Bacteria-free Cultures Research Articles

A mesophilic relative of common glacier algae, Ancylonema palustre sp. nov., provides insights into the induction of vacuolar pigments in zygnematophytes.

The green algae of the genus Ancylonema, which belong to the zygnematophytes, are prevalent colonizers of glaciers worldwide. They display a striking reddish-brown pigmentation in their natural environment, due to vacuolar compounds related to gallic acid. This pigmentation causes glacier darkening when these algae bloom, leading to increased melting rates. The Ancylonema species known so far are true psychrophiles, which hinders experimental work and limits our understanding of these algae. For instance, the biosynthesis, triggering factors, and biological function of Ancylonema's secondary pigments remain unknown. In this study, we introduce a mesophilic Ancylonema species, A. palustre sp. nov., from temperate moorlands. This species forms the sister lineage to all known psychrophilic strains. Despite its morphological similarity to the latter, it exhibits unique autecological and photophysiological characteristics. It allows us to describe vegetative and sexual cellular processes in great detail. We also conducted experimental tests for abiotic factors that induce the secondary pigments of zygnematophytes. We found that low nutrient conditions combined with ultraviolet B radiation result in vacuolar pigmentation, suggesting a sunscreen function. Our thriving, bacteria-free cultures of Ancylonema palustre will enable comparative genomic studies of mesophilic and extremophilic zygnematophytes. These studies may provide insights into how Ancylonema species colonized the world's glaciers.

Scent detection of Brucella abortus by African giant pouched rats (Cricetomys ansorgei)

BackgroundBrucellosis is a contagious zoonosis caused by bacteria of the genus Brucella. While the disease has been eradicated in most developed countries, it remains endemic in sub–Saharan Africa where access to reliable diagnostics is limited. African giant pouched rats (Cricetomys ansorgei) have been trained to detect the scent of Mycobacterium tuberculosis to increase case detection in sub-Saharan Africa. Given the similar diagnostic challenges facing brucellosis and tuberculosis, we explored the feasibility of training African giant pouched rats to detect Brucella.ResultsAfter 3 months of training, rats reliably identified cultured Brucella, achieving an average sensitivity of 93.56% (SD = 0.650) and specificity of 97.65% (SD = 0.016). Rats readily generalized to novel, younger Brucella cultures that presumably generated a weaker volatile signal and correctly identified at least one out of three fecal samples spiked with Brucella culture during a final test of feasibility.DiscussionTo our knowledge, these experiments are the first to demonstrate Brucella emits a unique odor profile that scent detection animals can be trained to identify. Importantly, cultured E. coli samples were included throughout training and test to ensure the rats learned to specifically identify Brucella bacteria rather than any bacteria in comparison to bacteria-free culture medium. E. coli controls therefore served a crucial function in determining to what extent Brucella abortus emits a unique odor signature. Further research is needed to determine if a Brucella-specific volatile signature is present within clinical samples. If confirmed, the present results suggest trained rats could serve as a valuable, novel method for the detection of Brucella infection.

The algal microbiome protects Desmodesmus intermedius from high light and temperature stress

The mutualistic impacts of bacterial communities on algal growth and biomass accumulation are well documented, but it is unknown how temperature and light stress influence these mutualistic interactions. Here, we generated a bacteria-free (axenic) culture of the green alga Desmodesmus intermedius C046 and compared its growth and yield in the laboratory to its native xenic counterpart - featuring a microbiome that has been maintained with its algal host for at least 5 years – under unstressed and high light, high temperature stress conditions. We then added exogenous microbiomes from high light marine environments to the axenic culture to quantify the contribution of these newly generated microbial consortia in improving D. intermedius growth, yield, and resilience to high light and heat. The native microbiome increased growth and biomass accumulation, under both unstressed and stressed conditions. Three of the five newly derived microbiomes had increased growth compared to the axenic and negligible, decreased, or increased growth impacts compared to the xenic culture. Stress conditions led to decreased algal exudation, larger algal cells when grown alone, but smaller algal cells with an associated microbiome. Our results suggest that the algal microbiome plays a role in microalgal response to heat and light stress, and efforts for screening new algal strains for high biomass productivity under those stress conditions should include a strategy for microbiome optimization.

Antibacterial Activity against Four Fish Pathogenic Bacteria of Twelve Microalgae Species Isolated from Lagoons in Western Greece.

Microalgae may produce a range of high-value bioactive substances, making them a promising resource for various applications. In this study, the antibacterial activity of twelve microalgae species isolated from lagoons in western Greece was examined against four fish pathogenic bacteria (Vibrio anguillarum, Aeromonas veronii, Vibrio alginolyticus, and Vibrio harveyi). Two experimental approaches were used to evaluate the inhibitory effect of microalgae on pathogenic bacteria. The first approach used bacteria-free microalgae cultures, whereas the second approach used filter-sterilized supernatant from centrifuged microalgae cultures. The results demonstrated that all microalgae had inhibitory effects against pathogenic bacteria in the first approach, particularly 4 days after inoculation, where Asteromonas gracilis and Tetraselmis sp. (red var., Pappas) exhibited the highest inhibitory activity, reducing bacterial growth by 1 to 3 log units. In the second approach, Tetraselmis sp. (red var., Pappas) showed significant inhibition against V. alginolyticus between 4 and 25 h after inoculation. Moreover, all tested cyanobacteria exhibited inhibitory activity against V. alginolyticus between 21 and 48 h after inoculation. Statistical analysis was performed using the independent samples t-test. These findings suggested that microalgae produce compounds with antibacterial activity, which could be useful in aquaculture.

Impact of early empirical antifungal therapy on prognosis of sepsis patients with positive yeast culture: A retrospective study from the MIMIC-IV database.

Mortality and other clinical outcomes of culture-negative and culture-positive among patients with fungal sepsis have not been documented, and whether antifungal therapy prior to fungal culture reports is related to decreased mortality among patients remains largely controversial. This study aimed to determine the mortality and other clinical outcomes of patients with positive yeast cultures and further investigate the effects of initial empiric antifungal therapy. A retrospective study was conducted among septic patients using the Medical Information Mart for Intensive Care (MIMIC)-IV database. Patients with sepsis were divided into two groups based on first fungal culture status during intensive care unit (ICU) stay, and initial empirical antifungal therapy was prescribed based on physician's experience prior to fungal culture reports within 48 h. The primary outcome was in-hospital all-cause mortality. The secondary outcomes were 30-day all-cause mortality, 60-day all-cause mortality, length of ICU stay and length of hospital stay. Multivariate logistic regression, propensity score matching (PSM), subgroup analyses and survival curve analyses were performed. This study included 18,496 sepsis patients, of whom 3,477 (18.8%) had positive yeast cultures. Patients with positive yeast cultures had higher in-hospital all-cause mortality, 60-day all-cause mortality, and longer lengths of ICU stay and hospital stay than those with negative yeast cultures after PSM (all p < 0.01). Multivariate logistic regression analysis revealed that positive yeast culture was a risk factor for in-hospital mortality in the extended model. Subgroup analyses showed that the results were robust among the respiratory infection, urinary tract infection, gram-positive bacterial infection and bacteria-free culture subgroups. Interestingly, empiric antifungal therapy was not associated with lower in-hospital mortality among patients with positive yeast cultures, mainly manifested in stratification analysis, which showed that antifungal treatment did not improve outcomes in the bloodstream infection (odds ratio, OR 2.12, 95% CI: 1.16-3.91, p = 0.015) or urinary tract infection groups (OR 3.24, 95% CI: 1.48-7.11, p = 0.003). Culture positivity for yeast among sepsis patients was associated with worse clinical outcomes, and empiric antifungal therapy did not lower in-hospital all-cause mortality in the bloodstream infection or urinary tract infection groups in the ICU.

Antibacterial activity in three Chaetoceros microalgae species cultures by using antibiotics

Diatoms, such as Chaetoceros, grow in a mutualistic relationship with bacteria. However, in some cases, it is necessary to grow them in bacteria-free cultures. To reduce bacterial load, antibiotics are used, and on certain occasions it is necessary to use a mixture with more than one antibiotic. This work aimed to obtain a quick and effective protocol to reduce the bacterial load and evaluate the response of three Chaetoceros species with aquacultural importance. Single and mix antibiotics were used. Microalgal and bacterial growth was measured. The growth parameters for diatoms showed that the significantly highest cell concentration was for C. muelleri (3.15 x106 cells mL-1) and the lowest values to C. calcitrans (2.98 x106 cells mL-1). The significantly highest growth rate was for C. calcitrans (0.77 divisions per day), and the lowest values for Chaetoceros sp. (0.60 divisions per day). The growth parameters for heterotrophic bacteria showed that the significantly highest bacterial load was for Chaetoceros sp. (19.16 x106 CFU (Colony-Forming Units) mL-1) and the lowest values were for C. calcitrans (12.23 x106 CFU mL-1). The growth rate of the heterotrophic bacteria present in Chaetoceros cultures was similar among the three studied species. Streptomycin® and sulfate G41® produced a partial reduction of bacterial load. The most effective treatment for all three species was the use of an antibiotic mix composed of ampicillin® (250 μg mL-1), kanamycin® (200 μg mL-1), neomycin® (50 μg mL-1), and streptomycin® (100 μg mL-1) for three days. The mix prepared with the highest antibiotic concentration produced a reduction of bacteria (100%) for three days; however, it also induced a significant reduction of the growth of the three Chaetoceros species.

Reactive oxygen species limit intestinal mucosa-bacteria homeostasis in vitro

Interactions between epithelial and immune cells with the gut microbiota have wide-ranging effects on many aspects of human health. Therefore, there is value in developing in vitro models capable of performing highly controlled studies of such interactions. However, several critical factors that enable long term homeostasis between bacterial and mammalian cultures have yet to be established. In this study, we explored a model consisting of epithelial and immune cells, as well as four different bacterial species (Bacteroides fragilis KLE1958, Escherichia coli MG1655, Lactobacillus rhamnosus KLE2101, or Ruminococcus gnavus KLE1940), over a 50 hour culture period. Interestingly, both obligate and facultative anaerobes grew to similar extents in aerobic culture environments during the co-culture period, likely due to measured microaerobic oxygen levels near the apical surface of the epithelia. It was demonstrated that bacteria elicited reactive oxygen species (ROS) production, and that the resulting oxidative damage heavily contributed to observed epithelial barrier damage in these static cultures. Introduction of a ROS scavenger significantly mitigated oxidative damage, improving cell monolayer integrity and reducing lipid peroxidation, although not to control (bacteria-free culture) levels. These results indicate that monitoring and mitigating ROS accumulation and oxidative damage can enable longer term bacteria-intestinal epithelial cultures, while also highlighting the significance of additional factors that impact homeostasis in mammalian cell-bacteria systems.

Fungal-fungal and fungal-bacterial interactions in aquatic decomposer communities: bacteria promote fungal diversity.

Fungi produce a variety of extracellular enzymes, making recalcitrant substrates bioavailable. Thus, fungi are central for the decomposition of dead organic matter such as leaf litter. Despite their ecological importance, our understanding of relationships between fungal species diversity and ecosystem functioning is limited, especially with regard to aquatic habitats. Moreover, fungal interactions with other groups of microorganisms such as bacteria are rarely investigated. This lack of information may be attributed to methodological limitations in tracking the biomass of individual fungal species in communities, impeding a detailed assessment of deviations from the overall performance expected from the sum of individual species' performances, so-called net diversity effects (NDEs). We used fungal species-specific biomolecular tools to target fungal-fungal and fungal-bacterial interactions on submerged leaves using four cosmopolitan aquatic fungal species and a stream microbial community dominated by bacteria. In microcosms, we experimentally manipulated fungal diversity and bacterial absence/presence and assessed functional performances and fungal community composition after 14d of incubation. Fungal community data were used to evaluate NDEs on leaf colonization. The individual fungal species were functionally distinct and fungal cultures were on average more efficient than the bacterial culture. In absence of bacteria, NDEs correlated with growth rate (negatively) and genetic divergence (positively), but were predominantly negative, suggesting that higher fungal diversity led to a lower colonization success (niche overlap). In both absence and presence of bacteria, the overall functional performances of the communities were largely defined by their composition (i.e., no interactions at the functional level). In the presence of bacteria, NDEs correlated with genetic divergence (positively) and were largely positive, suggesting higher fungal diversity stimulated colonization (niche complementarity). This stimulation may be driven by a bacteria-induced inhibition of fungal growth, alleviating competition among fungi. Resulting feedback loops eventually promote fungal coexistence and synergistic interactions. Nonetheless, overall functional performances are reduced compared to bacteria-free cultures. These findings highlight the necessity to conduct future studies, investigating biodiversity-ecosystem functioning relationships using artificial systems, without exclusion of key organisms naturally co-occurring in the compartment of interest. Otherwise, study outcomes might not reflect true ecological relationships and ultimately misguide conservation strategies.

The Influence of Bacteria on the Growth, Lipid Production, and Extracellular Metabolite Accumulation by Phaeodactylum tricornutum (Bacillariophyceae).

To examine the impact of heterotrophic bacteria on microalgal physiology, we co-cultured the diatom Phaeodactylum tricornutum with six bacterial strains to quantify bacteria-mediated differences in algal biomass, total intracellular lipids, and for a subset, extracellular metabolite accumulation. A Marinobacter isolate significantly increased algal cell concentrations, dry biomass, and lipid content compared to axenic algal cultures. Two other bacterial strains from the Bacteroidetes order, of the genera Algoriphagus and Muricauda, significantly lowered P. tricornutum biomass, leading to overall decreased lipid accumulation. These three bacterial co-cultures (one mutualistic, two competitive) were analyzed for extracellular metabolites via untargeted liquid chromatography mass spectrometry to compare against bacteria-free cultures. Over 80% of the extracellular metabolites differentially abundant in at least one treatment were in higher concentrations in the axenic cultures, in agreement with the hypothesis that the co-cultured bacteria incorporated algal-derived organic compounds for growth. Furthermore, the extracellular metabolite profiles of the two growth-inhibiting cultures were more similar to one another than the growth-promoting co-culture, linking metabolite patterns to ecological role. Our results show that algal-bacterial interactions can influence the accumulation of intracellular lipids and extracellular metabolites, and suggest that utilization and accumulation of compounds outside the cell play a role in regulating microbial interactions.

Effect of Streptococcus mutans on the flexural strength of resin-based restorative materials

There are a limited number of studies about the effects of microbial aging on the mechanical properties of restorative materials. Therefore, this study aimed to evaluate the effect of simulated aging with Streptococcus mutans on the flexural strength of different resin-based materials. This experimental study was performed on the blocks of different types of restorative materials including composite resin, giomer, and a resin-modified glass ionomer (RMGI). Moreover, three types of aging, such as 30-day storage in distilled water, S. mutans, and germ-free culture medium, were used in this study. The three-point bending flexural strength of the specimens before and after aging was measured according to the International Organization for Standardization-4049 standard. Data were analyzed by two-way ANOVA and post hoc Tukey's tests. A P < 0.05 was considered statistically significant. Results showed that the 30-day aging with the S. mutans significantly reduced the flexural strength of all three types of materials (P = 0.00). In all restorative materials, storage in a bacteria-free culture medium acted the same as distilled water, and there was no significant difference between these two solutions in terms of the flexural strength of the material, compared to the before-aging strength (P > 0.05). Furthermore, no significant difference was observed between S. mutans-based aging and distilled water aging regarding RMGI (P = 0.75). It can be concluded that aging by S. mutans reduced the flexural strength in all three restorative materials.

Management of microbial contaminants in the In Vitro Gene Bank: a case study of taro [Colocasia esculenta (L.) Schott

Ex situ conservation of vegetatively propagated crops by tissue culture is sometimes hampered by covert endophytic bacteria which become visible after repeated subculture over prolonged periods. In the present study, we identified bacterial contaminants and devised a strategy for their elimination from 20 in vitro conserved accessions of taro [Colocasia esculenta (L.) Schott] held in the In Vitro Gene Bank (IVGB) at ICAR-National Bureau of Plant Genetic Resources (NBPGR). Visually, the cultures were exhibiting white to dark brown exudation in tissue culture growth medium and undergoing slow degeneration as recorded by wilting and necrosis of leaves and shoots. On culturing the macerated pieces of shoots, corms, and leaf petioles from the contaminated taro cultures on bacterial indexing medium, six distinct bacterial colonies were observed. The identity of these bacteria was established through 16S rDNA sequencing as gram-negative strains (2) of Ralstonia spp. and gram-positive strains (4) of Paenibacillus spp. Thereafter, two strategies were adopted for elimination of contaminants from the cultures: (i) use of antibiotic supplemented media and (ii) hardening of micro-corms in field and re-establishment under in vitro conditions. Antibiotic supplemented media was not effective in eliminating contamination as reappearance of bacterial colonies was observed after subculturing in antibiotic-free growth medium. In contrast, re-establishment in vitro from hardened infected plants resulted in an average of 6.03% bacteria-free cultures in 14 accessions. The findings indicate that endophytic bacteria associated with the host (taro) plants under tissue culture conditions may efficiently be mitigated through periodic transfer of the tissue culture derived corms to ex vitro conditions and re-introduction of the bacteria-free shoot tip explants in vitro.

Phytotoxicity, Bioaccumulation, and Degradation of Nonylphenol in Different Microalgal Species without Bacterial Influences.

Nonylphenol (NP) is a contaminant that has negative impacts on aquatic organisms. To investigate its phytotoxicity, bioaccumulation, and degradation in algae without associated bacteria, six freshwater microalgae—Ankistrodesmus acicularis, Chlorella vulgaris, Chroococcus minutus, Scenedesmus obliquus, Scenedesmus quadricauda, and Selenastrum bibraianum—in bacteria-free cultures were studied. When exposed to 0.5–3.0 mg L−1 NP for 4 days, cell growth and photosynthesis, including maximal photochemistry (Fv/Fm), were suppressed progressively. The antioxidant responses of superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) showed species differences. While the antioxidant enzymes in C. vulgaris and S. obliquus were more active with the increase of NP (0–3 mg L−1), they dropped in the other four algae at concentrations of 1 and 1.5 mg L−1. Therefore, C. vulgaris and S. obliquus were designated as NP-tolerant species and showed more conspicuous and faster changes of antioxidant reactions compared with the four NP-sensitive species. All six species degraded NP, but A. acicularis was more reactive at low NP concentrations (<1 mg L−1), suggesting its possible application in sewage treatment for its potential for effective NP removal from water bodies in a suitable scope. Therefore, the conclusion is that biodegradation of NP by algae is species specific.

Extracts from cultures of Pseudomonas fluorescens induce defensive patterns of gene expression and enzyme activity while depressing visible injury and reactive oxygen species in Arabidopsis thaliana challenged with pathogenic Pseudomonas syringae.

We evaluated the ability of metabolic elicitors extracted from Pseudomonas fluorescens N21.4 to induce systemic resistance (ISR) in Arabidopsis thaliana against the pathogen Pseudomonas syringae DC3000. Metabolic elicitors were obtained from bacteria-free culture medium with n-hexane, ethyl acetate and n-butanol in three consecutive extractions. Each extract showed plant protection activity. The n-hexane fraction was the most effective and was used to study the signal transduction pathways involved by evaluating expression of marker genes of the salicylic acid (SA) signalling pathway (NPR1, PR1, ICS and PR2) and the jasmonic acid/ethylene (JA/ET) signalling pathway (PDF1, MYC2, LOX2 and PR3). In addition, the level of oxidative stress was tested by determining the activity of enzymes related to the ascorbate-glutathione cycle. N-hexane extracts stimulated both pathways based on overexpression of ICS, PR1, PR2, PDF1 and LOX2 genes. In addition, activity of the pathogenesis-related proteins glucanase (PR2) and chitinase (PR3), lipoxygenase and polyphenol oxidase was enhanced together with an increased capacity to remove reactive oxygen species (ROS). This was associated with less oxidative stress as indicated by a decrease in malondialdehyde (MDA), suggesting a causative link between defensive metabolism against P. syringae and ROS scavenging.

Evaluation of Galleria mellonella larvae for studying the virulence of Streptococcus suis.

Background Streptococcus suis is an encapsulated Gram-positive bacterium and the leading cause of sepsis and meningitis in young pigs, resulting in considerable economic losses in the porcine industry. S. suis is considered an emerging zoonotic agent with increasing numbers of human cases over the last years. In the environment, both avirulent and virulent strains occur in pigs, with no evidence for consistent adapatation of virulent strains to the human host. Currently, there is an urgent need for a convenient, reliable and standardised animal model to rapidly assess S. suis virulence. Wax moth (Galleria mellonella) larvae have successfully been used in human and animal infectious disease studies. Here, we developed G. mellonella larvae as a model to assess virulence of S. suis strains.ResultsFourteen isolates of S. suis belonging to different serotypes killed G. mellonella larvae in a dose-dependent manner. Larvae infected with the virulent serotype 2 strain, S. suis S3881/S10, were rescued by antibiotic therapy. Crucially, the observed virulence of the different serotypes and mutants was in agreement with virulence observed in piglets (Sus scrofa) and the zebrafish larval infection model. Infection with heat-inactivated bacteria or bacteria-free culture supernatants showed that in most cases live bacteria are needed to cause mortality in G. mellonella.ConclusionsThe G. mellonella model is simple, cost-efficient, and raises less ethical issues than experiments on vertebrates and reduces infrastructure requirements. Furthermore, it allows experiments to be performed at the host temperature (37 °C). The results reported here, indicate that the G. mellonella model may aid our understanding of veterinary microbial pathogens such as the emerging zoonotic pathogen S. suis and generate hypotheses for testing in the target animal host. Ultimately, this might lead to the timely introduction of new effective remedies for infectious diseases. Last but not least, use of the G. mellonella infection model to study S. suis virulence adheres to the principles of replacement, reduction and refinement (3Rs) and can potentially reduce the number of vertebrates used for experimental infection studies.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-016-0905-2) contains supplementary material, which is available to authorized users.

Removal and Biodegradation of Nonylphenol by Four Freshwater Microalgae.

The removal and biodegradation of nonylphenol (NP) by four freshwater microalgae, including three green algae (Scendesmus quadriauda, Chlorella vulgaris, and Ankistrodesmus acicularis) and one cyanobacterium (Chroococcus minutus) were studied in bacteria-free cultures exposed to different concentrations of NP for 5 days. All four algal species showed a rapid and high ability to remove NP (including bioaccumulation and biodegradation). Among these species, A. acicularis (Ankistrodesmus acicularis) had the highest NP removal rate (83.77%) at 120 h when exposed to different NP treatments (0.5–2.5 mg·L−1), followed by C. vulgaris (Chlorella vulgaris) (80.80%), S. quadriauda (Scendesmus quadriauda) (70.96%) and C. minutus (Chroococcus minutus) (64.26%). C. vulgaris had the highest NP biodegradation percentage (68.80%) at 120 h, followed by A. acicularis (65.63%), S. quadriauda (63.10%); and C. minutus (34.91%). The extracellular NP contents were lower than the intracellular NP contents in all tested algae. The ratio of the extracellular NP content and the intracellular NP content ranged from 0.04 to 0.85. Therefore, the removal of NP from the medium was mainly due to the algal degradation. These results indicate that A. acicularis and C. vulgaris are more tolerant to NP and could be used for treatment of NP contaminated aqueous systems effectively by bioremoval and biodegradation.

Inhibition of miR122a by Lactobacillus rhamnosus GG culture supernatant increases intestinal occludin expression and protects mice from alcoholic liver disease

Alcoholic liver disease (ALD) has a high morbidity and mortality. Chronic alcohol consumption causes disruption of intestinal microflora homeostasis, intestinal tight junction barrier dysfunction, increased endotoxemia, and eventually liver steatosis/steatohepatitis. Probiotic Lactobacillus rhamnosus GG (LGG) and the bacteria-free LGG culture supernatant (LGGs) have been shown to promote intestinal epithelial integrity and protect intestinal barrier function in ALD. However, little is known about how LGGs mechanistically works to increase intestinal tight junction proteins. Here we show that chronic ethanol exposure increased intestinal miR122a expression, which decreased occludin expression leading to increased intestinal permeability. Moreover, LGGs supplementation decreased ethanol-elevated miR122a level and attenuated ethanol-induced liver injury in mice. Similar to the effect of ethanol exposure, overexpression of miR122a in Caco-2 monolayers markedly decreased occludin protein levels. In contrast, inhibition of miR122a increased occludin expression. We conclude that LGGs supplementation functions in intestinal integrity by inhibition of miR122a, leading to occludin restoration in mice exposed to chronic ethanol.

English

Malathion degrading bacteria were isolated from a soil sample. Growth of mono and co-cultured strains of Pseudomonas in media containing the organophosphate insecticide, malathion as sole source of carbon was employed to assess degradability of the bacteria. On the seventh day of inoculation, bacteria free culture fluids as well as un-inoculated fluids were analyzed by gas chromatography and mass spectrometry (GC-MS). Malathion was not detected in the cell free culture fluids, instead four degradation products namely phoratoxon sulfone, malaoxon, butanedioic acid and ethyl methyl-methyl phosphonate made their appearance in the cultivation culture. All of the three strains used in this study, appeared efficient in degrading the insecticide especially in co-culturing conditions. These bacteria are promising for detoxifying the insecticide contaminated soils. Key words: Pseudomonas, detoxification, malathion.

The Mystery of Clade X: Orciraptor gen. nov. and Viridiraptor gen. nov. are Highly Specialised, Algivorous Amoeboflagellates (Glissomonadida, Cercozoa)

In freshwater ecosystems a vast diversity of elusive protists exists that specifically feed on microalgae. Due to difficulties in isolation and long-term maintenance, most of these are still poorly known. In this study stable, bacteria-free cultures of several limnetic, algivorous amoeboflagellates were investigated by light microscopy and molecular phylogenetic analyses. All strains represent naked, biflagellate cells, either occurring as rigid flagellates or as surface-attached amoebae. They perforate cell walls of certain Zygnematophyceae and Chlorophyceae (Viridiplantae) and phagocytose algal cell contents. Time-lapse microscopy revealed the feeding behaviour, locomotional processes and life histories of the amoeboflagellates. Clear differences in cell morphology and food range specificity led to the description of two new, monotypic genera Orciraptor and Viridiraptor, which occupy similar, but distinct ecological niches in aquatic ecosystems as 'necrophytophagous' and 'parasitoid' protists, respectively. Molecular phylogenetic analyses based on 18S rDNA sequence data demonstrated that Orciraptor and Viridiraptor belonged to 'clade X' within the order Glissomonadida (Cercozoa, Rhizaria). In conclusion, we established the phenotypic identity of a clade, which until now was exclusively known from environmental sequences, and erect the new family Viridiraptoridae for 'clade X'. Its algivorous members are compared with other glissomonads and nomenclatural, methodological and ecological aspects of these novel 'raptorial' amoeboflagellates are discussed.

Mechanisms Underlying Modulation of SLC26A3 Activity by Lactobacillus acidophilus‐secreted Soluble Factors

DRA (Down‐Regulated in Adenoma, SLC26A3) mediates apical membrane Cl−/HCO3− exchange activity in mammalian intestinal epithelial cells. Our previous studies showed that soluble factor(s) in the bacteria‐free culture supernatant (CS) of the probiotic Lactobacillus acidophilus (LA) stimulated Cl−/HCO3− exchange activity in Caco‐2 cells via increasing apical membrane DRA levels. Current studies were undertaken to elucidate the mechanisms of LA‐CS enrichment of functional DRA on the apical cell surface. Post‐confluent Caco‐2 monolayers were treated for 3 h with LA‐CS. DRA function was measured as DIDS‐sensitive 125I‐uptake, surface DRA assessed by cell‐surface biotinylation/confocal microscopy. Inhibitors of PI3 kinase and Rac1, as well as methyl‐β‐D‐cyclodextran, a lipid raft‐disrupting agent, abrogated LA‐CS effects on DRA function. LA‐CS enhanced DRA levels in the detergent insoluble (DI) fraction in Caco‐2 cells. Silencing the PDZ protein NHERF2 attenuated LA‐CS enhancement of apical membrane DRA. Molecular size fractionation showed that soluble factors in the LA‐CS that enhance functional DRA on the apical membrane are of low molecular mass (3–10 kD) and are heat‐stable. Our results define novel mechanisms involving lipid rafts, NHERF, and PI3K‐Rac1 pathway in altered trafficking of DRA induced by probiotic‐secreted bioactive factor(s) to modulate intestinal chloride absorption. [Grant support: VA/NIDDK/Bill &amp; Melinda Gates Foundation]

Cell Growth and Toxins' Content ofOstreopsiscf.Ovatain Presence and Absence of Associated Bacteria

Bacteria associated to benthic dinoflagellate Ostreopsis cf. ovata cultures were removed to assess their effects on algal growth and toxins' production. Bacteria were removed using an antibiotic cocktail (streptomycin, ciprofloxacin, gentamicin and penicillin G). The actual axenic status of antibiotic treated cultures and bacterial growth in control cultures were assessed by epifluorescence microscopy using SYBR gold dye. The removal of bacteria unaffected algal growth, except conferring a higher cell number at mid stationary phase. Toxin profile and quantification of each toxin (PLTX, OVTX-a, -b, -c, -d, -e) were performed by HR LC-MS on both cell pellet and growth medium extracts. No changes in toxins' profile nor in cell and extra-cellular toxins' concentrations were found between bacteria-free and control cultures at the early stationary phase. Whereas, in late stationary phase axenic cultures showed significant lower cell toxins' concentrations and higher extra-cellular toxins' values, though not significantly (total cell toxins' concentrations: 39.3 and 24.9 pg cell—1; total extra-cellular toxins' concentrations: 23.8 and 28.3 μgL—1 for control and bacteria-free cultures, respectively).