Modulation of the microbial ecology of the human colon by probiotics, prebiotics and synbiotics to enhance human health: an overview of enabling science and potential applications

The application of probiotics and prebiotics to the manipulation of the microbial ecology of the human colon has recently seen many scientific advances. The sequencing of probiotic genomes is providing a wealth of new information on the biology of these microorganisms. In addition, we are learning more about the interactions of probiotics with human cells and with pathogenic bacteria. An alternative means of modulating the colonic microbial community is by the use of prebiotic oligosaccharides. Increasing knowledge of the metabolism of prebiotics by probiotics is allowing us to consider specifically targeting such dietary intervention tools at specific populatiori groups and specific disease states. (c) 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

The impact of date palm fruits and their component polyphenols, on gut microbial ecology, bacterial metabolites and colon cancer cell proliferation

The fruit of the date palm (Phoenix dactylifera L.) is a rich source of dietary fibre and polyphenols. We have investigated gut bacterial changes induced by the whole date fruit extract (digested date extract; DDE) and its polyphenol-rich extract (date polyphenol extract; DPE) using faecal, pH-controlled, mixed batch cultures mimicking the distal part of the human large intestine, and utilising an array of microbial group-specific 16S rRNA oligonucleotide probes. Fluorescence microscopic enumeration indicated that there was a significant increase in the growth of bifidobacteria in response to both treatments, whilst whole dates also increased bacteroides at 24 h and the total bacterial counts at later fermentation time points when compared with DPE alone. Bacterial metabolism of whole date fruit led to the production of SCFA, with acetate significantly increasing following bacterial incubation with DDE. In addition, the production of flavonoid aglycones (myricetin, luteolin, quercetin and apigenin) and the anthocyanidin petunidin in less than 1 h was also observed. Lastly, the potential of DDE, DPE and metabolites to inhibit Caco-2 cell growth was investigated, indicating that both were capable of potentially acting as antiproliferative agents in vitro, following a 48 h exposure. This potential to inhibit growth was reduced following fermentation. Together these data suggest that consumption of date fruits may enhance colon health by increasing beneficial bacterial growth and inhibiting the proliferation of colon cancer cells. This is an early suggestion that date intake by humans may aid in the maintenance of bowel health and even the reduction of colorectal cancer development.

Microbial ecology of biotechnological processes

The investigation of phylogenetic diversity and functionality of complex microbial communities in relation to changes in the environmental conditions represents a major challenge of microbial ecology research. Nowadays, particular attention is paid to microbial communities occurring at environmental sites contaminated by recalcitrant and toxic organic compounds. Extended research has evidenced that such communities evolve some metabolic abilities leading to the partial degradation or complete mineralization of the contaminants. Determination of such biodegradation potential can be the starting point for the development of cost effective biotechnological processes for the bioremediation of contaminated matrices. This work showed how metagenomics-based microbial ecology investigations supported the choice or the development of three different bioremediation strategies. First, PCR-DGGE and PCR-cloning approaches served the molecular characterization of microbial communities enriched through sequential development stages of an aerobic cometabolic process for the treatment of groundwater contaminated by chlorinated aliphatic hydrocarbons inside an immobilized-biomass packed bed bioreactor (PBR). In this case the analyses revealed homogeneous growth and structure of immobilized communities throughout the PBR and the occurrence of dominant microbial phylotypes of the genera Rhodococcus, Comamonas and Acidovorax, which probably drive the biodegradation process. The same molecular approaches were employed to characterize sludge microbial communities selected and enriched during the treatment of municipal wastewater coupled with the production of polyhydroxyalkanoates (PHA). Known PHA-accumulating microorganisms identified were affiliated with the genera Zooglea, Acidovorax and Hydrogenophaga. Finally, the molecular investigation concerned communities of polycyclic aromatic hydrocarbon (PAH) contaminated soil subjected to rhizoremediation with willow roots or fertilization-based treatments. The metabolic ability to biodegrade naphthalene, as a representative model for PAH, was assessed by means of stable isotope probing in combination with high-throughput sequencing analysis. The phylogenetic diversity of microbial populations able to derive carbon from naphthalene was evaluated as a function of the type of treatment.

Enrichment of nitrifying microbial communities from shrimp farms and commercial inocula

Nitrifying bacteria were selected from shrimp farm water and sediment (natural seed) in Thailand and from commercial seed cultures. The microbial consortia from each source giving the best ammonia removal during batch culture pre-enrichments were used as inocula for two sequencing batch reactors (SBRs). Nitrifiers were cultivated in the SBRs with 100 mg NH4-N/I and artificial wastewater containing 25 ppt salinity. The two SBRs were operated at a 7 d hydraulic retention time (HRT) for 77 d after which the HRT was reduced to 3.5 d. The amounts of ammonia removed from the influent by microorganisms sourced from the natural seed were 85% and 92% for the 7 d HIRT and the 3.5 d HRT, respectively. The ammonia removals of microbial consortia from the commercial seed were 71% and 83% for these HRTs respectively. The quantity of ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) was determined in the SBRs using the most probable number (MPN) technique. Both AOB and NOB increased in number over the long-term operation of both SBRs. According to quantitative fluorescence in situ hybridisation (FISH) probing, AOB from the natural seed and commercial seed comprised 21 +/- 2% and 30 +/- 2%, respectively of all bacteria. NOB could not be detected with currently-reported FISH probes, suggesting that novel NOB were enriched from both sources. Taken collectively, the results from this study provide an indication that the nitrifiers from shrimp farm sources are more effective at ammonia removal than those from commercial seed cultures.

Kinetic and phylogenetic characterization of an anaerobic dechlorinating microbial community

The reductive dechlorination (RD) of tetrachloroethene (PCE) to vinyl chloride (VC) and, to a lesser extent, to ethene (ETH) by an anaerobic microbial community has been investigated by studying the processes and kinetics of the main physiological components of the consortium. Molecular hydrogen, produced by methanol-utilizing acetogens, was the electron donor for the PCE RD to VC and ETH without forming any appreciable amount of other chlorinated intermediates and in the near absence of methanogenic activity. The microbial community structure of the consortium was investigated by preparing a 1 6S rDNA clone library and by fluorescence in situ hybridization (FISH). The PCR primers used in the clone library allowed the harvest of 16SrDNA from both bacterial and archaeal members in the community. A total of 616 clones were screened by RFLP analysis of the clone inserts followed by the sequencing of RFLP group representatives and phylogenetic analysis. The clone library contained sequences mostly from hitherto undescribed bacteria. No sequences similar to those of the known RD bacteria like 'Dehalococcoides ethenogenes' or Dehalobacter restrictus were found in the clone library, and none of these bacteria was present in the RD consortium according to FISH. Almost all clones fell into six previously described phyla of the bacterial domain, with the majority (56(.)6%) being deep-branching members of the Spirochaetes phylum. Other clones were in the Firmicutes phylum (18(.)5%), the Chloroflexi phylum (16(.)4%), the Bacteroidetes phylum (6(.)3%), the Synergistes genus (11(.)1%) and a lineage that could not be affiliated with existing phyla (11(.)1%). No archaeal clones were found in the clone library. Owing to the phylogenetic novelty of the microbial community with regard to previously cultured microorganisms, no specific microbial component(s) could be hypothetically affiliated with the RD phenotype. The predominance of Spirochaetes in the microbial consortium, the main group revealed by clone library analysis, was confirmed by FISH using a purposely developed probe.

A soil-based microbial biofilm exposed to 2,4-D: bacterial community development and establishment of conjugative plasmid pJP4

A soil suspension was used as a source to initiate the development of microbial communities in flow cells irrigated with 2,4-dichlorophenoxyacetic acid (2,4-D) (25 mu g ml(-1)). Culturable bacterial members of the community were identified by 16S rRNA gene sequencing and found to be members of the genera Pseudomonas, Burkholderia, Collimonas and Rhodococcus. A 2,4-D degrading donor strain, Pseudomonas putida SM 1443 (pJP4::gfp), was inoculated into flow cell chambers containing 2-day old biofilm communities. Transfer of pJP4::gfp from the donor to the bacterial community was detectable as GFP fluorescing cells and images were captured using confocal scanning laser microscopy (GFP fluorescence was repressed in the donor due to the presence of a chromosomally located lacl(q) repressor gene). Approximately 5-10 transconjugant microcolonies, 20-40 mu m in diameter, could be seen to develop in each chamber. A 2,4-D degrading transconjugant strain was isolated from the flow cell system belonging to the genus Burkholderia.

Molecular investigation of the microbial populations of the pink sugarcane mealybug, Saccharicoccus sacchari

In an attempt to better understand the microbial diversity and endosymbiotic microbiota of the pink sugarcane mealybug (PSMB) Saccharicoccus sacchari Cockerell (Homoptera: Pseudococcidae), culture-independent approaches, namely PCR, a 16S rDNA clone library, and temperature gradient gel electrophoresis (TGGE) were used. Previous work has indicated that the acetic acid bacteria Gluconacetobacter sacchari, Gluconacetobacter diazotrophicus, and Gluconacetobacter liquefaciens represent only a small proportion of the microbial community of the PSMB. These findings were supported in this study by TGGE, where no bands representing G. sacchari, G. diazotrophicus, and G. liquefaciens on the acrylamide gel could be observed following electrophoresis, and by a 16S rDNA clone library study, where no clones with the sequence of an acetic acid bacterium were found. Instead, TGGE revealed that the mealybug microbial community was dominated by beta- and gamma-Proteobacteria. The dominant band in TGGE gels found in a majority of the mealybug samples was most similar, according to BLAST analysis, to the beta-symbiont of the craw mealybug Antonina crawii and to Candidatus Tremblaya princeps, an endosymbiont from the mealybug Paracoccus nothofagicola. The sequences of other dominant bands were identified as gamma-Proteobacteria, and were most closely related to uncultured bacterial clones obtained from soil samples. Mealybugs collected from different areas in Queensland, Australia, were found to produce similar TGGE profiles, although there were a few exceptions. A 16S rDNA clone library based on DNA extracted from a mealybug collected from sugarcane in the Burdekin region in Queensland, Australia, indicated very low levels of diversity among mealybug microbial populations. All sequenced clones were most closely related to the same members of the gamma-Proteobacteria, according to BLAST analysis.

Microbial diversity within the water column of a larval rearing system for the ornate rock lobster (Panulirus ornatus)

The ornate tropical rock lobster, Panulirus ornatus has substantial potential as an aquaculture species though disease outbreaks during the animal's extended larval lifecycle are major constraints for success. In order to effectively address such disease-related issues, an improved understanding of the composition and dynamics of the microbial communities in the larval rearing tanks is required. This study used flow cytometry and molecular microbial techniques (clone libraries and denaturing gradient gel electrophoresis (DGGE)) to quantify and characterise the microbial community of the water column in the early stages (developmental stage I-II) of a P. ornatus larval rearing system. DGGE analysis of a 5000 L larval rearing trial demonstrated a dynamic microbial community with distinct changes in the community structure after initial stocking (day I to day 2) and from day 4 to day 5, after which the structure was relatively stable. Flow cytometry analysis of water samples taken over the duration of the trial demonstrated a major increase in bacterial load leading up to and peaking on the first day of the initial larval moult (day 7), before markedly decreasing prior to when > 50% of larvae moulted (day 9). A clone library of a day 10 water sample taken following a mass larval mortality event reflected high microbial diversity confirmed by statistical analysis indices. Sequences retrieved from both clone library and DGGE analyses were dominated by gamma- and alpha-Proteobacteria affiliated organisms with additional sequences affiliated with beta- and epsilon-Proteobacteria, Bacteroidetes, Cytophagales and Chlamydiales groups. Vibrio affiliated species were commonly retrieved in the clone library, though absent from DGGE analysis.

The microbial ecology of cow slurry in vermiculture beds

The suitability of cow slurry as a substrate for vermicomposting by Eisenia fetida was investigated. Particular attention was given to the effects of the earthworm on the decomposition and stabilisation of the slurry; and to the interactions between E. fetida and the microflora of the substrate. Assessment of the chemical and microbiological changes in cow slurry stored under forced aeration, and subsequently in shallow trays, showed that neither method was suitable for the treatment of slurry. A comparison of two methods of vermicomposting showed that top-feeding of slurry was more efficient in promoting earthworm growth and cocoon production than the mixing of slurry with solid materials. Management practices were found to have an important influence on the efficiency of the process. An investigation o:f the effect of E. fetida. on the decomposition of slurry indicated that the presence of this earthworm enhanced the stabilisation of the substrate and increased the plant-available nitrogen content. Specific nutritional interactions were observed between E. fetida and micro-organisms in sand/cellulose microcosms. The earthworms were found to be feeding directly upon the cells of certain micro-organisms. Other species were found to be toxic to E. fetida.. A technique was developed :for the production of axenic E. fetida., and the use of such earthworms in :feeding experiments confirmed the importance of some micro-organisms in earthworm nutrition. The seeding of vermiculture beds with one such micro-organism stimulated earthworm growth and consumption of the substrate. Vermicomposted mixtures of cow slurry and spent mushroom compost were shown to have potential application as casing materials in mushroom cultivation. The findings of this study indicate the suitability of vermicomposting as a method for the stabilisation of intensively-produced cow slurry, and give some indication of the importance of micro-organisms in the nutrition of E. fetida.

Limitations of the widely used GAM42a and BET42a probes targeting bacteria in the Gammaproteobacteria radiation

The 23S rRNA-targeted probes GAM42a and BET42a provided equivocal results with the uncultured gammaproteobacterium 'Candidatus Competibacter phosphatis' where some cells bound GAM42a and other cells bound BET42a in fluorescence in situ hybridization (FISH) experiments. Probes GAM42a and BET42a span positions 1027-1043 in the 23S rRNAand differ from each other by one nucleotide at position 1033. Clone libraries were prepared from PCR products spanning the 16S rRNA genes, intergenic spacer region and 23S rRNA genes from two mixed cultures enriched in 'Candidatus C. phosphatis'. With individual clone inserts, the 16S rDNA portion was used to confirm the source organism as 'Candidatus C. phosphatis' and the 23S rDNA portion was used to determine the sequence of the GAM42a/BET42a probe target region. Of the 19 clones sequenced, 8 had the GAM42a probe target (T at position 1033) and 11 had G at position 1033, the only mismatch with GAM42a. However, none of the clones had the BET42a probe target (A at 1033). Non-canonical base-pairing between the 23S rRNA of 'Candidatus C. phosphatis' with G at position 1033 and GAM42a (G-A) or BET42a (G-T) is likely to explain the probing anomalies. A probe (GAM42_C1033) was optimized for use in FISH, targeting cells with G at position 1033, and was found to highlight not only some 'Candidatus C. phosphatis' cells, but also other bacteria. This demonstrates that there are bacteria in addition to 'Candidatus C. phosphatis' with the GAM42_C1033 probe target and not the BET42a or GAM42a probe target.