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South America is populated by a wide range of bumble bee species that represent an important source of biodiversity, supporting pollination services in natural and agricultural ecosystems. These pollinators provide unique specific microbial niches, populated by a wide number of microorganisms such as symbionts, environmental opportunistic bacteria, and pathogens. Recently, it was demonstrated how microbial populations are shaped by trophic resources and environmental conditions but also by anthropogenic pressure, which strongly affects microbes' functionality. This study is focused on the impact of different land uses (natural reserve, agroecosystem, and suburban) on the gut microbiome composition of two South American bumble bees, Bombus pauloensis and Bombus bellicosus. Gut microbial DNA extracted from collected bumble bees was sequenced on the Illumina MiSeq platform and correlated with land use. Nosema ceranae load was analyzed with qPCR and correlated with microbiome data. Significant differences in gut microbiome composition between the two wild bumble bee species were highlighted, with notable variations in α- and β-diversity across study sites. Bombus bellicosus showed a high abundance of Pseudomonas, a genus that includes environmental saprobes, and was found to be the second major taxa populating the gut microbiome, probably indicating the vulnerability of this host to environmental pollution. Pathogen analysis unveils a high prevalence of N. ceranae, with B. bellicosus showing higher susceptibility. Finally, Gilliamella exhibited a negative correlation with N. ceranae, suggesting a potential protective role of this commensal taxon. Our findings underscore the importance of considering microbial dynamics in pollinator conservation strategies, highlighting potential interactions between gut bacteria and pathogens in shaping bumble bee health. [ABSTRACT FROM AUTHOR]

The genus Serratia harbors opportunistic pathogenic species, among which Serratia marcescens is pathogenic for honeybees although little studied. Recently, virulent strains of S. marcescens colonizing the Varroa destructor mite's mouth were found vectored into the honeybee body, leading to septicemia and death. Serratia also occurs as an opportunistic pathogen in the honeybee's gut with a low absolute abundance. The Serratia population seems controlled by the host immune system, but its presence may represent a hidden threat, ready to arise when honeybees are weakened by biotic and abiotic stressors. To shed light on the Serratia pathogen, this research aims at studying Serratia's development dynamics in the honeybee body and its interactions with the co-occurring fungal pathogen Vairimorpha ceranae. Firstly, the degree of pathogenicity and the ability to permeate the gut epithelial barrier of three Serratia strains, isolated from honeybees and belonging to different species (S. marcescens, Serratia liquefaciens, and Serratia nematodiphila), were assessed by artificial inoculation of newborn honeybees with different Serratia doses (104, 106, and 108 cells/mL). The absolute abundance of Serratia in the gut and in the hemocoel was assessed in qPCR with primers targeting the luxS gene. Moreover, the absolute abundance of Serratia was assessed in the gut of honeybees infected with V. ceranae at different development stages and supplied with beneficial microorganisms and fumagillin. Our results showed that all tested Serratia strains could pass through the gut epithelial barrier and proliferate in the hemocoel, with S. marcescens being the most pathogenic. Moreover, under cage conditions, Serratia better proliferates when a V. ceranae infection is co-occurring, with a positive and significant correlation. Finally, fumagillin and some of the tested beneficial microorganisms could control both Serratia and Vairimorpha development. Our findings suggest a correlation between the two pathogens under laboratory conditions, a co-occurring infection that should be taken into consideration by researches when testing antimicrobial compounds active against V. ceranae, and the related honeybees survival rate. Moreover, our findings suggest a positive control of Serrata by the environmental microorganism Apilactobacillus kunkeei in a in vivo model, confirming the potential of this specie as beneficial bacteria for honeybees. [ABSTRACT FROM AUTHOR]

Bombus pauloensis is a native bumble bee species widely distributed over South America and a key pollinator for native plants and commercial crops. This species is affected by pathogens such as Nosema ceranae, Crithidia bombi and Apis mellifera Filamentous Virus (AmFV). This work aims to document the presence and intensity of exogenous pathogens on the native bumble bee B. pauloensis in different periods of the year during spring and summer. Bumble bees were sampled in four study areas with contrasting land uses to preliminary evaluate if anthropization levels can be related to the presence and intensity of pathogens. DNA was isolated from twenty individuals per sampling site and N. ceranae, C. bombi and AmFV pathogens load were quantified by quantitative PCR. The results showed a wide and ubiquitous prevalence of N. ceranae and C. bombi pathogens in all the sampled bumble bees throughout the year, with a pathogens load that did not differ significantly among the sampling sites. AmFV was undetected in any of the individuals analyzed in any sampling site. This suggests that human activities could equally impact all habitats populated by B. pauloensis. Honey bees were detected with a relevant abundance in all the sampling sites and could be one of the main anthropogenic drivers of pathogens spread and host switches in the analyzed sampling sites. This study analyzes the general occurrence of the prevalence and intensity of the pathogens in a native Argentinian bumble bee species and attempts the correlation with the land use [ABSTRACT FROM AUTHOR]

About 95% of global hydrogen production is made by fossil fuels using different technologies which are all characterized by high energy consumption and high carbon emissions. Alternatively, more sustainable production methods, such as biological fermentation processes, are under study. Dark fermentation, also called acidogenesis, entails the transformation of a great variety of organic substances into a mixture of organic and inorganic products, as well as gases (H2 and CO2). In this study we tested an exhausted fermentation broth, derived after Clostridium fermentation for H2 production, as a biostimulant via foliar application in an intensive apple orchard. Two different doses were applied upon dilution of the broth in water (100 mL L−1 and 10 mL L−1), evaluating the main fruit quality parameters (fresh weight, fruit diameter, dry matter, firmness, soluble solid content, color lightness, DA index) in addition to macro- and micro-nutrients and heavy metals concentrations. Chemical characterization of the broth showed a high amount of low-MW polypeptides (Trp-Glu-Lys, Ile-Pro-Ile, Phe-Pro-Lys, His-Pro) and organic acids (formic acid, butyric acid, butanedioic acid); moreover, quantitative analyses of inorganic ions showed no heavy metal detection but high concentrations of nitrogen, phosphorus and potassium, compatible with use in agriculture. The fruit quality parameters showed significantly higher mean fruit weight compared to the untreated trees, as well as higher dry matter. No statistical differences were recorded among the treatments for fruit firmness, diameter and yield. Soluble solids content in both treatments were significantly lower than the controls, whereas the DA index mean values were higher in both treatments compared to the controls, indicating a delay in fruit ripening probably due to the high nitrogen broth concentration. Regarding the chemical analyses of fruits, no particular differences were found among the treatments, except for Fe, which showed a significantly higher amount upon treatment with the lower dose. As concerns leaves, no phytotoxic symptoms were detected in both treatments, making the described exhausted broth a candidate for its use as a plant biostimulant. Additional studies are needed to evaluate the ideal application dose, identify further action targets and implement appropriate strategies to concentrate the biostimulant active compounds. [ABSTRACT FROM AUTHOR]

Introduction: Apis mellifera evolved mainly in African, Asian, and European continents over thousands of years, leading to the selection of a considerable number of honey bees subspecies that have adapted to various environments such as hot semi-desert zones and cold temperate zones. With the evolution of honey bee subspecies, it is possible that environmental conditions, food sources, and microbial communities typical of the colonized areas have shaped the honey bee gut microbiota. Methods: In this study the microbiota of two distinct lineages (mitochondrial haplotypes) of bees Apis mellifera ruttneri (lineage A) and Apis mellifera ligustica and carnica (both lineage C) were compared. Honey bee guts were collected in a dry period in the respective breeding areas (the island of Malta and the regions of Emilia-Romagna and South Tyrol in Italy). Microbial DNA from the honey bee gut was extracted and amplified for the V3-V4 regions of the 16S rRNA gene for bacteria and for ITS2 for fungi. Results: The analyses carried out show that the Maltese lineage A honey bees have a distinctive microbiota when compared to Italian lineage C honey bees, with the most abundant genera being Bartonellaceae and Lactobacillaceae, respectively. Lactobacillaceae in Maltese Lineage A honey bees consist mainly of Apilactobacillus instead of Lactobacillus and Bombilactobacillus in the lineage C. Lineage A honey bee gut microbiota also harbors higher proportions of Arsenophonus, Bombella, Commensalibacter, and Pseudomonas when compared to lineage C. Discussion: The environment seems to be the main driver in the acquisition of these marked differences in the gut microbiota. However, the influence of other factors such as host genetics, seasonality or geography may still play a significant role in the microbiome shaping, in synergy with the environmental aspects. [ABSTRACT FROM AUTHOR]

Members of Bacillus spp. have been widely used to enrich the soil/root interface to provide plant growth promoting activities. A new isolate, namely to Bacillus sp. VWC18, has been tested under greenhouse conditions in lettuce (Lactuca sativa L.) pots at different concentrations (103, 105, 107, and 109 CFU·mL−1) and application time (single inoculum at transplant and multiple inoculum every ten days) to evaluate the best application dose and frequency. Analysis of foliar yield, main nutrients, and minerals evidenced a significant response for all applications. The lowest (103 CFU·mL−1) and the highest doses (109 CFU·mL−1), applied every ten days until harvest, had the greatest efficacy; the nutrient yield (N, K, P, Na, Ca, Fe, Mg, Mn, Cu, and B) increased more than twice. A new randomized block design with three replicates was then performed in lettuce and basil (Ocinum basilicum L.), with the two best performing concentrations applied every ten days. In addition to previous analysis, root weight, chlorophyll, and carotenoids were also examined. Both experiments confirmed the previous results: inoculation of the substrate with Bacillus sp. VWC18 promoted plant growth, chlorophyll, and mineral uptake in both crop species. Root weight duplicated or triplicated compared to control plants, and chlorophyll concentration reached even higher values. Both parameters had a dose-dependent increase. [ABSTRACT FROM AUTHOR]

Millet is the sixth-highest yielding grain in the world and a staple crop for millions of people. Fermentation was applied in this study to improve the nutritional properties of pearl millet. Three microorganism combinations were tested: Saccharomyces boulardii (FPM1), Saccharomyces cerevisiae plus Campanilactobacillus paralimentarius (FPM2) and Hanseniaspora uvarum plus Fructilactobacillus sanfranciscensis (FPM3). All the fermentation processes led to an increase in minerals. An increase was observed for calcium: 254 ppm in FPM1, 282 ppm in FPM2 and 156 ppm in the unfermented sample. Iron increased in FPM2 and FPM3 (approx. 100 ppm) with respect the unfermented sample (71 ppm). FPM2 and FPM3 resulted in richer total phenols (up to 2.74 mg/g) compared to the unfermented sample (2.24 mg/g). Depending on the microorganisms, it was possible to obtain different oligopeptides with a mass cut off ≤10 kDalton that was not detected in the unfermented sample. FPM2 showed the highest resistant starch content (9.83 g/100 g) and a prebiotic activity on Bifidobacterium breve B632, showing a significant growth at 48 h and 72 h compared to glucose (p < 0.05). Millet fermented with Saccharomyces cerevisiae plus Campanilactobacillus paralimentarius can be proposed as a new food with improved nutritional properties to increase the quality of the diet of people who already use millet as a staple food. [ABSTRACT FROM AUTHOR]

Fermentation is one of the most ancient strategies to improve safety and extend shelf-life of the products. Starter cultures are mainly represented by lactic acid bacteria (LAB), which may also be bioprotective agents controlling the fermentation process, the native microbiota and pathogen outgrowth. This work aimed to select new LAB strains from spontaneously fermented sausages produced in different areas of Italy, which can be effective as starter cultures and bioprotective agents in fermented salami. The strains, mainly belonging to the Latilactobacillus sakei species, were characterized for their ability to inhibit major meat pathogens, the presence of antibiotic resistances and amine production. Moreover, technological performances, such as growth and acidification kinetics at increasing NaCl concentrations, were studied. As a result, new autochthonous Lat. sakei strains were obtained, lacking antibiotic resistance, possessing antimicrobial activity against Clostridium sporogenes, Listeria monocytogenes, Salmonella and Escherichia coli and with high growth performance under osmotic pressure. These strains have the potential for future application to improve the safety of fermented meats, even under conditions in which chemical preservatives are reduced or eliminated. Moreover, studies on autochthonous cultures are pivotal for guaranteeing specific characteristics of traditional products that represent an important cultural heritage. [ABSTRACT FROM AUTHOR]

Probiotic bacteria, capable of conferring benefits to the host, can present challenges in design, development, scale-up, manufacturing, commercialization, and life cycle management. Strain identification is one of the main quality parameters; nevertheless, this task can be challenging since established methodologies can lack resolution at the strain level for some microorganisms and\or are labor-intensive and time-consuming. Fourier transform infrared spectroscopy (FTIRS) has been largely used for the investigation of pathogenic species in the clinical field, whereas only recently has been proposed for the identification of probiotic strains. Within the probiotic industrial production, bacterial strains can be subjected to stressful conditions that may affect genomic and phenotypic characteristics; therefore, real-time monitoring of all the sequential growth steps is requested. Considering the fast, low-cost, and high-throughput features, FTIRS is an innovative and functional technology for typing probiotic strains from benchtop experiments to large-scale industrial production, allowing the monitoring of stability and identity of probiotic strains. In this study, the discriminatory power of FTIRS was assessed for four Lactiplantibacillus plantarum probiotic strains grown under different conditions, including temperatures (30 and 37◦C) and medium (broth and agar), after consecutive sub-culturing steps. A comparison between the generated spectra with pulsed-field gel electrophoresis (PFGE) profiles was also performed. FTIRS was not only able to distinguish the strains of L. plantarum under different growth conditions but also to prove the phenotypic stability of L. plantarum type strain LP-CT after six growing steps. Regardless of the growth conditions, FTIRS spectra related to LP-CT constituted a unique hierarchical cluster, separated from the other L. plantarum strains. These results were confirmed by a PFGE analysis. In addition, based on FTIRS data, broth cultures demonstrated a higher reproducibility and discriminatory power with respect to agar ones. These results support the introduction of FTIRS in the probiotic industry, allowing for the step-by-step monitoring of massive microbial production while also guaranteeing the stability and purity of the probiotic strain. The proposed novel approach can constitute an impressive improvement in the probiotic manufacturing process. [ABSTRACT FROM AUTHOR]

This study investigates the isolation, identification, and fermentation performance of autochthonous acetic acid bacteria (AAB) from local niche habitats on the Island of Gozo (Malta) and their further use for vinegar production, employing local raw materials. The bacteria were isolated from grapevine berries and vinegar produced in the cottage industry. Following phenotype and genotype identification, the AAB were ascribed to the genera Acetobacter, Gluconobacter, and Komagataeibacter. A mixture of selected AAB was tested as an inoculum for vinegar production in bench fermenters, under different conditions and substrates, namely, grapes, honey, figs, onions, prickly pear, and tomatoes. The bench fermenters were operated under semi-continuous fermentation where working volumes were maintained by discharging and subsequent recharging accordingly to maintain the acidity in fermenters by adding 30--50 g/l of acetic acid for optimal Acetobacteraceae performance. Finally, the vinegar products obtained from the different substrates were evaluated for their quality, including organoleptic properties, which showed the superior quality of wood-treated vinegar samples with respect to neat vinegar samples. [ABSTRACT FROM AUTHOR]

There has been growing consumer demand for the products and services of the short food supply chains (SFSCs) in recent times. A procedure was developed to identify the technological and nontechnological innovations that can improve the performance and competitiveness of the SFSCs. The needs of the SFSCs for innovative solutions were collected by interviewing 18 SFSCs from 9 countries. An inventory was prepared to contain 136 technological and non-technological innovations, meeting these needs. The innovations were collected from the good practices of the 18 SFSCs, experiences of the project partners and state of the art. The success factors and bottlenecks of each short food supply chain operation and their current value propositions were identified. From the inventory, those innovations were selected for each short food chain case study which can be applied to eliminate or reduce the bottlenecks or enhance the success factors leading to new, upgraded value propositions with increased added value for the consumers. The new, upgraded value propositions can serve as a starting point for developing a strategy for improving the competitiveness of a short food chain organisation through the application of innovations. [ABSTRACT FROM AUTHOR]

Particulate composites based on poly(lactic acid) (PLA) and poly(butylene succinate) (PBS) biopolymers have been formulated. Silver skin (SSK), the by‐product derived from the roasting of coffee beans, has been milled to micrometric size and added up to a 30 wt% of loading, without any previous treatment. The dynamic mechanical properties of the composites have been investigated, as well as the stability toward UV and IR radiation. The filler improves some mechanical properties such as the elastic modulus (E′) mostly in PBS. Photoaging experiments demonstrate that SSK, due to its polyphenols content and antioxidant activity, is able to protect both PLA and PBS from UV degradation, highlighting an added value of SSK waste‐based composites. [ABSTRACT FROM AUTHOR]

The structure and environmental distribution of Tn5271-like elements in bacteria strains found in the Niagara River watershed and elsewhere are described. Transposons formed from homologous components of the insertion element IS1071 and the operon cbaABC were observed on Tn5271 of strains in Niagara Falls, NY, and on pCPE3 of strains in Bologna, Italy. Differences in the junction sequences between the internal DNA and the IS1071L and IS1071R elements indicate that the transposons were formed independent of each other on the two plasmids.

Fourier transform infrared (FTIR) spectroscopy, a technology traditionally used in chemistry to determine the molecular composition of a wide range of sample types, has gained growing interest in microbial typing. It is based on the different vibrational modes of the covalent bonds between atoms of a given sample, as bacterial cells, induced by the absorption of infrared radiation. This technique has been largely used for the study of pathogenic species, especially in the clinical field, and has been proposed also for the typing at different subspecies levels. The high throughput, speed, low cost, and simplicity make FTIR spectroscopy an attractive technique also for industrial applications, in particular, for probiotics. The aim of this study was to compare FTIR spectroscopy with established genotyping methods, pulsed-field gel electrophoresis (PFGE), whole-genome sequencing (WGS), and multilocus sequence typing (MLST), in order to highlight the FTIR spectroscopy potential discriminatory power at strain level. Our study focused on bifidobacteria, an important group of intestinal commensals generally recognized as probiotics. For their properties in promoting and maintaining health, bifidobacteria are largely marketed by the pharmaceutical, food, and dairy industries. Strains belonging to Bifidobacterium longum subsp. longum and Bifidobacterium animalis subsp. lactis were taken into consideration together with some additional type strains. For B. longum subsp. longum , it was possible to discriminate the strains with all the methods used. Although two isolates were shown to be strictly phylogenetically related, constituting a unique cluster, based on PFGE, WGS, and MLST, no clustering was observed with FTIR. For B. animalis subsp. lactis group, PFGE, WGS, and MLST were non-discriminatory, and only one strain was easily distinguished. On the other hand, FTIR discriminated all the isolates one by one, and no clustering was observed. According to these results, FTIR analysis is not only equivalent to PFGE, WGS, and MLST, but also for some strains, in particular, for B. animalis subsp. lactis group, more informative, being able to differentiate strains not discernible with the other two methods based on phenotypic variations likely deriving from certain genetic changes. Fourier transform infrared spectroscopy has highlighted the possibility of using the cell surface as a kind of barcode making tracing strains possible, representing an important aspect in probiotic applications. Furthermore, this work constitutes the first investigation on bifidobacterial strain typing using FTIR spectroscopy. [ABSTRACT FROM AUTHOR]

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

Abstract Background Infantile colic is a common disturb within the first 3 months of life, nevertheless the pathogenesis is incompletely understood and treatment remains an open issue. Intestinal gas production is thought to be one of the causes of abdominal discomfort in infants suffering from colic. However, data about the role of the amount of gas produced by infants' colonic microbiota and the correlation with the onset of colic symptoms are scanty. The benefit of supplementation with lactobacilli been recently reported but the mechanisms by which they exert their effects have not yet been fully defined. This study was performed to evaluate the interaction between Lactobacillus spp. strains and gas-forming coliforms isolated from stools of colicky infants. Results Strains of coliforms were isolated from stools of 45 colicky and 42 control breastfed infants in McConkey Agar and identified using PCR with species-specific primers, and the BBL™ Enterotube™ II system for Enterobacteriaceae. Gas-forming capability of coliforms was assessed in liquid cultures containing lactose as sole carbon source. The average count of total coliforms in colicky infants was significantly higher than controls: 5.98 (2.00-8.76) log10 vs 3.90 (2.50-7.10) CFU/g of faeces (p = 0.015). The following strains were identified: Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Enterobacter aerogenes, Enterobacter cloacae and Enterococcus faecalis. Then, 27 Lactobacillus strains were tested for their antagonistic effect against coliforms both by halo-forming method and in liquid co-cultures. Lactobacillus delbrueckii subsp.delbrueckii DSM 20074 and L. plantarum MB 456 were able to inhibit all coliforms strains (halo-forming method), also in liquid co-cultures, thus demonstrating an antagonistic activity. Conclusions This study shows that two out of 27 strains of Lactobacillus examined possess an antimicrobial effect against six species of gas-forming coliforms isolated from colicky infants. Our findings may stimulate new researches to identify which Lactobacillus strains can improve colicky symptoms by acting on coliforms gut colonization.

Abstract Background Vanillin is one of the most important aromatic flavour compounds used in the food and cosmetic industries. Natural vanillin is extracted from vanilla beans and is relatively expensive. Moreover, the consumer demand for natural vanillin highly exceeds the amount of vanillin extracted by plant sources. This has led to the investigation of other routes to obtain this flavour such as the biotechnological production from ferulic acid. Studies concerning the use of engineered recombinant Escherichia coli cells as biocatalysts for vanillin production are described in the literature, but yield optimization and biotransformation conditions have not been investigated in details. Results Effect of plasmid copy number in metabolic engineering of E. coli for the synthesis of vanillin has been evaluated by the use of genes encoding feruloyl-CoA synthetase and feruloyl hydratase/aldolase from Pseudomonas fluorescens BF13. The higher vanillin production yield was obtained using resting cells of E. coli strain JM109 harbouring a low-copy number vector and a promoter exhibiting a low activity to drive the expression of the catabolic genes. Optimization of the bioconversion of ferulic acid to vanillin was accomplished by a response surface methodology. The experimental conditions that allowed us to obtain high values for response functions were 3.3 mM ferulic acid and 4.5 g/L of biomass, with a yield of 70.6% and specific productivity of 5.9 μmoles/g × min after 3 hours of incubation. The final concentration of vanillin in the medium was increased up to 3.5 mM after a 6-hour incubation by sequential spiking of 1.1 mM ferulic acid. The resting cells could be reused up to four times maintaining the production yield levels over 50%, thus increasing three times the vanillin obtained per gram of biomass. Conclusion Ferulic acid can be efficiently converted to vanillin, without accumulation of undesirable vanillin reduction/oxidation products, using E. coli JM109 cells expressing genes from the ferulic acid-degrader Pseudomonas fluorescens BF13. Optimization of culture conditions and bioconversion parameters, together with the reuse of the biomass, leaded to a final production of 2.52 g of vanillin per liter of culture, which is the highest found in the literature for recombinant strains and the highest achieved so far applying such strains under resting cells conditions.