Comparative proteomic analysis of foodborne Salmonella Enteritidis SE86 subjected to cold plasma treatment.

The increasing demand for high quality and safe food led to important technological innovations in food processing. Cold plasma appears as an emerging technology that has demonstrated efficiency in the removal of microbial contamination from fresh and minimally processed food. In this study, the proteomic profile of Salmonella Enteritidis SE86 subjected to cold plasma treatment was investigated. The number of viable S. Enteritidis SE86 cells was analyzed at different time intervals upon exposure to cold plasma and approximately 100 µg of S. Enteritidis SE86 protein extracts were analyzed by Multidimensional Protein Identification Technology (MudPIT). The results demonstrated that no significant changes in cell counts were detected for up to 20 min exposure to cold plasma, and 2 log reduction was achieved after 60 min. Overall, 1096 proteins were identified, with 249 detected only in plasma-treated samples, and 9 exclusive in non-treated control samples. The proteins uniquely detected in cold plasma-treated cells that showed higher abundance were glyoxalase I, ABC transporter substrate-binding protein and transcriptional activator OsmE, followed by some oxidoreductases. Proteins related with carbohydrate and nucleotide metabolism were mostly overexpressed in cold plasma treated cells, suggesting energy metabolism was increased.

High-level adherence to a Mediterranean diet beneficially impacts the gut microbiota and associated metabolome.

OBJECTIVES: Habitual diet plays a major role in shaping the composition of the gut microbiota, and also determines the repertoire of microbial metabolites that can influence the host. The typical Western diet corresponds to that of an omnivore; however, the Mediterranean diet (MD), common in the Western Mediterranean culture, is to date a nutritionally recommended dietary pattern that includes high-level consumption of cereals, fruit, vegetables and legumes. To investigate the potential benefits of the MD in this cross-sectional survey, we assessed the gut microbiota and metabolome in a cohort of Italian individuals in relation to their habitual diets. DESIGN AND RESULTS: We retrieved daily dietary information and assessed gut microbiota and metabolome in 153 individuals habitually following omnivore, vegetarian or vegan diets. The majority of vegan and vegetarian subjects and 30% of omnivore subjects had a high adherence to the MD. We were able to stratify individuals according to both diet type and adherence to the MD on the basis of their dietary patterns and associated microbiota. We detected significant associations between consumption of vegetable-based diets and increased levels of faecal short-chain fatty acids, Prevotella and some fibre-degrading Firmicutes, whose role in human gut warrants further research. Conversely, we detected higher urinary trimethylamine oxide levels in individuals with lower adherence to the MD. CONCLUSIONS: High-level consumption of plant foodstuffs consistent with an MD is associated with beneficial microbiome-related metabolomic profiles in subjects ostensibly consuming a Western diet. TRIAL REGISTRATION NUMBER: This study was registered at clinical trials.gov as NCT02118857.

Effect of Whole-Grain Barley on the Human Fecal Microbiota and Metabolome.

In this study, we compared the fecal microbiota and metabolomes of 26 healthy subjects before (HS) and after (HSB) 2 months of diet intervention based on the administration of durum wheat flour and whole-grain barley pasta containing the minimum recommended daily intake (3 g) of barley ß-glucans. Metabolically active bacteria were analyzed through pyrosequencing of the 16S rRNA gene and community-level catabolic profiles. Pyrosequencing data showed that levels of Clostridiaceae (Clostridium orbiscindens and Clostridium sp.), Roseburia hominis, and Ruminococcus sp. increased, while levels of other Firmicutes and Fusobacteria decreased, from the HSB samples to the HS fecal samples. Community-level catabolic profiles were lower in HSB samples. Compared to the results for HS samples, cultivable lactobacilli increased in HSB fecal samples, while the numbers of Enterobacteriaceae, total coliforms, and Bacteroides, Porphyromonas, Prevotella, Pseudomonas, Alcaligenes, and Aeromonas bacteria decreased. Metabolome analyses were performed using an amino acid analyzer and gas chromatography-mass spectrometry solid-phase microextraction. A marked increase in short-chain fatty acids (SCFA), such as 2-methyl-propanoic, acetic, butyric, and propionic acids, was found in HSB samples with respect to the HS fecal samples. Durum wheat flour and whole-grain barley pasta containing 3% barley ß-glucans appeared to be effective in modulating the composition and metabolic pathways of the intestinal microbiota, leading to an increased level of SCFA in the HSB samples.

Salivary microbiota and metabolome associated with celiac disease.

This study aimed to investigate the salivary microbiota and metabolome of 13 children with celiac disease (CD) under a gluten-free diet (treated celiac disease [T-CD]). The same number of healthy children (HC) was used as controls. The salivary microbiota was analyzed by an integrated approach using culture-dependent and -independent methods. Metabolome analysis was carried out by gas chromatography-mass spectrometry-solid-phase microextraction. Compared to HC, the number of some cultivable bacterial groups (e.g., total anaerobes) significantly (P < 0.05) differed in the saliva samples of the T-CD children. As shown by community-level catabolic profiles, the highest Shannon's diversity and substrate richness were found in HC. Pyrosequencing data showed the highest richness estimator and diversity index values for HC. Levels of Lachnospiraceae, Gemellaceae, and Streptococcus sanguinis were highest for the T-CD children. Streptococcus thermophilus levels were markedly decreased in T-CD children. The saliva of T-CD children showed the largest amount of Bacteroidetes (e.g., Porphyromonas sp., Porphyromonas endodontalis, and Prevotella nanceiensis), together with the smallest amount of Actinobacteria. T-CD children were also characterized by decreased levels of some Actinomyces species, Atopobium species, and Corynebacterium durum. Rothia mucilaginosa was the only Actinobacteria species found at the highest level in T-CD children. As shown by multivariate statistical analyses, the levels of organic volatile compounds markedly differentiated T-CD children. Some compounds (e.g., ethyl-acetate, nonanal, and 2-hexanone) were found to be associated with T-CD children. Correlations (false discovery rate [FDR], <0.05) were found between the relative abundances of bacteria and some volatile organic compounds (VOCs). The findings of this study indicated that CD is associated with oral dysbiosis that could affect the oral metabolome.

UV-C pre-adaptation of Salmonella: effect on cell morphology and membrane fatty acids composition.

The present study was carried out to evaluate the effects of ultraviolet radiations (UV-C) on the fatty acids composition of three serovars of Salmonella: S. typhimurium, S. hadar and S. zanzibar. Results obtained show that UV-C treatment increases significantly (P ≤ 0.05) the percentage of cyclic fatty acids. The atomic force microscopy was used to study the morphology and cell surface of irradiated strains. Results show that UV-C rays induce morphological changes and alter the bacterial cell surface (presence of grooves and irregularities).

High pressure homogenization vs heat treatment: safety and functional properties of liquid whole egg.

This research investigated the potential of multi-pass homogenization treatment for the inactivation of Salmonella enterica serovar Enteritidis inoculated at different levels in liquid whole egg (LWE) comparing the efficacy of this treatment with a traditional thermal one performed at 65 °C. Moreover, the effects of high pressure treatment (HPH) on structural and functional properties such as viscosity, microstructure and foaming abilities of LWE were investigated. The data obtained suggested that the multi-pass high pressure treatment at 100 MPa of S. enterica serovar Enteritidis inoculated in LWE at 7 and 4 log CFU/ml resulted in a first order inactivation kinetic, while the thermal inactivation curves of S. enterica serovar Enteritidis inoculated at 8 and 4 log CFU/ml presented a non-linear behaviour, with a marked tail after 3 min of treatment at 65 °C. Additionally, HPH treatment caused an increase in foaming capacity of LWE, with respect to the untreated samples, passing from values of 26% of the control to 50% of pressure treated samples.

Combined effect of atmospheric gas plasma and UVA light: A sustainable and green alternative for chemical decontamination and microbial inactivation of fish processing water.

The simultaneous use of UVA light irradiation coupled with low energy cold plasma generated by a dielectric barrier discharge prototype, results in significant enhancement of efficiency of the integrated process with respect to the sole plasma treatment. This effect has been demonstrated both on microbial inactivation of a food-borne pathogen, i.e. Listeria monocytogenes, and on the degradation of a compound of biological origin such as phenylalanine. In the latter case, the analysis of its reaction intermediates and the spectroscopic identification and quantification of peroxynitrites, allowed to propose mechanistic hypotheses on the nature of the observed synergistic effects. Moreover, it has been demonstrated that the process does not affect the quality of trout fillets, indicating its suitability as a chlorine-free, green, and sustainable tool for the decontamination of fish processing water.

Identification of volatile components of liverwort (Porella cordaeana) extracts using GC/MS-SPME and their antimicrobial activity.

Chemical constituents of liverwort (Porella cordaeana) extracts have been identified using solid-phase microextraction-gas chromatography mass spectrometry (SPME-GC/MS). The methanol, ethanol and ethyl acetate extracts were rich in terpenoids such as sesquiterpene hydrocarbons (53.12%, 51.68%, 23.16%), and monoterpene hydrocarbons (22.83%, 18.90%, 23.36%), respectively. The dominant compounds in the extracts were ß-phellandrene (15.54%, 13.66%, 12.10%) and ß-caryophyllene (10.72%, 8.29%, 7.79%, respectively). The antimicrobial activity of the extracts was evaluated against eleven food microorganisms using the microdilution and disc diffusion methods. The minimum inhibitory concentration (MIC) varied from 0.50 to 2.00 mg/mL for yeast strains (Saccharomyces cerevisiae 635, Zygosacharomyces bailii 45, Aerobasidium pullulans L6F, Pichia membranaefaciens OC 71, Pichia membranaefaciens OC 70, Pichia anomala CBS 5759, Pichia anomala DBVPG 3003 and Yarrowia lipolytica RO13), and from 1.00 to 3.00 mg/mL for bacterial strains (Salmonella enteritidis 155, Escherichia coli 555 and Listeria monocytogenes 56Ly). Methanol extract showed better activity in comparison with ethanol and ethyl acetate extracts. High percentages of monoterpene and sesquiterpene hydrocarbons could be responsible for the better antimicrobial activity.

Isolation and Identification of Wild Yeast from Malaysian Grapevine and Evaluation of Their Potential Antimicrobial Activity against Grapevine Fungal Pathogens.

Pathogenic fungi belonging to the genera Botrytis, Phaeomoniella, Fusarium, Alternaria and Aspergillus are responsible for vines diseases that affect the growth, grapevine yield and organoleptic quality. Among innovative strategies for in-field plant disease control, one of the most promising is represented by biocontrol agents, including wild epiphytic yeast strains of grapevine berries. Twenty wild yeast, isolated and molecularly identified from three different Malaysian regions (Perlis, Perak and Pahang), were evaluated in a preliminary screening test on agar to select isolates with inhibition against Botrytis cinerea. On the basis of the results, nine yeasts belonging to genera Hanseniaspora, Starmerella, Metschnikowia, Candida were selected and then tested against five grape berry pathogens: Aspergillus carbonarius, Aspergillus ochraceus, Fusarium oxysporum, Alternaria alternata and Phaeomoniella chlamydospora.Starmerella bacillaris FE08.05 and Metschnikowia pulcherrima GP8 and Hanseniaspora uvarum GM19 showed the highest effect on inhibiting mycelial growth, which ranged between 15.1 and 4.3 mm for the inhibition ring. The quantitative analysis of the volatile organic compound profiles highlighted the presence of isoamyl and phenylethyl alcohols and an overall higher presence of low-chain fatty acids and volatile ethyl esters. The results of this study suggest that antagonist yeasts, potentially effective for the biological control of pathogenic moulds, can be found among the epiphytic microbiota associated with grape berries.

High-Pressure Homogenization and Biocontrol Agent as Innovative Approaches Increase Shelf Life and Functionality of Carrot Juice.

Recently, application of high-pressure homogenization (HPH) treatments has been widely studied to improve shelf life and rheological and functional properties of vegetable and fruit juices. Another approach that has drawn the attention of researchers is the use of biocontrol cultures. Nevertheless, no data on their possible combined effect on fruit juices shelf life and functionality have been published yet. In this work, the microbial, organoleptic, and technological stability of extremely perishable carrot juice and its functionality were monitored for 12 and 7 days (stored at 4 and 10 °C, respectively) upon HPH treatment alone or in combination with a fermentation step using the biocontrol agent L. lactis LBG2. HPH treatment at 150 MPa for three passes followed by fermentation with L. lactis LBG2 extended the microbiological shelf life of the products of at least three and seven days when stored at 10 °C and 4 °C, respectively, compared to untreated or only HPH-treated samples. Moreover, the combined treatments determined a higher stability of pH and color values, and a better retention of ß-carotene and lutein throughout the shelf-life period when compared to unfermented samples. Eventually, use of combined HPH and LBG2 resulted in the production of compounds having positive sensory impact on carrot juice.

Influence of non-phosphate and low-sodium salt marination in combination with tumbling process on properties of chicken breast meat affected by white striping abnormality.

This study investigated the effects of non-phosphate and low-sodium (NPLS) marination on properties of white striping chicken breasts (WSCB). Chicken breasts were collected from slaughterhouse and classified as normal (NCB, n = 24) and severe WS (WSCB, n = 120). Sixty WSCB samples were vacuum-tumbled (30 min, 2 °C) with NPLS solution, containing 2.8% (w/v) potassium bicarbonate, 2.9% (w/v) potassium chloride, and 1.5% (w/v) sorbitol at the ratio of meat-to-marinade of 4 to 1 (w/w). The other 60 WSCB received no marination were assigned as nonmarinated WSCB. Properties of marinated (n = 12) and nonmarinated (n = 12) WSCB samples were determined at 0, 3, 7, 10, and 14 days of the storage at 4 °C. Properties of the NCB were also determined on day 0. Concerning day 0, the marinated WSCB exhibited higher (p < 0.05) pH, moisture content, total cooked yield, protein solubility, hardness, cohesiveness, and chewiness along with lower (p < 0.05) cooked loss, expressible water, and shear force than those of nonmarinated WSCB and NCB. Based on nuclear magnetic resonance spectroscopy, bound, intra-myofibrillar, and extra-myofibrillar water of cooked marinated WSCB were greater (p < 0.05) than those of cooked nonmarinated WSCB. The greater (p < 0.05) weight loss, moisture content, and total cooking yield were observed in marinated samples compared to those of nonmarinated WSCB throughout the storage period. Although microbial stability was reduced (p < 0.05), no difference (p ≥ 0.05) in lipid oxidation was detected between the treatments. The findings suggest the NPLS marination as a promising process for improving water holding capacity of the WSCB. PRACTICAL APPLICATION: This study presents the promising application of non-phosphate, low-sodium (NPLS) marination combined with vacuum-tumbling in improving water holding capacity of chicken breast meat affected with white striping condition. Although microbial stability of the marinated breast was negatively affected, no adverse impacts on lipid oxidation was observed during storage up to 14 days.

WHOLE-meal ancient wheat-based diet: Effect on metabolic parameters and microbiota.

BACKGROUND & AIMS: Ancient wheat varieties are considered to be healthier than modern ones, but the data are not univocal. We investigated changes in hematochemical parameters and evaluated microbiota data before and after a set period on a diet containing a whole-meal ancient wheat mix. PATIENTS AND METHODS: 29 cloistered nuns were recruited. The study comprised two consecutive 30-day periods; during the first one (T1), the nuns received wheat-based foods produced with refined "modern" flour ("Simeto"); during the second one (T2) received wheat-based foods produced with an unrefined flour mix composed of "ancient" cultivars. At entry to the study (T0) and at the end of T1 and T2 hematochemical parameters and fecal microbiota and metabolome were evaluated. RESULTS: At the end of T2, there was a significant reduction in serum iron, ferritin, creatinine, sodium, potassium, magnesium, total cholesterol, LDL- and HDL-cholesterol and folic acid. Furthermore, increased the abundance of cultivable enterococci, lactic acid bacteria and total anaerobes. The ability of the gut microbiome to metabolize carbohydrates increased after the period of diet containing ancient grain products. Several volatile organic compounds increased after the one month on the diet enriched with ancient grain products. CONCLUSIONS: Our data showed the beneficial effects deriving from a diet including ancient whole-meal/unrefined wheat flours.

Supplementation with Bifidobacterium breve BR03 and B632 strains improved insulin sensitivity in children and adolescents with obesity in a cross-over, randomized double-blind placebo-controlled trial.

BACKGROUND & AIMS: Variations in gut microbiota might impact metabolism leading to body weight excess. We assessed the impact of a probiotic supplementation in pediatric obesity on weight, metabolic alterations, selected gut microbial groups, and functionality. METHODS: Cross-over, double-blind, randomized control trial (BIFI-OBESE trial; NCT03261466). 101 youths (6-18 years, Tanner stage ≥2) with obesity and insulin-resistance on diet were randomized to 2 × 109 CFU/AFU/day of Bifidobacterium breve BR03 (DSM 16604) and B. breve B632 (DSM 24706) (51) or placebo (50) for 8 weeks with a 4-weeks wash-out period. RESULTS: All subjects (M/F 54/47) completed the first 8 weeks, and 82 (M/F 43/39) the last part without adverse events. Mixed-effects models revealed a carry-over effect on many variables in the entire study, narrowing the analysis to the first 8 weeks before the wash-out periods. All subjects improved metabolic parameters, and decreased weight and Escherichia coli counts. Probiotics improved insulin sensitivity at fasting (QUICKI, 0.013 CI95%0.0-0.03) and during OGTT (ISI, 0.654 CI95%-0.11-1.41). Cytokines, GLP1, and target microbial counts did not vary. Of 25 SCFAs, acetic acid and acetic acid pentyl-ester relative abundance remained stable in the probiotics, while increased in the placebo (p < 0.02). A signature of five butanoic esters identified three clusters, one of them had better glucose responses during probiotics. CONCLUSION: An 8 weeks treatment with B. breve BR03 and B632 had beneficial effects on insulin sensitivity in youths with obesity. Microbiota functionality could influence metabolic answers to probiotics. Long-term studies to confirm and enrich our findings are justified. Tailored probiotic treatments could be an additional strategy for obesity. TRIAL REGISTRATION: NCT03261466.

Diet influences the functions of the human intestinal microbiome.

Gut microbes programme their metabolism to suit intestinal conditions and convert dietary components into a panel of small molecules that ultimately affect host physiology. To unveil what is behind the effects of key dietary components on microbial functions and the way they modulate host-microbe interaction, we used for the first time a multi-omic approach that goes behind the mere gut phylogenetic composition and provides an overall picture of the functional repertoire in 27 fecal samples from omnivorous, vegan and vegetarian volunteers. Based on our data, vegan and vegetarian diets were associated to the highest abundance of microbial genes/proteins responsible for cell motility, carbohydrate- and protein-hydrolyzing enzymes, transport systems and the synthesis of essential amino acids and vitamins. A positive correlation was observed when intake of fiber and the relative fecal abundance of flagellin were compared. Microbial cells and flagellin extracted from fecal samples of 61 healthy donors modulated the viability of the human (HT29) colon carcinoma cells and the host response through the stimulation of the expression of Toll-like receptor 5, lectin RegIIIα and three interleukins (IL-8, IL-22 and IL-23). Our findings concretize a further and relevant milestone on how the diet may prevent/mitigate disease risk.

Molecular and phenotypic traits of in-vitro-selected mutants of Bifidobacterium resistant to rifaximin.

Nucleotide mutations inside a core region of the rpoB gene, encoding the beta subunit of RNA polymerase, were found in rifaximin-resistant mutants of Bifidobacterium. Five different missense mutations of codons 513, 516, 522 and 529 were identified. Further aspects of rifaximin resistance were investigated, using Bifidobacterium infantis BI07 as a model strain. Partial resistance of RNA polymerase of a BI07 mutant at a rifaximin concentration >10 microg/mL was observed by cell-free transcription assay. Mass spectrometry detection of rifaximin in the cellular pellet of the BI07 resistant mutant, as well as changes in biosynthesis of saturated and cyclopropane fatty acids during growth, suggested a reduction in membrane permeability for the antibiotic moiety.

Effect of a pre-treatment of milk with high pressure homogenization on yield as well as on microbiological, lipolytic and proteolytic patterns of "Pecorino" cheese.

The principal aim of this work was to compare Pecorino cheeses obtained from ewes' milk previously subjected to high pressure homogenization (HPH) at 100 MPa with those produced from raw and heat treated ewes' milk. The HPH milk treatment induced a significant increase of the cheese yield and caused a reduction of enterococci, lactococci and yeasts in the curds. Enterococci cell loads remained at lower levels in cheeses obtained from HPH milk over the ripening period. Analyses of free fatty acids, Sodium Dodecil Sulphate (SDS)-PAGE profiles, Gas Chromatography-Mass Spectrometry-Solid Phase Microextraction (GC-MS-SPME) measurements of volatile compounds and sensory traits evidenced that the pressure treatment can be regarded also as a useful tool to differentiate products obtained from the same raw material. In fact such a milk treatment induced a marked lipolysis, an early proteolysis, a relevant modification of the volatile molecule profiles and sensory properties of Pecorino cheese.

Fermented Nut-Based Vegan Food: Characterization of a Home made Product and Scale-Up to an Industrial Pilot-Scale Production.

Because of the impossibility to consume food of animal origin, vegan consumers are looking for substitutes that could enrich their diet. Among many substitutes, fermented nut products are made from different nut types and obtained after soaking, grinding, and fermentation. Although other fermented vegetable products have been deeply investigated, there are few data about the fermentative processes of nut-based products and the microbial consortia able to colonize these products are not yet studied. This study characterized a hand-made vegan product obtained from cashew nut. Lactic acid bacteria responsible for fermentation were identified, revealing a succession of hetero- and homo-fermentative species during process. Successively, some lactic acid bacteria isolates from the home-made vegan product were used for a pilot-scale fermentation. The products obtained were characterized and showed features similar to the home-made one, although the microbiological hazards have been prevented through proper and rapid acidification, enhancing their safety features. PRACTICAL APPLICATION: Spontaneous fermented products are valuable sources of microorganisms that can be used in many food processes as starter cultures. The lactic acid bacteria isolated in this research can be exploited by industries to develop new foods and therefore to enter new markets. The use of selected starter cultures guarantees good organoleptic characteristics and food safety (no growth of pathogens).

New signaling molecules in some gram-positive and gram-negative bacteria.

A new family of putative signaling molecules having a 2(5H)-furanone configuration has been described in this work. They were released during late exponential or stationary phase in different growth media by some gram-positive bacteria, such as Lactobacillus helveticus, Lactobacillus plantarum, Lactobacillus paraplantarum, Lactobacillus sanfranciscensis, Enterococcus faecalis, and a gram-negative species, i.e. Salmonella enterica. A pair of 2(5H)-furanones called furanones A and B occurred in all the conditioned media (CMs) of the species considered. These two molecules showed similar retention times and their spectral data shared the key fragments to include them in the 2(5H)-furanones family. However, some differences were observed in the mass fragmentation profiles. In particular the use of PCA analysis of all the mass fragments enabled the grouping of furanone A profiles of S. enterica, L. helveticus, L. plantarum, L. paraplantarum, L. sanfranciscensis and E. faecalis in one unique cluster with only few exceptions. On the other hand, the mass fragmentation profiles of furanone B of the major part of the species and strains could be grouped together and were differentiated from those of L. helveticus. The specific activity of cell-free supernatants of high density cultures of S. enterica confirmed that the release of active molecules, and specifically of furanones A and B, was cell density dependent. Moreover, a preliminary experiment suspending S. enterica cells into cell-free supernatants of L. helveticus previously exposed to an oxidative stress demonstrated that furanones A and B have a strong interspecific activity. In fact cell autolysis and cell envelope damages were observed with Scanning Electron Microscopy (SEM) in S. enterica.

Geochemistry and microbial diversity of cave waters in the gypsum karst aquifers of Emilia Romagna region, Italy.

Fifty-seven control points of waters (sinking streams, rivers in caves, and resurgences) hosted in gypsum karst areas in Emilia Romagna region (N-Italy) were sampled in the framework of a Project LIFE+08NAT/IT/000369 "Gypsum" in the period 2010-2014. The microbiology and chemistry of these waters have been analyzed to evaluate the impact of human activities or natural factors, in the gypsum karst systems. Waters have been analyzed for major chemistry (Ca, Mg, Na, K, SO4, HCO3, Cl, NO3) and some minor constituents (F, Br, NH4 and PO4), measuring pH, electric conductivity (EC), total dissolved solids (TDS) and temperature (T) in situ. The same samples have been analyzed with traditional microbiology techniques focused on total microbial count and on fecal microbiota, as index of human and/or animal contamination, and molecular biology techniques (sequencing of 16S rRNA segment and PCR-DGGE), focused on the characterization of microbial populations in the different sampling sites and determination of their variations and/or changes during the five years of the project. As expected, waters tend to be increasingly mineralized from sinking streams to resurgences, with only local and temporarily high contents in nitrates and ammonium, often related to the presence of bat colonies. PCR-DGGE revealed ecological changes, in terms of microbial populations present in the bulk water samples, in different sampling sites within the same cave. Although the impact of fecal microorganisms only rarely exceeded 2 log UFC/ml, the results evidenced fluctuations of these microorganisms mainly correlated to the season and to the biological activity of bats.