Probing Strategies

As well as affording design of PCR primers for specific bacterial populations, the improved 16S rRNA gene sequence information has greatly enhanced the development of probing strategies for gut micro-organisms. Two probing strategies have generally been employed, namely, dot-blot hybridization and fluorescent in situ hybridization (FISH). The nature of the 16S rRNA gene of bacteria also enables development of oligonucleotide probes targeting different taxonomic levels, i.e., domain level (Bact 338), group level (e.g., Chis 150), genus level (e.g., Bif 164) or species level (e.g., Bdis 656) (57,70). The last 5 years have seen enormous development and application of these strategies in gut microbiology (71-76).

A longitudinal study was performed with nine healthy human volunteers (five males, four females) monitoring the fecal microbiota using FISH (72). The results demonstrated that 90-100% of 40,6-diamidino-2-phenylindol dihydrochloride (DAPI)-stained cells were hybridized by the bacterial probe (Bact 338), and that the Clos. coccoides/Eub. rectale group (Erect 482) and Bacteroides group (Bfra 602 and Bdis 656) represented almost 50% of the total bacteria of healthy humans. In addition, the Low G + C #2 group (Lowgc2P) comprised 12% of the total bacteria, and Bifidobacterium (Bif 164) 3%. Initial data indicated that the Clostridium lituseburense group (Clit 135), the Clostridium histolyticum group (Chis 150), and the Streptococcus/Lactococcus group (Strc 493) all formed less than 1% of the total bacteria and so were not included in the longitudinal study. In general, the fecal microbiota of individuals was shown to fluctuate during the 8-month study. Interestingly, the greatest variation was seen in the bifidobacterial component of the microbiota. A more recent study from the same laboratory group employed a set of 15 probes to investigate the microbial composition of 11 healthy volunteers (73). Again, the Bacteroides group (27.7%) and the Clos. coccoides/Eub. rectale group (22.7%) were seen to be the numerically predominant bacterial components. In addition, three other predominant groups were identified: Atopobium group (11.9%), Eubacterium low G + C #2/Fuso. prausnitzii group (10.8%), and Ruminococcus and relatives (10.3%). Bifidobacterium (4.8%), Eub. hallii and relatives (3.8%), Lachnospira and relatives (3.6%), and Eubacterium cylindroides and relatives (1.8%) were also dominant members of the microbiota. However, Enterobacteriaceae, the Lactobacillus/Enterococcus group, Phascolarctobacterium and relatives, and Veillonella were all subdominant (each forming 1%). Taken together, this afforded 90.5% coverage of the total bacteria hybridized with the Bact 338 probe. (N.B.: Eub. hallii and relatives, and Lachnospira and relatives are subsets of the Clos. coccoides/Eub. rectale group, so were not included in summation). However, a large proportion of the DAPI-stained cells (~ 40%) were not accounted for by the Bact 338 probe. The question arises as to whether these cells are non-viable or metabolically inactive (low rRNA), impermeable, or represent novel bacterial groups whose 16S rRNA differs within the "conserved" region the Bact 338 probe targets or in the secondary structure surrounding it.

Other research groups have developed and validated additional oligonucleotide probes suitable for FISH, for potentially important members of the human GI tract microbiota (75,76). Ruminococcus obeum-like bacteria have been frequently identified in ribosomal clonal libraries of human fecal samples and the development of probing strategies was thus considered pertinent (76). Following validation, the Urobe 63 probe was used to examine the Rum. obeum group in three healthy Dutch males (three samples were collected from each subject over one month). FISH enumeration was performed both by epifluorescent microscopy and by flow cytometry (which require different handling and thus different protocols). The two methods gave comparable results, demonstrating that Rum. obeum-like bacteria comprise ~2.5% of the total bacterial count (Bact 338). A further six individuals (two males, four females) provided stool samples and the results were consistent in all subjects. In addition, counts of the Clos. coccoides/Eub. rectale group were made which indicated that the Rum. obeum group accounted for ~ 16% of this group (76). Similarly, the Fuso. prausnitzii cluster has been shown in numerous molecular analyses to be part of the dominant microbiota of healthy humans. As such, Suau, and coworkers (75) developed an oligonucleotide probe for this cluster which was applicable both for FISH and dot-blot hybridizations. Overall, 16.5% (range 5-28%) of the DAPI-stained cells hybridized with the Fprau 645 probe (n= 10 healthy adults). Samples from a further 10 healthy individuals were used for dot-blot analysis with the same probe and showed the Fuso. prausnitzii cluster accounted for 5.3% of the total bacterial 16S rRNA (range 1.5-9.5%). Unfortunately, these data are not comparable as different samples were used for each assay. In addition, the two assays provide distinctive enumeration: FISH provides counts of the number of cells in the sample (which can be represented as a percentage of total bacterial (Bact 338) cells or total cells (DAPI), whereas dot-blot provides an index of the percentage of total 16S rRNA the specific population forms. The index obtained by dot-blot is further complicated as it is not only proportional to the number of cells in the sample, but also the number of copies of the rRNA gene in each cell and the activity of the cells.

Dot-blot analyses of the healthy human fecal microbiota using an array of probes have, once again, highlighted the inter-individual variation (71,74). Both studies employed six oligonucleotide probes to monitor the predominant bacterial groups. The work by Sghir and colleagues (74) (n = 27 healthy adults; 13 males, 14 females) was consistent with earlier work which showed that the Bacteroides group (including Bacteroides, Prevotella and Porphyromonas; 37%), the Clos. leptum subgroup (16%) and the Clos. coccoides/Eub. rectale group (14%) were predominant, accounting for 67% of the total rRNA. Bifidobacterium and the enteric group each made up less than 1% of the total rRNA, whilst the low-G + C Gram-positive group (including Lactobacillus, Streptococcus and Enterococcus) represented 1% (74). Marteau and coworkers (71) similarly demonstrated the predominant fecal rRNA (n = 8 healthy adults; four males, four females) corresponded to the Clos. coccoides/Eub. rectale group (23%), the Clos. leptum subgroup (13%) and the Bacteroides group (8%) using the same probes as Sghir and colleagues (74). Although, using different probes, this later study indicated higher bifidobacterial and Lactobacillus/Enterococcus rRNA indices, 3% and 7%, respectively. Interestingly, Marteau and coworkers (71) compared the fecal rRNA indices of these bacterial groups and E. coli species with cecal rRNA indices. Overall, the indices for the Bacteroides group and the Clos. leptum subgroup were significantly higher in fecal samples than cecal samples, and the Lactobacillus/Enterococcus fecal rRNA index was significantly lower than that of the cecum. The Clos. coccoides/Eub. rectale rRNA index was higher in fecal samples than cecal samples, but the inter-individual variation meant that this was not statistically significant. Concurrent cultivation analysis monitoring total anaerobes, facultative anaerobes, bifidobacteria and Bacteroides demonstrated significantly higher levels of total anaerobes, bifidobacteria, and bacteroides populations in fecal samples compared to cecal samples (71).

Most recent developments in probing strategies include membrane-array and/or microarray methodologies (77,78). The results of these assays were in agreement with previous studies, demonstrating inter-individual variation in the fecal microbiota of different healthy human subjects. The predominant microbiota of healthy humans determined by the membrane-array technique (employing 60 oligonucleotide probes targeting 20 bacterial species) included Bacteroides species, Clos. clostridioforme, Clos. leptum, Fuso. prausnitzii, Pep. productus, Ruminococcus species, Bifidobacterium species and E. coli (78). In addition, analysis of the fecal microbiota of an individual suffering long-term diarrhea demonstrated a loss of a number of bacterial species common to the normal microbiota of healthy subjects. These results were replicated in a microarray study using the same probe array (77), where the probes were printed on aldehyde slides rather than applied to membranes.

Overall, probing and PCR-based strategies have been shown to afford good coverage of the predominant microbiota of the GI tract. This situation is likely to improve with continued development of specific primer sets and/or oligonucleotide probes, particularly in the light of increased diversity as elucidated by community analysis work. Indeed, such community profiling studies provide excellent direction for the development of novel probes and primers.

Pregnancy And Childbirth

Pregnancy And Childbirth

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