Brian Firek

Microbes in the neonatal intensive care unit resemble those found in the gut of premature infants

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.

Intestinal epithelial Toll-like receptor 4 regulates goblet cell development and is required for necrotizing enterocolitis in mice.

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 TLR4(loxp/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.

Crosslinking renders bacteriophage HK97 capsid maturation irreversible and effects an essential stabilization

In HK97 capsid maturation, structural change (‘expansion’) is accompanied by formation of covalent crosslinks, connecting residue K169 in the ‘E‐loop’ of each subunit with N356 on another subunit. We show by complementation experiments with the K169Y mutant, which cannot crosslink, that crosslinking is an essential function. The precursor Prohead‐II passes through three expansion intermediate (EI) states en route to the end state, Head‐II. We investigated the effects of expansion and crosslinking on stability by differential scanning calorimetry of wild‐type and K169Y capsids. After expansion, the denaturation temperature (Tp) of K169Y capsids is slightly reduced, indicating that their thermal stability is not enhanced, but crosslinking effects a major stabilization (ΔTp, +11°C). EI‐II is the earliest capsid to form crosslinks. Cryo‐electron microscopy shows that for both wild‐type and K169Y EI‐II, most E‐loops are in the ‘up’ position, 30 Å from the nearest N356: thus, crosslinking in EI‐II represents capture of mobile E‐loops in ‘down’ positions. At pH 4, most K169Y capsids remain as EI‐II, whereas wild‐type capsids proceed to EI‐III, suggesting that crosslink formation drives maturation by a Brownian ratchet mechanism.

Gut bacteria are rarely shared by co-hospitalized premature infants,regardless of necrotizing enterocolitis development

Premature infants are highly vulnerable to aberrant gastrointestinal tract colonization, a process that may lead to diseases like necrotizing enterocolitis. Thus, spread of potential pathogens among hospitalized infants is of great concern. Here, we reconstructed hundreds of high-quality genomes of microorganisms that colonized co-hospitalized premature infants, assessed their metabolic potential, and tracked them over time to evaluate bacterial strain dispersal among infants. We compared microbial communities in infants who did and did not develop necrotizing enterocolitis. Surprisingly, while potentially pathogenic bacteria of the same species colonized many infants, our genome-resolved analysis revealed that strains colonizing each baby were typically distinct. In particular, no strain was common to all infants who developed necrotizing enterocolitis. The paucity of shared gut colonizers suggests the existence of significant barriers to the spread of bacteria among infants. Importantly, we demonstrate that strain-resolved comprehensive community analysis can be accomplished on potentially medically relevant time scales. DOI: http://dx.doi.org/10.7554/eLife.05477.001

Lactobacillus rhamnosus HN001 decreases the severity of necrotizing enterocolitis in neonatal mice and preterm piglets: evidence in mice for a role of TLR9.

Necrotizing enterocolitis (NEC) is the leading cause of death from gastrointestinal disease in premature infants and develops partly from an exaggerated intestinal epithelial immune response to indigenous microbes. There has been interest in administering probiotic bacteria to reduce NEC severity, yet concerns exist regarding infection risk. Mechanisms of probiotic activity in NEC are unknown although activation of the microbial DNA receptor Toll-like receptor-9 (TLR9) has been postulated. We now hypothesize that the Gram-positive bacterium Lactobacillus rhamnosus HN001 can attenuate NEC in small and large animal models, that its microbial DNA is sufficient for its protective effects, and that protection requires activation of the Toll-like receptor 9 (TLR9). We now show that oral administration of live or UV-inactivated Lactobacillus rhamnosus HN001 attenuates NEC severity in newborn mice and premature piglets, as manifest by reduced histology score, attenuation of mucosal cytokine response, and improved gross morphology. TLR9 was required for Lactobacillus rhamnosus-mediated protection against NEC in mice, as the selective decrease of TLR9 from the intestinal epithelium reversed its protective effects. Strikingly, DNA of Lactobacillus rhamnosus HN001 reduced the extent of proinflammatory signaling in cultured enterocytes and in samples of resected human ileum ex vivo, suggesting the therapeutic potential of this probiotic in clinical NEC. Taken together, these findings illustrate that Lactobacillus rhamnosus HN001 is an effective probiotic for NEC via activation of the innate immune receptor TLR9 and that Lactobacillus rhamnosus DNA is sufficient for its protective effects, potentially reducing concerns regarding the infectious risk of this novel therapeutic approach.

Structure and Energetics of Encapsidated DNA in Bacteriophage HK97 Studied by Scanning Calorimetry and Cryo-electron Microscopy

Encapsidation of duplex DNA by bacteriophages represents an extreme case of genome condensation, reaching near-crystalline concentrations of DNA. The HK97 system is well suited to study this phenomenon in view of the detailed knowledge of its capsid structure. To characterize the interactions involved, we combined calorimetry with cryo-electron microscopy and native gel electrophoresis. We found that, as in other phages, HK97 DNA is organized in coaxially wound nested shells. When DNA-filled capsids (heads) are scanned in buffer containing 1 mM Mg(2+), DNA melting and capsid denaturation both contribute to the complex thermal profile between 82 degrees C and 96 degrees C. In other conditions (absence of Mg(2+) and lower ionic strength), DNA melting shifts to lower temperatures and the two events are resolved. Heads release their DNA at temperatures well below the onset of DNA melting or capsid denaturation. We suggest that, on heating, the internal pressure increases, causing the DNA to exit-probably via the portal vertex-while the capsid, although largely intact, sustains local damage that leads to an earlier onset of thermal denaturation. Heads differ structurally from empty capsids in the curvature of their protein shell, a change attributable to outwards pressure exerted by the DNA. We propose that this transition is sensed by the portal that is embedded in the capsid wall, whereupon the structure of the portal and its interactions with terminase, the packaging enzyme, are altered, thus signaling that packaging is at or approaching completion.

Mucosa-associated bacterial diversity in necrotizing enterocolitis.

Background Previous studies of infant fecal samples have failed to clarify the role of gut bacteria in the pathogenesis of NEC. We sought to characterize bacterial communities within intestinal tissue resected from infants with and without NEC. Methods 26 intestinal samples were resected from 19 infants, including 16 NEC samples and 10 non-NEC samples. Bacterial 16S rRNA gene sequences were amplified and sequenced. Analysis allowed for taxonomic identification, and quantitative PCR was used to quantify the bacterial load within samples. Results NEC samples generally contained an increased total burden of bacteria. NEC and non-NEC sample sets were both marked by high inter-individual variability and an abundance of opportunistic pathogens. There was no statistically significant distinction between the composition of NEC and non-NEC microbial communities. K-means clustering enabled us to identify several stable clusters, including clusters of NEC and midgut volvulus samples enriched with Clostridium and Bacteroides. Another cluster containing both NEC and non-NEC samples was marked by an abundance of Enterobacteriaceae and decreased diversity among NEC samples. Conclusions The results indicate that NEC is a disease without a uniform pattern of microbial colonization, but that NEC is associated with an abundance of strict anaerobes and a decrease in community diversity.

Identical bacterial populations colonize premature infant gut, skin, and oral microbiomes and exhibit different in situ growth rates

The initial microbiome impacts the health and future development of premature infants. Methodological limitations have led to gaps in our understanding of the habitat range and subpopulation complexity of founding strains, as well as how different body sites support microbial growth. Here, we used metagenomics to reconstruct genomes of strains that colonized the skin, mouth, and gut of two hospitalized premature infants during the first month of life. Seven bacterial populations, considered to be identical given whole-genome average nucleotide identity of >99.9%, colonized multiple body sites, yet none were shared between infants. Gut-associated Citrobacter koseri genomes harbored 47 polymorphic sites that we used to define 10 subpopulations, one of which appeared in the gut after 1 wk but did not spread to other body sites. Differential genome coverage was used to measure bacterial population replication rates in situ. In all cases where the same bacterial population was detected in multiple body sites, replication rates were faster in mouth and skin compared to the gut. The ability of identical strains to colonize multiple body sites underscores the habit flexibility of initial colonists, whereas differences in microbial replication rates between body sites suggest differences in host control and/or resource availability. Population genomic analyses revealed microdiversity within bacterial populations, implying initial inoculation by multiple individual cells with distinct genotypes. Overall, however, the overlap of strains across body sites implies that the premature infant microbiome can exhibit very low microbial diversity.

Acute appendicitis in children is associated with an abundance of bacteria from the phylum Fusobacteria

BACKGROUND Although luminal obstruction has traditionally been viewed as the underlying cause of appendicitis, recent evidence has suggested that the disease may result directly from invasion by specific pathogens, e.g. Fusobacterium nucleatum. The purpose of this study was to survey microbial communities within pediatric appendectomy specimens using a culture-independent approach. METHODS We performed 16S ribosomal gene sequence analysis to profile the microbiota present within luminal fluid obtained from 22 pediatric appendectomy specimens. These included 10 simple appendicitis cases, 5 perforated appendicitis cases, 2 interval appendectomies, and 5 incidental appendectomies. RESULTS Samples could be divided into 2 distinct clusters based upon the composition of the appendiceal bacterial communities. Appendicitis samples contained an increased abundance of Fusobacterium spp. and a reduced abundance of Bacteroides spp. relative to non-appendicitis cases. Appendicitis samples also contained variable amounts of other oral taxa such as Porphyromonas, Parvimonas, and Gemella, whereas these taxa were generally absent from non-appendicitis samples. CONCLUSIONS Acute appendicitis is associated with an abundance of Fusobacterium spp. and other pathogens commonly found in the oral cavity. Further research is needed to determine whether these organisms directly cause appendicitis or rather proliferate in the appendix as a secondary consequence of inflammation.

Mutational Analysis of a Conserved Glutamic Acid Required for Self-Catalyzed Cross-Linking of Bacteriophage HK97 Capsids

ABSTRACT The capsid of bacteriophage HK97 is stabilized by ∼400 covalent cross-links between subunits which form without any action by external enzymes or cofactors. Cross-linking only occurs in fully assembled particles after large-scale structural changes bring together side chains from three subunits at each cross-linking site. Isopeptide cross-links form between asparagine and lysine side chains on two subunits. The carboxylate of glutamic acid 363 (E363) from a third subunit is found ∼2.4 Å from the isopeptide bond in the partly hydrophobic pocket that contains the cross-link. It was previously reported without supporting data that changing E363 to alanine abolishes cross-linking, suggesting that E363 plays a role in cross-linking. This alanine mutant and six additional substitutions for E363 were fully characterized and the proheads produced by the mutants were tested for their ability to cross-link under a variety of conditions. Aspartic acid and histidine substitutions supported cross-linking to a significant extent, while alanine, asparagine, glutamine, and tyrosine did not, suggesting that residue 363 acts as a proton acceptor during cross-linking. These results support a chemical mechanism, not yet fully tested, that incorporates this suggestion, as well as features of the structure at the cross-link site. The chemically identical isopeptide bonds recently documented in bacterial pili have a strikingly similar chemical geometry at their cross-linking sites, suggesting a common chemical mechanism with the phage protein, but the completely different structures and folds of the two proteins argues that the phage capsid and bacterial pilus proteins have achieved shared cross-linking chemistry by convergent evolution.