Improving Characterization of Understudied Human Microbiomes Using Targeted Phylogenetics.

Whole-genome bacterial sequences are required to better understand microbial functions, niche-specific bacterial metabolism, and disease states. Although genomic sequences are available for many of the human-associated bacteria from commonly tested body habitats (e.g., feces), as few as 13% of bacterium-derived reads from other sites such as the skin map to known bacterial genomes. To facilitate a better characterization of metagenomic shotgun reads from underrepresented body sites, we collected over 10,000 bacterial isolates originating from 14 human body habitats, identified novel taxonomic groups based on full-length 16S rRNA gene sequences, clustered the sequences to ensure that no individual taxonomic group was overselected for sequencing, prioritized bacteria from underrepresented body sites (such as skin and respiratory and urinary tracts), and sequenced and assembled genomes for 665 new bacterial strains. Here, we show that addition of these genomes improved read mapping rates of Human Microbiome Project (HMP) metagenomic samples by nearly 30% for the previously underrepresented phylum Fusobacteria, and 27.5% of the novel genomes generated here had high representation in at least one of the tested HMP samples, compared to 12.5% of the sequences in the public databases, indicating an enrichment of useful novel genomic sequences resulting from the prioritization procedure. As our understanding of the human microbiome continues to improve and to enter the realm of therapy developments, targeted approaches such as this to improve genomic databases will increase in importance from both an academic and a clinical perspective.IMPORTANCE The human microbiome plays a critically important role in health and disease, but current understanding of the mechanisms underlying the interactions between the varying microbiome and the different host environments is lacking. Having access to a database of fully sequenced bacterial genomes provides invaluable insights into microbial functions, but currently sequenced genomes for the human microbiome have largely come from a limited number of body sites (primarily feces), while other sites such as the skin, respiratory tract, and urinary tract are underrepresented, resulting in as little as 13% of bacterium-derived reads mapping to known bacterial genomes. Here, we sequenced and assembled 665 new bacterial genomes, prioritized from a larger database to select underrepresented body sites and bacterial taxa in the existing databases. As a result, we substantially improve mapping rates for samples from the Human Microbiome Project and provide an important contribution to human bacterial genomic databases for future studies.

Genome Sequence of Christensenella minuta DSM 22607T.

Obesity influences and is influenced by the human gut microbiome. Here, we present the genome of Christensenella minuta, a highly heritable bacterial species which has been found to be strongly associated with obesity through an unknown biological mechanism. This novel genome provides a valuable resource for future obesity therapeutic studies.

Genome Sequences of 15 Gardnerella vaginalis Strains Isolated from the Vaginas of Women with and without Bacterial Vaginosis.

Gardnerella vaginalis is a predominant species in bacterial vaginosis, a dysbiosis of the vagina that is associated with adverse health outcomes, including preterm birth. Here, we present the draft genome sequences of 15 Gardnerella vaginalis strains (now available through BEI Resources) isolated from women with and without bacterial vaginosis.

Genome Sequences of 12 Bacterial Isolates Obtained from the Urine of Pregnant Women.

The presence of bacteria in urine can pose significant risks during pregnancy. However, there are few reference genome strains for many common urinary bacteria. We isolated 12 urinary strains of Streptococcus, Staphylococcus, Citrobacter, Gardnerella, and Lactobacillus These strains and their genomes are now available to the research community.

Genome Sequences of 11 Human Vaginal Actinobacteria Strains.

The composition of the vaginal microbiota is an important health determinant. Several members of the phylum Actinobacteria have been implicated in bacterial vaginosis, a condition associated with many negative health outcomes. Here, we present 11 strains of vaginal Actinobacteria (now available through BEI Resources) along with draft genome sequences.

Genome Sequences of 14 Firmicutes Strains Isolated from the Human Vagina.

Research on vaginal infections is currently limited by a lack of available fully sequenced bacterial reference strains. Here, we present strains (now available through BEI Resources) and genome sequences for a set of 14 vaginal isolates from the phylum Firmicutes These genome sequences provide a valuable resource for future research in understanding the role of Gram-positive bacteria in vaginal health and disease.

Genome Sequences of Nine Gram-Negative Vaginal Bacterial Isolates.

The vagina is home to a wide variety of bacteria that have great potential to impact human health. Here, we announce reference strains (now available through BEI Resources) and draft genome sequences for 9 Gram-negative vaginal isolates from the taxa Citrobacter, Klebsiella, Fusobacterium, Proteus, and Prevotella.

Extreme Sensory Complexity Encoded in the 10-Megabase Draft Genome Sequence of the Chromatically Acclimating Cyanobacterium Tolypothrix sp. PCC 7601.

Tolypothrix sp. PCC 7601 is a freshwater filamentous cyanobacterium with complex responses to environmental conditions. Here, we present its 9.96-Mbp draft genome sequence, containing 10,065 putative protein-coding sequences, including 305 predicted two-component system proteins and 27 putative phytochrome-class photoreceptors, the most such proteins in any sequenced genome.

Phenotypic and genotypic analysis of Clostridium difficile isolates: a single-center study.

Clostridium difficile infections (CDI) are a growing concern in North America, because of their increasing incidence and severity. Using integrated approaches, we correlated pathogen genotypes and host clinical characteristics for 46 C. difficile infections in a tertiary care medical center during a 6-month interval from January to June 2010. Multilocus sequence typing (MLST) demonstrated 21 known and 2 novel sequence types (STs), suggesting that the institution's C. difficile strains are genetically diverse. ST-1 (which corresponds to pulsed-field gel electrophoresis strain type NAP1/ribotype 027) was the most prevalent (32.6%); 43.5% of the isolates were binary toxin gene positive, of which 75% were ST-1. All strains were ciprofloxacin resistant and metronidazole susceptible, and 8.3% and 13.0% of the isolates were resistant to clindamycin and tetracycline, respectively. The corresponding resistance loci, including potential novel mutations, were identified from the whole-genome sequencing (WGS) of the resistant strains. Core genome single nucleotide polymorphisms (SNPs) determining the phylogenetic relatedness of the 46 strains recapitulated MLST types and provided greater interstrain differentiation. The disease severity was greatest in patients infected with ST-1 and/or binary gene-positive strains, but genome-wide SNP analysis failed to provide additional associations with CDI severity within the same STs. We conclude that MLST and core genome SNP typing result in the same phylogenetic grouping of the 46 C. difficile strains collected in a single hospital. WGS also has the capacity to differentiate those strains within STs and allows the comparison of strains at the individual gene level and at the whole-genome level.

Whole-genome analyses of Enterococcus faecium isolates with diverse daptomycin MICs.

Daptomycin (DAP) is a lipopeptide antibiotic frequently used as a "last-resort" antibiotic against vancomycin-resistant Enterococcus faecium (VRE). However, an important limitation for DAP therapy against VRE is the emergence of resistance during therapy. Mutations in regulatory systems involved in cell envelope homeostasis are postulated to be important mediators of DAP resistance in E. faecium. Thus, in order to gain insights into the genetic bases of DAP resistance in E. faecium, we investigated the presence of changes in 43 predicted proteins previously associated with DAP resistance in enterococci and staphylococci using the genomes of 19 E. faecium with different DAP MICs (range, 3 to 48 µg/ml). Bodipy-DAP (BDP-DAP) binding to the cell membrane assays and time-kill curves (DAP alone and with ampicillin) were performed. Genetic changes involving two major pathways were identified: (i) LiaFSR, a regulatory system associated with the cell envelope stress response, and (ii) YycFGHIJ, a system involved in the regulation of cell wall homeostasis. Thr120 → Ala and Trp73 → Cys substitutions in LiaS and LiaR, respectively, were the most common changes identified. DAP bactericidal activity was abolished in the presence of liaFSR or yycFGHIJ mutations regardless of the DAP MIC and was restored in the presence of ampicillin, but only in representatives of the LiaFSR pathway. Reduced binding of BDP-DAP to the cell surface was the predominant finding correlating with resistance in isolates with DAP MICs above the susceptibility breakpoint. Our findings suggest that genotypic information may be crucial to predict response to DAP plus ß-lactam combinations and continue to question the DAP breakpoint of 4 µg/ml.

Transferable vancomycin resistance in a community-associated MRSA lineage.

We report the case of a patient from Brazil with a bloodstream infection caused by a strain of methicillin-resistant Staphylococcus aureus (MRSA) that was susceptible to vancomycin (designated BR-VSSA) but that acquired the vanA gene cluster during antibiotic therapy and became resistant to vancomycin (designated BR-VRSA). Both strains belong to the sequence type (ST) 8 community-associated genetic lineage that carries the staphylococcal chromosomal cassette mec (SCCmec) type IVa and the S. aureus protein A gene (spa) type t292 and are phylogenetically related to MRSA lineage USA300. A conjugative plasmid of 55,706 bp (pBRZ01) carrying the vanA cluster was identified and readily transferred to other staphylococci. The pBRZ01 plasmid harbors DNA sequences that are typical of the plasmid-associated replication genes rep24 or rep21 described in community-associated MRSA strains from Australia (pWBG745). The presence and dissemination of community-associated MRSA containing vanA could become a serious public health concern.

Evolutionary Genomics of Salmonella enterica Subspecies.

ABSTRACT Six subspecies are currently recognized in Salmonella enterica. Subspecies I (subspecies enterica) is responsible for nearly all infections in humans and warm-blooded animals, while five other subspecies are isolated principally from cold-blooded animals. We sequenced 21 phylogenetically diverse strains, including two representatives from each of the previously unsequenced five subspecies and 11 diverse new strains from S. enterica subspecies enterica, to put this species into an evolutionary perspective. The phylogeny of the subspecies was partly obscured by abundant recombination events between lineages and a relatively short period of time within which subspeciation took place. Nevertheless, a variety of different tree-building methods gave congruent evolutionary tree topologies for subspeciation. A total of 285 gene families were identified that were recruited into subspecies enterica, and most of these are of unknown function. At least 2,807 gene families were identified in one or more of the other subspecies that are not found in subspecies I or Salmonella bongori. Among these gene families were 13 new candidate effectors and 7 new candidate fimbrial clusters. A third complete type III secretion system not present in subspecies enterica (I) isolates was found in both strains of subspecies salamae (II). Some gene families had complex taxonomies, such as the type VI secretion systems, which were recruited from four different lineages in five of six subspecies. Analysis of nonsynonymous-to-synonymous substitution rates indicated that the more-recently acquired regions in S. enterica are undergoing faster fixation rates than the rest of the genome. Recently acquired AT-rich regions, which often encode virulence functions, are under ongoing selection to maintain their high AT content. IMPORTANCE We have sequenced 21 new genomes which encompass the phylogenetic diversity of Salmonella, including strains of the previously unsequenced subspecies arizonae, diarizonae, houtenae, salamae, and indica as well as new diverse strains of subspecies enterica. We have deduced possible evolutionary paths traversed by this very important zoonotic pathogen and identified novel putative virulence factors that are not found in subspecies I. Gene families gained at the time of the evolution of subspecies enterica are of particular interest because they include mechanisms by which this subspecies adapted to warm-blooded hosts.

Genome sequence of Cronobacter sakazakii BAA-894 and comparative genomic hybridization analysis with other Cronobacter species.

BACKGROUND: The genus Cronobacter (formerly called Enterobacter sakazakii) is composed of five species; C. sakazakii, C. malonaticus, C. turicensis, C. muytjensii, and C. dublinensis. The genus includes opportunistic human pathogens, and the first three species have been associated with neonatal infections. The most severe diseases are caused in neonates and include fatal necrotizing enterocolitis and meningitis. The genetic basis of the diversity within the genus is unknown, and few virulence traits have been identified. METHODOLOGY/PRINCIPAL FINDINGS: We report here the first sequence of a member of this genus, C. sakazakii strain BAA-894. The genome of Cronobacter sakazakii strain BAA-894 comprises a 4.4 Mb chromosome (57% GC content) and two plasmids; 31 kb (51% GC) and 131 kb (56% GC). The genome was used to construct a 387,000 probe oligonucleotide tiling DNA microarray covering the whole genome. Comparative genomic hybridization (CGH) was undertaken on five other C. sakazakii strains, and representatives of the four other Cronobacter species. Among 4,382 annotated genes inspected in this study, about 55% of genes were common to all C. sakazakii strains and 43% were common to all Cronobacter strains, with 10-17% absence of genes. CONCLUSIONS/SIGNIFICANCE: CGH highlighted 15 clusters of genes in C. sakazakii BAA-894 that were divergent or absent in more than half of the tested strains; six of these are of probable prophage origin. Putative virulence factors were identified in these prophage and in other variable regions. A number of genes unique to Cronobacter species associated with neonatal infections (C. sakazakii, C. malonaticus and C. turicensis) were identified. These included a copper and silver resistance system known to be linked to invasion of the blood-brain barrier by neonatal meningitic strains of Escherichia coli. In addition, genes encoding for multidrug efflux pumps and adhesins were identified that were unique to C. sakazakii strains from outbreaks in neonatal intensive care units.

Characterizing a model human gut microbiota composed of members of its two dominant bacterial phyla.

The adult human distal gut microbial community is typically dominated by 2 bacterial phyla (divisions), the Firmicutes and the Bacteroidetes. Little is known about the factors that govern the interactions between their members. Here, we examine the niches of representatives of both phyla in vivo. Finished genome sequences were generated from Eubacterium rectale and E. eligens, which belong to Clostridium Cluster XIVa, one of the most common gut Firmicute clades. Comparison of these and 25 other gut Firmicutes and Bacteroidetes indicated that the Firmicutes possess smaller genomes and a disproportionately smaller number of glycan-degrading enzymes. Germ-free mice were then colonized with E. rectale and/or a prominent human gut Bacteroidetes, Bacteroides thetaiotaomicron, followed by whole-genome transcriptional profiling, high-resolution proteomic analysis, and biochemical assays of microbial-microbial and microbial-host interactions. B. thetaiotaomicron adapts to E. rectale by up-regulating expression of a variety of polysaccharide utilization loci encoding numerous glycoside hydrolases, and by signaling the host to produce mucosal glycans that it, but not E. rectale, can access. E. rectale adapts to B. thetaiotaomicron by decreasing production of its glycan-degrading enzymes, increasing expression of selected amino acid and sugar transporters, and facilitating glycolysis by reducing levels of NADH, in part via generation of butyrate from acetate, which in turn is used by the gut epithelium. This simplified model of the human gut microbiota illustrates niche specialization and functional redundancy within members of its major bacterial phyla, and the importance of host glycans as a nutrient foundation that ensures ecosystem stability.

The genome of Cyanothece 51142, a unicellular diazotrophic cyanobacterium important in the marine nitrogen cycle.

Unicellular cyanobacteria have recently been recognized for their contributions to nitrogen fixation in marine environments, a function previously thought to be filled mainly by filamentous cyanobacteria such as Trichodesmium. To begin a systems level analysis of the physiology of the unicellular N(2)-fixing microbes, we have sequenced to completion the genome of Cyanothece sp. ATCC 51142, the first such organism. Cyanothece 51142 performs oxygenic photosynthesis and nitrogen fixation, separating these two incompatible processes temporally within the same cell, while concomitantly accumulating metabolic products in inclusion bodies that are later mobilized as part of a robust diurnal cycle. The 5,460,377-bp Cyanothece 51142 genome has a unique arrangement of one large circular chromosome, four small plasmids, and one linear chromosome, the first report of a linear element in the genome of a photosynthetic bacterium. On the 429,701-bp linear chromosome is a cluster of genes for enzymes involved in pyruvate metabolism, suggesting an important role for the linear chromosome in fermentative processes. The annotation of the genome was significantly aided by simultaneous global proteomic studies of this organism. Compared with other nitrogen-fixing cyanobacteria, Cyanothece 51142 contains the largest intact contiguous cluster of nitrogen fixation-related genes. We discuss the implications of such an organization on the regulation of nitrogen fixation. The genome sequence provides important information regarding the ability of Cyanothece 51142 to accomplish metabolic compartmentalization and energy storage, as well as how a unicellular bacterium balances multiple, often incompatible, processes in a single cell.

The DNA sequence of human chromosome 7.

Human chromosome 7 has historically received prominent attention in the human genetics community, primarily related to the search for the cystic fibrosis gene and the frequent cytogenetic changes associated with various forms of cancer. Here we present more than 153 million base pairs representing 99.4% of the euchromatic sequence of chromosome 7, the first metacentric chromosome completed so far. The sequence has excellent concordance with previously established physical and genetic maps, and it exhibits an unusual amount of segmentally duplicated sequence (8.2%), with marked differences between the two arms. Our initial analyses have identified 1,150 protein-coding genes, 605 of which have been confirmed by complementary DNA sequences, and an additional 941 pseudogenes. Of genes confirmed by transcript sequences, some are polymorphic for mutations that disrupt the reading frame.

The male-specific region of the human Y chromosome is a mosaic of discrete sequence classes.

The male-specific region of the Y chromosome, the MSY, differentiates the sexes and comprises 95% of the chromosome's length. Here, we report that the MSY is a mosaic of heterochromatic sequences and three classes of euchromatic sequences: X-transposed, X-degenerate and ampliconic. These classes contain all 156 known transcription units, which include 78 protein-coding genes that collectively encode 27 distinct proteins. The X-transposed sequences exhibit 99% identity to the X chromosome. The X-degenerate sequences are remnants of ancient autosomes from which the modern X and Y chromosomes evolved. The ampliconic class includes large regions (about 30% of the MSY euchromatin) where sequence pairs show greater than 99.9% identity, which is maintained by frequent gene conversion (non-reciprocal transfer). The most prominent features here are eight massive palindromes, at least six of which contain testis genes.