Elisabeth Bik

Microbial Prevalence, Diversity and Abundance in Amniotic Fluid During Preterm Labor: A Molecular and Culture-Based Investigation

Background Preterm delivery causes substantial neonatal mortality and morbidity. Unrecognized intra-amniotic infections caused by cultivation-resistant microbes may play a role. Molecular methods can detect, characterize and quantify microbes independently of traditional culture techniques. However, molecular studies that define the diversity and abundance of microbes invading the amniotic cavity, and evaluate their clinical significance within a causal framework, are lacking. Methods and Findings In parallel with culture, we used broad-range end-point and real-time PCR assays to amplify, identify and quantify ribosomal DNA (rDNA) of bacteria, fungi and archaea from amniotic fluid of 166 women in preterm labor with intact membranes. We sequenced up to 24 rRNA clones per positive specimen and assigned taxonomic designations to approximately the species level. Microbial prevalence, diversity and abundance were correlated with host inflammation and with gestational and neonatal outcomes. Study subjects who delivered at term served as controls. The combined use of molecular and culture methods revealed a greater prevalence (15% of subjects) and diversity (18 taxa) of microbes in amniotic fluid than did culture alone (9.6% of subjects; 11 taxa). The taxa detected only by PCR included a related group of fastidious bacteria, comprised of Sneathia sanguinegens, Leptotrichia amnionii and an unassigned, uncultivated, and previously-uncharacterized bacterium; one or more members of this group were detected in 25% of positive specimens. A positive PCR was associated with histologic chorioamnionitis (adjusted odds ratio [OR] 20; 95% CI, 2.4 to 172), and funisitis (adjusted OR 18; 95% CI, 3.1 to 99). The positive predictive value of PCR for preterm delivery was 100 percent. A temporal association between a positive PCR and delivery was supported by a shortened amniocentesis-to-delivery interval (adjusted hazard ratio 4.6; 95% CI, 2.2 to 9.5). A dose-response association was demonstrated between bacterial rDNA abundance and gestational age at delivery (r2 = 0.42; P<0.002). Conclusions The amniotic cavity of women in preterm labor harbors DNA from a greater diversity of microbes than previously suspected, including as-yet uncultivated, previously-uncharacterized taxa. The strength, temporality and gradient with which these microbial sequence types are associated with preterm delivery support a causal relationship.

Dissecting biological "dark matter" with single-cell genetic analysis of rare and uncultivated TM7 microbes from the human mouth.

We have developed a microfluidic device that allows the isolation and genome amplification of individual microbial cells, thereby enabling organism-level genomic analysis of complex microbial ecosystems without the need for culture. This device was used to perform a directed survey of the human subgingival crevice and to isolate bacteria having rod-like morphology. Several isolated microbes had a 16S rRNA sequence that placed them in candidate phylum TM7, which has no cultivated or sequenced members. Genome amplification from individual TM7 cells allowed us to sequence and assemble >1,000 genes, providing insight into the physiology of members of this phylum. This approach enables single-cell genetic analysis of any uncultivated minority member of a microbial community.

Bacterial diversity in the oral cavity of 10 healthy individuals.

The composition of the oral microbiota from 10 individuals with healthy oral tissues was determined using culture-independent techniques. From each individual, 26 specimens, each from different oral sites at a single point in time, were collected and pooled. An 11th pool was constructed using portions of the subgingival specimens from all 10 individuals. The 16S ribosomal RNA gene was amplified using broad-range bacterial primers, and clone libraries from the individual and subgingival pools were constructed. From a total of 11 368 high-quality, nonchimeric, near full-length sequences, 247 species-level phylotypes (using a 99% sequence identity threshold) and 9 bacterial phyla were identified. At least 15 bacterial genera were conserved among all 10 individuals, with significant interindividual differences at the species and strain level. Comparisons of these oral bacterial sequences with near full-length sequences found previously in the large intestines and feces of other healthy individuals suggest that the mouth and intestinal tract harbor distinct sets of bacteria. Co-occurrence analysis showed significant segregation of taxa when community membership was examined at the level of genus, but not at the level of species, suggesting that ecologically significant, competitive interactions are more apparent at a broader taxonomic level than species. This study is one of the more comprehensive, high-resolution analyses of bacterial diversity within the healthy human mouth to date, and highlights the value of tools from macroecology for enhancing our understanding of bacterial ecology in human health.

Prevalence and diversity of microbes in the amniotic fluid, the fetal inflammatory response, and pregnancy outcome in women with preterm pre-labor rupture of membranes.

Citation DiGiulio DB, Romero R, Kusanovic JP, Gómez R, Kim CJ, Seok K, Gotsch F, Mazaki‐Tovi S, Vaisbuch E, Sanders K, Bik EM, Chaiworapongsa T, Oyarzún E, Relman DA. Prevalence and diversity of microbes in the amniotic fluid, the fetal inflammatory response, and pregnancy outcome in women with preterm pre‐labor rupture of membranes. Am J Reprod Immunol 2010; 64: 38–57

Rapid quantitative profiling of complex microbial populations

Diverse and complex microbial ecosystems are found in virtually every environment on earth, yet we know very little about their composition and ecology. Comprehensive identification and quantification of the constituents of these microbial communities—a ‘census’—is an essential foundation for understanding their biology. To address this problem, we developed, tested and optimized a DNA oligonucleotide microarray composed of 10 462 small subunit (SSU) ribosomal DNA (rDNA) probes (7167 unique sequences) selected to provide quantitative information on the taxonomic composition of diverse microbial populations. Using our optimized experimental approach, this microarray enabled detection and quantification of individual bacterial species present at fractional abundances of <0.1% in complex synthetic mixtures. The estimates of bacterial species abundance obtained using this microarray are similar to those obtained by phylogenetic analysis of SSU rDNA sequences from the same samples—the current ‘gold standard’ method for profiling microbial communities. Furthermore, probes designed to represent higher order taxonomic groups of bacterial species reliably detected microbes for which there were no species-specific probes. This simple, rapid microarray procedure can be used to explore and systematically characterize complex microbial communities, such as those found within the human body.

Distinct Distal Gut Microbiome Diversity and Composition in Healthy Children from Bangladesh and the United States

Background Our current understanding of the composition and stability of the human distal gut microbiota is based largely on studies of infants and adults living in developed countries. In contrast, little is known about the gut microbiota and its variation over time in older children and adolescents, especially in developing countries. Methodology/Principal Findings We compared the diversity, composition, and temporal stability of the fecal microbiota of healthy children, ages 9 to 14 years, living in an urban slum in Bangladesh with that of children of the same age range in an upper-middle class suburban community in the United States. We analyzed >8,000 near full-length 16S rRNA gene sequences and over 845,000 pyrosequencing reads of the 16S rRNA V1–V3 region. The distal gut of Bangladeshi children harbored significantly greater bacterial diversity than that of U.S. children, including novel lineages from several bacterial phyla. Bangladeshi and U.S. children had distinct fecal bacterial community membership and structure; the microbiota of Bangladeshi children was enriched in Prevotella, Butyrivibrio, and Oscillospira and depleted in Bacteroides relative to U.S. children (although similar to Bangladeshi adults). Furthermore, community membership and structure in Bangladeshi children was significantly less stable month-to-month than U.S. children. Conclusions/Significance Together, these results suggest that differing environmental or genetic factors may shape the microbiota of healthy children in the two countries. Further investigation is necessary to understand the mechanisms and factors that underlie these differences, and to incorporate these findings into new strategies for the prevention and treatment of childhood and adolescent diseases.

Host Species and Environmental Effects on Bacterial Communities Associated with Drosophila in the Laboratory and in the Natural Environment

The fruit fly Drosophila is a classic model organism to study adaptation as well as the relationship between genetic variation and phenotypes. Although associated bacterial communities might be important for many aspects of Drosophila biology, knowledge about their diversity, composition, and factors shaping them is limited. We used 454-based sequencing of a variable region of the bacterial 16S ribosomal RNA gene to characterize the bacterial communities associated with wild and laboratory Drosophila isolates. In order to specifically investigate effects of food source and host species on bacterial communities, we analyzed samples from wild Drosophila melanogaster and D. simulans collected from a variety of natural substrates, as well as from adults and larvae of nine laboratory-reared Drosophila species. We find no evidence for host species effects in lab-reared flies; instead, lab of origin and stochastic effects, which could influence studies of Drosophila phenotypes, are pronounced. In contrast, the natural Drosophila–associated microbiota appears to be predominantly shaped by food substrate with an additional but smaller effect of host species identity. We identify a core member of this natural microbiota that belongs to the genus Gluconobacter and is common to all wild-caught flies in this study, but absent from the laboratory. This makes it a strong candidate for being part of what could be a natural D. melanogaster and D. simulans core microbiome. Furthermore, we were able to identify candidate pathogens in natural fly isolates.

The evolution of epidemic Vibrio cholerae strains

The emergence of the novel Vibrio cholerae strain, O139 Bengal, which caused a large epidemic in Southeast Asia, underlines the adaptability of pathogenic microorganisms. Recent studies reveal that horizontal transfer of cell-wall polysaccharide genes played a central role in the emergence of this strain and that its genesis may not be as unique as initially believed.

Microbiome Assembly across Multiple Body Sites in Low-Birthweight Infants

ABSTRACT The purpose of this study was to evaluate the composition and richness of bacterial communities associated with low-birthweight (LBW) infants in relation to host body site, individual, and age. Bacterial 16S rRNA genes from saliva samples, skin swabs, and stool samples collected on postnatal days 8, 10, 12, 15, 18, and 21 from six LBW (five premature) infants were amplified, pyrosequenced, and analyzed within a comparative framework that included analogous data from normal-birthweight (NBW) infants and healthy adults. We found that body site was the primary determinant of bacterial community composition in the LBW infants. However, site specificity depended on postnatal age: saliva and stool compositions diverged over time but were not significantly different until the babies were 15 days old. This divergence was primarily driven by progressive temporal turnover in the distal gut, which proceeded at a rate similar to that of age-matched NBW infants. Neonatal skin was the most adult-like in microbiota composition, while saliva and stool remained the least so. Compositional variation among infants was marked and depended on body site and age. Only the smallest, most premature infant received antibiotics during the study period; this heralded a coexpansion of Pseudomonas aeruginosa and a novel Mycoplasma sp. in the oral cavity of this vaginally delivered, intubated patient. We conclude that concurrent molecular surveillance of multiple body sites in LBW neonates reveals a delayed compositional differentiation of the oral cavity and distal gut microbiota and, in the case of one infant, an abundant, uncultivated oral Mycoplasma sp., recently detected in human vaginal samples. IMPORTANCE Complications of premature birth are the most common cause of neonatal mortality. Colonization by the indigenous microbiota, which begins at delivery, may predispose some high-risk newborns to invasive infection or necrotizing enterocolitis (NEC), and protect others, yet neonatal microbiome dynamics are poorly understood. Here, we present the first cultivation-independent time series tracking microbiota assembly across multiple body sites in a synchronous cohort of hospitalized low-birthweight (LBW) neonates. We take advantage of archived samples and publically available sequence data and compare our LBW infant findings to those from normal-birthweight (NBW) infants and healthy adults. Our results suggest potential windows of opportunity for the dispersal of microbes within and between hosts and support recent findings of substantial baseline spatiotemporal variation in microbiota composition among high-risk newborns. Complications of premature birth are the most common cause of neonatal mortality. Colonization by the indigenous microbiota, which begins at delivery, may predispose some high-risk newborns to invasive infection or necrotizing enterocolitis (NEC), and protect others, yet neonatal microbiome dynamics are poorly understood. Here, we present the first cultivation-independent time series tracking microbiota assembly across multiple body sites in a synchronous cohort of hospitalized low-birthweight (LBW) neonates. We take advantage of archived samples and publically available sequence data and compare our LBW infant findings to those from normal-birthweight (NBW) infants and healthy adults. Our results suggest potential windows of opportunity for the dispersal of microbes within and between hosts and support recent findings of substantial baseline spatiotemporal variation in microbiota composition among high-risk newborns.

GENETIC ORGANIZATION AND FUNCTIONAL ANALYSIS OF THE OTN DNA ESSENTIAL FOR CELL-WALL POLYSACCHARIDE SYNTHESIS IN VIBRIO CHOLERAE O139

In 1992 a new Vibrio cholerae strain, designated V. cholerae O139 Bengal, emerged which has been responsible for large outbreaks of cholera in India and Bangladesh. Previously, we have shown that this strain arose from a V. cholerae O1 strain by the acquisition of novel DNA. Sequence analysis revealed that the novel DNA is flanked by two genes, rfaD and rfbQRS, which are also found in O1 strains. The mosaic structure of rfaDvco139 indicated that it was one of the regions involved in recombination between donor and acceptor DNA. However, sequence divergence between the O1 and O139 rfbQRS genes indicated that the second recombination site between donor and O1‐acceptor DNA is probably located downstream of rfbDvco139. The DNA region between rfaDvco139 and rfbQRSvco139, designated otn, contained seven open reading frames (ORFs). Two ORFs, otnA and otnB, showed homology with genes involved in cell‐wall polysaccharide synthesis. Mutations in otnA and otnB indicated that they are required for capsule synthesis but not lipopolysaccharide synthesis. The otn DNA is also found inV. cholerae O69 and O141 strains, and the organization of this DNA was essentially identical to that in the O139 strain. However, sequence divergence of the otnAB genes indicated that the O139 otn DNA region was not derived from the O69 or O141 strains. No antigenic relationship was found between the different V. cholerae serotypes carrying otn DNA, so the genes determining the antigenic specificity of the O antigen or capsule must be located outside the otn DNA. The O139 otn DNA contained a JUMPstart sequence, which is associated with polysaccharide‐synthetic genes in several bacterial species. Furthermore, a repeat motif was observed in extragenic regions. A number of observations suggest that these sequences may facilitate gene flow between V. cholerae strains and the assembly of clusters of functionally related genes.