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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Uncoupling protein-3 is a molecular determinant for the regulation of resting metabolic rate by thyroid hormone.

Thyroid hormones increase energy expenditure, partly by reducing metabolic efficiency. The control of specific genes at the transcriptional level is thought to be the major molecular mechanism. However, both the number and the identity of the thyroid hormone-controlled genes remain unknown, as do their relative contributions. Uncoupling protein-3, a recently identified member of the mitochondrial transporter superfamily and one that is predominantly expressed in skeletal muscle, has the potential to be a molecular determinant for thyroid thermogenesis. However, changes in mitochondrial proton conductance and resting metabolic rate after physiologically mediated changes in uncoupling protein-3 levels have not been described. Here, in a study on hypothyroid rats given a single injection of T(3), we describe a strict correlation in terms of time course between the induced increase in uncoupling protein-3 expression (at mRNA and protein levels) and decrease in mitochondrial respiratory efficiency, on the one hand, and the increase in resting metabolic rate, on the other. First, we describe our finding that uncoupling protein-3 is present and regulated by T(3) only in metabolically relevant tissues (such as skeletal muscle and heart). Second, we follow the time course (at 0, 6, 12, 24, 48, 65, 96, and 144 h) of both uncoupling protein-3 mRNA levels and mitochondrial uncoupling protein-3 density in gastrocnemius muscle and heart. In both tissues, the maximal (12-fold) increase in uncoupling protein-3 density was reached at 65 h. The resting metabolic rate [lO(2)(kg(0.75))(-1)h(-1)] showed the same time course, and at 65 h the increase vs. time zero was 45% (1.316 +/- 0.026 vs. 0.940 +/- 0.007; P < 0.001). At the same time point, gastrocnemius muscle mitochondria showed a significantly higher nonphosphorylating respiration rate (nanoatoms of oxygen per min/mg protein; increase vs. time zero, 40%; 118 +/- 4 vs. 85 +/- 9; P < 0.05), whereas the membrane potential decreased by 8% (168 +/- 2 vs. 182 +/- 4; P < 0.05). These data are diagnostic of mitochondrial uncoupling. The results reported here provide the first direct in vivo evidence that uncoupling protein-3 has the potential to act as a molecular determinant in the regulation of resting metabolic rate by T(3).

3,5-diiodo-L-thyronine regulates glucose-6-phosphate dehydrogenase activity in the rat.

Thyroid hormones influence the activity of lipogenic enzymes such as malic enzyme (ME) and glucose-6-phosphate dehydrogenase (G6PD). The effect of T3 on ME is exerted at the transcriptional level, but it is unclear if its effect on G6PD is also nuclear mediated. Furthermore, other iodothyronines that have been shown to possess biological activity (such as diiodothyronines) could contribute to this enzyme's regulation. In this study the effects of 3,5-diiodothyronine (T2) on the aforementioned enzymes were examined and compared with those of T3. Rats made hypothyroid by propylthiouracil and iopanoic acid treatment were used throughout. Enzyme activities were determined spectrophotometrically, and G6PD messenger RNA (mRNA) expression was analyzed by Northern blotting using a human G6PD complementary DNA probe. Injections of T2 to hypothyroid animals significantly enhanced the activity of both enzymes. The effect of T2 on ME was nuclear mediated and mimicked the effect of T3. The effects of T2 and T3 on G6PD differed. Injection of T3 into hypothyroid rats induced an increase in both enzyme activity and G6PD mRNA expression, indicating a nuclear-mediated effect. The effect of T2 on G6PD activity, on the other hand, was not nuclear mediated. The injection of T2 into hypothyroid animals did not change G6PD mRNA expression, and the strong increase in the enzyme's activity (from +70% to +300%) was unaffected by simultaneous injection of protein synthesis inhibitors. As the lowest dose of 1 microg T2/100 g BW affects G6PD activity 3-5 times more than the same dose of T3, these data provide the first evidence that T2 is a factor capable of regulating G6PD activity.

Expression of uncoupling protein-3 and mitochondrial activity in the transition from hypothyroid to hyperthyroid state in rat skeletal muscle.

We sought a correlation between rat skeletal muscle triiodothyronine (T3)-mediated regulation of uncoupling protein-3 (UCP3) expression and mitochondrial activity. UCP3 mRNA expression increased strongly during the hypothyroid-hyperthyroid transition. The rank order of mitochondrial State 3 and State 4 respiration rates was hypothyroid < euthyroid < hyperthyroid. The State 4 increase may have been due to the increased UCP3 expression, as the proton leak kinetic was stimulated in the hypothyroid-hyperthyroid transition and a good correlation exists between the State 4 and UCP3 mRNA level. As a significant proportion of an organism's resting oxygen consumption is dedicated to opposing the proton leak, skeletal muscle mitochondrial UCP3 may mediate part of T3's effect on energy metabolism.

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.

[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.

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.

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.

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.