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1.  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 β-lactam combinations and continue to question the DAP breakpoint of 4 μg/ml.
doi:10.1128/AAC.02686-14
PMCID: PMC4136017  PMID: 24867964
2.  The Common Marmoset Genome Provides Insight into Primate Biology and Evolution 
Worley, Kim C. | Warren, Wesley C. | Rogers, Jeffrey | Locke, Devin | Muzny, Donna M. | Mardis, Elaine R. | Weinstock, George M. | Tardif, Suzette D. | Aagaard, Kjersti M. | Archidiacono, Nicoletta | Rayan, Nirmala Arul | Batzer, Mark A. | Beal, Kathryn | Brejova, Brona | Capozzi, Oronzo | Capuano, Saverio B. | Casola, Claudio | Chandrabose, Mimi M. | Cree, Andrew | Dao, Marvin Diep | de Jong, Pieter J. | del Rosario, Ricardo Cruz-Herrera | Delehaunty, Kim D. | Dinh, Huyen H. | Eichler, Evan | Fitzgerald, Stephen | Flicek, Paul | Fontenot, Catherine C. | Fowler, R. Gerald | Fronick, Catrina | Fulton, Lucinda A. | Fulton, Robert S. | Gabisi, Ramatu Ayiesha | Gerlach, Daniel | Graves, Tina A. | Gunaratne, Preethi H. | Hahn, Matthew W. | Haig, David | Han, Yi | Harris, R. Alan | Herrero, Javier M. | Hillier, LaDeana W. | Hubley, Robert | Hughes, Jennifer F. | Hume, Jennifer | Jhangiani, Shalini N. | Jorde, Lynn B. | Joshi, Vandita | Karakor, Emre | Konkel, Miriam K. | Kosiol, Carolin | Kovar, Christie L. | Kriventseva, Evgenia V. | Lee, Sandra L. | Lewis, Lora R. | Liu, Yih-shin | Lopez, John | Lopez-Otin, Carlos | Lorente-Galdos, Belen | Mansfield, Keith G. | Marques-Bonet, Tomas | Minx, Patrick | Misceo, Doriana | Moncrieff, J. Scott | Morgan, Margaret B. | Muthuswamy, Raveendran | Nazareth, Lynne V. | Newsham, Irene | Nguyen, Ngoc Bich | Okwuonu, Geoffrey O. | Prabhakar, Shyam | Perales, Lora | Pu, Ling-Ling | Puente, Xose S. | Quesada, Victor | Ranck, Megan C. | Raney, Brian J. | Deiros, David Rio | Rocchi, Mariano | Rodriguez, David | Ross, Corinna | Ruffier, Magali | Ruiz, San Juana | Sajjadian, S. | Santibanez, Jireh | Schrider, Daniel R. | Searle, Steve | Skaletsky, Helen | Soibam, Benjamin | Smit, Arian F. A. | Tennakoon, Jayantha B. | Tomaska, Lubomir | Ullmer, Brygg | Vejnar, Charles E. | Ventura, Mario | Vilella, Albert J. | Vinar, Tomas | Vogel, Jan-Hinnerk | Walker, Jerilyn A. | Wang, Qing | Warner, Crystal M. | Wildman, Derek E. | Witherspoon, David J. | Wright, Rita A. | Wu, Yuanqing | Xiao, Weimin | Xing, Jinchuan | Zdobnov, Evgeny M. | Zhu, Baoli | Gibbs, Richard A. | Wilson, Richard K.
Nature genetics  2014;46(8):850-857.
A first analysis of the genome sequence of the common marmoset (Callithrix jacchus), assembled using traditional Sanger methods and Ensembl annotation, has permitted genomic comparison with apes and that old world monkeys and the identification of specific molecular features a rapid reproductive capacity partly due to may contribute to the unique biology of diminutive The common marmoset has prevalence of this dizygotic primate. twins. Remarkably, these twins share placental circulation and exchange hematopoietic stem cells in utero, resulting in adults that are hematopoietic chimeras.
We observed positive selection or non-synonymous substitutions for genes encoding growth hormone / insulin-like growth factor (growth pathways), respiratory complex I (metabolic pathways), immunobiology, and proteases (reproductive and immunity pathways). In addition, both protein-coding and microRNA genes related to reproduction exhibit rapid sequence evolution. This New World monkey genome sequence enables significantly increased power for comparative analyses among available primate genomes and facilitates biomedical research application.
doi:10.1038/ng.3042
PMCID: PMC4138798  PMID: 25038751
4.  Inactivation of SAM-Methyltransferase is the Mechanism of Attenuation of a Historic Louse Borne Typhus Vaccine Strain 
PLoS ONE  2014;9(11):e113285.
Louse borne typhus (also called epidemic typhus) was one of man's major scourges, and epidemics of the disease can be reignited when social, economic, or political systems are disrupted. The fear of a bioterrorist attack using the etiologic agent of typhus, Rickettsia prowazekii, was a reality. An attenuated typhus vaccine, R. prowazekii Madrid E strain, was observed to revert to virulence as demonstrated by isolation of the virulent revertant Evir strain from animals which were inoculated with Madrid E strain. The mechanism of the mutation in R. prowazekii that affects the virulence of the vaccine was not known. We sequenced the genome of the virulent revertant Evir strain and compared its genome sequence with the genome sequences of its parental strain, Madrid E. We found that only a single nucleotide in the entire genome was different between the vaccine strain Madrid E and its virulent revertant strain Evir. The mutation is a single nucleotide insertion in the methyltransferase gene (also known as PR028) in the vaccine strain that inactivated the gene. We also confirmed that the vaccine strain E did not cause fever in guinea pigs and the virulent revertant strain Evir caused fever in guinea pigs. We concluded that a single nucleotide insertion in the methyltransferase gene of R. prowazekii attenuated the R. prowazekii vaccine strain E. This suggested that an irreversible insertion or deletion mutation in the methyl transferase gene of R. prowazekii is required for Madrid E to be considered a safe vaccine.
doi:10.1371/journal.pone.0113285
PMCID: PMC4239044  PMID: 25412248
5.  Microbial sequencing to improve individual and population health 
Genome Medicine  2014;6(11):103.
Editorial summary
Recent advances in sequencing technologies are changing the face of infectious disease investigation and control. Personalized anti-infective therapies and surveillance of emergent pathogen outbreaks are just two examples of the potential benefits of merging the fields of genomics and infectious diseases.
doi:10.1186/s13073-014-0103-5
PMCID: PMC4262389  PMID: 25505493
6.  Next-generation pathogen genomics 
Genome Biology  2014;15(11):528.
doi:10.1186/s13059-014-0528-6
PMCID: PMC4283783  PMID: 25417942
7.  The conjunctival microbiome in health and trachomatous disease: a case control study 
Genome Medicine  2014;6(11):99.
Background
Trachoma, caused by Chlamydia trachomatis, remains the world’s leading infectious cause of blindness. Repeated ocular infection during childhood leads to scarring of the conjunctiva, in-turning of the eyelashes (trichiasis) and corneal opacity in later life. There is a growing body of evidence to suggest non-chlamydial bacteria are associated with clinical signs of trachoma, independent of C. trachomatis infection.
Methods
We used deep sequencing of the V1-V3 region of the bacterial 16S rRNA gene to characterize the microbiome of the conjunctiva of 220 residents of The Gambia, 105 with healthy conjunctivae and 115 with clinical signs of trachoma in the absence of detectable C. trachomatis infection. Deep sequencing was carried out using the Roche-454 platform. Sequence data were processed and analyzed through a pipeline developed by the Human Microbiome Project.
Results
The microbiome of healthy participants was influenced by age and season of sample collection with increased richness and diversity seen in younger participants and in samples collected during the dry season. Decreased diversity and an increased abundance of Corynebacterium and Streptococcus were seen in participants with conjunctival scarring compared to normal controls. Abundance of Corynebacterium was higher still in adults with scarring and trichiasis compared to adults with scarring only.
Conclusions
Our results indicate that changes in the conjunctival microbiome occur in trachomatous disease; whether these are a cause or a consequence is yet unknown.
Electronic supplementary material
The online version of this article (doi:10.1186/s13073-014-0099-x) contains supplementary material, which is available to authorized users.
doi:10.1186/s13073-014-0099-x
PMCID: PMC4256740  PMID: 25484919
8.  Whole Genome Sequence of the Treponema pallidum subsp. endemicum Strain Bosnia A: The Genome Is Related to Yaws Treponemes but Contains Few Loci Similar to Syphilis Treponemes 
Background
T. pallidum subsp. endemicum (TEN) is the causative agent of bejel (also known as endemic syphilis). Clinical symptoms of syphilis and bejel are overlapping and the epidemiological context is important for correct diagnosis of both diseases. In contrast to syphilis, caused by T. pallidum subsp. pallidum (TPA), TEN infections are usually spread by direct contact or contaminated utensils rather than by sexual contact. Bejel is most often seen in western Africa and in the Middle East. The strain Bosnia A was isolated in 1950 in Bosnia, southern Europe.
Methodology/Principal Findings
The complete genome of the Bosnia A strain was amplified and sequenced using the pooled segment genome sequencing (PSGS) method and a combination of three next-generation sequencing techniques (SOLiD, Roche 454, and Illumina). Using this approach, a total combined average genome coverage of 513× was achieved. The size of the Bosnia A genome was found to be 1,137,653 bp, i.e. 1.6–2.8 kbp shorter than any previously published genomes of uncultivable pathogenic treponemes. Conserved gene synteny was found in the Bosnia A genome compared to other sequenced syphilis and yaws treponemes. The TEN Bosnia A genome was distinct but very similar to the genome of yaws-causing T. pallidum subsp. pertenue (TPE) strains. Interestingly, the TEN Bosnia A genome was found to contain several sequences, which so far, have been uniquely identified only in syphilis treponemes.
Conclusions/Significance
The genome of TEN Bosnia A contains several sequences thought to be unique to TPA strains; these sequences very likely represent remnants of recombination events during the evolution of TEN treponemes. This finding emphasizes a possible role of repeated horizontal gene transfer between treponemal subspecies in shaping the Bosnia A genome.
Author Summary
Uncultivable treponemes represent bacterial species and subspecies that are obligate pathogens of humans and animals causing diseases with distinct clinical manifestations. Treponema pallidum subsp. pallidum causes sexually transmitted syphilis, a multistage disease characterized in humans by localized, disseminated, and chronic forms of infection, whereas Treponema pallidum subsp. pertenue (agent of yaws) and Treponema pallidum subsp. endemicum (agent of bejel) cause milder, non-venereally transmitted diseases affecting skin, bones and joints. The genetic basis of the pathogenesis and evolution of these microorganisms are still unknown. In this study, a high quality whole genome sequence of the T. pallidum subsp. endemicum Bosnia A strain was obtained using a combination of next-generation sequencing approaches and compared to the genomes of available uncultivable pathogenic treponemes. Relative to all known genomes of Treponema pallidum subspecies, no major genome rearrangements were found in the Bosnia A. The Bosnia A strain clustered with other yaws-causing strains, while syphilis-causing strains clustered separately. In general, the Bosnia A genome showed similar genetic characteristics to yaws treponemes but also contained several sequences thought to be unique to syphilis-causing strains. This finding suggests a possible role of repeated horizontal gene transfer between treponemal subspecies in shaping the Bosnia A genome.
doi:10.1371/journal.pntd.0003261
PMCID: PMC4222731  PMID: 25375929
9.  Coverage theories for metagenomic DNA sequencing based on a generalization of Stevens' theorem 
Journal of mathematical biology  2012;67(5):1141-1161.
Metagenomic project design has relied variously upon speculation, semi-empirical and ad hoc heuristic models, and elementary extensions of single-sample Lander–Waterman expectation theory, all of which are demonstrably inadequate. Here, we propose an approach based upon a generalization of Stevens' Theorem for randomly covering a domain. We extend this result to account for the presence of multiple species, from which are derived useful probabilities for fully recovering a particular target microbe of interest and for average contig length. These show improved specificities compared to older measures and recommend deeper data generation than the levels chosen by some early studies, supporting the view that poor assemblies were due at least somewhat to insufficient data. We assess predictions empirically by generating roughly 4.5 Gb of sequence from a twelve member bacterial community, comparing coverage for two particular members, Selenomonas artemidis and Enterococcus faecium, which are the least (∼3%) and most (∼12%) abundant species, respectively. Agreement is reasonable, with differences likely attributable to coverage biases. We show that, in some cases, bias is simple in the sense that a small reduction in read length to simulate less efficient covering brings data and theory into essentially complete accord. Finally, we describe two applications of the theory. One plots coverage probability over the relevant parameter space, constructing essentially a “metagenomic design map” to enable straightforward analysis and design of future projects. The other gives an overview of the data requirements for various types of sequencing milestones, including a desired number of contact reads and contig length, for detection of a rare viral species.
doi:10.1007/s00285-012-0586-x
PMCID: PMC3795925  PMID: 22965653
DNA sequencing; Coverage; Microbiome; Metagenomics
10.  High-Resolution Genetic Mapping in the Diversity Outbred Mouse Population Identifies Apobec1 as a Candidate Gene for Atherosclerosis 
G3: Genes|Genomes|Genetics  2014;4(12):2353-2363.
Inbred mice exhibit strain-specific variation in susceptibility to atherosclerosis and dyslipidemia that renders them useful in dissecting the genetic architecture of these complex diseases. Traditional quantitative trait locus (QTL) mapping studies using inbred strains often identify large genomic regions, containing many genes, due to limited recombination and/or sample size. This hampers candidate gene identification and translation of these results into possible risk factors and therapeutic targets. An alternative approach is the use of multiparental outbred lines for genetic mapping, such as the Diversity Outbred (DO) mouse panel, which can be more informative than traditional two-parent crosses and can aid in the identification of causal genes and variants associated with QTL. We fed 292 female DO mice either a high-fat, cholesterol-containing (HFCA) diet, to induce atherosclerosis, or a low-fat, high-protein diet for 18 wk and measured plasma lipid levels before and after diet treatment. We measured markers of atherosclerosis in the mice fed the HFCA diet. The mice were genotyped on a medium-density single-nucleotide polymorphism array and founder haplotypes were reconstructed using a hidden Markov model. The reconstructed haplotypes were then used to perform linkage mapping of atherosclerotic lesion size as well as plasma total cholesterol, triglycerides, insulin, and glucose. Among our highly significant QTL we detected a ~100 kb QTL interval for atherosclerosis on Chromosome 6, as well as a 1.4 Mb QTL interval on Chromosome 9 for triglyceride levels at baseline and a coincident 22.2 Mb QTL interval on Chromosome 9 for total cholesterol after dietary treatment. One candidate gene within the Chromosome 6 peak region associated with atherosclerosis is Apobec1, the apolipoprotein B (ApoB) mRNA-editing enzyme, which plays a role in the regulation of ApoB, a critical component of low-density lipoprotein, by editing ApoB mRNA. This study demonstrates the value of the DO population to improve mapping resolution and to aid in the identification of potential therapeutic targets for cardiovascular disease. Using a DO mouse population fed an HFCA diet, we were able to identify an A/J-specific isoform of Apobec1 that contributes to atherosclerosis.
doi:10.1534/g3.114.014704
PMCID: PMC4267931  PMID: 25344410
quantitative trait loci; multiparental models; lipoproteins; atherosclerosis; MPP; multiparental populations; Multiparent Advanced Generation Inter-Cross (MAGIC)
11.  Transferable Vancomycin Resistance in a Community-Associated MRSA Lineage 
The New England journal of medicine  2014;370(16):1524-1531.
SUMMARY
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.
doi:10.1056/NEJMoa1303359
PMCID: PMC4112484  PMID: 24738669
12.  Metagenomic analysis of double-stranded DNA viruses in healthy adults 
BMC Biology  2014;12(1):71.
Background
The Human Microbiome Project (HMP) was undertaken with the goal of defining microbial communities in and on the bodies of healthy individuals using high-throughput metagenomic sequencing analysis. The viruses present in these microbial communities, the ‘human virome,’ are an important aspect of the human microbiome that is particularly understudied in the absence of overt disease. We analyzed eukaryotic double-stranded DNA (dsDNA) viruses, together with dsDNA replicative intermediates of single-stranded DNA viruses, in metagenomic sequence data generated by the HMP. We studied 706 samples from 102 subjects were studied, with each subject sampled at up to five major body habitats: nose, skin, mouth, vagina, and stool. Fifty-one individuals had samples taken at two or three time points 30 to 359 days apart from at least one of the body habitats.
Results
We detected an average of 5.5 viral genera in each individual. At least one virus was detected in 92% of the individuals sampled. These viruses included herpesviruses, papillomaviruses, polyomaviruses, adenoviruses, anelloviruses, parvoviruses, and circoviruses. Each individual had a distinct viral profile, demonstrating the high interpersonal diversity of the virome. Some components of the virome were stable over time.
Conclusions
This study is the first to use high-throughput DNA sequencing to describe the diversity of eukaryotic dsDNA viruses in a large cohort of normal individuals who were sampled at multiple body sites. Our results show that the human virome is a complex component of the microbial flora. Some viruses establish long-term infections that may be associated with increased risk or possibly with protection from disease. A better understanding of the composition and dynamics of the virome may hold important keys to human health.
Electronic supplementary material
The online version of this article (doi:10.1186/s12915-014-0071-7) contains supplementary material, which is available to authorized users.
doi:10.1186/s12915-014-0071-7
PMCID: PMC4177058  PMID: 25212266
Metagenomics; Microbiome; Virome
13.  Genomic Epidemiology of Salmonella enterica Serotype Enteritidis based on Population Structure of Prevalent Lineages 
Emerging Infectious Diseases  2014;20(9):1481-1489.
Major lineages emerged during the 17th–18th centuries and diversified during the 1920s and 1950s.
Salmonella enterica serotype Enteritidis is one of the most commonly reported causes of human salmonellosis. Its low genetic diversity, measured by fingerprinting methods, has made subtyping a challenge. We used whole-genome sequencing to characterize 125 S. enterica Enteritidis and 3 S. enterica serotype Nitra strains. Single-nucleotide polymorphisms were filtered to identify 4,887 reliable loci that distinguished all isolates from each other. Our whole-genome single-nucleotide polymorphism typing approach was robust for S. enterica Enteritidis subtyping with combined data for different strains from 2 different sequencing platforms. Five major genetic lineages were recognized, which revealed possible patterns of geographic and epidemiologic distribution. Analyses on the population dynamics and evolutionary history estimated that major lineages emerged during the 17th–18th centuries and diversified during the 1920s and 1950s.
doi:10.3201/eid2009.131095
PMCID: PMC4178404  PMID: 25147968
Salmonella enterica serotype Enteritidis; genome sequencing; SNP; genomic epidemiology; subtyping, evolution, bacteria
14.  Genomic Encyclopedia of Bacteria and Archaea: Sequencing a Myriad of Type Strains 
PLoS Biology  2014;12(8):e1001920.
This manuscript calls for an international effort to generate a comprehensive catalog from genome sequences of all the archaeal and bacterial type strains.
Microbes hold the key to life. They hold the secrets to our past (as the descendants of the earliest forms of life) and the prospects for our future (as we mine their genes for solutions to some of the planet's most pressing problems, from global warming to antibiotic resistance). However, the piecemeal approach that has defined efforts to study microbial genetic diversity for over 20 years and in over 30,000 genome projects risks squandering that promise. These efforts have covered less than 20% of the diversity of the cultured archaeal and bacterial species, which represent just 15% of the overall known prokaryotic diversity. Here we call for the funding of a systematic effort to produce a comprehensive genomic catalog of all cultured Bacteria and Archaea by sequencing, where available, the type strain of each species with a validly published name (currently∼11,000). This effort will provide an unprecedented level of coverage of our planet's genetic diversity, allow for the large-scale discovery of novel genes and functions, and lead to an improved understanding of microbial evolution and function in the environment.
doi:10.1371/journal.pbio.1001920
PMCID: PMC4122341  PMID: 25093819
15.  Application of Next-Generation Sequencing to Identify Genes and Mutations Causing Autosomal Dominant Retinitis Pigmentosa (adRP) 
The goal of our research is to identify genes and mutations causing auto-somal dominant retinitis pigmentosa (adRP). For this purpose we established a cohort of more than 250 independently ascertained families with adRP in the Houston Laboratory for Molecular Diagnosis of Inherited Eye Diseases. Affected members of each family were screened for disease-causing mutations in genes and gene regions that are commonly associated with adRP. By this approach, we detected mutations in 65 % of the families, leaving 85 families that are likely to harbor mutations outside of the “common” regions or in novel genes. Of these, 32 families were tested by several types of next-generation sequencing (NGS), including (a) targeted polymerase chain reaction (PCR) NGS, (b) whole exome NGS, and (c) targeted retinal-capture NGS. We detected mutations in 11 of these families (31 %) bringing the total detected in the adRP cohort to 70 %. Several large families have also been tested for linkage using Afymetrix single nucleotide polymorphism (SNP) arrays.
doi:10.1007/978-1-4614-3209-8_16
PMCID: PMC4121110  PMID: 24664689
Retinitis pigmentosa; Next-generation sequencing; Linkage mapping; Mutation prevalence; Retinal gene capture; Whole-exome sequencing
16.  Virome genomics: a tool for defining the human virome 
Current opinion in microbiology  2013;16(4):479-484.
High throughput, deep sequencing assays are powerful tools for gaining insights into virus-host interactions. Sequencing assays can discover novel viruses and describe the genomes of novel and known viruses. Genomic information can predict viral proteins that can be characterized, describe important genes in the host that control infections, and evaluate gene expression of viruses and hosts during infection. Sequencing can also describe variation and evolution of viruses during replication and transmission. This review recounts some of the major advances in the studies of virus-host interactions from the last two years, and discusses the uses of sequencing technologies relating to these studies.
doi:10.1016/j.mib.2013.04.006
PMCID: PMC3755052  PMID: 23706900
17.  High-Resolution Quantification of Hepatitis C Virus Genome-Wide Mutation Load and Its Correlation with the Outcome of Peginterferon-Alpha2a and Ribavirin Combination Therapy 
PLoS ONE  2014;9(6):e100131.
Hepatitis C virus (HCV) is a highly mutable RNA virus and circulates as a heterogeneous population in individual patients. The magnitude of such population heterogeneity has long been proposed to be linked with diverse clinical phenotypes, including antiviral therapy. Yet data accumulated thus far are fairly inconclusive. By the integration of long RT-PCR with 454 sequencing, we have built a pipeline optimized for the quantification of HCV genome-wide mutation load at 1% resolution of mutation frequency, followed by a retrospective study to examine the role of HCV mutation load in peginterferon-alpha2a and ribavirin combination antiviral therapy. Genome-wide HCV mutation load varied widely with a range from 92 to 1639 mutations and presented a Poisson distribution among 56 patients (Kolmogorov-Smirnov statistic  = 0.078, p = 0.25). Patients achieving sustained virological response (n = 26) had significantly lower mutation loads than that in null responders (n = 30) (mean and standard derivation: 524±279 vs. 805±271, p = 0.00035). All 36,818 mutations detected in 56 patients displayed a power-law distribution in terms of mutation frequency in viral population. The low-frequency mutation load, but not the high-frequency load, was proportional firmly to the total mutation load. In-depth analyses revealed that intra-patient HCV population structure was shaped by multiple factors, including immune pressure, strain difference and genetic drift. These findings explain previous conflicting reports using low-resolution methods and highlight a dominant role of natural selection in response to therapeutic intervention. By attaining its signatures from complex interaction between host and virus, the high-resolution quantification of HCV mutation load predicts outcomes from interferon-based antiviral therapy and could also be a potential biomarker in other clinical settings.
doi:10.1371/journal.pone.0100131
PMCID: PMC4065037  PMID: 24950220
18.  Precise Dissection of an Escherichia coli O157:H7 Outbreak by Single Nucleotide Polymorphism Analysis 
Journal of Clinical Microbiology  2013;51(12):3950-3954.
The current pathogen-typing methods have suboptimal sensitivities and specificities. DNA sequencing offers an opportunity to type pathogens with greater degrees of discrimination using single nucleotide polymorphisms (SNPs) than with pulsed-field gel electrophoresis (PFGE) and other methodologies. In a recent cluster of Escherichia coli O157:H7 infections attributed to salad bar exposures and romaine lettuce, a subset of cases denied exposure to either source, although PFGE and multiple-locus variable-number tandem-repeat analysis (MLVA) suggested that all isolates had the same recent progenitor. Interrogation of a preselected set of 3,442,673 nucleotides in backbone open reading frames (ORFs) identified only 1 or 2 single nucleotide differences in 3 of 12 isolates from the cases who denied exposure. The backbone DNAs of 9 of 9 and 3 of 3 cases who reported or were unsure about exposure, respectively, were isogenic. Backbone ORF SNP set sequencing offers pathogen differentiation capabilities that exceed those of PFGE and MLVA.
doi:10.1128/JCM.01930-13
PMCID: PMC3838074  PMID: 24048526
19.  Widespread Colonization of the Lung by Tropheryma whipplei in HIV Infection 
Rationale: Lung infections caused by opportunistic or virulent pathogens are a principal cause of morbidity and mortality in HIV infection. It is unknown whether HIV infection leads to changes in basal lung microflora, which may contribute to chronic pulmonary complications that increasingly are being recognized in individuals infected with HIV.
Objectives: To determine whether the immunodeficiency associated with HIV infection resulted in alteration of the lung microbiota.
Methods: We used 16S ribosomal RNA targeted pyrosequencing and shotgun metagenomic sequencing to analyze bacterial gene sequences in bronchoalveolar lavage (BAL) and mouths of 82 HIV-positive and 77 HIV-negative subjects.
Measurements and Main Results: Sequences representing Tropheryma whipplei, the etiologic agent of Whipple’s disease, were significantly more frequent in BAL of HIV-positive compared with HIV-negative individuals. T. whipplei dominated the community (>50% of sequence reads) in 11 HIV-positive subjects, but only 1 HIV-negative individual (13.4 versus 1.3%; P = 0.0018). In 30 HIV-positive individuals sampled longitudinally, antiretroviral therapy resulted in a significantly reduced relative abundance of T. whipplei in the lung. Shotgun metagenomic sequencing was performed on eight BAL samples dominated by T. whipplei 16S ribosomal RNA. Whole genome assembly of pooled reads showed that uncultured lung-derived T. whipplei had similar gene content to two isolates obtained from subjects with Whipple’s disease.
Conclusions: Asymptomatic subjects with HIV infection have unexpected colonization of the lung by T. whipplei, which is reduced by effective antiretroviral therapy and merits further study for a potential pathogenic role in chronic pulmonary complications of HIV infection.
doi:10.1164/rccm.201211-2145OC
PMCID: PMC3734615  PMID: 23392441
human; microbiome; metagenome; 16S ribosomal RNA; bronchoalveolar lavage
20.  Comparison of the Respiratory Microbiome in Healthy Nonsmokers and Smokers 
Rationale: Results from 16S rDNA-encoding gene sequence–based, culture-independent techniques have led to conflicting conclusions about the composition of the lower respiratory tract microbiome.
Objectives: To compare the microbiome of the upper and lower respiratory tract in healthy HIV-uninfected nonsmokers and smokers in a multicenter cohort.
Methods: Participants were nonsmokers and smokers without significant comorbidities. Oral washes and bronchoscopic alveolar lavages were collected in a standardized manner. Sequence analysis of bacterial 16S rRNA-encoding genes was performed, and the neutral model in community ecology was used to identify bacteria that were the most plausible members of a lung microbiome.
Measurements and Main Results: Sixty-four participants were enrolled. Most bacteria identified in the lung were also in the mouth, but specific bacteria such as Enterobacteriaceae, Haemophilus, Methylobacterium, and Ralstonia species were disproportionally represented in the lungs compared with values predicted by the neutral model. Tropheryma was also in the lung, but not the mouth. Mouth communities differed between nonsmokers and smokers in species such as Porphyromonas, Neisseria, and Gemella, but lung bacterial populations did not.
Conclusions: This study is the largest to examine composition of the lower respiratory tract microbiome in healthy individuals and the first to use the neutral model to compare the lung to the mouth. Specific bacteria appear in significantly higher abundance in the lungs than would be expected if they originated from the mouth, demonstrating that the lung microbiome does not derive entirely from the mouth. The mouth microbiome differs in nonsmokers and smokers, but lung communities were not significantly altered by smoking.
doi:10.1164/rccm.201210-1913OC
PMCID: PMC3734620  PMID: 23491408
lung; microbiome; smoking; bronchoscopy; metagenome
21.  Exploration of bacterial community classes in major human habitats 
Genome Biology  2014;15(5):R66.
Background
Determining bacterial abundance variation is the first step in understanding bacterial similarity between individuals. Categorization of bacterial communities into groups or community classes is the subsequent step in describing microbial distribution based on abundance patterns. Here, we present an analysis of the groupings of bacterial communities in stool, nasal, skin, vaginal and oral habitats in a healthy cohort of 236 subjects from the Human Microbiome Project.
Results
We identify distinct community group patterns in the anterior nares, four skin sites, and vagina at the genus level. We also confirm three enterotypes previously identified in stools. We identify two clusters with low silhouette values in most oral sites, in which bacterial communities are more homogeneous. Subjects sharing a community class in one habitat do not necessarily share a community class in another, except in the three vaginal sites and the symmetric habitats of the left and right retroauricular creases. Demographic factors, including gender, age, and ethnicity, significantly influence community composition in several habitats. Community classes in the vagina, retroauricular crease and stool are stable over approximately 200 days.
Conclusion
The community composition, association of demographic factors with community classes, and demonstration of community stability deepen our understanding of the variability and dynamics of human microbiomes. This also has significant implications for experimental designs that seek microbial correlations with clinical phenotypes.
doi:10.1186/gb-2014-15-5-r66
PMCID: PMC4073010  PMID: 24887286
22.  Sixty years of genome biology 
Genome Biology  2013;14(4):113.
Sixty years after Watson and Crick published the double helix model of DNA's structure, thirteen members of Genome Biology's Editorial Board select key advances in the field of genome biology subsequent to that discovery.
doi:10.1186/gb-2013-14-4-113
PMCID: PMC3663092  PMID: 23651518
23.  Quantitation and Composition of Cutaneous Microbiota in Diabetic and Nondiabetic Men 
The Journal of Infectious Diseases  2013;207(7):1105-1114.
Background. Diabetic foot infections are a leading cause of lower extremity amputations. Our study examines the microbiota of diabetic skin prior to ulcer development or infection.
Methods. In a case-control study, outpatient males were recruited at a veterans hospital. Subjects were swabbed at 4 cutaneous sites, 1 on the forearm and 3 on the foot. Quantitative polymerase chain reaction (qPCR) with primers and probes specific for bacteria, Staphylococcus species, Staphylococcus aureus, and fungi were performed on all samples. High-throughput 16S ribosomal RNA (rRNA) sequencing was performed on samples from the forearm and the plantar aspect of the foot.
Results. qPCR analysis of swab specimens from 30 diabetic subjects and 30 control subjects showed no differences in total numbers of bacteria or fungi at any sampled site. Increased log10 concentrations of Staphylococcus aureus, quantified by the number of nuc gene copies, were present in diabetic men on the plantar aspect of the foot. High-throughput 16S rRNA sequencing found that, on the foot, the microbiota in controls (n = 24) was dominated by Staphylococcus species, whereas the microbiota in diabetics (n = 23) was more diverse at the genus level. The forearm microbiota had similar diversity in diabetic and control groups.
Conclusions. The feet of diabetic men had decreased populations of Staphylococcus species, increased populations of S. aureus, and increased bacterial diversity, compared with the feet of controls. These ecologic changes may affect the risk for wound infections.
doi:10.1093/infdis/jit005
PMCID: PMC3583274  PMID: 23300163
microbiota; microbiome; diabetic foot; cutaneous; Staphylococcus; Staphylococcus aureus
24.  Phylogenomics and the Dynamic Genome Evolution of the Genus Streptococcus 
Genome Biology and Evolution  2014;6(4):741-753.
The genus Streptococcus comprises important pathogens that have a severe impact on human health and are responsible for substantial economic losses to agriculture. Here, we utilize 46 Streptococcus genome sequences (44 species), including eight species sequenced here, to provide the first genomic level insight into the evolutionary history and genetic basis underlying the functional diversity of all major groups of this genus. Gene gain/loss analysis revealed a dynamic pattern of genome evolution characterized by an initial period of gene gain followed by a period of loss, as the major groups within the genus diversified. This was followed by a period of genome expansion associated with the origins of the present extant species. The pattern is concordant with an emerging view that genomes evolve through a dynamic process of expansion and streamlining. A large proportion of the pan-genome has experienced lateral gene transfer (LGT) with causative factors, such as relatedness and shared environment, operating over different evolutionary scales. Multiple gene ontology terms were significantly enriched for each group, and mapping terms onto the phylogeny showed that those corresponding to genes born on branches leading to the major groups represented approximately one-fifth of those enriched. Furthermore, despite the extensive LGT, several biochemical characteristics have been retained since group formation, suggesting genomic cohesiveness through time, and that these characteristics may be fundamental to each group. For example, proteolysis: mitis group; urea metabolism: salivarius group; carbohydrate metabolism: pyogenic group; and transcription regulation: bovis group.
doi:10.1093/gbe/evu048
PMCID: PMC4007547  PMID: 24625962
comparative genomics; phylogenetics; gene gain and loss; enrichment; lateral gene transfer
25.  Propionibacterium acnes strain populations in the human skin microbiome associated with acne 
The human skin microbiome plays important roles in skin health and disease. However, bacterial population structure and diversity at the strain level is poorly understood. We compared the skin microbiome at the strain level and genome level of Propionibacterium acnes, a dominant skin commensal, between 49 acne patients and 52 healthy individuals by sampling the pilosebaceous units on their noses. Metagenomic analysis demonstrated that while the relative abundances of P. acnes were similar, the strain population structures were significantly different in the two cohorts. Certain strains were highly associated with acne and other strains were enriched in healthy skin. By sequencing 66 previously unreported P. acnes strains and comparing 71 P. acnes genomes, we identified potential genetic determinants of various P. acnes strains in association with acne or health. Our analysis suggests that acquired DNA sequences and bacterial immune elements may play roles in determining virulence properties of P. acnes strains and some could be future targets for therapeutic interventions. This study demonstrates a previously unreported paradigm of commensal strain populations that could explain the pathogenesis of human diseases. It underscores the importance of strain level analysis of the human microbiome to define the role of commensals in health and disease.
doi:10.1038/jid.2013.21
PMCID: PMC3745799  PMID: 23337890

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