Characterization of the microbial community composition in Italian Cinta Senese sausages dry-fermented with natural extracts as alternatives to sodium nitrite.

Nitrite is widely used in meat products as a multifunctional additive, combining flavour and colour properties with antioxidant and antimicrobial effects. However, nitrite may form reaction products (i.e., nitrosamine) that are potentially carcinogenic to humans. The meat industry, in response to consumers' demands for nitrite-free products, is seeking natural alternatives to nitrite, such as plant-based extracts. Three types of dry-fermented sausages were manufactured: NIT, containing 30 ppm of sodium nitrite; GSE, containing grape seed extract and olive pomace hydroxytyrosol; and CHE, containing chestnut extract and olive pomace hydroxytyrosol. Next-generation sequencing (NGS) was used to analyse microbial consortia, which were correlated with physical and chemical parameters. The prokaryotic community composition was similar among treatments, with a high relative abundance of Staphylococcus xylosus and Lactobacillus sakei, collectively accounting for 87% of the total community. However, significant differences were observed in both operational taxonomic unit (OTU) presence/absence and relative abundance. Ten genera varied in abundance between treatments. The increase in Lactobacillaceae in CHE may explain the reduced pH levels detected in these samples. In conclusion, NGS analysis showed that the prokaryotic community composition was similar in GSE and NIT, while CHE varied in both the composition and relative abundance of different taxa.

Dietary supplementation of chestnut and quebracho tannins mix: Effect on caecal microbial communities and live performance of growing rabbits.

The interest in antimicrobial compounds as feed additives is currently increasing. Among different options, tannins seem to have several beneficial effects when employed in animals diet. The present study aimed at investigating the influence on caecal microbial communities of the supplementation of a chestnut and quebracho tannins mix in meat rabbit's diet, also considering animals live performances. Four groups of rabbits were fed with a different diet: a control diet (C); a control diet with coccidiostat (CC), and two experimental diets with 0.3% (T0.3) and 0.6% (T0.6) chestnut and quebracho tannins mix. For microbial analysis, culture-dependent and culture-independent methods were employed. Live performances were not significantly affected by tannins mix supplementations, as well as culturable microbial loads of E. coli, Enterobacteriaceae, Bacteroides spp. and Bifidobacterium spp. C. perfringens was always under the detection limit. A consistent result was obtained by qPCR. As for PCR-DGGE analysis, the Richness and evenness (Shannon-Weiner index) of bacterial communities in caecum resulted significantly higher in control samples (C and CC) than in those from rabbit fed with tannin-containing diets. Sequencing analysis revealed that the phylum Firmicutes was less represented in samples from control groups. As for the methanogen archaeal DGGE, no significant differences were found in richness and diversity among different groups, all dominated by Methanobrevibacter spp.. This work highlights the potential antimicrobial effect of chestnut and quebracho tannins mix in an in vivo system revealed by molecular analysis.

Effect of different types of olive oil pomace dietary supplementation on the rumen microbial community profile in Comisana ewes.

Olive oil pomace (OOP) is a bio-waste rich in highly soluble polyphenols. OOP has been proposed as an additive in ruminant feeding to modulate rumen fermentations. Three groups of ewes were fed the following different diets: a control diet and two diets supplemented with OOP, obtained with a two-phase (OOP2) or three-phase (OOP3) olive milling process. Rumen liquor (RL) showed a higher content of 18:3 cis9 cis12 cis15 (α-linolenic acid, α-LNA) with OOP2 inclusion, and of 18:2 cis9 trans11 (rumenic acid, RA) with OOP3 inclusion. The overall composition of the RL microbiota did not differ among treatments. Significant differences, between control and treated groups, were found for six bacterial taxa. In particular, RL microbiota from animals fed OOPs showed a reduction in Anaerovibrio, a lipase-producing bacterium. The decrease in the Anaerovibrio genus may lead to a reduction in lipolysis, thus lowering the amount of polyunsaturated fatty acids available for biohydrogenation. Milk from animals fed OOP showed a higher content of 18:1 cis9 (oleic acid, OA) but the α-LNA concentration was increased in milk from animals treated with OOP2 only. Therefore, inclusion of OOP in ruminant diets may be a tool to ameliorate the nutritional characteristics of milk.

Effect of increasing amounts of olive crude phenolic concentrate in the diet of dairy ewes on rumen liquor and milk fatty acid composition.

Agro-industrial by-products contain several secondary plant metabolites, such as polyphenols, tannins, saponins, and essential oils. The effects of these compounds on animal metabolism may vary significantly according to the dose, the chemical nature of the molecules, and the overall composition of the diet. In the Mediterranean area, the olive oil extraction is associated with 2 by-products: olive pomace and wastewater, both rich in polyphenols. In particular, wastewater may be further processed to obtain olive crude phenolic concentrate (OCPC). An experiment was carried out aiming to evaluate animal performance, milk fatty acid (FA) profile, diversity of rumen microbial population, and rumen liquor FA profile in dairy ewes fed diets containing extruded linseed (EL) and increasing doses of OCPC. Twenty-eight Comisana ewes in mid lactation were allotted to 4 experimental groups. The experiment lasted 5 wk after 3 wk of adaptation. Diets were characterized by lucerne hay administrated ad libitum and by 800 g/ewe and day of 4 experimental concentrates containing 22% of EL on dry matter and increasing dose of OCPC: 0 (L0), 0.6 (L0.6), 0.8 (L0.8), and 1.2 (L1.2) g of OCPC/kg of dry matter. Milk yield was daily recorded and milk composition was analyzed weekly. At the beginning and at the end of the experiment, samples of rumen liquor were collected to analyze FA profile, changes in rumen microbial population, and dimethylacetal (DMA) composition. The inclusion of OCPC did not affect milk yield and gross composition, whereas milk from L0.8 and L1.2 sheep contained higher concentrations of linoleic (+18%) and α-linolenic acid (+24%) and lower concentration of the rumen biohydrogenation intermediates. A similar pattern was observed for rumen liquor FA composition. No differences were found in the diversity of the rumen microbial population. Total amount of DMA did not differ among treatments, whereas significant differences were found in the concentration of individual DMA; in the diet with a higher amount of OCPC, DMA 13:0, 14:0, 15:0, and 18:0 increased, whereas DMA 16:0 decreased. Probably the presence of polyphenols in the diet induced a rearrangement of bacteria membrane phospholipids as a response to the rumen environment stimulus. Overall, the use of OCPC allowed a significant increase in the polyunsaturated FA content of milk, probably due to a perturbation of the rumen biohydrogenation process. Further studies are needed to understand the correlation between diet composition and the pattern of DMA in rumen liquor.

Effect of Dietary Chestnut or Quebracho Tannin Supplementation on Microbial Community and Fatty Acid Profile in the Rumen of Dairy Ewes.

Ruminants derived products have a prominent role in diets and economy worldwide; therefore, the capability to control the rumen microbial ecosystem, for ameliorating their quality, is of fundamental importance in the livestock sector. The aim of this study was to evaluate the effect of dietary supplementation with chestnut and quebracho tannins on microbial community and fatty acid profile, in the rumen fluid of dairy ewes. Multivariate analysis of PCR-DGGE profiles of rumen microbial communities showed a correlation among the presence of chestnut or quebracho in the diet, the specific Butyrivibrio group DGGE profiles, the increase in 18:3 cis9, cis12, and cis15; 18:2 cis9 and cis12; 18:2 cis9 and trans11; 18:2 trans11 and cis15; and 18:1 trans11 content, and the decrease in 18:0 concentration. Phylogenetic analysis of DGGE band sequences revealed the presence of bacteria representatives related to the genera Hungatella, Ruminococcus, and Eubacterium and unclassified Lachnospiraceae family members, suggesting that these taxa could be affected by tannins presence in the diets. The results of this study showed that tannins from chestnut and quebracho can reduce the biohydrogenation of unsaturated fatty acids through changes in rumen microbial communities.

Indole-3-acetic acid in plant-pathogen interactions: a key molecule for in planta bacterial virulence and fitness.

The plant pathogenic bacterium Pseudomonas savastanoi, the causal agent of olive and oleander knot disease, uses the so-called "indole-3-acetamide pathway" to convert tryptophan to indole-3-acetic acid (IAA) via a two-step pathway catalyzed by enzymes encoded by the genes in the iaaM/iaaH operon. Moreover, pathovar nerii of P. savastanoi is able to conjugate IAA to lysine to generate the less biologically active compound IAA-Lys via the enzyme IAA-lysine synthase encoded by the iaaL gene. Interestingly, iaaL is now known to be widespread in many Pseudomonas syringae pathovars, even in the absence of the iaaM and iaaH genes for IAA biosynthesis. Here, two knockout mutants, ΔiaaL and ΔiaaM, of strain Psn23 of P. savastanoi pv. nerii were produced. Pathogenicity tests using the host plant Nerium oleander showed that ΔiaaL and ΔiaaM were hypervirulent and hypovirulent, respectively and these features appeared to be related to their differential production of free IAA. Using the Phenotype Microarray approach, the chemical sensitivity of these mutants was shown to be comparable to that of wild-type Psn23. The main exception was 8 hydroxyquinoline, a toxic compound that is naturally present in plant exudates and is used as a biocide, which severely impaired the growth of ΔiaaL and ΔiaaM, as well as growth of the non-pathogenic mutant ΔhrpA, which lacks a functional Type Three Secretion System (TTSS). According to bioinformatics analysis of the Psn23 genome, a gene encoding a putative Multidrug and Toxic compound Extrusion (MATE) transporter, was found upstream of iaaL. Similarly to iaaL and iaaM, its expression appeared to be TTSS-dependent. Moreover, auxin-responsive elements were identified for the first time in the modular promoters of both the iaaL gene and the iaaM/iaaH operon of P. savastanoi, suggesting their IAA-inducible transcription. Gene expression analysis of several genes related to TTSS, IAA metabolism and drug resistance confirmed the presence of a concerted regulatory network in this phytopathogen among virulence, fitness and drug efflux.

Genomic and phenotypic characterization of the species Acinetobacter venetianus.

Crude oil is a complex mixture of hydrocarbons and other organic compounds that can produce serious environmental problems and whose removal is highly demanding in terms of human and technological resources. The potential use of microbes as bioremediation agents is one of the most promising fields in this area. Members of the species Acinetobacter venetianus have been previously characterized for their capability to degrade n-alkanes and thus may represent interesting model systems to implement this process. Although a preliminary experimental characterization of the overall hydrocarbon degradation capability has been performed for five of them, to date, the genetic/genomic features underlying such molecular processes have not been identified. Here we have integrated genomic and phenotypic information for six A. venetianus strains, i.e. VE-C3, RAG-1(T), LUH 13518, LUH 7437, LUH 5627 and LUH 8758. Besides providing a thorough description of the A. venetianus species, these data were exploited to infer the genetic features (presence/absence patterns of genes) and the short-term evolutionary events possibly responsible for the variability in n-alkane degradation efficiency of these strains, including the mechanisms of interaction with the fuel droplet and the subsequent catabolism of this pollutant.

High-throughput phenomics.

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.

Effect of stoned olive pomace on rumen microbial communities and polyunsaturated fatty acid biohydrogenation: an in vitro study.

BACKGROUND: Stoned olive pomace (SOP), which represents approximately 50% of the conversion process of olives to olive oil, is largely not utilised and creates costs for its disposal and has negative environmental impacts. In vitro trial experiments were employed to study the effect of feeds integrated with this bio-waste, which is rich in polyphenols, on rumen biohydrogenation, using sheep rumen liquor as inoculum. RESULTS: Fatty acid (FA) analysis and a polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE) approach aimed at characterising the microbial community indicated that including SOP in feeds at the level of 50 g/kg and 90 g/kg induced changes in the FA profile and microbial populations. The simultaneous decrease of Butyrivibrio proteoclasticus and accumulation of vaccenic acid was observed. A depression in the populations 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 alters the rumen bacterial community, including bacteria responsible for the hydrogenation of vaccenic acid to stearic acid, thereby modifying the FA profile of the rumen liquor. Hence, a use of SOP aimed to produce meat or dairy products enriched in functional lipids can be hypothesised.

Evaluation of reduced susceptibility to quaternary ammonium compounds and bisbiguanides in clinical isolates and laboratory-generated mutants of Staphylococcus aureus.

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.