Academic literature on the topic 'Bacteriology, Agricultural'

Born in Sydney in 1911, Sydney Dattilo Rubbo was educated at Sydney Boys? High School and the University of Sydney (BSc, 1934) before travelling to London to further his studies. He obtained a diploma in bacteriology from the London School of Hygiene and Tropical Medicine (1935) and was awarded a scholarship for microbiological research at the University of London (PhD, 1937). Returning to Australia in 1937, Rubbo took up an appointment as a senior lecturer in the Department of Bacteriology at the University of Melbourne where he taught students of medicine, dentistry, science and agricultural science. A ?brilliant and provocative lecturer?, he inspired a generation of students. He also studied and completed a medical degree (MB, BS, 1943) and in 1945, at the age of 33, was appointed Professor of Bacteriology (Microbiology from 1964), a position he held until 1969.

Bacteria in soil are very diverse, very numerous, and functionally important, and have historically been an important object of research by Canadian microbiologists. Only a small fraction of bacteria in soils are amenable to culturing in the laboratory, limiting the ability to study these organisms. Canadian scientists have contributed to the development and implementation of both nucleic acidbased and chemical biomarker-based methods now widely used for assessing soil microbial biodiversity without the need for isolation and cultivation. Pesticide degradation, and the cycling of nitrogen in soils are used here to illustrate the significance of bacterial biodiversity to soil functions relevant to human and environmental health, and crop production . There remains much to be discovered about the genetic and functional biodiversity of soil bacteria, and much to be gained from this knowledge. A number of recommendations are made for future research in soil bacteriology. Key words: Soil quality, bacteria, microbial biodiversity, pesticide biodegradation, nitrogen cycling.

Resilience is the capacity of systems to recover their initial state or functions after a disturbance. The concepts of resilience and resistance are complementary in ecology and both represent different aspects of the stability of ecosystems. However, antibiotic resilience is not used in clinical bacteriology whereas antibiotic resistance is a recognized major problem. To join the fields of ecology and clinical bacteriology, we first review the resilience concept from ecology, socio-ecological systems and microbiology where it is widely developed. We then review resilience-related concepts in microbiology, including bacterial tolerance and persistence, phenotypic heterogeneity and collective tolerance and resistance. We discuss how antibiotic resilience could be defined and argue that the use of this concept largely relies on its experimental measure and its clinical relevance. We review indicators in microbiology which could be used to reflect antibiotic resilience and used as valuable indicators to anticipate the capacity of bacteria to recover from antibiotic treatments.

Effective laboratory and classroom demonstration of microbiome size and shape, diversity, and ecological relationships is hampered by a lack of high-resolution, easy-to-use, readily accessible physical or digital models for use in teaching. Three-dimensional (3D) representations are, overall, more effective in communicating visuospatial information, allowing for a better understanding of concepts not directly observable with the unaided eye. Published morphology descriptions and microscopy images were used as the basis for designing 3D digital models, scaled at 20,000×, using computer-aided design software (CAD) and generating printed models of bacteria on mass-market 3D printers. Sixteen models are presented, including rod-shaped, spiral, flask-like, vibroid, and filamentous bacteria as well as different arrangements of cocci. Identical model scaling enables direct comparison as well as design of a wide range of educational plans.

Sulfate reducing bacteria utilize sulfate as their terminal electron acceptor and reduce it to sulphide. Acid mine drainage, by-products of mining activities, is an acidic sulfate-rich wastewater suitable habitat for sulfate reducing bacteria. Isolation and identification of sulfate reducing bacteria collected from Muara Enim coal mining, South Sumatra was carried out at Laboratory of Environmental Biotechnology, Indonesian Center for Biodiversity and Biotechnology (ICBB), Bogor, and Laboratory of Microbiology, Faculty of Veterinary, Bogor Agricultural University. Postgate B liquid media was used for isolation and purification via serial dilution. Physiological and biochemical characterization was done based on Bergeys Manual of Determinative Bacteriology. Fifteen pure isolates have been isolated with diverse characteristics. Eight isolates can sustain at pH 3, while the rest sustain at pH 4 or above. Sulfate reduction efficiency of each isolates were different, but increased as the pH increased. The bacteria are classified as Desulfovibrio sp., which is characterized straight rods, motile, non spore-forming and able to grow in simple organic carbon.

Tripartite rhizosphere (host, fungus, and rhizobacterial) interactions were studied to determine the mechanism(s) associated with lack of oospore germination and host colonization by Pythium aphanidermatum at soil temperatures below 27°C. Results indicate that rhizobacterial competition for nutrients was responsible for the general supression of pathogen activity at low soil temperature. In general less than 25% host colonization occurred at 20°C whereas greater than 90% colonization recorded at 27°C. However, when bacterial competition was reduced or eliminated by the addition of vancomycin, host colonization at 20°C increased to 83%. Competition between the fungus and the resident rhizobacterial population was also shown to occur prior to any significant increase in bacterial multiplication. The generation time of bacteria in undisturbed rhizosphere soil was estimated at about 8 hrs at both 20 and 27°C. Whereas both the rate and percentage germination of oospores were increased significantly at both 20 and 27°C in the rhizosphere soil if bacterial competition was inhibited by the addition of vancomycin.

Runoff from agricultural lands carrying microorganisms from livestock manure can contaminate the food and water supplies of both animals and humans. Planning and design of animal waste best management practices (BMPs) thus becomes more important as livestock populations become more concentrated. A computer model is proposed to predict the effects of animal waste BMPs on the bacteria concentration of runoff from agricultural lands. The model uses Monte Carlo simulation to combine the deterministic relationships resulting from previous modeling efforts with statistical knowledge concerning rainfall events and temperature variation. Model output is in the form of monthly maximum and minimum log bacteria concentrations of runoff resulting from a storm assumed to occur immediately after manure is applied to the land. The effects of implementing such BMPs as waste storage, filter strips, and incorporation of manure into the soil can be compared. Data and information collected from the Owl Run watershed in Fauquier County, Virginia is used to demonstrate the model applicability and potential. Long-term manure storage is determined to be the most appropriate practice for reducing bacteria concentrations for the study site. Incorporation of manure is as effective as long-term storage, but requires additional labor. Buffer strips significantly reduce bacteria concentrations, but not as effectively as long-term storage or incorporation. Additional efforts are needed to investigate the most influential variables and to make the temperature simulation submodel more computationally efficient. Once BMPs have been implemented on the study site, more data should be collected to test the accuracy of the model.
Master of Science

Orientador: Wanderley José de Melo
Coorientador: Lúcia Maria Carareto Alves
Banca: Thiago Assis Rodrigues Nogueira
Banca: Estevam Guilherme Lux Hoppe
Resumo: O lodo de esgoto é uma alternativa como fertilizante orgânico na agricultura, porém em sua composição pode apresentar patógenos que oferecem risco ao homem e ao ambiente. Objetivou-se, com o presente estudo, avaliar a fertilidade do solo, e a presença de ovos viáveis de helmintos, coliformes termotolerantes, Escherichia coli para os patótipos EHEC, EPEC e STEC e a atividade enzimática das enzimas protease, redutase do nitrato e urease no solo após dezoito anos de aplicações anuais de lodo de esgoto em um Latossolo Vermelho eutroférrico (LVef) e Latossolo Vermelho distrófico (LVd). O lodo utilizado foi obtido na SABESP de Franca, São Paulo e o experimento foi instalado em delineamento de blocos cazualiados, sendo 4 tratamentos e 5 repetições. Os tratamentos foram T1: controle, apenas com aplicação de adubação mineral, T2: 5, T3: 10 e T4: 20 Mg ha-1 de LE. Antes de ser incorporado ao solo, realizou-se análise do lodo para ovos viáveis de helmintos e coliformes termotolerantes. Aos 40 dias, coletou-se amostras de solo na profundidade de 0-10 cm para avalição de ovos viáveis de helmintos no solo. Aos 70 dias, coletou-se amostras de solo na profundidade de 0-20cm para a análise da fertilidade. Para a análise de coliformes termotolerantes, seguindo a técnica de tubos múltiplos, as amostras foram coletadas no dia 0, 26, 40 e aos 78 dias. Para a realização da reação em cadeia da polimerase (PCR) para identificar a presença de Escherichia coli, coletou-se amostras de solo antes do iní... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: The sewage sludge is an alternative as organic fertilizer to use in agriculture, but in its composition may have pathogens that offer to humans and the environment risks. The present study objective was to evaluate soil fertility, and the presence of viable helminth eggs, fecal coliforms, Escherichia coli for pathotypes EHEC, EPEC and STEC and the enzymatic activity of protease enzymes, nitrate reductase and urease in the soil after eighteen years of annual applications of sewage sludge in an Oxisol (LVef) and Oxisol (LVd). The sludge used was obtained in SABESP Franca, São Paulo and the experiment was installed in designing cazualiados blocks, 4 treatments and 5 repetitions. Treatments were T1: control, only with application of mineral fertilizer, T2: 5, T3: T4 10 and 20 Mg ha-1 LE. Before being incorporated into the soil, there was sludge analysis for viable helminth eggs and fecal coliforms. At 40 days, it is collected soil samples at a depth of 0-10 cm for viable helminth eggs evaluation in the soil. After 70 days it is collected soil samples at a depth of 0-20cm for fertility analysis. For fecal coliforms analysis, following the technique of multiple pipes, the samples were collected at day 0, 26, 40 and 78 days. To carry out the polymerase chain reaction (PCR) for the presence of Escherichia coli was collected from soil samples before the beginning of the experiment at day 0, after 26 days 40, 58, 78, 110 and 146 days . For the evaluation of enzyme activity, samples wer... (Complete abstract click electronic access below)
Mestre

L'eau et le sol sont considérés comme des sources potentielles de mycobactéries non-tuberculeuses (MNT). Parmi les infections humaines causées par les MNT d'origine environnementale, les infections pulmonaires et cutanées sont souvent décrites. Le manque de connaissances sur leur cycle de vie dans l'environnement requiert des outils analytiques, qui ne sont actuellement pas adaptés à ce type d'échantillons. Cette thèse vise donc premièrement à proposer des méthodes de quantification en bactériologie et en biologie moléculaire dans le but de déterminer les sources des MNT dans les bassins versants. Ainsi, la comparaison des méthodes d'isolement de MNT a montré que le traitement au chlorure de cetylpyridininium de l'eau suivi d'une culture en milieu riche supplémenté par un mélange d'antibiotiques (polymyxine B, amphotéricine, acide nalidixique, triméthoprime, carboxy-pénicilline) limitait la croissance des microorganismes interférents et éliminait moins de MNT que les autres méthodes comparées (Radomski et al. 2010, doi: 10.1128/AEM.00942-10). Bien que des espèces de MNT potentiellement pathogènes aient été isolées de l'eau de surface de la Seine en utilisant ces outils bactériologiques, la quantification des MNT ne s'est pas avérée reproductible. En conséquence, une méthode de quantification par polymérisation en chaîne en temps réel (qPCR) a été développée pour énumérer le genre Mycobacterium dans l'eau (Radomski et al. 2010, doi: 10.1128/AEM.02659-09). La nouvelle méthode développée, ciblant l'ARNr 16S, était plus spécifique que les autres méthodes qPCR publiées, ciblant un autre locus de l'ARNr 16S et le gène hsp65 (respectivement 100 % versus 44 % et 91 %). La comparaison des méthodes d'extraction d'ADN mycobactérien a montré que la lyse enzymatique combinée au bromure d'hexadécyltriméthylammonium était la procédure la plus efficace pour énumérer par qPCR les MNT dans des échantillons environnementaux. Ainsi, ces méthodes d'extraction d'ADN et de qPCR ont été utilisées pour étudier des sources de MNT dans des bassins versants. Dans un second temps, nous avons étudié trois sources potentielles de MNT : une ponctuelle et deux diffuses. Plus précisément, une station d'épuration (STEP) a été choisie comme source ponctuelle de MNT et a été étudiée en temps sec en fonction d'indicateurs de contamination fécale et des paramètres globaux habituellement contrôlés. Les MNT ont atteint 5,52×105±3,97×105 copies/L dans l'eau en entrée de STEP (84 % d'échantillons positifs), n'ont pas été détectées dans l'eau en sortie de STEP après décantation physico-chimique et biofiltration et ont été estimées à 1,04×106 ±1,75×106 copies/g dans les boues de STEP (50 % d'échantillons positifs). La plupart des MNT (98±2 %, correspondant à 2,45±0,78 log10) ont été éliminées par décantation physico-chimique et les MNT restantes (0,74×104 ±1,40×104 copies/L) ont été éliminées par biofiltration (53 % d'échantillons positifs). Ces résultats ont montré également que Mycobacterium, Escherichia coli et les entérocoques intestinaux possèdent des comportements significativement différents conduisant respectivement à trois modèles : hydrophobe, hydrophile et intermédiaire. Concernant les sources diffuses, la densité de MNT a été mesurée dans divers sols ruraux et urbains qui ont été caractérisés par différents paramètres physico-chimiques. Les densités de MNT les plus importantes ont été mesurées dans des sols de forêts tourbeuses (9,27×104±5,00×104 copies/g sec) et dans des sols faiblement urbanisés proches de marécages côtiers (1,71×106±2,85×106 copies/g sec) alors qu'aucune MNT n'a été détectée dans les autres types de sols étudiés. De plus, la densité de MNT a été significativement associée à des sols proches de zones acides et des teneurs fortes des sols en eau, matière organique et fer. Ces résultats suggéreraient que les MNT sont dépendantes de leur production intra et extracellulaire de chélateurs de fer et indiqueraient que les zones faiblement urbanisées pourraient être impactées par la proximité de marais acides. Afin d'étudier une autre source diffuse, les MNT et d'autres paramètres ont été mesurés lors d'événements pluvieux dans l'eau de surface de la Marne et de ses principaux affluents. Les densités de MNT ont été estimées à 2,16×105±2,36×105 copies/L dans environ 20 % des échantillons d'eau collectés, et elles ne différaient pas entre les zones péri-urbaines et rurales échantillonnées. Nos résultats ont montré que la pluviométrie et la durée de l'évènement expliquaient la diminution du nombre de MNT détectées dans l'eau de surface au cours de l'événement pluvieux de faible intensité (6,6 mm/h de pluviométrie cumulées en 5,5 h). Ces résultats ont souligné que certains affluents de la Marne pouvaient apporter des MNT en temps sec, mais qu'au cours de l'évènement pluvieux suivi les densités de MNT diminuaient.En guise d'amélioration à ces études appliquées, des réflexions sur les défis relatifs à la surveillance des microorganismes pathogènes dans l'environnement ont été explorées. En nous focalisant sur la MNT la plus pathogène, M. avium, nous avons discuté des défis de la détection et de l'énumération et proposé un guide d'adaptation des méthodes médicales aux échantillons environnementaux (Radomski et al. 2011, ed. A. Méndez-Vilas, Vol. 2). Ce guide se présente sous la forme d'un arbre de décision permettant de choisir les outils analytiques les plus appropriés pour surveiller les microorganismes pathogènes dans l'environnement. De plus, une stratégie in silico de comparaison de génomes bactériens totalement séquencés a été développée dans le but de décrire des nouvelles cibles de détection. L'analyse in silico des génomes totalement séquencés a permis de détecter 11 protéines présentant entre 80 % et 100 % de similarité dans les génomes mycobactériens et moins de 50 % de similarité dans les génomes non-mycobactériens des genres Corynebacterium, Nocardia et Rhodococcus. Sur la base d'alignements des séquences d'ADN de ces cibles potentielles, il a été possible de dessiner des amorces PCR et une sonde pour détecter le gène codant la sous-unité C de la synthase de l'adénosine triphosphate qui semble exclusivement conservée dans le génome mycobactérien. Le développement d'outils analytiques, en particulier la qPCR, a permis de montrer qu'une STEP éliminait efficacement les MNT et que le traitement des eaux usées est nécessaire pour préserver l'eau de surface de cette source ponctuelle de MNT. Il a été mis en évidence que les événements pluvieux diminuent la densité de MNT dans l'eau de surface et que les sols acides sont des sources naturelles majeures de MNT qui pourraient impacter des zones faiblement urbanisées en temps de pluie via le ruissellement. Concernant les réflexions sur la surveillance des microorganismes pathogènes dans l'environnement, l'arbre de décision des outils analytiques appropriés et la nouvelle stratégie in silico de détection de cibles moléculaires pourraient être appliqués pour l'étude d'autres microorganismes de l'environnement

Thesis (PhD)--Stellenbosch University, 2013.
ENGLISH ABSTRACT: Entomopathogenic nematodes have the potential to be outstanding biocontrol agents against agricultural pest insects. Combined with their bacterial symbionts, these biocontrol agents have proven to be very effective against numerous pests. The nematodes belong to the families Steinernematidae and Heterorhabditidae, and are ideal to be used in, and integrated with, pest management systems. There is a dire need for new and innovative methods to control agricultural pests, as numerous pest insects have developed resistance against broad-spectrum insecticides. Together with the environmental impact of these insecticides and the safety aspect regarding humans and animals, the need to develop new technologies, including entomopathogenic nematodes for pest management, is high. In this study, the associated symbiotic bacteria of three entomopathogenic nematodes species were isolated, and the potential of two nematode species to be successfully mass cultured in liquid medium was evaluated. Regarding the symbiotic bacteria, results from the study showed that bacteria species from all three nematode species, Heterorhabditis noenieputensis, Steinernema khoisanae and Heterorhabditis zealandica, were novel. Heterorhabditis noenieputensis was isolated in the Mpumalanga province during a previous survey conducted in citrus orchards. The bacterium isolated from this nematode belongs to the genus Photorhabdus, and bear closest similarity (98.6%) to the type strain of P. luminescens subsp laumondii (TT01T). Photorhabdus luminescens subsp. noenieputensis subsp. nov., derives its name from the area where the nematode was sourced, namely the farm Springbokvlei, near the settlement Noenieput close to the Namibian border. Thus far, 85 Steinernema spp. have been described worldwide, including S. khoisanae which was isolated in the Western Cape province of South Africa. Four S. khoisanae strains, namely SF87, SF80, SF362 and 106-C, were used for characterisating the new bacteria from different localities in South Africa. Using the neighbor-joining method, all the strains were aligned with 97% homology to the 16S rRNA sequences of several Xenorhabdus- type strains, indicating that they belonged to the same genus. The multigene approach was used to distinguish between the Xenorhabdus spp. and partial recA, dnaN, gltX, gyrB and infB gene sequences of the various strains were analysed. The bacterium species was named Xenorhabdus khoisanae sp. nov. after the nematode from which it was isolated. The results showed that the third bacterium species, which was isolated from H. zealandica, was new. The sequence of the bacteria strain clustered with the type strains of P. temperata and P. asymbiotica, indicate that it belonged to the genus Photorhabdus. This is the first study to show that H. zealandica associates with a luminescent Photorhabdus species, rather than with the known non-luminescent P. temperata. The potential of H. zealandica and Steinernema yirgalemense mass culture in liquid was investigated. Results illustrated that H. zealandica and its P. luminescens symbiont can be successfully cultured in liquid. However, two generations occurred during the process time, instead of the desirable one-generation. The growth curve of the symbiotic bacteria during the process time was measured, in order to determine when the stationary phase was reached, with the results showing this to occur after 36 h. Therefore, the optimum amount of time required for inoculating the IJs and for aiding in maximum infective juvenile (IJ) recovery is 36 h for adding the nematodes post pre-culturing of the bacteria. Future research goals should be to increase the percentage recovery in liquid culture, which would increase the number of nematodes produced per ml, which would, therefore, reduce the processing time significantly. The results from mass culturing the second nematode species, S. yirgalemense, indicated an asynchronous nematode development in the first generation. Growth curves were performed with the symbiotic bacteria that showed the exponential phase of Xenorhabdus started after 15 h, and that, after 42 h, the stationary phase was reached, with an average of 51 × 107 cfu·ml-1. Bioassays were performed to compare the virulence between in vitro- and in vivo-produced nematodes, with the results showing that the in vitro-produced nematodes were significantly less virulent than were the nematodes produced in vivo. The success obtained with the production of S. yirgalemense in liquid culture can serve as the first step in the optimising and upscaling of the commercial production of nematodes in industrial fermenters. The last aim of the current study was to determine when Xenorhabdus reached the stationary phase, when it is grown in a 20-L fermenter, as this would be the optimum time at which to add the IJs of S. yirgalemense. Such characteristics as the effect of stationary phase conditions on the bacterial cell density and on the DO2 rate in the fermenter were investigated. The results showed that the stationary phase of Xenorhabdus was reached after 36 h at 30˚C, which took 6 h less than did the same procedures followed with the Xenorhabdus sp. cultured in Erlenmeyer flasks on orbital shakers. This is the first step toward the liquid mass culturing of S. yirgalemense in industrial-size fermenters. Data from this study indicated the optimum amount of time that is required for adding nematodes to the bacterial culture in the fermenter, and for ensuring the optimum recovery of IJs, as well as a subsequent high yield of nematodes within a minimum processing time. This is the first report of its kind to investigate comprehensively the successful liquid culture of two South African entomopathogenic nematode species for the sole purpose of evaluating potential commercialisation. Results emanating from this study could be used as groundwork in future, in combination with similar research such as culturing nematodes intensively in large fermenters.
AFRIKAANSE OPSOMMING: Entomopatogeniese nematodes het die potensiaal om as doeltreffende biologiese beheeragente teen sleutelplaaginsekte gebruik te word. Elke nematood werk interaktief met ‘n spesifieke bakterium. Entomopatogeniese nematodes, behorende tot die families Steinernematidae en Heterorhabditidae, is ideale kandidate vir gebruik in ‘n geïntegreerde plaagbestuurprogram. Tans is daar ʼn behoefte vir nuwe metodes vir die beheer van plaaginsekte, omdat meeste insekte reeds weerstand opgebou het teen bestaande plaagdoders. As gevolg van die negatiewe impak van plaagdoders op die omgewing, asook kommer oor veiligheid vir die mens en diere, is die ontwikkeling en gebruik van alternatiewe plaagbeheermiddels noodsaaklik. In die eerste deel van die studie word drie nuwe bakterie spesies geïsoleer en beskryf. Resultate van hierdie studie het aangetoon dat die bakterië spesies vanuit die nematode spesies, Heterorhabditis noenieputensis, Steinernema khoisanae, en Heterorhabditis zealandica, tot dusver onbeskryf was. Eersgenoemde, H. noenieputensis, is afkomstig van ʼn sitrusboord in die Mpumalanga Provinsie. Die bakterie hieruit geïsoleer behoort tot die genus Photorhabdus en is biologies verwant (98.6%) aan P. luminescens subsp laumondii (TT01T). Die bakterie is benaam as Photorhabdus luminescens subsp. noenieputensis nov. en is na die nematood waaruit dit geïsoleer is vernoem. Tot dusver is wêreldwyd 82 spesies van Steinernema spp. beskryf, insluitende S. khoisanae van die Weskaap provinsie. Vier bakterie isolate is van S. khoisanae, SF87, SF80, SF362 en 106-C geïsoleer. Die buur-koppeling metode was gebruik om te bepaal dat hierdie bakterie isolate tot 97% ooreenstem met verskeie isolate van Xenorhabdus se 16S rRNA DNS volgordebepalings. Om tussen Xenorhabdus spp. te onderskei is ʼn multi-geen benadering gebruik deur gedeeltelike recA, dnaN, gltX, gyrB en infB DNS basispaar volgordebepalings van die verskeie isolate te bepaal. Hierdie bakterie isolaat is soortgelyk ook vernoem as, Xenorhabdus khoisanae sp. nov., na die nematood waaruit dit geïsoleer is. Die derde onbekende bakteriële spesie is uit H. zealandica geïsoleer. Die DNS basispaar volgordebepaling van die 16S geen van SF41 toon aan dat dit in dieselfde groep as P. temperata en P. asymbiotica val en sodoende aan die genus Photorhabdus behoort. Hierdie is die eerste studie met die bevinding dat H. zealandica ook met ʼn ander bakterie spesie geassosieer kan word buiten die normale P. temperata spesie. Die tweede deel van die studie gaan oor die teling van twee nematood spesies, H. zealandica en Steinernema yirgalemense, en hulle is geëvalueer vir hulle potensiaal om geteel te word in ʼn vloeibare medium. Die resultate het gewys dat H. zealandica met sy P. luminescens simbiont suksesvol in vloeistof aangeteel kan word, ten spyte van die feit dat daar twee generasies ontwikkel het, in plaas van die meer ideale enkel generasie. Die groeikurwe van die simbiotiese bakterie was gemonitor om te bepaal wanneer die stasionêre fase bereik word. Die resultate toon dat hierdie fase na 36 uur bereik was. Dus was die infektiewe nematode larwes eers na 36 uur tot die vloeibare medium waarin die bakterie geteel was bygevoeg. Navorsing in die toekoms moet dus gefokus wees om die persentasie herwinning van die infektiewe larwes te verhoog. Dit sal daartoe lei dat meer nematodes per ml geproduseer kan word en ook die prosesseringstyd van die nematodes verminder. ʼn Tweede nematode spesie, S. yirgalemense, was ook in vloeistof geteel. Hier het ʼn asinkroniese ontwikkeling in die eerste generasie plaasgevind wat problematies is. Groeikurwes is bepaal van die bakteriële simbiont en die resultate het gewys dat die groeifase van Xenorhabdus na 15 uur in aanvang geneem het en dat die stasionêre fase bereik was na 42 uur met ʼn gemiddelde van 51 × 107 selle·ml-1. Die virulensie van nematodes wat in vitro geteel is, is vergelyk met die virulensie van nematodes wat in vivo geteel is en die resultate het getoon dat die in vitro geteelde nematodes minder virulent was. Die teling van S. yirgalemense in vloeistof was oor die algemeen meer suksesvol as die teling van H. zealandica in dieselfde medium. Die doelwit van die laaste gedeelte van hierdie studie was om te bepaal wanneer Xenorhabdus die stasionêre fase bereik wanneer dit in ʼn 20-L fermenter gekweek word. Dit bepaal sodoende die optimale tyd wanneer die infektiewe larwes van S. yirgalemense bygevoeg behoort te word. Die uitwerking van die stasionêre fase op die bakteriële selle, asook die DO2-konsentrasie in die fermenter, was geëvalueer. Resultate het gewys dat die stasionêre fase van Xenorhabdus na 36 uur bereik was, wat 6 uur korter is as toe dit gekweek is in Erlenmeyer flesse. Hierdie studie is die eerste stap om die massa teling van S. yirgalemense in industriële fermenters suksesvol te bemeester. Die data wat verkry was, het aangedui wat die ideale tydsduur sal wees om die bakteriegetalle te vermeerder voordat die nematode bygevoeg word. Hierdie is die eerste studie wat die teling van twee Suid-Afrikaanse nematode spesies omvattend in vloeistof evalueer het. Die hoof doelwit is om die potensiaal van hierdie nematode spesies, met die oog op kommersiële gebruik, te meet. Die resultate van hierdie studie kan gekombineer word met toekomstige studies in hierdie spesifieke navorsingsveld.

Because of the potential health hazards associated with the use of sludge for agricultural purposes, Dudley et al (1980) published a scheme for the routine analysis of bacteria in municipal sewage sludge. In this study, the Dudley et al scheme (1980) was modified by updating some of the procedures. Aerobically digested sludge generated by the Bowling Green Wastewater Treatment Plant, Bowling Green, Kentucky, was analyzed using the modified scheme. Sludge samples were collected once every two months over a one-year period from October 1989 to August 1990. Egg yolk-free tryptose sulfite cycloserine agar in conjunction with the revewrse CAMP test was used to assay for Clostridium perfringens. This procedure improved the one proposed by Dudley et al. (1980) by achieving a higher confirmation rate, reducing testing time, allowing for easier interpretation of results, and increasing accuracy. Selective and differential media by Rippey and Cabelli (1979) were added to the scheme to isolate Aeromonas, Aeroomonas hydrophila and Aeromonas caviae were successfully isolated wand were identified using the system by Cunliffe and Adcock (1989) for speciating aeromonads. Baird-Parker medium was compared to mannitol salt agar for effectiveness in isolating Staphylococcus from sludge. Statistical analysis showed Baird-Parker medium to be significantly more effective than mannitol salt agar. However, neither agar reduced background flora to acceptable levels. Staphylococcus isolates were subject to species identification by the API Staph Ident system (Analytab Products, Plainview, New York). Staphylococcus xylosus, Staphylococcus haemolyticus, and Staphylococcus epidermidis were found to be present in the sludge. A procedure by Ottolenghi and Hamparian (1987) was employed to isolate Salmonella in sludge. No salmonellae were isolated over the one year period. Over the year-long study, bacterial numbers, with the exception of Clostridium perfringens and the total aerobic count, fluctuated with variations in the aerobic digester temperature. Numbers decreased as temperature increased. Clostridium perfringens counts were the most consistent throughout the year and exceeded fecal coliform and fecal streptococci counts in five of the six samplings.

L'objectif de mon travail fut de valider et d'optimiser la spectrométrie de masse de type MALDI-TOF pour l'identification et la classification d'un ensemble de bactéries pathogènes ou opportunistes chez l'homme, en enrichissant une base de données et en testant la robustesse de la méthode, afin d'obtenir une méthode rapide fixe et fiable d'acquisition de résultats. Les différents résultats obtenus ont permis la validation de la technique comme outil d'identification bactérienne fiable en routine. Elle permet désormais de caractériser les mélanges de deux bactéries voir même la différentiation d'espèces très proches comme les Shigella spp et E. coli. Nous avons montré que la technique sera encore améliorée par un outil supplémentaire de comparaison des souches pour une veille épidémiologique "en temps réel", sans investissement supplémentaire, en permettant plusieurs types d'économie. Elle apporte un gain réel dans la prise en charge du patient et le choix éclairé des antibiotiques testés pour l'antibiogramme. La technique peut aussi constituer un outil alternatif de sérotypage.

Engineered nanomaterials (ENMs) have become ubiquitous in consumer products and industrial applications, and consequently the environment. Much of the environmentally released ENMs are expected to enter terrestrial ecosystems via land application of nano-enriched biosolids to agricultural fields. Among the organisms most likely to encounter nano-enriched biosolids are the key soil bacteria known as plant growth promoting rhizobacteria (PGPR). I reviewed what is known concerning the toxicological effects of ENMs to PGPR and observed the need for high-throughput methods to evaluate lethal and sublethal toxic responses of aerobic microbes. I addressed this issue by developing high-throughput microplate assays which allowed me to normalize oxygen consumption responses to viable cell estimates. Oxygen consumption is a crucial step in cellular respiration which may be examined relatively easily along with viability and may provide insight into the metabolic/physiological response of bacteria to toxic substances. Because many of the most toxic nanomaterials (i.e. metal containing materials) exhibit some level of ionic dissolution, I first developed my methods by examining metal ion responses in the PGPR, Bacillus amyloliquefaciens GB03. I found this bacterium exhibits differential oxygen consumption responses to Ag+, Zn2+, and Ni2+. Exposure to Ag+ elicited pronounced increases in O2 consumption, particularly when few viable cells were observed. Also, while Ni2+ and Zn2+ are generally thought to induce similar toxic responses, I found O2 consumption per viable cell was much more variable during Ni2+ exposure and that Zn2+ induced increased O2 utilization to a lesser extent than Ag+. Additionally, I showed my method is useful for probing toxicity of traditional antibiotics by observing large increases in O2 utilization in response to streptomycin, which was used as a positive control due to its known effects on bacterial respiration. After showing the utility of my method for examining metal ion responses in a single species of PGPR, I investigated the toxicity of silver ENMs (AgENMs) and ions to three PGPR, B. amyloliquefaciens GB03, Sinorhizobium meliloti 2011, and Pseudomonas putida UW4. The ENM exposures consisted of untransformed, polyvinylpyrrolidone coated silver ENMs (PVP-AgENMs) and 100% sulfidized silver ENMs (sAgENMs), which are representative of environmentally transformed AgENMs. I observed species specific O2 consumption responses to silver ions and PVP-AgENMs. Specifically, P. putida exhibited increased O2 consumption across the observed range of viable cells, while B. amyloliquefaciens exhibited responses similar to those found in my first study. Additionally, S. meliloti exhibited more complex responses to Ag+ and PVP-AgENMs, with decreased O2 consumption when cell viability was ~50-75% of no metal controls and increased O2 consumption when cell viability was <50%. I also found the abiotically dissolved fraction of the PVP-AgENMs was likely responsible for most of the toxic response, while abiotic dissolution did not explain the toxicity of sAgENMs. My work has yielded a straightforward, cost-effective, and high-throughput method of evaluating viability and oxygen consumption in aerobic bacteria. I have used this method to test a broad range of toxic substances, including, metal ions, antibiotics, and untransformed and transformed ENMs. I observed species specific toxic responses to Ag+, PVP-AgENMs, and sAgENMs in PGPR. These results not only show the clear utility of the methodology, but also that it will be crucial to continue examining the responses of specific bacterial strains even as nanotoxicology, as a field, must move toward more complex and environmentally relevant systems.

Ready-to-eat vegetable salads containing lettuce as a main ingredient have become popular food items in recent years. Microorganisms associated with these products determine their shelf-life, sensory appeal and safety. This thesis investigates the bacterial ecology of lettuce, aspects of their pre-harvest contamination with microorganisms, and the presence of antimicrobial constituents in such produce. Commercial pesticides (insecticides, herbicides, fungicides), used during lettuce cultivation were examined as potential sources of microbial contaminants. None of the pesticide concentrates contained viable microorganisms. After reconstitution in water, two of the pesticides supported growth of inoculated species of Pseudomonas, Salmonella and Escherichia coli. Pesticides reconstituted in agricultural waters (bore, dam and river) supported the growth of microorganisms (e.g. Pseudomonas, Acinetobacter, Aeromonas spp. and coliforms) naturally present in these waters. Unless properly managed, pesticide application could contribute microbial contaminants to vegetable produce, thereby affecting their quality. Bacterial species associated with retail samples of lettuce were examined by plate culture on Tryptone Soy Agar and PCR-DGGE analysis. Macerates and rinses of lettuce sub-samples with and without addition of Tween 80 were examined to maximize bacterial recoveries. Predominant bacteria isolated by agar culture included species of Pseudomonas, Agrobacterium, Curtobacterium and Burkholderia, at populations of 103-106 cfu/g. PCR-DGGE was unable to recover the same incidence of species as agar culture and failed to detect bacteria in many samples. In some samples, PCR-DGGE detected species of Bacillus, Pseudomonas, Serratia and Acinetobacter, not found by culture. Failure of the PCR-DGGE analyses was attributed to interference by plant chloroplast DNA. Preparative agarose gel electrophoresis of lettuce macerates was necessary to remove chloroplast DNA before application of PCR-DGGE analysis. Thirty percent of lettuce samples contained Acinetobacter species at 101-104 cfu/g when examined after culture on minimal salts agar or enrichment in Baumann enrichment medium. Other Acinetobacter media failed to give reliable isolation of these species from lettuce and salad vegetables. Lettuce could be an environmental source of Acinetobacter nosocomial infections. Juices, solvent extracts and supercritical fluid carbon dioxide extracts of lettuce and capsicum samples did not exhibit antimicrobial action against a range of food spoilage and pathogenic bacteria.