Abstract We designed a 16S rRNA gene PCR-RFLP scheme to identify all currently described Bartonella spp. The 16S rRNA genes of all Bartonella spp. were in-silico analyzed in order to design a RFLP technique able to discriminate among different species. The restriction enzymes selected were MaeIII, MseI, Sau96I, BsaAI, DrdI, FokI, BssHII, BstUI, AluI, TspDTI and HphI which, according to a decision-making tree, facilitated the differentiation of all the currently described species of Bartonella.The technique was experimentally tested in different species of Bartonella, including human pathogenic B. bacilliformis and B. henselae with a 100% of concordance with the in-silico predicted patterns.This novel RFLP assay could be used to identify both human and non-human pathogenic Bartonella in diagnostic, phylogenetic and epidemiologic studies.

Parodontitis ist eine Erkrankung des Zahnhalteapparates, die durch einen komplexen bakteriellen Biofilm unterhalten wird. Neben Mikroorganismen wie A. actinomycetemcomitans, P. gingivalis, T. denticola und T. forsythensis werden Keime unbekannter Spezies in parodontalen Taschen ausfindig gemacht. Durch die Entschlüsselung von 16S rRNA-Gensequenzen konnte die orale Flora nahezu vollständig katalogisiert werden. Allerdings fehlen bei vielen Phylotypen die entsprechenden Typstämme für weitergehende phänotypische Analysen. Grundlage dieser Arbeit bildeten 59 Patientenisolate der Parodontitis-Stammsammlung des Instituts für Hygiene und Mikrobiologie bei denen partielle 16S rRNA Sequenzen keine Spezieszuordnung ermöglichten. Nahezu vollständige 16S rRNA-Sequenzen wurden erstellt und mit Datenbankeinträgen verglichen. Bei mehr als der Hälfte der Stämme konnte keine taxonomische Zuordnung auf Sequenzierebene getroffen werden. 43 Isolate wuchsen unter aerober Atmosphäre, 16 benötigten eine anaerobe Umgebung. Alle Kulturmorphologien und nach Gram gefärbten mikroskopischen Präparate wurden fotografisch dokumentiert und katalogisiert. Die hier untersuchten Stämme, die zufällig auf der Basis taxonomischer Fragestellungen ausgewählt wurden, waren zum überwiegenden Teil auf Amoxicillin und Metronidazol empfindlich. Diese Antibiotika finden alle ihre Verwendung bei der Parodontitistherapie. Ciprofloxacin, das wegen seiner intrazellulären Wirkung ein interessantes Agens ist, wies v.a. bei Actinomyceten und Streptokokken Wirkungslücken auf. Es bleibt zu diskutieren, ob dieser Umstand nachteilig ist, da auf der einen Seite diese Genera ein orales Reservoir für Gyrasehemmer-Resistenzen ausbilden können, auf der anderen Seite diese grampositiven Keime möglicherweise parodontalprotektiv wirken könnten. In dieser Studie konnte eine Stammsammlung charakterisiert werden, die zukünftig insbesondere angesichts der zu erwartenden Entschlüsselung des oralen Metagenoms für weitere funktionelle Untersuchungen von Interesse sein dürfte. / The aim of this study was the characterisation of the subgingival periodontal microbiota by cultural and molecular methods. Periodontitis is a bacterial disease, which is caused by different periodontal pathogens like Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Treponema denticola and Tannerella forsythensis. Furthermore there exist some species in the complex subgingival biofilm that are not cultured yet. They may participate in the progression or persistence of periodontal destruction. In this paper 59 Plaque samples were obtained from a previous study of the Institute for Hygiene and Microbiology from the University of Würzburg where the comparison of subgingival microorganisms with public databases didn´t allow a species identification. Almost complete 16S rRNA sequences were provided and compared with database entries. More than half of the bacterial isolates didn’t permit a taxonomic allocation. Macroscopic and microscopic features, the susceptibility against amoxicillin, ciprofloxacin and metronidazole are described here. 43 isolates grew under aerobic conditions, 16 needed anaerobic atmosphere. The morphology of the culture and the colouring according to Gram were documented photographically and listed. Most tested microorganisms were susceptible to amoxicillin and metronidazole. Except for Actinomyces and Streptococcus ciprofloxacin exhibits good results in the treatment of periodontal disease. In this study a collection of subgingival bacteria could be characterized, which might be of interest for further investigations in view of the decoding of the oral metagenome.

Optimizing the production of useful macromolecules from transgenic microorganisms is crucial to biopharmaceutical companies. Improving bacterial growth and replication depends largely on the efficiency of translation, which is rate-limited by initiation. Among the most important interactions between the mRNA translation initiation region (TIR) and the translation machinery is the association between the Shine-Dalgarno (SD) sequence in the TIR and the complementary anti-SD (aSD) sequence which is located within a short unstructured segment that includes the 3’ terminus (3’ TAIL) of the mature 16S rRNA. However, the mature 3’ TAIL has been poorly characterized in the majority of bacteria, rendering optimal SD/aSD pairing unclear in these species. In light of this, we established a novel strategy to characterize the mature 3’ TAILs of bacterial species that leverages the availability of publically stored RNA sequencing (RNA-Seq) data. In chapter 2, we devised a RNA-Seq-based approach to successfully recover the experimentally verified 3’ TAIL in E. coli (5’-CCUCCUUA-3’) and resolve inconsistencies surrounding the identity of the 3’ TAIL in Bacillus subtilis. In chapter 3 we improve the method introduced in chapter 2 to clearly and more reliably define the 3’ TAIL termini for 13 bacterial species with available protein abundance data. Our results reveal considerable heterogeneity in the termini of 3’ TAILs among closely related species and that sites downstream of the canonical CCUCC aSD motif are more important to initiation than previously believed. My research contributes to advance our understanding in microbial translation efficiency in two significant ways: 1) providing an RNA-Seq-based approach to characterize rRNA transcripts, and 2) elucidating optimal recognition between protein-coding genes and the rRNA translation machinery.

Infections of the cow udder leading to mastitis and lower milk quality are a critical challenge facing northeast organic dairy farmers. Limited mastitis treatment options are available to organic producers and bedding systems impact cow health, including mastitis risk. Composted bedded pack, a system touted for increased cow comfort and well-being, allows stratified accumulation of bedding and manure in the barn. This method is gaining popularity among organic producers, yet little is known about the microbiota of the accumulated pack and its interaction with the cow mammary gland. An in-depth single farm study was conducted that surveyed bedded pack (microbiome and microarthropod community), dipteran vectors of bacterial mastitis pathogens, and the teat skin and teat cistern milk microbiomes. Comparisons were made with four additional farms utilizing bedded packs to test generality of results. Few fly pests were observed in the bedded pack. However, bedding on all farms was found to harbor the mesostigmatid mite genus Glyptholaspis, a well-established predator of nematodes and muscid fly larvae, suggesting that predators may suppress populations of biting flies in bedded pack barns. Additionally, the fungivorous genus Rhizoglyphus was commonly abundant in all farms, suggesting that the mite community regulates microbial activity at multiple trophic levels. High-throughput sequencing of universal marker genes for bacterial and fungal communities was used to characterize the skin and milk microbiome of cows with both a healthy and infected quarter on the case study farm, and the composted bedded pack of all five farms. The bedded pack microbiome varied with bedding material and management style; fungal taxa were primarily yeasts of the Ascomycota; all farms additionally contained anaerobic fungi associated with the bovine rumen. Common bacterial genera included Acinetobacter and Pseudomonas, both of which were also commonly observed on teat skin and in milk. The udder microbiome varied through time and between skin and milk. Both healthy and infected milk microbiomes reflected a diverse group of microbial DNA sequences. Health status of the quarter changed whether taxa were shared between the teat skin, milk, and bedding. Proportion of taxa shared between healthy milk and skin was stable while taxa shared in infected quarters varied widely. Taxa shared among all habitats included yeast genus Debaryomyces and bacteria Acinetobacter guillouiaea. Results support an ecological interpretation of both the udder and the bedded pack environment and support the notion that mastitis can be described as an imbalance of the healthy mammary gland microbiome. Future work might compare udder health between common bedding practices, investigating the impact of bedding on the microbiota of the mammary gland in the healthy and diseased state.

Abstract Acetes is a kind of macroholozooplankton. We can use the "genetic analysis to know its evolution, zooplankton population structure and taxa. The step is :ampified the 16S rDNA of the mitochondrial DNA and the COI fragment DNA in Acetes, and then sequence. We get the genetic distance between species of the 16S rDNA is 2.65~13.16%, and it's 1.43~25.42% of the COI fragment gene in Acetes. These results show that the diversity of COI fragment DNA in Acetes is more than the 16S rDNA of the mitochondria DNA. If we translated the COI gene DNA to amino acid sequence, the genetic distance between species of the amino acid sequence is about 1.83%~8.97%. By the way, we know that the genetic distance between species of the amino acid sequences are clearly less then the DNA sequences. Using the genetic data, we constructure a phylogenetic tree. The tree divided the Acetes into two group : Japonicus group and Erythraeus group, we can find the Acetes japonicus is the most permitive species and the A. erythraeus is the last specation species in the Acetes under the DNA sequences. But under the tree which was made from the amino acid sequence, we found the Acetes erythraeus is the most permitive species in the Acetes. The genetic distance between A. intermedius and A. erythreaus were 5.44% and 18.01% of the 16S rDNA and COI gene which indicated that the A. intermedius is true species. The population of A. japonicus between Japon Sea and Taiwan showed 0.24% and 2.36% of the 16S rDNA and COI gene that reveled two population should have gene-flow. The populations diversity of the A. intermedius in Taiwan showed the same way which had no different and the populations in Taiwan should be a single population.

Abstract Recent researches have pointed out that most of marine bacteria are uncultivable. However, majority of prior researches about bacteria flora in cultivated ponds used cultivating method to researches. That means those researches ignored uncultivable bacteria in ponds and caused inaccuracy in counting bacteria. Therefore we used analyzing bacteria 16S rDNA sequences to study composition of bacteria in cultivated ponds in place of traditional bacteria taxonomy. The phylogenetic diversity of bacteria examined by analyzing the 16S rDNA sequences permits the characterization of environmental bacteria community without culturing and has been used widely. This research is to adopt both MMA medium cultivation and direct recovery of bacteria 16S rDNA sequences to investigate bacteria flora in a closed Penaeus monodon pond. We sampled from A2 (10¡Ñ8¡Ñ1.5m) test pond at the Department of Marine Resource in Sun Yat-Sen University on August 17, 1999. Then, we adopted AO (Acridine Orange) epifluorescent microscopic technique to count total direct count (TDC) and direct count of viable bacteria cell (DVC). Respective results were 2.846¡Ñ107/ml, 1.029¡Ñ107/ml, and plate count (PC) determined by MPN count method were 1.130¡Ñ105cfu/ml. In the part of cultivable bacteria, they could be separated into 9 groups by their morphologic after culturing in MMA plate at 25¢Jfor 5 days. We isolated 15 strains to analyze their 16S rDNA sequences, and separated respectively into 4 groups after comparing with the genebank. Those four groups are CFB group, low G+C Gram-positive bacteria, Alpha proteobacteria and Gamma proteobacteria. The genus Vibrio (47%) in the Gamma proteobacteria group is the dominant. In the part of uncultivable bacteria, we filtered bacteria from the water in the same pond, amplified the 16S rDNA by the polymerase chain reaction (PCR) and then cloned. After that, we randomly isolated 40 clones for sequence analysis. The bacteria belong to following groups, cyanobacteria, CFB group, Verrucomicrobia, Gram-positive eubacteria, Alpha proteobacterium, Beta proteobacterium, Gamma proteobacterium and Delta proteobacterium.

The purpose of this study was first to describe the clinical findings, case management and short-term outcome of horses with septic synovial structures over the last 25 years, and to identify risk factors and treatment modalities associated with specific short-term outcomes. Secondly, we wanted to evaluate a broad range real time polymerase chain reaction (RT PCR) assay for the diagnosis of septic synovitis from synovial fluid (SF) samples of horses, and compare its performance to currently available diagnostic methods. For the retrospective study, 367 cases met the inclusion criteria. Lavage of the synovial structure and endoscopic surgery were associated with an increased likelihood of discharge from hospital, however, none of the local antimicrobial delivery modalities were associated with a significant change in outcome. No significant improvement in hospital outcome of horses with septic synovitis was identified over the past 25 years. For the RT PCR study, 48 SF samples from horses with suspected septic synovitis were included, and RT PCR and microbial culture was performed on all samples. One additional RT PCR assay was performed on samples with discordant results or identification of dissimilar organisms. Thirty-eight percent of SF samples had positive culture results, and 42% of SF samples had positive RT PCR results. Sensitivity and specificity for the RT PCR assay relative to agreement of observers on the likelihood of infection were 87% and 72%, respectively, whereas for culture they were 56% and 86%, respectively (P=0.001). The combination of culture and RT PCR assay resulted in sensitivity and specificity of 85% and 81%, respectively. The broad range RT PCR assay was more sensitive than culture for identification of horses with septic synovitis. Further refinement of the RT PCR assay technique may facilitate use in a clinical setting. / Equine Guelph, University of Guelph

The gastrointestinal (GI) tract of cats and dogs is inhabited by many different types of microorganisms, known as the GI microbiota. Mounting evidence suggests that the administration of certain dietary and/or therapeutic agents can alter the composition and activity of the GI microbiota, thus influencing gastrointestinal health and disease. The aim of this study was to evaluate the gastrointestinal microbiota in response to dietary and therapeutic interventions in cats and dogs. A multi-species synbiotic formulation, containing a total of 5x109 colony forming units of a mixture of seven probiotic bacterial strains and a blend of prebiotics, was administered daily for 21 days to healthy cats and dogs. Fecal samples were collected before, during, and up to three weeks after discontinuation of the administration of the synbiotic. The fecal microbiota was analyzed using 454-pyrosequencing, denaturing gradient gel electrophoresis, quantitative real-time PCR, and 16S rRNA gene clone libraries. The results showed that the synbiotic led to increased concentrations of probiotic bacteria in the feces but did not alter the predominant bacterial phyla. Additionally, we investigated the effect of age, body weight, and baseline abundance of probiotic related bacterial genera, as potential predictors of intestinal colonization by the ingested microorganisms. The results suggested that cats having a low abundance of fecal probiotic genera before consuming probiotics may have a higher concentration of the probiotic groups in feces during consumption of the symbiotic formulation. Also, a proton-pump inhibitor, aimed at suppressing the secretion of gastric acid, was administered daily for 15 days to healthy dogs. Changes in the GI microbiota were analyzed using 454-pyrosequencing, fluorescent in situ hybridization, and quantitative real-time PCR. The results suggested that inhibition of gastric acid secretion can alter the abundance of several gastric, duodenal, and fecal bacterial groups. However, these changes were not associated with major qualitative modifications of the overall composition of the GI microbiota. These studies showed that dietary and therapeutic agents can alter the composition of the GI microbiota and suggest that these changes could be associated with particular characteristics of the host. The clinical significance of these results needs further investigation.

Arsenic in aquifers poisons more than 100 million people in Asia alone, as aquifers remain the primary source of water for drinking and farming. Previous studies have suggested a link between the mobilisation of arsenic in aquifers and biochemical processes. As a result of the complex interaction of microbes with arsenic bearing minerals, the relatively immobile arsenate [As(V)] is reduced to labile and more soluble arsenite [As(III)] in aquifers, resulting in elevated concentrations of the metalloid. The numerous microbial communities capable of multiple-metabolic activities colonising these arsenic impacted aquifers mean that the exact mechanism of arsenic mobilisation in aquifers remains poorly understood. To resolve this ambiguity, this study undertakes a combination of metaomic, geochemical, and statistical analyses of 75 aqueous and sediment samples (three sample sets) from 3 transects with arsenic impacted aquifers in Bangladesh and Cambodia. Key geochemical and physical properties including arsenic speciation, iron speciation, mineral and elemental compositions, pH and Eh were recorded using the state-of-the art techniques of XANES, XRF, ICP-MS and other in situ techniques. Next generation sequencing (NGS) platforms such as MiSeq, HiSeq, Nextseq and Pyrosequencing, were used to sequence and analyse DNA and RNA extracted from field samples, allowing characterisation the extent bacterial communities, including any arsenic related genes and transcripts found in these arsenic impacted aquifers. The biogeochemical findings suggest that direct As redox transformations are central to arsenic fate and transport, and that there is a residual reactive pool of both As(V) and Fe(III) in deeper sediments that could be released by microbial respiration in response to hydrologic perturbation, such as increased groundwater pumping that introduces reactive organic carbon to depth. The main findings of this molecular investigation are (i) the most abundant bacterial species belonging to the families of Comamonadaceae, Moraxellaceae, Rhodocyclaceae, Gallionellaceae etc, not known for dissimilatory arsenic reduction, might possess arrA genes and thus have the potential to mobilise arsenic through dissimilatory arsenate reduction; (ii) the bacterial community structure revealed through 16S rRNA gene based sequencing and analysis, resembles the family level community structure revealed through the WGS based community analysis; (iii) although arsenic resistant genes are found in many organisms, they are transcribed only in a few organisms; (iv) the application of O2-PLS analyses may be useful for not only identifying novel organisms associated with key biogeochemical process, but also has clear potential to predict the physical/chemical environment in situ associated with microbial samples via community profiling. In conclusion, the results obtained from this study help establish the identity of microorganisms potentially playing a role in arsenic mobilisation in aquifers, and help decipher the underpinning mechanisms. This deeper level of understanding will in turn help to better target measures that can be applied to arsenic mitigation.

Made available in DSpace on 2015-02-27T17:39:00Z (GMT). No. of bitstreams: 2 39.pdf: 933474 bytes, checksum: 32d444b6fff1f4ea75d1d816d67c7aa6 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2011 / Fundação Oswaldo Cruz. Instituto Nacional de Controle de Qualidade em Saúde / Os produtos que requerem a característica de esterilidade devem ser submetidos ao Ensaio de Esterilidade Bacteriana e Fúngica assim como o ambiente de execução desse teste, a fim de evitar resultados falso-positivos. A legislação recomenda a identificação de microrganismos provenientes dos Ensaios e do ambiente onde estes foram realizados. A dificuldade da identificação de vários gêneros por metodologias fenotípicas e identificações equivocadas de bastonetes Gram positivos esporulados (BGPE) têm sido relatadas em vários estudos e mostram a necessidade da utilização de metodologias moleculares mais adequadas a essa identificação. Para tal, a análise da sequência do gene 16S rRNA tem sido promissora uma vez que este gene é universal para bactérias e está disponível em bancos de dados em uma grande quantidade de sequências permitindo, assim, a comparação das sequências de isolados bacterianos desconhecidos. O objetivo do presente estudo foi avaliar isolados bacterianos de BGPE provenientes de testes de esterilidade de produtos farmacêuticos e do controle ambiental das áreas onde são realizados estes testes. Para atingir esse objetivo foram avaliados 83 dos isolados bacterianos encaminhados para o Setor de Identificação Bacteriana do Departamento de Microbiologia do Instituto Nacional de Controle de Qualidade em Saúde da Fundação Oswaldo Cruz utilizando duas metodologias fenotípicas, os sistemas comerciais API 50CHB e VITEK 32 cartão BAC e uma genotípica, a análise da sequência do gene 16S rRNA. O API e o VITEK identificaram a espécie ou as prováveis espécies de 54% dos isolados bacterianos. Através da análise da sequência do gene 16S rRNA foram identificados os gêneros de todos os isolados bacterianos, que são: Bacillus, Paenibacillus, Terribacillus, Lysinibacillus, Cohnella, Oceanobacillus e Paenisporosarcina. Essa análise também mostrou que 18% dos isolados bacterianos avaliados podem fazer parte de espécies ainda não descritas. / Os produtos que requerem a característica de esterilidade devem ser submetidos ao Ensaio de Esterilidade Bacteriana e Fúngica assim como o ambiente de execução desse teste, a fim de evitar resultados falso-positivos. A legislação recomenda a identificação de microrganismos provenientes dos Ensaios e do ambiente onde estes foram realizados. A dificuldade da identificação de vários gêneros por metodologias fenotípicas e identificações equivocadas de bastonetes Gram positivos esporulados (BGPE) têm sido relatadas em vários estudos e mostram a necessidade da utilização de metodologias moleculares mais adequadas a essa identificação. Para tal, a análise da sequência do gene 16S rRNA tem sido promissora uma vez que este gene é universal para bactérias e está disponível em bancos de dados em uma grande quantidade de sequências permitindo, assim, a comparação das sequências de isolados bacterianos desconhecidos. O objetivo do presente estudo foi avaliar isolados bacterianos de BGPE provenientes de testes de esterilidade de produtos farmacêuticos e do controle ambiental das áreas onde são realizados estes testes. Para atingir esse objetivo foram avaliados 83 dos isolados bacterianos encaminhados para o Setor de Identificação Bacteriana do Departamento de Microbiologia do Instituto Nacional de Controle de Qualidade em Saúde da Fundação Oswaldo Cruz utilizando duas metodologias fenotípicas, os sistemas comerciais API 50CHB e VITEK 32 cartão BAC e uma genotípica, a análise da sequência do gene 16S rRNA. O API e o VITEK identificaram a espécie ou as prováveis espécies de 54% dos isolados bacterianos. Através da análise da sequência do gene 16S rRNA foram identificados os gêneros de todos os isolados bacterianos, que são: Bacillus, Paenibacillus, Terribacillus, Lysinibacillus, Cohnella, Oceanobacillus e Paenisporosarcina. Essa análise também mostrou que 18% dos isolados bacterianos avaliados podem fazer parte de espécies ainda não descritas.