Influences of milk components on biofilm formation of Cronobacter spp. (Enterobacter sakazakii)

Aim:  To determine the critical component(s) of skim milk for biofilm formation of Cronobacter species.
Methods and Results:  Biofilm forming ability of 72 Cronobacter strains in skim milk preparation was assayed by crystal violet staining. The results revealed that whey protein and casein are more important determinants of skim milk for biofilm formation than lactose, although there was a wide variation in biofilm forming ability. Biofilm structure and capsular material of six strains exhibiting different biofilm forming ability was investigated via electron microscopes. Scanning electron microscopy showed visually that while the strong biofilm formers (E27B, FSM 30 and 2·82) resulted in almost complete coagulation of skim milk, the weak biofilm formers (55, FSM 290 and 2·84) caused less coagulation. No capsule was clearly delineated in transmission electron micrographs of either strong or weak biofilm formers.
Conclusion:  These results indicate that, for biofilm formation of Cronobacter species in skim milk, nitrogen source is probably a more important determinant than carbohydrate, and that strong biofilm formers are responsible for substantial coagulation of skim milk.
Significance and Impact of the Study:  This study provides information for better understanding of the underlying mechanisms by which Cronobacter species form biofilm in infant formula milk.     

Metabolic depression and spleen and liver enlargement in juvenile Arctic charr Salvelinus alpinus exposed to chronic parasite infection

The present study on the connection between standard metabolic rate ( R _S) and chronic Diplostomum spp. infection resulted in a decrease in R _S, and an enlargement in spleen and liver sizes in the infected juvenile Arctic charr Salvelinus alpinus compared to control fish. As splenic enlargement observed in infected fish was not due to condition-related changes in the spleen, it could most probably be explained by increased leucocyte synthesis. The higher liver masses in infected S. alpinus may have been related to disorders in energetic function, which could have had major effects on biochemical regulation by the liver. The proposed metabolic syndrome with a possible reduction in insulin sensitivity in tissues results in ineffective glucose and lipid metabolism and thus it is suggested that chronic Diplostomum infection in S. alpinus might not impose direct energetic costs, but it may weaken the efficiency of energy metabolism and thus lead to lowered R _S.     

SdrI of Staphylococcus saprophyticus is a multifunctional protein: localization of the fibronectin-binding site

The transferability of a large plasmid that harbors a tetracycline resistance gene tet (S), to fish and human pathogens was assessed using electrotransformation and conjugation. The plasmid, originally isolated from fish intestinal Lactococcus lactis ssp. lactis KYA-7, has potent antagonistic activity against the selected recipients ( Lactococcus garvieae and Listeria monocytogenes ), preventing conjugation. Therefore the tetracycline resistance determinant was transferred via electroporation to L . garvieae . A transformant clone was used as the donor in conjugation experiments with three different L. monocytogenes strains. To our knowledge, this is the first study showing the transfer of an antibiotic resistance plasmid from fish-associated lactic bacteria to L. monocytogenes , even if the donor L. garvieae was not the original host of the tetracycline resistance but experimentally created by electroporation. These results demonstrate that the antibiotic resistance genes in the fish intestinal bacteria have the potential to spread both to fish and human pathogens, posing a risk to aquaculture and consumer safety.     

Stomatal response to humidity in a sugarcane field: simultaneous porometric and micrometeorological measurements*

Abstract. Gas exchange data obtained with wellventilated leaf cuvettes provide clear evidence of a stomatal response to leaf-air vapour pressure difference (V). In contrast, remotely sensed leaf temperatures with specific assumptions regarding canopy boundary layer characteristics, have been interpreted to mean that stomata do not respond to V. We address this apparent discrepancy in a sugarcane field by simultaneous application of a single-leaf, porometric technique and a whole-canopy, Bowen ratioenergy balance technique. These methods indicated significant stomatal response to V in well-irrigated sugarcane. Stomatal responses to V in the field were obscured by strong covariance of major environmental parameters so that opening responses to light and closing responses to V tended to offset each other. Low boundary layer conductance significantly uncoupled V at the leaf surface (V_s) from V determined in the bulk atmosphere (V_a). This reduced the range of the stimulus, V_s, thereby reducing the range of the stomatal response, without indicating low stomatal sensitivity to V. Stomatal responses to V_a may be smaller than expected from V response curves in cuvettes, since V_s rather than the conventionally measured V_a is analogous to V in a well-stirred cuvette. Recently published conclusions that remotely sensed canopy temperatures are inconsistent with stomatal response to V may be based on erroneous estimates of canopy boundary layer conductance and thus of V_s, use of air saturation deficit rather than V to express evaporative demand, and investigation at higher levels of evaporative demand than those eliciting maximal stomatal response.     

Microplate assay for quantitation of neutral lipids in extracts from microalgae

Lipid quantitation is widespread in the algae literature, but popular methods such as gravimetry, gas chromatography and mass spectrometry (GC–MS), and Nile red cell staining suffer drawbacks, including poor quantitation of neutral lipids, expensive equipment, and variable results among algae species, respectively. A high-throughput microplate assay was developed that uses Nile red dye to quantify neutral lipids that have been extracted from algae cells. Because the algal extracts contained pigments that quenched Nile red fluorescence, a mild bleach solution was used to destroy pigments, resulting in a nearly linear response for lipid quantities in the range of 0.75 to 40 μg. Corn oil was used as a standard for quantitation, although other vegetable oils displayed a similar response. The assay was tested on lipids extracted from three species of Chlorella and resulted in close agreement with triacylglycerol (TAG) levels determined by thin layer chromatography. The assay was found to more accurately measure algal lipids conducive to biodiesel production and nutrition applications than the widely used gravimetric assay. Assay response was also consistent among different species, in contrast to Nile red cell staining procedures.     

Differences in Survival and Growth Among Tropical Rain Forest Pioneer Tree Seedlings in Relation to Canopy Openness and Herbivory

Although differences in canopy openness, herbivory and their interaction may promote species coexistence, how these factors affect pioneer tree species and potentially limit growth, and survival has been poorly studied, particularly in tropical South Asia. We monitored the effect of canopy openness and herbivore damage on seedling survival and growth of 960 individuals of six pioneer tree species: Dillenia triquetra, Macaranga indica, Macaranga peltata, Schumacheria castaneifolia, Trema orientalis, and Wendlandia bicuspidata. Seedlings were placed in four gap‐understory positions—center, outer gap edge, inner forest edge, and understory—in four large, natural gaps within the Sinharaja World Heritage Reserve, Sri Lanka. Canopy openness positively affected survival probability beyond the 550‐d experiment, while herbivory decreased survival and was highest in understory conditions. The relative order of species survival stayed fairly consistent between gap‐understory positions and followed their known shade tolerance rankings. When averaged across all experimental conditions, T. orientalis had the lowest survival probability estimate beyond the 550‐d experiment (0.05), but the greatest capacity for growth where it successfully established, while the species with highest averaged survival probability (0.79), D. triquetra, showed the lowest growth. One species, W. bicuspidata, responded positively to herbivory by re‐sprouting. Coexistence of D. triquetra, T. orientalis, and W. bicuspidata can be explained by a trade‐off among species in survival, growth, and response to herbivory. In addition to variation in canopy light environment, herbivory may be important in determining pioneer species distribution through fine‐scale niche partitioning and should be carefully considered in reforestation efforts.     

Competitive exclusion as a mode of action of a novel Bacillus cereus aquaculture biological agent

Aims: To determine the contribution of potential modes of action of a Bacillus cereus aquaculture biological control agent in inhibition of the fish pathogen, Aeromonas hydrophila. Methods and Results: When B. cereus was tested in plate well inhibition studies, no production of antimicrobial compounds was detected. Bacillus cereus had a high growth rate (0·96 h^?1), whereas Aer. hydrophila concentration decreased by c. 70% in co‐culture experiments. In nutrient limitation studies, B. cereus had a significantly higher growth rate when cultured under glucose (P < 0·05) and iron (P < 0·01) limitation in comparison with Aer. hydrophila. Bacillus cereus glucose (0·30 g l^?1 h^?1) and iron (0·60 mg l^?1 h^?1) uptake rates were also significantly higher (P < 0·01) than the Aer. hydrophila glucose (0·14 g l^?1 h^?1) and iron (0·43 mg l^?1 h^?1) uptake rates. Iron uptake was facilitated by siderophore production shown in time profile studies where relative siderophore production was c. 60% through the late exponential and sporulation phases. Conclusions: Competitive exclusion by higher growth rate, competition for organic carbon and iron, facilitated by siderophore production, could be identified as mechanisms of pathogen growth inhibition by B. cereus. Significance and Impact of the Study: This study is the first elucidation of the mechanism of action of our novel B. cereus biological agent in growth attenuation of pathogenic Aer. hydrophila. This study enhances the application knowledge and attractiveness for adoption of B. cereus NRRL 100132 for exploitation in aquaculture.     

Probiotic properties of Lactobacillus strains isolated from the feces of breast-fed infants and Taiwanese pickled cabbage

This study assessed potential probiotic Lactobacillus strains isolated from the feces of breast-fed infants and from Taiwanese pickled cabbage for their possible use in probiotic fermented foods by evaluating their (i) in vitro adhesive ability, resistance to biotic stress, resistance to pathogenic bacteria, and production of β-galactosidase; (ii) milk technological properties; and (iii) in vivo adhesive ability, intestinal survival and microbial changes during and after treatment. Five Lactobacillus isolates identified as Lactobacillus reuteri F03, Lactobacillus paracasei F08, Lactobacillus rhamnosus F14, Lactobacillus plantarum C06, and Lactobacillus acidophilus C11 that showed resistance to gastric juice and bile salts were selected for further evaluation of their probiotic properties. All the strains demonstrated the ability to adhere to Caco-2 cells, particularly, strain L. plantarum C06 and L. reuteri F03 showed satisfactory abilities, which were similar to that of the reference strain L. rhamnosus GG. The strains L. paracasei F08 and L. acidophilus C11 had the highest β-galactosidase activity. Most of the strains were resistant to aminoglycosides and vancomycin but sensitive to ampicillin, erythromycin, and penicillin. All the 5 strains elicited antibacterial activity against both Gram-positive (Bacillus cereus, Listeria monocytogenes and Staphylococcus aureus) and -negative (Escherichia coli and Salmonella enterica) pathogens. Moreover, the strains L. reuteri F03, L. paracasei F08, and L. plantarum C06 could grow rapidly in milk without nutrient supplementation and reached 10? cfu/mL after 24 h of fermentation at 37 °C. The viable cell counts of the 3 strains remained above 10? cfu/mL after 21 d of storage at 4 °C. In the animal feeding trial, the number of intestinal lactobacilli increased significantly after administration of milk fermented with the 3 strains, and the counts of fecal coliforms and Clostridium perfringens were markedly reduced. Lactobacillus strains could also survive in the ileal intestinal tissue of the treated rats. Technologically interesting Lactobacillus isolates may be used in the future as probiotic starter cultures for manufacturing novel fermented foods.