No description available.

No description available.

<i>Clostridioides difficile</i> is a highly antibiotic resistant and infectious endospore-forming bacterium, responsible for an estimated 450,000 cases per year. The formation of an endospore is necessary for the survival of the bacterium while in-transit between hosts and while passing through the toxic environment of the host's stomach. Essential to the endospore's resistance is a thick layer of highly modified peptidoglycan called the cortex. While the endospore cortex is forming, the enzymes CwlD and PdaA convert N-acetylmuramic acid (NAM) into muramic--lactam (MAL). MAL serves as a recognition element for germination-specific lytic enzymes that degrade cortex peptidoglycan layers during germination. Without the MAL residues the endospore cannot complete germination. Unique to the Peptostreptococcaceae family, which includes C. difficile, is the lipoprotein GerS, which is required for the function of CwlD. The interaction between these two proteins is poorly understood. In this work, attempts to complement a Bacillus subtilis cwlD mutant using C. difficile gerS and/or cwlD were unsuccessful. No MAL residues were produced, and spores produced were incapable of completing germination. In vitro assays of CwlD activity on purified peptidoglycan revealed binding and activity of C. difficile CwlD, which were significantly increased when in complex with GerS. The ability of C. difficile CwlD+GerS to function in vitro but not to complement in B. subtilis suggests that in vivo activity is blocked by some factor in this heterologous system. Such a factor might be the in vivo ionic environment or a failure to properly localize within the forespore in B. subtilis cells. Furthering the understanding of C. difficile's germination machinery will potentially provide new targets for therapies. / Master of Science / Antibiotics have saved countless lives since their initial discovery and subsequent use to kill harmful bacteria. However, they have also led to the rise of antibiotic associated diarrhea (AAD), which can be fatal. AAD is caused by antibiotic resistant bacteria that can infect the gut after a large number of bacteria, which exist normally in a healthy gut, are killed by antibiotics. The number one cause of AAD is Clostridioides difficile, which accounts for approximately 450,000 cases a year in the United States, and millions of cases worldwide. C. difficile is highly antibiotic resistant and can exist in the environment for decades as an endospore, protected from many types of disinfectants. This bacterium is commonly spread in hospital settings where it can survive many of the cleaning regiments to infect vulnerable patients. Our work focused on how one of the structures of the endospore is modified in C. difficile to better understand a part of the machinery necessary for causing infection. Studying how the bacteria produces an endospore can shed light on targets for new treatments.

Spores of various strains of Bacillus cereus are the causative agents of emetic and diarrheal foodborne illnesses. Typically, spores will survive thermal treatments that destroy vegetative cells, and then go on to germinate to form the vegetative cells that are associated with toxin production. The spore has to germinate in order to develop into the vegetative cells that produce toxins, hence a thorough understanding of the proteins and molecular mechanisms that underpin spore germination are of great significance from spore control perspectives. A major objective of this thesis was to use molecular genetic and fluorescence microscopy techniques to characterise the location and function of the GerP proteins in Bacillus cereus 14579. The GerP proteins have been identified from mutagenesis studies across the Bacilli as being implicated in spore germination, most likely by impacting upon the permeability of the spore coat. Data presented in this thesis reveal that the various GerP proteins all localise to the same inner-coat vicinity within the spore, as determined via the super-resolution ellipsoid localisation microscopy technique. The study also reveals that only the GerPA protein is required for the localisation of the other GerP proteins in the developing spore. A number of other coat and or germination associated proteins in B. cereus 14579 were examined in the course of this work. These include the GerN and GerT antiporters, which are both shown to have an involvement in inosine mediated spore germination in this strain. However, hypothetical interactions between antiporter proteins and the ‘linker-like’ N-terminal domain of the GerIA inosine-responsive germinant receptor protein appear unlikely since spores engineered with a truncated GerIA receptor subunit germinate normally. The protein encoded at locus BC1245 was also examined in this work, since it too had been implicated in spore germination. Data presented in this thesis indicate that this is not the case, and that the protein is a component of the spore coat. Overall, the work conducted in this project contributes to knowledge of spore assembly, spore structure and mechanisms that underpin germination, which ultimately, should permit the development of improved methodologies for spore control.

No description available.

Les endospores bactériennes sont l’une des formes les plus résistantes du vivant. Leur capacité à survivre aux traitements de décontamination et leur potentielle pathogénicité pose de réels problèmes aux industriels de l’agroalimentaire. L’usage de conservateurs ou de traitements thermiques reste aujourd’hui la seule solution pour empêcher leur développement dans les aliments. Cependant, les impacts négatifs de ces deux procédés sur les qualités nutritionnelles et la santé du consommateur poussent les industriels à se tourner vers des méthodes de décontamination alternatives.Le procédé de traitement par hautes pressions hydrostatiques (HP) est l’un des nouveaux procédés de décontamination athermique le plus rependu pour la pasteurisation des aliments. Cependant, ce procédé n’a que très peu d’effet sur les endospores et doit donc être combiné avec d’autres méthodes de décontamination. L’objectif de cette thèse a été d’évaluer le potentiel de la combinaison du traitement HP avec la biopréservation pour inactiver des spores thermorésistantes et pathogènes. Cette méthode de décontamination douce met en œuvre des bactéries lactiques protectrices ou les molécules antimicrobiennes qu’elles produisent, telles que la nisine. Des méthodes d’investigations globales telles que la microspectroscopie, la spectroscopie infrarouge et le dichroïsme circulaire, il a été démontré que les HP avaient un effet non négligeable sur l’effet antimicrobien de la nisine. En affectant les structures secondaires de cette protéine, la pression induit une diminution non négligeable de son action antimicrobienne. Cependant, présente dans le milieu de recouvrement de spores traitées en pression, la nisine peut induire une inactivation conséquente (>7 log) d’une population de spores thermophiles et pathogènes. Cette sensibilisation des spores à a nisine par les HP est due l’initiation des toutes premières phases de la germination des spores induites par la pression, non détectées par des méthodes d’analyses classiques. Ces résultats apportent ainsi de nouvelles données pour la compréhension de l’effet des HP sur les spores. Ils permettent aussi d’envisager l’utilisation conjointe des HP et de la nisine à l’échelle industrielle. / Bacterial endospores are one of the most resistant life form on earth. Their capacity to survive to decontamination processes and their potential pathogenicity represent a real problem for the food industry. Currently, the only way to prevent their development in foods is the application of thermal treatments or the use of preservatives. However, these two methods have negative impacts on the nutritional properties of foods and on the consumers’ health. High hydrostatic pressure (HP) is a non-thermal process widely used for commercial pasteurization of foods. However, this process has a very low effect on spores and has therefore to be combined with other decontamination processes to enhance its effectiveness. The objective of this work was to evaluate the potential of the use of biopreservation as an additional hurdle for the inactivation of thermoresistant and pathogenic foodborne spores by HP. Biopreservation is a gentle decontamination process involving protective culture or the antimicrobial agents they produce, like nisin. Thanks to global investigation methods such as microspectroscopy, infrared spectroscopy or circular dichroism, this study showed that HP treatment could affect the antimicrobial properties of nisin. By affecting the secondary structures of this protein, HP can induce a drastic drop in its antimicrobial activity. However when added in the recovery medium of HP-treated spores, nisin can induce their synergistic inactivation (> 7log). This HP-sensitization of spore to nisin is due to the induction of the very first steps of the germination process, usually not detected by the current methods of germination analysis. These results bring knew knowledges about the underlying mechanisms of spores germination under HP and gives new perspectives for the combined used of HP and nisin at the industrial scale.

Rapid, portable detection of biological threat agents such as Bacillus anthracis endospores (“spores”) is extremely important given the real and perceived threats of bioterrorism. Gas chromatography-mass spectrometry (GC-MS) is an excellent general means for chemical detection, although special sample preparation and specialized equipment are required to employ GC-MS for detecting biological agents such as spores in the field. A GC sample introduction probe consisting of a helical wire that can be retracted inside a syringe needle, called a coiled wire filament (CWF), was employed as a simple, passively-heated means to introduce the mixture of spores plus reagents for thermochemolysis methylation (TCM) into the pre-heated GC inlet. There, reactions between spore biomarkers and the TCM reagent mixture occur between 250-290°C. At these conditions, monomethyl sulfate salt mixtures are convenient and efficient TCM reagents for the rapid conversion of a key unique spore biomarker, dipicolinic acid (DPA), to its dimethyl ester (Me2DPA). By this process, Me2DPA yields from spores were quantitatively assessed for different combinations of tetramethylammonium hydroxide (TMA+OH−), sodium hydroxide (Na+OH−), and hydrogen monomethyl sulfate (H+MeSO4−). The best reagent mixture was found by varying the combinations of the ions within the neutral or basic system containing TMA+, Na+, OH−, and MeSO4− according to a novel scheme for design of experiments termed ionic mixtures design of experiments (IMDOE). A combination of the above ions was found that is near-quantitative in its methylation of DPA to Me2DPA; this mixture contained a 1:3:1:3 mole ratio of TMA+:Na+:OH−:MeSO4−. This yield of Me2DPA was approximately a ten-fold increase over the best performance observed at the same conditions with tetramethylammonium hydroxide alone, the TCM reagent widely-used for GC. The reactions involving MeSO4− and TMA+ as methylating reagents, plus relevant hydrolysis and methylation reactions involving acid and base plus water and methanol, were investigated. An overall model is presented and mechanisms are proposed for reasons why basic mixtures of MeSO4− salts are more effective in methylating DPA compared to TMA+ salts at the conditions employed.

Thesis (PhD (Food Science))--University of Stellenbosch, 2009. / ENGLISH ABSTRACT: Species of Alicyclobacillus are acid-tolerant and heat-resistant bacteria that cause spoilage of heat-treated fruit juices stored at room temperature. During the past decade, Alicyclobacillus spp. have become a major cause of spoilage in pasteurised fruit juices leading to significant economic losses world-wide. Spoilage has been reported in apple, pear, orange, peach, mango and white grape juice, as well as in fruit juice blends, fruit juice containing drinks and tomato products, such as tomato juice and canned tomatoes. Spoilage is characterised by a medicinal smell and guaiacol production. These endospore-formers have been shown to survive pasteurisation conditions of 95 °C for 2 min, grow at temperatures between 25° and 60 °C and a pH range of 2.5 to 6.0. Knowledge of this organism is limited, both locally and internationally and the route of contamination to the final product is not well established. In this study the fruit concentrate processing environment was investigated as a potential source and route of contamination for the final product. Species of Alicyclobacillus were isolated from orchard soil, various stages during processing and from fruit juice and concentrates. The isolates were identified based on morpholological, biochemical and physiological properties. Identification to species level was done by 16S ribosomal RNA gene sequencing and strain differentiation by RAPD-PCR. Results indicate that species of A. acidoterrestris and Alicyclobacillus acidocaldarius were found in orchard soil and throughout the processing environment. This is the first report on the isolation of these species from orchard soil, vinegar flies and the fruit processing environment. The 16 isolates identified as A. acidoterrestris grouped into four clusters based on RAPD-PCR banding patterns, suggesting that they belong to at least four genotypic groups. Isolates from the fruit concentrate, wash water and soil located outside of the fruit processing plant grouped into one cluster. Concluded from these results, A. acidoterrestris found in the wash water and soil outside of the factory could act as a potential reservoir of organisms for the contamination of the final fruit concentrate. Thus good manufacturing practices play an essential role in controlling incidence of spoilage caused by these bacteria. Fruit juices can be treated using ultraviolet (UV-C) light with a wavelength of 254 nm, which has a germicidal effect against micro-organisms. Alicyclobacillus acidoterrestris spores were inoculated into tap water, used wash water from a fruit processing plant and grape juice concentrate. Ultraviolet dosage levels (J L−1) of 0, 61, 122, 183, 244, 305 and 367 were applied using a novel UV-C turbulent flow system. The UV treatment method was shown to reliably achieve in excess of a 4 log10 reduction (99.99%) per 0.5 kJ L-1 of UV-C dosage in all the liquids inoculated with A. acidoterrestris. The applied novel UV technology could serve as an alternative to thermal treatments of fruit juices for the inactivation of Alicyclobacillus spores or in the treatment of contaminated processing wash water. Finally, the thermal inactivation at 95 °C for two strains of A. acidoterrestris isolated from contaminated fruit juice concentrates were investigated in a 0.1% (m/v) peptone buffer solution (pH 7.04) and grape juice (pH 4.02, 15.5 °Brix). The thermal inactivation of A. acidoterrestris spores followed first-order kinetics, suggesting that as the microbial population is exposed to a specific high temperature, the spores inactivated at a constant rate. D-values determined in the buffer solution were calculated to be 1.92 min and 2.29 min, while in grape juice D-values were found to be 2.25 min and 2.58 min for the two strains tested. From this study it is clear that the D-value is dependant on the strain tested, but also on the soluble solids of the solution the cells are suspended in. The results indicated that the spores of A. acidoterrestris isolated from South African fruit juice concentrate may survive after the pasteurisation treatment commonly applied during manufacturing. / AFRIKAANSE OPSOMMING: Spesies van Alicyclobacillus is suur-tolerante en hittebestande bakterieë wat bederf veroorsaak in hitte-behandelde vrugtesappe wat teen kamertemperatuur gestoor word. Gedurende die afgelope dekade het Alicyclobacillus spp. ‘n belangrike oorsaak van bederf in gepasteuriseerde vrugtesappe geword en beduidende ekonomiese verliese wêreldwyd veroorsaak. Bederf is aangeteken in appel-, peer-, lemoen-, perske-, mango- en witdruiwesap, sowel as in vrugtesapversnitte, vrugtesapbevattende drankies en in tamatieprodukte soos tamatiesap en ingemaakte tamaties. Bederf word gekenmerk deur ’n medisinale reuk en guaiacol produksie. Daar is gevind dat hierdie endospoorvormers pasteurisasie teen 95 °C vir 2 min kan oorleef en kan groei by temperature tussen 25° en 60 °C en ‘n pH van 2.5 to 6.0. Plaaslik sowel as internasionaal is kennis van hierdie organisme beperk en die roete van kontaminasie van produkte is nog nie goed vasgestel nie. In hierdie studie is die vrugtekonsentraat-verwerkingsmilieu ondersoek as ‘n moontlike bron en roete van kontaminasie van die finale produk. Spesies van Alicyclobacillus is vanuit vrugteboordgrond, verskeie verwerkingstadia en van vrugtesap en vrugtesapkonsentraat geïsoleer. Die isolate is op grond van morfologiese, biochemiese en fisiologiese eienskappe geïdentifiseer. Identifikasie tot spesiesvlak is deur 16S rDNS sekwensering gedoen en stam differensiasie deur RAPD-PKR. Resultate het aangetoon dat A. acidoterrestris en A. acidocaldarius in vrugteboordgrond sowel as in alle stadia van die verwerkingsmilieu voorkom. Dit is die eerste verslag van die isolering van hierdie spesies uit die Suid-Afrikaanse vrugteverwerkingsmilieu, vrugteboordgrond en asynvlieë. Die 16 isolate, geïdentifiseer as A. acidoterrestris en in vier groepe geplaas op grond van hul RAPD-PKR bandpatrone, dui aan dat hulle aan minstens vier genotipiese groepe behoort. Isolate afkomstig van die vrugtekonsentraat, waswater en die grond buitekant die vrugteverwerkingsaanleg het een groep gevorm. Uit hierdie resultate kan afgelei word dat A. acidoterrestris, wat in die waswater en grond buite die aanleg voorkom, as ‘n moontlike bron van organismes vir die kontaminering van die finale vrugtekonsentraat kan dien. Goeie vervaardigingspraktyke speel dus ‘n noodsaaklike rol in die beheer van bederf veroorsaak deur hierdie bakterieë. Vrugtesappe kan behandel word met ultravioletlig (UV-C) met ‘n golflengte van 254 nm wat ‘n dodende effek op mikro-organismes het. Kraanwater, gebruikte waswater van ‘n vrugtesapvervaardigingsaanleg en druiwesapkonsentraat is met A. acidoterrestris spore geïnokuleer. Ultraviolet toedieningsvlakke (J L−1) van 0, 61, 122, 183, 244, 305 en 367 is aangewend met behulp van ‘n nuwe UV-C drukvloei stelsel. Daar is aangetoon dat die UV-behandelingsmetode ‘n betroubare vermindering (99.99%) van meer as 4 log10 per 0.5 kJ L-1 van ‘n UV-C dosis gee in al die vloeistowwe wat geïnokuleer is met A. acidoterrestris. Die toegepaste nuwe UV-tegnologie kan gebruik word as ‘n alternatief tot die hittebehandeling van vrugtesap vir die deaktivering van Alicyclobacillus spore of in die behandeling van gekontamineerde waswater. Ten slotte is hitte-deaktivering teen 95 °C van twee stamme van A. acidoterrestris, geïsoleer uit gekontamineerde vrugtesapkonsentraat, in ‘n 0.1% (m/v) peptoonbufferoplossing (pH 7.04) en druiwesap (pH 4.02, 15.5 °Brix), ondersoek. Die hitte-deaktivering van A. acidoterrestris spore het eerste-orde kinetika gevolg, wat aandui dat die mikrobe-populasie teen ‘n konstante tempo afsterf, wanneer blootgestel aan ‘n spesifieke hoë temperatuur. Die D-waardes in die bufferoplossing is bereken as 1.92 min en 2.29 min, terwyl daar gevind is dat die D-waardes in druiwesap 2.25 min en 2.58 min is vir die twee betrokke stamme. Vanuit hierdie studie is dit duidelik dat die D-waardes afhang van die betrokke stam, maar ook van die oplosbare vaste stowwe van die oplossing waarin die selle opgelos is. Die resultate dui daarop dat die spore van A. acidoterrestris, wat geïsoleer is uit Suid-Afrikaanse vrugtesapkonsentraat, die pasteurisasiebehandeling wat algemeen tydens vervaardiging toegepas word, kan oorleef. Aangesien die toepassing van strenger hittebehandeling om spore van A. acidoterrestris te deaktiveer onaanvaarbare organoleptiese veranderinge in die produk tot gevolg het, word dit aanbeveel dat die risiko van bederf verminder behoort te word deur die gebruik van goeie vervaardigingspraktyke gedurende vrugteverwerking.

Phytonematodes are known to cause substantial losses in crop yields across the world. Since the middle of the last century, these pests have been adequately controlled by chemical nematicides. However, due to increasing public health concern, strict regulations in the EU and elsewhere have significantly reduced the usage of these environmentally not-so-safe chemicals. This has led us to look for reliable biological alternatives. The Pasteuria group of Gram-positive endospore-forming bacteria (phylum: Firmicutes) often associated with nematode-suppressive soils are potentially reliable nematode biocontrol agents. However, the highly specific interaction of Pasteuria to their nematode hosts poses a challenge to the management of heterogeneous populations of nematodes in the field; the mechanism behind this specificity remains unclear. One of the fundamental basis of host specificity is the attachment of Pasteuria endospores to the cuticle of their host nematodes which is the first and essential step in the infection process. Thus, understanding the molecular mechanisms that govern the attachment process is important in identifying suitable populations of Pasteuria for effective broad-range management of plant parasitic nematodes in soil. Previous studies suggest the presence of immunogenic collagen-like fibres and carbohydrates on the endospore coat of Pasteuria that may have a role in the initial interaction of the endospores with their nematode hosts. Published work on phylogeny relates Pasteuria to Bacillus spp. most of which have well annotated and characterized genomes while the genome of Pasteuria remains to be sequenced completely. In this thesis, I attempt to explore the endospore biology of obligate and fastidious Pasteuria spp. using the wide knowledgebase of well studied Bacillus endospores. The primary aim was to characterize the immunogeneic determinants that are possibly responsible for the attachment of Pasteuria endospores to the host nematode cuticle by a combination of computational and lab-based approaches. To approve the suggested phylogenetic closeness of Pasteuria to Bacillus, the first part of the study focused on phylogeny reconstruction of Pasteuria spp. amongst Bacillus spp. and other members of the phylum Firmicutes. This was followed by in silico studies to identify candidate collagen-like genes in P. penetrans; the putative functional proteins encoded by these candidate genes were then comparatively characterized with collagens from other organisms including the members of the genus Bacillus. The surface associated collagen-like proteins and other possible immunogens on the endospores of Pasteuria were characterized by protein immunoblotting, lectin blotting and immunofluorescence microscopy and comparisons were made with B. thuringiensis endospores. Lastly, endospore attachment assays were done to test the hypothesis that collagens and carbohydrates play a role in Pasteuria endospore attachment. The results of the computational analyses suggest a family of collagen coding putative genes in the Pasteuria genome, all of which are predicted to have varied biochemical properties and are seemingly of diverse evolutionary origin. The Western blot and microscopic analyses show that the endospores of P. penetrans and B. thuringiensis share some common immunodominant surface epitopes. The attachment assays confirm the involvement of collagens and at least one carbohydrate (N-acetylglucosamine) in the endospore attachment. However, the results also indicate possible involvement of other adhesins in the process; to support this, at the end of the thesis, I propose a new 'Multitype Adhesin Model' for initial interaction of Pasteuria endospores with the cuticle of their host nematodes. The outcomes of this project will help in identifying the molecular basis of the complex Pasteuria-nematode interaction. This will provide a basis to develop environmentally benign nematode bio-management strategies.

Methods for fast, simple detection of biomarkers to detect and differentiate closely related Bacillus endospores including Bacillus anthracis (BA), Bacillus thuringiensis (BT), Bacillus atrophaeus (BG), and Bacillus cereus (BC) using thermochemolysis and methylation (TCM), coiled wire filament (CWF), solid phase micro extraction (SPME) and gas chromatography-mass spectrometry (GC-MS) were developed. The main biomarkers detected and used for differentiation include dipicolinic acid methyl ester (DPAME), fatty acid methyl esters (FAMEs), 3-methyl-2-butenoic acid methyl ester (3M2BAME), 2-butenoic acid methyl ester (2BAME), and several methylated sugars. TCM of endospores was performed based on hydrolysis and methylation at elevated temperature after the endospores were mixed with sulfuric acid (H2SO4) with or without addition of tetramethylammonium hydroxide (TMAH) in methanol (MeOH). TCM products were then introduced into a heated GC injector port using a coiled wire filament (CWF) or solid phase microextraction (SPME) for detection and differentiation of the endospores by GC-MS.The CWF, which consisted of a tiny platinum helical wire coil attached to a retractable plunger that moved the coil in and out of a syringe needle housing, allowed for sampling to be accomplished by dipping the CWF in an endospore sample suspension, evaporating the suspension liquid, and then introducing the CWF into the injection port to enable on-line TCM. New SPME techniques, including half-half extraction, coated-needle extraction (CNE), and a new home-made polymer coated needle were used to speed up solid phase micro extraction of biomarkers produced from TCM. These simplified the detection of anthrose and other biomarkers. TCM with a CWF and TCM with SPME produced high intensity profiles of DPAME, FAMEs, 2BAME, 3M2BAME and methylated sugars. While the presence of DPAME can be used for the general detection of endospores (Bacillus and Clostridium) and the presence of 3M2BAME for the detection of BA, specific saturated and unsaturated C15, C16, and C17 fatty acid methyl esters and methylated sugars provide additional information for differentiating various Bacillus species grown at different temperatures and in different media. DPAME was detected in samples containing as few as 2,500 and 6,000 endospores using TCM-CWF with and without a concentration step, respectively. GC-MS peak area percent reproducibility for FAMEs using TCM and CWF varied from 3 to 13% (RSD). Better than 97% correct predictability of Bacillus species identity was obtained from a blind experiment consisting of 145 samples using DPAME and specific FAMEs. Conventional SPME and a modified form of "in-needle" extraction allow for detection of the biomarkers in less than 35 min. The detection limits with SPME sample introduction injection were approximately 5 x 103 endospores.Using these approaches, differentiation of Bacillus endospores and other biological agents grown under different conditions were based on the following characteristics: (1) presence of DPAME and specific FAMEs (iso or anteiso C15:0 and iso or anteiso C17:0) in Bacillus endospores, (2) unique presence of 3M2BAME (anthrose by-product) in BA, (3) absence of 2-butenoic acid methyl ester in BG, and (4) presence and absence of specific methylated monosaccharides in various Bacillus species. Clostridium endospores and non-sporulating bacteria, such as Yersinia pestis (YP) and Francisella tularensis (FT) could also be easily distinguished from Bacillus endospores based on the presence and absence of several specific sugar derivatives and fatty acid methyl esters (FAMEs), such as iso or anteiso C15:0 and iso or anteiso C17:0, and > C18 FAMEs which were simultaneously produced during TCM.