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A study was conducted to optically determine the viability of the Gram +, endospore-forming bacterial genera Bacillus using a hyperspectral reflectance spectrometer. Endospores are a dormant, differentiated cellular capsule form taken by select bacteria to ensure survival when environmental conditions become unfavorable. Two species of Bacillus were used for this study, Bacillus megaterium and Bacillus subtilis. These endospores were killed using a chemical treatment of sodium hypochlorite or a physical treatment of heat and pressure in an autoclave. The treated samples along with viable samples were lyophilized to form a powder. A reflectance spectrometer measuring 350 nm to 2500 nm (UV to shortwave infrared) was used to collect optical data on bulk powders. The resulting spectra were analyzed using several different methods, including integration and a Support Vector Machine (SVM) to obtain optimal separability of viable and nonviable endospores. Results of this study demonstrated the significant spectral separability of live and dead endospores with a level of confidence <0.05 in both species.

Alkaline glutaraldehyde (GTA) has been used as a high level chemical disinfectant and sterilant for many years and is known to kill a broad spectrum of organisms ranging from vegetative eukaryotes to bacterial endospores. Although the mechanism of sporicidal action has been studied on numerous occasions, GTA's exact mechanism(s) of action are still debated. In addition to the uncertainty of GTA's mechanism(s) of action, GTA has also shown significant variability in the time required to kill endospores and naked viruses. A better knowledge of the lethal mechanism(s) of GTA is needed to understand this discrepancy in kill times for GTA against spores of different species. Similar trends have been observed in GTA's activity against non-enveloped viruses. Based on previous work, one proposed major mechanism of GTA's sporicidal activity is related to the number of available primary amines located on the surface of microbes. In this study, we have compared the efficacy of GTA on spores from 5 Bacillus species. We have also developed a method for staining these spores with amine reactive dyes to create fluorescent profiles correlating to the abundance of free amino groups on each spore type. We also describe a method for staining non-enveloped viruses to identify exposed primary amino groups on capsid proteins that may act as targets for GTA, using amine reactive Gold nanoparticles. We found that GTA 6-Log10 reduction times for various spore types varied at both the batch and species level. Spore coat thickness and fluorescence were useful tools in predicting the susceptibility of spores to GTA. Amine reactive gold particles (AuNPs) also proved useful in identifying virus susceptibility to GTA. Ultimately, more reliable disinfection testing methods are needed, and caution should be used when trying to extrapolate data generated from surrogate organisms to other species.

BillerudKorsnäs is a manufacturer of fiber-based cartonboard and liquid packaging board. Microbial growth occurs at several steps in cartonboard production due to favourable environment and the good access to nutrients from the raw material, and additives such as starch. Vegetative bacteria are usually not harmful in the production and die in the hot drying end of the cartonboard machine. The most abundant microflora at paper- and cartonboard factories consists largely of sporeforming microorganisms from the genera Bacillus and Paenibacillus. The endospores are highly resistant and can stay in the final end product, which is undesirable. Levels of endospores from these species at BillerudKorsnäs production unit KM5 are usually low, but an occational increase can be seen when a new cartonboard product, KW1 is produced. Today, the method used for controlling the microbiology is by adding biocides to broke towers. This has shown to be both expensive and non-effective at KM5. A new method is needed for controlling the microbiology at KM5 that is more effective, costbeneficial and environmental friendly.The aim of this project was to test a hypothesis for spore formation at a paper board factory in lab-scale experiments. A suggestion of a technical change in the process would be made that could minimize spore formation and the use of biocides at KM5. A model organism Bacillus licheniformis (E-022052) was used to study effects of environmental conditions on spore formation. Experiments were also performed in controlled bioreactor trials, where methods to minimize spore formation were tested.The experiments showed that nutrient deficiency of a primary carbon source was the major reason for spore formation and should be avoided at KM5. Further, the experiments showed that oxygen limitation significantly decreases the endospore formation.The conclusion reached, was that spore formation could be minimized by a feed addition of glucose to Broke tower 1 during the few days production of KW1. A second alternative includes using a feed of concentrated pulp that could be used to minimize spore formation without the use of biocides and without the need for rebuilding of the mill.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第19779号 / 農博第2175号 / 新制||農||1041(附属図書館) / 学位論文||H28||N4995(農学部図書室) / 32815 / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 小川 順, 教授 阪井 康能, 教授 栗原 達夫 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

Bacterial spore peptidoglycan (PG) is very loosely cross-linked relative to vegetative PG. Theories suggest that loosely cross-linked spore PG may have a flexibility which contributes to the attainment of spore core dehydration. The structure of the PG found in fully dormant spores has previously been examined in wild type and many mutant strains. These analyses showed little correlation between the degree of spore PG cross-linking and core dehydration. However, these studies only examined the structure of PG from dormant spores and did not allow for the structural analysis of spore PG during sporulation when actual spore PG synthesis and core dehydration occur. Structural analyses of developing spore PG from wild type Bacillus subtilis and eight mutant strains are included in this study. Structural analyses of developing spore PG suggest the following: a) the germ cell wall PG is synthesized first next to the inner forespore membrane; b) cross-linking is relatively high in the first 10% of spore PG synthesized; b) a rapid decrease in cross-linking is observed during synthesis of the next 20% of the spore PG; and c) this decrease is followed by an eightfold rise in the degree of cross-linking during synthesis of the final 70% of the spore PG. This increasing gradient of cross-linking was previously predicted to contribute to the attainment of spore core dehydration. However, analyses of mutant strains indicate this cross-linking gradient is not required for the attainment of spore dehydration. / Master of Science

Penicillin-binding proteins (PBP's) are membrane-associated enzymes involved in the polymerization of peptidoglycan. PBP's are divided into three classes based upon their molecular weights and functional domains. Gene expression is regulated in the two differentiated cells in Bacillus subtilis, the mother cell and the forespore, by coordinated expression of different sigma factors that recognize specific promoters in each compartment. The functional and compartmental specificity of individual penicillin-binding proteins from the different classes of PBP's were examined during sporulation in B. subtilis. Analyses of three class A high molecular weight PBP's indicated that pbpF and pbpG must be expressed in the forespore to carry out their specific role during spore peptidoglycan synthesis. Expressing pbpD in either the forespore or the mother cell could not complement for the loss of pbpF and pbpG, suggesting that there must be additional sequence information in PBP2c and PBP2d that allows them to carry out their specific role during germ cell wall synthesis. Analyses of a low molecular weight PBP, PBP5*, suggested that expressing dacB in either the mother cell or in the forespore could regulate the level of spore peptidoglycan cross-linking to what is typical of wild type spore peptidoglycan. / Master of Science

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.