Microbial, chemical, and isotopic monitoring integrated approach to assess potential leachate contamination of groundwater in a karstic aquifer (Apulia, Italy).

Landfill sites are subjected to long-term risks of accidental spill of leachate through the soil and consequential contamination of the groundwater. Wide areas surrounding the landfill can seriously be threatened with possible consequences to human health and the environment. Given the potential impact of different coexisting anthropic pollution sources (i.e., agriculture and cattle farming) on the same site, the perturbation of the groundwater quality may be due to multiple factors. Therefore, it is a challenging issue to correctly establish the pollution source of an aquifer where the landfill is not isolated from other anthropic land uses, especially in the case of a karstic coastal aquifer. The present study is aimed at setting in place an integrated environmental monitoring system that included microbiological, chemical, and isotope methods to evaluate potential groundwater pollution in a landfill district in the south of Italy located in Murgia karstic aquifer. Conventional (microbial plate count and physical-chemical analyses) and advanced methods (PCR-ARISA, isotope analysis of δ18O, δ2H, 3H, δ 13C, δ 15N-NO3-, and δ 18O-NO3-) were included in the study. Through data integration, it was possible to reconstruct a scenario in which agriculture and other human activities along with seawater intrusion in the karst aquifer were the main drivers of groundwater pollution at the monitored site. The microbiological, chemical, and isotope results confirmed the absence of leachate effects on groundwater quality, showing the decisive role of fertilizers as potential nitrate sources. The next goal will be to extend long-term integrated monitoring to other landfill districts, with different geological and hydrogeological characteristics and including different sources of pollution, to support the ecological restoration of landfills.

The effect of substituting energy crop with agricultural waste on the dynamics of bacterial communities in a two-stage anaerobic digester.

The replacement of energy crops with agricultural waste in biogas production through anaerobic digestion (AD) is both an environmentally sustainable and economically profitable strategy. However, the change of feeding mix in AD might result in nutrient imbalance or increase of the ammonium concentration, negatively affecting the activity of the microbes responsible for the process. In the present study the structure and dynamics of the bacterial communities of a full-scale two-stage AD plant, composed of a hydrolysis/acidogenesis (H) and an acetogenesis/methanogenesis (M) tanks, was monitored during feedstock substitution. Energy crop (triticale) was replaced by poultry manure litter and olive mill pomace. The increase percentage of poultry manure litter (up to 8.6%) and olive mill pomace (up to 30.5%) in the recipe incremented the total solids (up to 21% in H) and, consequently, the nitrogen content in the digestate (6.7 g N/kg in the solid fraction in H and 4-5 g NH4+-N/L in the liquid fraction). This favored the growth of Lactococcus sp. with consequent increment of lactate production (∼ 1 mg L-1 last two days of the survey) and the establishment of Weissella and Lactobacillus spp. Syntrophic acetate-oxidizers, including Syntrophaceticus (6% ± 1.7%), were detected manly in M but were negatively affected by the addition of the poultry manure litter, while the sulfate-reducing bacteria correlated with the variations of the volatile fatty acids. Planctomycetes putatively capable of anammox process were also found in the H during the first two days of the survey and accounted for 0.3 ± 0.01% of the total bacterial community. The stability of the process during feedstock change is the result of the shift of bacterial populations of different functional groups that showed peculiar adaptation patterns in the two stages of the plant.

In vitro removal of ochratoxin A by two strains of Saccharomyces cerevisiae and their performances under fermentative and stressing conditions.

AIMS: The aim of this research was to study the effect of time, temperature, sugar content and addition of diammonium phosphate (DAP) on ochratoxin A (OTA) removal by two strains of Saccharomyces cerevisiae using a completely randomized design. METHODS AND RESULTS: The strains were grown in a medium containing OTA (2 µg l(-1)), two sugar levels (200 and 250 g l(-1)), with or without DAP (300 mg l(-1)), and incubated at 25-30°C. The yeasts were able to decrease the toxin amount by c. 70%, with the highest removing effect observed after 3 days at 30°C in the presence of 250 g l(-1) of sugars and with DAP; after 10 days, the toxin was partially released into the medium. The strains produced high ethanol and glycerol contents, showed high tolerance to single/combined stress conditions and possessed ß-d-glucosidase, pectinase and xylanase activities. CONCLUSIONS: Ochratoxin A removal was affected by time, temperature, sugar and addition of DAP. Moreover, the phenomenon was reversible. SIGNIFICANCE AND IMPACT OF THE STUDY: Ochratoxin A removal could be an interesting trait for the selection of promising strains; however, the strains removing efficiently the toxin could release it back; thus, the selection of the starter should take into account both the removal and the binding ability of OTA.

Expression of Lactobacillus brevis IOEB 9809 tyrosine decarboxylase and agmatine deiminase genes in wine correlates with substrate availability.

AIMS: Lactobacillus brevis IOEB 9809 is able to produce both tyramine and putrescine via tyrosine decarboxylase and agmatine deiminase enzymes, respectively, when cultured on synthetic media. The aims of this study were to assess the expression of L. brevis IOEB 9809 tdc and aguA1 genes, during wine fermentation and to evaluate the effect of substrate availability and pH on tdc and aguA1 expression, as well as on biogenic amine production and L. brevis viability. METHODS AND RESULTS: The relative expression of L. brevis IOEB 9809 tdc and aguA1 genes was analysed in wine by quantitative real-time RT-PCR (qRT-PCR) during a period of incubation of 30 days. Cell viability, pH values, putrescine and tyramine concentration were monitored throughout the experiments. CONCLUSIONS: The wine trials indicated that L. brevis IOEB 9809 is able to produce both tyramine and putrescine during wine fermentation. Increased cell viability was also observed in wine supplemented with tyrosine or agmatine. qRT-PCR analysis suggests a strong influence of substrate availability on the expression of genes coding for tyrosine decarboxylase and agmatine deiminase in L. brevis IOEB 9809. Less evident is the relationship between putrescine and tyramine production and tolerance to wine pH. SIGNIFICANCE AND IMPACT OF STUDY: To our knowledge, this study represents the first assessment of relative expression of L. brevis IOEB 9809 genes involved in biogenic amine production in wine. Furthermore, an effect of biogenic amine production on viability of L. brevis during wine fermentation was established.

[Immunity and cancer: the role of PSA IgM Immune complexes for prostate cancer]. / Immunità e cancro: ruolo degli immunocomplessi PSA-IGM nel cancro della prostata.

Immunoediting is a new concept in cancer surveillance. Immunity is involved in detecting cellular waste, and taking off transformed cells. In particular, natural IgM antibodies play an important role in immunosurveillance mechanisms against transformed cells in humans.? Scientific evidence indicates that biomarkers for different types of cancer, such as liver and colorectal cancer, circulate in blood associated with immunoglobulin M (IgM) to form complexes that improve diagnosis in comparison to circulating free biomarkers. In prostate cancer it has been demonstrated that testing for serum levels of the PSA-IgM immune complex improves the diagnostic performance of total PSA. Preliminary reports indicate that the combination of PSA-IgM with total PSA is the best approach to reduce the number of negative prostatic mapping thus improving the diagnosis of prostate cancer.

Occurrence of Vibrio vulnificus in mussel farms from the Varano lagoon environment.

AIMS: Monitoring the occurrence of the human pathogen Vibrio vulnificus in a mussel farm located in the lagoon of Varano (Italy). METHODS AND RESULTS: A total of 72 samples of mussel, water and sediment, collected from two locations of Varano lagoon in the Gargano peninsula, during a 7-month survey, were analysed. Isolation and PCR characterization of six V. vulnificus environmental genotype strains revealed that this pathogen was isolated when with T was above 22 °C and salinity ranged between 22.7 and 26.4‰. No significant correlation of the occurrence of V. vulnificus with water pH or salinity was observed. Moreover, 8% of mussel samples were found to be contaminated by V. vulnificus. All of that positive mussel samples originated from the same sampling station. CONCLUSION: It is suggested that warmer season are risky to eat raw or undercooked bivalve molluscs in the local area. SIGNIFICANCE AND IMPACT OF THE STUDY: To increase knowledge about environmental conditions that may affect the occurrence of waterborne pathogen Vibrio vulnificus in seafood.

Technological properties of Oenococcus oeni strains isolated from typical southern Italian wines.

AIMS: To isolate indigenous Oenococcus oeni strains suitable as starters for malolactic fermentation (MLF), using a reliable polyphasic approach. METHODS AND RESULTS: Oenococcus oeni strains were isolated from Nero di Troia wines undergoing spontaneous MLF. Samples were taken at the end of alcoholic fermentation and during MLF. Wine samples were diluted in a sterile physiological solution and plated on MRS and on modified FT80. Identification of O. oeni strains was performed by a polymerase chain reaction (PCR) experiment using strain-specific primers. Strains were further grouped using a multiplex RAPD-PCR analysis. Then, six strains were inoculated in two winelike media with two different ethanol concentrations (11 and 13% vol / vol) with a view to evaluate their capacity to grow and to perform MLF. In addition, a quantitative PCR (qRT-PCR) approach was adapted to monitor the physiological state of the strains selected. CONCLUSION: A positive correlation between the malolactic activity performance and the ability to develop and tolerate stress conditions was observed for two selected O. oeni strains. SIGNIFICANCE AND IMPACT OF THE STUDY: The results reported are useful for the selection of indigenous MLF starter cultures with desired oenological traits from typical regional wines. It should be the base for the improvement in organoleptic quality of typical red wine.

Squamous cell carcinoma antigen-1 (SERPINB3) polymorphism in chronic liver disease.

BACKGROUND: The serpin squamous cell carcinoma antigen (SCCA, SERPINB3) has been found over-expressed in primary liver cancer and at lower extent in cirrhosis and chronic hepatitis. A novel SCCA-1 variant (SCCA-PD), presenting a single mutation in the reactive centre (Gly351Ala), has been recently identified (rs3180227). AIM: To explore SCCA-1 polymorphism in patients with HCV infection as single etiologic factor and different extent of liver disease. METHODS: One hundred and fourty-eight patients with chronic HCV infection (45 chronic hepatitis, 53 cirrhosis, 50 HCC) and 50 controls were evaluated. SCCA-1 polymorphism was studied by restriction fragment length polymorphism and confirmed randomly by direct sequencing. Circulating SCCA-IgM complex was determined by ELISA. RESULTS: SCCA-PD was detected with higher frequency in cirrhotic patients (45.3%, odds ratio=2.62; 95%CI 1.13-6.10, p=0.038) than in patients with chronic hepatitis or in controls (24.4% and 24%, respectively). Intermediate figures were found in hepatocarcinoma (36.0%). SCCA-IgM in serum was lower in patients carrying SCCA-PD than in wild type patients and the difference was statistically significant in cirrhotic patients (mean+/-S.D.=117.45+/-54.45 U/ml vs. 268.52+/-341.27 U/ml, p=0.026). CONCLUSIONS: The newly identified SCCA-PD variant was more frequently found in liver cirrhosis, suggesting that patients carrying this polymorphism are more prone to develop progressive liver fibrosis.

Tumour-specific induction of immune complexes: DCP-IgM in hepatocellular carcinoma.

BACKGROUND: In the sera of liver, colorectal and prostate cancer patients, several biomarkers may be detected as IgM immune complexes. To determine whether the presence of immune complexes was correlated to an increase of IgMs, we measured the IgM content in the sera of patients with hepatocellular carcinoma (HCC) and cirrhosis, and evaluated the occurrence of des-gamma-carboxy prothrombin (DCP) as immune complexes (DCP-IgM) compared to the levels of DCP and alpha-fetoprotein (AFP). PATIENTS AND METHODS: Serum samples from 31 patients with cirrhosis, 33 untreated HCC patients diagnosed by ultrasound, computed tomography and/or magnetic resonance and confirmed by histopathology, when indicated, and 30 healthy controls were analysed. Concentrations of IgM and DCP-IgM were determined by ELISAs. RESULTS: Circulating IgM in patients with HCC (median level = 1.79 mg mL(-1)) and cirrhosis (1.09 mg mL(-1)) were not significantly different (P = 0.1376) while DCP-IgM were significantly higher in HCC patients (median level = 2171.2 AU mL(-1)) than in those with cirrhosis (1152 AU mL(-1), P = 0.0047). No correlation was found between DCP-IgM and IgM in HCC (r = 0.227) and cirrhosis patients (r = 0.475). DPC-IgM was positive in 55% (18/33) of HCC patients and in 26% (8/31) of cirrhosis patients compared to 39% and 26% for DCP and 48% and 13% for AFP. DCP-IgM, DCP and AFP tests had 100% specificity in healthy controls. CONCLUSIONS: DCP-IgM in HCC patients was not associated with an increase in IgM concentration. DCP-IgM was more frequently detected in HCC patients than DCP and AFP, strengthening the diagnostic role of IgM immune complexes for liver cancer.

Monitoring SCCA-IgM complexes in serum predicts liver disease progression in patients with chronic hepatitis.

About 30% of the patients with chronic hepatitis develop a progressive liver disease and one of the most intriguing issues is the detection of noninvasive markers for fibrosis stage and disease progression. High levels of squamous cell carcinoma antigen (SCCA)-immunoglobulin M (IgM) are detectable in hepatocellular carcinoma and their increase in cirrhotic patients can predict tumour development. As SCCA-IgM can also be detectable at low percentages in patients with chronic hepatitis, the aim of this study was to assess SCCA-IgM complexes in relation to disease outcome in this group of patients. An ELISA assay was used to determine the presence of SCCA-IgM in 188 patients with chronic hepatitis and in 100 controls. An additional serum sample was available after a median period of 6 years in 57 untreated patients: these patients were subdivided in group A, including eight patients with a fibrosis score increase > or =2 in a second liver biopsy and group B, including 49 patients without fibrosis progression during a similar follow up. SCCA-IgM complexes were detectable in 63 of 188 (33%) patients but in none of the controls. A significant increase of SCCA-IgM levels over time was observed in patients with fibrosis progression (mean +/- SD: 117 +/- 200 U/mL/year), but not in those without histologic deterioration (mean +/- SD: -8.8 +/- 31 U/mL/year, P < 0.0001). In conclusion, monitoring SCCA-IgM levels over time appears a useful approach to identify patients with chronic hepatitis at higher risk for cirrhosis development.

Migraine and epilepsy: clinical and pathophysiological relations.

Migraine and epilepsy are both chronic disorders characterised by recurrent neurological attacks, with a partial clinical and therapeutic overlap and frequently occurring together. Although still incompletely clarified, the possible existence of a link between migraine and epilepsy has long been debated. In this paper the epidemiologic evidence of migraine and epilepsy comorbidity, the possible occurrence of both disturbances in close temporal association, possible shared physiopathologic mechanisms and the rationale for antiepileptic drug use in migraine prophylaxis will be discussed.

Validation of AIDA Cefalee, a computer-assisted diagnosis database for the management of headache patients.

AIDA Cefalee is a database for the management of headache patients developed on behalf of the Italian Neurological Association for Headache Research (ANIRCEF). The system integrates a diagnostic expert system able to suggest the correct ICHD-II diagnosis once all clinical characteristics of a patient's headache have been collected. The software has undergone a multicentre validation study to assess: its diagnostic accuracy; the impact of using the software on visit duration; the userfriendliness degree of the software interface; and patients' acceptability of computer-assisted interview. Five Italian headache centres participated in the study. The results of this study validate AIDA Cefalee as a reliable diagnostic tool for primary headaches that can improve diagnostic accuracy with respect to the standard clinical method without increasing the time length of visits even when used by operators with basic computer experience.

Cloning and characterization of the hsp 18.55 gene, a new member of the small heat shock gene family isolated from wine Lactobacillus plantarum.

Using a molecular approach based on PCR, RT-PCR and northern blot analysis, a new member of the small heat shock family of wine, Lactobacillus plantarum, was cloned and characterized. The protein sequence deduced from the isolated gene had a calculated molecular mass of 18.548 kDa and was therefore named HSP 18.55. The gene codes for a protein homologous to the previously characterized HSP 19.3 and HSP 18.5 and is co-transcribed with an upstream gene of unknown function. Analysis of the 5' flanking region of the hsp 18.55 gene revealed the presence of putative cis elements able to bind alternative sigma factor sigma(B). Based on its structure, the gene was classified as belonging to class II of the heat shock genes according to Bacillus subtilis nomenclature for shock-responsive genes. Expression of the newly identified small heat shock gene, analyzed by RT-PCR and northern blot analysis, was induced by a wide range of abiotic stresses including heat, cold and ethanol, suggesting that the small family of heat shock genes is probably involved in the general stress response in wine L. plantarum. Moreover, the expression of hsp 18.5, hsp 18.55 and hsp 19.3 genes, analyzed over a complete culture cycle, revealed that early growing cells contained substantial amounts of hsp 18.5, hsp 18.55 and hsp 19.3 mRNAs, which rapidly declined upon entry into stationary phase.

A beta-glucosidase gene isolated from wine Lactobacillus plantarum is regulated by abiotic stresses.

AIMS: Little genetic information exists on the ability of wine lactic acid bacteria (LAB) to hydrolyse glycoconjugates during malolactic fermentation. We tried to fill this important gap by characterizing a gene codifying for a putative beta-glucosidase enzyme from wine Lactobacillus plantarum and from a commercial strain of Oenococcus oeni. METHODS AND RESULTS: The coding region of the putative beta-glucosidase gene is 1400 nucleotides long and started with an ATG codon. The gene is widespread among LAB and the highest identity was observed between the nucleotide of L. plantarum, Lactobacillus pentosus, Lactobacillus paraplantarum and O. oenibeta-glucosidase gene. The protein sequence deduced from the isolated genes has a calculated molecular mass of 61.19 kDa. Furthermore, the expression of the beta-glucosidase gene in L. plantarum strain was analysed, under several stress, by reverse transcriptase (RT)-PCR and Northern-blot analysis. The gene was apparently regulated by abiotic stresses such as temperature, ethanol and pH. CONCLUSIONS: The beta-glucosidase gene is widespread among LAB and its expression is probably regulated by a wide range of abiotic stresses. SIGNIFICANCE AND IMPACT OF THE STUDY: The inhibitory effect of temperature and ethanol on the L. plantarumbeta-glucosidase gene may be useful to explain the differences found in beta-glucosidase activity reported in wines by several authors.

Real-time PCR for the detection of Escherichia coli O157:H7 in dairy and cattle wastewater.

AIMS: Developing and evaluating a rapid real-time polymerase chain reaction (PCR) method for the identification of Escherichia coli O157:H7 in cattle and dairy wastewater samples produced from mozzarella cheese factories, without pre-enrichment step before DNA extraction. METHODS AND RESULTS: Wastewater samples were collected from a dairy farm producing mozzarella cheese and located in Puglia (south of Italy). Plate count and other microbial assays were performed 1 h after sampling. Wastewater samples were artificially inoculated with 10(4), 10(7) and 10(8) cells ml(-1) of E. coli O157:H7, strain EDL933. PCR protocols for stx1, stx2 and eae genes were first tested on pure DNA extracted from type strains, in order to optimize the amplification conditions and reagent concentration before real-time PCR experiments. Three specific fragments of ca 106, 150 and 200 bp corresponding to genes eae, stx1 and stx2, respectively, were obtained. Real-time PCR experiments were performed with DNA extracted from dairy and manure wastewater samples inoculated with 10(4), 10(7) and 10(8) colony-forming units (CFU) ml(-1) of E. coli O157:H7 strain EDL 933. The sensitivity limit of the assay was 10(-1) pg microl(-1) for eae, stx2 and 16SrRNA, and 1 pg microl(-1) for stx1 gene respectively. CONCLUSIONS: A real-time PCR protocol has been developed and used in order to identify potential pathogens in dairy wastewater, in which previous methods (including standard PCR) failed to work. SIGNIFICANCE AND IMPACT OF THE STUDY: Cattle and dairy wastewater samples produced from mozzarella cheese factories may harbour verocytotoxin-producing E. coli. The availability of rapid and sensitive molecular methods may be useful to monitor the persistence of verocytotoxin-producing E. coli in general and to assess the effectiveness of wastewater treatments.

Detection of circulating CEA-IgM complexes in early stage colorectal cancer.

We have recently shown that alpha fetoprotein (AFP) and squamous cell carcinoma antigen (SCCA), biomarkers associated with hepatocellular carcinoma, may be detected in patient sera as circulating immune complexes with IgM, and that assessment of serum levels of AFP-IgM and SCCA-IgM may be used for the detection of liver cancer. In this study we measured the levels of carcinoembryonic antigen (CEA) as free form (FCEA) and complexed to IgMs (CEA-IgM) in sera of patients affected by colorectal carcinoma (CRC) at different stages as well as in healthy subjects. FCEA levels were above the 5 ng/mL cutoff in 43% of CRC patients (31/72) and CEA-IgM levels were above the 200 AU/mL cutoff in 38% of CRC patients (27/72). Serum levels of CEA-IgM immune complexes (IC) and FCEA did not overlap and 64% of patients (46/72) were positive for at least one marker without compromising the detection specificity (94%). Early detection of CRC was significantly improved by CEA-IgM IC assay. CRC patients at an early stage (stage 1) had elevated CEA-IgM levels in 29% of cases (7/24), while FCEA levels were elevated in only 8% of cases (2/24). These results indicate that CEA-IgM is a complementary serological marker to FCEA which is much more sensitive for early stage CRC, and that the combination of these biomarkers may be useful in the early detection of colorectal cancer.

Improvement of liver cancer detection with simultaneous assessment of circulating levels of free alpha-fetoprotein (AFP) and AFP-IgM complexes.

We assessed the presence of alpha-fetoprotein (AFP) complexed with IgM (AFP-IgM IC) in serum of patients affected by hepatocellular carcinoma (HCC), cirrhosis and chronic hepatitis as well as in healthy subjects by means of a dedicated ELISA assay. The amount of AFP-IgM IC was expressed in arbitrary units (AU) on a reference standard curve. Free AFP (FAFP) levels were determined in parallel in each sample by means of an automated immunoassay system. The mean serum concentration of AFP-IgM IC was significantly higher in HCC patients (mean +/- SD: 1378.3 +/- 2935.7 AU/mL) than in cirrhotic patients (129.8 +/- 261.4 AU/mL) and in patients with chronic hepatitis (80.9 +/- 168.9 AU/mL) (p < 0.01). HCC patients had FAFP values above the 20 ng/mL cutoff in 44% of cases (22/50) and AFP-IgM IC values above the 120 AU/mL cutoff in 60% of cases (30/50). The occurrence of the free and IgM-complexed form of circulating AFP did not overlap, and 82% of patients (41/50) were positive for at least one marker. The results indicate that AFP-IgM IC is a complementary serological marker to FAFP and that the combination of these biomarkers may be useful in the diagnosis of liver cancer.

Molecular characterization of lactic acid populations associated with wine spoilage.

We have investigated the prevalence of spoilage lactic acid bacteria (LAB) in table wines produced in the Apulia region. The occurrence of LAB was evaluated in wines produced with low sulphur dioxide doses and not supplemented with selected malolactic starters such as Oenococcus oeni. About 150 strains were isolated from wine must and a molecular characterization was performed using PCR-based techniques. Most of the strains analysed belonged to Lactobacillus plantarum species. However, some of the strains were identified as Pediococcus damnosus and Leuconostoc sp. The amplified fragments of Pediococcus damnosus were cloned and sequenced. The coding sequence was highly homologous to that of the ropy plasmid confirming that the isolated strain was a ropy(+) Pediococcus damnosus. In all the samples analysed, the final must pH value reached was relatively high (from 3.78 to 3.90). The high pH values had probably negatively influenced (counteracted) the activity of sulphur dioxide added, allowing proliferation of spoilage wine microorganisms.

Expression analysis of putative arcA, arcB and arcC genes partially cloned from Lactobacillus plantarum isolated from wine.

AIMS: The aim of this paper was to study if homofermentative strains (Lacobacillus plantarum) capable of malolactic fermentation in wine can degrade arginine via the ADI pathway. METHODS AND RESULTS: Homofermentative lactic acid bacteria (LAB) isolated from a typical red wine were investigated for their ability to produce citrulline. Citrulline was formed suggesting that the arginine metabolism takes place via the arginine deiminase (ADI) pathway and not via the arginase/urease pathway. Ammonia was also detected with Nessler's reagent, and all the strains examined were able to produce ammonia. Identification of homofermentative LAB was performed using 16S ribosomal sequence analysis. The strains were further classified as belonging to L. plantarum species. Furthermore, the genes encoding for the three pathway enzymes (ADI, ornithine transcarbamylase, carbamate kinase) were partially cloned and gene expression was performed at two different pH values (3.6 and 4.5). CONCLUSIONS: The results suggest that citrulline production in wine, could be performed by homofermentative LAB. SIGNIFICANCE AND IMPACT OF THE STUDY: Homofermentative malolactic bacteria (L. plantarum) may degrade arginine through the ADI pathway.