Heat pasteurization is an effective water treatment to address the emerging plant pathogen issue associated with increased water recycling practices in the ornamental horticulture industry. The current protocol that recommends treating water at 95"C for 30 s, however, faces two major challenges: its energy cost and environmental footprint. We hypothesized that temperature required to inactivate major pathogens in re-circulated water may be substantially lowered from 95"C with extended exposure time. The goal of this study was to test this hypothesis and make this water decontamination technology economically more attractive while reducing its environmental impact. Specific objectives were to (1) examine the effect of water temperature on the survival of Phytophthora and bacterial species, two major groups of plant pathogens in water recycling systems, and (2) elucidate the underlying biological mechanisms by which plant pathogens are killed at those temperatures. Lab assays were performed to determine the survival of zoospores and chlamydospores of P. nicotianae, and oospores of P. pini as well as seven bacterial species after heat treatments at given periods of time. Greenhouse experiments were conducted to determine the applicability of the lab assay data to the real world using annual vinca (Catharanthus roseus) and P. nicotianae as a model system. The results of these studies indicated that the water temperature required to eliminate Phytophthora and bacterial species can be lowered to 48"C from 95"C if treatment time extends to 24 h. Two major steps were taken to elucidate the underlying biological mechanisms. Firstly, a scheme based on the DNA fingerprint and sequence analysis was developed for characterizing bacterial species in irrigation water, after comparing two typing strategies, three sample concentration methods, and evaluating conditions in denaturing gradient gel electrophoresis (DGGE) profiling. Bacterial species detected by culture-dependent and -independent strategies were rather different. The greater bacterial diversity was detected when water samples were concentrated by using both methods than centrifugation or filtration alone. As for DGGE profiling, 40 to 60% denaturant concentrations at 70 V for 16 h revealed the highest bacterial diversity. Secondly, water samples were taken from an irrigation reservoir in a local nursery and analyzed for bacterial diversity following heat treatments at 42 and 48"C. After these heat treatments "-proteobacteria, "-proteobacteria, and Firmicutes became dominant which presents a substantial shift of bacterial community structure compared to those in the control water at 25"C. Among the dominant in treated water were Bacillus, Pseudomonas, Paenibacillus, Brevibacillus, and Lysobacter species, which may have potential biocontrol activities against plant pathogens. This study provided the scientific basis for developing a more energy-efficient and environmentally sound heat pasteurization protocol for water decontamination. / Ph. D.

Thousands of parrots all over the world suffer from illnesses and medical complications that can result from interactions between their immune system and bacteria in their digestive tract. The aim of this master's thesis is to understand the link between symptoms of these medical issues, the composition of blood and gastrointestinal microbiota in parrots. Using the hematological methods, 198 blood samples representing 53 parrot species were analyzed. The composition of microbiome was defined by combination of a molecular approach using bacterial 16S rRNA gene sequencing in 132 fecal samples, 12 intestine samples, 228 cloacal swabs and 236 beak swabs representing in total 61 parrot species and a diagnostic approach by psittacine fecal Gram's stain method. Significant association of hematological parameters with individual, environmental and clinical factors was observed, as well as its considerable interspecific variability. Absolute heterophile and lymphocyte counts have been shown more useful for infectious and autoimmune disease monitoring than H/L ratio. Relative numbers of basophiles were the best indicator for behavioral disorders. In relation to hematological parameters, the effect of the bacterial family Flavobacteriaceae, as part of the oral microbiota, and the bacteria Escherichia or...

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.

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.

Morphology and ultrastructure of 25 oscillatorian strains was examined and phylogenetic analysis of 16S rDNA oscillatorian sequences was conducted. Genera Phormidium and Oscillatoria were shown to be polyphyletic. Although morphologically similar strains are found in different branches of the phylogenetic tree, considerable correlation between molecular, ultrastructural and some morphological and ecological traits was detected in several lineages.

Termites are known to harbor within their gut a diverse assemblage of symbiotic microorganisms. Little work has been done, however, to describe the diversity and function of the bacteria in the economically important eastern subterranean termite, Reticulitermes flavipes. The first object of this study was to characterize the bacterial diversity in the gut of R. flavipes using amplified rDNA restriction analysis (ARDRA) and 16S rRNA gene sequencing. It was determined that ARDRA was an effective technique for characterizing the diversity of the termite gut microbiota. Of the 512 clones analyzed in the ARDRA study, 261 different ARDRA profiles were found. Forty-two 16S rRNA gene sequences were also analyzed, resulting in 33 different ribotypes. Representatives from six major bacterial phyla, Proteobacteria, Spirochaetes, Bacteroidetes, Firmicutes, Actinobacteria, and the newly proposed "Endomicrobia," were discovered. Further analysis indicated that the gut of R. flavipes may harbor as many as 1,318 ribotypes per termite. The second objective was to determine if the gut bacterial diversity could be manipulated by changing the termite's food source. Using ARDRA analysis, I found no evidence that changing the food source affected the termite gut bacterial diversity. In addition, changing the food source did not induce aggression in nestmates fed on different food sources. The third objective was to search for patterns of coevolution between termites and their gut symbiotic bacteria. Using rRNA gene sequences from this study and sequences from public databases (1,450 sequences total), a neighbor-joining tree demonstrated strong evidence for coevolution of termites and their symbiotic bacteria, especially in the phyla Bacteroidetes, Actinobacteria, Spirochaetes, and "Endomicrobia." Many monophyletic clusters were entirely composed of phylotypes specific to Isoptera. / Ph. D.

Neste trabalho desenvolvemos métodos analíticos e moleculares para a detecção, isolamento e classificação filogenética de Alicyclobacillus spp. a partir de sucos de laranja e frutos ácidos, pelas técnicas: RT-PCR, nested RT-PCR, RAPD, seqüenciamento do 16S rRNA e análise por métodos eletroforéticos. A sensibilidade na detecção dos A. acidoterrestris foi melhorada por meio de reações de nested RT-PCR, utilizando primers internos (amplicon de 191 bp) que foram desenhados a partir do primeiro amplicom de 294 bp. O limite de detecção foi estudado com as reações RT-PCR e nested RT-PCR, sendo capazes de detectar concentrações de 0,1 UFC mL-1 para culturas puras e 2 UFC mL-1 em sucos de laranja artificialmente inoculados. A inibição de esporos de A. acidoterrestris também foi estudada para monitorar a diminuição da viabilidade com tratamento térmico e Sapindus saponaria (200 mg L-1), utilizando RT-PCR e nested RT-PCR. Com o tratamento térmico de 99 oC por 1 h o grau de inibição dos esporos foi de 96,3%. Enquanto que, com a fração purificada de S. saponaria (200 mg L-1) incubada à 45 oC por 2 dias foi de 93,6%, mas com incubação de 99 oC por 1 h foi de 98,7%, na mesma concentração de saponina. Todas as análises de quantificação de produtos de RT-PCR e nested RT-PCR foram analisadas por meio de eletroforese em gel de agarose e eletroforese capilar em microchip no Bioanalyzer 2100 (Agilent), com os kits DNA 500 e DNA 1000 LabChip®, obtendo-se maior sensibilidade nos microchips. A classificação molecular de dezenove cepas, isoladas a partir de diferentes sucos e frutos ácidos, foram estudadas utilizando RAPD-PCR e eletroforese capilar em microchips. Utilizando cinco primers aleatórios nas reações de RAPD, foi possível estudar os polimorfismos analisados nos microchips. Segundo as análises eletroforéticas, as cepas de suco concentrado e diluído de laranja (1, 2, 6) suco concentrado de limão (lim) e suco de laranja in natura (T2, T3), apresentaram similaridades genéticas com a A. acidoterrestris. O estudo de análise filogenética baseada na comparação de seqüências de DNA da região variável do gene 16S rRNA de Alicyclobacillus acidoterrestris, foi utilizado para identificar e agrupar onze cepas isoladas de superfícies e sucos de frutos ácidos. Na árvore filogenética gerada pelo método neighbor joining e bootstrap 1000x, as cepas analisadas mostraram similaridades de 99% entre todas elas, observando-se uma maior similaridade do controle A. acidoterretris (C2) com a cepa isolada de suco concentrado de limão (lim), e uma boa discriminação entre controles das espécies A. acidocaldarius, A. cicloheptanicus, A. sendaiensis e Sulfobacillus acidophilus. / In this work, we developed analytical and molecular methods for detection, isolation and phylogenetic classification of Alicyclobacillus spp. from orange juice and acid fruits using RT-PCR, nested RT-PCR, RAPD and sequencing of 16S rRNA techniques and electrophoretic methods of analysis. The sensitivity on the detection of A. acidoterrestris in orange juice was improved by nested RT-PCR, using internal primers (amplicon of 191 bp) that were designed after sequencing the first amplicon (294 bp). The detection limit was studied with RT-PCR and nested RT-PCR assay, it was able to detect concentrations of 0.1 UFC mL-1 for media culture and 2 UFC mL-1 in inoculated orange juice. The inhibition in spores from A. acidoterrestris was also studied to monitoring the diminution of viability with heat treatment and Sapindus saponaria (200 mg mL-1), using RT-PCR and nested RT-PCR assays. The inhibition by heat treatment at 99 oC for 1 h was 96.3%. However, incubation with S. saponaria at 45 oC for 2 days inhibited 93,6%, however, with incubation of 99 oC for 1 h was 98,7%, in the same concentration of saponin. The quantification of the RT-PCR and nested RT-PCR amplification product were accomplished by capillary electrophoresis in microchips using the Bioanalyzer 2100 in conjunction with the LabChip (TM) DNA 500 and DNA 1000. The molecular classification of nineteen strains isolated from different juice and acidic fruit, were studied using RAPD-PCR and capillary electrophoresis in microchips. Using five random primers in the RAPD assay, it was possible to study the polymorphisms analyzed in microchips. According to electrophoresis analyses, the strains from concentrated and diluted orange juices (1, 2, 6), lemon concentrated juice (lim) and natural orange juice (T2, T3), showed genetic similarities with the A. acidoterrestris. The study of the phylogenetic analyses based on DNA comparison sequences of the variable region of 16S rRNA gene from Alicyclobacillus acidoterrestris, was utilized for the identification and grouping of eleven strains isolated from surface and juice of acid fruits. In the phylogenetic tree produced by neighbor joining and bootstrap 1000, the strains showed similarities of 99% among all strains, showing a high similarity of A. acidoterrestris with a strain isolated from lemon concentrate juice (lim), and a good discrimination between the species A. acidocaldarius, A. cycloheptanicus, A. sendaiensis and Sulfobacillus acidophilus.