京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第21813号 / 農博第2326号 / 新制||農||1066(附属図書館) / 学位論文||H31||N5185(農学部図書室) / 京都大学大学院農学研究科応用生物科学専攻 / (主査)教授 左子 芳彦, 教授 澤山 茂樹, 准教授 吉田 天士 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

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Nanofluidics is an emerging field with many science and engineering applications. The physics of material transport through nanochannels are of interest in filtration, sensing, device miniaturization, and biomimetics. To address such ambitions with nanofluidic tools will require advancements in our understanding and control over nanofluidic systems. This work contributes to electrokinetic phenomena, characterization techniques, and applications in nanofluidics. Ion transport data through nanopipettes are used to validate a finite element model for nonlinear electrokinetic flows. With the model, we conclude that asymmetric surfaces induce fluid vortices and provide insight into supporting mathematical techniques. We then establish nanobubble-plugged nanopipettes as promising ionic devices due to the electrokinetic effects of three-phase interfaces. Using cryogenic transmission electron microscopy, ion current measurements, and extensive physical modeling, we conclude that nanobubble plugs are metastable, slow-growing, and induce strong current rectification and enhancement. All these insights let us study microbial surfaces using electrokinetic phenomena detected by a scanned nanopipette. Over immobilized Pseudomonas aerugonsa cells and Δphz-type biofilms, we detect topographic and surface charge properties due to voltage-dependent signals through a scanned nanopipette probe. Our efforts establish a fast hopping probe scanning ion conductance microscopy technique for long-range surface charge detection. Finally, we use an integrated carbon nanotube channel to demonstrate how solid-state charge can drive electrokinetic flows through Coulomb drag coupling.

The lung was recently identified to consist of a complex microenvironment made up of microorganisms that interact with one another and the host cells via direct and indirect interactions. As a result, understanding the dynamic of the microbiome in chronic respiratory diseases has become the focus of pulmonary researches. In cystic fibrosis (CF), chronic infections are a comorbidity associated with the genetic disorder. Recently, it was noted that the interactions of the fungus, Aspergillus fumigatus, and the bacterium, Pseudomonas aeruginosa together contribute to more severe disease outcomes in CF patients. In vitro co-cultures show that P. aeruginosa and A. fumigatus can affect one another’s growth and pathogenicity, but very few studies have attempted to model interactions of these microorganisms in vivo. Based on clinical and basic research, we developed a co-exposure model in which we could compare non-allergic and allergic animals co-exposed to Pseudomonas aeruginosa and Aspergillus fumigatus. While both groups had significant neutrophilia and production of acute phase response cytokines and chemokines, the allergic co-exposed group had a greater mortality with 34.8% of the animals expiring by 24h in comparison to 12.5% for the non-allergic co-exposed animals and 100% survival in the controls. A contributing factor to the more severe disease outcomes in the allergic co-exposed group is the increase in eosinophilic inflammation and IL-17A production, which only occurs when both microorganisms are viable. In addition, it was found that viable P. aeruginosa but not A. fumigatus causes interstitial inflammation, significant neutrophilia, and even death during co-exposures. The decline in health of animals co-exposed to the fungus and bacteria could be attributed not only to the host’s inflammatory response, but also to the spatial and temporal co-localization in the lung. To address this, we performed in vitro studies finding an aggregation of the microorganisms that could also be identified in vivo. This current research emphasizes the need for in vivo studies on polymicrobial interactions. / ND Agricultural Experiment Station; National Institute of Allergy and Infectious Diseases of the National Institutes of Health under Award Number R155AI137886

A common vector of nosocomial infections is Pseudomonas aeruginosa. This opportunistic pathogen is resistant against different classes of antibiotics as well as against biocides. Some P. aeruginosa strains are able to form biofilm. The biofilm formation on medical devices represent a common occurrence that can lead to serious illness and death. Bacteria in biofilms are more resistant than their planktonic counterparts. Therefore it is important to find disinfectants that will be effective also against biofilm. Bactericidal efficacy of four disinfectants has been evaluated on biofilm and on planktonic forms of P. aeruginosa.

Algal blooms caused by toxigenic cyanobacterial species are an increasing economic burden globally, as high anthropogenic inputs of nitrogen and phosphorous, coupled with rising levels of atmospheric CO2, promote eutrophication and enhance bloom proliferation. Of the freshwater bloom forming species, Microcystis aeruginosa has garnered the most attention due to the production of toxic secondary metabolites known as microcystins. These cyclic peptides are potent eukaryotic protein phosphatase 1 and 2A inhibitors, and can induce hepatic damage if concentration levels reach above the World Health Organization level of 1 µg/L. Current mitigation strategies of water column disruption or by use of broad acting chemicals, are limited in their range and may cause unwanted off target effects to the surrounding biota. Antisense oligonucleotides are short single-stranded DNA polymers that hybridize with transcribed mRNA, and suppress translation of protein products through steric hindrance of ribosomes, or by RNAse H degradation of the DNA/RNA bound complex. While antisense oligonucleotide applications have proven successful in the pharmaceutical industry, their potential remains largely unexplored in environmental contexts. For this reason, we investigated the knockdown of microcystin synthetase gene cluster mcyE in M. aeruginosa. We found that ionic charge neutralization coupled with heat shock were effective chemical competence based methods for delivery, mcyE transcript abundance in cells treated with phosphodiester linked antisense oligonucleotides significantly decreased in RT-qPCR analysis, and production of intracellular microcystin significantly decreased over a 24 hour period (-1.9 fg/cell). This work demonstrates a novel proof of concept for the potential use of exogenous antisense oligonucleotides to target M. aeruginosa in harmful algal bloom occurrences.

Publicación a texto completo no autorizada por el autor / Con la finalidad de esclarecer cual es la fuente de carbono con la que se produce mayor cantidad de RL, en esta investigación se compara la producción de RL por P. aeruginosa 6K11 en cinéticas de crecimiento manteniendo parámetros constantes usando 5 fuentes de carbono: glucosa (G) al 3%, 4% y 5%; glicerol (Y) al 3%, 4% y 5%; aceite de maíz (M) al 6%, 7% y 8%, aceite de pescado (P) al 2%, 3% y 4% y aceite de soya quemado (Q) al 4%, 5% y 6%. Para las cinéticas se usa el medio mineral base Sigmund y Wagner modificado a pH 6.8 y la incubación es a 35°C con 140 rpm de agitación por 250 horas. Los resultados confirman que existe una diferencia significativa (p<0.05) entre las producciones de RL al usar diversas fuentes de carbono. De igual manera se encuentra diferencia entre las diversas concentraciones de la misma fuente de carbono. El orden según producción de RL es: M7% > M8% > M6% > Y4% > Y5% > Q5% > Q6% > Q4% > Y3% > G4% > P2% > P3% > G5% > P4% > G3%. Así mismo se encuentra que el aceite de maíz al 7% es la fuente de carbono que genera la mayor producción de RL equivalente a 17.49 gRL/L. / Perú. Ministerio de la Producción. Programa Nacional de Innovación para la Competitividad y Productividad (Innóvate Perú). Fondo para la Innovación, la Ciencia y la Tecnología (FINCyT) / Tesis

The Pseudomonas aeruginosa ATCase was cloned and sequenced to determine the correct size, subunit composition and architecture of this pivotal enzyme in pyrimidine biosynthesis. During the course of this work, it was determined that the ATCase of Pseudomonas was not 360,000 Da but rather present in a complex of 484,000 Da consisting of two different polypeptides (36,000 Da and 44,000 Da) with an architecture similar to that of E. coli ATCase, 2(C3):3(r2). However, there was no regulatory polypeptide found in the Pseudomonas ATCase.

Introduction: Gram-negative prosthetic joint infections (GN-PJI) present unique challenges in management due to their distinct pathogenesis of biofilm formation on implant surfaces. The purpose of this study is to establish a clinically representative GN-PJI model that can reliably recapitulate biofilm formation on titanium implant surface in vivo. I hypothesized that biofilm formation on an implant surface will affect its ability to osseointegrate. Methods: The model was developed using 3D-printed titanium hip implants, to replace the femoral head of male Sprague-Dawley rats using a posterior surgical approach. GN-PJI was induced using two bioluminescent Pseudomonas aeruginosa (PA) strains: a reference strain (PA14-lux) and a mutant strain that is defective in biofilm formation (flgK-lux). Infection was assessed in real-time using the in vivo imaging system (IVIS) and Magnetic Resonance Imaging (MRI) and in vitro by quantifying bacterial loads on collected implants surface and in periprosthetic tissues as well as biofilm visualization using the Field emission scanning electron microscopy (FE-SEM). The implant stability, as an outcome, was directly assessed by quantifying the osseointegration in vitro using microCT scan, and indirectly assessed by identifying the gait pattern changes using DigiGaitTM system in vivo. Results: Bioluminescence detected by IVIS, was focused on the hip region, demonstrating localized-infection, with the ability of PA14-lux to persist in the model compared to flgK-lux defective in biofilm formation. This was corroborated by MRI as the PA14-lux induced relatively larger implant-related abscesses. Biofilm formation at the bone-implant-interface induced by the PA14-lux was visualized using FE-SEM versus defective-biofilm formation by flgK-lux. This could be quantitatively confirmed, by average viable-colony-count of the sonicated implants, 3.77x108CFU/ml versus 3.65x103CFU/ml for PA14-lux and flgK-lux, respectively (p=0.0025; 95%CI: -6.08x108 to -1.45x108). This difference in the ability to persist in the model was reflected significantly on the implant osseointegration with a mean intersection surface 4.1x106μm2 1.99x106 for PA14-lux versus 6.44x106μm2 2.53x106 for flgK-lux and 7.08x106μm2 1.55x106 for non-infected control (p=0.048). Conclusions: To date, the proposed in vivo biofilm-based model is the most clinically representative for GN-PJI since animals can bear weight on the implant and poor osseointegration correlates with biofilm formation. Clinical Relevance: The current model will allow for reliable testing of novel biofilm-targeting therapeutics.

Bacterial biofilms are 1,000 times harder to kill than planktonic bacteria. Spores are also harder to kill compared to vegetative cells. We developed a rapid model for establishing dry surface bacterial biofilms for disinfectant efficacy testing and challenged them with seven EPA-registered disinfectants. We also demonstrated that during the disinfection of surfaces contaminated with bacterial spores, disinfectant wipes may transfer spores from contaminated to uncontaminated surfaces.