Joseph F. Petrosino

Chimeric 16S rRNA sequence formation and detection in Sanger and 454-pyrosequenced PCR amplicons

Bacterial diversity among environmental samples is commonly assessed with PCR-amplified 16S rRNA gene (16S) sequences. Perceived diversity, however, can be influenced by sample preparation, primer selection, and formation of chimeric 16S amplification products. Chimeras are hybrid products between multiple parent sequences that can be falsely interpreted as novel organisms, thus inflating apparent diversity. We developed a new chimera detection tool called Chimera Slayer (CS). CS detects chimeras with greater sensitivity than previous methods, performs well on short sequences such as those produced by the 454 Life Sciences (Roche) Genome Sequencer, and can scale to large data sets. By benchmarking CS performance against sequences derived from a controlled DNA mixture of known organisms and a simulated chimera set, we provide insights into the factors that affect chimera formation such as sequence abundance, the extent of similarity between 16S genes, and PCR conditions. Chimeras were found to reproducibly form among independent amplifications and contributed to false perceptions of sample diversity and the false identification of novel taxa, with less-abundant species exhibiting chimera rates exceeding 70%. Shotgun metagenomic sequences of our mock community appear to be devoid of 16S chimeras, supporting a role for shotgun metagenomics in validating novel organisms discovered in targeted sequence surveys.

Microbiota modulate behavioral and physiological abnormalities associated with neurodevelopmental disorders

Neurodevelopmental disorders, including autism spectrum disorder (ASD), are defined by core behavioral impairments; however, subsets of individuals display a spectrum of gastrointestinal (GI) abnormalities. We demonstrate GI barrier defects and microbiota alterations in the maternal immune activation (MIA) mouse model that is known to display features of ASD. Oral treatment of MIA offspring with the human commensal Bacteroides fragilis corrects gut permeability, alters microbial composition, and ameliorates defects in communicative, stereotypic, anxiety-like and sensorimotor behaviors. MIA offspring display an altered serum metabolomic profile, and B. fragilis modulates levels of several metabolites. Treating naive mice with a metabolite that is increased by MIA and restored by B. fragilis causes certain behavioral abnormalities, suggesting that gut bacterial effects on the host metabolome impact behavior. Taken together, these findings support a gut-microbiome-brain connection in a mouse model of ASD and identify a potential probiotic therapy for GI and particular behavioral symptoms in human neurodevelopmental disorders.

The Placenta Harbors a Unique Microbiome

Metagenomic approaches demonstrate that the human placenta is not sterile but harbors a unique microbiome. Bacteria in Healthy Placentas Contrary to the prevailing idea of a “sterile” intrauterine environment, Aagaard and coauthors demonstrated the consistent presence of a microbiome in placentas from healthy pregnancies. This microbiome was consistently different from those reported in other parts of the body, including the skin and urogenital tract. The placental microbiome was most similar to that of the oral cavity, but the clinical implications of this finding remain to be explored. In addition, the authors identified associations between the composition of the placental microbiome and a history of remote antenatal infection, as well as preterm birth, raising the possibility that the placental microbiome may play a role in these events. Humans and their microbiomes have coevolved as a physiologic community composed of distinct body site niches with metabolic and antigenic diversity. The placental microbiome has not been robustly interrogated, despite recent demonstrations of intracellular bacteria with diverse metabolic and immune regulatory functions. A population-based cohort of placental specimens collected under sterile conditions from 320 subjects with extensive clinical data was established for comparative 16S ribosomal DNA–based and whole-genome shotgun (WGS) metagenomic studies. Identified taxa and their gene carriage patterns were compared to other human body site niches, including the oral, skin, airway (nasal), vaginal, and gut microbiomes from nonpregnant controls. We characterized a unique placental microbiome niche, composed of nonpathogenic commensal microbiota from the Firmicutes, Tenericutes, Proteobacteria, Bacteroidetes, and Fusobacteria phyla. In aggregate, the placental microbiome profiles were most akin (Bray-Curtis dissimilarity <0.3) to the human oral microbiome. 16S-based operational taxonomic unit analyses revealed associations of the placental microbiome with a remote history of antenatal infection (permutational multivariate analysis of variance, P = 0.006), such as urinary tract infection in the first trimester, as well as with preterm birth <37 weeks (P = 0.001).

A catalog of reference genomes from the human microbiome.

News from the Inner Tube of Life A major initiative by the U.S. National Institutes of Health to sequence 900 genomes of microorganisms that live on the surfaces and orifices of the human body has established standardized protocols and methods for such large-scale reference sequencing. By combining previously accumulated data with new data, Nelson et al. (p. 994) present an initial analysis of 178 bacterial genomes. The sampling so far barely scratches the surface of the microbial diversity found on humans, but the work provides an important baseline for future analyses. Standardized protocols and methods are being established for large-scale sequencing of the microorganisms living on humans. The human microbiome refers to the community of microorganisms, including prokaryotes, viruses, and microbial eukaryotes, that populate the human body. The National Institutes of Health launched an initiative that focuses on describing the diversity of microbial species that are associated with health and disease. The first phase of this initiative includes the sequencing of hundreds of microbial reference genomes, coupled to metagenomic sequencing from multiple body sites. Here we present results from an initial reference genome sequencing of 178 microbial genomes. From 547,968 predicted polypeptides that correspond to the gene complement of these strains, previously unidentified (“novel”) polypeptides that had both unmasked sequence length greater than 100 amino acids and no BLASTP match to any nonreference entry in the nonredundant subset were defined. This analysis resulted in a set of 30,867 polypeptides, of which 29,987 (~97%) were unique. In addition, this set of microbial genomes allows for ~40% of random sequences from the microbiome of the gastrointestinal tract to be associated with organisms based on the match criteria used. Insights into pan-genome analysis suggest that we are still far from saturating microbial species genetic data sets. In addition, the associated metrics and standards used by our group for quality assurance are presented.

Metagenomic Pyrosequencing and Microbial Identification

BACKGROUND The Human Microbiome Project has ushered in a new era for human metagenomics and high-throughput next-generation sequencing strategies. CONTENT This review describes evolving strategies in metagenomics, with a special emphasis on the core technology of DNA pyrosequencing. The challenges of microbial identification in the context of microbial populations are discussed. The development of next-generation pyrosequencing strategies and the technical hurdles confronting these methodologies are addressed. Bioinformatics-related topics include taxonomic systems, sequence databases, sequence-alignment tools, and classifiers. DNA sequencing based on 16S rRNA genes or entire genomes is summarized with respect to potential pyrosequencing applications. SUMMARY Both the approach of 16S rDNA amplicon sequencing and the whole-genome sequencing approach may be useful for human metagenomics, and numerous bioinformatics tools are being deployed to tackle such vast amounts of microbiological sequence diversity. Metagenomics, or genetic studies of microbial communities, may ultimately contribute to a more comprehensive understanding of human health, disease susceptibilities, and the pathophysiology of infectious and immune-mediated diseases.

Decade-long bacterial community dynamics in cystic fibrosis airways

The structure and dynamics of bacterial communities in the airways of persons with cystic fibrosis (CF) remain largely unknown. We characterized the bacterial communities in 126 sputum samples representing serial collections spanning 8–9 y from six age-matched male CF patients. Sputum DNA was analyzed by bar-coded pyrosequencing of the V3–V5 hypervariable region of the 16S rRNA gene, defining 662 operational taxonomic units (OTUs) from >633,000 sequences. Bacterial community diversity decreased significantly over time in patients with typically progressive lung disease but remained relatively stable in patients with a mild lung disease phenotype. Antibiotic use, rather than patient age or lung function, was the primary driver of decreasing diversity. Interpatient variability in community structure exceeded intrapatient variability in serial samples. Antibiotic treatment was associated with pronounced shifts in community structure, but communities showed both short- and long-term resilience after antibiotic perturbation. There was a positive correlation between OTU occurrence and relative abundance, with a small number of persistent OTUs accounting for the greatest abundance. Significant changes in community structure, diversity, or total bacterial density at the time of pulmonary exacerbation were not observed. Despite decreasing community diversity in patients with progressive disease, total bacterial density remained relatively stable over time. These findings show the critical relationship between airway bacterial community structure, disease stage, and clinical state at the time of sample collection. These features are the key parameters with which to assess the complex ecology of the CF airway.

Gastrointestinal Microbiome Signatures of Pediatric Patients With Irritable Bowel Syndrome

BACKGROUND & AIMS The intestinal microbiomes of healthy children and pediatric patients with irritable bowel syndrome (IBS) are not well defined. Studies in adults have indicated that the gastrointestinal microbiota could be involved in IBS. METHODS We analyzed 71 samples from 22 children with IBS (pediatric Rome III criteria) and 22 healthy children, ages 7-12 years, by 16S ribosomal RNA gene sequencing, with an average of 54,287 reads/stool sample (average 454 read length = 503 bases). Data were analyzed using phylogenetic-based clustering (Unifrac), or an operational taxonomic unit (OTU) approach using a supervised machine learning tool (randomForest). Most samples were also hybridized to a microarray that can detect 8741 bacterial taxa (16S rRNA PhyloChip). RESULTS Microbiomes associated with pediatric IBS were characterized by a significantly greater percentage of the class γ-proteobacteria (0.07% vs 0.89% of total bacteria, respectively; P < .05); 1 prominent component of this group was Haemophilus parainfluenzae. Differences highlighted by 454 sequencing were confirmed by high-resolution PhyloChip analysis. Using supervised learning techniques, we were able to classify different subtypes of IBS with a success rate of 98.5%, using limited sets of discriminant bacterial species. A novel Ruminococcus-like microbe was associated with IBS, indicating the potential utility of microbe discovery for gastrointestinal disorders. A greater frequency of pain correlated with an increased abundance of several bacterial taxa from the genus Alistipes. CONCLUSIONS Using 16S metagenomics by PhyloChip DNA hybridization and deep 454 pyrosequencing, we associated specific microbiome signatures with pediatric IBS. These findings indicate the important association between gastrointestinal microbes and IBS in children; these approaches might be used in diagnosis of functional bowel disorders in pediatric patients.

A Metagenomic Approach to Characterization of the Vaginal Microbiome Signature in Pregnancy

While current major national research efforts (i.e., the NIH Human Microbiome Project) will enable comprehensive metagenomic characterization of the adult human microbiota, how and when these diverse microbial communities take up residence in the host and during reproductive life are unexplored at a population level. Because microbial abundance and diversity might differ in pregnancy, we sought to generate comparative metagenomic signatures across gestational age strata. DNA was isolated from the vagina (introitus, posterior fornix, midvagina) and the V5V3 region of bacterial 16S rRNA genes were sequenced (454FLX Titanium platform). Sixty-eight samples from 24 healthy gravidae (18 to 40 confirmed weeks) were compared with 301 non-pregnant controls (60 subjects). Generated sequence data were quality filtered, taxonomically binned, normalized, and organized by phylogeny and into operational taxonomic units (OTU); principal coordinates analysis (PCoA) of the resultant beta diversity measures were used for visualization and analysis in association with sample clinical metadata. Altogether, 1.4 gigabytes of data containing >2.5 million reads (averaging 6,837 sequences/sample of 493 nt in length) were generated for computational analyses. Although gravidae were not excluded by virtue of a posterior fornix pH >4.5 at the time of screening, unique vaginal microbiome signature encompassing several specific OTUs and higher-level clades was nevertheless observed and confirmed using a combination of phylogenetic, non-phylogenetic, supervised, and unsupervised approaches. Both overall diversity and richness were reduced in pregnancy, with dominance of Lactobacillus species (L. iners crispatus, jensenii and johnsonii, and the orders Lactobacillales (and Lactobacillaceae family), Clostridiales, Bacteroidales, and Actinomycetales. This intergroup comparison using rigorous standardized sampling protocols and analytical methodologies provides robust initial evidence that the vaginal microbial 16S rRNA gene catalogue uniquely differs in pregnancy, with variance of taxa across vaginal subsite and gestational age.

The Complete Genome Sequence of Escherichia coli DH10B: Insights into the Biology of a Laboratory Workhorse

Escherichia coli DH10B was designed for the propagation of large insert DNA library clones. It is used extensively, taking advantage of properties such as high DNA transformation efficiency and maintenance of large plasmids. The strain was constructed by serial genetic recombination steps, but the underlying sequence changes remained unverified. We report the complete genomic sequence of DH10B by using reads accumulated from the bovine sequencing project at Baylor College of Medicine and assembled with DNAStars SeqMan genome assembler. The DH10B genome is largely colinear with that of the wild-type K-12 strain MG1655, although it is substantially more complex than previously appreciated, allowing DH10B biology to be further explored. The 226 mutated genes in DH10B relative to MG1655 are mostly attributable to the extensive genetic manipulations the strain has undergone. However, we demonstrate that DH10B has a 13.5-fold higher mutation rate than MG1655, resulting from a dramatic increase in insertion sequence (IS) transposition, especially IS150. IS elements appear to have remodeled genome architecture, providing homologous recombination sites for a 113,260-bp tandem duplication and an inversion. DH10B requires leucine for growth on minimal medium due to the deletion of leuLABCD and harbors both the relA1 and spoT1 alleles causing both sensitivity to nutritional downshifts and slightly lower growth rates relative to the wild type. Finally, while the sequence confirms most of the reported alleles, the sequence of deoR is wild type, necessitating reexamination of the assumed basis for the high transformability of DH10B.

The Gut Microbiome Modulates Colon Tumorigenesis

ABSTRACT Recent studies have shown that individuals with colorectal cancer have an altered gut microbiome compared to healthy controls. It remains unclear whether these differences are a response to tumorigenesis or actively drive tumorigenesis. To determine the role of the gut microbiome in the development of colorectal cancer, we characterized the gut microbiome in a murine model of inflammation-associated colorectal cancer that mirrors what is seen in humans. We followed the development of an abnormal microbial community structure associated with inflammation and tumorigenesis in the colon. Tumor-bearing mice showed enrichment in operational taxonomic units (OTUs) affiliated with members of the Bacteroides, Odoribacter, and Akkermansia genera and decreases in OTUs affiliated with members of the Prevotellaceae and Porphyromonadaceae families. Conventionalization of germfree mice with microbiota from tumor-bearing mice significantly increased tumorigenesis in the colon compared to that for animals colonized with a healthy gut microbiome from untreated mice. Furthermore, at the end of the model, germfree mice colonized with microbiota from tumor-bearing mice harbored a higher relative abundance of populations associated with tumor formation in conventional animals. Manipulation of the gut microbiome with antibiotics resulted in a dramatic decrease in both the number and size of tumors. Our results demonstrate that changes in the gut microbiome associated with inflammation and tumorigenesis directly contribute to tumorigenesis and suggest that interventions affecting the composition of the microbiome may be a strategy to prevent the development of colon cancer. IMPORTANCE The trillions of bacteria that live in the gut, known collectively as the gut microbiome, are important for normal functioning of the intestine. There is now growing evidence that disruptive changes in the gut microbiome are strongly associated with the development colorectal cancer. However, how the gut microbiome changes with time during tumorigenesis and whether these changes directly contribute to disease have not been determined. We demonstrate using a mouse model of inflammation-driven colon cancer that there are dramatic, continual alterations in the microbiome during the development of tumors, which are directly responsible for tumor development. Our results suggest that interventions that target these changes in the microbiome may be an effective strategy for preventing the development of colorectal cancer. The trillions of bacteria that live in the gut, known collectively as the gut microbiome, are important for normal functioning of the intestine. There is now growing evidence that disruptive changes in the gut microbiome are strongly associated with the development colorectal cancer. However, how the gut microbiome changes with time during tumorigenesis and whether these changes directly contribute to disease have not been determined. We demonstrate using a mouse model of inflammation-driven colon cancer that there are dramatic, continual alterations in the microbiome during the development of tumors, which are directly responsible for tumor development. Our results suggest that interventions that target these changes in the microbiome may be an effective strategy for preventing the development of colorectal cancer.