Legionella anisa or Legionella bozemanii? Traditional and molecular techniques as support in the environmental surveillance of a hospital water network.

Understanding the actual distribution of different Legionella species in water networks would help prevent outbreaks. Culture investigations followed by serological agglutination tests, with poly/monovalent antisera, still represent the gold standard for isolation and identification of Legionella strains. However, also MALDI-TOF and mip-gene sequencing are currently used. This study was conducted to genetically correlate strains of Legionella non pneumophila (L-np) isolated during environmental surveillance comparing different molecular techniques. Overall, 346 water samples were collected from the water system of four pavilions located in a hospital of the Apulia Region of Italy. Strains isolated from the samples were then identified by serological tests, MALDI-TOF, and mip-gene sequencing. Overall, 24.9% of water samples were positive for Legionella, among which the majority were Legionella pneumophila (Lpn) 1 (52.3%), followed by Lpn2-15 (20.9%), L-np (17.4%), Lpn1 + Lpn2-15 (7.1%), and L-np + Lpn1 (2.3%). Initially, L-np strains were identified as L. bozemanii by monovalent antiserum, while MALDI-TOF and mip-gene sequencing assigned them to L. anisa. More cold water than hot water samples were contaminated by L. anisa (p < 0.001). PFGE, RAPD, Rep-PCR, and SAU-PCR were performed to correlate L. anisa strains. Eleven out of 14 strains identified in all four pavilions showed 100% of similarity upon PFGE analysis. RAPD, Rep-PCR, and SAU-PCR showed greater discriminative power than PFGE.

Diversity of rhizospheric and endophytic bacteria isolated from dried fruit of Ficus carica.

There is currently an increasing demand for the characterization of endophytic bacteria isolated from different parts of plants (rhizosphere, roots, fruit, leaf) in order to improve the organic agriculture practices. The current research was performed to identify both rhizospheric bacteria isolated from the rhizosphere of Ficus carica in three different sites in the north of Tunisia and endophytic bacteria isolated from dried figs. We then characterized them for a diversity of plant growth-promoting (PGP) activities. A collection of 120 isolates from rhizospheric soil and 9 isolates from dried figs was obtained and purified. 16SrDNA gene amplification of rhizospheric bacteria revealed significant diversity and allowed for the assigning of the isolates to 6 phyla: Gammaproteobacteria, Alphaproteobacteria, Betaproteobacteria, Firmicutes, Actinobacteria, and Bacteroidetes. Representative strains of the collection (90 strains) were tested for numerous PGP activities and resistance to abiotic stresses. The most common PGP trait for all bacteria from the three regions was siderophore production (62%), followed by cellulase (38%), then protease activity (37%), then by lipases activity (17%) and lastly by solubilization of phosphates (9%). Twenty -three strains that showed most PGP traits were selected, 8 strains presented 12 or more, and 15 strains displayed between 7 and 11 of 17 PGP activities. The majority of the isolates manifested a possible adaptation to abiotic stress and unfavorable environments. PCR-DGGE analysis of soil rhizosphere of the three sites allowed also for the acquisition of a Cluster analysis of rhizospheric bacterial communities. Our current study identified and characterized for the first time in Tunisia rhizospheric and endophytic bacteria from dried fruit of Ficus carica.

Comparison of analytical approaches for identifying airborne microorganisms in a livestock facility.

An intensive study, applied to a site characterized by multiple sources of microorganisms, was aimed at understanding the best approach to study bioaerosol. Culture-based, molecular biological, and chemical methods were applied to Particulate Matter (PM) samples collected in a livestock facility, during spring and autumn seasons, in two different outdoor areas. The first one was close to a place where feed was stored and handled and the second next to an open cowshed. Qualitative analysis of bacteria was performed by sequencing techniques applied to DNA extracted from both isolated culturable bacteria and particulate matter samples. Quantification of microorganisms was achieved through three distinct approaches. Microorganism colonies were counted, after incubation at 28 °C, and expressed as colony-forming units (CFU) per m3. Chemical method consisted in the identification of individual biomarkers, and their conversion to number of microorganisms per m3, using proper conversion factors. Finally, qPCR was applied to DNA extracted from PM samples, and the results were expressed as total amount of bacteria present in the bioaerosol (UG/m3). The presence of airborne sterols was also studied to broaden the knowledge of bioaerosol components in atmosphere. Small seasonal differences and major sampling site differences occurred. Obviously, culture-dependent method identified less and different bacteria, than culture-independent approach. The chemical approach and the culture independent metagenomic method were in good agreement. As expected, CFU/m3 accounted for not more than 0.3% of bacteria calculated as the average of chemical and culture independent metagenomic methods. The complexity of the obtained results shows that the different approaches are complementary to obtain an exhaustive description of bioaresol in terms of concentration, speciation, viability, pathogenicity.

First insights into oxidative stress and theoretical environmental risk of Bronopol and Detarox® AP, two biocides claimed to be ecofriendly for a sustainable aquaculture.

Bronopol and Detarox® AP are broad spectrum antimicrobial biocides of growing interest for the aquaculture sector. While their effectiveness against aquatic pathogens has been demonstrated, toxicity data on wild or farmed species are still lacking, as is information on their potential environmental risk for aquatic ecosystems. With this study, we assessed the acute and sublethal toxicity of Bronopol and Detarox® AP in the freshwater bivalve Sinanodonta woodiana and their theoretical risk for aquatic ecosystem. The 96-h median lethal concentration (LC50) was determined using the acute toxicity test, while for the sublethal toxicity test the bivalves were exposed to two concentrations for 14 days of Bronopol (2.5 and 50 mg/L) and Detarox® AP (1.11 and 22.26 mg/L) followed by a 14-day withdrawal period. Biocide-mediated oxidative processes were investigated via a panel of oxidative stress biomarkers (malondialdehyde, superoxide dismutase, catalase, glutathione peroxidase, glutathione S-transferase). Theoretical environmental risk assessment of both biocides, with predicted concentration of no effect (PNEC), expected theoretical concentration (TEC) in the environment, and risk quotient (RQ) was performed. TEC was calculated using a model based on the size of the aquaculture facility and the receiving basin, the estimated quantity of biocide dissolved in water, and published data on biocide stability in water. Although the LC50 was higher for Bronopol (2440 mg/L) than for Detarox® AP (126 mg/L), fluctuations in oxidative stress biomarkers levels indicated that both biocides exert a slight oxidative pressure on S. woodiana. Theoretical environmental risk assessment suggested a muted risk with Detarox® AP and greater eco-sustainability compared to Bronopol.

Characterization of long-range transported bioaerosols in the Central Mediterranean.

Airborne bacteria were characterized over a 2-y period via high-throughput massive sequencing of 16S rRNA gene in aerosol samples collected at a background mountain European Monitoring and Evaluation Programme (EMEP) Network site (Monte Martano, Italy) located in the Central Mediterranean area. The air mass origin of nineteen samples was identified by air mass modelling and a detailed chemical analysis was performed. Four main origins (Saharan, North-western, North-eastern, and Regional) were identified, and distinct microbial communities were associated with these air masses. Samples featured a great bacterial diversity with Protobacteria being the most abundant phylum, and Sphingomonas followed by Acidovorax, Acinetobacter and Stenotrophomonas the most abundant genera of the dataset. Bacterial genera including potential human and animal pathogens were more abundant in European and in Regional samples compared to Saharan samples; this stressed the relevance of anthropic impact on bacterial populations transported by air masses that cross densely populated areas. The principal aerosol chemical characteristics and the airborne bacterial communities were correlated by cluster analysis, similarity tests and non-metric multidimensional scaling analysis, explaining most of the variability observed. However, the strong correlation between bacterial community structure and air mass origin hampered the possibility to disentangle the effects of variations in bacterial populations and in dust provenance on variations in chemical variables.

Plant-microorganisms interaction promotes removal of air pollutants in Milan (Italy) urban area.

Plants and phyllosphere microorganisms may effectively contribute to reducing air pollution in cities through the adsorption and biodegradation of pollutants onto leaves. In this work, during all seasons, we sampled atmospheric particulate matter (PM10) and leaves of southern magnolia Magnolia grandiflora and deodar cedar Cedrus deodara, two evergreen plant species widespread in the urban area of Milan where the study was carried out. We then quantified Polycyclic Aromatic Hydrocarbons (PAHs) both in PM10 and on leaves and used sequencing of 16S rRNA gene, shotgun metagenomics and qPCR analyses to investigate the microbial communities hosted by the sampled leaves. Taxonomic and functional profiles of epiphytic bacterial communities differed between host plant species and seasons and the microbial communities on leaves harboured genes involved in the degradation of hydrocarbons. Evidence collected in this work also suggested that the abundance of hydrocarbon-degrading microorganisms on evergreen leaves increased with the concentration of hydrocarbons when atmospheric pollutants were deposited at high concentration on leaves, and that the biodegradation on the phyllosphere can contribute to the removal of PAHs from the urban air.

Evaluation of Bioaerosol Bacterial Components of a Wastewater Treatment Plant Through an Integrate Approach and In Vivo Assessment.

Wastewater carries different pathogenic and non-pathogenic microorganisms that can be dispersed in the surrounding environment. Workers who frequent sewage treatment plants can therefore be exposed to aerosols that contain a high concentration of potentially dangerous biological agents, or they can come into direct contact with contaminated material. This can lead to allergies, infections and occupational health-associated diseases. A characterization of biological risk assessment of bioaerosol exposure is necessary. The aim of this study was to evaluate the application of an interdisciplinary method that combines chemical and biological approaches for the analysis of a bioaerosol derived from a wastewater treatment plant (WWTP) situated in Italy. Sampled filters were analyzed by HPLC-MS/MS spectroscopy that searched for different chemical biomarkers of airborne microorganisms. The analytical quantification was compared to the biological cultural method that revealed an underrated microbial concentration. Furthermore, next generation sequencing analysis was used also to identify the uncultivable species that were not detected by the culture dependent-method. Moreover, the simple animal model Caenorhabditis elegans was used to evaluate the pathogenicity of two isolates-Acinetobacter iwoffii and Micrococcus luteus-that showed multidrug-resistance. This work represents a starting point for the development of a multidisciplinary approach for the validation of bioaerosol exposure on WWTP workplaces.

Airborne bacteria and persistent organic pollutants associated with an intense Saharan dust event in the Central Mediterranean.

In this paper, we present a comprehensive taxonomic survey of the bacterial community and accurate quantification of polycyclic aromatic hydrocarbons (PAHs) associated with an intense Saharan dust advection, which impacted Central Mediterranean area in the whole 2014-2015 period. This work is part of an intensive field campaign at the EMEP regional background site of Monte Martano (Central Italy), considered well representative of long-range transport in the Central Mediterranean area. 22 samples have been characterized in their provenance region and have been considered for the chemical and biological characterization. The event described in the present paper was exceptionally intense at the sampling site allowing a detailed evaluation of the dust load on a regional scale, an estimation of the impact of PAH based on the Toxic Equivalency Factor methodology and a thorough characterization of the airborne bacterial fraction performed by High Throughput Sequencing approach. Afterward, we cultured viable bacteria and evaluated several enzymatic activities and conducted UV survival tests. Principal findings include: (i) the striking evidence that, during the Saharan dust event, a highly diverse and abundant bacterial community was associated with PAH concentrations higher than the yearly mean; (ii) the tangible presence of cultivable microbes; (iii) the proof that the isolates recovered from Saharan dust had the potential to be metabolically active and that almost all of them were able to persist following UV radiation exposure. Comparisons of results for the present case study with mean values for the 2014-2015 experimental campaign are presented. The bacterial community and chemical speciation associated with the Saharan dust advection were specific and very different from those associated with other air masses. The particular case of North-Western Atlantic, which represents one of the most typical advection route reaching the sampling site is discussed in detail.

Food-Associated Lactobacillus plantarum and Yeasts Inhibit the Genotoxic Effect of 4-Nitroquinoline-1-Oxide.

Lactic acid bacteria and yeasts, representing the prevailing microbiota associated with different foods generally consumed without any cooking, were identified and characterized in vitro for some functional properties, such as acid-bile tolerance and antigenotoxic activity. In particular, 22 Lactobacillus plantarum strains and 14 yeasts were studied. The gastro-intestinal tract tolerance of all the strains was determined by exposing washed cell suspensions at 37°C to a simulated gastric juice (pH 2.0), containing pepsin (0.3% w/v) and to a simulated small intestinal juice (pH 8.0), containing pancreatin (1 mg mL-1) and bile extract (0.5%), thus monitoring changes in total viable count. In general, following a strain-dependent behavior, all the tested strains persisted alive after combined acid-bile challenge. Moreover, many strains showed high in vitro inhibitory activity against a model genotoxin, 4-nitroquinoline-1-oxide (4-NQO), as determined by the short-term method, SOS-Chromotest. Interestingly, the supernatants from bacteria- or yeasts-genotoxin co-incubations exhibited a suppression on SOS-induction produced by 4-NQO on the tester strain Escherichia coli PQ37 (sfiA::lacZ) exceeding, in general, the value of 75%. The results highlight that food associated microorganisms may reach the gut in viable form and prevent genotoxin DNA damage in situ. Our experiments can contribute to elucidate the functional role of food-associated microorganisms general recognized as safe ingested with foods as a part of the diet.

Bacterial Composition, Genotoxicity, and Cytotoxicity of Fecal Samples from Individuals Consuming Omnivorous or Vegetarian Diets.

This study analyzes the composition of viable fecal bacteria and gut toxicology biomarkers of 29 healthy volunteers, who followed omnivorous, lacto-ovo-vegetarian, or vegan diets. In particular, the research was focused on the prevalence of some representative viable bacteria from the four dominant phyla (Firmicutes, Bacteroidetes, Proteobacteria, Actinobacteria) commonly present in human feces, in order to evaluate the relationship between microorganisms selected by the habitual dietary patterns and the potential risk due to fecal water (FW) genotoxicity and cytotoxicity, considered as biomarkers for cancer risk and protective food activity. The relative differences of viable bacteria among dietary groups were generally not statistically significant. However, compared to omnivores, lacto-ovo-vegetarians showed low levels of total anaerobes. Otherwise, vegans showed total anaerobes counts similar to those of omnivores, but with lower number of bifidobacteria and the highest levels of bacteria from the Bacteroides-Prevotella genera. FW genotoxicity of lacto-ovo-vegetarians resulted significantly lower either in relation to that of omnivores and vegans. Lacto-ovo-vegetarians also showed the lowest levels of cytotoxicity, while the highest were found for vegans. These results highlighted that lacto-ovo-vegetarian diet was particularly effective in a favorable modulation of microbial activity, thus contributing to a significant reduction of the genotoxic and cytotoxic risk in the gut.

Characterization of the Skin Microbiota in Italian Stream Frogs (Rana italica) Infected and Uninfected by a Cutaneous Parasitic Disease.

In human and wildlife populations, the natural microbiota plays an important role in health maintenance and the prevention of emerging infectious diseases. In amphibians, infectious diseases have been closely associated with population decline and extinction worldwide. Skin symbiont communities have been suggested as one of the factors driving the different susceptibilities of amphibians to diseases. The activity of the skin microbiota of amphibians against fungal pathogens, such as Batrachochytrium dendrobatidis, has been examined extensively, whereas its protective role towards the cutaneous infectious diseases caused by Amphibiocystidium parasites has not yet been elucidated in detail. In the present study, we investigated, for the first time, the cutaneous microbiota of the Italian stream frog (Rana italica) and characterized the microbial assemblages of frogs uninfected and infected by Amphibiocystidium using the Illumina next-generation sequencing of 16S rRNA gene fragments. A total of 629 different OTUs belonging to 16 different phyla were detected. Bacterial populations shared by all individuals represented only one fifth of all OTUs and were dominated by a small number of OTUs. Statistical analyses based on Bray-Curtis distances showed that uninfected and infected specimens had distinct cutaneous bacterial community structures. Phylotypes belonging to the genera Janthinobacterium, Pseudomonas, and Flavobacterium were more abundant, and sometimes almost exclusively present, in uninfected than in infected specimens. These bacterial populations, known to exhibit antifungal activity in amphibians, may also play a role in protection against cutaneous infectious diseases caused by Amphibiocystidium parasites.

In Vitro Inhibition of 4-Nitroquinoline-1-Oxide Genotoxicity by Probiotic Lactobacillus rhamnosus IMC501.

Inhibition of 4-nitroquinoline-1-oxide (4-NQO) genotoxicity by a probiotic strain of Lactobacillus rhamnosus (IMC501) was assessed by the prokaryotic short-term bioassay SOSChromotest, using Escherichia coli PQ37 as the target organism. Results showed the ability of strain IMC501 to rapidly and markedly counteract, in vitro, the DNA damage originated by the considered genotoxin. The inhibition was associated with a spectroscopic hypsochromic shift of the original 4-NQO profile and progressive absorbance increase of a new peak. IR-Raman and GC-MS analyses confirmed the disappearance of 4-NQO after contact with the microorganism, showing also the absence of any genotoxic molecule potentially available for metabolic activation (i.e., 4-hydroxyaminoquinoline-1-oxide and 4-nitrosoquinoline-1-oxide). Furthermore, we have shown the presence of the phenyl-quinoline and its isomers as major non-genotoxic conversion products, which led to the hypothesis of a possible pattern of molecular transformation. These findings increase knowledge on lactobacilli physiology and contribute to the further consideration of antigenotoxicity as a nonconventional functional property of particular probiotic strains.

Protective effects of probiotic Lactobacillus rhamnosus IMC501 in mice treated with PhIP.

The aim of the present study was to investigate the antigenotoxic properties of the probiotic Lactobacillus rhamnosus IMC501; DNA damage was induced by one representative food mutagen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). Mice were treated orally with suspensions of lactobacilli for 10 days before administration of food mutagen. During the treatment, the abundance of lactobacilli in feces, as assessed by qPCR analysis, increased, whereas ß-glucuronidase and N-acetyl-ß-glucosaminidase activities decreased. The extent of DNA damage was measured in colon and liver cells by comet assay. In colonocytes, diet supplementation with IMC501 resulted in a significant inhibition of DNA damage induced by PhIP. The results obtained in this in vitro study suggest that Lactobacillus rhamnosus IMC501 used as a dietary supplement can provide a useful integration of antimutagen food components of the normal diet, which are generally lower than the protective level.

Addition of maize stalks and soybean oil to a historically PCB-contaminated soil: effect on degradation performance and indigenous microbiota.

Objective of this study was to assess the single or combined effect of a plant oil and a lignocellulosic waste, namely soybean oil (SO) and maize stalks (MS), respectively, on resident microbiota and bioremediation performances of a soil historically contaminated by medium to highly chlorinated PCBs. Higher concentrations of both biphenyl- and chlorobenzoate-degrading cultivable bacteria were found in the MS-amended microcosms (MSM) than the non amended or SO-amended ones after 30 d incubation at 28°C. Fungal growth, instead, was strikingly stimulated in the microcosms that had undergone concomitant MS and SO supplementation (MS-SOM). Denaturing gradient gel electrophoresis analyses of 16S and 18S rRNA genes showed that both amendments promoted a remarkable increase in both bacterial and fungal biodiversity. The abundances of biphenyl-2,3-dioxygenase (bph) and that of catechol-2,3-dioxygenase (C230) genes in the non-amended contaminated soil were constant over time. Conversely, after 60 d incubation, bph and C230 abundances increased 2.8- and 61-fold in the MSM, respectively, and, in the MS-SOM, 1.4- and 46-fold, respectively, with respect to the zero time point. Although the overall PCB removal was not positively affected by the amendments, the concomitant presence of both MS and SO led to significantly higher depletions of hexa-, hepta-, octa- and nona-chlorinated congeners than in the non-amended microcosms (i.e. 24.6, 22, 20.5 and 9.5%, versus 19.4, 16.4, 14.7 and 6.1%, respectively). In all microcosms, PCB degradation was negatively correlated with hydrophobicity, organic matter/water partition coefficient, molecular weight and extent of chlorination of the pollutants with the notable exception of the MS-SOM ones where such a relationship was less stringent.

Aerobic treatment of dairy wastewater in an industrial three-reactor plant: effect of aeration regime on performances and on protozoan and bacterial communities.

An industrial three-reactor plant treating 45 m(3) d(-1) of dairy wastewater was monitored to investigate the effect of different aeration regimes on performance efficiency and to find relationships with bacterial and protozoan communities in the activated sludge. During the study, the plant was maintained at six different "on/off" cycles of the blower (45/15, 15/15, 15/45, 30/30, 30/45 and 30/60 min), providing between 30.2 and 90.6 kg O(2) d(-1), and the main chemical/biochemical parameters (COD, BOD, NH(4)(+), NO(2)(-), NO(3)(-), PO(4)(3-), etc.) were determined. When at least 45.4 kg O(2) d(-1) (30/45) were provided, COD removal efficiencies were always in the range 88-94% but decreased to about 70% under aeration regimes 15/45 and 30/60. Ammonium ion degradation performance was compromised only in the lowest aeration regime (15/45). Total number of protozoa and their species richness, and bacterial viable counts and denaturing gradient gel electrophoresis (DGGE) profiles were used to characterize the microbiota of the activated sludge. Cell abundances and community structures of protozoa and bacteria were very similar in the three aerated reactors but changed with the aeration regimes. In particular, the 15/45 and 30/60 regimes led to low protozoan diversity with prevalence of flagellates of the genus Trepomonas at the expense of the mobile and sessile forms and, thus, to a less efficient activated sludge as indicated by Sludge Biotic Index values (3 and 4.5 for the two regimes, respectively). The structure of the bacterial community strongly changed when the aeration regimes varied, as indicated by the low similarity values between the DGGE profiles. On the contrary, number of viable bacteria and values of the biodiversity index remained stable throughout the whole experimentation. Taken together, the results of the present study clearly indicate that aeration regime variations strongly influence the structure of both protozoan and bacterial communities and, above all, that a high biodiversity among protozoan populations in the activated sludge is prerequisite for high performances in dairy wastewater treatment.

Bioaugmentation of a historically contaminated soil by polychlorinated biphenyls with Lentinus tigrinus.

BACKGROUND: Several species belonging to the ecological group of white-rot basidiomycetes are able to bring about the remediation of matrices contaminated by a large variety of anthropic organic pollutants. Among them, polychlorobiphenyls (PCBs) are characterized by a high recalcitrance due to both their low bioavailability and the inability of natural microbial communities to degrade them at significant rates and extents. Objective of this study was to assess the impact of a maize stalk-immobilized Lentinus tigrinus CBS 577.79 inoculant combined with soybean oil (SO), as a possible PCB-mobilizing agent, on the bioremediation and resident microbiota of an actual Aroclor 1260 historically contaminated soil under unsaturated solid-phase conditions. RESULTS: Best overall PCB depletions (33.6 ± 0.3%) and dechlorination (23.2 ± 1.3%) were found after 60 d incubation in the absence of SO where, however, the fungus appeared to exert adverse effects on both the growth of biphenyl- and chlorobenzoate-degrading bacteria and the abundance of genes coding for both biphenyl dioxygenase (bph) and catechol-2,3-dioxygenase. A significant (P < 0.001) linear inverse relationship between depletion yields and degree of chlorination was observed in both augmented and control microcosms in the absence of SO; conversely, this negative correlation was not evident in SO-amended microcosms where the additive inhibited the biodegradation of low chlorinated congeners. The presence of SO, in fact, resulted in lower abundances of both biphenyl-degrading bacteria and bph. CONCLUSIONS: The PCB depletion extents obtained in the presence of L. tigrinus are by far higher than those reported in other remediation studies conducted under unsaturated solid phase conditions on actual site soils historically contaminated by Aroclor 1260. These results suggest that the bioaugmentation strategy with the maize stalk-immobilized mycelium of this species might be promising in the reclamation of PCB-contaminated soils. The addition of SO to matrices contaminated by technical PCB mixtures, such as Aroclor 1242 and Delor 103 and characterized by a large preponderance of low chlorinated congeners, might not be advisable.

Food borne yeasts as DNA-bioprotective agents against model genotoxins.

Yeasts isolated from Italian beverages and foods (wine and cheeses) were identified as Saccharomyces cerevisiae and Debaryomyces hansenii by sequencing the D1/D2 domain of the 26S rRNA gene and differentiated, at strain level, by microsatellite PCR fingerprinting and RAPD-PCR. All the strains showed antioxidant activity, as demonstrated by their ability to scavenge the free radical diphenyl-1-picrylhydrazyl (DPPH). Furthermore, tested strains revealed high in vitro inhibitory activity against two model genotoxins, 4-nitroquinoline-1-oxide (4-NQO) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), as showed by short-term methods with different target cells: SOS-Chromotest with Escherichia coli PQ37 and Comet assay with HT-29 enterocytes. High inhibitory activity towards 4-NQO was associated with cell viability, while heat-inactivated cells showed a reduced antigenotoxic capability. Surprisingly, high inhibition of MNNG genotoxicity was observed even with heat-treated cells. Moreover, the strains able to inhibit the genotoxins induced some changes in the spectroscopic properties of the original compound. This result perfectly agrees with the information obtained by the two bioassays. Interestingly, strains characterized for antioxidant and antigenotoxic properties, also presented acid-bile tolerance, indicating that food autochthonous yeasts could be expected to reach gut in viable form and thus prevent genotoxin DNA damage in situ.

Two-phase olive mill waste composting: community dynamics and functional role of the resident microbiota.

In this study, physico-chemical modifications and community dynamics and functional role of the resident microbiota during composting of humid husk from a two-phase extraction system (TPOMW) were investigated. High mineralization and humification of carbon, low loss of nitrogen and complete degradation of polyphenols led to the waste biotransformation into a high-quality compost. Viable cell counts and denaturing gradient gel electrophoresis (DGGE) profiling of the 16S rRNA genes showed that the thermophilic phase was characterized by the strongest variations of cell number, the highest biodiversity and the most variable community profiles. The isolation of tannin-degrading bacteria (e.g. Lysinibacillus fusiformis, Kocuria palustris, Tetrathiobacter kashmirensis and Rhodococcus rhodochrous) suggested a role of this enzymatic activity during the process. Taken together, the results indicated that the composting process, particularly the thermophilic phase, was characterized by a rapid succession of specialized bacterial populations with key roles in the organic matter biotransformation.

Short-term impact of dry olive mill residue addition to soil on the resident microbiota.

The short-term response of the resident soil bacterial and fungal communities to the addition of 5% (w/w) of either dry olive mill residue (DOR), DOR treated with Phlebia sp. (PTDOR) or DOR previously extracted with water (WEDOR) was investigated. As opposed to bacteria, the diversity of fungi increased upon the amendments as assessed by denaturing gradient gel electrophoresis of 18S rDNA. Over the first 30 days, phospholipid fatty acids analyses indicated a gradual decrease in the relative abundances of gram(+) bacteria (from 44.8% to 37.9%) and a concomitant increase of gram(-) bacteria (from 37.3% to 51.2%) in DOR-amended soil. A considerable increase in the fungal/bacterial ratio was observed after 7 days in DOR, WEDOR and PTDOR-amended soils with respect to the control (0.316, 0.165 and 0.265, respectively, vs. 0.011). The overall microbial activity was stimulated by the amendments as indicated by the higher activity levels of both dehydrogenase and fluorescein diacetate hydrolase. These results indicate that DOR at the application level examined is not toxic on soil microorganisms.

Selective targeting of death receptor 5 circumvents resistance of MG-63 osteosarcoma cells to TRAIL-induced apoptosis.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a tumor necrosis factor superfamily member, targets death receptors and selectively kills malignant cells while leaving normal cells unaffected. However, unlike most cancers, many osteosarcomas are resistant to TRAIL. To investigate this resistance, we characterized the response of MG-63 osteosarcoma cells and hPOB-tert osteoblast-like cells to TRAIL and agonist antibodies to death receptor 4 (DR4) and death receptor 5 (DR5). We found that MG-63 osteosarcoma cells and hPOB-tert osteoblast-like cells show no or very little response to TRAIL or a DR4 agonist, but MG-63 cells undergo apoptosis in response to a DR5 agonist. Analysis of TRAIL receptor expression showed that normal osteoblastic and osteosarcoma cells express a variety of TRAIL receptors but this does not correlate to TRAIL responsiveness. Production of the soluble decoy receptor osteoprotegerin also could not explain TRAIL resistance. We show that TRAIL activates the canonical caspase-dependent pathway, whereas treatment with cycloheximide increases the sensitivity of MG-63 cells to TRAIL and anti-DR5 and can also sensitize hPOB-tert cells to both agents. Proapoptotic and antiapoptotic protein expression does not significantly differ between MG-63 and hPOB-tert cells or change following treatment with TRAIL or anti-DR5. However, sequencing the death domain of DR4 in several osteoblast-like cells showed that MG-63 osteosarcoma cells are heterozygous for a dominant-negative mutation, which can confer TRAIL resistance. These results suggest that although the dominant-negative form of the receptor may block TRAIL-induced death, an agonist antibody to the active death receptor can override cellular defenses and thus provide a tailored approach to treat resistant osteosarcomas.