Donald C. Liu

Redefining the Role of Intestinal Microbes in the Pathogenesis of Necrotizing Enterocolitis

Neonatal necrotizing enterocolitis (NEC) remains an important cause of morbidity and mortality among very low birth weight infants. It has long been suspected that intestinal microbes contribute to the pathogenesis of NEC, but the details of this relationship remain poorly understood. Recent advances in molecular biology and enteric microbiology have improved our ability to characterize intestinal microbes from infants with NEC and from healthy unaffected newborns. The lack of diversity within the neonatal intestine makes it possible to study gut microbial communities at a high level of resolution not currently possible in corresponding studies of the adult intestinal tract. Here, we summarize clinical and laboratory evidence that supports the hypothesis that NEC is a microbe-mediated disorder. In addition, we detail recent technologic advances that may be harnessed to perform high-throughput, comprehensive studies of the gut microbes of very low birth weight infants. Methods for characterizing microbial genotype are discussed, as are methods of identifying patterns of gene expression, protein expression, and metabolite production. Application of these technologies to biological samples from affected and unaffected newborns may lead to advances in the care of infants who are at risk for the unabated problem of NEC.

Red death in Caenorhabditis elegans caused by Pseudomonas aeruginosa PAO1

During host injury, Pseudomonas aeruginosa can be cued to express a lethal phenotype within the intestinal tract reservoir—a hostile, nutrient scarce environment depleted of inorganic phosphate. Here we determined if phosphate depletion activates a lethal phenotype in P. aeruginosa during intestinal colonization. To test this, we allowed Caenorhabditis elegans to feed on lawns of P. aeruginosa PAO1 grown on high and low phosphate media. Phosphate depletion caused PAO1 to kill 60% of nematodes whereas no worms died on high phosphate media. Unexpectedly, intense redness was observed in digestive tubes of worms before death. Using a combination of transcriptome analyses, mutants, and reporter constructs, we identified 3 global virulence systems that were involved in the “red death” response of P. aeruginosa during phosphate depletion; they included phosphate signaling (PhoB), the MvfR–PQS pathway of quorum sensing, and the pyoverdin iron acquisition system. Activation of all 3 systems was required to form a red colored PQS+Fe3+ complex which conferred a lethal phenotype in this model. When pyoverdin production was inhibited in P. aeruginosa by providing excess iron, red death was attenuated in C. elegans and mortality was decreased in mice intestinally inoculated with P. aeruginosa. Introduction of the red colored PQS+Fe3+ complex into the digestive tube of C. elegans or mouse intestine caused mortality associated with epithelial disruption and apoptosis. In summary, red death in C. elegans reveals a triangulated response between PhoB, MvfR–PQS, and pyoverdin in response to phosphate depletion that activates a lethal phenotype in P. aeruginosa.

Lymphotoxin regulates commensal responses to enable diet-induced obesity

Microbiota are essential for weight gain in mouse models of diet-induced obesity (DIO), but the pathways that cause the microbiota to induce weight gain are unknown. We report that mice deficient in lymphotoxin, a key molecule in gut immunity, were resistant to DIO. Ltbr−/− mice had different microbial community composition compared to their heterozygous littermates, including an overgrowth of segmented filamentous bacteria (SFB). Furthermore, cecal transplantation conferred leanness to germ-free recipients. Housing Ltbr−/− mice with their obese siblings rescued weight gain in Ltbr−/− mice, demonstrating the communicability of the obese phenotype. Ltbr−/− mice lacked interleukin 23 (IL-23) and IL-22, which can regulate SFB. Mice deficient in these pathways also resisted DIO, demonstrating that intact mucosal immunity guides diet-induced changes to the microbiota to enable obesity.

Gut microbial gene expression in mother-fed and formula-fed piglets.

Background Effects of diet on the structure and function of gut microbial communities in newborn infants are poorly understood. High-resolution molecular studies are needed to definitively ascertain whether gut microbial communities are distinct in milk-fed and formula-fed infants. Methodology/Principal Findings Pyrosequencing-based whole transcriptome shotgun sequencing (RNA-seq) was used to evaluate community wide gut microbial gene expression in 21 day old neonatal piglets fed either with sows milk (mother fed, MF; n = 4) or with artificial formula (formula fed, FF; n = 4). Microbial DNA and RNA were harvested from cecal contents for each animal. cDNA libraries and 16S rDNA amplicons were sequenced on the Roche 454 GS-FLX Titanium system. Communities were similar at the level of phylum but were dissimilar at the level of genus; Prevotella was the dominant genus within MF samples and Bacteroides was most abundant within FF samples. Screened cDNA sequences were assigned functional annotations by the MG-RAST annotation pipeline and based upon best-BLASTX-hits to the NCBI COG database. Patterns of gene expression were very similar in MF and FF animals. All samples were enriched with transcripts encoding enzymes for carbohydrate and protein metabolism, as well as proteins involved in stress response, binding to host epithelium, and lipopolysaccharide metabolism. Carbohydrate utilization transcripts were generally similar in both groups. The abundance of enzymes involved in several pathways related to amino acid metabolism (e.g., arginine metabolism) and oxidative stress response differed in MF and FF animals. Conclusions/Significance Abundant transcripts identified in this study likely contribute to a core microbial metatranscriptome in the distal intestine. Although microbial community gene expression was generally similar in the cecal contents of MF and FF neonatal piglets, several differentially abundant gene clusters were identified. Further investigations of gut microbial gene expression will contribute to a better understanding of normal and abnormal enteric microbiology in animals and humans.

Pseudomonas aeruginosa virulence expression is directly activated by morphine and is capable of causing lethal gut-derived sepsis in mice during chronic morphine administration.

Objective: This study was designed to examine the effect of morphine administration on the intestinal mucus barrier and determine its direct effect on the virulence and lethality of Pseudomonas aeruginosa, one of the most frequent pathogens to colonize the gut of critically ill patients. Background Data: Surgical injury is associated with significant exposure of host tissues to morphine from both endogenous release and its use as a potent analgesic agent. Morphine use in surgical patients exposed to extreme physiologic stress is well established to result in increased infection risk. Although morphine is a known immunosuppressant, whether it directly induces virulence expression and lethality in microbes that colonize the human gut remains unknown. Methods: Mice were implanted with a slow release morphine or placebo pellet with and without intestinal inoculation of P. aeruginosa created by direct cecal injection. Mucus production and epithelial integrity was assessed in cecal tissue via Alcian blue staining and histologic analysis. In vivo and in vitro P. aeruginosa virulence expression was examined using reporter strains tagged to the epithelial barrier disrupting protein PA-I lectin. P. aeruginosa chemotaxis toward morphine was also assayed in vitro. Finally, the direct effect of morphine to induce PA-I lectin expression was determined in the absence and presence of methylnaltrexone, a &mgr; opioid receptor antagonist. Results: Mice intestinally inoculated with P. aeruginosa and implanted with a morphine pellet demonstrated significant suppression of intestinal mucus, disrupted intestinal epithelium, and enhanced mortality; whereas exposure of mice to either systemic morphine or intestinal P. aeruginosa alone enhanced intestinal mucus without mortality, suggesting a shift in P. aeruginosa during morphine exposure to a mucus suppressing, barrier disrupting, and lethal phenotype. Direct exposure of P. aeruginosa to morphine in vitro confirmed that morphine can transform P. aeruginosa to a more virulent phenotype that is attenuated in part by methylnaltrexone. Conclusions: Morphine administration shifts intestinal P. aeruginosa to express a virulent phenotype and may play a role in its ability to causes lethal gut-derived sepsis in a susceptible host.

Outcomes in pediatric patients undergoing straight vs J pouch ileoanal anastomosis: a multicenter analysis

BACKGROUND Outcomes remain controversial for patients undergoing straight (SIAA) vs J pouch (JPAA) ileoanal anastomosis, particularly in children where fewer such cases are performed. Our 3 centers have had extensive experience with both techniques. Thus, we had the unique opportunity to compare outcomes within the same centers. METHODS We retrospectively analyzed 250 children after proctocolectomy with either SIAA or JPAA, for the first 3 years after pull-through. A functional stooling score was developed to further assess outcomes. Data were analyzed using chi(2) tests and generalized linear mixed models for repeated measures. RESULTS Two hundred three patients had sufficient data for complete analysis (42% males; mean surgery age, 15 +/- 7years). Surgical indications were ulcerative colitis (168) and familial adenomatoid polyposis (35). Surgical procedures included SIAA (112) and JPAA (91). Daytime and nighttime stooling frequencies were significantly higher (P < .013) for SIAA patients at 1 to 24 months after pull-through; however, stooling frequencies began approximating each other by this time. Symptomatic pouchitis (compared to enteritis after SIAA) was significantly higher in JPAA patients (odds ratio, 4.5; confidence interval, 2.32-8.72). Frequency of pouchitis declined with time. There was no significant difference in the incidence of surgical complications between the 2 groups. Finally, continence rates were strikingly good in both groups compared to previously reported series. CONCLUSION Straight ileoanal anastomosis and JPAA are associated with considerable morbidity; SIAA has higher stool frequency and JPAA has increased pouchitis. Over time, we found that problems improved, and functional stooling scores became similar. JPAA had consistently lower stool frequency and better continence rates; however, these differences were small and may have minimal clinical significance. In addition, such differences need to be balanced against the high rate of pouchitis with JPAA. Continence was excellent regardless of the technique.

Improved infant swallowing after gastroesophageal reflux disease treatment: A function of improved laryngeal sensation?

Objective: The objective of this study was to describe improvements in pediatric swallowing after gastroesophageal reflux treatment.

Gram Negative Bacteria Are Associated with the Early Stages of Necrotizing Enterocolitis

Introduction Necrotizing enterocolitis (NEC) affects 5–10% of infants born weighing less than 1500 g. Most models of NEC recapitulate late-stage disease with gut necrosis and elevated inflammatory mediators. Evaluation of NEC at earlier, less lethal stages of disease will allow investigation of initial disease triggers and may advance our understanding of temporal relationships between factors implicated in NEC pathogenesis. In this manuscript, we describe our investigation of early NEC and test the hypothesis that bacteria and inflammatory mediators differ between animals with early NEC and disease free animals. Methods On DOL7 C3HeB/FeJ pups were fed liquid formula with 1×104 Streptococcus thoraltensis, Serratia marcescens, and Pseudomonas aeruginosa every 3 h. To initiate NEC, pups underwent asphyxia (100% N2 for 90 s) and hypothermia (4°C for 10 min) after feeding. Pups were euthanized at 72 h. Intestines were collected for histologic NEC scoring and DNA/RNA extraction. Bacterial populations were identified by 16S rRNA pyrosequencing and principal component analysis (PCA). RNA isolates underwent QRT-PCR for Toll-like Receptor 4 (TLR4) and inducible nitric oxide synthase (iNOS). Results Despite histologic, intestinal damage in mice with NEC, no gross necrosis was observed suggesting early disease. QRT-PCR yielded no difference between groups in TLR4 or iNOS mRNA levels. PCA demonstrated relative clustering of microbial communities based on presence or absence of NEC. 16S pyrosequencing demonstrated similar phyla between groups (Firmicutes and Proteobacteria predominated in all animals). However, the colonic microbiota of animals with NEC had more Citrobacter (p<0.01), Klebsiella (p<0.05), and Tatumella (p<0.05), while that of animals without NEC had more Streptococcus (p<0.01) and Enterococcus (p<0.01). Conclusion Citrobacter, Klebsiella, and Tatumella are associated with NEC. Differential colonic bacteria were identified despite the lack of inflammatory mediator elevation traditionally associated with NEC. This suggests a temporal relationship between bacteria and inflammatory mediators such that alterations in gut microbiota are associated with early NEC, while inflammatory mediator elevation is associated with advanced NEC.

The human microbiome and surgical disease.

Objective:The purpose of this review article is to summarize what is currently known about microbes associated with the human body and to provide examples of how this knowledge impacts the care of surgical patients. Background:Pioneering research over the past decade has demonstrated that human beings live in close, constant contact with dynamic communities of microbial organisms. This new reality has wide-ranging implications for the care of surgical patients. Methods and Results:Recent advances in the culture-independent study of the human microbiome are reviewed. To illustrate the translational relevance of these studies to surgical disease, we discuss in detail what is known about the role of microbes in the pathogenesis of obesity, gastrointestinal malignancies, Crohn disease, and perioperative complications including surgical site infections and sepsis. The topics of mechanical bowel preparation and perioperative antibiotics are also discussed. Conclusions:Heightened understanding of the microbiome in coming years will likely offer opportunities to refine the prevention and treatment of a wide variety of surgical conditions.

Laparoscopic versus open fundoplication in infants

BACKGROUND Laparoscopic esophagogastric fundoplication is an effective treatment for severe gastroesophageal reflux disease (GERD), although its role in the very young is still largely undetermined. We review our surgical outcome in infants with severe GERD, comparing laparoscopic (LNF) with open (ONF) Nissen fundoplication. METHODS This study reviewed 55 consecutive Nissen fundoplications performed for GERD on infants less than 1 year old at our institution between January 1996 and June 2000. The follow-up period for LNF averaged 14.2 months (range, 3.3-42 months), as compared with 16.5 months (range, 1-37.1 months) for ONF (p was not significant, t-test). Surgical outcome was compared in terms of the following parameters: average operative time, times to initiation and completion of feeding schedule, postoperative complications, and recurrence rates. RESULTS For the study, 53 infants were divided into two groups: LNF (n = 39; 73.6%) and ONF (n = 14; 26.4%). The average operating time for LNF was 120 +/- 24 min (range, 60-195 min), as compared with 91 +/- 21 min (range, 60-135 min) for ONF (p < 0.05, t-test). Time to initiation of postoperative feeding schedule was 1.3 +/- 0.3 days for LNF, as compared with 3 +/- 0.9 days for ONF (p < 0.05, t-test). Full feedings were reached in 1.7 +/- 0.6 days for LNF, as compared with 1.3 +/- 0.9 for ONF (p was not significant, t-test). During the short-term follow-up period, recurrent reflux developed in 2/14 ONF patients (14.3%) as compared with 1/39 LNF patients (2.6%) (p < 0.05). CONCLUSIONS We conclude that in addition to sparing infants the morbidity of celiotomy, laparoscopic Nissen fundoplication had a surgical outcome comparable to that of traditional open fundoplication in infants with severe GERD. Importantly, resumption of goal nutritional regimens was equally efficient in both groups.