Volker Mai

Fecal microbiota in premature infants prior to necrotizing enterocolitis.

Intestinal luminal microbiota likely contribute to the etiology of necrotizing enterocolitis (NEC), a common disease in preterm infants. Microbiota development, a cascade of initial colonization events leading to the establishment of a diverse commensal microbiota, can now be studied in preterm infants using powerful molecular tools. Starting with the first stool and continuing until discharge, weekly stool specimens were collected prospectively from infants with gestational ages ≤32 completed weeks or birth weights≤1250 g. High throughput 16S rRNA sequencing was used to compare the diversity of microbiota and the prevalence of specific bacterial signatures in nine NEC infants and in nine matched controls. After removal of short and low quality reads we retained a total of 110,021 sequences. Microbiota composition differed in the matched samples collected 1 week but not <72 hours prior to NEC diagnosis. We detected a bloom (34% increase) of Proteobacteria and a decrease (32%) in Firmicutes in NEC cases between the 1 week and <72 hour samples. No significant change was identified in the controls. At both time points, molecular signatures were identified that were increased in NEC cases. One of the bacterial signatures detected more frequently in NEC cases (p<0.01) matched closest to γ-Proteobacteria. Although this sequence grouped to the well-studied Enterobacteriaceae family, it did not match any sequence in Genbank by more than 97%. Our observations suggest that abnormal patterns of microbiota and potentially a novel pathogen contribute to the etiology of NEC.

Intestinal Microbial Ecology in Premature Infants Assessed with Non-Culture-Based Techniques

OBJECTIVES To use high throughput techniques to analyze intestinal microbial ecology in premature neonates, who are highly susceptible to perturbations of the luminal environment associated with necrotizing enterocolitis (NEC) and late-onset sepsis. STUDY DESIGN With non-culture-based techniques, we evaluated intestinal microbiota shortly after birth and during hospitalization in 23 neonates born at 23 to 32 weeks gestational age. Microbiota compositions were compared in 6 preterm infants in whom NEC, signs of systemic inflammation, or both developed with matched control subjects by using 16S ribosomal RNA pyrosequencing. RESULTS Microbial DNA was detected in meconium, suggesting an intrauterine origin. Differences in diversity were detected in infants whose mothers intended to breast feed (P = .03), babies born to mothers with chorioamnionitis (P = .06), and in babies born at <30 weeks gestation (P = .03). A 16S ribosomal RNA sequence analysis detected Citrobacter-like sequences only in cases with NEC (3 of 4) and an increased frequency of Enterococcus-like sequences in cases and Klebsiella in control subjects (P = .06). The overall microbiota profiles in cases with NEC were not distinguishable from that in control subjects. CONCLUSIONS Microbial DNA in meconium of premature infants suggests prenatal influences.

Intestinal Dysbiosis and Depletion of Butyrogenic Bacteria in Clostridium difficile Infection and Nosocomial Diarrhea

ABSTRACT Clostridium difficile infection (CDI) causes nearly half a million cases of diarrhea and colitis in the United States each year. Although the importance of the gut microbiota in C. difficile pathogenesis is well recognized, components of the human gut flora critical for colonization resistance are not known. Culture-independent high-density Roche 454 pyrosequencing was used to survey the distal gut microbiota for 39 individuals with CDI, 36 subjects with C. difficile-negative nosocomial diarrhea (CDN), and 40 healthy control subjects. A total of 526,071 partial 16S rRNA sequence reads of the V1 to V3 regions were aligned with 16S databases, identifying 3,531 bacterial phylotypes from 115 fecal samples. Genomic analysis revealed significant alterations of organism lineages in both the CDI and CDN groups, which were accompanied by marked decreases in microbial diversity and species richness driven primarily by a paucity of phylotypes within the Firmicutes phylum. Normally abundant gut commensal organisms, including the Ruminococcaceae and Lachnospiraceae families and butyrate-producing C2 to C4 anaerobic fermenters, were significantly depleted in the CDI and CDN groups. These data demonstrate associations between the depletion of Ruminococcaceae, Lachnospiraceae, and butyrogenic bacteria in the gut microbiota and nosocomial diarrhea, including C. difficile infection. Mechanistic studies focusing on the functional roles of these organisms in diarrheal diseases and resistance against C. difficile colonization are warranted.

Diarrheagenic Escherichia coli Infection in Baltimore, Maryland, and New Haven, Connecticut

BACKGROUND Diarrhea remains a common complaint among US patients who seek medical attention. METHODS We performed a prospective study to determine the etiology of diarrheal illness among patients and control subjects of all ages presenting to the emergency departments and outpatient clinics of 2 large academic hospitals in Baltimore, Maryland, and New Haven, Connecticut. We used molecular methods to detect the presence of diarrheagenic Escherichia coli pathotypes, including enteroaggregative E. coli (EAEC), as well as Shiga toxin-producing, cytodetaching, enterotoxigenic and enteropathogenic E. coli. RESULTS Of the pathotypes sought, only EAEC was found in an appreciable proportion (4.5%) of case patients, and it was found more frequently among case patients than control subjects (P<.02). Surprisingly, EAEC was the most common bacterial cause of diarrhea in our population. EAEC was common in all age strata and was not associated with foreign travel or immunodeficiency. EAEC infection is frequently accompanied by fever and abdominal pain, though this did not happen more frequently in patients with EAEC infection than in patients with diarrhea due to other causes. CONCLUSIONS Our data suggest that EAEC infection should be considered among persons with diarrhea that does not yield another known etiologic agent.

Culture-independent identification of gut bacteria correlated with the onset of diabetes in a rat model

Bacteria associated with the onset of type 1 diabetes in a rat model system were identified. In two experiments, stool samples were collected at three time points after birth from bio-breeding diabetes-prone (BB-DP) and bio-breeding diabetes-resistant (BB-DR) rats. DNA was isolated from these samples and the 16S rRNA gene was amplified using universal primer sets. In the first experiment, bands specific to BB-DP and BB-DR genotypes were identified by automated ribosomal intergenic spacer analysis at the time of diabetes onset in BB-DP. Lactobacillus and Bacteroides strains were identified in the BB-DR- and BB-DP-specific bands, respectively. Sanger sequencing showed that the BB-DP and BB-DR bacterial communities differed significantly but too few reads were available to identify significant differences at the genus or species levels. A second experiment confirmed these results using higher throughput pyrosequencing and quantitative PCR of 16S rRNA with more rats per genotype. An average of 4541 and 3381 16S rRNA bacterial reads were obtained from each of the 10 BB-DR and 10 BB-DP samples collected at time of diabetes onset. Nine genera were more abundant in BB-DP whereas another nine genera were more abundant in BB-DR. Thirteen and eleven species were more abundant in BB-DP and BB-DR, respectively. An average of 23% and 10% of all reads could be classified at the genus and species levels, respectively. Quantitative PCR verified the higher abundance of Lactobacillus and Bifidobacterium in the BB-DR samples. Whether these changes are caused by diabetes or are involved in the development of the disease is unknown.

Diarrhea in young children from low-income countries leads to large-scale alterations in intestinal microbiota composition

BackgroundDiarrheal diseases continue to contribute significantly to morbidity and mortality in infants and young children in developing countries. There is an urgent need to better understand the contributions of novel, potentially uncultured, diarrheal pathogens to severe diarrheal disease, as well as distortions in normal gut microbiota composition that might facilitate severe disease.ResultsWe use high throughput 16S rRNA gene sequencing to compare fecal microbiota composition in children under five years of age who have been diagnosed with moderate to severe diarrhea (MSD) with the microbiota from diarrhea-free controls. Our study includes 992 children from four low-income countries in West and East Africa, and Southeast Asia. Known pathogens, as well as bacteria currently not considered as important diarrhea-causing pathogens, are positively associated with MSD, and these include Escherichia/Shigella, and Granulicatella species, and Streptococcus mitis/pneumoniae groups. In both cases and controls, there tend to be distinct negative correlations between facultative anaerobic lineages and obligate anaerobic lineages. Overall genus-level microbiota composition exhibit a shift in controls from low to high levels of Prevotella and in MSD cases from high to low levels of Escherichia/Shigella in younger versus older children; however, there was significant variation among many genera by both site and age.ConclusionsOur findings expand the current understanding of microbiota-associated diarrhea pathogenicity in young children from developing countries. Our findings are necessarily based on correlative analyses and must be further validated through epidemiological and molecular techniques.

A large-scale benchmark study of existing algorithms for taxonomy-independent microbial community analysis

Recent advances in massively parallel sequencing technology have created new opportunities to probe the hidden world of microbes. Taxonomy-independent clustering of the 16S rRNA gene is usually the first step in analyzing microbial communities. Dozens of algorithms have been developed in the last decade, but a comprehensive benchmark study is lacking. Here, we survey algorithms currently used by microbiologists, and compare seven representative methods in a large-scale benchmark study that addresses several issues of concern. A new experimental protocol was developed that allows different algorithms to be compared using the same platform, and several criteria were introduced to facilitate a quantitative evaluation of the clustering performance of each algorithm. We found that existing methods vary widely in their outputs, and that inappropriate use of distance levels for taxonomic assignments likely resulted in substantial overestimates of biodiversity in many studies. The benchmark study identified our recently developed ESPRIT-Tree, a fast implementation of the average linkage-based hierarchical clustering algorithm, as one of the best algorithms available in terms of computational efficiency and clustering accuracy.

Associations between dietary habits and body mass index with gut microbiota composition and fecal water genotoxicity: an observational study in African American and Caucasian American volunteers

BackgroundAfrican Americans (AA) suffer from an increased incidence and mortality of colorectal cancer (CRC). Environmental exposures including dietary habits likely contribute to a high burden of CRC, however, data on the dietary habits of AA is sparse. Diet might change the composition and the activities of the intestinal microbiota, in turn affecting fecal genotoxicity/mutagenicity that is thought to be associated with carcinogenesis.MethodsWe assessed dietary habits by food frequency questionnaire and by food records in 52 AA and 46 CA residents of the Eastern Shore of MD. Fecal microbiota composition was determined using 16S rRNA based methods and fecal genotoxicity measured using the Comet assay.ResultsAA reported an increased intake of heterocyclic amines and a decreased dietary intake of vitamins including vitamin D (p < 0.05) that correlated with differences in fecal microbiota composition but not fecal genotoxicity. Intake of dietary fiber, calcium, total fat and heterocyclic amines correlated with differences in microbiota composition. Total bacterial counts/g of stool and raw counts of Bacteroides were increased in AA. In contrast to a previous study, BMI was not associated with proportions of Bacteroides.ConclusionDietary habits of African Americans, including increased HCA intake and decreased vitamin D intake might at least partially contribute to CRC through modifications of gut microbiota composition that result in changes of the intestinal milieu.

Distortions in Development of Intestinal Microbiota Associated with Late Onset Sepsis in Preterm Infants

Late onset sepsis (LOS) is a major contributor to neonatal morbidity and mortality, especially in premature infants. Distortions in the establishment of normal gut microbiota, commensal microbes that colonize the digestive tract, might increase the risk of LOS via disruption of the mucosal barrier with resultant translocation of luminal contents. Correlation of distortions of the intestinal microbiota with LOS is a necessary first step to design novel microbiota-based screening approaches that might lead to early interventions to prevent LOS in high risk infants. Using a case/control design nested in a cohort study of preterm infants, we analyzed stool samples that had been prospectively collected from ten preterm infants with LOS and from 18 matched controls. A 16S rRNA based approach was utilized to compare microbiota diversity and identify specific bacterial signatures that differed in their prevalence between cases and controls. Overall α-diversity (Chao1) was lower in cases two weeks before (p<0.05) but not one week before or at the time of diagnosis of LOS. Overall microbiota structure (Unifrac) appeared distinct in cases 2 weeks and 1 week before but not at diagnosis (p<0.05). Although we detected few operational taxonomic units (OTUs) unique or enriched in cases, we found many OTUs common in controls that were lacking in cases (p<0.01). Bifidobacteria counts were lower in cases at all time points. Our results support the hypothesis that a distortion in normal microbiota composition, and not an enrichment of potential pathogens, is associated with LOS in preterm infants.

Intestinal Microbial Ecology and Environmental Factors Affecting Necrotizing Enterocolitis

Necrotizing enterocolitis (NEC) is the most devastating intestinal disease affecting preterm infants. In addition to being associated with short term mortality and morbidity, survivors are left with significant long term sequelae. The cost of caring for these infants is high. Epidemiologic evidence suggests that use of antibiotics and type of feeding may cause an intestinal dysbiosis important in the pathogenesis of NEC, but the contribution of specific infectious agents is poorly understood. Fecal samples from preterm infants ≤32 weeks gestation were analyzed using 16S rRNA based methods at 2, 1, and 0 weeks, prior to diagnosis of NEC in 18 NEC cases and 35 controls. Environmental factors such as antibiotic usage, feeding type (human milk versus formula) and location of neonatal intensive care unit (NICU) were also evaluated. Microbiota composition differed between the three neonatal units where we observed differences in antibiotic usage. In NEC cases we observed a higher proportion of Proteobacteria (61%) two weeks and of Actinobacteria (3%) 1 week before diagnosis of NEC compared to controls (19% and 0.4%, respectively) and lower numbers of Bifidobacteria counts and Bacteroidetes proportions in the weeks before NEC diagnosis. In the first fecal samples obtained during week one of life we detected a novel signature sequence, distinct from but matching closest to Klebsiella pneumoniae, that was strongly associated with NEC development later in life. Infants who develop NEC exhibit a different pattern of microbial colonization compared to controls. Antibiotic usage correlated with these differences and combined with type of feeding likely plays a critical role in the development of NEC.