Occurrence of human pathogenic viruses in drinking water and in its sources: A review.

Since many waterborne diseases are caused by human pathogenic viruses, virus monitoring of drinking water (DW) and DW sources is crucial for public health. Therefore, the aim of this review was to describe the occurrence of human pathogenic viruses in DW and DW sources; the occurrence of two viruses proposed as novel indicators of human faecal contamination (Pepper mild mottle virus and Tobacco mosaic virus) was also reported. This research was focused on articles that assessed viral occurrence using molecular methods in the surface water used for DW production (SW-D), groundwater used for DW production (GW-D), DW and bottled-DW (BW). A total of 1544 studies published in the last 10 years were analysed, and 79 were ultimately included. In considering the detection methods, filtration is the most common concentration technique, while quantitative polymerase chain reaction is the most common quantification technique. Regarding virus occurrence in SW-D, GW-D, and DW, high percentages of positive samples were reported for adenovirus, polyomavirus and Pepper mild mottle virus. Viral genomes were frequently detected in SW-D and rarely in GW-D, suggesting that GW-D may be a safe DW source. Viral genomes were also detected in DW, posing a possible threat to human health. The lowest percentages of positive samples were found in Europe, while the highest were found in Asia and South America. Only three articles assessed viral occurrence in BW. This review highlights the lack of method standardization and the need for legislation updates.

Occurrence of antibiotic-resistant bacteria and resistance genes in the urban water cycle.

This study investigates the antibiotic resistance fate in the urban water cycle, evaluating the dynamics of antibiotic-resistant bacteria (ARB) and antibiotic-resistant genes (ARGs) in three different full-scale wastewater treatment plants (WWTPs) and two drinking water treatment plants (DWTPs) located in the same geographical area (North-West of Italy). ARB (tetracycline-, ampicillin-, and sulfonamide-resistant bacteria) were quantified by plate counting and the abundances of selected ARGs (i.e., tetA, blaTEM, and sulII) and intI1 gene were measured using quantitative real-time PCR (qPCR). Higher concentrations of ARB and ARGs were observed in the WWTPs with respect to the DWTPs identifying the WWTP as hotspot for the spread of antibiotic resistances. Although a significant reduction of ARB and ARGs was observed in WWTPs and DWTPs after the treatment, none of the detected ARB or ARGs was completely removed in drinking water. The stability of the antibiotic-resistant rates between inlet and outlet associated with the reduction of relative ARG abundances underlined that both the treatments (WWTs and DWTs) did not apply any selective pressure. The overall results highlighted the importance to investigate the antibiotic resistance dynamics in aquatic ecosystems involved in urban water cycle integrating the information obtained by culture-dependent method with the culture-independent one and the need to monitor the presence of ARB and ARGs mainly in drinking water that represents a potential route of transmission to human.

Comparison of UV, Peracetic Acid and Sodium Hypochlorite Treatment in the Disinfection of Urban Wastewater.

One source of water contamination is the release of wastewater that has not undergone efficient treatment. The aim of this study was to evaluate the reduction obtained with sodium hypochlorite (NaClO), UV and peracetic acid disinfection treatment of Salmonella spp., pathogenic Campylobacter, STEC and bacterial indicators in three full-scale municipal wastewater plants. A general reduction in Salmonella was observed after disinfection, but these bacteria were detected in one UV-treated sample (culture method) and in 33%, 50% and 17% of samples collected after NaClO, UV and PAA disinfection treatments, respectively (PCR method). A better reduction was also observed under NaClO disinfection for the microbial indicators. Independent of the disinfection treatment, E. coli O157:H7 was not detected in the disinfected samples, whereas some samples treated with UV and PAA showed the presence of the stx1 gene. No reduction in the presence of stx2 genes was verified for any of the disinfection treatments. Campylobacter was not detected in any of the analysed samples. The overall results highlight a better reduction in microbiological parameters with a NaClO disinfection treatment in a full-scale municipal wastewater plant compared with UV and PAA. However, the results indicate that a complete and specific monitoring program is necessary to prevent a possible risk to public health.

Editorial Comments to the Special Issue: "Legionella Contamination in Water Environment".

Legionella spp [...].

Traits of orthorexia nervosa and muscle dysmorphia in Italian university students: a multicentre study.

PURPOSE: This study aimed to evaluate the prevalence of traits of orthorexia nervosa (ON) and muscle dysmorphia (MD) in a group of undergraduates, investigate the associations between the risk of these conditions and the type of university course attended, the individual characteristics (gender, BMI, amount of physical activity, supplements and medicines use, dieting) and the risk of eating disorders (EDs). METHODS: A self-reported questionnaire consisting of a socio-demographic section and three tests validated for the evaluation of a risk of ON (ORTO-15), MD (MDDI-ITA) and EDs (EAT-26) was completed by 918 students from three Italian universities. RESULTS: 29.0% of participants demonstrated traits of ON and 5.0% of MD, without differences in prevalence in the three areas of study investigated (health-scientific, economic-humanistic, sport sciences); students of sport sciences exhibited a significantly higher score for MDDI-ITA (F = 6.493, p = 0.002). Participants with ON and MD traits were more on a diet (OR = 0.47, p ≤ 0.001 and OR = 0.428, p = 0.020, respectively) and showed a higher prevalence of EDs risk (OR = 3.55, p < 0.001 and OR = 10.23, p ≤ 0.001, respectively). The simultaneous presence of ON, MD, and EDs traits was seen in 5.4% of the students and the three test scores were correlated. CONCLUSIONS: The prevalence of ON and MD traits was found similar to that reported in the literature on undergraduates. Some associations observed improvement in the knowledge about these conditions, especially the association of participants with ON and MD traits with dieting and EDs traits and the correlation of the three test scores suggests a connection among these potential conditions. LEVEL OF EVIDENCE: Level V, descriptive cross-sectional survey.

Cryptosporidium Oocyst Contamination in Drinking Water: A Case Study in Italy.

The aim of this study was to evaluate the occurrence of Cryptosporidium oocysts in a drinking water treatment plant (DWTP) located in a rural area of northern Italy. Influent and effluent samples were collected at the DWTP over three years (2013-2016). In parallel, tap water samples from a public drinking fountain were collected as well. All samples were analyzed for the presence of Cryptosporidium spp. oocysts by a common method based on an immunomagnetic separation (IMS)/immunofluorescence assay (IFA), complemented by 4,6-diamidino-2-phenylindole (DAPI) staining. A reverse transcriptase-PCR (RT-PCR) protocol was added to evaluate oocyst viability. The results highlighted a high variability of oocyst concentrations across all samples (mean 4.3 ± 5.8/100 L) and a high variability in the percentage of DAPI-positive specimens (mean 48.2% ± 40.3%). Conversely, RT-PCR did not reveal the presence of viable C. parvum and C. hominis oocysts. A nested PCR targeting Cryptosporidium 18S ribosomal DNA, carried out in two water samples, confirmed the presence of a Cryptosporidium genotype associated with wild animals in the river and in tap water. The results obtained underline the vulnerability of the investigated surface water to Cryptosporidium spp. contamination. Although the recovered Cryptosporidium genotype is not a human pathogen, its presence demonstrates the existence of a potential pathogen Cryptosporidium spp. contamination risk. Moreover, these results underline the importance of also considering unconventional (not bacterial) biological contaminations (protozoa) in water resources in rural areas, including those of developed countries.

Shape-engineered titanium dioxide nanoparticles (TiO2-NPs): cytotoxicity and genotoxicity in bronchial epithelial cells.

The aim of this study was to evaluate cytotoxicity (WST-1 assay), LDH release (LDH assay) and genotoxicity (Comet assay) of three engineered TiO2-NPs with different shapes (bipyramids, rods, platelets) in comparison with two commercial TiO2-NPs (P25, food grade). After NPs characterization (SEM/T-SEM and DLS), biological effects of NPs were assessed on BEAS-2B cells in presence/absence of light. The cellular uptake of NPs was analyzed using Raman spectroscopy. The cytotoxic effects were mostly slight. After light exposure, the largest cytotoxicity (WST-1 assay) was observed for rods; P25, bipyramids and platelets showed a similar effect; no effect was induced by food grade. No LDH release was detected, confirming the low effect on plasma membrane. Food grade and platelets induced direct genotoxicity while P25, food grade and platelets caused oxidative DNA damage. No genotoxic or oxidative damage was induced by bipyramids and rods. Biological effects were overall lower in darkness than after light exposure. Considering that only food grade, P25 and platelets (more agglomerated) were internalized by cells, the uptake resulted correlated with genotoxicity. In conclusion, cytotoxicity of NPs was low and affected by shape and light exposure, while genotoxicity was influenced by cellular-uptake and aggregation tendency.

Mutagenic and genotoxic effects induced by PM0.5 of different Italian towns in human cells and bacteria: The MAPEC_LIFE study.

Particulate matter (PM) is considered an atmospheric pollutant that mostly affects human health. The finest fractions of PM (PM2.5 or less) play a major role in causing chronic diseases. The aim of this study was to investigate the genotoxic effects of PM0.5 collected in five Italian towns using different bioassays. The role of chemical composition on the genotoxicity induced was also evaluated. The present study was included in the multicentre MAPEC_LIFE project, which aimed to evaluate the associations between air pollution exposure and early biological effects in Italian children. PM10 samples were collected in 2 seasons (winter and spring) using a high-volume multistage cascade impactor. The results showed that PM0.5 represents a very high proportion of PM10 (range 10-63%). PM0.5 organic extracts were chemically analysed (PAHs, nitro-PAHs) and tested by the comet assay (A549 and BEAS-2B cells), MN test (A549 cells) and Ames test on Salmonella strains (TA100, TA98, TA98NR and YG1021). The highest concentrations of PAHs and nitro-PAHs in PM0.5 were observed in the Torino, Brescia and Pisa samples in winter. The Ames test showed low mutagenic activity. The highest net revertants/m3 were observed in the Torino and Brescia samples (winter), and the mutagenic effect was associated with PM0.5 (p < 0.01), PAH and nitro-PAH (p < 0.05) concentrations. The YG1021 strain showed the highest sensitivity to PM0.5 samples. No genotoxic effect of PM0.5 extracts was observed using A549 cells except for some samples in winter (comet assay), while BEAS-2B cells showed light DNA damage in the Torino, Brescia and Pisa samples in winter, highlighting the higher sensitivity of BEAS-2B cells, which was consistent with the Ames test (p < 0.01). The results obtained showed that it is important to further investigate the finest fractions of PM, which represent a relevant percentage of PM10, taking into account the chemical composition and the biological effects induced.

Health Risk Associated with Exposure to PM10 and Benzene in Three Italian Towns.

Air pollution in urban areas is a major concern as it negatively affects the health of a large number of people. The purpose of this study was to assess the inhalation health risk for exposure to PM10 and benzene of the populations living in three Italian cities. Data regarding PM10 and benzene daily measured by "traffic" stations and "background" stations in Torino, Perugia, and Lecce during 2014 and 2015 were compared to the limits indicated in the Directive 2008/50/EC. In addition, an inhalation risk analysis for exposure to benzene was performed for adults and children by applying the standard United States Environmental Protection Agency's (USEPA) methodology. The levels of PM10 detected in Torino exceeded the legal limits in both years with an increased mean concentration >10 µg/m³ comparing with background station. Benzene concentrations never exceeded the legislative target value. The increased cancer risk (ICR) for children exposed to benzene was greater than 1 × 10-6 only in the city of Torino, while for adults, the ICR was higher than 1 × 10-6 in all the cities. The results suggest the need for emission reduction policies to preserve human health from continuous and long exposure to air pollutants. A revision of legal limits would also be recommended.

Effectiveness of a neutral electrolysed oxidising water (NEOW) device in reducing Legionella pneumophila in a water distribution system: A comparison between culture, qPCR and PMA-qPCR detection methods.

Disinfection of hot water systems is critical for reducing Legionnaires' disease in high-risk buildings. The use of neutral electrolysed oxidising water (NEOW) is a promising method for the control of microorganisms in hot water systems. However, full-scale evaluations of the efficacy of NEOW devices to control Legionella pneumophila are currently lacking. The aim of this study was to assess the effectiveness of a NEOW device in reducing L. pneumophila in a hotel water network. Water samples (n = 67) were collected from different sites of a hotel distribution system before and after the installation of the NEOW device at the 1st, 4th, 8th and 12th week. Detection of L. pneumophila was performed comparing culture, qPCR and PMA-qPCR methods. Total bacterial counts (22 °C and 37 °C), Pseudomonas spp. and physico-chemical parameters were also monitored. The NEOW treatment resulted in a reduction of the amount of L. pneumophila positive samples (-32%) and of the number of heavily contaminated points (>104 CFU/L and >103 CFU/L) (-100% and -96%, respectively). Treatment maintained L. pneumophila at low levels (<102 CFU/L), which do not require specific intervention measures. The effectiveness of the disinfection system was also confirmed by PMA-qPCR (p < 0.001). The use of PMA resulted in a signal decrease in almost all samples upon the disinfection treatment. The NEOW disinfection device appears to be a promising approach to reduce the colonisation of hot water systems by L. pneumophila; however, further investigations are needed to ascertain its efficiency over longer time periods.

Buccal micronucleus cytome assay in primary school children: A descriptive analysis of the MAPEC_LIFE multicenter cohort study.

BACKGROUND: Recent data support the hypothesis that genetic damage occurring early in life during childhood can play an important role in the development of chronic diseases in adulthood, including cancer. OBJECTIVES: The objective of this paper, part of the MAPEC_LIFE project, is to describe the frequency of micronuclei and meta-nuclear alterations in exfoliated buccal cells of 6-8year-old Italian children recruited in five Italian towns (i.e., Brescia, Torino, Pisa, Perugia and Lecce) with different air pollution levels. METHODS: About 200 children per town were recruited from primary schools. Biological samples were collected twice from the same children, in two different seasons (winter 2014-15 and late spring 2015). Cytogenetic damage was evaluated by the buccal micronucleus cytome assay. RESULTS: Overall,n = 1046 children represent the final cohort of the MAPEC_LIFE study. On the whole, the results showed a higher mean MN frequency in winter (0.42 ±â€¯0.54‰) than late-spring (0.22 ±â€¯0.34‰). MN frequency observed among the five Italian towns showed a trend that follows broadly the levels of air pollution in Italy: the highest MN frequency was observed in Brescia during both seasons, the lowest in Lecce (winter) and Perugia (late-spring). CONCLUSIONS: To the best of our knowledge, the number of recruited children included in the analysis (n = 1046) is the highest compared to previous studies evaluating the frequency of MN in exfoliated buccal cells so far. MN frequency was associated with winter season and living in towns at various levels of air pollution, suggesting an important role of this exposure in determining early cytogenetic effects.

Viability of Legionella pneumophila in Water Samples: A Comparison of Propidium Monoazide (PMA) Treatment on Membrane Filters and in Liquid.

Legionella pneumophila is a ubiquitous microorganism widely distributed in aquatic environments and can cause Legionellosis in humans. A promising approach to detect viable cells in water samples involves the use of quantitative polymerase chain reaction (qPCR) in combination with photoactivatable DNA intercalator propidium monoazide (PMA). However, the PMA efficiency could be different depending on the experimental conditions used. The aim of this study was to compare two PMA exposure protocols: (A) directly on the membrane filter or (B) in liquid after filter washing. The overall PMA-induced qPCR means reductions in heat-killed L. pneumophila cells were 2.42 and 1.91 log units for exposure protocols A and B, respectively. A comparison between the results obtained reveals that filter exposure allows a higher PMA-qPCR signal reduction to be reached, mainly at low concentrations (p < 0.05). This confirms the potential use of this method to quantify L. pneumophila in water with low contamination.

Socio-Economic and Environmental Factors Associated with Overweight and Obesity in Children Aged 6-8 Years Living in Five Italian Cities (the MAPEC_LIFE Cohort).

The prevalence of obesity among Italian children has reached such alarming levels as to require detailed studies of the causes of the phenomenon. A cross-sectional study was carried out in order to assess the weight status of 1164 Italian children aged 6-8 years (the Monitoring Air Pollution Effects on Children for Supporting Public Health Policy (MAPEC_LIFE) cohort) and to identify any associations between selected socio-economic and environmental factors and overweight/obesity. The data were obtained by means of a questionnaire given to parents, and any associations were examined by binomial logistic regression analyses. Overweight was found to be positively associated with male gender, parents of non-Italian origin, and parents who smoke, and negatively associated with the parents' level of education and employment. In addition, the frequency of overweight varied in relation to the geographical area of residence, with a greater prevalence of overweight children in the cities of central-southern Italy. This study highlights the need to implement appropriate obesity prevention programs in Italy, which should include educational measures concerning lifestyle for parents from the earliest stages of their child's life.

Assessing Methanobrevibacter smithii and Clostridium difficile as not conventional faecal indicators in effluents of a wastewater treatment plant integrated with sludge anaerobic digestion.

Wastewater treatment plants (WWTP) are an important source of surface water contamination by enteric pathogens, affecting the role of environmental water as a microbial reservoir. We describe the release to the environment of certain anaerobes of human and environmental concern. The work was focused on emerging microbial targets. They are tracing, by RT-qPCR, on WWTP effluents, both liquid and solid, when an anaerobic digestion step is included. The focus is placed on Clostridium spp. with the specific quantification of Clostridium perfringens, as typical bioindicator, and Clostridium difficile, as emerging pathogen not only confined into nosocomial infection. Moreover methanogens were quantified for their involvement in the anaerobic digestion, and in particular on Methanobrevibacter smithii as major methanogenic component of the human gut microbiome and as not conventional faecal indicator. In the water samples, a reduction, statistically significant, in all microbial targets was observed (p < 0.01), 2 log for the total bacteria, 1.4 log for the Clostridium spp. and M. smithii, 1 log for total methanogens, C. perfringens and C. difficile. The AD process contribute to a significant change in microbial levels into the sludge for total bacteria and total methanogens (p < 0.01), both when the input sludge are primary and secondary, while for the presence of Clostridium spp. and C. difficile there was not a significant change. The produced data are innovative showing which is the diffusion of such anaerobic microorganisms throughout the WWTP and opening a discussion on the implementation of possible techniques for a more efficient microbial removal from effluents, particularly bio-solids, to reduce the potential release of pathogens into the environment.

Inactivation of Escherichia coli and Staphylococcus aureus by pulsed electric fields increases with higher bacterial population and with agitation of liquid medium.

Inactivation of Escherichia coli, E. coli O157:H7, and Staphylococcus aureus in liquid media by pulsed electric fields (PEF) was conducted at varying bacterial populations with and without sample agitation. A laboratory-scale PEF batch unit with a rectangular electric pulse was used, operating under the following conditions: 25 kV/cm (E. coli, E. coli O157:H7) and 30 kV/cm (S. aureus) electric field strengths, 1-µs pulse width, 1-Hz pulse repetition rate, and 20 to 350 pulses for all samples. Not surprisingly, bacterial inactivation (for all three strains) increased with increasing pulse number, achieving the highest reduction at 350 pulses. Log CFU per milliliter microbial inactivation increased commensurately with increasing bacterial population (P < 0.05) but only when samples were treated with more than 200 pulses. For example, when E. coli was treated with 200 pulses at 10(5) CFU/ml, inactivation was only 3.0 Log versus 4.8 Log at the 10(10) inoculation level. When E. coli O157:H7 was treated with 200 pulses at 10(5) CFU/ml, inactivation was only 2.5 Log versus 4.6 Log at the 10(10) inoculation level. When S. aureus was treated with 200 pulses at 10(6) CFU/ml, inactivation was only 2.6 Log versus 4.8 Log at the 10(10) inoculation level. Inactivation of populations was also found to be statistically greater (P < 0.05) when liquid samples were agitated, in comparison to nonagitated samples. Because PEF inactivation activity is influenced by bacterial population and sample agitation, future studies should carefully consider these factors in experimental designs and/or scaled-up industry application.

Photocatalytic bacterial inactivation by TiO2-coated surfaces.

The aim of this study was the evaluation of the photoactivated antibacterial activity of titanium dioxide (TiO2)-coated surfaces. Bacterial inactivation was evaluated using TiO2-coated Petri dishes. The experimental conditions optimized with Petri dishes were used to test the antibacterial effect of TiO2-coated ceramic tiles. The best antibacterial effect with Petri dishes was observed at 180, 60, 30 and 20 min of exposure for Escherichia coli, Staphylococcus aureus, Pseudomonas putida and Listeria innocua, respectively. The ceramic tiles demonstrated a photoactivated bactericidal effect at the same exposure time. In general, no differences were observed between the antibacterial effect obtained with Petri dishes and tiles. However, the photochemical activity of Petri dishes was greater than the activity of the tiles.Results obtained indicates that the TiO2-coated surfaces showed a photoactivated bactericidal effect with all bacteria tested highlighting that the titania could be used in the ceramic and building industry for the production of coated surfaces to be placed in microbiologically sensitive environments, such as the hospital and food industry.

Development of a PCR protocol for the detection of Escherichia coli O157:H7 and Salmonella spp. in surface water.

Escherichia coli O157:H7 and Salmonella are pathogenic microorganisms that can cause severe gastrointestinal illness in humans. These pathogens may be transmitted in a variety of ways, including food and water. The presence of Salmonella and E. coli O157:H7 in surface waters constitutes a potential threat to human health when used for either drinking or recreation. As with most waterborne pathogens, Salmonella and E. coli O157:H7 are difficult to detect and enumerate with accuracy in surface waters due to methodological limitations. The aim of this study was to develop a protocol for the detection of Salmonella spp., E. coli O157:H7 and E. coli virulence genes (stx (1), stx (2) and eae) in water using a single enrichment step and PCR. In spiked water samples, PCR results showed high sensitivity (<3 CFU/L) for both microorganisms. The protocol developed in this study has been applied in different surface waters in association with microbiological and physical analysis. The frequency of PCR positive samples was 33% for Salmonella and 2% for E. coli O157:H7 producing intimin (eae) and Shiga-like toxin I (stx (1)). Moreover, the finding of amplicons corresponding to eae and stx (1) genes in the absence of E. coli O157:H7 suggested the possible presence of other pathogenic bacteria that carry these genes (e.g. EHEC, Shigella strains). The results obtained showed that the developed protocol could be applied as a routine analysis of surface water for the evaluation of microbiological risks.

First evidence of a membrane-bound, tyramine and beta-phenylethylamine producing, tyrosine decarboxylase in Enterococcus faecalis: a two-dimensional electrophoresis proteomic study.

The soluble and membrane proteome of a tyramine producing Enterococcus faecalis, isolated from an Italian goat cheese, was investigated. A detailed analysis revealed that this strain also produces small amounts of beta-phenylethylamine. Kinetics of tyramine and beta-phenylethylamine accumulation, evaluated in tyrosine plus phenylalanine-enriched cultures (stimulated condition), suggest that the same enzyme, the tyrosine decarboxylase (TDC), catalyzes both tyrosine and phenylalanine decarboxylation: tyrosine was recognized as the first substrate and completely converted into tyramine (100% yield) while phenylalanine was decarboxylated to beta-phenylethylamine (10% yield) only when tyrosine was completely depleted. The presence of an aspecific aromatic amino acid decarboxylase is a common feature in eukaryotes, but in bacteria only indirect evidences of a phenylalanine decarboxylating TDC have been presented so far. Comparative proteomic investigations, performed by 2-DE and MALDI-TOF/TOF MS, on bacteria grown in conditions stimulating tyramine and beta-phenylethylamine biosynthesis and in control conditions revealed 49 differentially expressed proteins. Except for aromatic amino acid biosynthetic enzymes, no significant down-regulation of the central metabolic pathways was observed in stimulated conditions, suggesting that tyrosine decarboxylation does not compete with the other energy-supplying routes. The most interesting finding is a membrane-bound TDC highly over-expressed during amine production. This is the first evidence of a true membrane-bound TDC, longly suspected in bacteria on the basis of the gene sequence.

Microbiological and chemical characterization of a typical Italian cheese: Robiola di Roccaverano.

Robiola di Roccaverano is a traditional Italian goat's milk cheese carrying a Protected Designation of Origin (PDO). The present work studied both cheese microflora and cheese physicochemical characteristics to obtain a more accurate description of this PDO product. Multivariate statistical analysis (PCA) was performed to evaluate the influence of cheesemaking (artisanal and industrial), ripening time, and season of production on cheese characteristics. Multiplex PCR and fatty acid methyl esters (FAMEs) were used to identify the kind of milk employed by Robiola di Roccaverano producers. The results obtained highlight some product differences between the artisanal and industrial products. These differences were most evident in the microbiological data. The use of PCA allowed cheese samples to cluster on the basis of their age (fresh or ripened), the origin of production (artisanal and industrial), and even the season of production. Gross composition, microbiological parameters, and gas chromatographic analyses of FAMEs provided the most important parameters for Robiola di Roccaverano cheese characterization.

Microbiological characterisation of Robiola di Roccaverano cheese using PCR-DGGE.

The aim of this study was the microbiological characterisation of a typical Italian cheese "Robiola di Roccaverano" with Protected Designation of Origin (PDO). Fresh and ripened robiola samples were collected from four artisanal and one industrial producer. Artisanal producers used raw goat's milk and natural fermentation, whilst the industrial producer used mixed cow-goat's milk and selected starters. The microbial communities were monitored during different seasons and ripening times with PCR-DGGE and culture-dependent methods. The cluster analysis showed that the DGGE bacterial patterns were related to the different manufacturing and climatic conditions, revealing the occurrence of species associated to Robiola di Roccaverano. The DGGE profiles of yeasts were affected by ripening in summer season. Moreover, the results obtained allowed the identification of microbial species such as Lactococcus lactis subsp. lactis, Geotricum spp. and Kluyveromyces lactis that are related to the production of this typical cheese.