Soils are inhabited by many bacteria from phylogenetic groups that are poorly studied because representatives are rarely isolated in cultivation studies. Part of the reason for the failure to cultivate these bacteria is the low frequency with which bacterial cells in soil form visible colonies when inoculated onto standard microbiological media, resulting in low viable counts. We investigated the effects of three factors on viable counts, assessed as numbers of CFU on solid media, and on the phylogenetic groups to which the isolated colony-forming bacteria belong. These factors were inoculum size, growth medium, and incubation time. Decreasing the inoculum size resulted in significant increases in the viable count but did not appear to affect colony formation by members of rarely isolated groups. Some media that are traditionally used for soil microbiological studies returned low viable counts and did not result in the isolation of members of rarely isolated groups. Newly developed media, in contrast, resulted in high viable counts and in the isolation of many members of rarely isolated groups, regardless of the inoculum size. Increased incubation times of up to 3 months allowed the development of visible colonies of members of rarely isolated groups in conjunction with the use of appropriate media. Once isolated, pure cultures of members of rarely isolated groups took longer to form visible colonies than did members of commonly isolated groups. Using these new media and extended incubation times, we were able to isolate many members of the phyla Acidobacteria (subdivisions 1, 2, 3, and 4), Gemmatimonadetes, Chloroflexi, and Planctomycetes (including representatives of the previously uncultured WPS-1 lineage) as well as members of the subclasses Rubrobacteridae and Acidimicrobidae of the phylum Actinobacteria.

Most soil bacteria belong to family-level phylogenetic groups with few or no known cultivated representatives. We cultured a collection of 350 isolates from soil by using simple solid media in petri dishes. These isolates were assigned to 60 family-level groupings in nine bacterial phyla on the basis of a comparative analysis of their 16S rRNA genes. Ninety-three (27%) of the isolates belonged to 20 as-yet-unnamed family-level groupings, many from poorly studied bacterial classes and phyla. They included members of subdivisions 1, 2, 3, and 4 of the phylum Acidobacteria, subdivision 3 of the phylum Verrucomicrobia, subdivision 1 of the phylum Gemmatimonadetes, and subclasses Acidimicrobidae and Rubrobacteridae of the phylum Actinobacteria. In addition, members of 10 new family-level groupings of subclass Actinobacteridae of the phylum Actinobacteria and classes Alphaproteobacteria, Betaproteobacteria, and Gammaproteobacteria of the phylum Proteobacteria were obtained. The high degree of phylogenetic novelty and the number of isolates affiliated with so-called unculturable groups show that simple cultivation methods can still be developed further to obtain laboratory cultures of many phylogenetically novel soil bacteria.For over 80 years it has been known that there is a large discrepancy between the number of bacterial colonies that form on solid media when soil is used as an inoculum and the total number of bacterial cells actually present in that same soil (9,23,40). This discrepancy has limited our understanding of the species diversity of soil bacterial communities. In the past decade, this limitation has been partially overcome through the application of molecular ecological techniques. In particular, comparative analysis of 16S rRNAs or 16S rRNA genes derived from nucleic acids extracted directly from soil has revealed the presence of many new groups of bacteria that were previously undetected in cultivation studies (2, 11-13, 16, 19, 21, 26, 28, 32, 35). Some of these groups appear to be important within soils, at least in terms of relative abundance of 16S rRNAs or 16S rRNA genes. However, these numerically abundant bacteria are rarely, if ever, isolated in cultivation experiments, which instead tend to result in the isolation of bacteria that appear to be minor components of the soil bacterial community (1,12,16,35). As a consequence, traditional cultivation techniques such as plate counting methods have been increasingly considered inadequate, and new, more sophisticated techniques have been developed for the isolation of novel bacteria from complex microbial habitats. These methods include the use of micromanipulators and optical (laser) tweezers (14), the construction of simulated natural environments (24), and cell encapsulation in gel microdroplets (43). Such methods are specialized and unlikely to be adopted by the broader scientific community, and so the challenge of obtaining cultures of the large number of "unculturable" bacterial groups seems a daunting one. The only avenues currently availab...

A more diverse intestinal microbiota in the first week of life is associated with a reduced risk of subsequent eczema in infants at increased risk of allergic disease. Interventions that enhance microbial diversity in early life may provide an effective means for the prevention of eczema in high-risk infants.

We have recently shown that maternal administration of Lactobacillus rhamnosus GG (LGG) during late pregnancy can have beneficial effects on the early development of infant gut microbiota, promoting a bifidobacteria profile similar to that of a healthy breastfed infant. It is uncertain, however, whether such probiotic supplementation could influence the diversity of infant gut microbiota. We investigated the effect of pre-natal LGG on gut microbial diversity in the early post-natal period. Day-7 faecal samples were collected from 98 infants at high risk of allergic disease, whose mothers participated in a pre-natal probiotic eczema prevention study. Faecal microbial diversity was assessed by terminal restriction fragment length polymorphism using restriction enzymes Sau96I and AluI. A greater number of peaks represent greater diversity of bacterial communities. Administration of LGG to mothers during late pregnancy had no effects on the mean number of peaks in faecal samples from 1-wk-old infants as compared to placebo (AluI 14.4 vs. 15.5, p = 0.17, 95% CI -0.4, 2.5; Sau96I 17.3 vs. 15.8, p = 0.15, 95% CI -3.5, 0.5). Prenatal LGG failed to modulate diversity of early infant gut microbiota despite promoting a beneficial bifidobacteria profile.

Infant colic is a distressing condition of unknown etiology. An aberrant gastrointestinal microbiota has been associated, and Lactobacillus reuteri supplementation has been shown to reduce crying and/or fussing time (‘crying time’) in some infants with colic. The relationship between L. reuteri gut colonization and crying time has not been examined. We investigated the relationship between L. reuteri colonization and fecal microbiota (microbial diversity and Escherichia coli), intestinal inflammation, and crying time in infants with colic, using a subset of 65 infants from the Baby Biotics trial, which randomized healthy term infants aged <13 weeks with infant colic to receive probiotic L. reuteri DSM 17938 (1 × 108 colony forming units) or placebo daily for 28 days. We observed an overall reduction in median crying time, regardless of L. reuteri colonization status (n = 14 colonized). There were no differences in E. coli colonization rates or densities, microbial diversity or intestinal inflammation by L. reuteri colonization status. We found that L. reuteri density positively correlated with crying time, and E. coli density negatively correlated with microbial diversity. As density of L. reuteri was associated with increased crying time, L. reuteri supplementation may not be an appropriate treatment for all infants with colic.