Evaluation of Potential Probiotic Properties and In Vivo Safety of Lactic Acid Bacteria and Yeast Strains Isolated from Traditional Home-Made Kefir.

Probiotics are known for their health-promoting resources and are considered as beneficial microorganisms. The current study focuses on the isolation, and on a complete in vitro and in vivo characterization, of yeast and lactic acid bacteria acquired from traditional homemade kefir in order to assess their potentiality as probiotic candidates. In particular, the isolates Pichia kudriavzevii Y1, Lactococcus lactis subsp. hordniae LAB1 and Lactococcus lactis subsp. lactis LAB2 were subjected to in vitro characterization to evaluate their suitability as probiotics. Resistance to acid and bile salts, auto-aggregation, co-aggregation, hydrophobicity, and biofilm production capability were examined, as well as their antioxidant activity. A safety assessment was also conducted to confirm the non-pathogenic nature of the isolates, with hemolysis assay and antibiotic resistance assessment. Moreover, mortality in the invertebrate model Galleria mellonella was evaluated. Current findings showed that P. kudriavzevii exhibited estimable probiotic properties, placing them as promising candidates for functional foods. Both lactic acid bacteria isolated in this work could be classified as potential probiotics with advantageous traits, including antimicrobial activity against enteric pathogens and good adhesion ability on intestinal cells. This study revealed that homemade kefir could be a beneficial origin of different probiotic microorganisms that may enhance health and wellness.

Profiling microbial communities in an extremely acidic environment influenced by a cold natural carbon dioxide spring: A study of the Mefite in Ansanto Valley, Southern Italy.

The Ansanto Valley's Mefite, one of the Earth's largest non-volcanic CO2 gas emissions, is distinguished by its cold natural carbon dioxide springs. These emissions originate from the intricate tectonics and geodynamics of the southern Apennines in Italy. Known for over two millennia for its lethal concentration of CO2 and other harmful gases, the Mefite has a reputation for being toxic and dangerous. Despite its historical significance and unique geological features, there is a lack of information on the microbial diversity associated with the Mefite's gas emissions. This study presents an integrated exploration of the microbial diversity in the mud soil, using high-throughput sequencing of 16S rRNA (Prokaryotes) and ITS2 (Fungi), alongside a geochemical site characterisation. Our findings reveal that the Mefite's unique environment imposes a significant bottleneck on microbial diversity, favouring a select few microbial groups such as Actinobacteria and Firmicutes for Prokaryotes, and Basidiomycota for Fungi.

Ecotoxicological assessment of waste-derived organic fertilizers and long-term monitoring of fertilized soils using a multi-matrix and multi-species approach.

The present study investigates the ecotoxicity of 7 biofertilizers, including biowaste-derived organic matrices. Real-field tests were conducted to assess the impacts of soil fertilization with sewage sludge digestate from high-solid thermophilic anaerobic digestion (HSTAD) compared to those obtained on non-amended and urea-fertilized soils. The physical-chemical and ecotoxic impact of HSTAD digestate on soil was monitored for 12 months, at 5 time points and 2 soil depths, on a maize field divided in 3 portions (non-treated, fertilized with urea, amended with digestate). The chemical and physical characteristics of the soil were previously analyzed for 3 years to provide a long-term outlook of the impacts of biofertilizer application. Seven bioindicators were utilized for direct (on whole soil) and indirect (on soil elutriates) ecotoxicological tests on fertilizers and amended soils, including plant seeds (Lepidium sativum, Sorghum saccharatum, and Sinapsis alba), the aquatic organism Daphnia magna, the alga Raphidocelis subcapitata, the luminescent bacterium Aliivibrio fischeri, and the Nematode Caenorhabditis elegans. No serious negative effects on soil fertilized with HSTAD digestate were evidenced. Conversely, bioassays rather showed positive effects, encouraging the utilization of HSTAD digestate in agriculture, considering the proper concentrations of use. The obtained data were interpolated and a test battery integrated index was generated, confirming the absence of ecotoxicological risk for the soils amended with the applied fertilizers. The long-term evolution of the physical-chemical soil characteristics (including the concentrations of potential contaminants) was similar for both HSTAD digestate and urea application as well as for non-fertilized soil, indicating no negative effects due to digestate application on land. On the contrary, digestate application improved the content of stabilized organic matter and nutrients in soil. This study proposes a more correct approach to ecotoxicity assessment of fertilized soils for biofertilizer evaluation and demonstrates the long-term safe application of HSTAD digestate on agricultural soil.

Simultaneous Irradiation with UV-A, -B, and -C Lights Promotes Effective Decontamination of Planktonic and Sessile Bacteria: A Pilot Study.

Surfaces in highly anthropized environments are frequently contaminated by both harmless and pathogenic bacteria. Accidental contact between these contaminated surfaces and people could contribute to uncontrolled or even dangerous microbial diffusion. Among all possible solutions useful to achieve effective disinfection, ultraviolet irradiations (UV) emerge as one of the most "Green" technologies since they can inactivate microorganisms via the formation of DNA/RNA dimers, avoiding the environmental pollution associated with the use of chemical sanitizers. To date, mainly UV-C irradiation has been used for decontamination purposes, but in this study, we investigated the cytotoxic potential on contaminated surfaces of combined UV radiations spanning the UV-A, UV-B, and UV-C spectrums, obtained with an innovative UV lamp never conceived so far by analyzing its effect on a large panel of collection and environmental strains, further examining any possible adverse effects on eukaryotic cells. We found that this novel device shows a significant efficacy on different planktonic and sessile bacteria, and, in addition, it is compatible with eukaryotic skin cells for short exposure times. The collected data strongly suggest this new lamp as a useful device for fast and routine decontamination of different environments to ensure appropriate sterilization procedures.

Possible Molecular Mechanisms Underlying the Decrease in the Antibacterial Activity of Protamine-like Proteins after Exposure of Mytilus galloprovincialis to Chromium and Mercury.

Natural bioactive compounds represent a new frontier of antimicrobial molecules, and the marine ecosystem represents a new challenge in this regard. In the present work, we evaluated the possibility of changes in the antibacterial activity of protamine-like (PL) proteins, the major nuclear basic protein components of Mytilus galloprovincialis sperm chromatin, after the exposure of mussels to subtoxic doses of chromium (VI) (1, 10, and 100 nM) and mercury (1, 10, and 100 pM) HgCl2, since these metals affect some properties of PL. After exposure, we analyzed the electrophoretic pattern of PLs by both acetic acid-urea polyacrylamide gel electrophoresis (AU-PAGE) and SDS-PAGE and determined the MIC and MBC of these proteins on different gram+ and gram- bacteria. PLs, particularly after mussels were exposed to the highest doses of chromium and mercury, showed significantly reduced antibacterial activity. Just at the highest doses of exposure to the two metals, changes were found in the electrophoretic pattern of PLs, suggesting that there were conformational changes in these proteins, which were confirmed by the fluorescence measurements of PLs. These results provide the first evidence of a reduction in the antibacterial activity of these proteins following the exposure of mussels to these metals. Based on the results, hypothetical molecular mechanisms that could explain the decrease in the antibacterial activity of PLs are discussed.

Anti-Biofilm Activity of Phenyllactic Acid against Clinical Isolates of Fluconazole-Resistant Candida albicans.

Commonly found colonizing the human microbiota, Candida albicans is a microorganism known for its ability to cause infections, mainly in the vulvovaginal region, and is responsible for 85% to 90% of vulvovaginal candidiasis (VVC) cases. The development of drug resistance in C. albicans isolates after long-term therapy with fluconazole is an important complication to solve and new therapeutic strategies are required to target this organism and its pathogenicity. In the present study, phenyllactic acid (PLA) an important broad-spectrum antimicrobial compound was investigated for its antifungal and antivirulence activities against clinical isolates of C. albicans. Previously characterized strains of C. albicans isolates from women with VVC and C. albicans ATCC90028 were used to evaluate the antimicrobial and time dependent killing assay activity of PLA showing a MIC 7.5 mg mL-1 and a complete reduction of viable Candida cells detected by killing kinetics after 4 h of treatment with PLA. Additionally, PLA significantly reduced the biomass and the metabolic activity of C. albicans biofilms and impaired biofilm formation also with changes in ERG11, ALS3, and HWP1 genes expression as detected by qPCR. PLA eradicated pre-formed biofilms as showed also with confocal laser scanning microscopy (CLSM) observations. Furthermore, the compound prolonged the survival rate of Galleria mellonella infected by C. albicans isolates. These results indicate that PLA is a promising candidate as novel and safe antifungal agents for the treatment of vulvovaginal candidiasis.

VT-1161-A Tetrazole for Management of Mono- and Dual-Species Biofilms.

VT-1161 is a novel tetrazole antifungal agent with high specificity for fungal CYP51 (compared to human CYP enzymes) which has been proven to have fewer adverse effects and drug-drug interaction profiles due to fewer off-target inhibitors. In this study, we evaluated the anti-biofilm potential of VT-1161 against mono- and dual-species biofilms of Candida albicans, Klebsiella pneumoniae and Staphylococcus aureus. VT-1161 inhibited planktonic growth of all three strains, with an MIC value of 2 µg mL-1 for C. albicans and 0.5 µg mL-1 for K. pneumoniae and S. aureus, and killed 99.9% of the microbial populations, indicating a cytocidal action. Additionally, VT-1161 showed an excellent anti-biofilm action, since it inhibited mono-microbial biofilms by 80% at 0.5 µg mL-1, and dual-species biofilms of C. albicans/K. pneumoniae and C. albicans/S. aureus by 90% at the same concentration. Additionally, the eradication of mature biofilms after 24 h of VT-1161 exposure was excellent, reaching 90% at 2 µg mL-1 for both mono- and dual-species biofilms. In such mixed biofilms, the use of VT-1161 was revealed to be an alternative treatment because it was able to reduce the number of cells of each species during both inhibition and eradication. Since long-term therapy is necessary for most fungal biofilm infections due to their recurrence and obstinacy, VT-1161 showed low cytotoxicity against normal human cell lines and also against the invertebrate model Caenorhabditis elegans. Considering the excellent anti-biofilm potential and its GRAS (generally recognized as safe) status, VT-1161 may find use in the prevention or therapeutic treatment of mono- or poly-microbial biofilms.

Biological and metabolic effects of the association between the microalga Galdieria sulphuraria and the fungus Penicillium citrinum.

Contamination of microalgae cultures can reduce their productivity and affect the quality of biomass and valuable bioproducts. In this article, after having isolated and identified for the first time the filamentous fungus Penicillium citrinum from heterotrophic cultures of the red polyextremophilic microalga Galdieria sulphuraria, we investigated the biological and metabolic significance of this alga-fungus association. In the same medium, both organisms grow better in each other's presence than separately. Both cell density and cell size of G. sulphuraria increase in co-cultures compared to pure alga cultures. In co-cultures, despite very severe growth conditions, the load of P. citrinum increases compared to pure fungus cultures. Optical microscope images have shown physical contact between cells of P. citrinum hyphae and G. sulphuraria which, however, retain their morphology and cell wall intact. GC-MS-based metabolomics analysis of metabolites excreted in the culture medium shows that pure cultures of the fungus and alga and co-cultures of alga plus fungus can be easily differentiated based on their metabolic products. Indeed, a richer assortment of extracellular metabolites (comprising both products of primary and secondary metabolism) is a distinct feature of co-cultures compared to both pure alga and pure fungus cultures.

A model for microbial interactions and metabolomic alterations in Candida glabrata-Staphylococcus epidermidis dual-species biofilms.

The fungus Candida glabrata and the bacterium Staphylococcus epidermidis are important biofilm-forming microorganisms responsible of nosocomial infections in patients. In addition to causing single-species disease, these microorganisms are also involved in polymicrobial infections leading to an increased antimicrobial resistance. To expand knowledge about polymicrobial biofilms, in this study we investigate the formation of single- and dual-species biofilms of these two opportunistic pathogens employing several complementary approaches. First, biofilm biomass, biofilm metabolic activity and the microbial composition in single- and dual-species biofilms were assessed and compared. Then, the expression of three genes of C. glabrata and three genes of S. epidermidis positively related to the process of biofilm formation was evaluated. Although S. epidermidis is a stronger biofilm producer than C. glabrata, both biological and genetic data indicate that S. epidermidis growth is inhibited by C. glabrata which dominates the dual-species biofilms. To better understand the mechanisms of the interactions between the two microorganisms, a broad GC-MS metabolomic dataset of extracellular metabolites for planktonic, single- and dual-species biofilm cultures of C. glabrata and S. epidermidis was collected. As demonstrated by Partial Least Squares Discriminant Analysis (PLS-DA) of GC-MS metabolomic data, planktonic cultures, single- and dual-species biofilms can be sharply differentiated from each other by the nature and levels of an assortment of primary and secondary metabolites secreted in the culture medium. However, according to our data, 2-phenylethanol (secreted by C. glabrata) and the synergistically combined antifungal activity of 3-phenyllactic acid and of the cyclic dipeptide cyclo-(l-Pro-l-Trp) (secreted by S. epidermidis) play a major role in the race of the two microorganisms for predominance and survival.

An Ecotoxicological Evaluation of Four Fungal Metabolites with Potential Application as Biocides for the Conservation of Cultural Heritage.

Biocides based on chemical synthetic compounds have been commonly used to counteract damages caused by microorganisms on stone cultural heritage. However, in the last few years, the use of commercial and traditional biocides has been banned and/or limited due to their dangerous profile for the environment, as well as human and animal health. Natural products could be used as suitable alternatives for cultural heritage purposes, as they have low toxicity and stability compared with synthetic pesticides. Even if most of the investigated solutions have already shown promising results, their efficiency, ecotoxicological, and chemical features are poorly investigated. In this manuscript, we aimed to evaluate the ecotoxicological profile of four fungal metabolites-namely, cavoxin, epi-epoformin, seiridin, and sphaeropsidone-with potential antimicrobial properties for monumental artworks. A battery of ecotoxicological tests using Aliivibrio fischeri (bacterium), Raphidocelis subcapitata (alga), Raphanus sativus L. (macrophyte), Daphnia magna (crustacean), and Caenorhabditis elegans (nematode) revealed a relative lower toxicity of these compounds, especially when compared with Preventol® and Rocima®, commercial biocides mainly used for the conservation of cultural heritage.

Exposure of Buffalo Milkers to Pathogenic Bacteria and Characterization of Isolated Methicillin-Resistant Staphylococcus spp.

The research was focused on the surveillance of the exposure of buffalo milkers in contact with both animals and potentially contaminated equipment, pointing attention on the diffusion of antibiotic-resistant Staphylococcus spp. The monitoring was performed for 12 months, allowing the collection of 600 raw milk and buffalo udder surface samples, 192 milking lanes, 400 milking clusters, 160 personal protective equipment (PPEs) and electronic devices surface samples in contact with the workers of four milking parlors located in Southern Italy. The analysis of the milk samples evidenced the highest exposure to the bacteria considered (and mainly to S. aureus) from late winter-spring seasons onward. The possible risk arising from buffalo udder, milking clusters, and lines were instead considered rather stable along the entire period of sampling. The PPEs turned out to be a source of contamination for milkers mainly during the spring and summer periods. The analysis for oxacillin/methicillin resistance revealed in all the farms enrolled an overall amount of 37.5% of Staphylococci strains (belonging to S. aureus, S. haemolyticus, S. pseudintermedius, S. chromogenes species) resistant both to methicillin and oxacillin. The investigation demonstrated that the potential transfer of pathogenic bacteria to humans would have a better chance to occur at milk resumption time (since late winter-spring onward) when the number of animals to be milked is greater and the activity in the milking parlor is more challenging. At the same time, the findings seem to point out that the potential risk may be worsened by a significant presence of oxacillin/methicillin-resistant Staphylococci, potentially resulting from irrational use of antibiotics.

A sequential utilization of the UV-A (365 nm) fluence rate for disinfection of water, contaminated with Legionella pneumophila and Legionelladumoffii.

Legionella species are the etiological agent of Legionnaires' disease, a pathology easily contracted from water circuits and by the inhalation of aerosol droplets. This bacterium mainly proliferates in water: Legionella pneumophila is the most commonly isolated specie in water environments and consequently in water system, although further Legionella species have frequently been isolated, including Legionella dumoffii. The simultaneous presence of the two species in the water system can therefore lead to the simultaneous infection of several people, giving rise to harmful outbreaks. Ultraviolet inactivation of waterborne microorganisms offers a rapid and effective treatment technique and recently is getting more attention mostly to eliminate unsafe level of contamination. To tackle the issue, the inactivation of the two species of Legionella spp., namely L. pneumophila and L. dumoffii, by means of UV-A light emitting diodes (UV-A LED) system is explored. We used a commercially available UV-A LED at 365 nm wavelength, and the UV-A dose is given incrementally to the Legionellae with a concentration of 106 CFU/mL in 0.9% NaCl (aq) solution. In this study, with a UV-A-dose of 1700 mJ/cm2, the log-reduction of 3-log (99.9% inactivation) for L. pneumophila and 2.1-log (99.1% inactivation) for L. dumoffii of the contaminated water are achieved. The Electrical Energy per Order (EEO) is evaluated and showed this system is more economic and efficient in comparison with UV-C and UV-B LEDs. Following the support of this preliminary study with additional tests, aiming to validate the technology, we expect this device may be installed in water plants such as cooling systems or any water purification station in either industrial or home scales to reduce the risk of this infectious disease, preventing consumers' health.

Evaluation of the Pathogenic-Mixed Biofilm Formation of Pseudomonas aeruginosa/Staphylococcus aureus and Treatment with Limonene on Three Different Materials by a Dynamic Model.

BACKGROUND: Biofilms have been found growing on implantable medical devices. This can lead to persistent clinical infections. The highly antibiotic-resistant property of biofilms necessitates the search for both potent antimicrobial agents and novel antibiofilm strategies. Natural product-based anti-biofilm agents were found to be as efficient as chemically synthesized counterparts with fewer side effects. In the present study, the effects of limonene as an antibiofilm agent were evaluated on Pseudomonas aeruginosa and Staphylococcus aureus biofilm formed on different surfaces using the CDC model system in continuous flow. The flgK gene and the pilA gene expression in P. aeruginosa, and the icaA gene and eno gene in S. aureus, which could be considered as efficient resistance markers, were studied. METHODS: Mono- and dual-species biofilms were grown on polycarbonate, polypropylene, and stainless-steel coupons in a CDC biofilm reactor (Biosurface Technologies, Bozeman, MT, USA). To evaluate the ability of limonene to inhibit and eradicate biofilm, a sub-MIC concentration (10 mL/L) was tested. The gene expression of P. aeruginosa and S. aureus was detected by SYBR Green quantitative Real-Time PCR assay (Meridiana Bioline, Brisbane, Australia). RESULTS: The limonene added during the formation of biofilms at sub-MIC concentrations works very well in inhibiting biofilms on all three materials, reducing their growth by about 2 logs. Of the same order of magnitude is the ability of limonene to eradicate both mono- and polymicrobial mature biofilms on all three materials. Greater efficacy was observed in the polymicrobial biofilm on steel coupons. The expression of some genes related to the virulence of the two microorganisms was differently detected in mono- and polymicrobial biofilm. CONCLUSIONS: These data showed that the limonene treatment expressed different levels of biofilm-forming genes, especially when both types of strains alone and together grew on different surfaces. Our findings showed that limonene treatment is also very efficient when biofilm has been grown under shear stress causing significant and irreversible damage to the biofilm structure. The effectiveness of the sanitation procedures can be optimized by applying antimicrobial combinations with natural compounds (e.g., limonene).

Antioxidant response to heavy metal pollution of Regi Lagni freshwater in Conocephalum conicum L. (Dum.).

Conocephalum conicum L. is a cosmopolitan liverwort species able to respond to local environmental pollution by changing its biological features. In the present study, we assessed the different biological responses in C. conicum to heavy metal contamination of Regi Lagni channels, a highly polluted freshwater body. As for the in field experiment, we set up moss bags containing collected samples of the local wild growing C. conicum, from the upstream site (non-polluted area), and we exposed them in the three selected sites characterized by different and extreme conditions of heavy metal pollution. In addition, to better understand the contribution of heavy metals to the alterations and response of the liverwort, we performed in vitro tests, using the same concentration of heavy metals measured in the sites at the moment of the exposition. In both experimental settings, bioaccumulation, ultrastructural damage, reactive oxygen species production and localization, antioxidant enzymes activity (superoxide dismutase, catalase and glutathione S-transferases), glutathione (reduced and oxidized) levels, localization of compounds presenting thiol groups and phenolic content were investigated. The results showed that the samples from different sites and conditions (for in vitro tests) showed significant differences. In particular, the ultrastructural alterations show a trend correlated to the different exposure situations; ROS contents, glutathione, antioxidant enzyme activities, and phenolic contents were increased showing an enhancement of the antioxidant defense both by the enzymatic way and by using the synthesis of antioxidant phenolic compounds. This study confirms the ability of C. conicum to respond to heavy metal pollution and the responses studied are, at least partially, correlated to the presence of heavy metals. All the responses considered respond consistently with the pollution trend and they can be proposed as pollution biomarkers. Therefore, we suggest the use of C. conicum to identify local hot spots of pollution in further investigation.

Hydrochemical, isotopic and microbiota characterization of telese mineral waters (Southern Italy).

The study deals with the analyses of springs and wells at the base of Montepugliano Hill that represents the SE edge of the wide carbonate Matese massif (Campania, southern Italy). At the base of the hill, from west to east and for almost one kilometre, cold springs HCO3-Ca type (Grassano springs, ~ 4.5 m3/s; TDS: about 0.45 g/L) pass to hypothermal, HCO3-Ca type, sulphurous and CO2-rich springs (~ 1 m3/s with TDS > 1 g/L). Some of the latter are widely used in Telese Spa and Centro Relax Spa. Chemical and isotopic analyses carried out for this study support the hypothesis that all these waters (mineral and non-mineral) have the same catchment area, which is located in the Matese massif. As regards the sulphurous springs, they receive both meteoric waters infiltration and uprising of deeper waters rich in endogenous CO2 and H2S gases through important faults systems. Far from these faults, the chemistry of groundwater is scarcely (or not at all) affected by these deep fluid enrichment processes. This scheme is very significant; in fact, when very important groundwater resources are present, it is possible to use both mineral waters in Spa and, in areas far from the faults, those not yet mineralized. Finally, at Montepugliano Hill, in the final stage of the flow path, groundwater is also affected by change in the microbiome: this could provide a basis for comparison between various mineral waters.

Biological responses to heavy metal stress in the moss Leptodictyum riparium (Hedw.) Warnst.

Leptodictyum riparium, a widely distributed aquatic moss, can both tolerate and accumulate very high concentrations of toxic heavy metals, with only slight apparent damage. Here we report the effects on photosynthetic yield, glutathione (GSH), phytochelatin (PCn) synthesis, nitrogen metabolism and cellular localization of molecules rich in SH groups in L. riparium exposed in vitro to heavy metals. We simulated the concentrations of Cu, Zn, Cd, Pb detected in Regi Lagni, Italy, one of the most contaminated freshwater sites in Southern Europe, in the laboratory to test how the moss responds to heavy metal contamination. There was a steady decrease of photosynthetic efficiency correlated with the heavy metal concentrations and ultrastructural organization. All PCn levels increased significantly as the concentration of heavy metals increased, while the GSH levels did not appear to be particularly affected. A significant increase of GDH and NADH-GOGAT activities increased with increasing heavy metal concentration. Immunoblotting analysis revealed an increase of the chl-GS2 while no significant increase was detected in the cyt-GS1. These results give insight into the molecular events underlying the metal-tolerance of the aquatic moss L. riparium exposed to environmental heavy metal concentrations.

Hygienic assessment of digestate from a high solids anaerobic co-digestion of sewage sludge with biowaste by testing Salmonella Typhimurium, Escherichia coli and SARS-CoV-2.

Anaerobic digestion is a consolidated technology to convert sewage sludge and other organic wastes into biogas and a nutrient-rich fertilizer (i.e. digestate). The origin of sewage sludge does not exclude the potential presence of pathogens (e.g. Salmonella spp. and SARS-CoV-2) in mature digestate that hence could represent a source of sanitary concerns when it is spread on soil for agriculture purpose. Therefore, an experimental study aimed at proving the sanitizing effect of a full scale thermophilic high solids anaerobic digestion process was conducted by monitoring the hygienic characteristics of mature digestate. Although Salmonella spp. was detected in the sewage sludge fed to the full scale plant, the anaerobic digestion treatment demonstrated sanitization capacity since the monitored pathogens were never found in the mature digestate over the entire duration of the monitoring survey. Furthermore, tests on the regrowth of Salmonella Typhimurium and Escherichia coli, artificially inoculated on mature digestate, were also conducted under both anaerobic and aerobic conditions with the aim to assess the effectiveness of mature digestate as microbial growth medium. Concentrations of Salmonella Typhimurium and Escherichia coli were drastically reduced after a short time of incubation under anaerobic process and the two microorganisms already resulted undetectable after 24-48 h, whereas, under aerobic conditions, two microorganisms' concentrations were stably high for longer than 10 days. The combination of no free oxygen, high temperature, anaerobic metabolites (e.g. total ammonium nitrogen, and volatile fatty acids) production, bacteria competition and lack of nutritional elements in mature digestate considerably reduced in 24-48 h the sanitary risks associated to accidently contaminated digestate. Furthermore, a SARS-CoV-2 monitoring survey on mature digestate during 13 months, resulted in the absence of the virus RNA in the analyzed digestate.

Screening and isolation of microbes from a Mud Community of Ischia Island Thermal Springs: preliminary analysis of a bioactive compound.

INTRODUCTION: Balneotherapy centers of Ischia island (Italy) offer treatments for different dermatological diseases (psoriasis, acne, atopic dermatitis) and upper respiratory tract infections. In this study, we integrated morphological and molecular approaches to give a focus on isolation and screening of extremophile bacteria from Ischia thermal mud for potential antimicrobial applications. METHODS: Samples were collected during 2019 at four sites. Some bacterial strains ATCC for antibacterial and antibiofilm activity were tested. After morphological characterization, screening for antagonistic isolates was made. The colonies isolated from thermal mud samples were submitted to molecular characterization. Susceptibility testing by dilution spotting was carried out and antibacterial efficacies of most active isolate were evaluated with a Minimal inhibition concentration assay. Biofilm formation, inhibition, eradication were examined. Statistical analyses were carried out utilizing Microsoft® Excel 2016/XLSTAT©-Pro. RESULTS: We isolated a natural compound with antimicrobial and antibiofilm activities. CONCLUSIONS: The results obtained in this study are discussed in the context of how hydrothermal systems are important environmental source of uncharted antimicrobial and antibiofilm compounds. In conclusion, to the most effective of our knowledge, this work presents the primary report on the preliminary investigation of thermophile microbial diversity and their antimicrobial and antibiofilm activities for future biotechnological interest.

A preliminary study on a novel bioaugmentation technique enhancing lactic acid production by mixed cultures fermentation.

The paper is a preliminary study on the selection of lactic acid producing microorganisms from a mixed microbial population via bioaugmentation. The bioaugmentation technique is based on pH sudden variations occurring in sequential batch steps of a dark fermentation process applied to simple substrates. Different conditions are tested and compared. The structure of microbial communities and concentrations of metabolic intermediates are analyzed to study the possible substrate conversion routes. Obtained results indicate that the initial mixed culture produced a lactic acid percentage of 5% in terms of CODLA/CODPRODUCTS. In the most favourable conditions, the selected culture produced a lactic acid percentage of 59%. The analysis of the composition of microbial communities before and after the bioaugmentation processes, indicates that lactic acid production mainly results from the population change to bacteria belonging to the genus Bacillus. Indeed, the relative abundance of Bacilli increased from 0.67%, to 8.40% during the bioaugmentation cycle.

An Integrated Analysis of Intracellular Metabolites and Virulence Gene Expression during Biofilm Development of a Clinical Isolate of Candida tropicalis on Distinct Surfaces.

Emergence of Candida tropicalis, which causes potential life-threatening invasive candidiasis, is often associated with colonization of medical devices as biofilm. Biofilm plays an important role in the virulence of the pathogen because of its complex structure, which provides resistance to conventional antimicrobials. In this study, the metabolic response of a clinical strain of C. tropicalis colonizing three distinct surfaces (polytetrafluoroethylene (PTFE), polystyrene, and polycarbonate) as well as the expression of virulence and stress related genes (ALS3, Hsp21, SAP1, SAP2, SAP3, and CYR1), were explored. Our results showed that lesser biofilm was developed on PTFE compared to polystyrene and polycarbonate. GS-MS metabolic analysis identified a total of 36 metabolites in the intracellular extract of cells grown on polystyrene, polycarbonate, and PTFE, essentially belonging to central carbon metabolism, amino acids, and lipids metabolism. The metabolic analysis showed that saturated and unsaturated fatty acids are preferentially produced during biofilm development on polycarbonate, whereas trehalose and vitamin B6, known as cellular protectors against a variety of stressors, were characteristic of biofilm on PTFE. The results of the transcriptomic analysis consider the different degrees of colonization of the three substrates, being CYR1, which encodes the component of signaling pathway of hyphal formation-cAMP-PKA, downregulated in PTFE biofilm compared to polycarbonate or polystyrene biofilms, while Hsp21 was upregulated in concomitance with the potential unfavorable conditions for biofilm formation on PTFE. Overall, this work provides new insights into the knowledge of C. tropicalis biofilm development on surfaces of medical relevance in the perspective of improving the management of Candida infections.