Developing a novel and versatile approach to study populations of microbes on surfaces

Spatial structure, for example regarding antibiotic gradients, is an important topic of investigation in microbial ecology and evolution. Experiments investigating pop- ulation dynamics in spatially-structured environments are often performed on agar plates. Whilst inexpensive and straightforward, these provide only rudimentary temporal and spatial control of environmental conditions. In chemostats and microfluidic devices, for well-mixed and micrometre-scale environments, respectively, regulating media inflow and outflow enables environ- mental control. We combine proven use of agar surfaces with such flow-enabled control in a novel, low-cost fluidic device; the device comprises an elastomer supporting base with a thin agar sheet on top on which microbes grow. Indented channels in the base allow flow of media/antibiotics below the agar surface. A Raspberry-Pi-operated camera allows for time-lapse imaging suitable for quantita- tive image analysis. As a proof of principle, we used our device for extended and robust growth of non-motile E. coli and motile P. aeruginosa maintaining the initial speed with which colonies propagate over three days, whilst a continual speed decrease occurred on agar plates. Guided by simulations of flow and diffusion, we then used the device to create stable antibiotic gradients within the agar. Along these gradients, we found P. aeruginosa exhibit unique microbial growth patterns with local adaptations. Because flow below the agar surface can be controlled spatially and temporally, the device promises a range of applications for studying microbial ecology and evolution in spatially continuous environments at a substrate-air interface.Engineering and Physical Sciences Research Council (EPSRC

An Optical Density Detection Platform with Integrated Microfluidics for In Situ Growth, Monitoring, and Treatment of Bacterial Biofilms

Systems engineering strategies utilizing platform-based design methodologies are implemented to achieve the integration of biological and physical system components in a biomedical system. An application of this platform explored, in which an integrated microsystem is developed capable of the on-chip growth, monitoring, and treatment of bacterial biofilms for drug development and fundamental study applications. In this work, the developed systems engineering paradigm is utilized to develop a device system implementing linear array charge-coupled devices to enable real time, non-invasive, label-free monitoring of bacterial biofilms. A novel biofilm treatment method is demonstrated within the developed microsystem showing drastic increases in treatment efficacy by decreasing both bacterial biomass and cell viability within treated biofilms. Demonstration of this treatment at the microscale enables future applications of this method for the in vivo treatment of biofilm-associated infections

Structure-Property Relationships of Polymer Films and Hydrogels to Control Bacterial Adhesion

The emergence and spread of antibiotic resistance across microbial species necessitates the need for alternative approaches to mitigate the risk of infection without relying on commercial antibiotics. Biofilm-related infections are a class of notoriously difficult to treat healthcare-associated infections that frequently develop on the surface of implanted medical devices. As biofilm formation is a surface-associated phenomenon, understanding how the intrinsic properties of materials affect bacterial adhesion enables the development of structure-property relationships that can guide the future design of infection-resistant materials. Despite lacking visual, auditory, and olfactory perception, bacteria still manage to sense and attach to surfaces. Previously, it has been reported that bacteria can detect and differentiate the surface chemistry and topography of surfaces; however, the influence of the stiffness and thickness on bacterial-surface interactions remains unknown. In this thesis, the effect that the fundamental material properties of polymer films and hydrogels (stiffness, thickness, and chemistry) have on the adhesion and surface-associated transport of bacteria was investigated. By decoupling the effect of the hydrogel’s stiffness and thickness from their chemistry, we suggest a key takeaway design rule: to optimize fouling-resistance, hydrogel coatings should be thick and soft. Two chemically distinct hydrogels, poly(ethylene glycol) and agar, were synthesized over a 1-1000 kPa range of Young’s modulus. Static adhesion experiments, conducted on 150 µm thick hydrogels, determined that Escherichia coli and Staphylococcus aureus colony surface coverage correlated positively with an increase in Young’s modulus. Notably, a substantial increase in adhesion occurred for both bacteria when the thickness of the hydrogels was reduced to 10 µm. The stiffness of poly(ethylene glycol) brushes and hydrogels was also found to influence the length and frequency of Staphylococcus aureus surface-associated transport via dynamic shear flow experiments. Furthermore, a universal hydrogel functionalization platform was developed for instances where mechanical properties of hydrogels are not adjustable. The incorporation of the fouling-resistant polymer zwitterion, poly(2-methacryloyloxyethyl phosphorylcholine), enhanced resistance to bacterial adhesion without altering the mechanical properties of covalently or physically crosslinked hydrogels. This thesis demonstrates that by combining structure-property relationships with fouling-resistant zwitterionic chemistry, the adhesion of proteins and microorganisms to polymer hydrogels is reduced

Der Einfluß von Umweltfaktoren auf die Virulenz von Pseudomonas aeruginosa

Research into the importance of phosphate as an environmental signal molecule for multicellular behavior in the Pseudomonads P. aureofaciens and P. fluorescens recently uncovered a role of the Pho regulon in the formation of bacterial biofilms. Most interestingly, it was clearly demonstrated that PhoB activates a phosphodiesterase which decreases the intracellular c-di-GMP levels in P. fluorescens, thus providing a link between the Pho regulon and c-di-GMP signaling pathways that impact on biofilm formation. In this study we present evidence that in the opportunistic pathogen P. aeruginosa the Pho regulon inhibits biofilm formation and is required for the repression of the TTSS. We furthermore identified an EAL domain protein, referred to as RapA, as a down-stream effector of the Pho regulon, which at least partially mediated the observed inhibition. Although the Pho regulon contributed to inhibition of biofilm formation, it had no effect on CupA fimbriae expression or Psl exopolysaccharide expression, the common factors contribute to biofilm formation in P. aeruginosa. Interestingly, in contrast to P. aeruginosa PAO1 strain, the observed inhibition of the biofilm formation was Pho regulon-dependent but independent of the availability of inorganic phosphate in PA14 strain. The Pho regulon-dependent repression of TTSS was also independent of the availability of inorganic phosphate in PA14 strain. These results add even more complexity to the regulation of the bacterial behavior by environmental cues. We demonstrated that a wild-type derived flgF mutant exihibited an enhanced biofilm formation as compared to the wild-type as shown by CSLM. Moreover, ExoT expression was strongly enhanced in the flgF mutant as compared to the wild-type indicating that the FlgF, similar to Pho regulon, negatively influences virulence phenotype (inhibition of biofilm formation and repression of the type three secretion system). We found that Pho regulon has no influence on the FlgF or flagella. Although the transcriptome analysis of the flgF mutant compared to the wild-type did not uncover an influence of FlgF on Pho regulon, it confirmed that the TTSS is negative regulated by the FlgF because most of genes encoding for TTSS were up-regulated. Moreover, pyochelin encoding genes were strongly down-regulated. We illustrated that both of the Pho regulon and the FlgF affect the production of siderophores. The pyochelin production was enhanced by PhoB and FlgF. The pyoverdine production was enhanced by PhoB but inhibited by FlgF. We also found that both of the Pho regulon and the FlgF are regulating the same virulence phenotype but independently. Our results add more complexity to the regulation of biofilm formation and TTSS expression in P. aeruginosa.Untersuchungen über die Bedeutung von Phosphat als umweltbedingtes Signalmolekül für ein multizelluläres Verhalten der Pseudomonaden P. aureofaciens und P. fluorescens haben kürzlich die Rolle des Pho Regulons in der Ausbildung von Biofilmen enthüllt. Interessanterweise wurde eindeutig gezeigt, dass PhoB eine Phosphodiesterase aktiviert, die den intrazellulären c-di-GMP Spiegel in P. fluorescens absenkt. Hierdurch wird eine Verknüpfung zwischen dem Pho Regulon und c-di-GMP Signalwegen, welche ebenfalls einen Einfluss auf die Biofilmbildung haben, erstellt. In dieser Arbeit wurde nachgewiesen, dass das Pho Regulon auch in dem opportunistischen Krankheitserreger P. aeruginosa die Biofilmbildung inhibiert und für die Unterdrückung des Typ-Drei-Sekretionssystemes benötigt wird. Weiterhin wurde als ein Downstream Effektor des Pho Regulons ein EAL-Domänen Protein identifiziert, welches RapA genannt wurde und zumindest teilweise die beobachtete Inhibierung vermittelte. Obwohl das Pho Regulon zu einer Inhibierung der Biofilmbildung beitrug, hatte es keinen Effekt auf die Expression von CupA Fimbrien oder Psl Polysacchariden - allgemeinen Faktoren die zur Ausbildung von Biofilmen in P. aeruginosa beitragen. Im Gegensatz zu dem Stamm PAO1 war die beobachtete Hemmung der Biofilmbildung in dem Stamm PA14 zwar abhängig von dem Pho Regulon, aber unabhängig von Vorkommen an anorganischem Phosphat. Die Pho abhängige Unterdrückung des Typ-Drei-Sekretionssystems in PA14 war ebenfalls unabhängig von Vorkommen an anorganischen Phosphat. Diese Ergebnisse unterstreichen die Komplexität der Regulation bakteriellen Verhaltens durch Einflüsse aus der Umwelt. Weiterhin konnten wir durch CLSM zeigen, das eine vom Wildtyp abstammende flgF Mutante eine erhöhte Biofilmbildung im Vergleich zum Wildtyp aufweist. Außerdem war die ExoT Expression in der flgF Mutante im Vergleich zum Wildtyp stark erhöht. Das deutet darauf hin, dass FlgF, ähnlich wie das Pho-Regulon, den Virulenz-Phänotyp negativ beeinflusst (Inhibierung der Biofilmbildung und Unterdrückung des Typ-Drei-Sekretionssystems). Nachforschungen haben ergeben, dass das Pho Regulon keinen Einfluss auf FlgF oder Flagellen besitzt. Eine Transkriptionsanalyse der flgF Mutante im Vergleich zum Wildtyp ließ ebenfalls keinen Einfluss von FlgF auf das Pho-Regulon erkennen. Es wurde jedoch die negative Regulation des Typ-Drei-Sekretionssystemes durch FlgF bestätigt, da die meisten Gene, die für das Typ-Drei-Sekretionssystem kodieren, in der flgF Mutante hochreguliert waren. Zusätzlich waren Pyochelin kodierende Gene stark herunterreguliert. Schließlich konnten wir zeigen, dass beides, das Pho Regulon und FlgF, die Produktion von Siderophoren beeinflusst. Während die Pyochelinsynthese durch PhoB und FlgF erhöht wurde, wurde die Produktion von Pyoverdin durch PhoB erhöht, durch FlgF jedoch gehemmt. Wir konnten ebenfalls demonstrieren, dass das Pho Regulon und FlgF den selben Virulenz- Phänotyp regulieren, jedoch unabhängig voneinander

3D Bioprinted Engineered Living Materials for Continuous Organophosphorus Compound Detoxification

Engineered living materials (ELMs) are a rapidly emerging class of materials, demonstrating a wide range of functionalities, including responsive morphing, self-healing, and bio-catalysis. 3D bioprinted hydrogels have been used for the fabrication of high resolution, compartmentalised, and load-bearing structures suitable for hosting microbial metabolism, and accordingly represent an ideal environment for ELMs. The interactions between material frameworks, such as hydrogels, and encapsulated life are now beginning to be investigated.Herein, by 3D printing a hydrogel-encapsulated population of Escherichia coli, a chemically inducible, metabolically active, microbial ELM was fabricated. The material was characterised using a wide range of techniques, including fluorescence microscopy and cryogenic electron microscopy. Toxic organophosphorus compound (OPC) detoxifying capabilities were conveyed to the material through inducible expression of Agrobacterium radiobacter phosphotriesterase (arPTE). The reaction diffusion process occurring at the interface of the OPC detoxifying ELM was investigated using continuous fluorescence imaging of Coumaphos hydrolysis.. Principal component analysis was then used to uncover spatial and temporal features within this data, with relevance for future optimisation of catalytic microbial ELMstructures. To further demonstrate the applicability of this 3D printable microbial ELM, the material was incorporated into an entirely 3D printed flow reactor, demonstrating effective, cyclical detoxification of an OPC solution at high flow rate.Looking towards the future of ELM design, a novel, 3D printable, contractile-thermosensitive,double-network hydrogel was used to create thermo-responsive OPC degrading bioreactors, capable of autonomously controlling their performance

Development of techniques for the analysis of acetic acid bacteria populations and their interaction in different food environments

El desenvolupament de tècniques moleculars per a la detecció, identificació i tipificació de bacteris acètics és la clau per a un millor enteniment de la coexistència d’aquesta microbiota relacionada amb productes alimentaris. S’han aplicat diferents tècniques dependents e independents de cultiu per a la identificació i quantificació de bacteris acètics que es troben en raïm i vi procedents de les Illes Canàries, així com en biofilm de vinagre de maduixa. Es van detectar espècies i generes de bacteris acètics que no s’havien descrit prèviament en aquests nínxols ecològics. El disseny d’encebadors específics i sondes TaqMan-MGB en la regió del espaiador intergènic (ITS) entre els gens 16S-23S rRNA va permetre la detecció de dues espècies molt relacionades com són Acetobacter malorum i Acetobacter cerevisiae mitjançant l’ús de PCR en temps real. L’ús d’encebadors degenerats va fer possible la detecció de la presencia del gen que codifica la cel•lulosa sintasa i correlacionar aquest gen amb el fenotip de producció de cel•lulosa en bacteris acètics de diferents espècies. D’altra banda, l’escombratge funcional realitzat sobre el genoma de Komagataeibacter europaeus Gae02 va permetre la detecció de una proteïna, amb seqüència homòloga a la activitat prefenat deshidratasa, que va presentar activitat “quorum quenching”.El desarrollo de técnicas moleculares para la detección, identificación y tipificación de bacterias acéticas es la clave para una mejor comprensión de la coexistencia de esta microbiota relacionada con productos alimentarios. Se han aplicado diferentes técnicas dependientes e independientes de cultivo para la identificación y cuantificación de bacterias acéticas que se encuentran en uvas sanas y vino procedentes de las Islas Canarias, así como en biofilm de vinagre de fresa. Se detectaron especies y géneros de bacterias acéticas que no habían sido anteriormente descritos en estos nichos. El diseño de cebadores específicos y sondas TaqMan-MGB sobre la región del espaciador intergénico (ITS) entre los genes 16S-23S rRNA permitió la detección de dos especies muy relacionadas como son Acetobacter malorum y Acetobacter cerevisiae mediante PCR en tiempo real. El uso de cebadores degenerados hizo posible la detección de la presencia del gen que codifica la celulosa sintasa y correlacionar este gen con el fenotipo de producción de celulosa en bacterias acéticas de diferentes especies. Por otra parte, el screening funcional realizado sobre el genoma de Komagataeibacter europaeus Gae02 permitió la detección de una proteína, con secuencia homóloga a la actividad prefenato dehidratasa, que presentó actividad quorum quenching.The development of molecular techniques for detection, identification and typing of Acetic Acid Bacteria is the key for a better understanding of the coexistence of this microbiota in food products. Different culture-dependent and -independent techniques were applied for the identification and the enumeration of Acetic Acid Bacteria microbiota present on healthy grapes and in wine of the Canary Islands as well as in strawberry vinegar biofilm. Species and genera of AAB not previously reported in these niches were detected. The design of specific primers and TaqMan-MGB probes in the 16S-23S rRNA gene internal transcribed region was successfully performed for the detection of the closely related species of Acetobacter malorum and Acetobacter cerevisiae using Real-Time PCR. The use of degenerated primers allowed the detection of cellulose synthase gene and the correlation of this gene with the cellulose production phenotype in AAB strains of different species. On the other hand, the functional screening in the genome of Komagataeibacter europaeus Gae02 allowed the detection of one protein, homologous to prephenate dehydratase, with quorum quenching activity

Biophysical mechanisms of antimicrobial resistance in swarming B. subtilis

Swarms and biofilms are the two major modes of bacterial collectives and confer cells with emergent properties that lack as individuals, such as an increase in antibiotic tolerance. Swarming is a rapid type of surface colonization, and therefore its ability to withstand high antibiotic concentrations could lead to the subsequent establishment of high lyre- silient biofilms orgenetically resistant bacteria in regions that could not otherwise have been colonized. However, whether the development of biofilms or resistant microcolonies by swarms is possible is unknown. Using swarming Bacillus subtilis, we reveal that a biophysical mechanism, reminiscent of motility-induced phase separation (MIPS),under- pins a swarming-to-biofilm transition through a localized dynamic phase transition. This transition, triggered by an external stressor, is underpinned by a localized multilayer formation. Inspired by the thermodynamic properties of active matter, we demonstrated that such multilayer formation forms through a nucleation and growth process near an antibiotic gradient, and through spinodal decomposition in absence of stress. The nucle- ation and growth of multiple layers near the antibiotic, triggers waves of bacteria that move towards the antibiotic source, suggesting a novel mechanism of bacterial transport. When swarming to biofilm transition was prohibited by the environmental conditions, the swarm uses an alternative strategy to cope with the antibiotic gradient by developing re- sistant microcolonies. Quantification of this resistance displayed that the higher resistance to kanamycinis acquired together with resistance to other antibiotics targeting the same process. Inspired by the medical motivation of this project, we suggested solutions to both the emergence of the biofilm and the development of resistant bacteria by using the insight that we gained throughout the study. In particular, we proved that the biofilm formation can be reduced when splitting the total dose of antibiotics in two different time steps: the first triggers the multi layer formation and the second targets this key region in the swarm. Alternatively, when the swarm develops resistant colonies, we observed that these become more vulnerable to other drugs, so a tratement using certain sequence of antibiotics could be highly effective to kill multi drug resistant bacteria. These could lead to new strategies to tackle antimicrobial resistance

Study of cell-cell communication using 3D living cell microarrays

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Electrical Engineering and Computer Science, 2007.This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.Includes bibliographical references (p. 135-152).Cellular behavior is not dictated solely from within; it is also guided by a myriad of external cues. If cells are removed from their natural environment, apart from the microenvironment and social context they are accustomed to, it is difficult to study their behavior in any meaningful way. To that end, I describe a method for using optical trapping for positioning cells with submicron accuracy in three dimensions, then encapsulating them in hydrogel, in order to mimic the in vivo microenvironment. This process has been carefully optimized for cell viability, checking both prokaryotic and eukaryotic cells for membrane integrity and metabolic activity. To demonstrate the utility of this system, I have looked at a model "quorum sensing" system in Vibrio Fischeri, which operates by the emission and detection of a small chemical signal, an acyl-homoserine lactone. Through synthetic biology, I have engineered plasmids which express "sending" and "receiving" genes. Bacteria containing these plasmids were formed into complex 3D patterns, designed to assay signaling response. The gene expression of the bacteria was tracked over time using fluorescent proteins as reporters. A model for this system was composed using a finite element method to simulate signal transport through the hydrogel, and simple mass-action kinetic equations to simulate the resulting protein expression over time.by Winston Timp.Ph.D