Probiotics are viable microorganisms that show beneficial effects on the health of the host by improving their intestinal microflora. The microorganisms applied as probiotics mainly include Lactobacillus and Bifidobacterium species. Probiotics can inhibit the bacterial pathogens, reduce serum cholesterol levels, improve lactose tolerance and stimulate the immune response. They also have other properties such as / tolerance to acid and bile salts, adherence to gastrointestinal cells for colonization, resistance to antibiotics and &beta / -galactosidase acitivity. The properties of probiotic products are determined by the characteristics of the microorganisms they contain. For that reason, isolation and characterization of new strains having probiotic properties is an important issue. New strains are generally isolated from their natural habitats which are fermented dairy products such as kefir. In order to exert beneficial health affects in the digestive system, commercial probiotic products should contain adequate numbers of viable cells. Probiotic microorganisms should protect their viability during their shelf storage. Therefore, the viability of probiotics is especially important for food manufacturers that search for new probiotic strains with good survival and stability properties upon storage. In this study, probiotic microorganisms were isolated from traditional kefir grains known as a &lsquo / complex probiotic&rsquo / . The isolates were firstly identified using biochemical tests, then the putative species belonging to &lsquo / Lactobacillus acidophilus group&rsquo / were identified with 16S rRNA gene sequencing. Analysis of sequencing resulted in differentiation of &ldquo / L. acidophilus group&rdquo / organisms, namely L. amylovorus and L. acidophilus. Moreover, typing of commercial and traditional L. acidophilus strains and L. amylovorus strains were performed with RAPD-PCR by using primer M13. While several L. acidophilus strains showed different RAPD fingerprints most of the L. acidophilus and L. amylovorus strains could not be differentiated due to high similarity of their RAPD fingerprints. Following identification, survival of these isolates in probiotic yogurt preparations were investigated and compared to the survival of commercial probiotics. Consequently, although the survival of kefir grain isolates were less than commercial probiotics, they sustained the minimum recommended level for probiotics (106 cfu/ml) during cold storage. Such level of survival makes them considerably good candidates to be used as commercial probiotic cultures.

Pienarūgščių bakterijų paieška ir jų identifikavimas migruojančių didžiųjų ančių (Anas platyrhynchos) žarnyne naudojant dalinių 16S rRNR geno sekų analizę ir kultivavimu paremtus metodus Klaidas VARNA Vilniaus Universiteto Ekologijos Institutas, Hidrobiontų Ekologijos ir Fiziologijos Laboratorija bei Populiacinės Genetikos Laboratorija, Akademijos-2, Vilnius-21, 08412, Lietuva. Šiame tyrime pavasarinių ir rudeninių didžiųjų ančių (Anas platyrhynchos) migrantų iš Nemuno deltos virškinamojo trakto pieno rūgšties bakterijų įvairovė buvo ištirta naudojant molekulinius metodus (polimerazės grandininės reakcijos amplifikacija ir dalinių 16S rRNR geno sekų sekvenavimas) ir kultivavimu paremtus metodus. Migruojančių didžiųjų ančių (Anas platyrhynchos) pieno rūgšties bakterijų paieška buvo atlikta pirmą kartą. Rudeniniai didžiųjų ančių migrantai plonojo žarnyno sienelėse (1.2×107 iki 2.1×107 k.f.v./g) ir jų turinyje (nuo 3.4×107 iki 1.1×108 k.f.v./g) turi didesnį pieno rūgšties bakterijų skaičių nei pavasariniai migrantai (atitinkamai nuo 3.2×106 iki 4.8×106 k.f.v./g ir nuo 1.0×107 iki 2.2×107 k.f.v./g). Tiek rudeninių tiek ir pavasarinių didžiųjų ančių migrantų plonojo žarnyno sienelėse ir jų turinyje dominavo kokinės pieno rūgšties bakterijų formos (atitinkamai 65% ir 83.5% bei 81.4% ir 91.6%), o lazdelių buvo mažiau (atitinkamai 35% ir 16.5% bei 18.6% ir 8.4%). Manoma, kad minėtus skirtumus įtakoja keli veiksniai: ilgai trunkanti migracija, perėjimo periodas, skirtingas maistas ir... [toliau žr. visą tekstą] / Identification of lactic acid bacteria in the migrant mallard ducks Anas platyrhynchos intestinal tract by partial 16S rRNA gene sequence analysis and using culture-based techniques Klaidas VARNA Institute of Ecology of Vilnius University, Laboratory of Hydrobionts Ecology and Physiology, Laboratory of Population Genetics, Akademijos-2, Vilnius-21, 08412, Lithuania. In this study the lactic acid bacteria diversity of the intestinal tract content of the vernal and autumnal migrant mallard ducks (Anas platyrhynchos) from Nemuno delta has been investigated by molecular methods: polymerase chain reaction amplification and sequencing of partial 16S rRNA genes and using culture-based techniques. The investigation of the lactic acid bacteria of the migrant mallard ducks has been performed the first time. Autumnal migrant mallard ducks in the small intestine walls (from 1.2×107 until 2.1×107 c.f.u./g) and in their content (from 3.4×107 until 1.1×108 c.f.u./g have the greatest number of the lactic acid bacteria then vernal migrants (respectively from 3.2×106 until 4.8×106 c.f.u./g and from 1.0×107 until 2.2×107 c.f.u./g). In the small intestine walls and in their content of the autumnal and vernal migrant mallard ducks, dominated cocci-shaped lactic acid bacteria (respectively 65% and 83.5%, 81.4% and 91.6%), whereas rod-shaped was under (respectively 35% and 16.5%, 18.6% and 8.4%). Supposedly, that these defferences determine some factors: a long migration, period of incubate... [to full text]

abstract: Obesity is a worldwide epidemic accompanied by multiple comorbidities. Bariatric surgery is currently the most efficient treatment for morbid obesity and its comorbidities. The etiology of obesity is unknown, although genetic, environmental, and most recently, microbiome elements have been recognized as contributors to this rising epidemic. The role of the gut microbiome in weight-loss or weight-gain warrants investigation, and bariatric surgery provides a good model to study influences of the microbiome on host metabolism. The underlying goals of my research were to analyze (i) the factors that change the microbiome after bariatric surgery, (ii) the effects of different types of bariatric surgeries on the gut microbiome and metabolism, (iii) the role of the microbiome on the success of bariatric surgery, and (iv) temporal and spatial changes of the microbiome after bariatric surgery. Roux-en-Y gastric bypass (RYGB) rearranges the gastrointestinal tract and reduces gastric acid secretions. Therefore, pH could be one of the factors that change microbiome after RYGB. Using mixed-cultures and co-cultures of species enriched after RYGB, I showed that as small as 0.5 units higher gut pH can aid in the survival of acid-sensitive microorganisms after RYGB and alter gut microbiome function towards the production of weight loss-associated metabolites. By comparing microbiome after two different bariatric surgeries, RYGB and laparoscopic adjustable gastric banding (LAGB), I revealed that gut microbiome structure and metabolism after RYGB are remarkably different than LAGB, and LAGB change microbiome minimally. Given the distinct RYGB alterations to the microbiome, I examined the contribution of the microbiome to weight loss. Analyses revealed that Fusobacterium might lessen the success of RYGB by producing putrescine, which may enhance weight-gain and could serve as biomarker for unsuccessful RYGB. Finally, I showed that RYGB alters the luminal and the mucosal microbiome. Changes in gut microbial metabolic products occur in the short-term and persist over the long-term. Overall, the work in this dissertation provides insight into how the gut microbiome structure and function is altered after bariatric surgery, and how these changes potentially affect the host metabolism. These findings will be helpful in subsequent development of microbiome-based therapeutics to treat obesity. / Dissertation/Thesis / Doctoral Dissertation Microbiology 2016

Made available in DSpace on 2015-04-20T12:31:09Z (GMT). No. of bitstreams: 1 Juliana Vianna Pereira.pdf: 4570056 bytes, checksum: c660fca0452deacfa10d23ee9bc79a2b (MD5) Previous issue date: 2009-08-26 / The complex microbiota of the oral cavity has been intensively studied and saliva is characterized by microorganisms which colonize different regions of mouth, such as tongue, supragengival and subgingival biofilm. Considering this, the purpose of this study was to evaluate the bacterial diversity of the saliva of patients with different levels of oral hygiene according to the Silness, Löe index. In this research, two genomic libraries of saliva source from 15 patients each were constructed. The pooled samples differ in the average index of Silness, Löe being considered as high or low index within the rate 1.0 to 3.0 and 0 to 0.5, respectively. The DNA saliva was extracted by phenol / chloroform method and 16S rRNA gene for the microorganisms of each sample were amplified and cloned. The sequences obtained were compared to those from sequences of the GenBank NCBI and RDP. The library resultant from saliva of patients with high level of dental biofilm showed 23 OTUs grouped as five known genus: Streptococcus, Granulicaella, Gemella, Peptostreptococcus and Veillonella besides 33.3% of uncultured bacteria. The Library made from saliva of patients with low level of dental biofilm, was significantly different from its counterpart (p = 0.000) and was composed by 42 OTUs, distributed among 11 known genus: Streptococcus, Granulicaella, Gemella, Veillonella, Oribacterium, Haemophilus, Escherichia, Neisseria, Prevotella, Capnocytophaga, Actinomyces, and 24.87% of uncultured bacteria. The genus Streptococcus was the more prevalent in the two libraries, constituting 79.08% of the first and 73.64% the second. In conclusion, patients with low dental biofilm index have saliva with higher bacterial diversity than patients with high dental biofilm index, and despite most uncultivated species aggregate with Steptococcus, they still are new and unknown microorganisms / A complexa microbiota da cavidade bucal tem sido intensivamente estudada e a saliva destaca-se por apresentar microrganismos de diferentes regiões, como a língua, biofilme subgengival e supragengival. Diante disto, o objetivo do presente estudo foi avaliar a diversidade bacteriana da saliva de pacientes com diferentes índices de higiene bucal e para isto, foram construídas duas bibliotecas genômicas da saliva, que foram constituídas por amostras de 15 pacientes cada uma, com a média de índice de biofilme de Silness; Löe diferenciado, sendo a primeira com índice de 1,0 a 3,0 (denominada alto índice) e a segunda, entre 0 a 0,5 (denominada baixo índice). O DNA da saliva foi extraído pelo método fenol/clorofórmio e o gene 16S rRNA para cada biblioteca foi amplificado e clonado. As sequências obtidas foram comparadas com aquelas armazenadas no GenBank do NCBI e RDP. A biblioteca composta pela saliva de pacientes com Alto índice de biofilme dental apresentou cinco Gêneros conhecidos: Streptococcus, Granulicaella, Gemella, Veillonella e Peptostreptococcus e 33,3% de bactérias não-cultivadas, agrupados em 23 OTUs. A Biblioteca, composta pela saliva de pacientes com Baixo índice de biofilme dental, foi diferente siguinificativamente da primeira (p=0,000) e foi composta de 42 OTUs, distribuídas em 11 Gêneros conhecidos: Streptococcus, Granulicaella, Gemella, Veillonella, Oribacterium, Haemophilus, Escherichia, Neisseria, Prevotella, Capnocytophaga, Actinomyces, alem de 24,87% de bactérias não-cultivadas. O Gênero Streptococcus foi o mais prevalente nas duas bibliotecas, constituindo 79,08% da primeira e 73,63% da segunda. Conclui-se que existe maior diversidade bacteriana na saliva de pacientes com Baixo índice de biofilme dental em relação à pacientes com Alto índice de biofilme dental e que apesar da maioria das espécies não-cultivadas agruparem-se com os Streptococcus, ainda contituem-se de microrganismos novos e desconhecidos

The giant barrel sponge, Xestospongia muta is an iconic and essential species of the coral reefs in South Florida. The sponge has primary roles providing ecosystem services and creating unique habitats for diverse microbial communities. On April 27, 2012 an outbreak of Sponge Orange Band Disease (SOB) was detected off the coast of South Florida. The disease begins with sponge bleaching, followed by mesohyl or “mesohyl” necrosis and often total mesohyl disintegration. Sampling from two diseased populations at Boynton Beach and Fort Lauderdale, FL took place on May 11th and May 29th, 2012. Each of the nine diseased sponges from Boynton Beach and the five diseased sponges from Fort Lauderdale had three separate mesophyl samples collected to examine the effects of disease progression on the microbial community. These included healthy mesohyl from a diseased sponge (HoD), the boundary layer which captured the advancing line of diseased mesohyl (BL) and diseased mesohyl from a diseased sponge (D). Mesohyl from three sponges with no visible signs of SOB disease were also collected from each sampling location to use for healthy controls (HC). Sequencing of the V4 region of the 16S rRNA gene was performed on all of these samples via the “454” pyrosequencing on a Titanium GS FLX platform. The microbial communities associated with the diseased samples revealed a microbiome shift that followed the progression of Sponge Orange Band Disease (SOB) and was dominated by Bacteroidetes, Protebacteria and Chloroflexi. No singular or group of microbes were solely found within the infected mesohyl of Xestospongia muta from both sampling site populations; therefore there is no unequivocal candidate as a definite microbial causative SOB agent. But there were bacteria associated with disease progression that included Armatimonadetes, Caldithrix, Chlorobi, Fibrobacteres, Fusobacteria, GN02, KSB3, OP1, OP2, OP8, Planctomycetes, SR1, TM6, Tenericutes, Verrucomicrobia, WPS-2 and ZB3. Verrucomicrobia and Plantomycetes increased significantly within the D and the BL populations, which was consistent within all the diseased sponges. This study provides a deep sequencing profile of microbial communities within Xestospongia muta affected with SOB Disease and provides a new insight into the sponge healthy microbiome.

Doctor of Philosophy / Division of Biology / Brett K. Sandercock / Gastrointestinal microbiota play a vital role in maintaining organismal health, through facilitating nutrient uptake, detoxification and interactions with the immune system. Shorebirds vary widely in life-history characteristics, such as habitat, migration and breeding system, but the dynamics of their gut microbial communities are unknown. In my dissertation, I investigated composition and dynamics of gut microbiota in migratory shorebirds from embryos to 10 day old chicks, and determined environment and host-related factors affecting gut microbial communities of adults. First, I tested whether precocial chicks from three species of arctic-breeding shorebirds acquire gut microbiota before or after hatching using next-generation sequencing. In addition, I documented the dynamics of gut microbial establishment. I showed that gut microbiota were absent in shorebird embryos before hatching, but that stable gut communities established within the first three days after hatching. In addition, gut microbiota of young shorebird chicks were more similar to the environmental microbiome than later in life, suggesting that the environment is a likely source for microbial recruitment. After reaching adulthood, shorebirds migrate long distances, potentially exposing them to a wide range of microorganisms. Host phylogeny and environmental factors have both been identified as drivers of gut microbiota composition in birds in previous studies. The second part of my project aimed to compare the relative importance of host and environmental factors that underlie variation in gut microbiota composition in eight species of migratory shorebirds sampled across the North American Arctic. I found that sampling site was the main driver of variation in gut microbiota of Arctic-breeding shorebirds, and that site-related variation in gut microbiota of shorebirds was a result of differences in core bacterial taxa that occurred in more than half of the analyzed samples. A relatively large influence of local environment on gut microbiota composition of chicks and adults lead to the question: how does site affect pathogen prevalence in shorebirds? Migratory behavior has been hypothesized to have evolved as a response to variation in climatic conditions and food availability, to avoid predation, and to reduce risk of exposure to pathogens. The migratory escape hypothesis predicts avoidance of high disease prevalence areas through migration, and has been proposed as one of the main reasons that many bird species migrate to the Arctic for breeding. To test the migratory escape hypothesis in shorebirds, I screened for prevalence of seven known avian pathogens in shorebirds at different stages of migration. I did not detect the majority of pathogens we tested for, with the exception of Campylobacter jejuni and C. coli. Prevalence of C. jejuni in shorebirds was linked to sampling sites but not shorebird species. My dissertation is the first comprehensive study to broadly characterize the gut microbiota in shorebirds. Overall, local environment emerged as an important factor in shaping microbiota composition in Arctic-breeding shorebirds throughout my dissertation research. The role of local environment in shaping gut microbiota invites future investigations of the interactions among shorebirds and the microorganisms present in their environment, as well as the functions gut microbiota perform within their shorebird hosts.

The main goal of this thesis was to design and implement a tool that would be able to classify the sequences of the 16S rRNA gene into taxonomic categories using the properties of the 16S rRNA gene. The created tool analyzes all input sequences simultaneously, which differs from common classification approaches, which classify input sequences individually. This tool relies on the fact that bacteria contain several copies of the 16S rRNA gene, which may differ in sequence. The main contribution of this work is design, implementation and evaluation of the capabilities of this tool. Experiments have shown that the proposed tool is able to identify the corresponding bacteria for smaller datasets and determine the correct ratios of their abundances. However, with larger datasets, the state space becomes very large and fragmented, which requires further improvements in order for it to search the state space in an efficient way.

Seventeen bacterial strains were isolated from 9 marine mud samples from the inshore environments of the East Sea. Four bacterial strains showed an inhibition against all tested microorganisms Staphylococcus aureus ATCC10832, Escherichia coli JM109, and Fusarium oxysporum. 16S rRNA sequences of four bacterial strains were obtained by PCR using specific primers. PCR products were cloned into E. coli DH5a using pJET1.2 blunt vector. The recombinant plasmids were sequenced and the lengths of these 16S rRNA sequences were ~930bp. The 16S rRNA sequence from the four bacterial DB1.2, DB1.2.3, DB4.2 and DB5.2 strain showed a high identity of 97 to 99% with the 16S rRNA sequence from Photobacterium sp., Oceanisphaera sp., Shigella sp., Stenotrophomonas sp, respectively. / Mười bảy chủng vi khuẩn đã được phân lập từ 9 mẫu bùn biển từ các vùng ven bờ biển Việt Nam. Bốn chủng vi khuẩn được ghi nhận có khả năng ức chế mạnh sự sinh trưởng và phát triển của các chủng vi khuẩn Staphylococcus aureus ATCC10832, Escherichia coli JM109, và thậm chí cả nấm Fusarium oxysporum. Trình tự gene 16S rRNA của bốn chủng vi khuẩn này đã được khuếch đại bằng PCR sử dụng cặp mồi đặc hiệu. Sản phẩm PCR được nối ghép vào vector pJET1.2 blunt sử dụng T4 ligase, hình thành plasmid tái tổ hợp và biến nạp vào E. coli DH5α. Khuẩn lạc có plasmid mang phân đoạn DNA chèn được nuôi cấy và tách plasmid. Trình tự 16S rRNA từ 4 chủng DB1.2, DB1.2.3, DB4.2 and DB5.2 chỉ ra có sự tương đồng 97 ÷ 99% so với trình tự 16S rRNA tương ứng của các chủng vi sinh vật biển trên ngân hàng gene thế giới là Photobacterium sp., Oceanisphaera sp., Shigella sp., và Stenotrophomonas sp.

The overall aim of this study was to investigate the potential influence of chitosan, a biodegradable and antimicrobial compound, on termite hindgut symbionts. For this purpose, a morphological quantifying technique was conducted on the protist community’s hindgut after feeding termites on chitosan-treated wood. The aim was to characterize the diversity of protist species in the economically important dark southern subterranean termite, Reticulitermes virginicus. A molecular phylogenetic analysis of the V3 and V4 hyper-variable regions of 16S ribosomal RNA (rRNA) gene of the bacterial community in the hindgut of R. virginicus was performed on termites exposed to chitosan treatment. Light microscopy visualization of protist species residing in the hindgut of workers showed presence of ten protist species both in the control sample and in termites fed a low concentration of chitosan. In this study, the coexistence of two species of the genus Trichonympha (T. agilis and T. burlesquei) is reported for the first time in R. virginicus. Monocercomonas sp. and Trichomitus trypanoides were the only two protists found in termites exposed to wood treated with higher chitosan concentration solutions and the absence of wood fragments in their food vacuoles was clear. This feature indicates that these two protists may not be involved in the digestion of the wood fragments impregnated with chitosan. The results of this study indicated that the potential effect of chitosan caused elimination of the protist species in termite hindguts. The genomic DNA of bacterial hindgut community of R. virginicus were profiled using sequences which amplified theV3-V4 sub-regions of 16S rRNA gene. Sequences were analyzed using a taxonomic analysis tool, Quantitative Insights Into Microbial Ecology (OIIME 2), in order to infer the effect of chitosan on the composition of the bacterial fauna in the hindgut. The richness and evenness results indicated that the most diversity was observed in the bacteria from termites not being exposed (UNX) to treatment compared to other treatment groups. On the other hand, the lowest richness and evenness were determined for chitosan-treated wood (CTE) and starved termites (STV). Of 28 bacterial phyla, Bacteroidetes, Firmicutes, Elusimicrobia, and Proteobacteria were the most dominant phyla across all the treatment groups. The results suggest that chitosan treated wood led to the microbial community shifts in R. virginicus. In addition, lack of a nutrition source and other changes in termite’s food affect the termite hindgut bacterial diversity.

This dissertation documents efforts to evaluate metabolic variables and the fecal microbiome in adult horses fed grass hay. In the first study, eight Arabian geldings limit-fed an 18% vs. 12% non-structural carbohydrate (NSC) hays in a cross-over design during two 28-day periods were included to evaluate the influence of grass hay NSC on serum insulin and plasma glucose concentrations. Serum insulin concentrations was higher in geldings fed the 18% NSC hay; however, this difference was only detected on day 7 and none of the geldings developed hyperinsulinemia. Blood glucose concentrations did not differ between hay groups. The second and third studies were extensions of the first and were conducted to use denaturing gradient gel electrophoresis (DGGE) and real-time PCR in evaluating the effect of forage carbohydrates on equine fecal bacteria diversity and abundance, respectively. Fecal microbiomes were similar (80.5-87.9%) between geldings. The abundance of bacteria belonging to the Firmicutes phylum increased (p = 0.02) in the feces of geldings fed 12% NSC hay (mean 8.06 range [8.03-8.11] log10 copies/g feces) compared to the feces of the same geldings when fed the 18% NSC hay (7.97 [7.97-7.98] log₁₀ copies/g feces). The Firmicutes (43.7%), Verrucomicrobia (4.1%), Proteobacteria (3.8%), and Bacteroidetes (3.7%) phyla dominated the fecal microbiomes. This work was the first to report the presence of the Actinobacteria, Cyanobacteria, and TM7 phyla in the equine fecal or gut microbiome. There was a high abundance (38%) of unclassified bacterial sequences in the gelding fecal microbiome. In the fourth study, 5 overweight adult mixed-breed mares and 5 adult mixed-breed mares in moderate condition, limit-fed a grass hay, were used to evaluate the effect of body condition on diet digestibility, plasma and fecal volatile fatty acid (VFA) concentrations, and fecal bacterial abundance. Hay, fecal, and blood samples were taken daily for 4 days after a 10 day adaptation period. A difference in hay digestibility, fecal VFA concentration, or bacterial abundance was not detected between overweight mares and mares in moderate condition. Plasma acetate, a product of microbial fermentation of fiber, was higher in the overweight mare group. / Ph. D.