Studies on the effect of system retention time on bacterial populations colonizing a three-stage continuous culture model of the human large gut using FISH techniques

Abstract

Fluorescence in situ hybridization was used to quantitate bacteria growing in a three-stage continuous culture system inoculated with human faeces, operated at two system retention times (60 and 20 h). Twenty-three different 16S rRNA gene oligonucleotide probes of varying specificities were used to detect bacteria. Organisms belonging to genera Bacteroides and Bifidobacterium, together with the Eubacterium rectale/Clostridium coccoides group, the Atopobium, Faecalibacterium prausnitzii and Eubacterium cylindroides groups, as well as the segmented filamentous bacteria, the Roseburia intestinalis group and lactic acid bacteria, were all present in high numbers in the continuous culture system. Other groups and species such as Ruminococci and Enterobacteria also persisted in the model, though not always at levels that allowed reliable quantitation. Some organisms such as Streptococci and Corynebacteria, present in the faecal inoculum, did not colonize the system. Other probes specific for Eubacterium lentum and for members of the genus Desulfovibrio did not detect these organisms at any time. Short chain fatty acid production was always highest in vessel I of the continuous culture system, however, a marked increase in acetate formation and a reduction in butyrate production occurred when system retention time was reduced to 20 h, which correlated with reductions in the numbers of butyrate-producing Roseburia.

Selection of fast and slow growing bacteria from fecal microbiota using continuous culture with changing dilution rate

“…Modification of the dilution rate in vitro has been shown to affect the establishment of certain types of bacterial communities together with the changes in their metabolic activities [17]. For instance, dilution rate 0.08 1/h increased the proportion of lactobacilli and bifidobacteria compared to that at 0.04 1/h [18] and in another study more organic acids were produced at D  = 0.1 1/h than at D  = 0.04 1/h [19].…”

Adherence and Cytokine Induction in Caco-2 Cells by Bacterial Populations from a Three-Stage Continuous-Culture Model of the Large Intestine

“…The final medium was adjusted to pH 5.5 by adding 1 M HCl prior to autoclaving it. Fecal material from a healthy 30-year-old male volunteer who had not taken antibiotics for at least 12 months prior to commencement of the study was used to inoculate the CCS, using methods described previously (7,24). Short-chain fatty acid (SCFA) analysis was used to confirm that the model was operating under steady-state growth conditions (7,24).…”

“…However, ethical considerations have hindered study of the structure of the normal gut microbiota and its metabolic activities in different parts of the large bowel, and most investigations have been done on fecal material. To overcome this, an in vitro three-stage continuousculture system (CCS) model was developed to investigate the behavior of the microbiota under environmental conditions simulating those in the proximal and distal gut (7,24). This system provides a controlled operator-defined environment that can be maintained in a steady state and that simulates the complexity and diversity of the microbiota (7,29,37).…”

Composition and Metabolic Activities of Bacterial Biofilms Colonizing Food Residues in the Human Gut

“…The range of fluorescent 16S rRNA gene oligonucleotide probes used in this study, which targeted all of the major populations of gut bacteria, has been described previously (7). In particular, probe Bif164 (5Ј-CA TCCGGCATTACCACCC-3Ј) was used to identify bifidobacteria (24), probe Ent1 (5Ј-CCGCTTGCTCTCGCGAG-3Ј) was used for enterobacteria (25), probe Bac 303 (5Ј-CCAATGTGGGGGACCTT-3Ј) was used for Bacteroides and Prevotella (32), probe Erec 482 (5Ј-GCTTCTTAGTCAGGTACCG-3Ј) was used for the Eubacterium rectale-Clostridium coccoides group (17), and the universal eubacterial probe Eub338 (5Ј-GCTGCCTCCCGTAGGAGT-3Ј) was used for total eubacteria (2).…”