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BackgroundThe source inoculum of gastrointestinal tract (GIT) microbes is largely influenced by delivery mode in full-term infants, but these influences may be decoupled in very low birth weight (VLBW, <1,500 g) neonates via conventional broad-spectrum antibiotic treatment. We hypothesize the built environment (BE), specifically room surfaces frequently touched by humans, is a predominant source of colonizing microbes in the gut of premature VLBW infants. Here, we present the first matched fecal-BE time series analysis of two preterm VLBW neonates housed in a neonatal intensive care unit (NICU) over the first month of life.ResultsFresh fecal samples were collected every 3 days and metagenomes sequenced on an Illumina HiSeq2000 device. For each fecal sample, approximately 33 swabs were collected from each NICU room from 6 specified areas: sink, feeding and intubation tubing, hands of healthcare providers and parents, general surfaces, and nurse station electronics (keyboard, mouse, and cell phone). Swabs were processed using a recently developed ‘expectation maximization iterative reconstruction of genes from the environment’ (EMIRGE) amplicon pipeline in which full-length 16S rRNA amplicons were sheared and sequenced using an Illumina platform, and short reads reassembled into full-length genes. Over 24,000 full-length 16S rRNA sequences were produced, generating an average of approximately 12,000 operational taxonomic units (OTUs) (clustered at 97% nucleotide identity) per room-infant pair. Dominant gut taxa, including Staphylococcus epidermidis, Klebsiella pneumoniae, Bacteroides fragilis, and Escherichia coli, were widely distributed throughout the room environment with many gut colonizers detected in more than half of samples. Reconstructed genomes from infant gut colonizers revealed a suite of genes that confer resistance to antibiotics (for example, tetracycline, fluoroquinolone, and aminoglycoside) and sterilizing agents, which likely offer a competitive advantage in the NICU environment.ConclusionsWe have developed a high-throughput culture-independent approach that integrates room surveys based on full-length 16S rRNA gene sequences with metagenomic analysis of fecal samples collected from infants in the room. The approach enabled identification of discrete ICU reservoirs of microbes that also colonized the infant gut and provided evidence for the presence of certain organisms in the room prior to their detection in the gut.

Previous studies in our laboratory have shown that the Staphylococcus aureus LytSR two-component regulatory system affects murein hydrolase activity and autolysis. A LytSR-regulated dicistronic operon has also been identified and shown to encode two potential membrane-associated proteins, designated LrgA and LrgB, hypothesized to be involved in the control of murein hydrolase activity. In the present study, a lrgAB mutant strain was generated and analyzed to test this hypothesis. Zymographic and quantitative analysis of murein hydrolase activity revealed that the lrgAB mutant produced increased extracellular murein hydrolase activity compared to that of the wild-type strain. Complementation of the lrgAB defect by providing the lrgAB genes in trans restored the wild-type phenotype, indicating that these genes confer negative control on extracellular murein hydrolase activity. In addition to these effects, the influence of the lrgAB mutation on penicillin-induced lysis and killing was examined. These studies demonstrated that the lrgAB mutation enhanced penicillininduced killing of cells approaching the stationary phase of growth, the time at which the lrgAB operon was shown to be maximally expressed. This effect of the lrgAB mutation on penicillin-induced killing was shown to be independent of cell lysis. In contrast, the lrgAB mutation did not affect penicillin-induced killing of cells growing in early-exponential phase, a time in which lrgAB expression was shown to be minimal. However, expression of the lrgAB operon in early-exponential-phase cells inhibited penicillin-induced killing, again independent of cell lysis. The data generated by this study suggest that penicillin-induced killing of S. aureus involves a novel regulator of murein hydrolase activity.

BACKGROUND & AIMS Little is known about factors that regulate intestinal epithelial differentiation; microbial recognition receptors such as Toll-like receptor (TLR)4 might be involved. We investigated whether intestinal TLR4 regulates epithelial differentiation and is involved in development of necrotizing enterocolitis (NEC) of the immature intestine. METHODS Mice with conditional disruption of TLR4 in the intestinal epithelium and TLR4 knockout (TLR4−/−) mice were generated by breeding TLR4loxp/loxp mice with villin-cre and Ella-cre, respectively. Enterocytes that did not express or overexpressed TLR4 were created by lentiviral or adenoviral transduction. Intestinal organoids were cultured on tissue matrices. Bile acids were measured by colorimetric assays, and microbial composition was determined by 16S pyrosequencing. NEC was induced in 7- to 10-day-old mice by induction of hypoxia twice daily for 4 days. RESULTS TLR4−/− mice and mice with enterocyte-specific deletion of TLR4 were protected from NEC; epithelial differentiation into goblet cells was increased via suppressed Notch signaling in the small intestinal epithelium. TLR4 also regulates differentiation of goblet cells in intestinal organoid and enterocyte cell cultures; differentiation was increased on deletion of TLR4 and restored when TLR4 was expressed ectopically. TLR4 signaling via Notch was increased in intestinal tissue samples from patients with NEC, and numbers of goblet cells were reduced. 16S pyrosequencing revealed that wild-type and TLR4-deficient mice had similar microbial profiles; increased numbers of goblet cells were observed in mice given antibiotics. TLR4 deficiency reduced levels of luminal bile acids in vivo, and addition of bile acids to TLR4-deficient cell cultures prevented differentiation of goblet cells. CONCLUSIONS TLR4 signaling and Notch are increased in intestinal tissues of patients with NEC and required for induction of NEC in mice. TLR4 prevents goblet cell differentiation, independently of the microbiota. Bile acids might initiate goblet cell development.