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The main objective of this study was to evaluate the efficacy of thymol in controlling environmental contamination in food processing facilities. The effect of thymol was tested as an agent to prevent planktonic and bacterial biofilm growth of twenty-five Listeria monocytogenes isolates from a variety of foods and five Escherichia coli isolates from a farm. The E. coli isolates were positive for extended spectrum &beta, lactamase (ESBL) genes. All isolates and reference strains were susceptible to thymol at Minimum inhibitory concentration (MIC) values ranging from 250 to 800 &mu, g/mL. An interesting activity of interference with biofilm formation of L. monocytogenes and E. coli was found for thymol at sub-MIC concentrations of 200, 100, 75, and 50 &mu, g/mL. Anti-biofilm activity ranging from 59.71% to 66.90% against pre-formed 24-h-old L. monocytogenes biofilms at concentrations of 500 or 800 &micro, g/mL, corresponding to 2&times, MIC, was determined against free-living forms of six isolates chosen as the best or moderate biofilm producers among the tested strains. The property of thymol to attack L. monocytogenes biofilm formation was also observed at a concentration of 100 &micro, g/mL, corresponding to 1/4 MIC, by using a stainless-steel model to simulate the surfaces in food industries. This study gives information on the use of thymol in food processing setting.

Resistance to carbapenems in Enterobacteriaceae, including Klebsiella pneumoniae, represents a major clinical problem given the lack of effective alternative antibiotics. Bacteriophages could provide a valuable tool to control the dissemination of antibiotic resistant isolates, for the decolonization of colonized individuals and for treatment purposes. In this work, we have characterized a lytic bacteriophage, named vB_Kpn_F48, specific for K. pneumoniae isolates belonging to clonal group 101. Phage vB_Kpn_F48 was classified as a member of Myoviridae, order Caudovirales, on the basis of transmission electron microscopy analysis. Physiological characterization demonstrated that vB_Kpn_F48 showed a narrow host range, a short latent period, a low burst size and it is highly stable to both temperature and pH variations. High throughput sequencing and bioinformatics analysis revealed that the phage is characterized by a 171 Kb dsDNA genome that lacks genes undesirable for a therapeutic perspective such integrases, antibiotic resistance genes and toxin encoding genes. Phylogenetic analysis suggests that vB_Kpn_F48 is a T4-like bacteriophage which belongs to a novel genus within the Tevenvirinae subfamily, which we tentatively named &ldquo, F48virus&rdquo, Considering the narrow host range, the genomic features and overall physiological parameters phage vB_Kpn_F48 could be a promising candidate to be used alone or in cocktails for phage therapy applications.

Antibiotic resistance in bacterial pathogens has currently reached very high and alarming levels [...]

© 2017, Springer International Publishing AG, part of Springer Nature. Aims: Biological soil crusts (biocrusts) are microbial communities commonly found in the upper layer of arid soils. These microorganisms release exopolysaccharides (EPS), which form the exopolymeric matrix (EPM), allowing them to bond soil particles together and survive long periods of dryness. The aim of this work is to develop methods for measuring metabolite retention by biocrust EPM and EPS. Methods: We report new methods for the investigation of metabolite sorption on biocrusts compared to the underlying unconsolidated subcrust fraction. A13C–labeled bacterial lysate metabolite mixture was incubated with biocrust, subcrust and biocrust-extracted EPS. Non-sorbed metabolites were extracted and analyzed by liquid chromatography/mass spectrometry. Results: This simple and rapid approach enabled the comparison of metabolite sorption on the biocrust EPM or EPS versus mineral sorption on the underlying soils. Our results suggest that the biocrust (and its extracted EPS) sorb more metabolites, especially amino acids and organic acids, than the underlying subcrust. Conclusions: This study demonstrates a useful method to highlight the essential role of biocrust (especially the EPM), which acts as a passive nutrient filter, sequestering metabolites released by microbes during wetting events. This may facilitate recovery of the community upon wetting and further enhance biocrust survival and nutrient retention.

Over the past decades, Salmonella 4,[5],12:i:- has rapidly emerged and it is isolated with high frequency in the swine food chain. Although many studies have documented the epidemiological success of this serovar, few investigations have tried to explain this phenomenon from a genetic perspective. Here a comparative whole-genome analysis of 50 epidemiologically unrelated S. 4,[5],12:i:-, isolated in Italy from 2010 to 2016 was performed, characterizing them in terms of genetic elements potentially conferring resistance, tolerance and persistence characteristics. Phylogenetic analyses indicated interesting distinctions among the investigated isolates. The most striking genetic trait characterizing the analyzed isolates is the widespread presence of heavy metals tolerance gene cassettes: most of the strains possess genes expected to confer resistance to copper and silver, whereas about half of the isolates also contain the mercury tolerance gene merA. A functional assay showed that these genes might be useful for preventing the toxic effects of metals, thus supporting the hypothesis that they can contribute to the success of S. 4,[5],12:i:- in farming environments. In addition, the analysis of the distribution of type II toxin-antitoxin families indicated that these elements are abundant in this serovar, suggesting that this is another factor that might favor its successful spread.

Antimicrobial resistance is a global threat caused by the rapid spread of multiresistant microorganisms. Antimicrobial stewardship (AS) is a coordinated intervention designed to improve the appropriate use of antimicrobials by promoting the selection of the optimal drug regimen, dose, duration of therapy and route of administration. AS programs have proved effective in reducing antimicrobial resistance, inappropriate antimicrobial use and in improving patient outcomes. Recently developed rapid diagnostic technologies in microbiology (RDTM) allows a faster and etiological diagnosis of infection and a reduction in the use of unnecessary empirical therapies. This may result in important advancement in time-critical care pathways for septic patients. Nevertheless, RDTM are costly and if not rationally positioned may consume resources and hinder the efficacy of AS programs. In this regard, Tuscany Region is engaged in designing, through a systemic approach, an effective high-quality clinical microbiological service grid. In order to develop a sustainable and equitable model for integrating diagnostic and antimicrobial stewardship we conducted a survey in the regional network of 14 microbiological laboratories. The results shows that in order to develop a sustainable service we need to improve the communication at the interface between laboratories and care unit, harmonize the time windows for processing samples and to devise a robust score for stratifying patient with suspected sepsis.

Tellurite (TeO32−) is a hazardous and toxic oxyanion for living organisms. However, several microorganisms can bioconvert TeO32− into the less toxic form of elemental tellurium (Te0). Here, Rhodococcus aetherivorans BCP1 resting (non-growing) cells showed the proficiency to produce tellurium-based nanoparticles (NPs) and nanorods (NRs) through the bioconversion of TeO32−, depending on the oxyanion initial concentration and time of cellular incubation. Te-nanostructures initially appeared in the cytoplasm of BCP1 cells as spherical NPs, which, as the exposure time increased, were converted into NRs. This observation suggested the existence of an intracellular mechanism of TeNRs assembly and growth that resembled the chemical surfactant-assisted process for NRs synthesis. The TeNRs produced by the BCP1 strain showed an average length (>700 nm) almost doubled compared to those observed in other studies. Further, the biogenic TeNRs displayed a regular single-crystalline structure typically obtained for those chemically synthesized. The chemical-physical characterization of the biogenic TeNRs reflected their thermodynamic stability that is likely derived from amphiphilic biomolecules present in the organic layer surrounding the NRs. Finally, the biogenic TeNRs extract showed good electrical conductivity. Thus, these findings support the suitability of this strain as eco-friendly biocatalyst to produce high quality tellurium-based nanomaterials exploitable for technological purposes.

Hepatic encephalopathy (HE) is a debilitating neuropsychiatric condition often associated with acute liver failure or cirrhosis. Advanced liver diseases are characterized by a leaky gut and systemic inflammation. There is strong evidence that the pathogenesis of HE is linked to a dysbiotic gut microbiota and to harmful microbial by-products, such as ammonia, indoles, oxindoles and endotoxins. Increased concentrations of these toxic metabolites together with the inability of the diseased liver to clear such products is thought to play an important patho-ethiological role. Current first line clinical treatments target microbiota dysbiosis by decreasing the counts of pathogenic bacteria, blood endotoxemia and ammonia levels. This review will focus on the role of the gut microbiota and its metabolism in HE and advanced cirrhosis. It will critically assess data from different clinical trials measuring the efficacy of the prebiotic lactulose, the probiotic VSL#3 and the antibiotic rifaximin in treating HE and advanced cirrhosis, through gut microbiota modulation. Additionally data from Randomised Controlled Trials using pre-, pro- and synbiotic will be also considered by reporting meta-analysis studies. The large amount of existing data showed that HE is a clear example of how an altered gut microbiota homeostasis can influence and impact on physiological functions outside the intestine, with implication for host health at the systems level. Nevertheless, a strong effort should be made to increase the information on gut microbiota ecology and its metabolic function in liver diseases and HE.

Silver nanoparticles (AgNPs), embedded into a specific polysaccharide (EPS), were biogenerated by Klebsiella oxytoca DSM 29614 under aerobic (AgNPs-EPSaer) and anaerobic conditions (AgNPs-EPSanaer). Both AgNPs-EPS matrices were tested by MTT assay for cytotoxic activity against human breast (SKBR3 and 8701-BC) and colon (HT-29, HCT 116 and Caco-2) cancer cell lines, revealing AgNPs-EPSaer as the most active, in terms of IC50, with a more pronounced efficacy against breast cancer cell lines. Therefore, colony forming capability, morphological changes, generation of reactive oxygen species (ROS), induction of apoptosis and autophagy, inhibition of migratory and invasive capabilities and proteomic changes were investigated using SKBR3 breast cancer cells with the aim to elucidate AgNPs-EPSaer mode of action. In particular, AgNPs-EPSaer induced a significant decrease of cell motility and MMP-2 and MMP-9 activity and a significant increase of ROS generation, which, in turn, supported cell death mainly through autophagy and in a minor extend through apoptosis. Consistently, TEM micrographs and the determination of total silver in subcellular fractions indicated that the Ag+ accumulated preferentially in mitochondria and in smaller concentrations in nucleus, where interact with DNA. Interestingly, these evidences were confirmed by a differential proteomic analysis that highlighted important pathways involved in AgNPs-EPSaer toxicity, including endoplasmic reticulum stress, oxidative stress and mitochondrial impairment triggering cell death trough apoptosis and/or autophagy activation.

This preliminary report shows eight complexes of triorganotin(IV): Ph3SnOH and (CH3)3SnOH with four chelating imines on new synthesis. Of these ligands, two are salen-like (four coordination sites, two imidic, two phenoxidic) [1], one is a tetradentate pyrrole derivative [2] while the fourth, a vita- min B6 derivative, is pentadentate [3]. Ligands have been characterized by means of FT-IR, UV-Vis, Fluorescence, 1H- and 13C-NMR, LC-MS ESI triple quadrupole; complexes by means FT-IR, 1H- and 119Sn-NMR, LC-MS ESI, using the isotopic distribution pattern as a discriminant [4]. Geometry and nature of coordination complexes have been also evaluated using the 119Sn chemical shifts. Solid-state synthesis of the complexes (with a ball mill [5]) was also explored; such method reduces both solvent consumption and time – from 8-10 h under controlled atmosphere to about 1 h, with results identical to the wet synthesis. Antitumoral and antibacterial activities of the triorganotin (IV) complexes (BS01M, BS01P, BS02M, BS02P, BS03M, BS03P, BS04M, BS04P) were tested in vitro. In both analyses, the Shiff bases alone showed no biological activity. Antimicrobial activity was evaluated by Kirby-Bauer method against the Gram-negative Escherichia coli, and the two Gram-positive Kocuria rizophila and Staphylococcus aureus strains. All the ML2 complexes were active in inhibiting bacterial growth, with BS02P and BS03P showing the best antibacterial performance. Among the ML complexes, BS01M was not active, BS02M showed a weak antibacterial activity only against the Gram-positive bacteria, BS04M was mainly active against the Gram-negative E. coli and BS03M was active against all the tested strains. Antitumor activity was evaluated by MTT assay against cervical (HeLa), colon adenocarcinoma (HT- 29) and breast (MDA-MB231) cancer cell lines. Results showed that ML2 complexes are more active than ML ones, with HeLa cells more sensitive to treatments. These complexes (especially the ML2) showed promising results; their mechanism of action is under investigation. References 1. K. Tayade, S.K. Sahoo, S. Chopra, N. Singh, Inorganica Chimica Acta, 421, 538-543 (2014); https://doi.org/10.1016/j. ica.2014.05.014 2. S. Meghdadi, M. Amirnasr, K. Mereiter, Polyhedron, 30, 1651-1656 (2011); https://doi.org/10.1016/j.poly.2011.03.041 3. D. Sharma, S.K. Sahoo, S. Chaudhary, R. Kanta Bera, J.F. Callan, Analyst, 138, 3646-3650 (2013); https://doi.org/10.1039/ C3AN00199G 4. G. Lawson, R.H. Dahm, N. Ostah, E.D. Woodland, Applied Organometallic Chemistry, 10, 125-133 (1996); https://doi. org/10.1002/(SICI)1099-0739(199603)10:23.0.CO;2-1 5. G.A. Bowmaker, Chemical Communications, 49, 334-348 (2013); http://doi.org/10.1039/C2CC35694E 6. R. Di Stefano, M. Scopelliti, C. Pellerito, G. Casella, T. Fiore, G.C. Stocco, R. Vitturi, L. Ronconi, I.D. Sciacca, L. Pellerito, Journal of Inorganic Biochemistry, 98, 534-546 (2004); https://doi.org/10.1016/j.jinorgbio.2003.12.013 7. C. Pellerito, L. Nagy, L. Pellerito, A. Szorcsik, Journal of Organometallic Chemistry, 691, 1733-1747 (2006); https://doi. org/10.1016/j.jorganchem.2005.12.025

The transition from commensalism to pathogenicity of Candida albicans reflects both the host inability to mount specific immune responses and the microorganism’s dimorphic switch efficiency. In this study, we used whole genome sequencing and microarray analysis to investigate the genomic determinants of the phenotypic changes observed in two C. albicans clinical isolates (YL1 and YQ2). In vitro experiments employing epithelial, microglial, and peripheral blood mononuclear cells were thus used to evaluate C. albicans isolates interaction with first line host defenses, measuring adhesion, susceptibility to phagocytosis, and induction of secretory responses. Moreover, a murine model of peritoneal infection was used to compare the in vivo pathogenic potential of the two isolates. Genome sequence and gene expression analysis of C. albicans YL1 and YQ2 showed significant changes in cellular pathways involved in environmental stress response, adhesion, filamentous growth, invasiveness, and dimorphic transition. This was in accordance with the observed marked phenotypic differences in biofilm production, dimorphic switch efficiency, cell adhesion, invasion, and survival to phagocyte-mediated host defenses. The mutations in key regulators of the hyphal growth pathway in the more virulent strain corresponded to an overall greater number of budding yeast cells released. Compared to YQ2, YL1 consistently showed enhanced pathogenic potential, since in vitro, it was less susceptible to ingestion by phagocytic cells and more efficient in invading epithelial cells, while in vivo YL1 was more effective than YQ2 in recruiting inflammatory cells, eliciting IL-1β response and eluding phagocytic cells. Overall, these results indicate an unexpected isolate-specific variation in pathways important for host invasion and colonization, showing how the genetic background of C. albicans may greatly affect its behavior both in vitro and in vivo. Based on this approach, we propose that the co-occurrence of changes in sequence and expression in genes and pathways driving dimorphic transition and pathogenicity reflects a selective balance between traits favoring dissemination of the pathogen and traits involved in host defense evasion. This study highlights the importance of investigating strain-level, rather than species level, differences, when determining fungal–host interactions and defining commensal or pathogen behavior.

Tuberculosis is an infectious disease caused by Mycobacterium tuberculosis. The development of strains resistant to antitubercular drugs, makes it necessary to find new compounds against this pathogen in order to treat such disease. G-quadruplexes (G-4s), secondary structures that may form in G-rich regions, were found in promoter regions of several human and viral genes where they influence processes such as transcription and replication of DNA. G-4s are considered as promising targets for anticancer and antiviral chemotherapy thanks to the discovery of small molecules that bind and stabilize their structure, decreasing gene expression levels (G-4 ligands). Recently, bioinformatics analysis identified in M. tuberculosis 10,000 motifs with the potential to fold into G-4 structure, 45 of them are localized upstream or overlapping the transcription start site of genes distributed among different functional categories. Analysis in vitro (circular dichroism, UV spectroscopy and Taq polymerase stop assay) confirmed the formation of G4 structures in four of these genes: mosR, zwf1, clpX and ndhA. To test the possibility to use G-quadruplex structures as therapeutic targets, we evaluated the effect of two G-4 ligands (BRACO-19 e c-exNDI 2) on M. tuberculosis. Results showed that these compounds can inhibit the mycobacterial growth with a minimal inhibitory concentration (MIC) in the micromolar range and make the bacteria more sensitive to oxidative stress. We also evaluated the interactions between BRACO-19 and two well characterized antitubercular drugs (ethambutol and isoniazid), while interaction with isoniazid was shown to be addictive, that with ethambutol was indifferent. To study the influence of G4 structures on gene transcription, the zwf1, mosR, clpX, ndhA expression was evaluated in presence of BRACO-19. In particular the G4 sequence in zwf1 promoter was better characterized. Single strand oligonucleotides containing the sequence of the putative G-4 localized at the promoter region of zwf1 with different mutations were analyzed by circular dichroism, allowing us to identify a single point mutation able to disrupt the G-4 structure. This mutation was introduced in the zwf1 promoter and the final construct was cloned upstream a promoterless eGFP gene in a reporter plasmid. eGFP expression from this plasmid was compared to that of a similar plasmid in which the reporter gene was transcribed from the wild-type zwf1 promoter in physiologic conditions or after treatment of bacterium with BRACO-19 and H2O2.The results suggested that the G-quadruplex structure upstream of zwf1, at least in the observed conditions, hasn’t a preeminent role in gene regulation.

The development of highly active anti-retroviral therapy (HAART) has considerably improved life expectancy of HIV-1 positive patients by transforming this infection, which once was lethal, into a manageable chronic illness. Although a significant suppression of viral replication under undetectable level is guaranteed following a constant therapeutic adherence, this therapy fails to completely eliminate the infection due to the persistence of HIV-1 into reservoirs, which represent therefore the main obstacle to a definite cure. Furthermore, a lifelong adherence to treatment is associated with drug toxicities and persistent immune dysfunction, which can lead to discontinuation of therapy and the onset of drug resistance. These hurdles, together with the high economic costs of providing HAART to more than 35 million people, which are currently affected by HIV-1, contribute to render HIV-1/AIDS pandemic one of the most important global health challenge. In this scenario, the search for a curative strategy is necessary. Recent successes in inherited immune deficiencies treatment and cancer immunotherapy have raised interest in gene and cell modification to treat HIV-1 infection with the final aim of inducing permanent resistance to HIV-1. In particular, anti HIV-1 gene therapy (GT) protocols, based on engineering of autologous T cells or their progenitors, such as the CD34+ hematopoietic stem cells (HSCs), appear a promising approach to repopulate the immune system after a single therapeutic intervention. Long-term HIV-1 remission in the “patient of Berlin”, who received an heterologous stem cell transplant for acquired immunodeficiency syndrome-related lymphoma from a CCR5 homozygous null HLA-matched donor (CCR5 -/-), even after discontinuation of conventional therapy, has energized the field. However, due to the limited chance of finding matching Δ32 CCR5 donors, recapitulating this clinical success on a large scale appears to be difficult. Moreover, autologous regimens are potentially less toxic, as they may not require full bone marrow ablation or subsequent immune suppression for engraftment. In this setting, the goal would be to disable the CCR5 gene in enough target cells to confer benefit and transplanted back into the patient. Different GT strategies to artificially disrupt the CCR5 gene or transcript have proved to be successful in primary T cells, HSCs, as well as in humanized mice, and are recently been tested in clinical trials. Inhibitors of entry or of early step of viral replication prior the virus integration are expected to lead an advantage selection of gene modified cells, thus preventing the establishment of chronic HIV-1 infection and limit the continued replenishment of viral reservoirs. However, a major goal of gene therapy is to target simultaneously multiple viral sites and endogenous host factors, interfering with different steps of viral cycle, hoping to reduce the onset of virus variants. Among antiviral agents, small interfering RNAs (siRNAs) are less immunogenic than protein-based ones and represent a powerful tool to silence gene expression post-transcriptionally in a sequence specific manner. In order to obtain a stable and long expression, necessary for a chronic disease, multiple anti-HIV-1 siRNAs can be accommodated into self-inactivating (SIN) lentiviral vectors, which are currently preferred for their ability to efficiently transduce target cells, and then because they confer a potentially safer integration site profile, compared to gammaretroviral vectors. For these reasons, different combinatorial platforms based on SIN lentiviral vectors were previously developed to express multiple siRNAs against highly conserved regions of cellular and viral genes, including the cellular co-receptor CCR5, the vif and tat/rev viral factors, involved in different phases of HIV-1 replication and pathogenesis. These siRNAs were placed under the control of different human Polymerase III promoters (such as U6, 7SK and H1) either as independent transcriptional units or as extended short hairpin RNA (e-shRNA), able to express the three siRNA under the control of a single promoter. The most potent antiviral activity in transduced human primary CD4+ T lymphocytes was conferred by two effective anti-HIV-1 combinatorial vectors (i.e. pLL3.7 U6shCCR5-7SKshvif-H1lhtat/rev and pLL3.7 H1e-shRNA). However, HIV-1 can use an alternative coreceptor (i.e. CXCR4) for entering into target cells, that is less favored as target of siRNA or novel gene-editing technologies, because its disruption can compromise fundamental physiological functions, especially the maturation of HSCs. At the same time, CXCR4 tropic viruses are relevant in the pathogenesis of AIDS. Thus, to improve the efficacy of this approach, the two selected vectors were optimized by the insertion of a small membrane anchored C-peptide (maC46) fusion inhibitor, which has been shown to protect against a broad range of HIV-1 isolates and it has been tested in a phase 1 clinical trial. When expressed on target cell surface, the maC46 peptide, which derives from the C-terminal heptad repeat (HR2) of the HIV-1 gp41 envelope glycoprotein, blocks the membrane fusion by interacting with the N-terminal coiled coil domain of the gp41 intermediate structure and preventing the six-helix bundle formation. In these constructs, the expression of the peptide, either fused in frame with the enhanced green fluorescence protein (eGFP) or alone, is driven by the human Elongation Factor 1 promoter (EF1), a cellular-derived enhancer/promoter, which has been shown to confer high level of transgene expression in HSCs and a more safety profile, since decreased cross-activation of nearby promoters. Additionally, the new developed lentiviral vectors carries an optimized version of the Woodchuck hepatitis virus post-transcriptional regulatory element (WPRE*), which enhances the vector titer and lacks oncogenic properties. Furthermore, control vectors either lacking the maC46 encoding sequence or characterized by a scrambled sequence in place of the siRNA encoding ones were generated. Once transfected into different cell lines, the developed vectors led to the expression of the maC46 peptide, that correctly localized at the plasma membrane, as shown with immunofluorescence cell staining. Furthermore, recombinant lentiviral particles (RLVPs) were produced and titrated either using Reverse Transcriptase (RT) Assay and Fluorescence-activated cell sorting (FACS). The efficacy of the maC46 peptide fusion inhibitor, in combination with the silencing activity of the expressed siRNAs, was evaluated in challenge experiments, in which transduced T lymphoblastoid Jurkat cells were infected with HIV-1 HXBc2 Vpr+/Vpu+/Nef+ R4-tropic molecular clone at different multeplicity of infection (M.O.I.). In parallel, in collaboration with the Baum’s group at the Department of Experimental Hematology of the Hannover Medical School, the mutagenic potential of these vectors was assessed by means of in vitro immortalization assay. The results obtained so far clearly show that the optimized vectors, combining for the first time a potent fusion inhibitor with three short hairpin (sh)RNAs, appear to be extremely promising as anti-HIV-1 approach. Importantly, these vectors showed a strongly reduced insertional transformation potential compared to a positive gammaretroviral control vector. This strategy could be now extended to primary cells and in particular to HSCs, the ultimate target of this gene therapy approach, with the final aim of accomplishing a high level of protection from HIV-1 infection over sustained lengths of time, without cell toxicity, and loss of stemness, proliferation capability and differentiation potential for possible future clinical applications.

Background Rett syndrome (RTT) is a neurological disorder mainly caused by mutations in MeCP2 gene. It has been shown that MeCP2 impairments can lead to cytokine dysregulation due to MeCP2 regulatory role in T-helper and T-reg mediated responses, thus contributing to the pro-inflammatory status associated with RTT. Furthermore, RTT subjects suffer from an intestinal dysbiosis characterized by an abnormal expansion of the Candida population, a known factor responsible for the hyper-activation of pro-inflammatory immune responses. Therefore, we asked whether the intestinal fungal population of RTT subjects might contribute the sub-inflammatory status triggered by MeCP2 deficiency. Methods We evaluated the cultivable gut mycobiota from a cohort of 50 RTT patients and 29 healthy controls characterizing the faecal fungal isolates for their virulence-related traits, antifungal resistance and immune reactivity in order to elucidate the role of fungi in RTT’s intestinal dysbiosis and gastrointestinal physiology. Results Candida parapsilosis, the most abundant yeast species in RTT subjects, showed distinct genotypic profiles if compared to healthy controls’ isolates as measured by hierarchical clustering analysis from RAPD genotyping. Their phenotypical analysis revealed that RTT’s isolates produced more biofilm and were significantly more resistant to azole antifungals compared to the isolates from the healthy controls. In addition, the high levels of IL-1β and IL-10 produced by peripheral blood mononuclear cells and the mixed Th1/Th17 cells population induced by RTT C. parapsilosis isolates suggest the capacity of these intestinal fungi to persist within the host, being potentially involved in chronic, pro-inflammatory responses. Conclusions Here we demonstrated that intestinal C. parapsilosis isolates from RTT subjects hold phenotypic traits that might favour the previously observed low-grade intestinal inflammatory status associated with RTT. Therefore, the presence of putative virulent, pro-inflammatory C. parapsilosis strains in RTT could represent an additional factor in RTT’s gastrointestinal pathophysiology, whose mechanisms are not yet clearly understood. Electronic supplementary material The online version of this article (10.1186/s12876-018-0785-z) contains supplementary material, which is available to authorized users.

Stenotrophomonas maltophilia SeITE02 and Ochrobactrum sp. MPV1 were isolated from the rhizosphere soil of the selenium-hyperaccumulator legume Astragalus bisulcatus and waste material from a dumping site for roasted pyrites, respectively. Here, these bacterial strains were studied as cell factories to generate selenium-nanostructures (SeNS) under metabolically controlled growth conditions. Thus, a defined medium (DM) containing either glucose or pyruvate as carbon and energy source along with selenite (SeO23−) was tested to evaluate bacterial growth, oxyanion bioconversion and changes occurring in SeNS features with respect to those generated by these strains grown on rich media. Transmission electron microscopy (TEM) images show extra- or intra-cellular emergence of SeNS in SeITE02 or MPV1 respectively, revealing the presence of two distinct biological routes of SeNS biogenesis. Indeed, the stress exerted by SeO23− upon SeITE02 cells triggered the production of membrane vesicles (MVs), which surrounded Se-nanoparticles (SeNPsSeITE02−G_e and SeNPsSeITE02−P_e with average diameter of 179 ± 56 and 208 ± 60 nm, respectively), as highlighted by TEM and scanning electron microscopy (SEM), strongly suggesting that MVs might play a crucial role in the excreting mechanism of the SeNPs in the extracellular environment. On the other hand, MPV1 strain biosynthesized intracellular inclusions likely containing hydrophobic storage compounds and SeNPs (123 ± 32 nm) under pyruvate conditioning, while the growth on glucose as the only source of carbon and energy led to the production of a mixed population of intracellular SeNPs (118 ± 36 nm) and nanorods (SeNRs; average length of 324 ± 89). SEM, fluorescence spectroscopy, and confocal laser scanning microscopy (CLSM) revealed that the biogenic SeNS were enclosed in an organic material containing proteins and amphiphilic molecules, possibly responsible for the high thermodynamic stability of these nanomaterials. Finally, the biogenic SeNS extracts were photoluminescent upon excitation ranging from 380 to 530 nm, whose degree of fluorescence emission (λem = 416–640 nm) was comparable to that from chemically synthesized SeNPs with L-cysteine (L-cys SeNPs). This study offers novel insights into the formation, localization, and release of biogenic SeNS generated by two different Gram-negative bacterial strains under aerobic and metabolically controlled growth conditions. The work strengthens the possibility of using these bacterial isolates as eco-friendly biocatalysts to produce high quality SeNS targeted to possible biomedical applications and other biotechnological purposes.

La betulla dell’Etna (Betula aetnensis Rafin), è una delle specie arboree che durante le ultime glaciazioni si è spinta verso sud e dove oggi costituisce popolazioni pressoché relitte localizzate esclusivamente in Sicilia sulla sommità dell’Etna. I betulleti sono presenti sui versanti orientali ed occidentali dell’Etna, ad un’altitudine compresa tra 1.400 e 2.000 m s.l.m. Si tratta di popolamenti pionieri che colonizzano substrati vulcanici inalterati e/o poco evoluti dove costituiscono a volte dei piccoli boschi puri o misti a pino laricio. Questi boschi in passato erano sottoposti a ceduazioni, che dal 1987 non sono state più eseguite a seguito della istituzione del Parco Regionale dell’Etna. Negli ultimi anni è stato osservato un progressivo declino della specie reso evidente dalla limitata presenza di rinnovazione naturale e dall’incremento di processi di deperimento a carico delle piante adulte. Molti popolamenti risultano infatti attaccati da patogeni fungini (ad esempio del genere Heterobasidion e Armillaria) che ne stanno causando la sparizione. In ragione del notevole interesse scientifico e biogeografico di Betula aetnensis, e considerando le crescenti minacce che la caratterizzano, è stata avviata una indagine multidisciplinare per studiare diversi aspetti. dell’ecologia della specie, ivi compreso l’entità ed il ruolo delle simbiosi micorriziche e tenendo in considerazione anche i rischi per la conservazione della Betulla, tra i quali i cambiamenti climatici. È noto, infatti, che i funghi micorrizici possono avere molteplici effetti positivi sulla crescita di specie minacciate, sulla produttività, e sul loro stato di salute. Tuttavia lo stato delle simbiosi micorriziche della Betulla dell’Etna, e della maggior parte delle specie minacciate, non è stato ad oggi valutato. Obiettivo del presente lavoro è stato quello di valutare il grado di deperimento dei betulleti, le interazioni della betulla con i funghi simbionti (endomicorrize ed ectomicorrize), e verificare se realmente i cambiamenti climatici incidano sulla crescita e la sopravvivenza della pianta. Le aree di saggio in cui sono presenti popolamenti puri di betulla o mista al pino laricio e in buone condizioni sono state confrontate con aree con evidenti effetti del declino. Il rilievo della rinnovazione in ciascuna area di studio ha evidenziato la totale mancanza di rinnovazione nei substrati più alterati confermando il ruolo pioniero della specie, Il campionamento dendrocronologico ha fornito informazioni sull’età delle piante, sulla dinamica degli accrescimenti e la sensibilità delle betulle ai fattori climatici quali le piogge e le temperature. Etna birch (Betula aetnensis Rafin) is one of the tree species that during the last glaciations has moved southward, where currently it establishes populations that can be considered relicts because they are present exclusively on the summit of Etna Mount. Etna birch stands are present on the eastern and western slopes of Etna, at an altitude between 1,400 and 2,000 m a.s.l.. These are pioneer populations that colonize unaltered and/or poorly developed volcanic substrates where they establishes small, pure or mixed stands together with Pinus nigra subsp. calabrica (Loud.) A.E. Murray. In the past, such woods were subject to coppice management, but only until 1987, following the establishment of the Etna Regional Park. In recent years, a progressive decline in Etna birch natural populations has been observed, resulting evident from the limited occurrence of natural regeneration and from the increase in decay processes of adult plants. Many natural populations are attacked by fungal pathogens (for example of the genus Heterobasidion and Armillaria) that are causing their disappearance. Due to the remarkable scientific and biogeographical interest of Betula aetnensis, and considering the growing threats that characterize it, a multidisciplinary investigation has been launched to study various aspects of the ecology of the species, including the extent and role of mycorrhizal symbioses, and taking into account also the risks for the conservation of the species, among which also climatic change. In fact, it is well known that mycorrhizal fungi can have multiple positive effects on the growth of endangered species, on their productivity, as well as on plant health. However, up to now the mycorrhizal status of Etna birch, and that of most of threatened species, has not yet been evaluated. The aim of the present work is to evaluate the degree of decay of Betula aetnensis, its interactions with symbiotic fungi (endomycorrhizae and ectomycorrhizae), and to verify whether or not climatic change actually affects the plant growth and survival. The study areas in which pure birch or mixed stands with Calabrian pine are in good conditions have been compared to areas with clear effects of decay. The survey of the regeneration in each study area showed the total lack of regeneration in the most altered substrates, confirming the strong pioneer role of the species. The dendrochronological sampling provided information on plant age, growth dynamics and the sensitivity of birches to climate factors such as rainfall and temperatures.

Background Mycobacterium tuberculosis (MTB), the aetiological agent of tuberculosis (TB), is capable of interfering with the phagosome maturation pathway, by inhibiting phagosome-lysosome fusion and the autophagic process to ensure survival and replication in macrophages. Thus, it has been proposed that the modulation of autophagy may represent a therapeutic approach to reduce MTB viability by enhancing its clearance. Objective The aim of this study was to investigate whether transglutaminase type 2 (TG2) is involved in the pathogenesis of MTB. Results We have shown that either genetic or pharmacological inhibition of TG2 leads to a marked reduction in MTB replicative capacity. Infection of TG2 knockout mice demonstrated that TG2 is required for MTB intracellular survival in macrophages and host tissues. The same inhibitory effect can be reproduced in vitro using Z-DON, a specific inhibitor of the transamidating activity of TG2. Massive cell death observed in macrophages that properly express TG2 is hampered by the absence of the enzyme and can be largely reduced by the treatment of wild-type macrophages with the TG2 inhibitor. Our data suggest that reduced MTB replication in cells lacking TG2 is due to the impairment of LC3/autophagy homeostasis. Finally, we have shown that treatment of MTB-infected murine and human primary macrophages with cystamine, a TG2 inhibitor already tested in clinical studies, causes a reduction in intracellular colony-forming units in human macrophages similar to that achieved by the anti-TB drug capreomycin. Conclusion These results suggest that inhibition of TG2 activity is a potential novel approach for the treatment of TB.

Objectives Pseudomonas aeruginosa is a major cause of severe healthcare-associated infections and often shows MDR phenotypes. Ceftolozane/tazobactam is a new cephalosporin/β-lactamase inhibitor combination with potent activity against P. aeruginosa. This survey was carried out to evaluate the susceptibility of P. aeruginosa, circulating in Italy, to ceftolozane/tazobactam and comparators and to investigate the molecular epidemiology of carbapenemase-producing strains. Methods Consecutive non-replicate P. aeruginosa clinical isolates (935) from bloodstream infections and lower respiratory tract infections were collected from 20 centres distributed across Italy from September 2013 to November 2014. Antimicrobial susceptibility testing was performed by broth microdilution and results were interpreted according to the EUCAST breakpoints. Isolates resistant to ceftolozane/tazobactam were investigated for carbapenemase genes by PCR, and for carbapenemase activity by spectrophotometric assay. WGS using an Illumina platform was performed on carbapenemase-producing isolates. Results Ceftolozane/tazobactam was the most active molecule, retaining activity against 90.9% of P. aeruginosa isolates, followed by amikacin (88.0% susceptibility) and colistin (84.7% susceptibility). Overall, 48 isolates (5.1%) were positive for carbapenemase genes, including blaVIM (n = 32), blaIMP (n = 12) and blaGES-5 (n = 4), while the remaining ceftolozane/tazobactam-resistant isolates tested negative for carbapenemase production. Carbapenemase producers belonged to 10 different STs, with ST175 (n = 12) and ST621 (n = 11) being the most common lineages. Genome analysis revealed different trajectories of spread for the different carbapenemase genes. Conclusions Ceftolozane/tazobactam exhibited potent in vitro activity against P. aeruginosa causing invasive infections in Italy. Carbapenemase production was the most common mechanism of resistance to ceftolozane/tazobactam.

Bioremediation, that uses microorganisms to remove environmental pollutants, is the best way of restoring the environment due to its low cost and sustainability. Immobilization of microorganisms capable of degrading specific contaminants significantly promotes bioremediation processes. An innovative ready to use bioremediation system to clean up oil-contaminated water was developed immobilizing highly performant marine and soil HC degrading bacteria, on biodegradable oil-absorbing carriers. Two soil Actinobacteria (Gordonia sp. SoCg, Nocardia sp. SoB) and two marine Gammaproteobacteria (Alcanivorax sp. SK2, Oleibacter sp.5), were immobilized on biopolymeric membranes prepared by electrospinning (polylactic acid, PLA and polycaprolactone, PCL). These carriers are characterized by high uptake capacity, oil retention, buoyancy, durability, reusability and recoverability of the oil absorbed. The morphology of the carriers and microbial adhesion and proliferation were evaluated using scanning electron microscopy (SEM). A high capacity of adhesion and proliferation of bacterial cells was observed on membranes after 5 days. The bioremediation efficiency of the carrier-bacteria systems was tested on crude oil by GC-FID analysis and compared whit planktonic cells. The bacterial immobilization on PLA and PCL membranes was a promoting factor for biodegradation, increasing hydrocarbon removal up to 20%, in respect to planktonic cells. Biofilm-mediated bioremediation is a versatile tool to be developed for in situ and ex situ bioremediation of aquatic systems. Several applications can be designed to exploit both the high oil uptake capacity of the carriers, and the biodegradation potential of autochtonous microrganisms and/or of selected microorganisms that are immobilized on the carriers before exposure to the contaminated site.

Enterococcus faecium E35048, a bloodstream isolate from Italy, was the first strain where the oxazolidinone resistance gene optrA was detected outside China. The strain was also positive for the oxazolidinone resistance gene cfr. WGS analysis revealed that the two genes were linked (23.1 kb apart), being co-carried by a 41,816-bp plasmid that was named pE35048-oc. This plasmid also carried the macrolide resistance gene erm(B) and a backbone related to that of the well-known Enterococcus faecalis plasmid pRE25 (identity 96%, coverage 65%). The optrA gene context was original, optrA being part of a composite transposon, named Tn6628, which was integrated into the gene encoding for the ζ toxin protein (orf19 of pRE25). The cfr gene was flanked by two ISEnfa5 insertion sequences and the element was inserted into an lnu(E) gene. Both optrA and cfr contexts were excisable. pE35048-oc could not be transferred to enterococcal recipients by conjugation or transformation. A plasmid-cured derivative of E. faecium E35048 was obtained following growth at 42°C, and the complete loss of pE35048-oc was confirmed by WGS. pE35048-oc exhibited some similarity but also notable differences from pEF12-0805, a recently described enterococcal plasmid from human E. faecium also co-carrying optrA and cfr; conversely it was completely unrelated to other optrA- and cfr-carrying plasmids from Staphylococcus sciuri. The optrA-cfr linkage is a matter of concern since it could herald the possibility of a co-spread of the two genes, both involved in resistance to last resort agents such as the oxazolidinones.

Staphylococcus aureus is the major cause of foodborne diseases worldwide. In this retrospective study, 84 S. aureus strains were characterized. The collection comprises 78 strains isolated during 1998 and 2014 from dairy products and tissue samples from livestock bred for dairy production in Sicily. One isolate was obtained from a pet (dog), one from an exotic animal (a circus elephant), and four human isolates were obtained during a severe food poisoning outbreak that occurred in Sicily in 2015. All the strains were characterized by pulsed-field gel electrophoresis (PFGE), for antibiotic resistance and presence of toxin genes. PFGE results showed 10 different pulsotypes, with three relatively frequent and three unique. The antibiotic resistance profiling showed that penicillin G (35.7%) and tetracycline (20.2%) resistance is largely spread. Most isolates contained at least one toxin gene making them a potential threat for public health. Enterotoxin sec gene was observed in 28.6% and seg in 23.8% of the strains, respectively; the human isolates were the only ones to concurrently harbor both seg and sei genes. In addition, 24 isolates were randomly selected and analyzed by multilocus sequence typing. Interestingly, the analysis showed the presence of 12 sequence types (STs), of which 6 were novel. One of them, ST700, was detected in 29% of the isolates and was found to be spread throughout Sicily. ST700 has been present in the island for almost 16 years (1998–2014) and it shows no host preference since it was isolated from different ruminant species. Four human isolates shared both the pulsotype (PT10) and the sequence type (ST9), as well as the virulence genes (seg-sei); this observation suggests that the isolates originated from a single clone, although they were obtained from two different individuals.

Nel progetto la costruzione di modelli di sviluppo aziendale che adottano tecniche e misure di adattamento capaci di preparare le aree più a rischio.

Freshwater ecosystems represent 0.01% of the water on Earth, but they support 6% of global biodiversity that is still mostly uncharacterized. Here, we describe the genome sequences of three strains belonging to novel species in the Pseudomonas , Flavobacterium , and Sediminibacterium genera recovered from a water sample of Lake Garda, Italy.

Soil inoculation with cyanobacteria (cyanobacterization) is a biotechnological method widely studied to improve soil quality and productivity. During their growth on soil, cyanobacteria excrete exopolysaccharides (EPSs) which glue trichomes to soil particles, in a three-dimensional extracellular polymeric matrix. EPS productivity is an important screening parameter to select proficient inoculants and is affected by growth conditions and abiotic stresses. In this study, we evaluated the capability of the cyanobacterium Schizothrix cf. delicatissima AMPL0116 to form biocrusts when inoculated in sand microcosms under stressing conditions, and the characteristics of the synthesized polymeric matrix. In parallel, we evaluated the characteristics of exopolysaccharidic exudates of the strain when grown in liquid culture, under optimal growth setting. Our results pointed out at significant differences of the exopolymers produced in the two conditions in terms of monosaccharidic composition and molecular weight distribution, and proved the capability of S. cf. delicatissima AMPL0116 to form stable bioaggregates on sandy soils.

Soil inoculation with cyanobacteria to promote the formation of biocrusts is considered a potential eco-friendly method to counteract desertification spread in drylands. Research is needed to increase the number of proficient cyanobacterial strains, selected for their capability to survive in harsh conditions and to form stable biocrusts quickly. We hereby present a microcosm study to assess the capability of Leptolyngbya ohadii, native to the Negev Desert, to form biocrusts on sand collected in the same environment, during a three-month incubation period. Inoculation was carried out in sand-filled microcosms without nutrient addition and a limited water supply (equivalent to desert dew input). Parameters related to biocrusts growth and to their physico-chemical attributes were measured, and the exopolysaccharides (EPS) synthesized by the strain during biocrust formation were quantified and characterized. After 15 days of incubation, L. ohadii was able to form biocrusts with a thickness and a physical stability superior to other test strains of cyanobacteria, and typical of much older natural biocrusts. Biocrust characteristics were dependent on the synthesis of EPS, and on the capability to migrate in the sand, stabilizing sand aggregates at different locations within the microcosms. In contrast to other tested strains, L. ohadii produced compositionally complex EPS during the entire incubation period despite the lack of nutrients, producing biocrusts with an amphiphilic extracellular matrix, a character effective in conferring stability to sand aggregates, chelating nutrients and maintaining hydration. Overall, this study shows that L. ohadii is a promising inoculant that may be considered to promote the formation of biocrusts in natural desert settings.

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Streptomyces coelicolor is a Gram-positive microorganism often used as a model of physiological and morphological differentiation in streptomycetes, prolific producers of secondary metabolites with important biological activities. In the present study, we analysed Streptomyces coelicolor growth and differentiation in the presence of the hypo-methylating agent 5′-aza-2′-deoxycytidine (5-aza-dC) in order to investigate whether cytosine methylation has a role in differentiation. We found that cytosine demethylation caused a delay in spore germination, aerial mycelium development, sporulation, as well as a massive impairment of actinorhodin production. Thus, we searched for putative DNA methyltransferase genes in the genome and constructed a mutant of the SCO1731 gene. The analysis of the SCO1731::Tn5062 mutant strain demonstrated that inactivation of SCO1731 leads to a strong decrease of cytosine methylation and almost to the same phenotype obtained after 5-aza-dC treatment. Altogether, our data demonstrate that cytosine methylation influences morphological differentiation and actinorhodin production in S. coelicolor and expand our knowledge on this model bacterial system.

This chapter was inadvertently published with an incorrect spelling of the author's name as V. Arriza whereas it should be V. Arizza. In addition to this the affiliation of one of the chapter authors Elisse Sutton was published incorrectly and it has now been corrected to read as Department of Biological Sciences, Macquarie University, Sydney, NSW, Australia.

Naphthenic acids (NAs) are an important group of toxic organic compounds naturally occurring in hydrocarbon deposits. This work shows that Rhodococcus aetherivorans BCP1 cells not only utilize a mixture of eight different NAs (8XNAs) for growth but they are also capable of marked degradation of two model NAs, cyclohexanecarboxylic acid (CHCA) and cyclopentanecarboxylic acid (CPCA) when supplied at concentrations from 50 to 500 mgL−1 . The growth curves of BCP1 on 8XNAs, CHCA, and CPCA showed an initial lag phase not present in growth on glucose, which presumably was related to the toxic effects of NAs on the cell membrane permeability. BCP1 cell adaptation responses that allowed survival on NAs included changes in cell morphology, production of intracellular bodies and changes in fatty acid composition. Transmission electron microscopy (TEM) analysis of BCP1 cells grown on CHCA or CPCA showed a slight reduction in the cell size, the production of EPS-like material and intracellular electrontransparent and electron-dense inclusion bodies. The electron-transparent inclusions increased in the amount and size in NA-grown BCP1 cells under nitrogen limiting conditions and contained storage lipids as suggested by cell staining with the lipophilic Nile Blue A dye. Lipidomic analyses revealed significant changes with increases of methyl-branched (MBFA) and polyunsaturated fatty acids (PUFA) examining the fatty acid composition of NAs-growing BCP1 cells. PUFA biosynthesis is not usual in bacteria and, together with MBFA, can influence structural and functional processes with resulting effects on cell vitality. Finally, through the use of RT (Reverse Transcription)- qPCR, a gene cluster (chcpca) was found to be transcriptionally induced during the growth on CHCA and CPCA. Based on the expression and bioinformatics results, the predicted products of the chcpca gene cluster are proposed to be involved in aerobic NA degradation in R. aetherivorans BCP1. This study provides first insights into the genetic and metabolic mechanisms allowing a Rhodococcus strain to aerobically degrade NAs.&nbsp

The gellan hydrogel is used as a cleaning agent for paper artworks. The purposes of this work were to give the gellan hydrogel both a microbial resistance and a cytocidal action against the biodeteriogens using hydrolates. Broth microdilution assays were performed to identify the fungicidal action of 16 hydrolates vs 3 fungal strains. The study concerned the Minimal Fungicidal Concentration and pH, colour, and ultra-structural variations on all paper samples, by placing them in contact with GELYD (GELlan + hYDrolat). The in vitro tests showed that eight out of 16 hydrolates strongly inhibited fungal growth, but only two of them (Monarda fistulosa, Citrus aurantium subsp amara) showed in situ fungicidal action. Data indicate a new and interesting eco-innovative use of hydrolates, since GELYD is able not only to clean paper artworks, but also to kill the fungal cells present on paper that are potentially harmful for paper and operators.

Breast microbiota compositions are not well understood, and a few recent reports have begun to explore the correlation between breast tissue dysbiosis and cancer. Given that various methods for breast microbiota detection were used, the aim of the present paper was to clarify which hypervariable region of the 16S-rRNA gene (V2, V3, V4, V6 + 7, V8, and V9) is the most informative for breast tissue microbiota. Core needle biopsies (CNBs) were compared with surgical excision biopsies (SEBs) to find a less invasive form of recovery useful for the analysis of a larger statistical population and potentially for diagnostic use of breast tissue microbiota. Finally, this study was the first to analyse the breast microbiota of tumours and paired normal tissues of a Mediterranean population. Our findings showed that the V3 region is the most informative for breast tissue microbiota, accounting for 45% of all reads. No significant differences were found between CNB and SEB specimens in terms of total reads and numbers of Operational Taxonomic Units (OTUs). Moreover, we find that more similarities than differences exist between tumours and adjacent normal tissues. Finally, the presence of the Ralstonia genus is associated with breast tissue.

Objectives To characterize a novel phenicol-oxazolidinone-tetracycline resistance gene, named poxtA, identified in a previously described MRSA strain that was highly resistant to linezolid and also carried the cfr gene. Methods The poxtA gene was identified by bioinformatic analysis of the whole genome sequence of Staphylococcus aureus AOUC-0915. The poxtA gene was cloned in a shuttle plasmid vector and expressed in Escherichia coli, S. aureus and Enterococcus faecalis to investigate the protein function. Comparative sequence analyses at the protein and genetic levels were carried out using standard procedures. Results The poxtA gene encodes a protein that is 32% identical to OptrA and exhibits structural features typical of the F lineage of the ATP-binding cassette (ABC) protein superfamily that cause antibiotic resistance by ribosomal protection. Expression of poxtA in E. coli, S. aureus and E. faecalis was able to decrease susceptibility to phenicols, oxazolidinones and tetracyclines. A database search identified the presence of poxtA in E. faecalis, Enterococcus faecium and Pediococcus acidilactici strains, mostly of animal origin, and revealed the presence of poxtA homologues in the genomes of some Clostridiales. Analysis of the genetic context revealed that poxtA was located in a composite transposon-like structure containing two IS1216 elements. Conclusions A novel resistance gene, named poxtA, encoding a protein of the antibiotic resistance (ARE) ABC-F lineage, was identified in the genome of an MRSA of clinical origin. PoxtA can confer decreased susceptibility to phenicols, oxazolidinones and tetracyclines and is associated with a putative mobile element that could contribute to its horizontal dissemination.

Background Klebsiella oxytoca DSM 29614 - isolated from acid mine drainages - grows anaerobically using Fe(III)-citrate as sole carbon and energy source, unlike other enterobacteria and K. oxytoca clinical isolates. The DSM 29614 strain is multi metal resistant and produces metal nanoparticles that are embedded in its very peculiar capsular exopolysaccharide. These metal nanoparticles were effective as antimicrobial and anticancer compounds, chemical catalysts and nano-fertilizers. Results The DSM 29614 strain genome was sequenced and analysed by a combination of in silico procedures. Comparative genomics, performed between 85 K. oxytoca representatives and K. oxytoca DSM 29614, revealed that this bacterial group has an open pangenome, characterized by a very small core genome (1009 genes, about 2%), a high fraction of unique (43,808 genes, about 87%) and accessory genes (5559 genes, about 11%). Proteins belonging to COG categories “Carbohydrate transport and metabolism” (G), “Amino acid transport and metabolism” (E), “Coenzyme transport and metabolism” (H), “Inorganic ion transport and metabolism” (P), and “membrane biogenesis-related proteins” (M) are particularly abundant in the predicted proteome of DSM 29614 strain. The results of a protein functional enrichment analysis - based on a previous proteomic analysis – revealed metabolic optimization during Fe(III)-citrate anaerobic utilization. In this growth condition, the observed high levels of Fe(II) may be due to different flavin metal reductases and siderophores as inferred form genome analysis. The presence of genes responsible for the synthesis of exopolysaccharide and for the tolerance to heavy metals was highlighted too. The inferred genomic insights were confirmed by a set of phenotypic tests showing specific metabolic capability in terms of i) Fe2+ and exopolysaccharide production and ii) phosphatase activity involved in precipitation of metal ion-phosphate salts. Conclusion The K. oxytoca DSM 29614 unique capabilities of using Fe(III)-citrate as sole carbon and energy source in anaerobiosis and tolerating diverse metals coincides with the presence at the genomic level of specific genes that can support i) energy metabolism optimization, ii) cell protection by the biosynthesis of a peculiar exopolysaccharide armour entrapping metal ions and iii) general and metal-specific detoxifying activities by different proteins and metabolites. Electronic supplementary material The online version of this article (10.1186/s12866-018-1330-5) contains supplementary material, which is available to authorized users.

Human cytomegalovirus (HCMV) is a leading cause of severe disease in immunocompromised individuals, including AIDS and transplanted patients, and in congenitally infected newborns. Despite the availability of several drugs, pharmacological treatment of HCMV infections is associated with poor bioavailability, toxicity and the emergence of resistant strains. Furthermore, no vaccine is available and no drugs are approved to prevent vertical transmission during pregnancy, therefore, it is essential to identify new potential targets of therapeutic intervention. HCMV DNA polymerase accessory protein UL44 plays an essential role in viral replication, conferring processivity to the DNA polymerase catalytic subunit UL54 by tethering it to the DNA. Binding of UL44 to dsDNA occurs in the absence of ATP and clamp loaders, and depends on UL44 homodimerization. Indeed, our research group previously demonstrated that the protein can dimerize in cells and point mutations disrupting protein self-interaction also prevent DNA binding and abolish viral OriLyt-dependent DNA replication in transcomplementation assay. Therefore, disruption of UL44 homodimerization represents an attractive target for the development of new antivirals. Based on these observations, using the recently published crystal structure of UL44 hodimers our research group previously performed a virtual screening with the Glide software in combination with a library of 1.3 x 106 small molecules (SMs) to identify SMs potentially interfering with UL44 homodimerization. After three rounds of screening (HTVS: high-throughput virtual screening, SP: standard precision, XP: extra precision), followed by an in depth analysis of compounds chemical properties, 18 SMs were selected for further analysis. Selected compounds were obtained from a commercial supplier, to be tested in a variety of assays for their ability to inhibit UL44 homodimerization both in cell and in vitro, as well as on HCMV replication. For this purpose, we applied two in vitro methods to monitor the effect of our candidates on UL44 dimerization such GST-pulldown Assay and Thermal Shift Assay (TSA). Furthermore, Plaque Reduction Assay (PRA) and Fluorescence Reduction Assay (FRA) were performed to study their inhibitory effects on viral replication. Initially we validated that GST-pulldown assay was suitable to study UL44 dimerization and its perturbation caused by SMs. Therefore, we performed a first screening to test the effect of the 18 SMs on UL44 dimerization, and we identified 3 of these which reproducibly inhibited the dimerization. Prompted by these results, we selected such SMs to investigate a possible dose-response relationship between the dimerization inhibition and the compounds concentration. Unfortunately, data analysis revealed a high variability, and lack of correlation between the inhibition and the concentration of SMs used in the assays. In parallel, PRAs were performed to validate the effect of selected SMs, but these were unable to inhibit viral replication with high potency. Subsequently, two recombinant HCMV viruses (TB4-IE2-EYFP and TB4-UL83-EYFP) were obtained from Michael Winkler (Leibniz-Institut für Primatenforschung Goettingen, Germany) in order to study the effect of our molecules on HCMV replication by FRAs. Therefore, after a screening of the 18 SMs, 4 of these were identified as possible inhibitors of viral replication and were selected to further studies. We investigated their and 50% effective dose (ED50) and 50% cytotoxic concentration (CC50) values and their effects on the viral gene expression. Only 2 SMs reproducibly inhibited expression of Early and Late genes. Then we evaluated an alternative in vitro assay and among the various possibilities, we have chosen TSA that is able to inform whether a SM induce the disruption of constitutive oligomeric interfaces. In a first moment we validated that the assay allow the discrimination between monomeric and dimeric forms of UL44, suggesting its potential for the screening of our SMs. As result of the SMs screening by TSA, one molecule revealed a possible inhibition of dimerization.

We have recently shown that Pseudomonas aeruginosa, an opportunistic pathogen that chronically infects the lungs of patients with cystic fibrosis (CF) and other forms of lung disease, is extremely efficient in recruiting zinc from the environment and that this capability is required for its ability to cause acute lung infections in mice. To verify that P. aeruginosa faces zinc shortage when colonizing the lungs of human patients, we analyzed the expression of three genes that are highly induced under conditions of zinc deficiency (zrmA, dksA2 and rpmE2), in bacteria in the sputum of patients with inflammatory lung disease. All three genes were expressed in all the analyzed sputum samples to a level much higher than that of bacteria grown in zinc-containing laboratory medium, supporting the hypothesis that P. aeruginosa is under zinc starvation during lung infections. We also found that the expression of several virulence traits that play a central role in the ability of P. aeruginosa to colonize the lung is affected by disruption of the most important zinc importing systems. Virulence features dependent on zinc intake include swarming and swimming motility and the ability to form biofilms. Furthermore, alterations in zinc assimilation interfere with the synthesis of the siderophore pyoverdine, suggesting that zinc recruitment could modulate iron uptake and affect siderophore-mediated cell signaling. Our results reveal that zinc uptake is likely to play a key role in the ability of P. aeruginosa to cause chronic lung infections and strongly modulates critical virulence traits of the pathogen. Taking into account the recent discovery that zinc uptake in P. aeruginosa is promoted by the release of a small molecular weight molecule showing high affinity for zinc, our data suggest novel and effective possibilities to control lung infections by these bacteria.

Neonatal hypoxic ischemic encephalopathy (HIE) in the perinatal period can lead to significant neurological deficits in later life. Total body cooling (TBC) is a neuroprotective strategy used in the treatment of HIE and has been shown to reduce seizures and improve neurodevelopmental outcomes in treated infants. Little is known, however, about the effects of HIE/TBC on the developing gut microbiota composition and subsequent metabolic profile. Ten term infants with HIE who received TBC at 33.5°C for 72h were recruited. A control group consisted of nine healthy full term infants. Faecal samples were collected from both groups at 2 years of age and stored at -20°C. 16S rRNA amplicon Illumina sequencing was carried out to determine gut microbiota composition and 1H NMR analysis was performed to determine the metabolic profile of faecal water. The gut microbiota composition of the HIE/TBC infants were found to have significantly lower proportions of Bacteroides compared to the non-cooled healthy control group. Alpha diversity measures detected significantly lower diversity in microbial richness in the HIE/TBC infant group compared to the control infants (Shannon index

In this study the functional status of bacterial biomass within a vertical subsurface flow (VSSF) constructed wetland was examined with the aim to understand the relationship between viable and dead bacteria in soil and influent/effluent wastewater and elucidate the large amount of dead cells in the soil which may affect the long-term behavior of the system. The quantification of viable and dead bacteria in influent and effluent wastewater and in the soil of a VSSF was performed at single-cell level by flow cytometry (FCM). An optimised pre-treatment was applied to soil samples using sodium pyrophosphate and ultrasonication at a specific energy of 80 kJ/L. Viable and dead cells were detected on the basis of cellular membrane integrity coupling SYBR-Green I and Propidium Iodide. The bacteria profile in the VSSF soil depends on the depth and the material grain size. In the upper 0–10 cm sand layer the number of total bacteria per gram of dry weight (DW) was higher (1.82 × 10 9 cells/gDW) than in the deeper 40–50 cm (4.8 × 10 8 cells/gDW) probably due to the vertical feeding and a sieving effect of influent in the top layers. Bacterial biomass in the entire VSSF depth was 0.082 mgVSS/gDW or 144 gVSS/m 3 (per cubic meter of VSSF bed). Size of viable bacteria in the VSSF was smaller (0.16 μm 3 /cell) than typical size of activated sludge (0.23 μm 3 /cell), due to lower nutrient conditions and a longer retention time of viable bacteria in the bed, estimated at around 130 days by mass balance. Dead bacteria were prevalent in the VSSF soil with a viable/dead bacteria ratio (V/D) of 0.52. The content of dead bacteria might be higher in the soil due to the presence of unsaturated zones not reached by fresh influent wastewater (“dead-zones”), where moisture and substrate are not so available and bacteria may die. Conversely, the higher V/D ratio (3.3) in the effluent reflects the enrichment of wastewater with viable bacteria during the passage through the VSSF bed and along preferential water flow, with higher water content and substrate availability, where the bacterial growth is favored.

Forty rhizobial strains were isolated from root nodules of Medicago littoralis Rhode and Melilotus indicus (L.) harvested from the sandy soils of Touggourt’s oases in the Oued Righ Valley, Algerian Sahara. The isolates were studied for their cultural, biochemical and symbiotic effectiveness. All of them were fast-growing bacteria; utilized a wide range of carbon sources, produced abundant extracellular polysaccharides, tolerated high concentrations of NaCl (up to 2.5 %), grew at temperatures between 28 and 45 °C and at pH values between 4.5 and 9. The isolates were sensitive to the antibiotics kanamycin, tetracycline and rifampicin but showed resistance to neomycin and erythromycin. All the isolates induced the formation of effective nodules on their host plants. On the basis of the physiological, biochemical and symbiotic effectiveness, we selected six strains MD05, MD09, MD12, ML08, ML17 and ML22 for genotypic characterization. Phylogenetic analysis of the selected strains based on 16S ribosomal RNA gene showed that these strains of bacteria were affiliated to the Ensifer meliloti group.

Arbuscular mycorrhizal fungi (AMF) may promote plant invasion by enhancing plant performance and competitiveness. However, only a small number of studies have considered the interactions between local soil microbial communities and invasive plants, and even fewer have focused on alien trees. Ailanthus altissima is a serious problem in the Mediterranean Basin, where it has invaded many habitats. We investigated the symbiosis between A. altissima and indigenous AMF in two invaded, ecologically different Mediterranean woodlands. Mycorrhizal infection was high at both sites (> 60% of the root fragments were mycorrhizal), indicating that A. altissima roots may be infected by AMF under different ecological conditions. In a greenhouse experiment, A. altissima seedling growth was evaluated in untreated soil (natural soil containing the entire soil microbial community), in sterilized soil, and in sterilized soil containing the entire microbial community except AMF. Seedling growth was greater in the natural soil than in the other two soils. Seedlings in natural soil but not in the other two soils were extensively colonized by AMF, indicating that AMF may have been responsible for the greater growth in the natural soil. Growth was poorer in the sterile soil that had been inoculated with a soil suspension lacking AMF than in the sterile soil, suggesting that the natural soil also contained pathogens and that the AMF countered the effects of these pathogens. Overall, the results indicate that AMF are an important component of Mediterranean woodlands invaded by A. altissima and AMF may facilitate A. altissima's invasion in these woodlands.

The aim of the present study is to investigate the efficiency of the combined pulsed electric fields and high pressure carbon dioxide (PEF + HPCD) treatment on the Gram-negative pathogen Salmonella Typhimurium in a liquid medium, by means of both plate count technique and flow cytometry (FCM). PEF was applied at two conditions: (1) 1 single pulse of 1 ms length at 30 kV/cm and (2) 12 pulses of 4 ms length at 30 kV/cm, while HPCD at 12 MPa, 22 °C and 35 °C for different treating times (0–45 min). At both temperatures, the application of PEF as HPCD pre-treatment was demonstrated to enhance the inactivation kinetics and to decrease the treatment time to inactivate S. Typhimurium if compared to HPCD alone. Further, the approach based on FCM permitted to investigate the functional status of bacterial cells after PEF and HPCD treatments distinguishing among viable bacteria (considered as intact cells), permeabilised cells and depolarised cells simultaneously. It has been demonstrated that the synergistic effect is due to the electroporation effect of PEF which lead to changes in the cell membrane potential but also in a partial structural damage, favoring the subsequent CO2 penetration into the cells and increasing the inactivation kinetics, thus improving the efficiency of the entire process.

BACKGROUND The modulation of gut microbiota is considered to be the first target to establish probiotic efficacy in a healthy population. OBJECTIVE This study was conducted to determine the impact of a probiotic on the intestinal microbial ecology of healthy volunteers. METHODS High-throughput 16S ribosomal RNA gene sequencing was used to characterize the fecal microbiota in healthy adults (23-55 y old) of both sexes, before and after 4 wk of daily consumption of a capsule containing at least 24 billion viable Lactobacillus paracasei DG cells, according to a randomized, double-blind, crossover placebo-controlled design. RESULTS Probiotic intake induced an increase in Proteobacteria (P = 0.006) and in the Clostridiales genus Coprococcus (P = 0.009), whereas the Clostridiales genus Blautia (P = 0.036) was decreased; a trend of reduction was also observed for Anaerostipes (P = 0.05) and Clostridium (P = 0.06). We also found that the probiotic effect depended on the initial butyrate concentration. In fact, participants with butyrate >100 mmol/kg of wet feces had a mean butyrate reduction of 49 ± 21% and a concomitant decrease in the sum of 6 Clostridiales genera, namely Faecalibacterium, Blautia, Anaerostipes, Pseudobutyrivibrio, Clostridium, and Butyrivibrio (P = 0.021), after the probiotic intervention. In contrast, in participants with initial butyrate concentrations

Volcanic and geothermal systems emit endogenous gases by widespread degassing from soils, including CH4, a greenhouse gas twenty-five times as potent as CO2. Recently, it has been demonstrated that volcanic or geothermal soils are not only a source of methane, but are also sites of methanotrophic activity. Methanotrophs are able to consume 10–40 Tg of CH4 a−1 and to trap more than 50% of the methane degassing through the soils. We report on methane microbial oxidation in the geothermally most active site of Pantelleria (Italy), Favara Grande, whose total methane emission was previously estimated at about 2.5 Mg a−1 (t a−1). Laboratory incubation experiments with three top-soil samples from Favara Grande indicated methane consumption values of up to 59.2 nmol g−1 soil d.w. h−1. One of the three sites, FAV2, where the highest oxidation rate was detected, was further analysed on a vertical soil profile, the maximum methane consumption was measured in the top-soil layer, and values greater than 6.23 nmol g−1 h−1 were still detected up to a depth of 13 cm. The highest consumption rate was measured at 37 °C, but a still detectable consumption at 80 °C (> 1.25 nmol g−1 h−1) was recorded. The soil total DNA extracted from the three samples was probed by Polymerase Chain Reaction (PCR) using standard proteobacterial primers and newly designed verrucomicrobial primers, targeting the unique methane monooxygenase gene pmoA; the presence of methanotrophs was detected at sites FAV2 and FAV3, but not at FAV1, where harsher chemical–physical conditions and negligible methane oxidation were detected. The pmoA gene libraries from the most active site (FAV2) pointed to a high diversity of gammaproteobacterial methanotrophs, distantly related to Methylocaldum-Metylococcus genera, and the presence of the newly discovered acido-thermophilic Verrucomicrobia methanotrophs. Alphaproteobacteria of the genus Methylocystis were isolated from enrichment cultures under a methane-containing atmosphere at 37 °C. The isolates grow at a pH range of 3.5 to 8 and temperatures of 18–45 °C, and consume 160 nmol of CH4 h−1 mL−1 of culture. Soils from Favara Grande showed the largest diversity of methanotrophic bacteria detected until now in a geothermal soil. While methanotrophic Verrucomicrobia are reported as dominating highly acidic geothermal sites, our results suggest that slightly acidic soils, in high-enthalpy geothermal systems, host a more diverse group of both culturable and uncultivated methanotrophs.