The authors note that on page 12527, left column, second full paragraph, line 7, "Sequences meeting the above criteria were further classified by the Ribosomal Database Project (RDP) Naive Bayesian Classifier version 2.5 using training set 9 (46) from phylum to genus level." should instead appear as "Sequences meeting the above criteria were further classified by the Ribosomal Database Project (RDP) Naive Bayesian Classifier version 2.2 using training set 6 (46) from phylum to genus level."www.pnas.org/cgi

Objective The technology for the growth of human intestinal epithelial cells is rapidly progressing. An exciting possibility is that this system could serve as a platform for individualized medicine and research. However, to achieve this goal, human epithelial culture must be enhanced so that biopsies from individuals can be used to reproducibly generate cell lines in a short time frame so that multiple, functional assays can be performed (i.e., barrier function and host-microbial interactions). Design We created a large panel of human gastrointestinal epithelial cell lines (n = 65) from patient biopsies taken during routine upper and lower endoscopy procedures. Proliferative stem/progenitor cells were rapidly expanded using a high concentration of conditioned media containing the factors critical for growth (Wnt3a, R-spondin and Noggin). A combination of lower conditioned media concentration and Notch inhibition was used to differentiate these cells for additional assays. Results We obtained epithelial lines from all accessible tissue sites within two weeks of culture. The intestinal cell lines were enriched for stem cell markers and rapidly grew as spheroids that required passage at 1:3–1:4 every 3 days. Under differentiation conditions, intestinal epithelial spheroids showed region-specific development of mature epithelial lineages. These cells formed functional, polarized monolayers covered by a secreted mucus layer when grown on Transwell membranes. Using two-dimensional culture, these cells also demonstrated novel adherence phenotypes with various strains of pathogenic Escherichia coli. Conclusion This culture system will facilitate the study of inter-individual, functional studies of human intestinal epithelial cells, including host-microbial interactions.

Summary Background Gut bacteria might predispose to or protect from necrotising enterocolitis, a severe illness linked to prematurity. In this observational prospective study we aimed to assess whether one or more bacterial taxa in the gut differ between infants who subsequently develop necrotising enterocolitis (cases) and those who do not (controls). Methods We enrolled very low birthweight (1500 g and lower) infants in the primary cohort (St Louis Children’s Hospital) between July 7, 2009, and Sept 16, 2013, and in the secondary cohorts (Kosair Children’s Hospital and Children’s Hospital at Oklahoma University) between Sept 12, 2011 and May 25, 2013. We prospectively collected and then froze stool samples for all infants. Cases were defined as infants whose clinical courses were consistent with necrotising enterocolitis and whose radiographs fulfilled criteria for Bell’s stage 2 or 3 necrotising enterocolitis. Control infants (one to four per case; not fixed ratios) with similar gestational ages, birthweight, and birth dates were selected from the population after cases were identified. Using primers specific for bacterial 16S rRNA genes, we amplified and then pyrosequenced faecal DNA from stool samples. With use of Dirichlet multinomial analysis and mixed models to account for repeated measures, we identified host factors, including development of necrotising enterocolitis, associated with gut bacterial populations. Findings We studied 2492 stool samples from 122 infants in the primary cohort, of whom 28 developed necrotising enterocolitis; 94 infants were used as controls. The microbial community structure in case stools differed significantly from those in control stools. These differences emerged only after the first month of age. In mixed models, the time-by-necrotising-enterocolitis interaction was positively associated with Gammaproteobacteria (p=0·0010) and negatively associated with strictly anaerobic bacteria, especially Negativicutes (p=0·0019). We studied 1094 stool samples from 44 infants in the secondary cohorts. 18 infants developed necrotising enterocolitis (cases) and 26 were controls. After combining data from all cohorts (166 infants, 3586 stools, 46 cases of necrotising enterocolitis), there were increased proportions of Gammaproteobacteria (p=0·0011) and lower proportions of both Negativicutes (p=0·0013) and the combined Clostridia–Negativicutes class (p=0·0051) in infants who went on to develop necrotising enterocolitis compared with controls. These associations were strongest in both the primary cohort and the overall cohort for infants born at less than 27 weeks’ gestation. Interpretation A relative abundance of Gammaproteobacteria (ie, Gram-negative facultative bacilli) and relative paucity of strict anaerobic bacteria (especially Negativicutes) precede necrotising enterocolitis in very low birthweight infants. These data offer candidate targets for interventions to prevent necrotising enterocolitis, at least among infants born at less than 27 weeks’ gestation. Funding Nation...

As it descended from Escherichia coli O55:H7, Shiga toxin (Stx)-producing E. coli (STEC) O157:H7 is believed to have acquired, in sequence, a bacteriophage encoding Stx2 and another encoding Stx1. Between these events, sorbitol-fermenting E. coli O157:H ؊ presumably diverged from this clade. We employed PCR and sequence analyses to investigate sites of bacteriophage integration into the chromosome, using evolutionarily informative STEC to trace the sequence of acquisition of elements encoding Stx. Contrary to expectations from the two currently sequenced strains, truncated bacteriophages occupy yehV in almost all E. coli O157:H7 strains that lack stx 1 (stx 1 -negative strains). Two truncated variants were determined to contain either GTT or TGACTGTT sequence, in lieu of 20,214 or 18,895 bp, respectively, of the bacteriophage central region. A single-nucleotide polymorphism in the latter variant suggests that recombination in that element extended beyond the inserted octamer. An stx 2 bacteriophage usually occupies wrbA in stx 1 ؉ /stx 2 ؉ E. coli O157:H7, but wrbA is unexpectedly unoccupied in most stx 1 -negative/stx 2 ؉ E. coli O157:H7 strains, the presumed progenitors of stx 1 ؉ /stx 2 ؉ E. coli O157:H7. Trimethoprim-sulfamethoxazole promotes the excision of all, and ciprofloxacin and fosfomycin significantly promote the excision of a subset of complete and truncated stx bacteriophages from the E. coli O157:H7 strains tested; bile salts usually attenuate excision. These data demonstrate the unexpected diversity of the chromosomal architecture of E. coli O157:H7 (with novel truncated bacteriophages and multiple stx 2 bacteriophage insertion sites), suggest that stx 1 acquisition might be a multistep process, and compel the consideration of multiple exogenous factors, including antibiotics and bile, when chromosome stability is examined.Shiga toxins 1 and 2 (Stx1 and Stx2) are cardinal virulence factors of Escherichia coli O157:H7. Stx1 is nearly identical to Stx, the principal extracellular cytotoxin of Shigella dysenteriae serotype 1 (8). Stx2 has 56% identity to Stx1 (36). The stx 1 and stx 2 A and B subunit genes exist as tandem open reading frames (ORFs) in the central portion of lambdoid bacteriophages in E. coli O157:H7 (19). In Sakai and EDL933, the two E. coli O157:H7 strains that have been completely sequenced, the bacteriophage that encodes Stx1 is integrated into yehV (57), which encodes a protein that positively regulates curli expression (7), and is flanked by duplications of CCCTGT CACGTTACGCGCGTG. The bacteriophage that encodes Stx2 is integrated into wrbA (32, 40), which encodes a novel multimeric flavodoxin-like protein (13), and is flanked by duplications of GACATATTGAAAC. Almost all E. coli O157:H7 strains possess stx 2 , and approximately three-quarters contain, in addition, stx 1 (strains lacking stx 1 are referred to hereafter as stx 1 -negative strains) (23,38,45). Most human non-O157:H7 Stx-producing E. coli (STEC) strains possess stx 1 but lack stx 2 (5, 6, 28, 51). Except for an Stx2...