Role of Intestinal Microbiota in Ulcerative Colitis
Role of Intestinal Microbiota in Ulcerative Colitis Role of Intestinal Microbiota in Ulcerative Colitis
Methodology part 6. Methodology, considerations and choices time above 24 hours is not recommended, since fermentation products are not removed in batch systems, they can cause inhibition and result in a decline of microbial population and death of bacteria (Coles et al., 2005). Inocula The samples used as inocula in in vitro fermentation systems are often fresh feces to insure viable bacteria cells. However, in the in vitro fermentation studies performed in this thesis, frozen fecal samples stored in glycerol (25%) were used (Paper 2‐6). The use of frozen samples was due to different delivery days of samples from volunteers prior to the in vitro experiments. Use of frozen samples in in vitro fermentation studies has previously been validated by Rose et al. (2010). The study showed that during 150 hours of fermentation in a continuous system, the SCFA profiles were the same for samples originated from fresh or frozen feces. Furthermore, Rose et al. (2010) demonstrated that the composition of the microbiota in terms of viable cells was unaffected by freezing. This was in line with a previous study by Crowther (1971), who demonstrated that the viability of Gram‐negative bacteria and Gram‐positive bacteria was the same, no matter if the feces were frozen in glycerol or fresh. Validation of the in vitro method When using an in vitro system for screening, it has to be considered robust and repeatable, hence it needs to demonstrate reproducibility within reasonable limits of statistical variation (Coles et al., 2005). Thus, repeatable in vitro experiments were conducted using the parameters mentioned above prior to the screening of prebiotic candidates described in the experimental part of this thesis. In vitro fermentations were carried out at five different occasions in an anaerobic cabinet (containing 10% H2, 10% CO2, and 80% N2) using frozen fecal samples as inocula from three healthy subjects and FOS (5 g/l) as substrate in minimal medium. Each fermentation experiment of each subject was carried out in triplicates. After 24 hours of incubation, fermentation samples were taken out for DNA extraction, and levels of Bacteroidetes, Firmicutes, bifidobacteria and lactobacilli were quantified using quantitative Real‐Time PCR (qPCR). The results demonstrated no significant difference in the levels of bacterial groups at the different occasions (Appendix 1). This implied that the fermentation set up was reproducible with a minimum of inocula from three different subjects. However, due to high inter‐individual variation, additional subjects were added 36
Methodology part 6. Methodology, considerations and choices to the system, if the amount of testing compound was sufficient. The in vitro fermentation procedure is further described in the following manuscripts; Paper 3 ‐ 6. 6.3. Continues systems Dynamic in vitro digestion methods are suited models to study the microbial ecology of the gastrointestinal tract. One of the most known physio‐chemical models is the validated Simulator of the Human Intestinal Microbial Ecosystem (SHIME). This model is multi‐compartmental and consists of a series of 5 vessels, simulating the stomach, small intestine and the three colon regions; ascending, transverse and descending (Molly et al., 1993). Hence, measurements of fermentation activity and composition of the colon microbial community in the different colonic regions can be performed (Yoo and Chen, 2006). The SHIME is unable to simulate selective absorption of metabolites and fluids, and can only simulate the growth of the luminal bacteria, whereas the adhesion of bacteria to mucus is omitted. However, Abbeele et al. (2011a) have recently developed a dynamic in vitro gut model incorporating mucin‐covered microcosms into the SHIME. This model named M‐SHIME simulates both the lumen and mucus environment, and allows studying the mucosal microbiota and interaction between luminal and mucosal microbial communities. Other in vitro models have previously been used to screen the adhering potency of intestinal microbes. They include adhesion assays to various components of the intestinal surface: e.g. intestinal mucus (Ouwehand et al., 2002b), mucins (Van den Abbeele et al., 2009), colonic tissue (Ouwehand et al., 2002a) or cell lines (Laparra and Sanz, 2009). However, they often only provide short‐term information regarding axenic cultures and tend to ignore the microbial interaction between and within the luminal and mucosal microbiota. The M‐SHIME set up used in the experimental part of this thesis consisted of two vessels simulating the stomach and the small intestine, and six ascending colon vessels, which were run in parallel without the transverse and descending colon. All ascending colon vessels were modified by incorporating mucin‐covered microcosms into the luminal suspension. The procedure for the M‐SHIME is described in details in Paper 2. 6.4. DNA extraction DNA extraction is a key factor affecting any approach for analyzing microbial diversity. Several methods have been reported for the isolation of microbial DNA from human stool samples (Machiels et al., 2000;Clement and Kitts, 2000;Zhang et al., 2006). However, problems can arise in 37
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Methodology part<br />
6. Methodology, considerations and choices<br />
time above 24 hours is not recommended, s<strong>in</strong>ce fermentation products are not removed <strong>in</strong> batch<br />
systems, they can cause <strong>in</strong>hibition and result <strong>in</strong> a decl<strong>in</strong>e <strong>of</strong> microbial population and death <strong>of</strong><br />
bacteria (Coles et al., 2005).<br />
Inocula<br />
The samples used as <strong>in</strong>ocula <strong>in</strong> <strong>in</strong> vitro fermentation systems are <strong>of</strong>ten fresh feces to <strong>in</strong>sure viable<br />
bacteria cells. However, <strong>in</strong> the <strong>in</strong> vitro fermentation studies performed <strong>in</strong> this thesis, frozen fecal<br />
samples stored <strong>in</strong> glycerol (25%) were used (Paper 2‐6). The use <strong>of</strong> frozen samples was due to<br />
different delivery days <strong>of</strong> samples from volunteers prior to the <strong>in</strong> vitro experiments. Use <strong>of</strong> frozen<br />
samples <strong>in</strong> <strong>in</strong> vitro fermentation studies has previously been validated by Rose et al. (2010). The<br />
study showed that dur<strong>in</strong>g 150 hours <strong>of</strong> fermentation <strong>in</strong> a cont<strong>in</strong>uous system, the SCFA pr<strong>of</strong>iles<br />
were the same for samples orig<strong>in</strong>ated from fresh or frozen feces. Furthermore, Rose et al. (2010)<br />
demonstrated that the composition <strong>of</strong> the microbiota <strong>in</strong> terms <strong>of</strong> viable cells was unaffected by<br />
freez<strong>in</strong>g. This was <strong>in</strong> l<strong>in</strong>e with a previous study by Crowther (1971), who demonstrated that the<br />
viability <strong>of</strong> Gram‐negative bacteria and Gram‐positive bacteria was the same, no matter if the<br />
feces were frozen <strong>in</strong> glycerol or fresh.<br />
Validation <strong>of</strong> the <strong>in</strong> vitro method<br />
When us<strong>in</strong>g an <strong>in</strong> vitro system for screen<strong>in</strong>g, it has to be considered robust and repeatable, hence<br />
it needs to demonstrate reproducibility with<strong>in</strong> reasonable limits <strong>of</strong> statistical variation (Coles et al.,<br />
2005). Thus, repeatable <strong>in</strong> vitro experiments were conducted us<strong>in</strong>g the parameters mentioned<br />
above prior to the screen<strong>in</strong>g <strong>of</strong> prebiotic candidates described <strong>in</strong> the experimental part <strong>of</strong> this<br />
thesis. In vitro fermentations were carried out at five different occasions <strong>in</strong> an anaerobic cab<strong>in</strong>et<br />
(conta<strong>in</strong><strong>in</strong>g 10% H2, 10% CO2, and 80% N2) us<strong>in</strong>g frozen fecal samples as <strong>in</strong>ocula from three<br />
healthy subjects and FOS (5 g/l) as substrate <strong>in</strong> m<strong>in</strong>imal medium. Each fermentation experiment <strong>of</strong><br />
each subject was carried out <strong>in</strong> triplicates. After 24 hours <strong>of</strong> <strong>in</strong>cubation, fermentation samples<br />
were taken out for DNA extraction, and levels <strong>of</strong> Bacteroidetes, Firmicutes, bifidobacteria and<br />
lactobacilli were quantified us<strong>in</strong>g quantitative Real‐Time PCR (qPCR). The results demonstrated no<br />
significant difference <strong>in</strong> the levels <strong>of</strong> bacterial groups at the different occasions (Appendix 1). This<br />
implied that the fermentation set up was reproducible with a m<strong>in</strong>imum <strong>of</strong> <strong>in</strong>ocula from three<br />
different subjects. However, due to high <strong>in</strong>ter‐<strong>in</strong>dividual variation, additional subjects were added<br />
36