Beneficial Bacteria and Plant Extracts Promote Honey Bee Health and Reduce Nosema ceranae Infection.

The research aims to give new insights on the effect of administering selected bacterial strains, isolated from honey bee gut, and/or a commercial plant extract blend (HiveAlive®) on Nosema ceranae. Analyses were first performed under laboratory conditions such as different infective doses of N. ceranae, the effect of single strains and their mixture and the influence of pollen administration. Daily survival and feed consumption rate were recorded and pathogen development was analysed using qPCR and microscope counts. Biomarkers of immunity and physiological status were also evaluated for the different treatments tested using one bacterial strain, a mixture of all the bacteria and/or a plant extract blend as treatments. The results showed an increase of abaecin transcript levels in the midgut of the honey bees treated with the bacterial mixture and an increased expression of the protein vitellogenin in the haemolymph of honey bees treated with two separate bacterial strains (Bifidobacterium coryneforme and Apilactobacillus kunkeei). A significant effectiveness in reducing N. ceranae was shown by the bacterial mixture and the plant extract blend regardless of the composition of the diet. This bioactivity was seasonally linked. Quantitative PCR and microscope counts showed the reduction of N. ceranae under different experimental conditions. The antiparasitic efficacy of the treatments at field conditions was studied using a semi-field approach which was adapted from research on insecticides for the first time, to analyse antiparasitic activity against N. ceranae. The approach proved to be reliable and effective in validating data obtained in the laboratory. Both the mixture of beneficial bacteria and its association with Hive Alive® are effective in controlling the natural infection of N. ceranae in honey bee colonies.

Screening of Dietary Ingredients against the Honey Bee Parasite Nosema ceranae.

Nosema ceranae is a major pathogen in the beekeeping sector, responsible for nosemosis. This disease is hard to manage since its symptomatology is masked until a strong collapse of the colony population occurs. Conversely, no medicaments are available in the market to counteract nosemosis, and only a few feed additives, with claimed antifungal action, are available. New solutions are strongly required, especially based on natural methods alternative to veterinary drugs that might develop resistance or strongly pollute honey bees and the environment. This study aims at investigating the nosemosis antiparasitic potential of some plant extracts, microbial fermentation products, organic acids, food chain waste products, bacteriocins, and fungi. Honey bees were singularly infected with 5 × 104 freshly prepared N. ceranae spores, reared in cages and fed ad libitum with sugar syrup solution containing the active ingredient. N. ceranae in the gut of honey bees was estimated using qPCR. The results showed that some of the ingredients administered, such as acetic acid at high concentration, p-coumaric acid, and Saccharomyces sp. strain KIA1, were effective in the control of nosemosis. On the other hand, wine acetic acid strongly increased the N. ceranae amount. This study investigates the possibility of using compounds such as organic acids or biological agents including those at the base of the circular economy, i.e., wine waste production, in order to improve honeybee health.

Honeybee Exposure to Veterinary Drugs: How Is the Gut Microbiota Affected?

Several studies have outlined that a balanced gut microbiota offers metabolic and protective functions supporting honeybee health and performance. The present work contributes to increasing knowledge on the impact on the honeybee gut microbiota of the three most common veterinary drugs (oxytetracycline, sulfonamides, and tylosin). The study was designed with a semi-field approach in micro-hives containing about 500 honeybees. Micro-hives were located in an incubator during the day and moved outdoors in the late afternoon, considering the restrictions on the use of antibiotics in the open field but allowing a certain freedom to honeybees; 6 replicates were considered for each treatment. The absolute abundance of the major gut microbial taxa in newly eclosed individuals was studied with qPCR and next-generation sequencing. Antimicrobial resistance genes for the target antibiotics were also monitored using a qPCR approach. The results showed that the total amount of gut bacteria was not altered by antibiotic treatment, but qualitative variations were observed. Tylosin treatment determined a significant decrease of α- and ß-diversity indices and a strong depletion of the rectum population (lactobacilli and bifidobacteria) while favoring the ileum microorganisms (Gilliamella, Snodgrassella, and Frischella spp.). Major changes were also observed in honeybees treated with sulfonamides, with a decrease in Bartonella and Frischella core taxa and an increase of Bombilactobacillus spp. and Snodgrassella spp. The present study also shows an important effect of tetracycline that is focused on specific taxa with minor impact on alfa and beta diversity. Monitoring of antibiotic resistance genes confirmed that honeybees represent a great reservoir of tetracycline resistance genes. Tetracycline and sulfonamides resistance genes tended to increase in the gut microbiota population upon antibiotic administration. IMPORTANCE This study investigates the impact of the three most widely used antibiotics in the beekeeping sector (oxytetracycline, tylosin, and sulfonamides) on the honeybee gut microbiota and on the spread of antibiotic resistance genes. The research represents an advance to the present literature, considering that the tylosin and sulfonamides effects on the gut microbiota have never been studied. Another original aspect lies in the experimental approach used, as the study looks at the impact of veterinary drugs and feed supplements 24 days after the beginning of the administration, in order to explore perturbations in newly eclosed honeybees, instead of the same treated honeybee generation. Moreover, the study was not performed with cage tests but in micro-hives, thus achieving conditions closer to real hives. The study reaches the conclusion that the most common veterinary drugs determine changes in some core microbiota members and that incidence of resistance genes for tetracycline and sulfonamides increases following antibiotic treatment.

Kombucha Beverage from Green, Black and Rooibos Teas: A Comparative Study Looking at Microbiology, Chemistry and Antioxidant Activity.

Kombucha is usually obtained from the fermentation of black or green tea by a consortium of acetic acid bacteria and yeasts. In this study, kombucha was prepared from the same starter consortium using green and black teas as well as, for the first time, an infusion of rooibos leaves (Aspalathus linearis). Microbial diversity was analysed during fermentation both in the biofilm and in the corresponding kombuchas, using culture-dependent and -independent methods. Polyphenols, flavonoids, ethanol, and acids were quantified and anti-oxidant activities were monitored. All of the Kombuchas showed similarity in bacterial composition, with the dominance of Komagataeibacter spp. Beta diversity showed that the yeast community was significantly different among all tea substrates, between 7 and 14 days of fermentation and between biofilm and kombucha, indicating the influence of the substrate on the fermenting microbiota. Kombucha from rooibos has a low ethanol concentration (1.1 mg/mL), and a glucuronic acid amount that was comparable to black tea. Although antioxidant activity was higher in black and green kombucha compared to rooibos, the latter showed an important effect on the recovery of oxidative damage on fibroblast cell lines against oxidative stress. These results make rooibos leaves interesting for the preparation of a fermented beverage with health benefits.

Polysaccharides from by-products of the Wonderful and Laffan pomegranate varieties: New insight into extraction and characterization.

The main crude polysaccharides (CPS), extracted from two widely cultivated pomegranate varieties, Laffan and Wonderful, were studied and characterized. We obtained the highest CPS extraction yield (approximatively 10% w/w on dried matter) by 1h of decoction (ratio 1/40w/v). The predominant polymers (75-80%) of the CPS samples showed a hydrodynamic volume close to 2000kDa by size exclusion chromatography and the exocarp and mesocarp profiles were very similar. The proton spectra (1H NMR), according to sugar composition and gelling ability, confirmed the main polysaccharide fractions were pectin with different acylation and methylation degree. The CPS from Laffan and Wonderful mesocarp showed prebiotic properties in vitro with Lactobacillus and Bifidobacterium strains. The composition of the decoction (12% ellagitannins and 10% of CPS) obtained by a green extraction process of pomegranate by-products, makes it a suitable component of functional food formulations.

Effect of Bifidobacterium breve on the Intestinal Microbiota of Coeliac Children on a Gluten Free Diet: A Pilot Study.

Coeliac disease (CD) is associated with alterations of the intestinal microbiota. Although several Bifidobacterium strains showed anti-inflammatory activity and prevention of toxic gliadin peptides generation in vitro, few data are available on their efficacy when administered to CD subjects. This study evaluated the effect of administration for three months of a food supplement based on two Bifidobacterium breve strains (B632 and BR03) to restore the gut microbial balance in coeliac children on a gluten free diet (GFD). Microbial DNA was extracted from faeces of 40 coeliac children before and after probiotic or placebo administration and 16 healthy children (Control group). Sequencing of the amplified V3-V4 hypervariable region of 16S rRNA gene as well as qPCR of Bidobacterium spp., Lactobacillus spp., Bacteroides fragilis group Clostridiumsensu stricto and enterobacteria were performed. The comparison between CD subjects and Control group revealed an alteration in the intestinal microbial composition of coeliacs mainly characterized by a reduction of the Firmicutes/Bacteroidetes ratio, of Actinobacteria and Euryarchaeota. Regarding the effects of the probiotic, an increase of Actinobacteria was found as well as a re-establishment of the physiological Firmicutes/Bacteroidetes ratio. Therefore, a three-month administration of B. breve strains helps in restoring the healthy percentage of main microbial components.

Nonylphenol polyethoxylate degradation in aqueous waste by the use of batch and continuous biofilm bioreactors.

An aerobic bacterial consortium (Consortium A) was recently obtained from textile wastewater and was capable of degrading 4-nonylphenol and nonylphenol polyethoxylates (NPnEOs). In the perspective of developing a biotechnological process for the treatment of effluents from activated sludge plants fed with NPnEO contaminated wastewater, the capability of Consortium A of biodegrading an industrial mixture of NPnEOs in the physiological condition of immobilized cells was investigated. Two identically configured packed bed reactors were developed by immobilizing the consortium on silica beads or granular activated carbon. Both reactors were tested in batch and continuous mode by feeding them with water supplemented with NPnEOs. The two reactors were monitored through chemical, microbiological and molecular integrated methodology. Active biofilms were generated on both immobilization supports. Both reactors displayed comparable NPnEO mineralization under batch and continuous conditions. FISH analyses evidenced that the biofilms evolved with time by changing the reactor operation mode and the organic load. Taken together, the data collected in this study provide a preliminary strong indication on the feasibility of Consortium A-based biofilm technology for the decontamination of NPnEO containing effluents.

Characterization of four olive-mill-wastewater indigenous bacterial strains capable of aerobically degrading hydroxylated and methoxylated monocyclic aromatic compounds.

Seven aerobic bacterial strains capable of degrading several of the monocyclic aromatic compounds occurring in the phenolic fraction of olive-mill wastewaters (OMWs) were isolated from an Italian OMW. The results of the 16S rDNA restriction analysis evidenced that these strains are distributed among four different groups. One strain of each group was taxonomically characterized by sequencing the amplified 16S rDNA, and the four strains were assigned to the genera Comamonas (strain AV1A), Ralstonia (strain AV5BG), Pseudomonas (strain AV2A) and Sphingomonas (strain AV6C). The four strains, when checked for the ability to degrade nine monocyclic aromatic compounds abundant in OMWs, were found to significantly metabolize five to eight of them, both as resting cells and growing cells. Specific enzyme analyses of the same selected strains showed: (1) the occurrence of O-demethylating activities towards four methoxylated mono-aromatic acids in three of the four studied strains (strains AV1A, AV5BG and AV6C), (2) ring-cleavage activity towards protocatechuic acid in all of the strains, and (3) a ring-cleavage activity towards catechol in strain AV6C. The isolates described here exhibit a biodegradation potential towards monocyclic aromatic compounds of OMWs that is markedly broader and higher than that displayed by other aerobic bacteria described previously. These features make them excellent candidates for removing the low-molecular-weight phenolic compounds persisting in the effluent following anaerobic digestion of OMWs.