RESUMO
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.
Assuntos
Bactérias , Kefir , Metagenômica , Probióticos , RNA Ribossômico 16S , Animais , Abelhas/microbiologia , Kefir/microbiologia , RNA Ribossômico 16S/genética , Metagenômica/métodos , Bactérias/isolamento & purificação , Bactérias/genética , Bactérias/classificação , DNA Bacteriano/análise , Biodiversidade , DNA Ribossômico/genética , Ração Animal/microbiologiaRESUMO
Two species of Spiroplasma (Mollicutes) bacteria were isolated from and described as pathogens of the European honey bee, Apis mellifera, ~30 years ago but recent information on them is lacking despite global concern to understand bee population declines. Here we provide a comprehensive survey for the prevalence of these two Spiroplasma species in current populations of honey bees using improved molecular diagnostic techniques to assay multiyear colony samples from North America (U.S.A.) and South America (Brazil). Significant annual and seasonal fluctuations of Spiroplasma apis and Spiroplasma melliferum prevalence in colonies from the U.S.A. (n = 616) and Brazil (n = 139) occurred during surveys from 2011 through 2013. Overall, 33% of U.S.A. colonies and 54% of Brazil colonies were infected by Spiroplasma spp., where S. melliferum predominated over S. apis in both countries (25% vs. 14% and 44% vs. 38% frequency, respectively). Colonies were co-infected by both species more frequently than expected in both countries and at a much higher rate in Brazil (52%) compared to the U.S.A. (16.5%). U.S.A. samples showed that both species were prevalent not only during spring, as expected from prior research, but also during other seasons. These findings demonstrate that the model of honey bee spiroplasmas as springtime-restricted pathogens needs to be broadened and their role as occasional pathogens considered in current contexts.
Assuntos
Abelhas/microbiologia , Spiroplasma/isolamento & purificação , Animais , Carga Bacteriana , Brasil , Estações do Ano , Spiroplasma/classificação , Spiroplasma/genética , Estados UnidosRESUMO
Paenibacillus larvae is a gram-positive spore-forming bacteria, causative agent of American Foulbrood (AFB), a severe disease affecting larvae of the honeybee Apis mellifera. In an attempt to detect potential virulence factors secreted by P. larvae, we identified an enolase among different secreted proteins. Although this protein is a cytosolic enzyme involved in glycolytic pathways, it has been related to virulence. The aim of the present work was to evaluate its role during the infection of honeybee larvae. Toxicity assays showed that enolase was highly toxic and immunogenic to honeybee larvae. Its production was detected inside P. larvae vegetative cells, on the surface of P. larvae spores and secreted to the external growth medium. P. larvae enolase production was also confirmed in vivo, during the infection of honeybee larvae. This protein was able to hydrolyze milk proteins as described for P. larvae, suggesting that could be involved in larval degradation, maybe through the plasmin(ogen) system. These results suggest that P. larvae enolase may have a role in virulence and could contribute to a general insight about insect-pathogen interaction mechanisms.
Assuntos
Abelhas/microbiologia , Paenibacillus/enzimologia , Fosfopiruvato Hidratase/metabolismo , Fatores de Virulência/metabolismo , Animais , Abelhas/efeitos dos fármacos , Abelhas/imunologia , Imunofluorescência , Soros Imunes/análise , Hibridização in Situ Fluorescente , Larva/efeitos dos fármacos , Larva/microbiologia , Proteínas do Leite/metabolismo , Fosfopiruvato Hidratase/genética , Fosfopiruvato Hidratase/isolamento & purificação , Fosfopiruvato Hidratase/toxicidade , Esporos Bacterianos/enzimologia , Fatores de Virulência/genética , Fatores de Virulência/isolamento & purificação , Fatores de Virulência/toxicidadeRESUMO
American Foulbrood is a severe disease affecting larvae of honeybee Apis mellifera, causing significant decrease in the honeybee population, beekeeping industries and agricultural production. In spite of its importance, little is known about the virulence factors secreted by Paenibacillus larvae during larval infection. The aim of the present work was to perform a first approach to the identification and characterization of P. larvae secretome. P. larvae secreted proteins were analyzed by SDS-PAGE and identified by MALDI-TOF. Protein toxicity was evaluated using an experimental model based on feeding of A. mellifera larvae and immunogenicity was evaluated by Western blot, using an antiserum raised against cells and spores of P. larvae. Ten different proteins were identified among P. larvae secreted proteins, including proteins involved in transcription, metabolism, translation, cell envelope, transport, protein folding, degradation of polysaccharides and motility. Although most of these proteins are cytosolic, many of them have been previously detected in the extracellular medium of different Bacillus spp. cultures and have been related to virulence. The secreted proteins resulted highly toxic and immunogenic when larvae were exposed using an experimental model. This is the first description of proteins secreted by the honeybee pathogen P. larvae. This information may be relevant for the elucidation of bacterial pathogenesis mechanisms.
Assuntos
Proteínas de Bactérias/metabolismo , Abelhas , Paenibacillus/patogenicidade , Animais , Proteínas de Bactérias/toxicidade , Abelhas/efeitos dos fármacos , Abelhas/microbiologia , Feminino , Larva/efeitos dos fármacos , Camundongos , Paenibacillus/enzimologia , Paenibacillus/metabolismo , Peptídeo Hidrolases/metabolismo , Fatores de Virulência/metabolismoRESUMO
Paenibacillus larvae is the causative agent of American Foulbrood (AFB), the most severe bacterial disease that affects honeybee larvae. AFB causes a significant decrease in the honeybee population affecting the beekeeping industry and agricultural production. After infection of larvae, P. larvae secretes proteases that could be involved in the pathogenicity. In the present article, we present the secretion of different proteases by P. larvae. Inhibition assays confirmed the presence of metalloproteases. Two different proteases patterns (PP1 and PP2) were identified in a collection of P. larvae isolates from different geographic origin. Forty nine percent of P. larvae isolates showed pattern PP1 while 51% exhibited pattern PP2. Most isolates belonging to genotype ERIC I - BOX A presented PP2, most isolates belonging to ERIC I - BOX C presented PP1 although relations were not significant. Isolates belonging to genotypes ERIC II and ERIC III presented PP2. No correlation was observed between the secreted proteases patterns and geographic distribution, since both patterns are widely distributed in Uruguay. According to exposure bioassays, isolates showing PP2 are more virulent than those showing PP1, suggesting that difference in pathogenicity could be related to the secretion of proteases.
Assuntos
Abelhas/microbiologia , Infecções por Bactérias Gram-Positivas/veterinária , Larva/microbiologia , Metaloproteases/metabolismo , Paenibacillus , Animais , Infecções por Bactérias Gram-Positivas/enzimologia , Infecções por Bactérias Gram-Positivas/microbiologia , Interações Hospedeiro-Patógeno , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Larva/enzimologia , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Paenibacillus/enzimologia , Paenibacillus/genética , Paenibacillus/patogenicidade , Virulência , Fatores de VirulênciaRESUMO
This work describes the first molecular-genetic evidence for viruses in Brazilian honey bee samples. Three different bee viruses, Acute bee paralysis virus (ABPV), Black queen cell virus (BQCV), and Deformed wing virus (DWV) were identified during a screening of RNAs from 1920 individual adult bees collected in a region of southeastern Brazil that has recently shown unusual bee declines. ABPV was detected in 27.1% of colony samples, while BQCV and DWV were found in 37% and 20.3%, respectively. These levels are substantially lower than the frequencies found for these viruses in surveys from other parts of the world. We also developed and validated a multiplex RT-PCR assay for the simultaneous detection of ABPV, BQCV, and DWV in Brazil.