Gastrointestinal microbiology - part 7 ppsx

different chemostat- and non-chemostat-type models have major structural differences, but the batch fermentors are generally similarly structured, small-scale bottle fermentors. The chemostat models can be run using inocula in either an in vitro steady-state (the exponential growth of the bacterial has stabilized) achieved with several days of pre- fermentation of the fecal inoculum or after a short (16-24 hours) pre-fermentation. Batch-Type Simulators The simplest and most commonly used in vitro method in microbiological studies is the use of batch fermentation with intestinal fluid or fecal slurry to study the effects of different added ingredients. These chemostat are typically anaerobically sealed bottles with fecal, caecal or rumen material and these models simulate only a certain part of the animal’s GIT, e.g., mouse cecum or cow’s rumen. The transit times of the intestinal fluids through those areas are relatively short and therefore the run-times in batch fermenting simulations range from 2–24 hours (3–7). The accumulation of fermentation products (e.g., SCFAs) can change the conditions in the batch fermentation from the microbially balanced starting point to a more competitive environment for the fermentative microbiota, thus affecting the in vivo relevance in longer simulations. More complex fermentation models with several vessels and fluid transitions between vessels either continuously or semi-continuously avoid this accumulation of metabolites and depletion of nutrients. Chemostat-Type Simulators The in vitro colon simulators were introduced for the first time in 1981 (8), and all models functioning today have a lot in common with this model. Rumney and Rowland reviewed the first decade of in vitro simulators in their excellent article (3). Of the models reviewed by Rumney and Rowland, the Reading model introduced by Gibson and co-workers in 1988 (9), revised 1998 by Macfarlane and co-workers (10), is still actively being used and two new interesting models have been described in the literature. Of these, the SHIME (Simulator for Human Intestinal Microbiological Ecosystem) model introduced by Molly et al. in 1993 (11) and the EnteroMix w colon simulator introduced by Ma ¨ kivuokko et al. Figure 1 The Reading model. This model represents the human colon in three vessels: V1 proximal, V2 transverse, and V3 distal colon. Media is pumped to system continuously, and at the same time there is a continuous overflow from vessel to vessel. Source: From Ref. 9. Ma ¨ kivuokko and Nurminen238 in 2005 (12), together with the Reading model, are structurally chemostat models having 3–6 sequentially attached fermenting vessels with computer controlled fluid transition systems (Fig. 1) and (Table 1). The Reading model and the EnteroMix w model both simulate only the human colon, and a similar artificial simulator media described by Macfarlane et al. (10) is used in them to simulate the fluid entering the colon from the small intestine. The SHIME model simulates the whole human GIT from stomach to colon using artificial SHIME media, which has much in common with the medium described by Macfarlane and co-workers (10). These three models have three different designs in fluid transition. Fluids are either pumped semi-continuously to the subsequent vessels in three- hour intervals (EnteroMix w model), there is a continuous overflow of fluids between vessels (the Reading model), or the model can be a combination of these two types (SHIME). Reading Simulator The Reading simulator (Fig. 1) simulates the gut using a 3 stage continuous culture with three glass vessels (220 ml, 320 ml and 320 ml) and different pH in each vessel (5.8, 6.2, and 6.8); mimicking the human proximal, transverse, and distal colon, respectively. In the beginning of the simulation, each vessel is inoculated with 100 ml of 20% (wt/vol) of human feces. The system is incubated in a batch overnight, after which a continuous pumping of fresh simulator fluid to the first vessel is started. At the same time a continuous overflow from vessel to vessel begins and the system is run for at least 14 days in order achieve a steady-state condition in the vessels. The excess fluid from the third vessel is collected to a waste container. The total retention time of the system can vary, e.g., between 27 and 67 hours (10). The viability of the microbiota is determined by taking samples at regular intervals from the vessels. After the incubation period, the test substance is added to the system mixed in the fresh simulation fluid and the system is then run to new steady state [e.g., for 22 days (9)]. The last phase is the washout period [e.g., for 50 days (9)] with the original simulation fluid to determine how long the changes induced by the test substance can still be measured in the absence of the substrate itself. SHIME Model The current SHIME model is a single six-stage system, where the first three glass vessels simulate stomach and small intestine and the subsequent three glass vessels the large intestine (11a). The original SHIME model (Fig. 2) (11) was a single five-stage system without the stomach compartment. Working volumes in these vessels are 300 ml for stomach and small intestine, 1000 ml for ceacum and ascending colon, 1600 ml for Table 1 Colon Simulator Models Reading SHIME EnteroMix w TIM 1 TIM 2 Simulation area Colon Stomach to colon Colon Stomach to ileum Colon Vessel volumes 220–320 ml 300–1600 ml 6–15 ml 200 ml 200 ml pH levels 5.8–6.8 5.0–7.0 5.5–7.0 1.8–6.5 5.8 Running times 14 days to steady state 30 days per cycle 2 days w1 day w3 days Abbreviations: SHIME, Simulator for Human Intestinal Microbiological Ecosystem; TIM, TNO Intestinal Model. In Vitro Methods to Model the Gastrointestinal Tract 239 transverse colon, and 1200 ml for descending colon. pH is controlled in vessels 2, 3, 4, 5, and 6 in the ranges 5.0–6.5, 6.5–7.0, 5.5–6.0, 6.0–6.5 and 6.5–7.0, respectively. The system is inoculated by introducing 10 ml supernatant of a human western diet suspension per day to the three first vessels for eight successive days. The remaining three vessels 4–6 representing the different compartments of the colon are inoculated with 50 ml of fecal suspension for 10 successive days. The contents of these three vessels are pumped continuously from vessel to vessel and finally to a discard bottle. The transit time of the whole system is 84 hours. In the beginning of the simulation, 200 ml of fresh SHIME media (11) is added to vessel 1 (stomach) three times per day. Every 2–3 hours, the acidic (pH 2.0) contents of the first vessel is pumped to vessel 2 (duodenumCjejunum) along with 100 ml of pancreatic juice, supplemented with bile, to neutralize the acidity of the gastric effluent. After four hours the contents of vessel 2 is pumped to vessel 3 (ileum). After eight days of using SHIME media only, the actual test substrate mixed with the SHIME media is introduced to the system. Feeding of the substrate is continued for 12 days, followed by another SHIME media-only period for 8–10 days. This cycle of three periods is repeated for all the studied substrates and samples are taken after each period. The EnteroMix w Colon Simulator The EnteroMix w model (Fig. 3) has four parallel units each comprising four glass vessels, allowing four simulations to be run simultaneously using the same fecal inoculum (12). EnteroMix w model vessels 1, 2, 3, and 4 have the smallest working volumes (6, 8, 10, and 12 ml, respectively) of the three models presented here (Table 1). The pH levels in the vessels (5.5, 6.0, 6.5, and 7.0, respectively) are similar to the other models. Because of the small volumes of vessels, a 40 ml inoculum of 25% wt/vol human feces and only 4 g of test substrate is needed for four parallel 48-hour simulations. The simulation begins by filling the vessels of each of the four units with 0.9 mM anaerobic NaCl (3, 5, 7, and 9 ml to vessels 1, 2, 3, and 4, respectively) and inoculating the Effluent pump pump pump pump pump pump pump pump N 2 acid Vessel 1 Vessel 2 Vessel 3 Vessel 4 Vessel 5 pH control pH control pH control Pancreatic juice Figure 2 The original SHIME model. Vessels 1–5 in the figure mimic the different compartments of the human GIT: duodenum C jejunum, ileum, caecum C ascending colon, transverse colon and distal colon, respectively. In the revised version of this system, a vessel representing the stomach has been added before vessel 1. First five pumps work semi-continuously, and pumps between vessels, 3–5 and effluent work continuously. Source: From Ref. 11. Ma ¨ kivuokko and Nurminen240 first vessel with 10 ml of fecal inoculum. The inoculum is mixed in the vessel with NaCl and 10 ml of the mixed culture is pumped to the next vessel. This procedure continues through the vessels and finally the excess inoculum is pumped to waste container from the fourth vessel. After three hours of the incubation, 3 ml of fresh simulator media with (three test channels) or without (one control channel) test substance is pumped to the first vessel. The media is fermented in the first vessel for three hours, after which 3 ml of the fermented media is transferred to the second vessel, and 3 ml of fresh media is pumped to the first vessel. This procedure of transferring liquid to the next vessel continues through all the vessels, so that finally after 15 hours, when 3 ml of fermented fluid has been transferred from vessel four to the waste container for the first time, vessels 1, 2, 3, and 4 have respective volumes of 6, 8, 10, and 12 ml of fermenting fluid. The fermentation and three- hourly fluid transfers continue for 48 hours, after which the system is stopped and samples are collected from each vessel. Other Simulators In addition to simulate different parts of the GIT, chemostat-type simulators have also been used to simulate the oral cavity, in particular to investigate plaque formation (13); and to simulate the urinary bladder to investigate antibiotic sensitivity of urinary tract infection–causing pathogens (14). These simulators usually consist of a single chemostat. Non-Chemostat Models The third type of model is actually comprised of two complementary parts, the TIM (TNO Intestinal Model) systems 1 and 2 introduced by Minekus et al. in 1995 (15) and 1999 (16). The TIM 1 system (Fig. 4) comprises eight sequentially attached glass modules and mimics the stomach and small intestine, while the TIM 2-system consists of four glass modules in a loop mimicking the proximal colon of monogastric animals (Fig. 5). These N 2 +NH 3 +37°C N 2 N 2 N 2 N 2 SS S S S S S S S S S S +4°C Effluent +4°C Fresh medium V4V3V2V1 3 ml 5 ml 7 ml 9 ml 7.06.55.5 6.0 Volume pH Figure 3 The EnteroMix w model. The figure represents the initial volumes of the system before fresh medium is added to begin the simulation. The vessels V1 to V4 are mimicking different sections of the human colon: caecumCascending, transverse, descending, and distal colon, respectively. pH controlling and semi-continuous fluid transitions are operated via opening and closing of computer controlled valves (S). In Vitro Methods to Model the Gastrointestinal Tract 241 dynamic models differ from the three previously presented models in two main aspects: fluid transportation from vessel to vessel is executed via peristaltic valve-pumps and there is a constant absorption of water and fermentation products through dialysis membranes. In both systems the peristaltic movement of the intestinal fluid flowing in a flexible tube in the middle of the modules is achieved by changing the pressure of the 378C heated water circulating between the module walls and the flexible tube. The peristaltic pressure around the flexible tube is controlled via computer-controlled valves to mimic the gastric emptying times. For the simulation of intestinal absorption TIM 1 has two integrated 5 kDa dialysis membranes, after jejunal and ileal modules, and TIM 2 has one, a hollow- fiber membrane (molecular mass cut-off value 50 kDa) in the lumen of the system. The TIM 1 dialysis membranes allow real-time collection of absorbable metabolites and water that would be absorbable in the human jejunum and ileum. In the tube membrane of TIM 2 circulates dialysis fluid allowing absorption of e.g., water, and short-chain fatty acids. The pH-values are monitored in each compartment. In a TIM 1 simulation, a homogenized human meal is introduced into the gastric compartment in pre-set times. From the stomach, the fluid is pumped through the following six compartments. During the simulation, the secretion of enzymes, bile, and pancreatic juice and the pH-controlling of the stomach (a pH gradient from 5.0 to 1.8 in 80 minutes from the beginning) and duodenum (constant pH 6.5) is regulated via computer. In a TIM 2 simulation the model is first inoculated with 200 ml of fecal inoculum. Microbiota is allowed to adapt to the conditions for 16 hours, after which the actual simulation is started by adding ileal medium semi-continuously with or without the tested substrate to the system. The pH is constantly maintained constant at 5.8 representing the pH-level in the proximal colon. Samples can be taken both from the lumen of the simulator and from the dialysis liquid during the simulation. 7 7 7 8 8 4 3 2 5 1 6 Figure 4 TIM 1 model. The model is mimicking the different sections of the human small intestine: the gastric compartment (1), duodenum (2), jejunum (3) and ileum (4). Gastric (5) and intestinal secretions (6), peristaltic valve pumps (7) and dialysis devices (8) are also included in this simulator. Source: From Ref. 17. Ma ¨ kivuokko and Nurminen242 Comparison of the Models The four colon simulation models presented here have structural and functional differences (Table 1), but the solutions used to reproduce the critical conditions that influence the microbiology of the colon are similar in all four models. Firstly the colonic microbiota is simulated in each model using fecal samples from a single donor or several donors in a pooled sample, because more realistic samples of gastrointestinal tract bacteria from the ileum or cecum of humans are very difficult to obtain both ethically and technically. Secondly all the colon simulators use similar growth media that originate from media originally published by Gibson et al. in 1988 (9) mimicking the ileal fluids obtained from sudden-death victims. Thirdly all the colon models have strictly anaerobic conditions, similar pH set-points representing the in vivo situation in the colon of healthy humans (19) and all the functions of these systems are computer-controlled. The Reading model and the SHIME system are both run until a steady state in microbial growth is reached, while TIM 2 and the EnteroMix w model are run for a pre-determined time (2 or 5 days). The SHIME system is the only one of the above- mentioned four systems having a continuous line from stomach to distal colon, thus enabling the simulation of the whole GI-tract in one run. The simulated ileal fluid coming from TIM 1 can also be used indirectly as growth medium in TIM 2. The EnteroMix w model has the smallest working volumes (Table 1) in the vessels, enabling the simulation of small concentrations of the tested substrate. On the other hand the f g e j i b a g g d d h c Figure 5 TIM 2 model: The model represents the human proximal colon in one loop-shaped system: peristaltic mixing with flexible walls inside (a), pH electrode (b), alkaline pump (c), dialysis system (d), fluid level sensor (e), nitrogen inlet (f), peristaltic valves (g), sample port (h), gas sampling (i) and ileal medium reservoir. Source: From Ref. 18. In Vitro Methods to Model the Gastrointestinal Tract 243 small volumes do not allow any samplings during the simulation run, which is possible in all the other models, because the volume of microbiota would be too heavily affected in the vessels. The EnteroMix w model is also the only model having parallel channels in the same simulator allowing four parallel simulations to be run at the same time with the same fecal inoculum. SIMULATING THE RUMEN Although thesimulators described above are mainly aimed at simulating the human GIT, the models can also be used to simulate the GIT of other monogastric animals. However for the simulation of the ruminant GIT different factors have to be taken into consideration; in particular the different functioning of the rumen, retaining and fermenting solid material while liquid phase is allowed to pass on into the GIT. The anaerobic environment of the rumen is heterogeneous in nature: a large volume of free liquid, a complex solid mass of digesta, and a gas phase. Within this mixture, the diverse microbial population of bacteria, protozoa, and anaerobic fungi can be described as occurring in four different compartments (1) the microbes living free in suspension, (2) the microbes loosely associated with the solid material, (3) the microbes that are trapped in the solid material, and (4) the microbes close to or attached to the rumen wall (20). The complexity is still increased due to the different removal rates of the solid and liquid portions of rumen contents, revealing the dynamic nature of the rumen. Rumen Simulators The artificial rumen techniques developed over the past five decades for investigation of rumen physiology as well as evaluation of feed rations, have ranged from batch fermentations to more complicated continuous incubations. In addition, the absorption function of the rumen wall has been included in some designs, in which a semi-permeable membrane is applied for removal of the fermentation end products. Batch Culture The most simplistic, in vitro fermentations representing the rumen were performed in different kinds of tubes (21–23). Another way to conduct a static, batch simulation is to use closed glass serum bottles. As an example, in the study of Lopez et al. (24) 0.2 g of diet (ground to pass through 1 mm screen) was weighed into the 120 ml serum bottles and the fermentation process started by dispensing 50 ml of strained, 1:4 (v/v) buffered rumen fluid under CO 2 flushing. The bottles were sealed with butyl rubber stoppers and aluminium caps and incubated in a shaking water bath at C398C. After 24-hour incubation, total gas production and pH were measured and samples for methane, hydrogen, and short chain fatty acid analysis taken. The durations of the reported batch fermentations employing rumen microbes have varied from six (25) to 96 hours (26) or even up to 168 hours (27). The buffer systems applied in batch simulations are quite often adopted from by Menke et al. (28), McDougall (29), or Goering and van Soest (30). Due to the fact that gas production has been used as an indirect measure of digestibility and fermentation kinetics of ruminant feeds, a scaled glass syringe (volume of 100–150 ml) has also been used as a fermentation vessel (28,37). The piston is allowed to move upward without restrain and thus indicates the amount of gas released due to Ma ¨ kivuokko and Nurminen244 microbial activity. The more sophisticated ways to measure gas production kinetics have been reported, for example the syringe/electronic pressure transducer-equipment (32), which measured and released the accumulated gas. However more automated systems were, both an apparatus which combined electronic pressure transducers and electric micro-valves (33) and the automated pressure evaluation system (APES) (34) where the overpressure was released by use of pressure sensitive switches and solenoid valves. Semi-Continuous Culture (Rusitec) The structure of semi-continuous rumen simulation technique Rusitec (Fig. 6), which was described by Czerkawski and Breckenridge (35), provides three of the four microbial compartments mentioned earlier. A Rusitec reaction vessel with capacity ofone liter consisted of a Perspex cylinder (254!76) with an inlet at the bottom. The cylinder was sealed by flat Perspex cover provided with a screw flange for easy access. The cover is provided with two outlets, one for sampling and the other for effluent overflow and gas collection. The solids (feed or digesta) were placed in nylon bags (pore size 50–100 mm) inside a perforated container.This “cage”then slid up and down inside the reaction vessel, allowing the effluent to flush the solids. At the bottom of the vessel, the artificial saliva (29) was continuously infused and the excess liquid and the gases are forced out through an overflow by a slight positive S V G F R L C N T I O M E Figure 6 A schematic diagram of semi-continuous Rusitec unit: driving shaft (S), sampling valve (V), gas-tight gland (G), flange (F), main reaction vessel (R), rumen fluid (L), perforated food container (C), nylon gauze bag (N), rigid tube (T), inlet of artificial saliva (I), outlet through overflow (O), line to gas-collection bag (M), vessel for collection of effluent (E). Source: From Ref. 35. In Vitro Methods to Model the Gastrointestinal Tract 245 pressure in the gas space. The proper fermentation temperature was maintained by incubating the reaction vessel in water bath at 398C during the experiment. The fermentation in Rusitec was started by placing solid rumen digesta in one nylon bag and an equal amount of feed to be used in a second nylon bag. The reaction vessel was filled up to overflow with strained diluted rumen contents. After 24 hours the inoculum bag was removed and replaced with a new bag of food. Removal of the oldest bag (48 hours) and adding a new bag was repeated each day. At the beginning of the experiment and during feeding, the gas space was flushed with the mixture of CO 2 and N 2 (5:95 v/v). The removed bag is drained, placed in a plastic bag and the solids washed twice with the artificial saliva. This rumination mimicking process includes gentle pressing of the solids and squeezing out excess liquid, which is combined and returned to the reaction vessel. The Rusitec technique has been quite widely applied as such. It has been used by a number of authors to study, for example, decreased methanogenesis (36,37) and efficiency of recovery of particle-associated microbes from ruminal digesta (38). In reported Rusitec studies at least up to 16 reaction vessels have been applied simultaneously (39). The running times of sample collection periods have exceeded from five (40) to 36 days (36) after stabilizing the microbial population for 12 hours (39) to 17 days (40). Continuous Culture One of the earliest reports of continuous culture apparatus (Fig. 7) is the work of Stewart et al. (41). With the device designed by Quinn (42) the incubation time could exceed more beyond 24 hours because of the pH control system. In these simulation systems the relay solenoid outflow value sampling device outflow receptacle timed periodic impulse solenoid inflow valve stirring motor vent thermometer float with electrical contacts water bath water bath heater magnetic stirring motor magnet ice bath constant CO 2 pressure culture substrate Figure 7 One of the earliest continuous culture systems for studying rumen fermentation. Source: From Ref. 41. Ma ¨ kivuokko and Nurminen246 water insoluble substrates were continuously delivered to the vessel in the form of a slurry. One of the few devices taking the absorption of fermentation end products into account was developed by Rufener et al. (43) and improved by Slyter et al. (44). The apparatus (Fig. 8) consisted of six independent fermentation chambers (500 ml) with accessories providing anaerobiosis, constant volume, agitation of the fermentation mixture and collection of effluents and gases. For controlling the pH, this system included a dialysis bag containing a mixture of ion-exchange resins, which absorbed the short chain fatty acids. The fermentors were reported to reach the steady state in three to Q T K M U N R L O B C D E F H G A S V P I J Figure 8 A continuous culture apparatus providing absorption of fermentation products: centrifugal water pump (A), gas-sampling port (B), fermentor (C), feeding port (D), water-drainage pipe (E), Plexiglas reservoir (F), drainage tube (G), magnetic stirrer (H), water bath (I), dialysis sac with cation-exchange resin (J), saliva-inflow ground-glass joint (K), fermentor stirring device (L), gas-outlet tube (M), fermentor port (N), sampling glass tube and resin holder (O), liquid-effluent collection funnel (P), peristaltic pump (Q), effluent outlet (R), effluent rubber tubing (S), saliva-water reservoir (T), gas-collection bladder (U), feed-input apparatus (V). Ports D and N are shown 908 out of phase from their actual position to simplify the drawing. Source: From Ref. 44. 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