Bacterial diversity using metagenomics of 16s rDNA in water kefir, an innovative source of probiotics for bee nutrition.

Water kefir is a sparkling, slightly acidic fermented beverage made from sugar, water, and water kefir grains, which are a mixture of yeast and bacteria. These grains produce a variety of fermentation compounds such as lactic acid, acetaldehyde, acetoin, ethanol and carbon dioxide. In this study, a high-throughput sequencing technique was used to characterize the bacterial composition of the original water kefir from which potential probiotics were obtained. We studied the bacterial diversity of both water kefir grains and beverages. DNA was extracted from three replicate samples of both grains and beverages using the Powerlyzer Microbial Kit. The hypervariable V1-V2 region of the bacterial 16S ribosomal RNA gene was amplified to prepare six DNA libraries. Between 1.4M and 2.4M base-pairs were sequenced for the library. In total, 28721971 raw reads were obtained from all the samples. Estimated species richness was higher in kefir beverage samples compared to grain samples. Moreover, a higher level of microbial alpha diversity was observed in the beverage samples. Particularly, the predominant bacteria in beverages were Anaerocolumna and Ralstonia, while in grains Liquorilactobacillus dominated, with lower levels of Leuconostoc and Oenococcus. Although the bacterial diversity in kefir grains was low because only three genera were the most represented, all of them are LAB bacteria with the potential to serve as probiotics in the artificial feeding of bees.

Probiotics and in-hive fermentation as a source of beneficial microbes to support the gut microbial health of honey bees.

Managed populations of honey bees (Apis mellifera Linnaeus; Hymenoptera: Apidae) are regularly exposed to infectious diseases. Good hive management including the occasional application of antibiotics can help mitigate infectious outbreaks, but new beekeeping tools and techniques that bolster immunity and help control disease transmission are welcome. In this review, we focus on the applications of beneficial microbes for disease management as well as to support hive health and sustainability within the apicultural industry. We draw attention to the latest advances in probiotic approaches as well as the integration of fermented foods (such as water kefir) with disease-fighting properties that might ultimately be delivered to hives as an alternative or partial antidote to antibiotics. There is substantial evidence from in vitro laboratory studies that suggest beneficial microbes could be an effective method for improving disease resistance in honey bees. However, colony level evidence is lacking and there is urgent need for further validation via controlled field trials experimentally designed to test defined microbial compositions against specific diseases of interest.

Microbiological and Chemical characterization of water kefir: An innovative source of potential probiotics for bee nutrition / Caracterización microbiológica y química del kéfir de agua: una fuente innovadora de potenciales probióticos para la nutrición de las abejas

Abstract We evaluated the microbial composition of water kefir grains and beverage overthe course of one year to determine whether the number and type of microorganisms changedover the time. Bacteria and yeast colonies with different morphologies were isolated fromwater kefir and their antimicrobial activity was evaluated against Paenibacillus larvae andAscosphaera apis. A chemical characterization of kefir was also carried out. Our results con-firmed that bacteria and yeasts were more numerous in kefir grains compared with those in thebeverage. The counts of microorganisms declined, although an important microbial community was still present in kefir after the long storage period. Eleven strains which inhibited bee pathogens were isolated from kefir. Genotypic results demonstrated that these isolates included Lentilactobacillus hilgardii, Lentilactobacillus buchneri and Saccharomyces cerevisiae. Thus, water kefir may be an innovative source of potential probiotic strains for bee nutrition in order to control honeybee diseases.

Resumen Evaluamos la composición microbiana del kéfir de agua durante un ano para determinar si la cantidad y el tipo de microorganismos cambiaban con el tiempo. Se aislaron colonias de bacterias y de levaduras con diferentes morfologías, y su actividad antimicrobiana se evaluó frente a Paenibacillus larvaey Ascosphaeraapis. También se realizó una caracterización química del kéfir. Nuestros resultados confirmaron que las bacterias y las levaduras eran más numerosas en los gránulos de kéfir en comparación con la parte líquida. Los recuentos de microorganismos disminuyeron, aunque una cantidad igualmente importante se encontró en el kéfir después de un año. Se aislaron del kéfir once cepas que inhibieron los mencionados patógenos de abejas. Los resultados genotípicos demostraron que estos aislamientos eran Lentilactobacillus hilgardii, Lentilactobacillus buchneri y Saccharomyces cerevisiae. Por lo tanto, el kéfir de agua podría ser una fuente innovadora de potenciales cepas probióticas para contribuir a la nutrición y sanidad de las abejas.

Microbiological and chemical characterization of water kefir: An innovative source of potential probiotics for bee nutrition.

We evaluated the microbial composition of water kefir grains and beverage over the course of one year to determine whether the number and type of microorganisms changed over the time. Bacteria and yeast colonies with different morphologies were isolated from water kefir and their antimicrobial activity was evaluated against Paenibacillus larvae and Ascosphaera apis. A chemical characterization of kefir was also carried out. Our results confirmed that bacteria and yeasts were more numerous in kefir grains compared with those in the beverage. The counts of microorganisms declined, although an important microbial community was still present in kefir after the long storage period. Eleven strains which inhibited bee pathogens were isolated from kefir. Genotypic results demonstrated that these isolates included Lentilactobacillus hilgardii, Lentilactobacillus buchneri and Saccharomyces cerevisiae. Thus, water kefir may be an innovative source of potential probiotic strains for bee nutrition in order to control honeybee diseases.

Traceability of potential enterotoxigenic Bacillus cereus in bee-pollen samples from Argentina throughout the production process.

Bee-pollen is a functional food sold for human and animal consumption but also is a favorable microhabitat for many spore-forming bacteria. Among them, Bacillus cereus can produce several toxins and other virulence factors, causing an emetic or diarrheal syndrome after ingestion. The study involved 36 bee-pollen samples obtained from different sampling points throughout the production process (collecting, freezing, drying, and cleaning) in Argentina. Fifty isolates of B. cereus yielded 24 different fingerprint patterns with BOX and ERIC primers. Only three fingerprint patterns were maintained throughout the production process. In contrast, others were lost or incorporated during the different steps, suggesting that cross-contamination occurred as shown by differences in fingerprint patterns after freezing, drying, and cleaning steps compared to the initial collection step. Genes encoding for cereulide (ces), cytotoxin K (cytK), sphingomyelinase (sph), the components of hemolysin BL (hblA, hblB, hblC, hblD) and non-hemolytic complex (nheAB) were studied. All the isolates displayed one or more enterotoxin genes. The most frequent virulence genes detected belong to the HBL complex, being the most abundant hblA (98%), followed by hblD (64%), hblB (54%), and hblC (32%), respectively. Ten strains (20%), present at all sampling points, carried all the subunits of the HBL complex. The non-hemolytic enterotoxic complex (nheAB) was found in 48 strains (96%), while seven strains (14%) present at all sampling points showed the amplification product for sphingomyelinase (sph). One cereulide-producer was isolated at the cleaning step; this strain contained all the components for the hemolytic enterotoxin complex HBL, the NHE complex, and cytotoxin K related to the foodborne diarrhoeal syndrome. In total, 11 different virulence patterns were observed, and also a correlation between rep-fingerprint and virulence patterns. The results suggest that bee-pollen can be contaminated at any point in the production process with potential enterotoxic B. cereus strains, emphasizing the importance of hygienic processing.

Microbiological quality of honey from the Pampas Region (Argentina) throughout the extraction process / Calidad microbiológica de la miel en la Región Pampeana (Argentina) a lo largo del proceso de extracción

The microbiological quality of honey obtained from different processing points and the environmental quality within honey houses were assessed in the Pampas Region (Argentina). Mold and yeast (MY), culturable heterotrophic mesophilic bacteria (CHMB), the number of spore-forming bacteria as well as the presence of Shigella spp., Salmonella spp. and fecal coliforms were evaluated in 163 samples. These samples were taken from eight honey houses. Results showed that 89 samples had ≤10 CFU of MY/g honey, 69 ranged from 10 to 50 CFU/g and two reached 65.5 CFU/g. Eighty one percent of the samples showed ≤30 CFU of CHMB/g honey and only seven samples had between 50 and 54.25 CFU/g. Thirty six honey samples were obtained from drums: in 25 samples (69.4%) CHMB counts were less than ≤30 CFU/g of honey; in 20 samples (55.5%) the values of MY were between 10 and 50 CFU/g honey and total coliforms were only detected in 20 samples. Fecal coliforms, spores of clostridia as well as Salmonella spp. and Shigella spp were not detected and less than 50 spores of Bacillus spp. per g were observed in the honey from drums. Therefore, the microbiological honey quality within the honey houses did not show any sanitary risks. Our results were reported to honey house owners to help them understand the need to reinforce proper honey handling and sanitation practices.

Este estudio evaluó la calidad microbiológica de la miel dentro de varias plantas de extracción de miel y la calidad del medio ambiente de las mismas en la Región Pampeana (Argentina). Se trabajó con 163 muestras de miel provenientes de 8 plantas de extracción. Se cuantificaron hongos y levaduras, bacterias aeróbicas mesófilas, bacterias esporuladas y esporas de clostridios. Asimismo, se determinó la presencia de Salmonella spp., Shigella spp. y coliformes fecales. Los resultados mostraron que por g de miel, 89 muestras tuvieron menos de 10 UFC de hongos y levaduras, 69 tuvieron entre 10 y 50 UFC y 2 alcanzaron 65,5 UFC. Ochenta y uno por ciento de las muestras presentaron menos de 30 UFC de bacterias aeróbicas mesófilas por g de miel mientras que solo 7 tuvieron entre 50 y 54,25 UFC. Se obtuvieron 36 muestras de miel directamente de tambor: los conteos de bacterias aeróbicas mesófilas fueron ≤ 30 UFC/g de miel en 25 muestras (69,4%); los valores de hongos y levaduras estuvieron entre 10 y 50 UFC en 20 muestras (55,5%) y solo se detectaron coliformes totales. No se observaron coliformes fecales, esporas de clostridios así como tampoco Salmonella spp. y Shigella spp. y se obtuvieron menos de 50 esporas de Bacillus spp./g en miel de los tambores. Se concluye que la calidad microbiológica de la miel en las plantas de extracción no presentó riesgo sanitario. Los resultados fueron entregados a los dueños de las mismas como aporte para que valoren la importancia de reforzar la aplicación de buenas prácticas de manejo y saneamiento.

Microbiological quality of honey from the Pampas Region (Argentina) throughout the extraction process.

The microbiological quality of honey obtained from different processing points and the environmental quality within honey houses were assessed in the Pampas Region (Argentina). Mold and yeast (MY), culturable heterotrophic mesophilic bacteria (CHMB), the number of spore-forming bacteria as well as the presence of Shigella spp., Salmonella spp. and fecal coliforms were evaluated in 163 samples. These samples were taken from eight honey houses. Results showed that 89 samples had ≤10CFU of MY/g honey, 69 ranged from 10 to 50CFU/g and two reached 65.5CFU/g. Eighty one percent of the samples showed ≤30CFU of CHMB/g honey and only seven samples had between 50 and 54.25CFU/g. Thirty six honey samples were obtained from drums: in 25 samples (69.4%) CHMB counts were less than ≤30CFU/g of honey; in 20 samples (55.5%) the values of MY were between 10 and 50CFU/g honey and total coliforms were only detected in 20 samples. Fecal coliforms, spores of clostridia as well as Salmonella spp. and Shigella spp were not detected and less than 50 spores of Bacillus spp. per g were observed in the honey from drums. Therefore, the microbiological honey quality within the honey houses did not show any sanitary risks. Our results were reported to honey house owners to help them understand the need to reinforce proper honey handling and sanitation practices.