Dissertationen zum Thema „Bacteria isolated“

Thesis (MSc)--Stellenbosch University, 2012.
ENGLISH ABSTRACT: The deterioration of harvested sugarcane as a result of bacterial growth causes major losses of sucrose and a build-up of exopolysaccharides (EPS). Polysaccharides present during production increase the massecuite viscosity, which negatively influences evaporation and crystallisation. In this study 38 culturable EPSproducing bacteria were isolated from milled sugarcane. Analysis of the EPS showed the ubiquitous presence of glucose, however, 14 polysaccharides also contained mannose, fructose or galactose. In vitro treatment using Chaetomium erraticum dextranase to evaluate is effectiveness indicated that 37 of the EPS were hydrolysed to some extent. There were 21 polysaccharides that were only partially digested. The capacity of the isolates to produce EPS on different sugars indicated a correlation between sucrose and polysaccharide formation in 37 isolates. The results indicate there are more species involved in EPS production than previously thought as well as the presence of non-dextran polysaccharides.
AFRIKAANSE OPSOMMING: Bakteriële groei veroorsaak ‘n afname in gehalte, sukrose en ‘n verhoging in die hoeveelheid van eksternepolisakkeriede (EPS). Die verhoogde konsentrasie van polysakkariede gedurende die verwerkingsprosses veroorsaak ‘n verhoging in “massecuite” viskositeit. Hierdie verskynsel het ‘n nadelige uitwerking op die verdamping en kristalvorming van die produk. In gemaalde skuikerriet was 38 groeibare EPS-produserende bakterieë geisoleer. Die geanaliseerde EPS van hierdie bogenoemde bakterieë was daar in almal glukose teenwoordig. In 14 van hulle was mannose, fruktose en galaktose ook gevind. Die in vitro effektiwieteit van Chaetomium erraticum dekstranase op die EPS het gewys dat 37 het tot ‘n mate gehidroliseer maar 21 was net gedeeltelik verteer. As gevolg van die bo-genoemde resultate was daar gevind dat sukrose was ‘n noodsaaklike subtraat vir EPS produksie in die geisoleerde bakterieë. In hierdie studie was bevestig ‘n groter verskiedenheid EPS-produserende bakterieë gevind was en dat hulle assosiasie aan sukierriet prossering meer kompleks is as wat vooreen gedink was.

As part of a large study aimed at searching for patterns of diversity in the genus Persephonella along the north to south geochemical gradient of the ELSC, ten novel strains of Alphaproteobacteria were isolated unexpectedly. Using defined media under microaerophilic conditions to enrich for Persephonella from chimney samples collected at the seven vent fields on the ELSC and the dilution to extinction by serial dilution method to purify cultures, a total of ten strains belonging to the Alphaproteobacteria were isolated. Two of these isolates, designate MN-5 and TC-2 were chosen for further characterization and are proposed as two new species of a novel genus to be namedThermopetrobacter. Both strains are aerobic, capable of chemoautotrophic growth on hydrogen and grow best at 55°C, pH 6 and 3.0% NaCl. Strain MN-5 is capable of heterotrophic growth on pyruvate and malate and TC-2 is only able to grow heterotrophically with pyruvate. The GC content of MN-5 is 69.1 and TC-2 is 67 mol%. GenBank BLAST results from the 16S rRNA gene reveal the most closely related sequence to MN-5 is 90% similar and the most closely related sequence to strain TC-2 is 89% similar. Sampling at a shallow marine vent on the coast of Vulcano Island, Italy in 2007 led to the isolation of a novel species of Hydrogenothermus, a genus within the Hydrogenothermaceae family. This isolate, designated NV1, represents the secondHydrogenothermusisolated from a shallow marine vent. NV1 cells are rod-shaped, approximately 1.5μm long and 0.7μm wide, motile by means of a polar flagellum and grow singularly or in short chains. Cells grow chemoautotrophically using hydrogen or thiosulfate as electron donors and oxygen as the sole electron acceptor. Growth was observed between 45 and 75°C with an optimum of 65°C (doubling time 140 min), pH 4.0-6.5 and requires NaCl (0.5-6.0% w/v). The G+C content of total DNA is 32 mol%.

Contamination from various sources has a huge impact on soil health and microbial community composition. Metal contamination of soil in mining scenarios is of concern and is not adequately addressed, particularly with respect to the microbial community. The mining industry is one of the largest contributors to heavy metal contamination of soil in South Africa, especially since the country is one of the major mining countries in the world. Platinum mining is of special importance, since the largest percentage of the world’s reserves of platinum group metals are found and mined in South Africa. Metals from mining activities become irreversibly immobilized in soil systems because they cannot be degraded and has a huge impact on soil systems. In this study, bacteria was isolated from soil samples collected from a platinum mine tailings dam outside Rustenburg. During the warm sampling season (March 2006) most isolates were found, especially in sites 3 and 4. During the colder and drier season (May 2006) there were less isolates. Most of the isolated cultures also displayed a wide temperature growth range, mostly between 24°C - 37°C. Paenibacillus lautus and Bacillus subtilus DN-10 had a growth range between 5°C - 40°C. Culturable metal tolerant bacteria were isolated, purified and identified using 16S rDNA sequences. Nine different species were found namely Paenibacillus lautus strain DS19, Paenibacillus lautus, Paenibacillus sp. C15, uncultured Paenibacillaceae, Bacillus subtilis strain DN-10, Bacillus sp. KDNB5, Bacillus cereus, Stenotrophomonas maltophilia and Alcaligenes sp. DJWH 146-2. The ability of these strains to tolerate metal concentrations were explored by determining their minimum inhibitory concentrations for a selection of metals e.g. aluminum, barium, cobalt, chromium, cadmium, copper, iron, lead, manganese, nickel and mercury. Most isolates were able to tolerate >5mM of the Al\Ni alloy and cobalt. Transmission electron microscopy was used to determine the location of metals inside bacterial cells and electron dispersive X-ray analysis was used to determine the levels of metals inside microbial cells. Bacillus subtilis DN-10 (LDK0306) showed a high MIC (>5mM) for most metals used, except Hg. This strain also had a high percentage (10.26%) of Pb detected in its cells by EDX. This was the highest percentage detected. Plasmids were extracted from the identified strains and can help gain a better understanding of metal tolerance mechanisms used by these isolates.
Thesis(MSc (Environmental Sciences))--North-West University, Potchefstroom Campus, 2013

The most important resistance mechanism to beta-lactam antibiotics is the plasmid-mediated beta-lactamase and the common criterion for the epidemiology of these enzymes is the determination of their biochemical characteristics. Surveys of plasmid-encoded beta-lactamases of Gram-negative bacteria, used to investigate their relative clinical importance, have been poorly performed and rarely conducted outside the developed world. A survey of uropathogenic strains and of salmonellae and shigellae, isolated in south India in 1984, revealed a higher incidence of ampicillin resistance (minimum inhibitory concentration [MIC] > 10mg/1) than had ever been reported before (Enterobacteriacea 82%, salmonellae 90%, shigellae 60%). Only the enterobacterial strains showed any significant resistance to the first generation cephalosporin, cephalosporin, cephaloridine (MIC > 10mg/1). However, 66% of the salmonellae strains were cefuroxime resistant. A small proportion of all species conferred resistance to third generation cephalosporins. In the individual species, there was a very high incidence of ampicillin resistance (E. coli 76% Klebsiella spp 96%) and cephaloridine resistance (E.coli 57% Klebsiella spp 69%). Many of the ampicillin resistant strains harboured either auto-transferable or mobilisable plasmids (40%). Characterisation of the plasmid DNA from the E.coli transconjugants revealed the existence of 37 different plasmids types. The transconjugants from klebsiella, salmonella and shigella possessed fewer plasmids types than those from E. coli. Most plasmids possessed resistance genes to aminoglycosides and to six or more drugs. Beta-lactamase studies revealed that TEM-1 was the most predominant enzyme in all transconjugant strains followed by OXA-1, SHV-1, TEM-2, OXA-2 and the novel enzyme SAR-2. The SAR-2 enzyme was fully characterised and had a higher pI (8.3) than any previously characterised plasmid-mediated beta-lactamase. It had a broad-spectrum activity with the molecular weight of 36000. In addition the unusual observations of E.coli strains producing both the PSE-1 and PSE-2 beta-lactamases and strains hyperproducing the TEM-1 were made and these strains were studied further. The development and mechanisms of resistance to beta-lactam/beta-lactamase inhibitor combinations (ampicillin and clavulanic acid) have been performed with laboratory strains possessing the ampicillin resistance plasmid R1. The results show that challenge with clavulanic acid alone did not affect the expression or integrity of the beta-lactamase whereas challenge with the combination of ampicillin and clavulanic acid caused radical changes with the expression of the beta-lactamase. In some cases there were multiple copies of genes which resulted in hyperproduction of TEM-1 enzyme and this was sufficient to resist the combinations. Unfortunately, these variants also conferred resistance to second and third generation cephalosporins. Evidence of this type of resistance to clavulanic acid is now emerging in clinical practice.

Lactic acid bacteria (LAB) were isolated during the production and the ripening of Greek dry fermented sausages. Samples were taken at different stages, and 150 "wild' strains were isolated. The majority of the strains isolated were assigned to the species Lactobacillus plantarum biotype (1) (43.3 %) followed by Lb. curvatus, Lb. pentosus (10.7 %), Lb. brevis biotype (1) (8.7 %), Lactococcus lactis subsp. lactis biotype (1) (6.7 %) and Leuconostoc mesenteroides subsp. mesenteroid.es biotype (2) (5.3 %). The possibility of bacteriocin production was tested using the agar well diffusion assay (AWDA). One strain was found to produce bacteriocin (Leuconostoc mesenteroides E131) and its purification was attempted using precipitation with ammonium sulphate, cation exchange, and reverse phase chromatography. Moreover, the purification of curvaticin L442 was attemped, a bacteriocin produced by Lactobacillus curvatus L442, isolated from Greek traditional fermented sausage prepared without addition of starters. The bacteriocin was purified by 50% ammonium sulphate precipitation, cation exchange, reverse phase and gel filtration chromatography. Lb sakei I154, a bacteriocin producing strain isolated from Italian fermented sausage, and the semi-purified bacteriocin from Leuconostoc mesenteroides E131 were validated via industrial trials to evaluate whether the product (fermented sausages) maintains the technological characteristics and the traditional quality characteristics. Three fermentations under controlled conditions were conducted and at the end of these fermentations, products were sliced and packaged under controlled atmosphere (80% N2 + 20% CO2) and stored at 4+/-2 °C for 12 weeks to determine the shelf life of the product. Finally, from the previous industrial trials the proper production parameters were determined as well as the most effective packaging techniques, resulting in the conduction of a Standard Operation Procedures Guide concerning the whole production of traditional fermented sausages.

There is a growing recognition that biofilms are the principal cause of chronicity or persistence in infections. Biofilms have been implicated in chronic wounds as a cause of delayed healing. However, only few wound management strategies treat wounds with the assumption that biofilm may be the cause of failure to heal. The fact is biofilms are difficult to treat because of their resistance to antimicrobial agent. Biofilm formation is known to be a three stage process that has been found in dental plaque to be influenced by cell to cell recognition also called coaggregation. The main aim in this study was to investigate the ability of bacteria isolated from chronic wounds to form biofim in vitro and the possible role of coaggregation in the establishment of biofilm. 164 pairs of clinical bacteria were tested for ability to coaggregate. Out of 71 isolates 58 (81.69%) gave positive coaggregation score and 13 (18.3%) did not coaggregate. The presence of biofilm was tested using 59 of the 71 bacteria and all isolates (100%) indicated an ability to form biofilm in vitro with various degrees of adherence. 74.57% were strongly adherent, 15.26% moderate, and 10.17% weakly adherent There was a significant association between the ability of isolates to coaggregate and to form biofilm (p<0.05) Using isolates that had been recovered from the same patients, matrices were constructed from 5 patients to investigate the structure of the network. The probability that a node will be connected was high (p>0.01) indicating that bacteria in chronic wounds are highly connected to one another. Random and selected node removal from the network revealed that bacteria in chronic wounds may be strongly dependent on one another and favour a polymicrobial rather than a monospecies infection. Network analysis also demonstrated coaggregation ability of some bacteria to act as pioneers in the establishment of the biofilm and provided support for the idea that coaggregation might influence biofilm formation in chronic wounds.

This thesis reports the results obtained during the three years PhD course focused on the study of culturable bacterial endophytes of Vitis vinifera Glera and their beneficial activities. The study, part of a large project named “EndoFlorVit project” (FEARS-UE and Regione Del Veneto), aims at investigate the biodiversity and the plant growth promoting activities of culturable endophytes isolated from Glera grapevine in vineyards of Conegliano-Valdobbiadene DOCG production area. This thesis reports the results of the isolation of culturable bacterial endophytes from surface-sterilized Glera grapevine tissues. 381 culturable strains were successfully isolated from roots, shoots and leaves of Vitis vinifera Glera, sampled from six different vineyards in the Conegliano-Valdobbiadene DOCG area (Veneto, Italy). The community was investigated by Amplified Ribosomal DNA Restriction Analysis (ARDRA) and nucleotide sequencing to identify the most representative genera of the Glera microbiome. Approximately 30% of the isolates belonged to the genus Bacillus, which was the most represented; other genera such as Staphylococcus, Microbacterium, Paenibacillus, Curtobacterium, Stenotrophomonas, Variovorax, Micrococcus and Agrococcus were identified. The composition of the communities isolated from different vineyards was not the same; moreover we reported that endophyte biodiversity inside plants was influenced by the season. After molecular characterization, we focused our attention to investigate the plant growth promoting abilities of the culturable strains. Using biochemical tests we assayed some of the most important and effective properties in order to investigate the physiology of these bacteria and identify some strains that could have a strong beneficial effect on plant nutrition and growth. In this work, using Carboxymethyl Cellulose degradation test, we demonstrated that 85 strains secreted cellulolytic enzymes; this trait could confer to these bacteria an advantage in plant penetration and tissues colonization. By qualitative biochemical assays, we demonstrated that many strains were able to solubilize phosphate (127 strains), produce ammonia (142 strains) and secrete siderophores (155 strains). Using the colorimetric Salkowsky assay, we determined that 17 strains produced the phytohormone Indol 3-acetic acid (IAA) ; using Arabidopsis thaliana DR5:GUS, where the β-glucuronidase reporter gene is expressed under control of a IAA-induced promoter, we demonstrated that bacterial IAA was recognised by the Arabidopsis plants and caused morphological alteration on the root architecture. It is known that IAA is not the only bacterial molecule that influence the plant growth and the root morphology. To investigate the effects of the Glera endophytes on the plant morphology we used the model system Arabidopsis thaliana co-cultured in vitro with every single strain. Morphological parameters (root length, surface and diameter) were measured by a software and statistically analysed by cluster analysis. Plants were thus clustered according with the effect of the strain on the root parameters, demonstrating the effects of the strains on roots morphology. In particular, some strains caused an enhanced root length displaying a plant growth promotion effect. By this large-scale characterization we selected two of the most promising strains, one for the putative plant growth effect (Pantoea agglomerans GL83) and one as putative biocontrol agent (Bacillus licheniformis GL174) that were transformed with a DNA cassette containing a gfp reporter gene. Using Laser Scanning Confocal Microscopy, we demonstrate the colonization of the stem endosphere of Glera cuttings 20 and 30 days after the inoculum of the fluorescent strains. In this thesis, the evidences of the colonization are reported demonstrating that Pantoea agglomerans GL83 and Bacillus licheniformis GL174 are true Glera endophytes able to colonize cuttings when re-inoculated. After we demonstrated that B. licheniformis GL174 is a true endophyte of Glera, we investigated the biocontrol abilities of the strain. Results of antagonism tests against plant pathogenic fungi are shown, demonstrating that the strain is able to reduce and inhibit the mycelia growth of the grapevine pathogens Phaeoacremonium aleophilum, Phaeomoniella spp., Botryosphaeria spp., Botrytis cinerea and for the more generic plant pathogens Sclerotinia sclerotiorum and Phytophthora infestans. After that, we demonstrated by PCR and DNA sequencing that GL174 has the operons coding for lipopeptide synthetase enzymes and that the strain produced cyclic lipopeptide belonging to surfactin and lichenysin families. These molecules with antimicrobial effects were identified and characterized by mass spectrometric analysis and the results are reported and discussed in this thesis. The genome of the strain was thus sequenced to better investigate the strain and the sequences were preliminary analysed identifying the presence of many genes coding for lytic enzymes. The production of lipopeptides, the inhibition of fungal growth and the ability to colonize inner tissues of Glera indicated GL174 as a good candidate for biocontrol. Another aspect of endophyte-plant symbiosis that is not well explained is the ecology of these bacterial strains and the interactions between different bacterial species in the rhizosphere and inside plants are poorly described. Bacteria-bacteria interactions are likely to be an important factor that defines the composition of the endophyte community. The study of these interactions is essential to understand plant-bacteria relationship; moreover, the study of how the native community of rhizobacteria and endophytes may change after the inoculation of other bacteria is important for a safe and aware use of commercial biocontrol or bio-fertilizer products containing endophytes. A preliminary ecological study is presented in this thesis: some ecological aspects of endophyte of grapevine, endophytes of other plant species and some bacteria commercialized as beneficial strains, called “biofector strains” were analysed using tomato, a model plant for agriculture and horticulture. In this work, we demonstrated that these strains were able to colonize tomato plants and the population densities of the diverse tissues sampled are reported in the Chapter number 5. This work, that is still ongoing, aims to evaluate the impact of these endophytic strains investigating if the inoculum of the bacteria on tomato plants leads to a different endophytic community in comparison to uninoculated plants. This study is essential to unravel the effects of the bio effector strains on natural endophytic populations of plants: from peeled stems of all the inoculated plants the total DNA was extracted; this material will be used as template for the 16S rDNA amplification of all the endophytes present in the plants. Many sets of primers are being tested to select the best combination for this approach. The amplicons will be sequenced and analysed to determine if the community of endophytes has been changed by the inoculum of the external endophytic bio-effector strain. In conclusion, the results presented in this thesis are an overview of the composition of the endophytic community of Glera plants cultivated in the Conegliano-Valdobbiadene DOCG area. The isolation of the culturable strains has provided a large collection of bacteria that, during the PhD course, was characterized investigating plant growth promoting activities and bacteria effects on plant morphology considering different mechanisms underlying plant-microbe interactions. The evidence obtained in this work describes a clear and novel background to understand Glera endophytes biology and ecology and, when confirmed in planta by field trials, will permit the selection of some efficient strains to use as safe endophytic bio-fertilizers and biocontrol agents, for a sustainable production of Glera grapes.
Questo lavoro di tesi presenta e discute i risultati sperimentali ottenuti durante il corso di dottorato in Biologia Evoluzionistica presso la Scuola di Dottorato di Bioscienze e Biotecnologie dell’Università degli Studi di Padova. Questa ricerca, parte di un progetto più ampio denominato “EndoFlorVit” (FEARS-UE e Regione del Veneto), ha come scopo la caratterizzazione molecolare e lo studio delle proprierà di promozione della crescita vegetale e di bio-controllo di batteri endofiti isolati da piante di Vitis vinifera di cultivar Glera, coltivate nell’area di produzione del Prosecco di Conegliano-Valdobbiadene DOCG. Le comunità di microrganismi isolate sono state studiate utilizzando la tecnica ARDRA (Amplified Ribosomal DNA Restriction Analysis) e il sequenziamento di porzioni del 16S rDNA identificando che circa il 30% dei ceppi isolati appartengono al genere Bacillus, il quale risulta essere il più rappresentato nelle piante campionate. Altri generi a cui appartengono numerosi ceppi isolati sono Staphylococcus, Microbacterium, Paenibacillus, Curtobacterium, Stenotrophomonas, Variovorax, Micrococcus e Agrococcus. La composizione delle comunità endofite isolate da differenti piante non è uniforme: esse variano nei differenti vigneti e sono inoltre influenzate dalla stagionalità. Oltre alla descrizione dei ceppi isolati, in questo lavoro di tesi sono presentati e discussi i risultati dello studio delle proprietà di promozione della crescita vegetale dei ceppi isolati. Utilizzando saggi biochimici sono state investigate alcune delle principali attività benefiche che hanno un effetto di miglioramento della nutrizione vegetale. Mediante il test di degradazione della carbossimetil-cellulosa sono stati identificati 85 ceppi capaci di secernere enzimi degradanti la cellulosa: questa capacità può conferire ai ceppi che la esprimono un vantaggio nella colonizzazione dei tessuti vegetali facilitando loro il processo di penetrazione nei tessuti della pianta. Attraverso saggi biochimici qualitativi è stato possibile dimostrare che numerosi ceppi sono in grado di solubilizzare il fosfato insolubile (127 ceppi), produrre ammoniaca (142 ceppi) e secernere siderofori (155 ceppi). Inoltre, utilizzando il saggio di Salkowski, è stato dimostrato che 17 ceppi batterici producono l’ormone vegetale Acido 3-indolacetico (IAA). Per investigare l’effetto dei ceppi produttori di IAA sulla fisiologia e morfologia della pianta è stato utilizzata la pianta modello Arabidopsis thaliana DR5:GUS, una linea mutante esprimenti l’enzima β-glucuronidasi sotto controllo di un promotore indotto da IAA. Utilizzando questo sistema sperimentale è stato dimostrato che l’IAA prodotto dai batteri viene riconosciuto dalle piante di Arabidopsis e causa alterazioni alla morfologia e architettura radicale. È noto tuttavia che l’IAA non è l’unica molecola batterica che influenza la crescita vegetale e la morfologia vegetale. In tal senso, è stato valutato l’effetto di ciascun ceppo isolato sulla pianta Arabidopsis thaliana Col-0 wild tipe. Tre parametri morfologici della radice (lunghezza superficie e diametro) sono considerati e analizzati statisticamente mediante cluster analysis. Le piante quindi sono state raggruppate secondo gli effetti che i batteri hanno provocato sull’apparato radicale assegnando in questo modo a ciascun ceppo l’effetto corrispondente. In questo modo è stato dimostrato che alcuni ceppi hanno causato allungamento della radice, un effetto ascrivibile come promozione della crescita. Da questa caratterizzazione ed analisi su larga scala dei ceppi isolati sono stati selezionati due ceppi particolarmente promettenti per la promozione della crescita vegetale e per il biocontrollo: Pantoea agglomerans GL83 e Bacillus licheniformis GL174. Questi due ceppi sono stati trasformati geneticamente con un costrutto contenente il gene che esprime la proteina fluorescente GFP. Utilizzando tecniche di microscopia confocale è stato dimostrato che entrambi i ceppi fluorescenti sono in grado di ricolonizzare talee di vite Glera quando inoculate e che persistono all’interno dei tessuti del fusto dopo 20 e 30 giorni dopo l’inoculo. In questa tesi quindi sono presentate le evidenze sperimentali che questi due ceppi, Pantoea agglomerans GL83 e Bacillus licheniformis GL174, sono veri endofiti di vite Glera e risultano quindi interessanti per le loro proprietà benefiche. Dopo aver confermato che GL174 è endofita della vite Glera, il ceppo è stato investigato per evidenziarne alcune capacità utili per il biocontrollo dei patogeni. In questo lavoro di tesi sono presentati i risultati di saggi di antagonismo in vitro nei quali il ceppo in esame ha effetto di inibizione della crescita del micelio di alcuni funghi patogeni della vite (Phaeoacremonium aleophilum, Paeomoniella spp., Botryosphaeria spp., Botrytis cinerea) e di due patogeni più generalisti (Sclerotinia sclerotiorum e Phytophtora infestans). Lo studio del ceppo GL174 si è successivamente focalizzato sulla capacità del batterio di produrre i lipopeptidi ciclici, una classe di molecole con forte attività antimicrobica e surfattante. Per prima cosa, attraverso PCR e sequenziamento del DNA, è stata identificata la presenza nel genoma batterico degli operoni codificanti per alcune lipopepide sintetasi, gli enzimi deputati alla sintesi di queste molecole. La produzione di lipopeptidi è stata successivamente dimostrata utilizzando tecniche di spettrometria di massa; le quali hanno permesso di identificare le molecole prodotte e di ricostruirne la struttura chimica. La produzione di queste molecole e la capacità inibitoria di funghi patogeni rendono il ceppo GL174 un buon candidato come agente di biocontrollo nella coltivazione della vite e di altre specie economicamente rilevanti. L’ ecologia dei batteri endofiti è un tema che ancora non è stato del tutto investigato all’interno dello studio dell’interazione tra piante ed endofiti. Inoltre, le interazioni che avvengono a livello di rizosfera ed endosfera non sono ancora ben descritte ma evidenze sperimentali suggeriscono che esse siano un fattore importante nella definizione della composizione delle comunità endofite. Lo studio di come un inoculo batterico esogeno può modificare la composizione della comunità nativa di endofiti è essenziale per un uso consapevole di formulati commerciali a base di batteri endofiti. In questa tesi viene quindi presentato un lavoro preliminare che analizza l’ecologia di ceppi isolati da Glera, ceppi isolati da altre specie vegetali, e batteri commercializzati come biostimolanti. La colonizzazione di piante di pomodoro, pianta modello per lo studio delle specie orticole, da parte di questi ceppi microbici è stata dimostrata e quantificata per radici, fusto e foglie di piante coltivate per 3 e 5 settimane. Questo lavoro ha come scopo inoltre l’analisi delle comunità di endofiti di queste piante inoculate per confrontarne la composizione con piante non inoculate. Lo studio, che è ancora in corso, ha comportato l’estrazione del DNA totale della endosfera di porzioni di fusto; da questo DNA saranno amplificati i 16S rDNA dei batteri endofiti presenti e sequenziati con tecniche di NGS. In conclusione, i risultati presentati in questa tesi descrivono la composizione delle comunità di endofiti coltivabili isolate da piante di vite Glera della zona del Prosecco Conegliano-Valdobbiadene DOCG. L’isolamento di tali batteri ha fornito una larga collezione di ceppi batterici, le cui proprietà benefiche che promuovono la crescita vegetale e gli effetti dei batteri sulla morfologia radicale di Arabidopsis thaliana sono stati analizzati e presentati criticamente coinvolgendo più aspetti importanti nell’interazione pianta-endofiti. I risultati ottenuti in questo lavoro descrivono le proprietà di alcuni ceppi isolati da Glera che, quando confermato da prove sperimentali in campo, potranno essere utilizzati in sicurezza come agenti endofiti di biofertilizzazione e/o biocontrollo nella produzione dell’uva Glera e di altre specie vegetali economicamente importanti.

Bacterial contamination of platelet concentrates (PCs) poses the major transfusion-associated infectious risk. Coagulase negative staphylococci (CoNS), the predominant platelet contaminants, are recognized as one of the leading causes of hospital-acquired infections due to their ability to form biofilms (surface-attached aggregates). In this study, 29 CoNS strains were characterized for their growth and biofilm formation abilities in media and PCs. Twenty-five strains were aerobic including Staphylococcus epidermidis, S. capitis, and S. chromogenes, while four were identified as the anaerobe S. saccharolyticus. Biofilm-associated icaA and icaD genes were amplified from eight strains. Interestingly, only six of those strains were biofilm-positive. Sequencing of S. capitis icaD revealed no mutations that could explain differences in biofilm phenotypes. Growth of CoNS in PCs varied significantly between strains. This study provides preliminary evidence that slow-growing biofilm-positive S. epidermidis are more likely to be missed during platelet culture, highlighting the need for improved screening methods.

Since the 1970s, a growing body of research has suggested that bacteria play an active role in precipitation. These bacteria are capable of catalyzing the formation of ice at relatively warm temperatures utilizing a specific protein family which aids in the binding of water molecules. However, the overall biodiversity, concentration, and relationship of ice nucleation active (ice+) bacteria with air mass trajectories and precipitation chemistry is not well studied. Precipitation events were collected over 15 months in Blacksburg, VA and ice+ bacteria were isolated from these samples. From these samples, 33,134 total isolates were screened for ice nucleation activity (INA) at -8 °C. A total of 593 of these isolated positively confirmed for INA at the same temperature in subsequent tests. The precipitation events had a mean concentration of 384±147 colony forming units per liter. While the majority of confirmed ice+ bacteria belonged to the gammaproteobacteria, a well-studied class of bacteria, including ice+ species of Pseudomonas, Pantoea, and Xanthomonas, two isolates were identified as Lysinibacillus, a Gram-positive member of the Firmicute phylum. These two isolates represent the first confirmed non-gammaproteobacteria with INA. After further characterization, the two isolates of Lysinibacillus did not appear to use a protein to freeze water. Instead, the Lysinibacillus isolates used a secreted, nanometer-sized molecule that is heat, lysozyme, and proteinase resistant. In an attempt to identify the mechanism responsible for this activity, species type strains were tested for INA and UV mutants were generated to knock out the ice+ phenotype. Based on these results, only members of the species L. parviboronicapiens exhibit INA and the genes responsible for the activity may lie within a type-1 polyketide synthase/non-ribosomal peptide synthase gene cluster. This gene cluster is absent from the genomes of all non-ice+ strains of Lysinibacillus, and contains mutations in five of the nine ice nucleation inactive mutants generated from the rain isolated strain. To better understand the phylogenetic relationship among ice+ Lysinibacillus, a comprehensive reference guide was compiled to provide the most up-to-date information regarding the genus and each of its species. This reference will be available to other researchers investigating Lysinibacillus species or other closely related genera.
PHD

Objectives: The aims of this study was to investigate the RC microbiota in CCF canine teeth in the domestic dogs (Canis familiaris) and cheetahs (Acinonyx jubatus), identify the possible factors related to the presence of aerobic or anaerobic bacteria and evaluate and evaluate antibiotic susceptibility of bacteria isolated. Animals: Thirty nine animals suffering from CCF of their canine teeth were included in this study, of which 20 were dogs and 19 were cheetahs. Procedures: Evaluation of the oral cavity of animals while under general anaesthesia was performed and those without necrotic pulps or those that had received antibiotic therapy in the previous two weeks were excluded. Microbial samples were taken from 63 RC of which 27 were from dogs and 36 were from cheetahs. Strict anaerobic and aerobic techniques were used in parallel for plating, incubation and identification of the bacteria isolated in this manner. In an attempt to evaluate the sensitivity of the culture media and anaerobic technique used, additional samples were collected after the samples for bacterial isolation had been taken from the last eight pulps. These comprised those from six cheetahs and two dogs and were analysed using culture techniques and an initial screening with the 16S rRNA-specific PCR. Results: • Dogs: A total of 49 cultivable isolates were recovered belonging to 19 different bacterial species and 13 different genera. Individual RC yielded a maximum of four bacterial species. Of the bacterial isolates, 4.08 % were strict anaerobes, being represented by Clostridium acetobulitycum (2.04 %) and Prevotella melalinogenica (2.04 % ). The incidence of aerobic bacteria and facultative anaerobic bacteria in this study were 18.36 % and 77.56 %respectively of all the bacterial isolates. Of these Pasteurella multocida ( 10.20 % ), Corynebacterium spp. (10.20 %), Moraxella spp. (8.17 %), Bacillus spp. (6.12 %), Aeromonas salmonicida (6.12 %), Escherichia coli (6.12 %) and Pseudomonas aeruginosa (6.12 %) were the bacteria most frequently isolated. In summary, the RC microflora was found to be predominantly Gram negative facultative anaerobic microorganisms. The antibiotic agents that showed the highest efficacy in vitro against the different bacteria isolates were Enrofloxacin (85.21 % ), Gentamicin (92.39 %), Chloramphenicol (89.13 %). • Cheetahs: A total of 59 cultivable isolates, belonging to 19 different microbial species and 13 different genera were recovered from 36 RC sampled. Thirty-two (54.49 %) of the cultivable isolates were Gram positive while 27 (45.71 %) were Gram negative. Individual root canals each yielded a maximum of six species. Four RC had no cultivable bacteria. The bacterial micro flora recovered from the RC of the animals showed a higher number of facultative anaerobes (62.72 % of all the bacterial isolates). Aerobic isolates were 28.81 %, and strict anaerobes 8.47 % of all the isolates. The latter species comprised Clostridium sordelli (5.08 % ), and Clostridium septicum (3.38 % ). The species with the highest isolation frequency were Bacillus spp. (22.13 %), Pasteurella multocida (10.16 %), Corynebacterium spp. (8.47 %), Enterococcus spp. (8.47 %), Moraxella spp. (8.47 %) and Pseudomonas aeruginosa (5.25 %). In summary, the bacteria isolated from the RC were Gram positive facultative anaerobic bacteria. The antibiotics, which showed the highest efficacy in vitro against the different bacteria isolates, were Enrofloxacin (91.96 %), Gentamicin (86.37 %) and Orbifloxacin (86.28 %). • Nucleic Acid-Base detection: In dogs, Gram negative and Gram positive bacterial species were equally represented. Anaerobic bacterial species predominated at 83.3 % (5/6) of the species detected. On the other hand, in cheetahs, the bacterial species isolated by the PCR method showed a prevalence of anaerobic bacteria (60.8 %, 14/23), while facultative anaerobes were isolated in 30.2 % (7 /23) of cases and aerobic bacteria in 8.6 % (2/23). Conclusions and Clinical Relevance: This study has indicated that the microbial flora in any single infected RC is much more diverse than it has been shown using cultural techniques alone and can contain potentially uncultivable bacterial species. Some of these species may represent potentially new phylotypes, which may be involved in endodontic infections and ultimatelyin periradicular periodontitis, and should therefore be considered in any future studies involved in defining endodontic pathogens. Copyright
Dissertation (MSc)--University of Pretoria, 2012.
Companion Animal Clinical Studies
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Thesis (MSc)--University of Stellenbosch, 2005.
ENGLISH ABSTRACT: Thirty lactic acid bacteria were isolated from the intestinal tract of Drosophila simulans Stuvervant and nine lactic acid bacteria from Merlot grapes collected from the same winery in the Stellenbosch region, South Africa. The isolates were grouped according to morphological, biochemical and physiological characteristics. Isolates selected from each group were identified to species level by PCR with species-specific primers, PCR-based DGGE and 16S rDNA sequencing. The majority of isolates from the intestinal tract of Drosophila simulans Stuvervant belonged to the species Lactobacillus plantarum, but Lactobacillus paracasei, Lactobacillus sanfranciscensis, Leuconostoc mesenteroides subsp. mesenteroides, Lactococcus lactis subsp. lactis, Enterococcus faecalis and Pediococcus pentosaceus were also identified. As far as we could determine, this is the first report on the isolation of L. paracasei, L. sanfranciscensis, L. mesenteroides subsp. mesenteroides, L. lactis subsp. lactis, E. faecalis and P. pentosaceus from vinegar flies. Lactobacillus plantarum has previously been isolated from Merlot grapes. The genotypic relatedness among isolates of L. plantarum isolated from the intestinal tract of vinegar flies and from Merlot grapes were determined by RAPD-PCR. The isolates were grouped into four genotypically well-separated clusters. Thirteen isolates from grape must and five from flies yielded identical RAPD-PCR banding patterns and grouped into one cluster, suggesting that they are descendants from the same strain. This suggests that L. plantarum has the ability to use vinegar flies as a vector.
AFRIKAANSE OPSOMMING: Dertig melksuurbakterieë is vanuit die dermkanaal van Drosophila simulans Stuvervant geïsoleer en nege melksuurbakterieë vanuit Merlot-druiwe. Die druiwe is afkomstig van dieselfde wynkelder in die Stellenbosch-area van Suid-Afrika. Die isolate is volgens morfologiese, biochemiese en fisiologiese eienskappe gegroepeer. Verteenwoordigende isolate vanuit die fenotipiese groepe is tot spesievlak met behulp van lukraak ge-amplifiseerde polimorfe-DNA (RAPD) polimerase ketting-reaksie (PKR), PKR met spesie-spesifieke inleiers, PKR-gebaseerde denaturerende gradient-jel elektroforese (DGGE) en 16S rDNA sekwensering geïdentifiseer. Die meerderheid isolate uit die ingewande van Drosophila simulans Stuvervant is as Lactobacillus plantarum geklassifiseer. Stamme van Lactobacillus paracasei, Lactobacillus sanfranciscensis, Leuconostoc mesenteroides subsp. mesenteroides, Lactococcus lactis subsp. lactis, Enterococcus faecalis en Pediococcus pentosaceus is ook geïdentifiseer. Sover bekend, is dit die eerste keer dat L. paracasei, L. sanfranciscensis, L. mesenteroides subsp. mesenteroides, L. lactis subsp. lactis, E. faecalis en P. pentosaceus uit asynvlieë geïsoleer is. Lactobacillus plantarum is voorheen uit Merlot-druiwe geïsoleer. Die genotipiese ooreenkoms tussen die stamme van L. plantarum wat uit die asynvlieë en Merlot-druiwe geïsoleer is, is deur middel van RAPD-PKR bepaal. Hiervolgens is die stamme in vier genotipies goed-gedefinieerde groepe geplaas. Dertien isolate vanuit druiwemos en vyf vanuit asynvlieë het identiese RAPD-PKR bandpatrone vertoon en het in een groep gesorteer. Hierdie resultate dui daarop dat die stamme heel moontlik uit een voorouer ontstaan het en dat asynvlieë heel moontlik as vektor vir L. plantarum dien.

Thesis (MSc)--Stellenbosch University, 2003.
ENGLISH ABSTRACT: The winemaking process involves a complex microbial flora where the interaction of yeasts, lactic acid bacteria and acetic acid bacteria play an important role in the quality and wholesomeness of the final product. Yeasts are primarily responsible for alcoholic fermentation. Malolactic fermentation follows alcoholic fermentation and is conducted by lactic acid bacteria. These bacteria are important in winemaking and can have a positive or negative effect on the wine quality. Biogenic amines are one of the compounds produced by lactic acid bacteria, which affect the hygienic quality and wholesomeness of the wine negatively and directly pose a health risk to the consumer. The demand of consumers for higher quality and healthier foods has led to renewed interest in studies on biogenic amines. Biogenic amines occur in a wide variety of food products, such as cheese, dried sausage, sauerkraut, fishery products, chocolates, wine and beer. This thesis focussed on the presence of biogenic amines in wine. The first objective of the study was to determine the ability of lactic acid bacteria isolated from South African wine to produce biogenic amines, using a decarboxylase screening plate method. The potential to produce the biogenic amines histamine, tyramine, putrescine and cadaverine was investigated. The results obtained showed that Lactobacillus species (Lactobacillus brevis and Lactobacillus hilgardil) might be the lactic acid bacteria responsible for tyramine and putrescine production and that it can contribute significantly to the overall biogenic amine content in wines. The results also suggest that amine production is strain dependent and not species specific. None of the lactic acid bacteria tested had the ability to produce histamine or cadaverine. It is important to remember that the ability of the lactic acid bacteria to produce biogenic amines has only been investigated in synthetic media and that it does not necessarily imply similar behaviour in wine. Wine represents a complex environment with a wide number of factors influencing microbial growth and decarboxylase activity and, thus, further investigation is necessary to determine if these amine-producing bacteria behave similarly in wine conditions. In addition, the polymerase chain reaction (PCR) amplification method was used for the identification of the tyrosine decarboxylase (TOe) gene in some of the tyramine-producing lactic acid bacteria. This was followed by the sequencing of the amplified products, which are partial TOe gene sequences, of two L. brevis strains and of a L. hilgardii strain. Only one tdc gene sequence has been described for bacteria (Enterococcus faecalis), while a partial TOC gene sequence from L. brevis lOEB 9809 was described. An amino acid sequence alignment of the three TOe gene fragments, obtained in this study, with the known TOe gene fragment of L. brevis lOEB 9809 and the tdc gene of E. faecalis showed a high degree of relatedness and conserved regions. To meet consumer demands, procedures are necessary to prevent the formation of amines in food products. One way of preventing the formation of biogenic amines is to relate amine production with certain lactic acid bacteria species involved in the winemaking process. Another possible way would be to develop a rapid detection method for bacteria carrying amino acid decarboxylase genes. The results of this study provide knowledge about which lactic acid bacteria in the winemaking process could contribute to the production of biogenic amines and the sequencing of additional partial TOe genes could possibly assist in the development of a rapid detection method for tyramine-producing lactic acid bacteria in food products.
AFRIKAANSE OPSOMMING: Die wynmaakproses behels 'n komplekse mikrobiese flora waar die interaksie van giste, melksuurbakterieë en asynsuurbakterieë 'n belangrike rol speel in die kwaliteit en heilsaamheid van die finale produk. Giste is primêr verantwoordelik vir alkoholiese fermentasie. Appelmelksuurgisting volg op alkoholiese fermentasie en word deur melksuurbakterieë uitgevoer. Hierdie bakterieë is belangrik in die maak van wyn en kan 'n positiewe of negatiewe uitwerking op die kwaliteit van wyn hê. Biogeniese amiene is een van die komponente wat deur melksuurbakterieë geproduseer kan word en wat die higiëniese kwaliteit en heilsaamheid van die wyn benadeel. Dit hou ook 'n gesondheidsrisiko vir die verbruiker in. Die vereiste van verbruikers vir hoër kwaliteit en gesonder voedselprodukte het nuwe belangstelling in studies op biogeniese amiene ontlok. Biogeniese amiene kom in 'n wye verskeidenheid voedselprodukte voor, soos kaas, droëwors, suurkool, vis, sjokolade, wyn en bier. Hierdie tesis fokus op die teenwoordigheid van biogeniese amiene in wyn. Die eerste doelwit van die studie was om melksuurbakterieë, wat uit Suid- Afrikaanse wyn geïsoleer is, se vermoë te bepaal om biogeniese amiene op dekarboksilase-agarplate te produseer. Die potensiaal om die biogeniese amiene histamien, tiramien, putresien en kadawerien te produseer, is bestudeer. Die resultate wat verkry is, toon dat Lactobacillus-spesies (Lactobacillus brevis en Lactobacillus hilgardit) vir tiramien- en putresienproduksie verantwoordelik is en dat hulle 'n belangrike bydrae kan lewer tot die totale biogeniese amienkonsentrasie in wyn. Die resultate dui ook daarop dat die produksie van amiene afhanklik is van die ras, en nié 'n spesifieke spesie nie. Geen melksuurbakterieë wat getoets is, het die vermoë getoon om histamien of kadawerien te produseer nie. Dit is belangrik om in ag te neem dat die vermoë van die melksuurbakterieë om amiene te produseer slegs in sintetiese media bestudeer is en dat dit nie noodwendig dieselfde gedrag in wyn sal toon nie. Wyn is 'n komplekse omgewing met 'n wye verskeidenheid faktore wat die mikrobiese groei en dekarboksilase-aktiwiteit kan beïnvloed, daarom is verdere studie nodig om vas te stelof hierdie amien-produserende bakterieë dieselfde gedrag in wyn sal toon. Die polimerase-kettingreaksie (PKR) amplifikasie-metode is vir die identifikasie van die tirosiendekarboksilase-geen (TDK) in sommige van die tiramienproduserende melksuurbakterieë gebruik. Dit is gevolg deur die volgordebepaling van die geamplifiseerde produkte, wat gedeeltelike TDK-geenvolgordes is, van twee L. brevis- en van een L. hilgardii-ras. Slegs een tdk-geenvolgorde is al voorheen vir bakterieë beskryf, nl. Enterococcus faecalis, asook 'n gedeeltelike TDK-geenvolgorde vir L. brevis lOEB 9809. 'n Vergelyking van die aminosuurvolgordes van die drie TDK-geenfragmente wat in die studie verkry is, het 'n hoë graad van ooreenkoms en gekonserveerde areas met die bekende TDK-geenfragment van L. brevis lOEB 9809 en die tdk-geen van E. faecalis getoon. Om verbruikers se behoeftes te bevredig, is dit noodsaaklik dat die vorming van amiene in voedselprodukte voorkom word. Een manier van voorkoming is om amienproduksie aan sekere melksuurbakterieë wat in die wynmaakproses betrokke is, te koppel. 'n Ander manier sal wees om 'n vinnige metode te ontwikkel vir die opsporing van bakterieë wat aminosuurdekarboksilase-gene dra. Die resultate van die studie verskaf kennis van watter melksuurbakterieë in die wynmaakproses tot die produksie van biogeniese amiene kan bydra. Die volgordebepaling van addisionele gedeeltelike TDK-gene kan moontlik tot die ontwikkeling van 'n vinnige opsporingsmetode van tiramien-produserende melksuurbakterieë in voedselprodukte bydra.

Thesis (MScAgric.)--University of Stellenbosch, 2001.
ENGLISH ABSTRACT: Changes in the bacterial population throughout the malting process of two barley cultivars, i.e. Clipper (local cultivar) and Prisma (imported cultivar), malted at Southern Associated Maltsters (SAM), Caledon, South Africa, were studied. Samples were taken from four individual runs of each cultivar at ten different stages, i.e. dry barley before steep, water from the first steep water-stand, barley after draining the first steep, water from the second steep water-stand, barley from the second steep water-stand, barley after draining of the second steep, barley from the first, second and third days of germination in the germination vessels (GV), and malt after kilning. Emphasis was placed on the taxonomy and composition of the lactic acid bacteria (LAB) isolated from the ten different phases. The LAB were identified to species level by using numerical analysis of total soluble cell protein patterns, RAPD-PCR banding patterns and 16S rRNA sequencing. The Gram-negative bacteria were identified to genus level by using the API 20E system and included Citrobacter spp., Enterobacter spp., Pantoea spp., Proteus spp., Seratia spp., Kluyvera spp., Klebsiella spp., Vibrio spp. and Escherichia coli. The number of viable bacteria throughout the malting process of the two cultivars did not differ significantly, although the LAB counts in the barley before steep and on the kilned malt were higher in Prisma than in Clipper. Leuconostoc argentinum, Leuconostoc laetis and Weissella confusa were the most predominant in both cultivars. A few strains of Weissella paramesenteroides, Lactobacillus casei, Lactococcus laetis and Lactobacillus rhamnosus were also isolated. Lb. casei and Lb. rhamnosus were not isolated from the Prisma cultivar, whilst W paramesenteroides and Le. laetis were absent in the Clipper cultivar. Kilned malt of the Clipper cultivar contained predominantly Le. argentinum, whereas the Prisma cultivar contained mainly Le. lactis. The effect of these bacteria on the fermenting ability of the brewer's yeast Saccharomyces cerevisiae SAB 05, was also studied. Fermentations were conducted in wort prepared from Clipper and Prisma malt. Yeast in combination with the different bacteria were used in the fermentation studies. Wort with only yeast was used as control. Emphasis was placed on the effect the bacteria has on the gravity, pH, yeast- and bacterial- counts and the different volatile aroma compounds produced throughout the fermentations. The presence of LAB and Gram-negative bacteria had no effect on the yeast to reduce the gravity of the fermenting wort, whilst the LAB caused a decrease in the pH of the fermentations in both Clipper and Prisma wort. The cell numbers of the Gram-negative bacteria decreased throughout the fermentations, whilst the LAB cell numbers remained constant. Comparisons could be drawn between the volatile aroma compounds produced in the control fermentation and fermentations with yeast and Gram-negative bacteria, yeast and Lactobacillus spp. and yeast and Weissella spp. Leuconostoc spp. had a much greater influence on the aromatic composition of fermented malt, with much more clear variations between Prisma and Clipper. No major differences were recorded in the aroma profiles of Prisma and Clipper malt fermented in the presence and absence of Lactococcus spp. The Gram-negative bacteria had no significant effect on the volatile aroma compounds produced by the yeast, whilst the LAB had a definite effect on aroma composition in both cultivars. The levels of four of the five principle aroma compounds, present in beer, were in the acceptable concentration range on the fmal day of fermentation. The compounds with the highest concentrations were iso-amyl alcohol, acetic acid and acetoin, with acetic acid being present in the highest concentration in all the fermentations.
AFRIKAANSE OPSOMMING: Veranderinge in die bakteriese populasie van die gars kultivars, Clipper (plaaslik) en Prisma (ingevoer), vermout by Southern Associated Maltsters (SAM), Caledon, Suid Afrika, is ondersoek. Monsters is van vier individuele lopies van elke kultivar en tydens tien verskillende fases van die vermoutingsproses geneem. Die tien verskillende stadia het die volgende ingesluit: Droë gars voor benatting, water van die eerste benattingsfase, gars nadat water van die eerste benattingsfase gedreineer is, water van die tweede benattingsfase, gars van die tweede benattingsfase, gars na die dreinering van water in die tweede benattings fase, gars na die eerste, tweede en derde dag van ontkieming binne die ontkiemingstenke, en mout na droging. Klem is geplaas op die taksonomie en samestelling van melksuurbakterieë (MSB) wat tydens die tien verskillende fases geïsoleer is. Die MSB is tot spesievlak geïdentifiseer deur gebruik te maak van numeriese analise van totale oplosbare selproteïen bandpatrone, RAPD-PKR bandpatrone en 16S rRNA volgorde-bepaling. Gram-negatiewe bakterieë is tot op genusvlak geïdentifiseer deur gebruik te maak van die API 20E toetssisteem. Spesies van die genera Citrobacter, Enterobacter, Pantoea, Proteus, Seratia, Kluyvera, Klebsiella, Vibrio asook Escherichia coli is geïdentifiseer. Tydens die vermoutingsproses van die twee kultivars is geen beduidende verskille in die lewensvatbare bakterietellings gevind nie, alhoewel die MSB-tellings in die gars voor benatting en mout na droging in Prisma hoër was as in Clipper. Leuconostoc argentinum, Leuconostoc laetis en Weissella confusa het die meeste voorgekom in beide kultivars. Kleiner hoeveelhede van Weissella paramesenteroides, Lactobacillus casei, Lactococcus laetis en Lactobacillus rhamnosus is ook geïsoleer. Lb. casei en Lb. rhamnosus het nie in die Prisma-kultivar voorgekom nie, terwyl W paramesenteroides en Le. laetis nie in die Clipper-kultivar teenwoordig was nie. Le. argentinum het meestal in die gedroogde mout van die Clipper-kultivar voorgekom, terwyl Le. laetis meestal in die Prisma-kultivar waargeneem is. Die effek van hierdie bakterieë op die fermentasievermoë van die brouersgis Saccharomyces cerevisiae SAB 05 is ook bestudeer. Die fermentasies is in Clipper- en Prisma- wort gedoen. Vir die fermentasiestudies is gis in kombinasie met verskillende bakterieë gebruik. Wort met slegs gis het as kontrole gedien. Klem is geplaas op die effek van die bakterieë op die digtheid, pH, gis- en bakterietellings en die verskillende vlugtige komponente wat tydens die fermentasies geproduseer is. Die teenwoordigheid van MSB en Gram-negatiewe bakterieë het geen effek gehad op die vermoë van die gis om die digtheid van die gefermenteerde wort te verlaag nie. Die MSB het wel 'n verlaging van die pH in beide Clipper- en Prisma- wort teweeggebring. Tydens die fermentasie het die Gramnegatiewe bakterietellings verminder, terwyl die MSB-tellings konstant gebly het. 'n Verband is gevind tussen vlugtige komponente geproduseer in die kontrole-fermentasie en fermentasies met gis en Gram-negatiewe bakterieë, gis en Lactobacillus spp. en gis en Weissella spp. Leuconostoc spp. het groter veskille in die samestelling van die gefermenteerde wort teweeg gebring met duidelike verskille tussen Clipper en Prisma. Die teenwoordigheid van Lactococcus spp. het nie groot verskille in die samestelling van die gefermenteerde wort getoon nie. Op die laaste dag van die fermentasies was die vlakke van vier uit die vyfbelangrikste vlugtige aroma komponente wat in bier voorkom in die kontrole fermentasies in aanvaarbare konsentrasies teenwoordig. Die Gramnegatiewe bakterieë het geen beduidende invloed gehad op die vlugtige aroma komponente wat deur die gis geproduseer is nie, terwyl die MSB 'n besliste effek in die aroma-samestelling van beide die kultivars gehad het. Die komponente met die hoogste konsentrasies was, isoamiel-alkohol, asynsuur en asetoin. Asynsuur was in al die fermentasies in die hoogste konsentrasie teenwoordig.

This study was conducted on twenty-eight men at Halley Base, Antarctica, in total physical isolation from all other human contact from beginning March to end December 1983. Aims of study: observe S. aureus carriage in this community; monitor effects on carriage of topical antibacterials. Initially, weekly nasal, axillary and perineal swabs taken. From week 24 throat swabs taken from known nasal carriers. Two courses of antibacterials given to all subjects, regardless of carrier status. Two further courses given to known carriers. Eight subjects consistently carried own phage type throughout study, despite application of antibacterials. Eradication appeared successful in two, possibly three individuals, but after significant interval (39 weeks in one) S. aureus found of phage type either not isolated before in study, or not found for prolonged period. May reflect inadequacy of conventional sampling methods. S. aureus in throat of nine of twelve nasal carriers. No consistent skin carriers. Seven subjects intermittent nasal carriers. Four probably acquired strain from consistent carriers. Approximately 90% of stored isolates revived for phage on return to UK. Two consistent carriers and one intermittent carrier yielded non-typable strains. Alternative typing method developed. All phage types indistinguishable by polyacrylamide gel electrophoresis (PAGE) of whole cell extracts. Insufficient protein in supernatants for PAGE. Western Blotting of supernatants using normal human plasma as anti-staphylococcal antibody source distinguished between different phage types, but non-typable strains still indistinguishable. Conclusions: 1) Individuals' carrier status stable over many months. Living in proximity to persistent carriers, some individuals never gave positive swab. 2) Throat may be significant carriage site. 3) Topical antibacterial application unlikely to eradicate S. aureus from nose, particularly in persistent carriers. 4) Apparent eradication may represent suppression. 5) Western Blotting of culture supernatants may provide alternative typing method, also information on strains of direct clinical significance.

Rat hsc70 and human hsp70 have been expressed in bacteria using the T7 polymerase system. The recombinant proteins, which were the major proteins in E.coli, had the same molecular weights as the tissue-isolated proteins and were immunoactive with hsc70/hsp70 antibodies. ATP binding assay by equilibrium dialysis showed a K$\sb{\rm d}$ for ATP of 0.44 $\mu$M. At saturation, 0.4 mole of ATP was bound per mole of hsc70. Both recombinant and tissue-isolated hsc70/hsp70 have ATPase activities. The denatured substrate, reduced carboxyl methylated $\alpha$-lactalbumin (RCMLA), stimulated ATPase rates of bovine tissue-isolated hsc70/hsp70, but the ATPase rates of rat skeletal muscle and recombinant hsc70 were not changed upon the adding of RCMLA. The analysis of two-dimensional gels showed hsc70/hsp70 isolated from different sources had different isoform patterns. It is speculated that each isoform may have its own substrate specificity.

Background Information The global misuse and overuse of antibiotics in human medicine and the animal production industry is contributing to the development of antibiotic resistance in bacteria. This is a serious threat to modern medicine and public health. Antibiotic resistant organisms can cause severe infections in humans which are difficult to treat, and in some cases impossible to resolve which can lead to premature death. Several studies have been conducted across the globe to assess the use of antibiotics in the seafood industry and the associated health risks, however, limited studies have recently explored this risk in an Australian setting. Aims This thesis aimed to investigate the presence of antibiotic residues in seafood sold in Western Australia. Furthermore, the occurrence of antibiotic resistance in Gram negative bacteria isolated from fish sold in Perth, Western Australia was assessed. The impact of country of origin on the presence of antibiotic residues and antibiotic resistant bacteria in seafood samples has also been considered. Methodology Historical data was accessed from the Local Health Authorities Analytical Committee regarding the presence of eight antibiotic types in 253 seafood samples purchased throughout Western Australia between May and June 2017. Forty-four fish samples, a mix of local and imported from Asian countries, were sourced from retail shops located in the metropolitan area of Perth between September and November 2017. Gram negative bacteria were isolated by homogenisation of the fish with a Luria Bertani Broth and incubation on media selective for Gram negative bacteria. A series of preliminary microbial identification tests were conducted on selected bacterial isolates. Matrix Assisted Laser Desorption Ionization-Time of Flight confirmed the identification of the bacteria to species level. The identified bacteria (n = 35) were analysed for antibiotic susceptibility to eight antibiotic types using the standard disc diffusion method. Results The majority of seafood samples were free from antibiotic residue contamination and compliant with Australian legislation. A single non-compliant sample contained antibiotic residues below the level required to pose an immediate health risk to the consumer. This result suggests the Australian consumer has limited risk of consuming antibiotic residues in seafood. Thirty-five Gram negative bacterial isolates from ten genera were identified. The majority of the antibiotic resistance observed in the bacteria was either explained by intrinsic resistance or was similar to previous reports. Potential acquired antibiotic resistance was observed in four Acinetobacter species and a Rhizobium isolate which were isolated from commonly farmed fish from Australia (n = 1), China (n = 1) and Vietnam (n = 3). It is possible the fish may have been exposed to antibiotics during the production cycle. However, this result must be read with caution since there are limited standardised breakpoint guidelines for these particular species and, therefore the results were inferred using guidelines for other, similar, bacterial species. From these results, it appears that there is limited risk to consumer health from exposure to antibiotic resistant bacteria via consumption of seafood, however, only a limited number of samples were assessed, and Gram positive bacteria were not evaluated in this study. These results are reassuring but suggest that vigilance is required to ensure that the risk to consumers is minimised. Where antibiotics are used inappropriately in environmental settings, the risk of environmental bacteria developing further antibiotic resistance will remain. Routine surveillance of antibiotic resistance in pathogenic bacteria in domestic and imported food of animal origin is recommended to monitor this potential risk to human public health.

>Magister Scientiae - MSc
The negative environmental impact of fossil fuels and growing concerns about petroleum supplies has driven the search for alternative, renewable transportation fuels. An 'ideal' fuel replacement would be a biofuel produced from lignocellulosic biomass. Unfortunately, the presence of lignin in plant cell walls impedes the breakdown of cell wall polysaccharides into simple sugars and the subsequent conversion of these sugars into useable fuels. One of the most common fates of lignin in nature is to be metabolized by lignin peroxidases (LiPs), predominantly of microbial origin. This study aims to isolate and characterise microorganism(s) involved in the degradation of lignocellulose. Thermophilic bacteria were isolated from straw-based compost and screened for lignin peroxidase activity. One isolate, CP11, showed significant lignin peroxidase activity and based on 16S rRNA gene sequence analysis, the isolate was found to be most closely related to Bacillus thermoamylovorans. Morphological, physiological and biochemical characterisation was conducted to determine whether the isolate was a novel species. Morphologically, CP11 was characterised as an endospore-forming, Gram positive rod. In addition, the isolate was found to be a facultative anaerobe, catalase positive and capable of utilising a range of carbon sources including glucose, sucrose and arabinose. Isolate CP11 was moderately thermotolerant and grew between 37°C and 55°C, with an optimum growth temperature of 45°C. Based on its phenotypic characteristics CP11 could be clearly distinguished from its closest phylogenetic neighbours. Preliminary characterisation of the lignin peroxidase was conducted using crude enzyme extract and Azure B dye as the substrate. Activity was detected in the supernatant only and a growth curve was constructed to determine the growth phase of lignin peroxidase production. In order to identify the gene encoding the lignin peroxidase a small insert library was constructed and screened for ligninase activity using Azure B as the substrate.
National Research Foundation

Two abandoned mine tailings sites (Calumet near Ottawa, and Potter near Timmins, Ontario) have been shown to support active populations of iron- (IRB) and sulfate-reducing bacteria (SRB). The competition between IRB and SRB for similar electron donors was however never assessed. The present study looked into the potential competition between IRB and SRB isolated from those 2 sites since they represent different pH conditions and mineralogy. The Potter tailings are acidic to slightly acidic and contain large quantities of sulfides, whereas the Calumet tailings are alkaline and contain less pyrite and more carbonate minerals. Batch experiments were designed to test the competition between IRB and SRB for 3 electron donors (acetate, formate and lactate) and to determine the role of abiotic Fe(III) reduction. Results from abiotic systems indicated that Fe(II) was released overtime in the various systems due to the reduction of Fe(III)-rich minerals by the organic acids present in the medium and due to the chemical oxidation of pyrite by ferric iron. IRB could only grow in the Calumet systems containing inhibited SRB. In these systems, IRB growth was favored in the presence of acetate. In the systems containing active SRB, IRB growth was minimal in the presence of all electron donors, suggesting that they cannot compete with SRB under the conditions used in the systems. SRB present in all systems were capable of oxidizing all 3 electron donors, including acetate. Our results also showed that complete and incomplete lactate oxidizers were present in the Calumet and Potter systems.

Sorghum plants excrete phenolic acids which reduce subsequent crop yields. These acids accumulate in field soil by combining with soil and clay particles to form stable complexes which remain until degraded by bacterial metabolism. The amount of phenolic acids in soil samples were obtained by gas chromatography measurements, while Azotobacter populations were obtained by plate counts in 40 sorghum field samples from Denton County, Texas. One can conclude that increasing the Azotobacter population in the soil increased the degradation rate of phenolic acids proportionally. It is proposed that seed inoculation will introduce selected strains of Azotobacter into the soil. The presence of Azotobacter should increase crop size in subsequent plantings.

This study has contributed to the patented technology of biocement (Microbial Biocementation, WO/2006/066326). Biocementation or biogrout is a sand consolidation technology, in which the carbonate released from microbial urea hydrolysis precipitates with an excess of calcium ions to form in-situ calcite (CaCO3) precipitation. Under the right conditions this can result in soil solidification and has found significant commercial interest. This study has enriched and isolated highly urease active bacteria, particularly suitable for the fermentation process. Six strains with different properties relevant for biocementation were isolated. The most urease active strain (strain MCP11) produced sufficient urease to allow the use of the non-concentrated cell suspension for biocementation experiments. Activities and specific activities were 11-28 mM urea hydrolysed.min-1 and 2.2-5.6 mM urea hydrolysed.min-1.OD-1 respectively. A separate strain (strain MCP4) showed spontaneous flocculation at the end of the batch growth, showing its increased tendency to attach to surfaces. This can be useful for effective cell concentration and for improved attachment during the cementation process. The possibility of causing cementation by using enrichments rather than pure strains has been documented. This may allow a cheaper production of the urease than by traditional pure culture processes. Urease production was optimised by increasing the concentration of yeast extract and the addition of Ni2+ ions to the growth media, resulting in increasing urease activity as the reproducible urease yield. This was accomplished by the addition of 10 ¦ÌM Ni 2+ ions and increasing the level of yeast extract to 20 g.L-1 Some of the isolated strains were suitable for biocementation process producing mechanical strength (¡Ý0.6 MPa) within several hours depending on the rate of urea conversion. This mechanical strength enhancement of the cemented columns was performed without a large decrease in the permeability. The formation of CaCO3 crystals in the presence of high concentration of calcium and urea was monitored. This crystal growth was monitored over time by video recording the ureolytic reaction on a microscopic slide. The crystals also were examined through SEM. It was found that two types of CaCO3 precipitates were formed; these precipitates were calcite rhombohedral crystals and spheroids. Video clips showed that the rhombohedral crystals originated from the spheroids. These spheroids were fragile, not stable and were considered to be vaterite. This study suggested that the strength of the cemented column was caused mostly due to the point-to-point contacts of rhombohedral CaCO3 crystals and adjacent sand grains. A method of producing high strength cemented samples from sand was developed. This method first attaches the cells into the sand-column by growing them in the presence of calcium ions as little as 6 mM. Then, the cells were incubated in-situ for about 48 hours to enable attachment to the surface of the sand granules. Then the cells were reused over 20-times by continuous supply of cementation solution (equi-molar concentration of calcium and urea). This method produced a mechanical strength of up to 30 MPa, which is equivalent to construction cement. The mechanical strength could be increased by supplying the bacteria in-situ with a food source and 10 ¦ÌM Ni2+ ions, allowing some measures of reaction rate control in-situ. To our knowledge, this study was the first study to use biological cementation to produce strength comparable to that of traditional cemented construction materials such as sandstone and concrete. The key factors for the optimal CaCO3 precipitation (strength production) in-situ were examined. It was found that in-situ urease activity was the key factor for strength production. The maximum in-situ urease activity was achieved by supplementing the cementation solution with growth media, and the use of 0.5 M urea and Ca2+ as cementation solution. The in-situ urease activity differed according to the different bacterial strains which tolerated the cementation conditions differently. One of the advantages of the present study was that cementation of porous media could be achieved without clogging the injection end. The injection end could be clogged by CaCO3 precipitation due to cementation reaction (cells, calcium and urea). By sequentially flushing the cells and cementation solution, clogging of the injection end could be avoided and high penetration depth was achieved as long as there was sufficient passage of cementation solution. Uniform cementation along 1 m packed sand-column was obtained. This uniformity was confirmed by the urease activity measurement, calcite precipitation and mechanical strength production. For finer sand, homogenous cementation proved more difficult.

Thesis (MSc (Biomedical Technology))--Cape Peninsula University of Technology, 2017.
Background: It is well established that Klebsiella pneumoniae (K. pneumoniae) is an opportunistic pathogenic organism that has been frequently identified as the cause of nosocomial and community acquired infections. Furthermore, studies have shown that over the last few decades strains of the genus Klebsiella have systematically developed resistance to numerous antibiotics. Aims and Methods: The primary aim of this study was to investigate the prevalence of K. pneumoniae in nosocomial and community isolates in the Western Cape province of South Africa. Various identification techniques such as the polymerase chain reaction (PCR) using the API 20 E, the VITEK®2 system, primers specific for the 16S-23S rDNA ITS region and the Matrix-assisted laser desorption/ionization Time of Flight Mass Spectrometry (MALDI-TOF MS) were compared for the identification of this pathogen. The VITEK 2 system was used to detect antibiotic resistant profiles of the K. pneumoniae isolates and to identify the extended spectrum beta-lactamase (ESBL) phenotypic among these isolates. The PCR was used to detect Beta-lactam genes viz. CTX-M (blaCTX-M), TEM (blaTEM) and SHV (blaSHV) respectively in both the genome and plasmid DNA of K. pneumoniae using gene specific primers. Results: In total 57 agar plate bacterial cultures or glycerol stock bacterial cultures were obtained during 2011. Of the 57 isolates, the API 20 E test identified 47 (82.5%) of the isolates (n = 57) as K. pneumoniae while 10 isolates (17.5%) were identified as Raoultella species. The VITEK 2 method and PCR identified all 57 isolates as K. pneumoniae (100%). Of the isolates, 82.5% (47/57) were positively identified as Klebsiella species, 14% (8/57) were identified as Klebsiella variicola and 3.5% (2/57) were shown as no reliable identification (NRI) when using the MALDI-TOF MS. Examination of the 57 isolates using primers specific for the CTX-M (blaCTX-M), TEM (blaTEM) and SHV (blaSHV) respectively showed the following: PCR amplicons for the TEM gene were produced successfully for 46 (81%) of the 57 isolates included in this project, while 11 (19%) of the samples did not yield any TEM amplicons; PCR amplicons for the blaSHV gene were obtained successfully for 56 (98%) of the 57 DNA samples, while 1 sample (2%) did not yield any SHV amplicons; and PCR amplicons for the blaCTX-M gene were produced successfully by 89% (n = 51) of the DNA samples included in this project, while 11% (n = 6) did not yield any CTX-M amplicon. Extended-spectrum beta-lactamase phenotypes had been confirmed in 84% (n = 48) K. pneumoniae isolates while nine isolates were found to be non-ESBL. Resistance rates for these 48 isolates were high and showed resistance patterns of: Amoxicillin/Ampicillin, Amoxycillin/Clavulanate, Ceftriaxone/Cefotaxime, Cefuroxime/Cefprozil and Ceftazidime (100%, n = 48); Piperacillin/Tazobactam and Cefoxitin (98%, 47/48); Cefepime (96%, 46/48); Aztreonam (94%; 45/48); Tobramycin (81%, 39/48); Gentamycin and Ciprofloxacin (77%, 37/48); Trimethoprim/Sulfamethoxazole (67%, 32/48); and Tigecycline (25% 12/48). Conclusion: For the analysis by all four methods employed, a total agreement of 68.4% was obtained, indicating the positive identification of K. pneumoniae in 39 of the 57 samples analysed. An average agreement of 28.1% was then obtained for the comparison of results generated for three of the methods utilised, while a 3.5% average agreement was obtained for at least two methods. Furthermore, all four methods agreed that 82.5% of the isolates were Klebsiella species while three methods agreed that 17.5% of the isolates were Klebsiella species. Based on the results obtained in the current study, PCR and VITEK 2 were the methods of choice for the identification of K. pneumoniae. The current study also showed, that ESBL-K. pneumoniae strains are present in the Western Cape province, South Africa; with high resistance profiles to numerous antibiotics including the Cephalosporins.

This study has contributed to the patented technology of biocement (Microbial Biocementation, WO/2006/066326). Biocementation or biogrout is a sand consolidation technology, in which the carbonate released from microbial urea hydrolysis precipitates with an excess of calcium ions to form in-situ calcite (CaCO3) precipitation. Under the right conditions this can result in soil solidification and has found significant commercial interest. This study has enriched and isolated highly urease active bacteria, particularly suitable for the fermentation process. Six strains with different properties relevant for biocementation were isolated. The most urease active strain (strain MCP11) produced sufficient urease to allow the use of the non-concentrated cell suspension for biocementation experiments. Activities and specific activities were 11-28 mM urea hydrolysed.min-1 and 2.2-5.6 mM urea hydrolysed.min-1.OD-1 respectively. A separate strain (strain MCP4) showed spontaneous flocculation at the end of the batch growth, showing its increased tendency to attach to surfaces. This can be useful for effective cell concentration and for improved attachment during the cementation process. The possibility of causing cementation by using enrichments rather than pure strains has been documented. This may allow a cheaper production of the urease than by traditional pure culture processes. Urease production was optimised by increasing the concentration of yeast extract and the addition of Ni2+ ions to the growth media, resulting in increasing urease activity as the reproducible urease yield. This was accomplished by the addition of 10 μM Ni 2+ ions and increasing the level of yeast extract to 20 g.L-1 Some of the isolated strains were suitable for biocementation process producing mechanical strength (≥ 0.6 MPa) within several hours depending on the rate of urea conversion. This mechanical strength enhancement of the cemented columns was performed without a large decrease in the permeability. The formation of CaCO3 crystals in the presence of high concentration of calcium and urea was monitored. This crystal growth was monitored over time by video recording the ureolytic reaction on a microscopic slide. The crystals also were examined through SEM. It was found that two types of CaCO3 precipitates were formed; these precipitates were calcite rhombohedral crystals and spheroids. Video clips showed that the rhombohedral crystals originated from the spheroids. These spheroids were fragile, not stable and were considered to be vaterite. This study suggested that the strength of the cemented column was caused mostly due to the point-to-point contacts of rhombohedral CaCO3 crystals and adjacent sand grains. A method of producing high strength cemented samples from sand was developed. This method first attaches the cells into the sand-column by growing them in the presence of calcium ions as little as 6 mM. Then, the cells were incubated in-situ for about 48 hours to enable attachment to the surface of the sand granules. Then the cells were reused over 20-times by continuous supply of cementation solution (equi-molar concentration of calcium and urea). This method produced a mechanical strength of up to 30 MPa, which is equivalent to construction cement. The mechanical strength could be increased by supplying the bacteria in-situ with a food source and 10 μM Ni2+ ions, allowing some measures of reaction rate control in-situ. To our knowledge, this study was the first study to use biological cementation to produce strength comparable to that of traditional cemented construction materials such as sandstone and concrete. The key factors for the optimal CaCO3 precipitation (strength production) in-situ were examined. It was found that in-situ urease activity was the key factor for strength production. The maximum in-situ urease activity was achieved by supplementing the cementation solution with growth media, and the use of 0.5 M urea and Ca2+ as cementation solution. The in-situ urease activity differed according to the different bacterial strains which tolerated the cementation conditions differently. One of the advantages of the present study was that cementation of porous media could be achieved without clogging the injection end. The injection end could be clogged by CaCO3 precipitation due to cementation reaction (cells, calcium and urea). By sequentially flushing the cells and cementation solution, clogging of the injection end could be avoided and high penetration depth was achieved as long as there was sufficient passage of cementation solution. Uniform cementation along 1 m packed sand-column was obtained. This uniformity was confirmed by the urease activity measurement, calcite precipitation and mechanical strength production. For finer sand, homogenous cementation proved more difficult.

This study has contributed to the patented technology of biocement (Microbial Biocementation, WO/2006/066326). Biocementation or biogrout is a sand consolidation technology, in which the carbonate released from microbial urea hydrolysis precipitates with an excess of calcium ions to form in-situ calcite (CaCO3) precipitation. Under the right conditions this can result in soil solidification and has found significant commercial interest. This study has enriched and isolated highly urease active bacteria, particularly suitable for the fermentation process. Six strains with different properties relevant for biocementation were isolated. The most urease active strain (strain MCP11) produced sufficient urease to allow the use of the non-concentrated cell suspension for biocementation experiments. Activities and specific activities were 11-28 mM urea hydrolysed.min-1 and 2.2-5.6 mM urea hydrolysed.min-1.OD-1 respectively. A separate strain (strain MCP4) showed spontaneous flocculation at the end of the batch growth, showing its increased tendency to attach to surfaces. This can be useful for effective cell concentration and for improved attachment during the cementation process. The possibility of causing cementation by using enrichments rather than pure strains has been documented. This may allow a cheaper production of the urease than by traditional pure culture processes. Urease production was optimised by increasing the concentration of yeast extract and the addition of Ni2+ ions to the growth media, resulting in increasing urease activity as the reproducible urease yield. This was accomplished by the addition of 10 μM Ni 2+ ions and increasing the level of yeast extract to 20 g.L-1 Some of the isolated strains were suitable for biocementation process producing mechanical strength (≥ 0.6 MPa) within several hours depending on the rate of urea conversion. This mechanical strength enhancement of the cemented columns was performed without a large decrease in the permeability. The formation of CaCO3 crystals in the presence of high concentration of calcium and urea was monitored. This crystal growth was monitored over time by video recording the ureolytic reaction on a microscopic slide. The crystals also were examined through SEM. It was found that two types of CaCO3 precipitates were formed; these precipitates were calcite rhombohedral crystals and spheroids. Video clips showed that the rhombohedral crystals originated from the spheroids. These spheroids were fragile, not stable and were considered to be vaterite. This study suggested that the strength of the cemented column was caused mostly due to the point-to-point contacts of rhombohedral CaCO3 crystals and adjacent sand grains. A method of producing high strength cemented samples from sand was developed. This method first attaches the cells into the sand-column by growing them in the presence of calcium ions as little as 6 mM. Then, the cells were incubated in-situ for about 48 hours to enable attachment to the surface of the sand granules. Then the cells were reused over 20-times by continuous supply of cementation solution (equi-molar concentration of calcium and urea). This method produced a mechanical strength of up to 30 MPa, which is equivalent to construction cement. The mechanical strength could be increased by supplying the bacteria in-situ with a food source and 10 μM Ni2+ ions, allowing some measures of reaction rate control in-situ. To our knowledge, this study was the first study to use biological cementation to produce strength comparable to that of traditional cemented construction materials such as sandstone and concrete. The key factors for the optimal CaCO3 precipitation (strength production) in-situ were examined. It was found that in-situ urease activity was the key factor for strength production. The maximum in-situ urease activity was achieved by supplementing the cementation solution with growth media, and the use of 0.5 M urea and Ca2+ as cementation solution. The in-situ urease activity differed according to the different bacterial strains which tolerated the cementation conditions differently. One of the advantages of the present study was that cementation of porous media could be achieved without clogging the injection end. The injection end could be clogged by CaCO3 precipitation due to cementation reaction (cells, calcium and urea). By sequentially flushing the cells and cementation solution, clogging of the injection end could be avoided and high penetration depth was achieved as long as there was sufficient passage of cementation solution. Uniform cementation along 1 m packed sand-column was obtained. This uniformity was confirmed by the urease activity measurement, calcite precipitation and mechanical strength production. For finer sand, homogenous cementation proved more difficult.

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
Bactérias ácido lácticas (BAL) foram isoladas do ambiente de produção de leite e avaliadas quanto ao potencial benéfico. Testes preliminares e análise por PCR foram aplicados para selecionar e identificar através de sequenciamento de rRNA 16S 15 cepas de BAL: Lactobacillus (n = 11; Lb. casei MSI1, Lb. casei MSI5, Lb. casei MRUV1, Lb. casei MRUV6, Lb. acidophilus MVA3, Lb. nagelli MSIV4, Lb. harbinensis MSI3, Lb. harbinensis MSIV2, Lb. fermentum SIVGL1, Lb. plantarum MLE5 e Lb. plantarum MSI2), Pediococcus (n = 2; P. pentosaceus MLEV8 e P. acidilactici MSI7) e Weissella (n = 2; W. paramesenteroides MRUV3 e W. paramesenteroides MSAV5). Todas as linhagens selecionadas apresentaram resistência ao baixo pH e à presença de sais biliares. O teste API ZYM foi realizado para caracterizar a atividade enzimática entre as cepas e foi observada elevada atividade β-galactosidase em 13 delas. Todas as cepas apresentaram alta taxa de sobrevivência ao suco gástrico e as condições intestinais simulados, capacidade de auto-agregação e co- agregação com micro-organismos indicadores e alta hidrofobicidade da superfície celular. A maioria das cepas foi positiva para os genes de adesão map e EFTu. Os resultados de deconjugação de sais biliares mostraram forte desconjugação para todas as cepas. Todas as cepas mostraram bons resultados para assimilar lactose. Após esta etapa de caracterização do potencial benéfico, as 15 BAL foram avaliadas quanto ao potencial de virulência e de resistência antimicrobiana. A produção de fatores de virulência (hemólise, gelatinase, lipase, desoxirribonuclease e aminas biogênicas: lisina, tirosina, histidina e a ornitina) foi avaliada por métodos fenotípicos, a 25 °C e 37 °C, bem como a resistência a 17 antibióticos. Os isolados foram também submetidos à análise de PCR para identificar a presença de 49 genes associados a fatores de virulência. Nenhuma das cepas apresentou atividade hemolítica, produção de gelatinase, lipase, desoxirribonuclease e aminas biogênicas. Das 15 cepas selecionadas, para 12 tipos de antibióticos no método de difusão em disco, todas as amostras foram resistentes à oxacilina e sulfa/trimetoprim, 14 foram resistentes a gentamicina, 11 foram resistentes a clindamicina, nove cepas foram resistentes à vancomicina, oito cepas para rifampicina, cinco foram resistentes a eritromicina, quatro foram resistentes à tetraciclina, duas cepas foram resistentes à ampicilina, uma cepa foi resistente ao cloranfenicol e nenhuma apresentou resistência ao imipenem. Para um teste quantitativo do antibiograma, 5 antibióticos em fitas Etest® (bioMérieux) foram selecionados. Todas as 15 cepas foram resistentes à vancomicina, duas para rifampicina, uma para gentamicina e uma para o cloranfenicol. Em relação aos genes relacionados com virulência, 19 dos 49 genes testados estavam presentes em algumas cepas. Após a caracterização do potencial virulento das 15 BAL, estas foram avaliadas quanto ao potencial tecnológico para aplicação na indústria de laticínios. Todas as cepas apresentaram capacidade de acidificação, atingindo valores de pH entre 0.73 e 2.11 em 24 horas: Lb. casei MRUV6 apresentou maior capacidade de acidificação (pH 2.11 após 24 h). Dez cepas foram capazes de produzir diacetil a 37 °C, com exceção de Lb. casei MSI1, Lb. harbinensis MSI3, Lb. fermentum SIVGL1, Lb. plantarum MLE5 e W. paramesenteroides MRUV3. Todas as cepas foram capazes de produzir exopolissacarídeos, e apenas duas cepas apresentaram atividade proteolítica (Lb. casei MSI5 e W. paramesenteroides MSAV5). Com base nessa caracterização, Lb. casei MRUV6 foi selecionado para produzir o leite fermentado, armazenado a 4 °C e 10 °C e monitorado até 35 dias de vida útil. As amostras foram submetidas a métodos fenotípicos e moleculares para avaliar a presença de Lb. casei MRUV6 (plaqueamento convencional e RT-PCR, verificando a expressão de gapdh, um gene housekeeping) e verificar a expressão do gene bsh, relacionado à resistência à sais biliares (RT-PCR). A população de Lb. casei MRUV6 se apresentou estável durante todo o período de armazenamento a 4 °C e 10 °C a níveis em torno de 9.9 log UFC/g e também pelo monitoramento da expressão do controle endógeno GAPDH. No entanto, o gene bsh não foi expresso durante o período de armazenamento. O estudo demonstrou o potencial uso da cepa de Lb. casei MRUV6 isolada de um ambiente lácteo para a produção de um produto lácteo fermentado e sua estabilidade durante o armazenamento a 4 °C e 10 °C. Todos os isolados do estudo apresentaram características benéficas, segurança para utilização em alimentos e potencial tecnológico para utilização na indústria de laticínios. Além disso, os mesmos podem ainda ser submetidos a estudos adicionais para avaliações in vivo e realizar a caracterização como probióticos.
Lactic acid bacteria isolated from dairy environment were evaluated for beneficial potential. Preliminary screening and PCR analysis were applied to select and identified through 16s rRNA sequencing 15 LAB strains: Lactobacillus (n = 11; Lb. casei MSI1, Lb. casei MSI5, Lb. casei MRUV1, Lb. casei MRUV6, Lb. acidophilus MVA3, Lb. nagelli MSIV4, Lb. harbinensis MSI3, Lb. harbinensis MSIV2, Lb. fermentum SIVGL1, Lb. plantarum MLE5 and Lb. plantarum MSI2), Pediococcus (n = 2; P. pentosaceus MLEV8 and P. acidilactici MSI7) and Weissella (n = 2; W. paramesenteroides MRUV3 and W. paramesenteroides MSAV5). All selected strains showed resistance to acidic pH and to presence of bile salt. API ZYM test characterized enzymatic activity of the strains and high β-galactosidase activity was observed in 13 strains. All strains presented high values for survival rate to simulated gastric and intestinal conditions, ability to auto and co-aggregate with indicators microorganisms and high cell surface hydrophobicity. Most of the strains were positive for map and EFTu beneficial genes. Strong bile salts deconjugation was applied for all strains and all strains showed good results for assimilating lactose. After this first part of the study, the 15 BAL were evaluated for potential virulence and antimicrobial resistance. The production of virulence factors (hemolysis, gelatinase, lipase, deoxyribonuclease and biogenic amines: lysine, tyrosine, histidine and ornithine) was assessed by phenotypic methods at 25 °C and 37 °C, as well as the resistance to 17 antimicrobials. The isolates were also subjected to PCR to identify the presence of 49 genes associated with virulence factors. None of the strains presented hemolytic activity or the production of gelatinase, lipase, deoxyribonuclease and tested biogenic amines. Of the 15 selected cultures, for 12 types of antibiotics in the disc diffusion method, all strains were resistant for oxacillin and sulfa/trimethoprim, 14 were resistant to gentamicin, 11 were resistant to clindamycin, nine strains were resistant to vancomycin, eight strains to rifampicin, five were resistant to erythromycin, four were resistant to tetracycline, two strains were resistant to ampicillin, one strain was resistant to chloramphenicol and none was resistant for imipenem. For a quantitative test of the antibiogram, five antibiotics were selected in Etest ® strips (bioMérieux). All 15 strains were resistant to vancomycin, two for rifampicin, one for gentamicin and one for chloramphenicol. Regarding the virulence related genes, 19 genes from 49 tested were present in some strains. Results showed that five cultures showed the presence of the int gene, four cultures showed the presence of the ant(4')-Ia gene, three cultures were positive for vanC2, cpd and tdc, two cultures for vanA, tet(K), tet(S), ermA, bcrR, mur-2ed, asa1 and ccf, and one culture was positive for vanC1, ermB, aph(3')-IIIa, aac(6’)-le-aph(2”)-Ia, bcrB and hyl. After characterizing the virulent potential of the 15 BAL, these strains were evaluated for the technological potential for application in the dairy industry. All strains presented acidification capacity, reaching pH values between 0.73 and 2.11 in 24 hours: Lb. casei MRUV6 presented the highest acidification ability (pH 2.11 after 24 h). Ten strains were able to produce diacetyl at 37 °C, except by Lb. casei MSI1, Lb. harbinensis MSI3, Lb. fermentum SIVGL1, Lb. plantarum MLE5 and W. paramesenteroides MRUV3. All strains were able to produce exopolysaccharides, and only two strains presented proteolytic activity (Lb. casei MSI5 and W. paramesenteroides MSAV5). Based on this characterization, Lb. casei MRUV6 was selected for producing fermented milk, stored at 4 °C and 10 °C and monitored until 35 days of shelf life. Samples were subjected to phenotypical and molecular methods to quantify the presence of Lb. casei MRUV6 (conventional plating and RT-PCR, by checking the expression of gapdh, a housekeeping gene) and to verify the expression of bsh gene, related to resistance to bile salts (RT-PCR). Lb. casei MRUV6 population was stable during storage period at 4 and 10 °C at levels around 9.9 log CFU/g, and by monitoring the expression of gapdh gene. However, bsh gene was not expressed during storage period. The study demonstrated the potential use of the beneficial strain Lb. casei MRUV6 isolated from a dairy environment for the production of a fermented milk product, and its stability during storage at 4 and 10 °C. All isolates from the study presented beneficial characteristics, safety for use in food and technological potential for use in the dairy industry. In addition, they may further be subjected to further studies for in vivo evaluations and characterization as probiotics.

I batteri lattici costituiscono un ampio ed eterogeneo gruppo di microrganismi storicamente utilizzati per le loro importanti proprietà. Basti pensare ai diversi settori in cui vengono utilizzati: industria alimentare e delle bevande per la produzione di prodotti tradizionali e innovativi fermentati e non, produzione di metaboliti rilevanti a livello industriale e, infine, come organismi probiotici per migliorare la salute e rafforzare il sistema immunitario dell'ospite. Negli ultimi anni quest'ultimo aspetto è stato valutato anche nel settore dell'apicoltura, integrando questi microrganismi nella dieta delle api mediante sciroppi di zucchero, al fine di valutare un possibile aumento della resistenza agli organismi patogeni. In questa tesi di dottorato sono stati utilizzati batteri lattici appartenenti alla specie Lactobacillus plantarum, recentemente riclassificata come Lactiplantibacillus plantarum, isolati dal pane d'api e dal tratto digestivo di Apis mellifera ligustica. Uno screening preliminare si è basato sulla capacità di 61 ceppi di L. plantarum di inibire alcuni dei principali patogeni per le api, come Peanibacillus larvae e Ascosphaera apis, responsabili rispettivamente della peste americana e della malattia di Chalkbrood. Sulla base dei risultati ottenuti da questo test, sono stati successivamente selezionati cinque ceppi e utilizzati per valutare la loro possibile applicazione nel settore dell'apicoltura come probiotici. I dati registrati in questo studio hanno evidenziato la capacità di questi ceppi, con gradi diversi, di inibire i due patogeni, di produrre EPS e di formare biofilm in condizioni e concentrazioni di zucchero differenti. La caratterizzazione biochimica dei ceppi ha mostrato la presenza di pattern enzimatici e di assimilazione dei carboidrati in grado di migliorare la digestione e l'assimilazione dei nutrienti da parte delle api. Inoltre, due dei cinque ceppi testati hanno mostrato un'elevata auto-aggregazione e adesione agli idrocarburi, due importanti prerequisiti per la colonizzazione e la protezione del tratto digestivo dell'ospite. Infine, quasi tutti i ceppi testati sono riusciti a sopravvivere alle condizioni di stress date da elevate concentrazioni di zucchero. Sulla base di queste conoscenze, potrebbero essere sviluppati nuovi approcci biotecnologici per migliorare la salute delle api e la qualità dei prodotti dell'alveare.
Lactic acid bacteria constitute a broad heterogeneous group of microorganisms historically used for their important properties. It suffices to think of the different sectors in which they are used: food and drink industry for the production of traditional and innovative fermented and non-fermented products, production of industrially relevant metabolites, and finally as probiotic organisms to improve health and strengthen of the host immune system. In the last years, this last aspect has been evaluated also in the beekeeping sector, integrating these microorganisms in the diet of bees by means of sugar syrups, in order to evaluate a possible increase in the resistance to pathogenic organisms. In this PhD thesis, lactic acid bacteria belonging to the species Lactobacillus plantarum, recently reclassified as Lactiplantibacillus plantarum, previously isolated from bee bread and from the digestive tract of Apis mellifera ligustica, were used A preliminary screening was based on the of ability of 61 L. plantarum strains to inhibit some of the main pathogens for bees, such as Peanibacillus larvae and Ascosphaera apis, responsible for the American Foulbrood disease and the Chalkbrood Disease, respectively. Based on the results obtained by this test, five strains were subsequently selected and used to evaluate their possible applicability in the beekeeping sector as probiotics. Data registered in this study highlighted the ability of these strains, with different degrees, to inhibit the two pathogens, to produce EPS and to form biofilm in different conditions and sugar concentrations. The biochemical characterization of the strains showed the presence of enzymatic patterns and carbohydrates assimilation that can improve the digestion and assimilation on nutrients by bees. Moreover, two out of five tested strains showed high auto-aggregation and adhesion to hydrocarbons, two important prerequisites for colonization and protection of the host digestive tract. Finally, almost all tested strains were able to survive the stress conditions given by high sugar concentrations. Based on this knowledge, new biotechnological approaches could be developed to improve the bee health and the quality of hive products.

Caulobacters are gram-negative bacteria that have a biphasic life cycle consisting of a swarmer and a stalked stage. As a result they have elicited interest as a simple developmental model. Less attention has focussed on their role in the environment, although they have been found in almost every aquatic environment as well as in many soils. Caulobacters are often described as oligotrophic bacteria because of their prevalence in pristine waters but have now been isolated from the relatively nutrient-rich wastewater environment. In order to learn more about this population some basic characterization was carried out and an assay system to determine their prevalence in sewage plants was designed. Most of the organisms isolated from sewage treatment facilities had similar gross morphological features, but differed in holdfast composition, total protein profile, antibiotic resistance and restriction fragment length polymorphism, thereby indicating a greater diversity than originally assumed. Most of the organisms hybridized with flagellin and surface array genes that had previously been cloned, and only one of 155 non-Caulobacter sewage isolates hybridized with the flagellin gene probe; consequently these were used in a DNA-based enumeration strategy. DNA was isolated directly from sewage and probed with the flagellin and the surface array gene probes. The signals obtained were compared to standards made up of pooled Caulobacter DNA from the sewage isolates and non-Caulobacter DNA from organisms also present in sewage. Using this assay Caulobacters could only be detected above the 1% level, which was higher than their proportion in the wastewater environment. It appears that this approach will not be useful in monitoring Caulobacters in treatment plants unless a more highly conserved or higher copy number probe is found.
Science, Faculty of
Microbiology and Immunology, Department of
Graduate

Norbert Pfennig, a German microbiologist, isolated the first true acidophilic purple bacterium in 1969. He named the organism Rhodoblastus acidophilus. Since the original work of Pfennig, no study has examined the phylogeny and physiology of the original strains of R. acidophilus or isolated any new strains. In this thesis six new strains of acidophilic purple nonsulfur bacteria were isolated from a Canadian Sphagnum peat bog. Moreover, three original Pfennig strains of R. acidophilus and two uncharacterized strains (previously isolated by Michael Madigan) were included in experiments aimed to describe the new isolates and further our understanding of the species Rhodoblastus acidophilus. Although pigmentation varied, all of the strains studied were very similar. The 16S rRNA genes of the new bog isolates and the original strains of R. acidophilus and Rhodoblastus sphagnicola, another acidophilic purple phototroph isolated from a Sphagnum peat bog in Russia, showed a 16S rRNA gene sequence similarity greater than or equal to 97%. All isolates were acidophilic and grew best photoheterotrophically on certain organic or fatty acids, or alcohols as carbon sources. Despite subtle physiological differences, all of the strains shared many characteristics. This indicates that R. acidophilus is a reasonably homogenous species and suggests that diversity of the acidophilic phototrophs may be low.

碩士
元智大學
生物科技與工程研究所
97
PHAs (polyhydroxyalkanoates) is biodegradability polymers which is synthesized by microorganisms, some bacteria will synthesize PHAs and accumulated in vivo when the environment have excess carbon source and lack of some nutrients (ex: nitrogen, phosphate or sulfate). PHB (polyhydroxybutyrate) is the most common type of PHAs which is accumulated by microorganisms in the environment. The enteric bacteria can be commonly found in many environments; however, there is no report on their ability for PHAs production. From complete genome sequences of some enteric bacteria (e.g. E. coli and Salmonella), there was also no evidence of genes related to PHS synthesis in their chromosomes. Most of these sequenced strains are from culture collection and clinical samples. During the process of screening PHA-producing microorganisms in the environment, we have isolated some Gram negative bacteria that can accumulate PHB and presumably belong to enteric bacteria according to their 16S rDNA sequences analysis. In this study, we examined if these environmental enteric bacteria can accumulate PHB.

Tese de mestrado, Microbiologia Aplicada, Universidade de Lisboa, Faculdade de Ciências, 2019
A fermentação tradicional de queijos e realizada em muitos países do mundo, nomeadamente Portugal, sendo que consiste no aproveitamento dos microrganismos naturalmente presentes no leite cru. Estes microrganismos encontram-se no leite devido as diferentes etapas de colheita e manuseamento durante e apos esse processo. Sendo que, alteram as moléculas presentes no leite através da fermentação dando novas propriedades ao produto final que será posteriormente curado. As condições destas fases de fermentação e maturação diferem entre tipos de queijos e zonas de produção, determinando as propriedades organoleticas do produto final. Os queijos de Azeitão e Nisa fazem parte dos 10 queijos portugueses que possuem a categoria de denominação de origem protegida (DOP). As zonas de produção dos queijos de Azeitão são Palmela, Sesimbra e Setúbal e as de Nisa são Nisa, Crato, Castelo de Vide, Marvão, Portalegre, Monforte, Arronches e Alter do Chão. Ambos são produzidos com leite de ovelha cru. No caso de Nisa o leite provém de uma raça de ovelha chamada Merina Branca e no caso de Azeitão não se especifica uma raça. O coalho vegetal usado na produção de ambos queijos é obtido de Cynara cardunculus L. Devido às diferentes fases e condições de produção o queijo de Azeitão consiste numa pasta semi-mole e amanteigada, de cor branca ou ligeiramente amarelada com um sabor ácido e salgado. O queijo de Nisa consiste numa pasta semi-dura de cor branca amarelada com um sabor ligeiramente ácido e um cheiro intenso. As bactérias ácido lácticas (BAL) são um grupo de vários géneros que partilham características em comum como serem gram-positivas, catálase negativas, não formam esporos, anaeróbicas facultativas e terem um nível G+C baixo. Para além disto, o seu nome provém da sua capacidade para fermentar açúcares, transformando-os em ácido láctico através de homo- ou heterofermentacao. Este grupo e o predominante em leite cru e, portanto, em queijos produzidos de forma artesanal e essas bactérias vão desempenhar diversos papéis ao longo da fermentação e maturação deste tipo de queijo. Os géneros de BAL que predominam em comidas fermentadas como os queijos são Lactococcus, Streptococcus, Pediococcus, Leuconostoc, Lactobacillus e Enterococcus. Tendo em conta que estas bactérias formam parte tanto da cadeia alimentar como da microbiota de animais e seres humanos podem servir como veículo de transmissão de genes ao existir uma transferência genética entre espécies ou géneros, como descrito na literatura. A problemática desta possível transferência ocorre quando esses genes conferem resistência a antibióticos ou fatores de virulência que anteriormente essas bactérias não possuíam. Essa transferência pode ocorrer para algumas bactérias que formam parte da nossa microbiota e são responsáveis por infecções oportunistas ou para bactérias patogénicas presentes no nosso corpo devido a uma infecção. Resistências adquiridas a antibióticos tem sido estudadas e observadas em BAL, em concreto em Enterococcus spp. devido ao seu papel patogénico oportunista. Foram descritas resistências a antibióticos de diferentes classes como ϐ-lactâmicos, cefalosporinas, aminoglicosidos, lincosamidas e estreptograminas. Em concreto, resistências a tetraciclina e eritromicina são das mais preocupantes devido a importância destes antibióticos e porque esta resistência tem sido atribuída ao uso indevido de antibióticos em comida de animais. Este trabalho teve como objetivo continuar o estudo de queijos de Azeitão e Nisa DOP que começou numa dissertação anterior onde se caracterizaram estes queijos pela sua microbiota e propriedades físico-químicas. Seguido deste estudo foi também analisada a diversidade dos microbiomas destes produtos como parte de outra dissertação. Assim, o presente trabalho consistiu em comparar os resultados anteriores de diversidade e caracterização microbiológica assim como completar essa análise com a procura de resistências a antibióticos e fatores de virulência. Durante o ano de 2018 foram recolhidos queijos de seis queijarias de Azeitão e de duas queijarias de Nisa. A partir destes foram feitas contagens de unidades formadoras de colonias (UFC) e isoladas as bactérias de diferentes grupos, nomeadamente, BAL isoladas com meio MRS (maioritariamente Lactobacillus spp.), Lactococcus spp. com meio M17 e Enterococcus spp. isoladas com meio SBA. Depois foi extraído o ADN desses isolados para realizar a técnica de RAPD-PCR e análise de dendrogramas criados com os perfis de bandas obtidos. A partir estes dendrogramas foi analisada a semelhança entre os isolados dos distintos queijos e foram obtidos índices de diversidades dos diferentes grupos de bactérias e queijarias estudadas. Esta diversidade e as contagens de bactérias foram comparadas as obtidas noutras dissertações deste mesmo projeto nas quais se estudaram anos de produção anteriores. Partindo destes mesmos dendrogramas, foram escolhidos isolados representantes de cada queijaria e realizados testes de resistência a antibióticos assim como a presença de factores de virulência. Relativo aos diferentes anos de produção estudados não foi observada uma tendência clara de contagens de UFC quando comparados os anos ou queijarias, isto e, não houve queijos que tivessem consistentemente maior ou menor numero de bactérias sendo que este numero foi variável ao longo dos anos. Enquanto a diversidade, foram observadas poucas coincidências nas queijarias com maior ou menor índice de diversidade. No caso dos lactococos foi observado o menor índice correspondente aos anos 2018 e 2016 na mesma queijaria, A4. Nos enterococos foi também encontrada a menor diversidade na mesma queijaria, N9, nos queijos dos anos 2018 e 2016. Nas amostras de 2018 o grupo com maior diversidade foi o dos enterococos mas cabe destacar que os três grupos bacterianos tiveram índices muito parecidos. A identificação dos representantes deste grupo de bactérias dos queijos de 2017 foi realizada com uma multiplex PCR e a maioria de isolados foi identificado como E. faecium, seguido por E. faecalis y E. durans estando igualmente representados em número de isolados. Foram encontradas diferenças significativas no numero de resistências ao longo dos anos nos grupos Lactococcus spp e Enterococcus spp. No caso deste último género, ocorreram também diferenças significativas entre o número de isolados resistentes de diferentes queijarias durante os três anos estudados. As resistências a antibióticos encontradas no grupo de BAL foram a clindamicina e eritromicina, no caso dos lactococos as resistências que foram observadas neste trabalho são consideradas intrínsecas pelo que não haveria possibilidade de transferência dessas resistências para outras bactérias. As resistências extrínsecas encontradas nos enterococos foram a teicoplanina, ciprofloxacina, tetraciclina, cloranfenicol, eritromicina e vancomicina. Apesar de terem sido observados isolados resistentes a três antibióticos de três classes diferentes estes não cumpriam todos os requisitos de modo a serem considerados multirresistentes. No estudo de fatores de virulência foi detetada a capacidade de hemólise em 11% dos representantes de 2016, 6% nos de 2017 e 12% nos de 2018. No teste para identificar a produção de gelatinase so foram observados dois resultados positivos e esses pertenciam a isolados de queijos de 2017. Concluindo, a diversidade do microbioma e as contagens dos queijos de Azeitão e Nisa estudados não seguiram nenhum padrão nos anos que foram comparados. O número de Enterococcus spp. resistentes diminuiu desde 2016 ate ao ultimo ano estudado neste trabalho, 2018, mas as resistências encontradas nos isolados de 2018 foram a antibióticos mais relevantes para a saúde pública como são a vancomicina, eritromicina e tetraciclina. No mesmo sentido, também não foram encontrados isolados multirresistentes e os fenótipos dos fatores de virulência estudados não foram observados em grande quantidade. Contudo, deve ser referido que o estudo destes queijos continuara e serão feitos mais testes tanto no âmbito da diversidade como do potencial patogénico dos isolados das bactérias acido lácticas presentes.
Azeitão and Nisa cheeses are products with protected designation of origin (PDO) traditionally manufactured in Portugal with sheep’s raw milk. The predominant group of bacteria in cheeses is lactic acid bacteria (LAB) which includes genera like Lactococcus spp. and Enterococcus spp., these are the microorganisms our work will be focusing on. The fact that these bacteria have important roles both in foods and the gut of animals (including humans) makes them conductors for both positive impact, such as probiotic features, and negative impact, such as transference of virulence factors or antibiotic resistances. Our aim with this work was to analyse the diversity of LAB in Azeitão and Nisa cheeses from several origins and years of production and assess the negative traits these bacteria could have and transfer such as antibiotic resistances, hemolysis and production of gelatinase. Enumeration of previously mentioned bacteria was performed, and results were compared to other years of production. During the years studied no unit had consistently higher CFU counts. Lactococcus spp. was the group with highest bacteria counts in the majority of units during the three years studied and Enterococcus spp. was the one with the lowest CFU. For diversity analysis, RAPD-PCR were performed in order to create dendrograms for each cheesemaking unit and bacterial group. Just like for bacterial enumeration, no specific trends were observed in the diversity values for the units throughout the studied years. Concerning 2018 cheeses, the group with the highest diversity was Enterococcus spp. even though it was also the one with less CFU. This independence between CFU counts and diversity was noted all through our work in different years, units and groups of bacteria. Identification of 2017 enterococci representatives was performed using a multiplex PCR and showed a predominance of E. faecium, followed by E. faecalis and E. durans that were equally represented. Pathogenic potential of representative isolates was assessed through the search of antibiotic resistances and virulence factors. Antibiotic susceptibility was studied through disc diffusion assays. Significant differences were observed in the number of resistances found in studied years for lactococci and enterococci. Between units there was also significant differences in the total number of resistances during 2016, 2017 and 2018 in enterococci. In LAB isolates resistances considered non intrinsic were found, to clindamycin and erythromycin. Furthermore, enterococci isolates resistant to teicoplanin, ciprofloxacin, tetracycline, chloramphenicol, erythromycin and vancomycin were observed. Isolates resistant to three or more antimicrobial agents were observed but these didn’t comply with the characteristics necessary to be classified as multidrug-resistant (MDR) bacteria. Virulence factors were studied and hemolysis was detected in 11% of representative isolates from 2016, 6% from 2017 and 12% in isolates from 2018 cheeses. Furthermore, only two positive results for gelatinase were found in 2017 representative isolates. In conclusion, our results showed that no pattern of bacterial enumeration or microbiome diversity is present throughout different years of production in artisanal cheeses such as the ones studied. Moreover, although alarming resistances were found in enterococci no multi-drug resistance isolates were observed. Further work should be performed to continue the characterization of the pathogenic potential of isolates present in these cheeses.

Polyhydroxyalkanoates (PHAs) are biodegradable polyesters and environmentally friendly thermoplastics, which are accumulated as carbon and energy storage materials in various bacteria in limited growth conditions with excess carbon sources. In this study, bacteria were isolated from samples taken from various marine ecosystems in the Archipelago of Madeira in the Atlantic Ocean, and screened for their ability to accumulate polyhydroxyalkanoates. These samples were taken from the seabed at depths of 30 and 1,700 meters to obtain a larger diversity of microorganisms and therefore, in an attempt to obtain new structures of PHAs. Strains were directly isolated from 612 mother plates where marine samples had been initially plated. A total of 724 isolates from mother plates were obtained, of which 174 were found PHA-positive using Nile red viable-colony screening. All synthesized intracellular inclusions during growth on starch carbon source. Twenty-five bacterial isolates in 25 mL-scale cultivation were proven promising for PHA production with PHA storage maximum 17.71 % for MD12-107 and 9.30 % for MD12-581 strain. The inclusions were predominantly identified as poly-β-hydroxybutyrate (PHB) using gas chromatography. Strains MD12-107 and MD12-581 were tested in 100 mL-scale and bioreactor cultivation. The best results were achieved with strain MD12-581 accumulated PHA storage 15.40 % in less than 6.5 hours with 5.5 g/L of cell dry weight and a specific growth rate was 0.24 h-1 when grown in medium containing 40 g/L of starch, 8 g/L of yeast extract and 4 g/L of peptone during batch cultivation.
Os Polihidroxialcanoatos (PHAs) são biopoliésteres biodegradáveis e termoplásticos ecológicos, que são acumulados sob a forma de materiais de armazenamento de energia em várias bactérias em condições de crescimento limitado e fonte de carbono em excesso. Neste estudo, as bactérias foram isoladas a partir de amostras recolhidas a partir de vários ecossistemas marinhos no arquipélago da Madeira, no Oceano Atlântico e seleccionadas pela sua capacidade de acumular poli-hidroxialcanoatos. Estas amostras foram extraídas do fundo do mar a uma profundidade de 30 e 1,700 metros para se obter uma maior diversidade de microorganismos e, por conseguinte, numa tentativa de obter novas estruturas de PHAs. As estirpes foram directamente isoladas de 612 placas mãe onde as amostras marinhas tinham sido previamente espalhadas. Na totalidade foram isoladas 724 estirpes a partir das placasmãe, das quais 174 demonstraram resultado positivo durante o screening com o corante Vermelho do Nilo. Todas as estirpes com resultado positivo sintetizaram inclusões intracelulares durante o crescimento em amido como fonte de carbono. Vinte e cinco isolados bacterianos foram testados em cultura de 25 mL com resultados promissores quanto à produção de PHA com armazenamento de 17,71 % para a estirpe MD12-107 e 9,30 % para a estirpe MD12-581. As inclusões foram analisadas através da técnica de cromatografia gasosa como sendo predominantemente poli-β-hidroxibutirato (PHB). A produção a partir das estirpes MD12-107 e MD12-581 foi testada em 100 ml e em bioreactor. Os melhores resultados foram alcançados pela estirpe MD12-581 com um armazenamento de PHA de 15,40% em menos de 6,5 horas, com 5,5 g/L de concentração de biomassa e uma taxa específica de crescimento de 0,24 h-1 quando cultivada num meio contendo 40 g/L de amido, 8 g/L de extracto de levedura e 4 g/L de peptona durante a produção em bioreactor.

碩士
國立高雄海洋科技大學
海洋生物技術研究所
105
In this study, we collected samples from the freshwater environments in Taiwan to research on bacterial diversity and bacterial taxonomic characterization. The potential novel species bacteria were screened and subjected to identification and classification. Freshwater samples were collected from a calla-lily field of Yangming Mountain in Taipei , an amaranth field of Luwild Township in Taitung , a taro field of in Luye Township in Taitung , the Beishi river in Fangliu Township in Pingtung , the farmland irrigation water of East stone Township in Chiayi , a paddy field of East stone Township in Chiayi. There were 20 bacterial strains isolated that containing one Gram-positive bacterium and nineteen Gram-negative bacteria. Phylogenetic analyses based on 16S rRNA gene sequences indicate that these Gram-negative bacteria belong to the class Gammaproteobacteria, class Betaproteobacteria, class Alphaproteobacteria or class Flavobacteria. Among them, strains TPY-10T、Teta-03T、Jyi-02T、Dbr-01T、Npb-02T、Tese-01T、Tese-05T and Tese-07T might be the potential novel species bacteria and strains TPY-10T、Teta-03T、Jyi-02T、Dbr-01T、Npb-02T and Tese-05T were subjected to further identification and classification in this study. Strain Npb-02T was isolated from the water of the Beishi river of Fangliu Township in Pingtung. Cells are Gram negative rod bacteria. Colonies are white colored, circular, round and smooth with entire margins. Phylogenetic analyses based on 16S rRNA gene sequences showed that the closest neighbor strain is Vogesella perlucida DS-28T with the highest sequence similarity 98.35%. On the basis of the genotypic, chemotaxonomic and phenotypic data, strain Npb-02T represents a novel species in the genus Vogesella, of which the name Vogesella amnigena sp. nov. is proposed. The type strain is Npb-02T (=BCRC80887T =LMG28419T). Strain Teta-03T was isolated from the water of a taro field in the Luye Township in Taitung. Cells are Gram negative rod bacteria. Colonies are soil yellow colored, circular, convex round and smooth with entire margins. Phylogenetic analyses based on 16S rRNA gene sequences showed that the closest neighbor strain is Novosphingobium soli CC-TPE-1T with the highest sequence similarity 96.88%. On the basis of the genotypic, chemotaxonomic and phenotypic data, strain Teta-03T represents a novel species in the genus Novosphingobium, of which the name Novosphingobium colocasiae sp. nov. is proposed. The type strain is Teta-03T (=BCRC80538T =LMG27385T =KCTC32255T). Strain Jyi-02T was isolated from the farmland irrigation water of East stone Township in Chiayi. Cells are Gram negative rod bacteria. Colonies are orange yellow colored, circular, convex round and smooth with entire margins. Phylogenetic analyses based on 16S rRNA gene sequences showed that the closest neighbor strain is Novosphingobium Soli CC-TPE-1T with the highest sequence similarity 97.63%. On the basis of the genotypic, chemotaxonomic and phenotypic data, strain Jyi-02T represents a novel species in the genus Novosphingobium, of which the name Novosphingobium arvoryzae sp. nov. is proposed. The type strain is Jyi-02T (=BCRC80537T =KCTC32422T). Strain Dbr-01T was isolated from the water of a paddy field of East stone Township in Chiayi. Cells are Gram negative rod bacteria. Colonies are yellow colored, circular, convex round and smooth with entire margins. Phylogenetic analyses based on 16S rRNA gene sequences showed that the closest neighbor strain is Novosphingobium mathurense SM117T with the highest sequence similarity 97.04%. On the basis of the genotypic, chemotaxonomic and phenotypic data, strain Dbr-01T represents a novel species in the genus Novosphingobium, of which the name Novosphingobium oryzaihumi sp. nov. is proposed. The type strain is Dbr-01T (=BCRC80536T =LMG27387T =KCTC32257T). Strain Tese-05T was isolated from the water of an amaranth field of Luwild Township in Taitung. Cells are Gram negative rod bacteria. Colonies are bright yellow colored, circular, convex round and smooth with entire margins. Phylogenetic analyses based on 16S rRNA gene sequences showed that the closest neighbor strain was Novosphingobium mathurense SM117T with the highest sequence similarity 96.11%. On the basis of the genotypic, chemotaxonomic and phenotypic data, strain Tese-05T represents a novel species in the genus Novosphingobium, of which the name Novosphingobium ipomoeae sp. nov. is proposed. The type strain is Tese-05T (=BCRC80904T =LMG28838T =KCTC42656T). Strain TPY-10T was isolated from the water of a calla-lily field of Yangming Mountain in Taipei. Cells are Gram negative rod bacteria. Colonies are cream white colored, circular, convex round and smooth with entire margins. Phylogenetic analyses based on 16S rRNA gene sequences showed that the closest neighbor strain is Cellvibrio mixtus subsp. mixtus ACM 2601T with the highest sequence similarity 97.75%. On the basis of the genotypic, chemotaxonomic and phenotypic data, strain TPY-10T represents a novel species in the genus Cellvibrio, of which the name Cellvibrio zantedeschiae sp. nov. is proposed. The type strain is TPY-10T (=BCRC80525T =LMG27291T =KCTC32239T).

碩士
大葉大學
生物產業科技學系
105
The use of land for agriculture is limited, therefore soil maintainess and application become more and more important. The use of chemical fertilizers (such as Nitrogen, phosphorus and potassium fertilizer) can cause damage to the land and increase soil pollution. Microbial fertilizer can help to reduce the use of chemical fertilizer and restore the soil condition. In this study, five strains of plant growth promoting rhizobacteria (PGPR) were isolated from soil and characterized. Based on 16S rDNA sequences, these five strains were found closed to Bacillus arbutinivorans, Streptomyces flavortricini, Bacillus megaterium, Pesudomonas putida and Micrococcus sp., respectively. Thus the name of Bacillus arbutinivorans MG-9, Streptomyces flavortricini MG-10, Bacillus megaterium MG-24, Pesudomonas putida MG-45, Micrococcus sp. MG-47 were given. These five strains were then subjected to plant growth promoting activity analysis. Methods applied in this thesis included Indole-3acetate production, phosphosolubitlity activity, siderophore production and ACC deaminase activity analysis. Nevertheless, salt tolerance analysis was also applied. The results showed that all these five bacterial strains have the ability to promote plant growth. At last, plant growth promoting activity of these five bacterial strains were proven by using corn and green onion planting experiments. All together of the results, this thesis discovered five PGPR strains, and have great potential on plants growth promotion.