Development and validation of an automated, microscopy-based method for enumeration of groups of intestinal bacteria

An automated microscopy-based method using fluorescently labelled 16S rRNA-targeted oligonucleotide probes directed against the predominant groups of intestinal bacteria was developed and validated. The method makes use of the Leica 600HR. image analysis system, a Kodak MegaPlus camera model 1.4 and a servo-controlled Leica DM/RXA ultra-violet microscope. Software for automated image acquisition and analysis was developed and tested. The performance of the method was validated using a set of four fluorescent oligonucleotide probes: a universal probe for the detection of all bacterial species, one probe specific for Bifidobacterium spp., a digenus-probe specific for Bacteroides spp. and Prevotella spp. and a trigenus-probe specific for Ruminococcus spp.. Clostridium spp. and Eubacterium spp. A nucleic acid stain, 4',6-diamidino-2-phenylindole (DAPI), was also included in the validation, In order to quantify the assay-error, one faecal sample was measured 20 times using each separate probe. Thereafter faecal samples of 20 different volunteers were measured following the same procedure in order to quantify the error due to individual-related differences in gut flora composition. It was concluded that the combination of automated microscopy and fluorescent whole-cell hybridisation enables distinction in gut flora-composition between volunteers at a significant level. With this method it is possible to process 48 faecal samples overnight, with coefficients of variation ranging from 0.07 to 0.30. (C) 1999 Elsevier Science B.V. All rights reserved

Escherichia coli as a probiotic?

The influence of oral treatment with a suspension of non-pathogenic Escherichia coli cells (commercially available as: Symbioflor II(R)) on the morphological composition of the gut microflora and on the systemic humoral immune response (the IgG-, IgA- and IgM-isotype) against the bacterial cells in the Symbioflor II preparation was measured. After a pretreatment period of 21 days, ten healthy human volunteers ingested 1*10(8) cells of E. coli daily for 14 days. Thereafter a follow-up period of 28 days completed the study The results of this study indicated that no effect of the treatment on the composition of the gut microflora could be observed. However, the immune-fluorescence measurements revealed a significant increase in circulating amounts of IgG directed against the administered E. coli cells. It is concluded that the treatment only resulted in a specific humoral immune response, while the gut microflora is not modulated

Escherichia coli as a probiotic?

The influence of oral treatment with a suspension of non-pathogenic Escherichia coli cells (commercially available as: Symbioflor II(R)) on the morphological composition of the gut microflora and on the systemic humoral immune response (the IgG-, IgA- and IgM-isotype) against the bacterial cells in the Symbioflor II preparation was measured. After a pretreatment period of 21 days, ten healthy human volunteers ingested 1*10(8) cells of E. coli daily for 14 days. Thereafter a follow-up period of 28 days completed the study The results of this study indicated that no effect of the treatment on the composition of the gut microflora could be observed. However, the immune-fluorescence measurements revealed a significant increase in circulating amounts of IgG directed against the administered E. coli cells. It is concluded that the treatment only resulted in a specific humoral immune response, while the gut microflora is not modulated

Development of 16S rRNA-based probes for the Coriobacterium group and the Atopobium cluster and their application for enumeration of Coriobacteriaceae in human feces from volunteers of different age groups

Two 16S rRNA-targeted probes were developed: one for the Coriobacterium group and the other for the Atopobium cluster (which comprises most of the Coriobacteriaceae species, including the Coriobacterium group). The new probes were based on sequences of three new Coriobacteriaceae strains isolated from human feces and clinical material and sequences from databases. Application of the probes to fecal samples showed that formula-fed infants had higher numbers of Coriobacterium group cells in their feces than breast-fed infants. In addition, based on the presented results, it is hypothesized that with the increasing age of a person, the diversity of Atopobium cluster species present in the feces increases