Candida species using molecular approaches with high discriminatory power is important in determining adequate measures for interruption of transmission of this yeast.

High potential of adhesion to abiotic and biotic materials in fish aquaculture facility by Vibrio alginolyticus strains.

AIMS: The ability of Vibrio alginolyticus strains isolated from Sparus aurata and Dicentrarchus labrax nursery to adhere to epithelial cell lines (Hep-2 and Caco-2), fish mucus and their ability to form a biofilm on different surfaces (glass, polystyrene, polyethylene and polyvinyl-chloride) was investigated in this study. METHODS AND RESULTS: The extracellular products were rich in enzymes and the strains were haemolytic on Wagatsuma agar and possessed several hydrolytic exoenzymes such as proteases, DNase and lipases. Most strains tested were multiresistant to the 17 antibiotics tested including those used in the farm to treat vibriosis. CONCLUSIONS: These bacteria were able to form a biofilm on all the surfaces tested and the cell density was the highest on the PVC surface followed by that on the glass slides, polystyrene and the polyethylene surface. More than 50% of the tested strains were adhesive to the epithelial cell lines (Hep-2 and Caco-2). SIGNIFICANCE AND IMPACT OF THE STUDY: These properties allow these bacteria to survive, proliferate and persist in all stages of fish rearing nursery even after seawater treatment with UV light.

Multiplex PCR detection of the antibiotic resistance genes in Staphylococcus aureus strains isolated from auricular infections.

Thirty-five Staphylococcus aureus strains from auricular infections were isolated. The identification of strains was confirmed by Api ID 32 Staph strips, the antibiotic susceptibility test was performed using ATB Staph kit. PCR assay was used to detect the oxacillin resistance gene (mecA) and the erythromycin genes (ermA, ermB, ermC, msrA and mef). The susceptibility profile of all strains revealed a low resistance level to oxacillin and erythromycin. The PCR results show that 60 % of the strains are mecA positive. The frequency of erythromycin genes was: ermA (+) 22.8 %, ermB (+) 45.7, ermC (+) 17.1, msrA (+) 28.6. The mef gene was not detected in any strain. No correlations between genotypic and phenotypic methods for the determination of oxacillin and erythromycin resistance was found. However, multiplex PCR technique was shown to be a fast, practical and economic technique for the detection of methicillin-and erythromycin-resistant staphylococci.

Pulsed-light system as a novel food decontamination technology: a review.

In response to consumer preferences for high quality foods that are as close as possible to fresh products, athermal technologies are being developed to obtain products with high levels of organoleptic and nutritional quality but free of any health risks. Pulsed light is a novel technology that rapidly inactivates pathogenic and food spoilage microorganisms. It appears to constitute a good alternative or a complement to conventional thermal or chemical decontamination processes. This food preservation method involves the use of intense, short-duration pulses of broad-spectrum light. The germicidal effect appears to be due to both photochemical and photothermal effects. Several high intensity flashes of broad spectrum light pulsed per second can inactivate microbes rapidly and effectively. However, the efficacy of pulsed light may be limited by its low degree of penetration, as microorganisms are only inactivated on the surface of foods or in transparent media such as water. Examples of applications to foods are presented, including microbial inactivation and effects on food matrices.

Phenotypic variations and molecular identification of Salmonella enterica serovar Typhimurium cells under starvation in seawater.

In seawater, enteric bacteria evolve toward a stressed state that is difficult to identify because of major alterations of their phenotype. In this study, we incubated four reference strains of S. enterica serovar Typhimurium in seawater microcosms for 10 months and studied the modifications of their main phenotypic characters. All of the strains lost some key characters used for traditional identification of the Salmonella genus. They became able to produce acetoin, and tryptophane deaminase activity became positive, but they lost the capacity to use rhamnose. We were able to show some modifications of the level of enzymatic profile as well as in their antibiotic susceptibility. The atypical cells of S. enterica serovar Typhimurium were identified by polymerase chain reaction (PCR) methods using the internal transcribed spacer region, and they were confirmed by multiplex PCR after the simultaneous amplification of the phoP, Hin, and H-li genes.

Immobilization of E. coli bacteria in three-dimensional matrices for ISFET biosensor design.

In recent years, cell-based biosensors (CBBs) have been very useful in biomedicine, food industry, environmental monitoring and pharmaceutical screening. They constitute an economical substitute for enzymatic biosensors, but cell immobilization remains a limitation in this technology. To investigate into the potential applications of cell-based biosensors, we describe an electrochemical system based on a microbial biosensor using an Escherichia coli K-12 derivative as a primary transducer to detect biologically active agents. pH variations were recorded by an ion-sensitive field effect transistor (ISFET) sensor on bacteria immobilized in agarose gels. The ISFET device was directly introduced in 100 ml of this mixture or in a miniaturized system using a dialysis membrane that contains 1 ml of the same mixture. The bacterial activity could be detected for several days. The extracellular acidification rate (ECAR) was analyzed with or without the addition of a culture medium or an antibiotic solution. At first, the microorganisms acidified their micro-environment and then they alkalinized it. These two phases were attributed to an apparent substrate preference of bacteria. Cell treatment with an inhibitor or an activator of their metabolism was then monitored and streptomycin effect was tested.

The anaerobic treatment of sewage and granule formation in upflow anaerobic sludge blanket reactor.

The granulation process was examined using synthetic wastewater containing glucose in a 1 liter laboratory upflow anaerobic sludge blanket (UASB) reactor. The anaerobic biotransformation of glucose was investigated during the granulation process. Anaerobic unacclimated sludge and glucose were used as seed and primary substrate, respectively. Massive initial granules were developed after three months of start-up. The effect of operational parameters such as influent glucose concentrations, pH, Volatile Fatty Acids (VFA) were also considered during granulation. The presence of a large concentration of sulphate in the sludge of a mesophilic (37 degrees C+/-1 degrees C) UASB reactor treating sewage resulted in severe process disturbance, with a complete inhibition of the propionate-degrading ability of the sludge. Severe inhibition of acetate removal was also observed, with concentrations of propionic acid and acetic acid in the reactor effluent of 1.72% and 18.88%, respectively. Hydrogen sulfide (H2S) is formed from the anaerobic decomposition of organic matter containing sulfate by sulfate-reducing bacteria. This gas is toxic at rate exceeding 2% tolerable by a reactor. The result shows the rate of hydrogen sulfide production was 3.8 %.

Virulence and enterobacterial repetitive intergenic consensus PCR of Vibrio alginolyticus strains isolated from Tunisian cultured gilthead sea bream and sea bass outbreaks.

Vibrio alginolyticus was isolated from the internal organs of diseased gilthead sea bream (Sparus aurata) and sea bass (Dicentrarchus labrax) cultured in two fish farms located on the Tunisian Mediterranean coast, from 2003 to 2005. After phenotypic characterisation, a selection of 34 isolates from gilthead sea bream and sea bass were molecularly typed by repetitive intergenic consensus PCR (ERIC-PCR) showing a high polymorphism among the isolated strains (19 genotypes). Most of the isolates were resistant to atleast two antimicrobial agents. All the tested strains were resistant to ampicillin. However, 91.17% were resistant to nitrofurantoin and 35.29% to tetracycline. Several strains isolated from diseased gilthead sea bream and sea bass were tested for virulence in both fish species by intraperitoneal injection. The selected isolates (n=7) were pathogenic for gilthead sea bream and sea bass. LD(50) values ranged from 5.01 x 10(4) to 6.20 x 10(7)CFU/fish. This is the first report on characterisation and virulence of V. alginolyticus for sea bass and sea bream in Tunisia.

Detection of icaA and icaD loci by polymerase chain reaction and biofilm formation by Staphylococcus epidermidis isolated from dialysate and needles in a dialysis unit.

Staphylococcus epidermidis, a coagulase-negative staphylococcus, is a major cause of infections associated with indwelling medical devices. Certain strains produce slime and form biofilm on polymer surfaces, where their pathogenicity is associated with biofilm formation. In this report, we investigated the presence or absence of the intercellular adhesion icaA and icaD genes by polymerase chain reaction, and phenotypic biofilm production was examined by qualitative Congo red agar (CRA) assay. A total of 32 strains of S. epidermidis were identified from dialysates and needles 4h after the initiation of dialysis. Qualitative biofilm production revealed that 16 (50%) strains produced slime on CRA plates. Among the 23 strains positive for the ica operon, 15 were biofilm positive on CRA, eight were biofilm negative, and one was icaA and icaD negative but produced slime. These results show that the ability of S. epidermidis to produce slime is not associated with the presence of icaA and icaD genes.

[Effect of previous culture conditions and the presence of the rpoS gene on the survival of Salmonella typhimurium in sea water exposed to sunlight]. / Influence des conditions de culture préalables et de la présence du gène rpoS pour la survie de Salmonella typhimurium en eau de mer exposée à la lumière solaire.

The effect of sunlight exposure on Salmonella typhimurium isogenic strains harboring an rpoS gene functional (rpoS+) or not functional (rpoS-) was investigated in microcosms of sterile sea water at 20 degrees C. The two strains rapidly lost their ability to produce colonies on solid culture media. The detrimental action of sunlight was more important when the salinity of sea water increased. The survival of stationary phase cells was influenced by RpoS. Bacteria grown in media with high salinity or osmolarity and transferred to sea water in stationary phase were more resistant to irradiation than those grown in media with low salinity. Prior growth under oxidative (0.2 mmol/L of H2O2) or amino acid starved (minimal medium) conditions did not modify the survival of either strain when they were exposed to sunlight. Bacteria were more resistant when cells were incubated in sea water in the dark prior to being exposed to sunlight. The resistance to sunlight irradiation was also greater in clones of both strains isolated from microcosms exposed to sunlight for 90 min, then further inoculated into sea water and reexposed to sunlight.

[Prevalence and characterization of uropathogenic Escherichia coli producing cytotoxic necrotizing factor type 1 (CNF1) in the center of Tunisia]. / Prévalence et caractérisation des Escherichia coli uropathogènes producteurs de facteur cytotoxique nécrosant type 1 (CNF1) dans le centre de la Tunisie.

During a three-year survey of the prevalence in central Tunisia of Escherichia coli producing CNF1 toxin (NTEC), 716 samples have been investigated by PCR for cnf1 gene. All samples were isolated from urine of adult and children patients presenting significant bacteriuria (> 10(5) colony-forming units/mL), independently of the severity of the clinical presentation; 328 strains were found harboring cnf1 gene, they were distributed into three clinical categories: 219 (66.76%) from patients with symptomatic bacteriuria, 76 (23.17%) from patients with uncomplicated cystitis and 33 (10.06%) from patients with acute pyelonephritis and complicated urinary tract infections. 98.78% (324) of CNF1 strains presented hemolytic activity. All 328 CNF1 strains harbored both sfa and pap genes and expressed MRHA activity. They belonged to 16 different serotypes. The most common serotypes, in order of frequency, were O6 (25.91%), O4 (17.98%), O2 (12.5%), O75 (9.14%), O78 (8.35%), and O83 (3.65%). Two strains (0.6%) were O168; a serotype shown to be associated to CNF2 producing bovine strains. The frequency of uropathogenic CNF1 strains in center Tunisia was about 45.81% and increased from 26.08% in 1998 to 58.16% in 2000. We showed that E. coli producing cytotoxic necrotizing factor (CNF1) was implicated in urinary tract infections in center Tunisia but no difference was shown between strains isolated from patients with complicated or uncomplicated urinary tract infections. The presence of CNF1 toxin with various associated virulence factors seemed to increase the risk for severe forms of urinary tract infections.

[Salmonella identification after incubation in the soil]. / De l'identification des salmonelles après incubation dans le sol.

We have tried to study the fate of Salmonella strains in soil. We have looked at their biochemical modifications during their evolution to dormant state and during their reviviscence. The beta galactosidase which is negative in the parent strain became positive after two weeks of cells starvation in soil. Stressed cells became able to produce acetoin. Some stressed cells did not produce the lysine decarboxylase, which is positive in parent cells. These modifications are reversible and depend on cultural conditions. Incubation of stressed cells in nutrient broth for more than four weeks helped them to reverse to normal forms. Simultaneous search for atypical Salmonella was done in dissect sludge of a domestic wastewater treatment plant and in soil irrigated with treated water. Atypical strains of Salmonella are found. We have seen that, after incubation in nutrient broth for more than four weeks, atypical strains characters evolved generally to their parental characters. All modifications of Salmonella in soil samples can make their identification very difficult and perhaps impossible.

[Changes in culture and biochemical characteristics of Salmonella paratyphi B after incubation in seawater]. / Modification des caractères culturaux et biochimiques du Salmonella paratyphi B après incubation dans l'eau de mer.

After incubation in seawater Salmonella paratyphi B cells rapidly became unable to grow on bacteriological media. Previous adaptation to high osmolarity conditions greatly slowed down this process. Strains isolated from seawater microcosms after varying incubation periods were qualitatively different and showed changes in some of their growth (colony shape and size) and biochemical properties (acidification of some sugars, gelatinase activity, acetoin production, nitrate reduction). Because of these modifications, the bacteria showed atypical profiles and could not be identified as members of the Salmonella genus. The alteration of the phenotype, although reversible, could explain some of the false-negative results obtained upon isolation and identification of these bacteria in seawater samples.