University of Groningen Probiotic Bacteria and Their Encapsulation Evaluated in Advanced Co-culture Models Yuan, Lu

University of Groningen

Probiotic Bacteria and Their Encapsulation Evaluated in Advanced Co-culture Models

Yuan, Lu

DOI:

10.33612/diss.160691131

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Yuan, L. (2021). Probiotic Bacteria and Their Encapsulation Evaluated in Advanced Co-culture Models. University of Groningen. https://doi.org/10.33612/diss.160691131

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95

CHAPTER 5

General Discussion, Summary/Samenvattig/!"

96

GENERAL DISCUSSION

The gut microbiome plays an important role in the development of various disorders, such as inflammatory diseases, irritable bowel syndrome, cancer, diabetes, obesity, Alzheimer’s diseases, among others 1_{. The balance between the prevalence of healthy and pathogenic}

bacteria in the gut is therewith crucial in health and disease. In 1907 Elie Metchnikoff hypothesized that the consumption of lactic acid bacteria reduced the number of harmful microbes in the gut 2_{. Nowadays, consumption of probiotic bacteria by oral administration is}

used for the prevention and treatment of a variety of disorders considered to originate from a disbalance in the gut microbiome 3_{. Oral administration of probiotics is not trivial however,}

because during passage through the gastrointestinal tract with its low pH and high bile-salt concentration, the number of viable probiotic bacteria that reaches the gut is greatly reduced. Good evaluation methods for probiotic efficacy should mimic the in vivo situation and include tissue cells requiring protection, pathogenic and probiotic bacteria, all at the same time. Therefore, this thesis explored the use of two advanced co-culture models, the transwell and the microfluidic device, for the evaluation of probiotic bacteria for infection-control purposes and possible benefits of protective encapsulation of probiotic bacteria by surface-engineered artificial shells.

Co-culture Infection Models

Clinical trials have shown promising results of probiotics consumption 3_{, but clinical evaluation}

is difficult as probiotics are less powerful than many drugs and mostly applicable as life-style drugs. Therefore, advanced in vitro systems are required, particularly for mechanistic studies of proof of potential, mostly long-term, efficacy. First, we have chosen the transwell system, which has been widely applied in co-culture studies, to set up an intestinal epithelial infection model (Chapter 2). Although the transwell system possesses advantages such as high-throughput and ease of handling, the intestinal epithelial cell layers lack a villi structure due to the static conditions in a transwell. Villi are important intestinal structures for the absorption of nutrients. Therefore we have also set-up a microfluidic device (gut(organ)-on-a-chip model) to study Bifidobacterium breve protection of intestinal epithelial cell layers against challenges with pathogenic Escherichia coli (Chapter 3). Intestinal epithelial cells formed clear villi structures under dynamic flow conditions in a microfluidic device. Yet, microfluidic devices possessed also drawbacks, since alginate hydrogel encapsulated probiotic bacteria could not be perfused through a microfluidic device unless special precautions were taken (Chapter 4). Moreover, microfluidic devices are expensive and stable cell layers are time-consuming to prepare compared with their growth in transwell systems. Therefore, transwell systems are preferable

General discussion

97 for high-throughput initial screening after which a microfluidic device can be applied for more in-depth evaluation of promising candidates and possibly replacement of animal experiments4,5_.

Non-invasive Techniques to Study Co-culture Infections

Non-invasive methods are needed for continuous, real-time evaluation of probiotic efficacy on intestinal epithelial cell layers under pathogen challenge. For the transwell system, we have used widely applied, transepithelial electrical resistance (TEER) measurements for non-invasive evaluation of the intestinal epithelial barrier integrity. The measurement of the barrier integrity at different time points showed two-stages in TEER value changes. The two-stage TEER changes were explained as mild and severe damage to cells by bacterial challenges. Interestingly, the mild damage of the cell layer, i.e. during the stage of increasing TEER values, could be cured by antibiotic treatment. Another non-invasive method for gut-on-a-chip evaluation is optical coherence tomography (OCT) images of epithelial layers and villi structure real time. OCT had not yet been applied to gut(organ)-on-a-chip models before. Compared with TEER, OCT can be easily applied without opening of a microfluidic device or staining of the cells and bacteria and it can be used in high throughput. OCT can also be build-in the microfluidic device set-up 6 _{for online following the challenges with pathogenic bacteria,}

introducing antibiotics or regular probiotic introduction in the device. However, OCT does not provide high-resolution details of cell-bacteria interactions, but that may become possible with the ongoing development of high-resolution OCT 7,8_.

Encapsulation of Probiotics for Improved Efficacy

It is essential to maintain the viability of probiotics on their way to their intestinal target site in order to exert their beneficial effects on the host. However, the harsh environment that oral-administrated probiotics encounter as e.g. gastric acids, hamper arrival of viable probiotic bacteria in the gut. Generally, the capsules applied for oral-administration contain a high number of (freeze-dried) probiotics (equivalent to 109 _{– 10}10 _{CFU) and it is estimated that only}

20-40% survive during passage of the gastrointestinal tract 9,10_{. The survival rate of probiotics}

in feces of healthy, human volunteers after drinking a fermented dairy product supplemented with Lactobacillus GG, was only 0.1% 9,11_{. In order to protect probiotic bacteria from such a}

harsh environment, we have encapsulated B. breve to protect them against gastric acids and antibiotics. For encapsulation, different methods were selected, based on the different degrees of bacteria-shell interaction. Alginate encapsulation yielded more efficacious probiotic protection, pathogen killing and tissue protection than more modern nanobiomaterial encapsulations. Unfortunately, alginate encapsulated probiotics were too large to be used in a microfluidic device, constituting a severe drawback of microfluidic devices.

98

Mechanisms of Probiotic Protection against Pathogens

Mechanisms of probiotic protection include biosurfactant and bacteriocin production, competitive adhesion to intestinal epithelial cell layers, modulation of tight junctions and others, as summarized in this thesis 12_{. In combination with antibiotics, alginate encapsulated}

probiotics could inhibit growth of antibiotic-resistant pathogens and prevent damage to intestinal epithelial layers, without affecting the density of microvilli (Figures 1A and 1B). B.

breve encapsulated with a ZIF-8 metal-organic frame-work did not survive the antibiotic

presence and less microvilli (Figure 1C) were visible due to antibiotic-resistant, surviving pathogens.

FIGURE 1. Phalloidin-FITC (F-actin) stained intestinal epithelial cell layers (apical surface). (A) unchallenged cell layer, (B) cells co-cultured with B. breve@Alginate for 4 h, and challenged with

E. coli for 2 h followed by a tetracycline (10 µg mL-1_{) treatment for 3 h, and cultured in fresh culture} medium without antibiotics for 19 h. (C) The same as panel (B) but now co-cultured with B.

breve@ZIF-8. White arrows indicated the microvilli (green dots) on the apical side of intestinal

epithelial cells. The inserts represent a schematic drawing of the villi structure of the intestinal epithelial cells in the related panel.

Microvilli are important for absorption and secretory functions as e.g. absorption of nutrients including electrolytes and carbohydrates 13_{, but also contain many enzymes including}

sucrase and maltase for digestion of food 14_{. Therewith, the loss of the microvilli may lead to}

diarrhea 15_{, that we here demonstrate can be prevented using probiotic bacteria. Encapsulation}

of probiotic bacteria is particularly useful for the prevention of antibiotic-associated diarrhea 16_.

Our in vitro results are consistent with the clinical outcome that live bacteria showed better

General discussion

99 prevention of antibiotics-associated diarrhea than heat-killed probiotics 17 _{and attests to the}

value of the co-culture models used.

Future Research

The human intestinal epithelium is composed of seven different cell types with different functions including goblet cells producing mucins that prevent most gut bacteria from penetrating through the intestinal epithelial barrier and Paneth cells which are the main supplier of anti-microbial defensins 18_{. However, in our study, we only used enterocytes Caco-2 BBe,}

derived from colorectal adenocarcinoma which exhibit many of the characteristics of mature enterocytes but are deficient in mucus-secretion. For future investigations, in order to mimic the human intestinal epithelium better, a stem-cell-derived intestinal organoid-on-a-chip might be used for studying the mechanisms of host-microbiome interactions 19_{. As the host}

“permissiveness” or “resistance” to probiotics varies between individuals 20_{, it will be an}

advantage to use a patient’s own stem cells in order to develop a personalized gut-on-chip that can be used to collect personalized data for providing personalized instructions of probiotics-consumption to its users. Moreover, as the human intestinal lumen is an almost completely anaerobic environment 21,22 _{and many probiotics are anaerobic, a microfluidic device with an}

oxygen gradient for co-cultures with cells, probiotics and pathogens may be considered as well in future studies 23_.

Ideally, it is expected that orally-administered probiotics can survive passage through the gastrointestinal tract, establish themselves permanently in the small intestines and colon, and provide specific health benefits to the host by the production of biosurfactant, bacteriocins and short-chain fatty acids. However, many probiotics are unable to colonize host intestinal epithelial tissue 24_{. Therefore it is important to select bacteria that can adhere strongly to}

intestinal epithelium 2_{. Alternatively, the probiotic’s cell surface could be modified by}

surface-engineered encapsulating shells equipped with specific functionalities that enhance the adhesion of encapsulated probiotics to intestinal cell layers. Such functional groups might involve thiols 25_{, acrylates} 26_{, catechols} 27 _{or boronates} 28 _{to improve the muco-adhesiveness}

100

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mechanism mediated by the cooperative action of three enteropathogenic Escherichia coli-injected effector proteins. Proc. Natl. Acad. Sci. 103, 1876–1881 (2006).

16. Blaabjerg, S., Artzi, D. M. & Aabenhus, R. Probiotics for the prevention of antibiotic-associated diarrhea in outpatients- a systematic review. Antibiotics 6, 21 (2017).

17. Wenus, C. et al. Prevention of antibiotic-associated diarrhoea by a fermented probiotic milk drink. Eur. J. Clin. Nutr. 62, 299–301 (2008).

18. Dieterich, W., Schink, M. & Zopf, Y. Microbiota in the gastrointestinal tract. Med. Sci. 6, 116 (2018).

19. Richmond, C. A. & Breault, D. T. Move over Caco-2 cells: Human-induced organoids meet gut-on-a-chip. Cell. Mol. Gastroenterol. Hepatol. 5, 634–635 (2018).

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101 44, 3–12 (2017).

23. Jalili-Firoozinezhad, S Gazzaniga, F. S. et al. A complex human gut microbiome cultured in an anaerobic intestine-on-a-chip. Nat. Biomed. Eng. 3, 520–531 (2019).

24. Laparra, J. M. & Sanz, Y. Comparison of in vitro models to study bacterial adhesion to the intestinal epithelium. Lett. Appl. Microbiol. 49, 695–701 (2009).

25. Mun, E. A., Williams, A. C. & Khutoryanskiy, V. V. Adhesion of thiolated silica nanoparticles to urinary bladder mucosa: Effects of PEGylation, thiol content and particle size. Int. J.

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102

SUMMARY

It is expected that antimicrobial-resistant bacterial infection is becoming the main cause of death by the year 2050. New strategies for infection control are needed including the development of new antibiotics as well as of renewed strategies, such as the use of probiotics. In Chapter 1, we summarized the available in vitro co-culture models in 2D and 3D systems, their critical factors and useful outcomes in novel antimicrobial evaluations in order to address the question up to what extent co-culture models can replace animal evaluation of novel antibiotics and infection control strategies. Proper dosing of antimicrobials involves finding the balance between bacterial killing and collateral tissue damage, which inevitably arises when antimicrobials are over-dosed. In order to study the infection-prone, highly complex ecosystem of the human intestines, a well-defined co-culture system is needed. Therefore, the aim of this thesis was to explore the use of advanced co-culture models for the evaluation of probiotic bacteria for intestinal infection control purposes and possible benefits of their surface-engineered encapsulation.

In Chapter 2, we developed a two-stage interpretative model of the time-dependence of the TEER (transepithelial electrical resistance) of epithelial layers grown in a transwell system during Escherichia coli challenges in the absence or presence of adhering bifidobacteria. E. coli adhesion in absence or presence of adhering bifidobacteria was enumerated using selective plating. After 4-8 h, E. coli challenges increased TEER to a maximum due to bacterial adhesion and increased expression of a tight-junction protein (ZO-1), concurrent with a less dense layer structure, that was indicative of mild epithelial layer damage. Before the occurrence of a TEER maximum, decreases in electrical conductance (i.e. the reciprocal TEER) did not relate with para-cellular dextran-permeability but after occurrence of TEER maxima, dextran-permeability and conductance increased linearly, indicative of more severe epithelial layer damage. Within 24 h after the occurrence of a TEER maximum, TEER decreased to below the level of unchallenged epithelial layers demonstrating microscopically-observable holes and apoptosis. Under probiotic protection by adhering bifidobacteria, TEER maxima were delayed or decreased in magnitude due to later transition from mild to severe damage, but similar linear relations between conductance and dextran permeability were observed as in absence of adhering bifidobacteria. Based on the time-dependence of the TEER and the relation between conductance and dextran-permeability, it is proposed that bacterial adhesion to epithelial layers first causes mild damage, followed by more severe damage after the occurrence of a TEER maximum. The mild damage caused by E. coli prior to the occurrence of TEER maxima was reversible upon antibiotic treatment, but the severe damage after occurrence of TEER maxima could not be reverted by antibiotic treatment. Thus,

Summary

103 single-time TEER is interpretable in two ways, depending whether increasing to or decreasing from its maximum. Adhering bifidobacteria elongate the time-window available for antibiotic treatment to repair initial pathogen damage to intestinal epithelial layers.

The transwell system for co-cultures is simple and inexpensive, but has the disadvantage that intestinal cells cannot form villi structures under static conditions, which are of importance for the absorption of nutrients and secretion of enzymes. Therefore in Chapter 3, we have used a microfluidic device to set-up a gut-on-a-chip model, with dynamic flow in which intestinal epithelial cells develop villi-like structures. In a microfluidic device, we studied

Bifidobacterium breve protection of epithelial cell layers against pathogenic E. coli challenges.

Imaging of cell layers in a gut-on-a-chip systems in the literature has been confined to 2D-imaging through conventional light microscopy and confocal laser scanning microscopy (CLSM) yielding 3D- and 2D-cross-sectional reconstructions. However, CLSM requires staining and is unsuitable for longitudinal visualization. Here, we compare merits of optical coherence tomography (OCT) with those of CLSM and light microscopy for visualization of intestinal epithelial layers during protection by a probiotic B. breve strain and a simultaneous challenge by a pathogenic E. coli strain. OCT cross-sectional images yielded film thicknesses that coincided with end-point thicknesses from cross-sectional CLSM images. Light microscopy on histological sections of epithelial layers at end-point yielded smaller layer thicknesses than OCT and CLSM. Protective effects of B. breve adhering to an epithelial layer against an E. coli challenge included preservation of layer thickness and membrane surface coverage by epithelial cells. OCT does not require staining or sectioning, making OCT suitable for longitudinal visualization of biological films, but as a drawback, OCT does not allow an epithelial layer to be distinguished from bacterial biofilms adhering to it. Thus, OCT is ideal to longitudinally evaluate epithelial layers under probiotic protection and pathogen challenges, but proper image interpretation requires application of a second method at end-point to distinguish bacterial and epithelial films.

B. breve has been shown to protect intestinal cells (Chapter 2 and Chapter 3), against

pathogenic E. coli challenges. However, oral administration of probiotics can severely hamper their viability when passing the gastro-intestinal tract during the exposure to acidic conditions. In Chapter 4, weencapsulated probiotic bacteria to enhance their functionality, when using probiotics in combination with antibiotics for treating intestinal infections. The interaction strength of encapsulating shells with bacterial cell surfaces however, varies amongst different encapsulation methods and may impact the efficacy of encapsulation. We compared the protection offered by encapsulating shells with different interaction strength towards probiotic

B. breve against simulated gastric fluid and tetracycline, including protamine-assisted SiO2

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(intermediate-interaction due to hydrogen binding) and ZIF-8 mineralization (strong-interaction due to coordinate covalent binding). Presence of encapsulating shells was demonstrated using X-ray photoelectron spectroscopy and particulate microelectrophoresis. Strong-interaction upon ZIF-8 encapsulation caused demonstrable cell wall damage to B. breve and reduced bacterial viability. Cell wall damage and reduced viability did not occur upon encapsulation with weakly-interacting yolk-shells. Only alginate-hydrogel-based shells yielded protection against simulated gastric acid and tetracycline. Accordingly, only alginate-hydrogel encapsulated B.

breve operated synergistically with tetracycline in killing tetracycline-resistant E. coli adhering

to intestinal epithelial layers and maintaining surface coverage of transwell membranes by epithelial cell layers and their barrier integrity. This synergy between alginate-hydrogel encapsulated B. breve and an antibiotic warrants further studies for treating antibiotic-resistant

E. coli infections in the gastro-intestinal tract.

In the general discussion (Chapter 5), the results of this thesis were discussed as specifically related with the aim of this thesis and suggestions for future research are proposed.

Samenvattig

105

SAMENVATTING

Verwacht wordt dat in 2050 de meeste sterfgevallen wereldwijd te wijten zullen zijn aan infecties veroorzaakt door antibiotica-resistente bacteriën. Daarom zijn nieuwe strategieën voor het voorkomen en behandelen van infecties nodig, inclusief nieuwe antibiotica. Echter ook oude gebruiken, zoals het gebruik van probiotica, dienen wederom ingezet te worden. Het meeste onderzoek naar nieuwe strategieën richt zich op het bestrijden van pathogenen, zonder naar de nevenschade op weefsels te kijken. Het samenspel tussen weefselcellen, bacteriën en antibiotica in zogenaamde co-cultuurmodellen is vaak onderbelicht. In Hoofdstuk 1 hebben we beschikbare in vitro co-cultuurmodellen in zowel 2D- als 3D-systemen samengevat en geëvalueerd of co-cultuurmodellen dierstudies voor het testen van nieuwe antimicrobiële strategieën kunnen vervangen.

Om het complexe ecosysteem in het menselijke maag-darm kanaal te onderzoeken zijn goed gedefinieerde co-cultuurmodellen nodig. In geval van darminfecties, zijn een goede keuze en dosering van antibiotica belangrijk, hetgeen inhoudt dat er een balans moet zijn tussen bacteriedodende effecten en het beperken van schade aan omliggende weefsels en aan de gezonde microflora van de darm. Dat laatste is echter vaak onvermijdelijk.

Het doel van dit proefschrift was om te onderzoeken of geavanceerde co-cultuurmodellen gebruikt kunnen worden om het effect van probiotische bacteriën op het voorkomen en behandelen van maag-darm infecties te evalueren. Tevens werd onderzocht of beschermende laagjes om probiotische bacteriën een voordeel op zou kunnen leveren om het maag-darm kanaal te passeren tijdens antibiotica behandeling.

In hoofdstuk 2 hebben we een model gepresenteerd dat de tijdsafhankelijkheid van de elektrische weerstand van darm-epitheellagen beschrijft gedurende infectie en de behandeling van infectie met behulp van probiotica (transepithelial electrical resistance; TEER). TEER is een indicator voor de integriteit van epitheellagen die verandert naarmate de cellen blootgesteld worden aan pathogenen. Er is een transwellsysteem gebruikt om epitheellagen te produceren. Een transwellsysteem is een systeem waarin twee compartimenten gescheiden worden door een membraan waarop in dit geval een epitheellaag gekweekt is. Deze epitheellaag wordt vervolgens blootgesteld aan Escherichia coli al dan niet in aanwezigheid van probiotische bifidobacteriën. Na vier tot acht uur blootstelling aan E. coli bereikte de TEER waarde een maximum. Het maximum werd geïnitieerd door hechting van bacteriën aan de epitheelcellen en door een verhoogde expressie van het ‘tight-junction’ eiwit (ZO-1). Tegelijkertijd leidde dit tot een minder dichte structuur van de epitheellaag. Dit laatste is indicatief voor schade aan de epitheellaag. Een parameter om de barrièrefunctie van de

106

epitheellaag te evalueren is de dextran doorlaatbaarheid van de laag. Vóórdat de maximale TEER bereikt wordt, lijkt het erop dat een afname in elektrische geleiding niet correspondeert met de doorlaatbaarheid van dextran, terwijl na het bereiken van de maximale TEER, deze parameters tegelijkertijd lineair toenemen. Dit betekent dat er ernstige schade aan het epitheel is aangebracht. Tijdens de eerste 24 uur na het bereiken van de maximale TEER, daalde de TEER waarde tot onder het niveau van onaangetaste epitheellagen en waren er daadwerkelijk gaten en dode cellen in de epitheellaag te zien. Om de epitheellaag te beschermen tegen pathogene bacteriestammen kunnen probiotische bifidobacteriën gebruikt worden. Deze bacteriën vertragen het bereiken van de maximale TEER of verlagen het maximum, terwijl de lineaire verbanden tussen elektrische geleiding en doorlaatbaarheid van dextran aanwezig blijven. Gebaseerd op de tijdsafhankelijkheid van TEER en de relatie tussen de geleiding en doorlaatbaarheid van dextran, stelden we dat bacteriële aanhechting aan epitheellagen eerst milde schade toebrengt, om later, na het bereiken van een maximale TEER, ernstige schade toe te brengen aan de epitheellaag. Het stadium van milde schade voor het bereiken van de maximale TEER waarde, bleek reversibel door behandeling met antibiotica. De ernstige schade, na het bereiken van een maximale TEER waarde, kon echter niet meer teruggedraaid worden. We concluderen daarom dat de TEER waarden tweevoudig interpreteerbaar zijn. De toename naar het maximum en afname vanaf het maximum vertegenwoordigen twee verschillende stadia van schade ten gevolge van infectie. Aangehechte bifidobacteriën spelen een belangrijke rol doordat ze de tijd tot het bereiken van een maximale TEER waarde verlengen, zodat er meer tijd is om een antibiotica behandeling in te zetten en ernstige schade aan de epitheellagen te voorkomen.

Het transwellsysteem voor co-culturen is simpel en goedkoop, maar er zit een belangrijk nadeel aan. Darmcellen karakteriseren zich met uitstulpingen (darmvlokken) die belangrijk zijn voor het absorberen van nutriënten en het afscheiden van enzymen. Bij het kweken van darmcellen in transwellsystemen ontstaan niet de karakteristieke uitstulpingen, mogelijk door het ontbreken van stroming. Om deze reden hebben we in Hoofdstuk 3 een microstroomkamer gebruikt om een ‘darm-op-een-chip model’ te maken. Hierin kunnen we voldoen aan de stromings-condities die nodig zijn voor het aanmaken van uitstulpingen door de darmepitheelcellen. In deze ‘darm-op-een-chip’ hebben we gekeken naar de bescherming van darmepitheelcellen door Bifidobacterium breve tegen E. coli kolonisatie. Het is echter lastig om een complexe 3D-structuur bestaande uit cellagen, bedekt met probiotische dan wel pathogene bacteriën te visualiseren. Standaard worden cellagen in microstroomkamers met conventionele lichtmicroscopie of met ‘confocal laser scanning microscopy’ (CLSM) gevisualiseerd. CLSM, en vaak ook conventionele lichtmicroscopie, kunnen echter niet longitudinaal als functie van de tijd toegepast worden. In dit hoofdstuk vergeleken we de

Samenvattig

107 voordelen van ‘optical coherence microscopy’ (OCT) met die van CLSM en conventionele lichtmicroscopie om de darmepitheellagen te bekijken, terwijl ze enerzijds beschermd werden door de probiotische B. breve en anderzijds op de proef gesteld werden door E. coli. OCT dwarsdoorsneden leverden dezelfde laagdiktes op als CLSM dwarsdoorsneden, maar histologische secties van de cellaag met gehechte bacteriën leverden dunnere laagdikten op dan de OCT en CLSM. Door deze technieken te combineren konden we laten zien dat probiotisch B. breve een dusdanig beschermend effect op de epitheellaag heeft dat de epitheellaag ongeveer even dik blijft. Terugkomend op de beeldvormende technieken heeft OCT de voorkeur voor longitudinale visualisatie, omdat er geen coupes gemaakt hoeven te worden en ook kleuring niet nodig is. OCT-beelden kunnen echter geen onderscheid maken tussen epitheellagen en de aanhechtende bacteriële laag. Het is dus aan te raden om longitudinaal een epitheellaag te volgen met behulp van OCT, maar voor een volledige interpretatie van de beelden zijn meerdere beeldvormende technieken vereist. Hiertoe volstaat echter een analyse “at end-point”.

We hebben in Hoofdstuk 2 en Hoofdstuk 3 laten zien, dat B. breve darmepitheelcellen kan beschermen tegen pathogene E. coli kolonisatie. Doordat probiotica via de mond in het maag-darm kanaal terecht komen, en daarmee de zure omgeving van de maag moeten overleven, behoeven ze een beschermend laagje. Zo’n laagje is ook noodzakelijk om ze te beschermen tijdens een antibiotica behandeling, die ook de gezonde darmflora en probiotische bacteriën kan doden. In Hoofdstuk 4 hebben we probiotische bacteriën op verschillende manieren in een laagje verpakt om ze te kunnen beschermen tegen maagzuur. Zo kunnen de gezondheidsvoordelen van probiotica behouden blijven tijdens antibiotica behandeling van darminfecties. Echter, sterke interacties tussen de probiotica met de beschermende laagjes kunnen de probiotica mogelijk inactiveren. De sterkte van interactie hangt van de materialen af waarmee het laagje gemaakt wordt, en daarmee bepaalt het materiaal de uiteindelijke werkzaamheid. Zodoende hebben we verschillende beschermende laagjes vergeleken om te bepalen welke de beste bescherming biedt aan de probiotische B. breve tegen gesimuleerd maagzuur en tetracycline, een antibioticum. We hebben verschillende beschermende laagjes onderzocht, variërend van laagjes met een zwakke interactie tot een sterke interactie met de probiotica. De aanwezigheid van de beschermende laagjes werd aangetoond met ‘X-ray photoelectron spectroscopy’ en micro-elektroforese. Het laagje dat de sterkste interactie met de B. breve had, maakte de celwand van de probiotische bacteriën kapot. Dit leidde tot verminderde overleving van B. breve. Het laagje dat de zwakste interactie had met de probiotische bacteriën had dit effect niet. Echter, alleen het alginaat hydrogel laagje, met een middelmatig sterke interactie, was resistent tegen maagzuur en tetracycline. Het alginaat hydrogel laagje met daarin de probiotische B. breve in combinatie met tetracycline werd

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toegepast in het in Hoofdstuk 2 beschreven transwellsysteem. Het bleek, dat deze combinatie werkte om tetracycline-resistente E. coli van de darmepitheelcellen te doden, en zodoende de integriteit van de epitheellaag te behouden. B. breve met een beschermend alginaat laagje en tetracycline werkten derhalve synergistisch in de behandeling van antibiotica-resistente E. coli-infecties in het maag-darm kanaal.

In de algemene discussie (Hoofdstuk 5) worden de conclusies van de verschillende hoofdstukken in samenhang en met betrekking op het doel van dit proefschrift besproken. Ook worden voorstellen voor toekomstig onderzoek gedaan ter behandeling van antibiotica-resistente bacteriële infecties in het maag-darmsysteem.

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Dear friends and readers, finally at this page; I still remember the first day I was in the Biomedical Engineering Department and how it felt. Everything was new and exciting, even the coffee machine seemed to be exciting☺ Thank you, from the bottom of my heart to the lovely BMEers and other people outside of the department over the past years for making my Ph.D. journey an unforgettable experience.

Dear Henny, my first promotor, thank you so much for the help on my papers and thesis, as well as help me to build up the professional working styles to work precisely, although still a long way for me to go, but here I started. I am so grateful for your suggestions and encouragements in our meetings, always being able to answer my questions despite your busy schedules. I really appreciated that you shared your passion, experience and ideas on research and life. I learned the way you supervised and helped me, which I hope I can provide to my students in the future.

Dear Henk, thank you for offering me the opportunity to study in Groningen. I am really grateful for your mentoring and supervision for my Ph.D. I really appreciate that you encouraged me to think critically, to organize the results coherently, and to present them in an interesting way, which will benefit along with my career. Also, thank you for organizing the English classes in the BME department to help the communication and understanding with fewer language barriers. I cannot forget the running events that we run together in Wuhan and many in Groningen, a lot of fun, and my sweet memories in Groningen.

Dear Brandon, thank you for helping me with the statistical analysis of the results. I cannot forget how many times I knocked on your door and discussed experiments and results. Also, thank you for the suggestions on giving a presentation, as well as many beers at your home and the Paterswoldsemeer barbecues every summer.

I would like to express my very warm gratitude to the reading committee. Prof. Yijin Ren, I am so happy that you accepted to read my thesis, and thank you for inviting me to be part of the editorial board of Kolff newspaper. Prof. Paul de Vos and Prof. Yunwei Wei, thank you for taking the time to read and review my thesis.

I am very grateful to the people in Pharmacy Department for their help and kindness that I have had a happy time over there. Sabeth, thank you for your generosity to allow me to work in your laboratory. Pim, thank you for your help and suggestions on the gut-on-a-chip project; without your help, the project cannot proceed smoothly. Thank you, JP and Patty for helping me out so many times in the lab.

Hao, thank you for the nice discussions and help on chapter 4. Also the hard work for the last chapter, especially in the year 2020 with a lot of uncertainties with traveling.

Acknowledgements

113

Inge, thank you for the suggestions on cell biology. I am grateful to the entire scientific staff, including Patrick, Prashant, Romana, Theo, Chris, Jelmer for the scientific discussion in the lunch meeting as well as the coffee-break chats.

Yangyang, I am so happy that I met you in Groningen, a lot of happy memories that we lived together, took care of Xiaohei, biked around Groningen, and joined many running events together. Happy memories we traveled to Croatia, enjoyed the delicious local foods and sunshine.

Huaiying, my dear hometown girl, thank you for taking care of Xiaohei during the holidays when we were in Croatia. I am really grateful for your comfort during the tough days and all the supports over the years in Groningen; also many thanks to Hao for the suggestions on the job searching in China.

Valentina, thank you for the support and help over the past years inside of the BME department and outside. We run Astrea team-marathon together, 4mijl Groningen, Dutch class, and much more. All these experiences brought us together and I am lucky to have you around. Jeroen, thank you for helping me with the Dutch summary, also the introduction of birds to the beginner, still remember the first time I saw the starring feather under sunshine. Special thanks for these unforgettable memories of “working” on the farm of Jan.

Gwenda, thank you for motivating me to run (many many times) and sharing many useful suggestions on searching for a postdoc as well as the useful tips to get rid of hurt during the running.

Special thanks for the cat-lovers, Aryan, thank you for sharing your knowledge on cats and bringing me the nice cookies and croissant. Simon, thank you for helping me on finding and bring a cat back home, as well as all the fun and relaxing moments with your sense of humor. Thanks to my wonderful neighbours Guangyue and Jingjin for all the supports and help during Covid time; life was wonderful to live near you two. Hongping, thank you for the warm words and encouragements, also happy BBQ/dinner time together with you and Xinghong; we can enjoy the delicious food together back to Chengdu in the near future. I cannot forget the kindness and warmth from Bingran and Feifei for taking care of me when I was sick, and helping me to move around in Groningen. Thank you, Yong for the help and suggestions on my projects, as well as many scientifc and non-scientfic chats. Chen, thank you so much for the help on my first year in Groningen. Yuchen, thank you for the kindness to be the photographer during AstreaRun and many other events. Lei, thank you for giving so many tips on studying and living in Groningen. JP, thank you for offering me a room to stay in the first half-year in Groningen. Zhiwei, thank you for taking care of Xiaohei when I was in China. I also

114

had so much fun during lunchtime with Ke, Lei, Chengxiong, Yuanfeng, Qihui, Yiruo, Kecheng, Ruifang, Yafei, Linzhu, thank you for sharing these delicious foods, recharging me with new energy to start working again. Also, many thanks to my officemates Xiaoxiang and Liang, many nice talks together in the office over the years; and thank you, Liang, for helping me moving into the new apartment.

Special thanks for the accompany during the traveling time. Can, a happy to travel in Glasgow and Edinburg, also many happy times we biked together with Derly at the beginning of our Ph.D. Yanyan, thank you for your support over the past years; and the trip together in the Crete; Damla, Mari, Luciani and Nathali, what a wonderful memory and a beautiful pink beach.

I would also express my gratitude to the people accompany during the coffee break and help in the lab: Torben, Abby, Maria, Olga, Lu, Linyan, Kaiqi, Zhengya, Yiwen, Runrun, Yue, Yiyang, Chuang, Neda, Lina, Lais, Thea, Tjitze, Colin, Rene, Vera, Rebecca, Thais, Matteus, Yori, Kiran, Alina, Thamir, Alejandro. Thanks for sharing the stories with me, I learned a lot about different countries and cultures.

Ina, thank you for the kindness and warmth in the BME department. Wya, thank you for helping with the financial forms, and wish you all the best after retirement in the near future.

Over the past years, I am helped out countless times by our technical staff: Reinier, thank you for all the help inside and outside of the department, useful suggestions on drawing the schematic figures, and funny jokes. Willem, thank you for these nice suggestions and discussion on the projects, and always helped me out in the lab. Ed, thank you for the help in OCT image processing; I cannot forget how many versions of Thorlabs I have. Hans, thank you for helping me with ADSAP and DLS measurements, and kindly explained the details to me. Willy, thank you for the help on chapter 4, also the introduction of lekker oliebollen. Minie, thank you for the training on cell culture and the introduction of your beautiful garden. Betsy, thank you for all the help on the ordering. Gesinda, thank you for the help on the OCT measurement. Joop, thank you for the introduction of AFM. Corien, thanks for your introduction and help on the microtome. Jelly and Marja, dank jullie wel dat Nederlands met me hebben spreken; veel plezier.

Over the past few years, I am very happy to be involved in the projects of undergraduates and masters. Really happy to work with these young students: Rebecca, Famke, Alienke, Levi, Nienke and Samuel, thanks for these nice memories in the lab.

Acknowledgements 115

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