Carlo Viti

INO - Istituto Nazionale di Ottica, Florens, Tuscany, Italy

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Publications (65)157.69 Total impact

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    ABSTRACT: Exposure to biocides may result in cross-resistance to other antimicrobials. Changes in biocide and antibiotic susceptibilities, metabolism, and fitness costs are studied in biocide-selected Escherichia coli and Klebsiella pneumoniae mutants. E. coli and K. pneumoniae mutants with various degrees of triclosan susceptibility were obtained after exposure to triclosan (TRI), benzalkonium-chloride (BKC), chlorhexidine (CHX) or sodium hypochlorite (SHC), and ampicillin, or ciprofloxacin. Mutant alterations in antimicrobial susceptibility and metabolism were tested using Phenotype Microarrays. Expression of AcrAB pump and global regulators (SoxR, MarA, RamA) was measured by RT-PCR and fabI gene was sequenced. Fitness costs of resistance were assessed by relative growth rates comparison. Triclosan-resistant (TRI(R)) and triclosan-hypersusceptible (TRI(HS)) mutants of E. coli and K. pneumoniae were obtained after selection with biocides and/or antibiotics. E. coli TRI(R) mutants, including those with changes in fabI gene, or in the expression of acrB and marA, exhibited variable susceptibility to TRI, CHX and antibiotics. TRI(R) mutants with a high MICTRI value presented improved metabolism of carboxylic acids, amino acids, and carbohydrates. In K. pneumoniae TRI(R) mutants, resistance to one antimicrobial provoked hyper-susceptibility to another one(s). TRI(R) mutants had fitness costs, particularly in marA- (E. coli) or ramA-overexpressing (K. pneumoniae) mutants. TRI, BKC, and CIP exposure frequently yielded TRI(R) mutants exhibiting alterations in AraC-like global regulators (MarA, SoxR, RamA), AcrAB-TolC and/or FabI, and influencing antimicrobial susceptibility, fitness, and metabolism. These variable phenotypes suggest a trade-off of different selective processes shaping the evolution towards antibiotic-biocide resistance, and influencing other adaptive traits. Copyright © 2015, American Society for Microbiology. All Rights Reserved.
    Antimicrobial Agents and Chemotherapy 03/2015; 59(6). DOI:10.1128/AAC.00187-15 · 4.45 Impact Factor
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    ABSTRACT: There is a growing concern by regulatory authorities for the selection of antibiotic resistance caused by the use of biocidal products. We aimed to complete the detailed information on large surveys by investigating the relationship between biocide and antibiotic susceptibility profiles of a large number of Staphylococcus aureus isolates using four biocides and antibiotics commonly used in clinical practice. The minimal inhibitory concentration (MIC) for most clinically-relevant antibiotics was determined according to the standardized methodology for over 1600 clinical S. aureus isolates and compared to susceptibility profiles of benzalkonium chloride, chlorhexidine, triclosan, and sodium hypochlorite. The relationship between antibiotic and biocide susceptibility profiles was evaluated using non-linear correlations. The main outcome evidenced was an absence of any strong or moderate statistically significant correlation when susceptibilities of either triclosan or sodium hypochlorite were compared for any of the tested antibiotics. On the other hand, correlation coefficients for MICs of benzalkonium chloride and chlorhexidine were calculated above 0.4 for susceptibility to quinolones, beta-lactams, and also macrolides. Our data do not support any selective pressure for association between biocides and antibiotics resistance and furthermore do not allow for a defined risk evaluation for some of the compounds. Importantly, our data clearly indicate that there does not involve any risk of selection for antibiotic resistance for the compounds triclosan and sodium hypochlorite. These data hence infer that biocide selection for antibiotic resistance has had so far a less significant impact than feared.
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    ABSTRACT: We performed a longitudinal study (repeated observations of the same sample over time) to investigate both the composition and structure of temporal changes of bacterial community composition in soil mesocosms, subjected to three different treatments (water and 5 or 25 mg kg(-1) of dried soil Cd(2+)). By analogy with the pan genome concept, we identified a core bacteriome and an accessory bacteriome. Resident taxa were assigned to the core bacteriome, while occasional taxa were assigned to the accessory bacteriome. Core and accessory bacteriome represented roughly 35 and 50 % of the taxa detected, respectively, and were characterized by different taxonomic signatures from phylum to genus level while 15 % of the taxa were found to be unique to a particular sample. In particular, the core bacteriome was characterized by higher abundance of members of Planctomycetes, Actinobacteria, Verrucomicrobia and Acidobacteria, while the accessory bacteriome included more members of Firmicutes, Clamydiae and Proteobacteria, suggesting potentially different responses to environmental changes of members from these phyla. We conclude that the pan-bacteriome model may be a useful approach to gain insight for modeling bacterial community structure and inferring different abilities of bacteria taxa.
    Antonie van Leeuwenhoek 01/2015; 107(3). DOI:10.1007/s10482-014-0372-4 · 2.14 Impact Factor
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    ABSTRACT: In nature, biofilms are the most common form of bacterial growth. In biofilms, bacteria display coordinated behaviour to perform specific functions. Here, we investigated electrical signalling as a possible driver in biofilm sociobiology. Using a multi-electrode array system that enables high spatio-temporal resolution, we studied the electrical activity in two biofilm-forming strains and one non-biofilm-forming strain. The action potential rates monitored during biofilm-forming bacterial growth exhibited a one-peak maximum with a long tail, corresponding to the highest biofilm development. This peak was not observed for the non-biofilm-forming strain, demonstrating that the intensity of the electrical activity was not linearly related to the bacterial density, but was instead correlated with biofilm formation. Results obtained indicate that the analysis of the spatio-temporal electrical activity of bacteria during biofilm formation can open a new frontier in the study of the emergence of collective microbial behaviour.
    Journal of The Royal Society Interface 01/2015; 12(102). DOI:10.1098/rsif.2014.1036 · 3.86 Impact Factor
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    ABSTRACT: Standard protocols are available in order to apply Phenotype MicroArray (PM) technology to characterize different groups of microorganisms. Nevertheless, there is the need to pay attention to several crucial steps in order to obtain high-quality and reproducible data from PM, such as the choice of the Dye mix, the type and concentration of the carbon source in metabolic experiments, the use of a buffered medium. A systematic research of auxotrophies in strains to be tested should be carefully evaluated before starting with PM experiments. Detailed protocols to obtain defined and reproducible phenotypic profiles for bacteria and yeasts are shown. Moreover, the innovative software opm R packages and DuctApe suite for the analysis of kinetic data produced by PM and panphenome description are reported.
    Methods in Molecular Biology 01/2015; 1231:99-123. DOI:10.1007/978-1-4939-1720-4_7 · 1.29 Impact Factor
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    ABSTRACT: The aim of the study was to evaluate milk fatty acid (FA) profile, animal performance, and rumen microbial population in response to diets containing soybean oil supplemented or not with chestnut and quebracho tannins in dairy ewes. Eighteen Comisana ewes at 122 ± 6 d in milking were allotted into 3 experimental groups. Diets were characterized by chopped grass hay administered ad libitum and by 800 g/head and day of 3 experimental concentrates containing 84.5 g of soybean oil/kg of dry matter (DM) and 52.8 g/kg of DM of bentonite (control diet), chestnut tannin extract (CHT diet), or quebracho tannin extract (QUE diet). The trial lasted 4 wk. Milk yield was recorded daily, and milk composition and blood parameters were analyzed weekly. At the end of the experiment, samples of rumen fluid were collected to analyze pH, volatile fatty acid profile, and the relative proportions of Butyrivibrio fibrisolvens and Butyrivibrio proteoclasticus in the rumen microbial population. Hepatic functionality, milk yield, and gross composition were not affected by tannin extracts, whereas milk FA composition was characterized by significant changes in the concentration of linoleic acid (CHT +2.77% and QUE +9.23%), vaccenic acid (CHT +7.07% and QUE +13.88%), rumenic acid (CHT -1.88% and QUE +24.24%), stearic acid (CHT + 8.71% and QUE -11.45%), and saturated fatty acids (CHT -0.47% and QUE -3.38%). These differences were probably due to the ability of condensed versus hydrolyzable tannins to interfere with rumen microbial metabolism, as indirectly confirmed by changes in the relative proportions of B. fibrisolvens and B. proteoclasticus populations and by changes in the molar proportions of volatile fatty acids. The effect of the CHT diet on the milk FA profile and microbial species considered in this trial was intermediate between that of QUE and the control diet, suggesting a differential effect of condensed and hydrolyzable tannins on rumen microbes. Compared with control animals, the presence of B. fibrisolvens increased about 3 times in ewes fed CHT and about 5 times in animals fed QUE. In contrast, the abundance of B. proteoclasticus decreased about 5- and 15-fold in rumen liquor of ewes fed CHT and QUE diets, respectively. The use of soybean oil and a practical dose of QUE or CHT extract in the diet of dairy ewes can be an efficient strategy to improve the nutritional quality of milk. Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.
    Journal of Dairy Science 11/2014; 98(2). DOI:10.3168/jds.2014-8651 · 2.55 Impact Factor
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    ABSTRACT: Background The stoned olive pomace (SOP), which represents approximately 50% of the conversion process of olive in oil, is largely not-utilized and creates costs for its disposal and problems related to environmental impact. In-vitro trial experiments were employed to study the effect of feeds integrated with this bio-waste rich in polyphenols on rumen biohydrogenation, using sheep rumen liquor as inoculum.ResultsFatty acid (FA) analysis and polymerase chain reaction denaturing gradient gel electrophoresis (DGGE) approach aimed to microbial community characterization showed that including SOP in feeds at the level of 50 g/kg and 90 g/kg induced changes in FA profile and in microbial populations. The contemporary decrease of Butyrivibrio proteoclasticus and the accumulation of vaccenic acid was observed. A depression of Neisseria weaveri, Ruminobacter amylophilus and other unclassified bacteria related to members of the Lachnospiraceae and Pasteurellaceae families was detected, suggesting that these microbial groups may be involved in rumen biohydrogenation.Conclusions Supplementation of feeds with SOP changes rumen bacterial community, including bacteria responsible for vaccenic acid hydrogenation to stearic acid, thus modifying FA profile of rumen liquor. Hence, an use of SOP aimed to produce meat or dairy products enriched in functional lipids could be hypothesized.
    BMC Veterinary Research 11/2014; 10(1):271. DOI:10.1186/s12917-014-0271-y · 1.74 Impact Factor
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    ABSTRACT: Bacterial infections causing mastitis in sheep can result in severe economic losses for farmers. A large survey of milk samples from ewes with mastitis in Sardinia, Italy, indicated an increasing prevalence of Pseudomonas aeruginosa infections. It has been shown previously that during chronic, biofilm-associated infections P. aeruginosa populations diversify. We report the phenotypic and genomic characterisation of two clonal P. aeruginosa isolates (PSE305 and PSE306) from a mastitis infection outbreak, representing distinct colony morphology variants. In addition to pigment production, PSE305 and PSE306 differed in phenotypic characteristics including biofilm formation, utilisation of various carbon and nitrogen sources, twitching motility. We found higher levels of expression of genes associated with biofilm formation (pelB) and twitching motility (flgD) in PSE305, compared to the biofilm and twitching-defective PSE306. Comparative genomics analysis revealed single nucleotide polymorphisms (SNPs) and minor insertion/deletion variations between PSE305 and PSE306, including a SNP mutation in the pilP gene of PSE306. By introducing a wild-type pilP gene we were able to partially complement the defective twitching motility of PSE306. There were also three larger regions of difference between the two genomes, indicating genomic instability. Hence, we have demonstrated that P. aeruginosa population divergence can occur during an outbreak of mastitis, leading to significant variations in phenotype and genotype, and resembling the behaviour of P. aeruginosa during chronic biofilm-associated infections.
    Veterinary Microbiology 11/2014; 175(1). DOI:10.1016/j.vetmic.2014.11.011 · 2.73 Impact Factor
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    ABSTRACT: Flor yeasts of Saccharomyces cerevisiae have an innate diversity of FLO11 which codes for a highly hydrophobic and anionic cell-wall glycoprotein with a fundamental role in biofilm formation. In this study, 380 nitrogen compounds were administered to three S. cerevisiae flor strains handling FLO11 alleles with different expression levels. S. cerevisiae strain S288c was used as the reference strain as it cannot produce FLO11p. The flor strains generally metabolized amino acids and dipeptides as the sole nitrogen source, although with some exceptions regarding L-histidine and histidine containing dipeptides. L-histidine completely inhibited growth and its effect on viability was inversely related to FLO11 expression. Accordingly, L-histidine did not affect the viability of the Δflo11 and S288c strains. Also, L-histidine dramatically decreased air-liquid biofilm formation and adhesion to polystyrene of the flor yeasts with no effect on the transcription level of the FLO11 gene. Moreover, L-histidine modified the chitin and glycans content on the cell-wall of flor yeasts. These findings reveal a novel biological activity of L-histidine in controlling the multicellular behavior of yeasts.
    PLoS ONE 11/2014; 9(11):e112141. DOI:10.1371/journal.pone.0112141 · 3.53 Impact Factor
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    ABSTRACT: Efflux pumps are membrane proteins involved in the active extrusion of a wide range of structurally dissimilar substrates from cells. A multidrug efflux pump named TetA belonging to the major facilitator superfamily (MFS) of transporters was identified in the Streptococcus thermophilus DSM 20617T genome. The tetA-like gene was found in the genomes of a number of S. thermophilus strains sequenced to date and in S. macedonicus ACA-DC 198, suggesting a possible horizontal gene transfer event between these two Streptococcus species, which are both adapted to the milk environment. Flow cytometry (single-cell) analysis revealed bistable TetA activity in the S. thermophilus population, and tetA-like gene over-expression resulted in a reduced susceptibility to ethidium bromide, tetracycline, and other toxic compounds even when the efflux pump was over-expressed in a strain naturally lacking tetA-like gene.This article is protected by copyright. All rights reserved.
    FEMS Microbiology Letters 04/2014; 355(1). DOI:10.1111/1574-6968.12449 · 2.72 Impact Factor
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    ABSTRACT: Staphylococcus aureus strains harboring QacA, QacB, QacC, QacG transporters and norA promoter up-regulating mutations were characterized by Phenotype Microarray (PM), standard methods for susceptibility testing, and ethidium bromide efflux assays, in order to increase knowledge on phenotypes associated to efflux pumps and their substrates. PM data and standard susceptibility testing lead to the identification of new potential efflux targets, such as guanidine hydrochloride or 8-hydroxyquinoline for QacA and QacC pumps, respectively. The identification of compounds to which the presence of efflux pumps induced increased susceptibility opens new perspectives for potential adjunct anti-resistance treatment (i.e. strains bearing QacB transporters showed increased susceptibility to thioridazine, amitriptyline and orphenadrine). Although the tested isolates were characterized by high degree of heterogeneity, a hallmark of clinical isolates, direct ethidium bromide efflux assays were effective in highlighting differences in efflux efficiency among strains. These data add to characterization of substrate specificity in the different classes of staphylococcal multi drug efflux systems conferring specific substrate profiles and efflux features to each of them.
    Microbiological Research 01/2014; DOI:10.1016/j.micres.2014.07.001 · 1.94 Impact Factor
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    ABSTRACT: Hexavalent chromium [Cr(VI)] contamination is one of the main problems of environmental protection because the Cr(VI) is hazardous to human health. Cr(VI) form is highly toxic, mutagenic and carcinogenic, and it spreads widely beyond the site of initial contamination because of its mobility. Cr(VI), crossing the cellular membrane via the sulfate uptake pathway, generates active intermediates Cr(V) and/or Cr(IV), free radicals, and Cr(III) as the final product. Cr(III) affects DNA replication, causes mutagenesis, and alters the structure and activity of enzymes, reacting with their carboxyl and thiol groups. To persist in Cr(VI)-contaminated environments, microorganisms must have efficient systems to neutralize the negative effect of this form of chromium. The systems involve detoxification or repair strategies such as Cr(VI) efflux pumps, Cr(VI) reduction to Cr(III) and activation of enzymes involved in the ROS detoxifying processes, repair of DNA lesions, sulfur metabolism, and iron homeostasis. This review provides an overview of the processes involved in bacterial and fungal Cr(VI) resistance that have been identified through "omics" studies. A comparative analysis of the described molecular mechanisms is offered and compared to the cellular evidence obtained with classical microbiological approaches. This article is protected by copyright. All rights reserved.
    FEMS microbiology reviews 11/2013; 38(4). DOI:10.1111/1574-6976.12051 · 13.81 Impact Factor
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    ABSTRACT: The MICs and minimum bactericidal concentrations (MBCs) for the biocides benzalkonium chloride and chlorhexidine were determined against 1,602 clinical isolates of Staphylococcus aureus. Both compounds showed unimodal MIC and MBC distributions (2 and 4 or 8 mg/liter, respectively) with no apparent subpopulation with reduced susceptibility. To investigate further, all isolates were screened for qac genes, and 39 of these also had the promoter region of the NorA multidrug-resistant (MDR) efflux pump sequenced. The presence of qacA, qacB, qacC, and qacG genes increased the mode MIC, but not MBC, to benzalkonium chloride, while only qacA and qacB increased the chlorhexidine mode MIC. Isolates with a wild-type norA promoter or mutations in the norA promoter had similar biocide MIC distributions; notably, not all clinical isolates with norA mutations were resistant to fluoroquinolones. In vitro efflux mutants could be readily selected with ethidium bromide and acriflavine. Multiple passages were necessary to select mutants with biocides, but these mutants showed phenotypes comparable to those of mutants selected by dyes. All mutants showed changes in the promoter region of norA, but these were distinct from this region of the clinical isolates. Still, none of the in vitro mutants displayed fitness defects in a killing assay in Galleria mellonella larvae. In conclusion, our data provide an in-depth comparative overview on efflux in S. aureus mutants and clinical isolates, showing also that plasmid-encoded efflux pumps did not affect bactericidal activity of biocides. In addition, current in vitro tests appear not to be suitable for predicting levels of resistance that are clinically relevant.
    Antimicrobial Agents and Chemotherapy 08/2013; 57(8):3488-97. DOI:10.1128/AAC.00498-13 · 4.45 Impact Factor
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    ABSTRACT: Background: The acquisition of resistance may produce fitness costs (FC) impacting the possibility of spread of resistant microorganisms. Thus, measuring these fitness costs is relevant for predicting the emergence of resistance. Objectives: To determine FC of Escherichia coli (Ec), Klebsiella pneumoniae (Kpn) and Salmonella enterica (Sal) mutants selected with biocides (triclosan, TRI; benzalkonium chloride, BZC; chlorhexidine, CHX) or antibiotics (ampicilin, Amp; ciprofloxacin, Cip) Methods: 21 mutants exhibiting different phenotypes of biocides or antibiotics resistance were studied. FC was determined by calculating their relative growth rates in comparison with wild-types stains. Expression of gapA, acrB, acrF, acrD, marA, ramA, and soxS was measured by quantitative RT-PCR. Phenotype microarrays of carbon sources were performed for Ec. Results and conclusions: Two of the 5 Ec mutants, presenting changes in TRI and ceftazidime susceptibility, showed high FC (26 and 75%). Six out of 9 Kpn mutants with decreased susceptibility to TRI and BKC had relevant FC (4-66%). The Kpn mutant with the highest cost exhibited reduced susceptibility to several antibiotics. Four of the 7 Sa mutants presented relevant FC. Mutants with the highest FC showed increased expression of marA (Ec) or ramA transcriptional regulators (Kpn and Sal). Ec mutants with the highest MIC to TRI and high FC metabolize carboxylic acids, aminoacids, and carbohydrates better than wild-type. FC of biocide resistance depends on the type of mutation involved. The finding that mutants with high FC can metabolize better some carbon sources indicate that FC may depend on the environment and the available nutrients.
    5th Federation of European Microbiological Societies, Leipzig; 07/2013
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    ABSTRACT: We report the draft genome sequence of 34, a Cr(VI)-hyperresistant and biofilm-producing bacterium that might be used for the bioremediation of chromate-polluted soils. The genome sequence might be helpful in exploring the mechanisms involved in chromium resistance and biofilm formation.
    Genome Announcements 01/2013; 1(1). DOI:10.1128/genomeA.00125-12
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    2nd International Conference on Antimicrobial Research (ICAR); 11/2012
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    ABSTRACT: The pneumococcal chromosome encodes about 140 transporters, many of which predicted to be involved in efflux. In order to critically evaluate pneumococcal efflux, a series of transporter mutants were constructed, and their phenotypes were assayed by disk diffusion, microdilution drug susceptibility testing (MIC), growth cultures at subMIC concentrations and Phenotype Microarray analysis. Mutants for seven ABC transporters, three MATE efflux pumps and one MFS transporter were obtained in S. pneumoniae strain DP1004. The susceptibility of these eleven mutants to over 250 different substances was compared to that of the parent strain. Out of the tested transporters, only the ABC transporter PatAB (SP2073-5) presented a clear MDR profile as the mutant showed significantly increased susceptibility to ethidium bromide, acriflavin and berberine. Among the other transporters analysed, the mutants devoid of the MATE efflux pump SP2065 exhibited reduced susceptibility to novobiocin and the DinF MATE family transport system (SP1939) exhibited increased susceptibility to moxifloxacin, ciprofloxacin and levofloxacin. This change in quinolone MIC was found to be independent form the competence mediated effect of quinolones on the cinA-recA-dinF operon. Furthermore the dinF mutant allowed, in contrast to the parental strain, to select for quinolone resistant mutants when exposed to moxifloxacin. These data confirm the clear MDR profile of the PatAB ABC transporter and propose for the MATE DinF a phenotype associated to quinolone susceptibility, in particular for moxifloxacin.
    Antimicrobial Agents and Chemotherapy 10/2012; 57(1). DOI:10.1128/AAC.01298-12 · 4.45 Impact Factor
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    ABSTRACT: A rapid method for screening the metabolic susceptibility of biofilms to toxic compounds was developed by combining the Calgary Biofilm Device (MBEC device) and Phenotype MicroArray (PM) technology. The method was developed using Pseudomonas alcaliphila 34, a Cr(VI)-hyper-resistant bacterium, as the test organism. P. alcaliphila produced a robust biofilm after incubation for 16 h, reaching the maximum value after incubation for 24 h (9.4 × 106 ± 3.3 × 106 CFU peg−1). In order to detect the metabolic activity of cells in the biofilm, dye E (5×) and menadione sodium bisulphate (100 μM) were selected for redox detection chemistry, because they produced a high colorimetric yield in response to bacterial metabolism (340.4 ± 6.9 Omnilog Arbitrary Units). This combined approach, which avoids the limitations of traditional plate counts, was validated by testing the susceptibility of P. alcaliphila biofilm to 22 toxic compounds. For each compound the concentration level that significantly lowered the metabolic activity of the biofilm was identified. Chemical sensitivity analysis of the planktonic culture was also performed, allowing comparison of the metabolic susceptibility patterns of biofilm and planktonic cultures.
    Biofouling 10/2012; 28(9):1023-1032. DOI:10.1080/08927014.2012.726352 · 3.70 Impact Factor
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    ABSTRACT: The effect of long-term (8 years) compost treatments (compost or compost plus mineral fertilizer) on genetic structure of bacterial and fungal populations in both bulk soil and rhizosphere of grapevine (Vitis vinifera) was analyzed in respect to a control constituted by the soil treated with mineral fertilization. Soils were sampled in early summer (July), mid-summer (August), and before harvest (October). Bacterial and fungal populations were characterized by genetic fingerprints generated by the application of 16S rDNA and ITS rDNA Multiplex Terminal Fragment Length Polymorphism (M-TRFLP) technique. Compost induced no significant differences at any time on microbial communities from bulk soil samples, whereas seasonal variations significantly affected both bacterial and fungal populations as indicated by the Multi Dimensional Scaling (MDS) ordination method of the M-TRFLPs results. MDS analysis of grapevine rhizosphere M-TRFLPs showed that temporal separation was significant for the bacterial population only. Results suggested that soil microbial populations in vineyard productive ecosystems may be sensitive to environmental changes induced by seasonal variations and show a certain degree of resilience to different agricultural practices.
    Geomicrobiology 08/2012; 29(6):506-519. DOI:10.1080/01490451.2011.582928 · 1.80 Impact Factor

Publication Stats

804 Citations
157.69 Total Impact Points

Institutions

  • 2015
    • INO - Istituto Nazionale di Ottica
      Florens, Tuscany, Italy
  • 1997–2015
    • University of Florence
      • • Dipartimento di Scienze delle Produzioni Agroalimentari e dell'Ambiente (DISPAA)
      • • Dipartimento di Biologia
      Florens, Tuscany, Italy
  • 2009
    • University of Bologna
      Bolonia, Emilia-Romagna, Italy
  • 1993–2000
    • National Research Council
      Roma, Latium, Italy