About the Authors: Lance B. Price * E-mail: lprice@gwu.edu Affiliations Milken Institute School of Public Health, George Washington University, Washington DC, United States of America, Division of ...Pathogen Genomics, Translational Genomics Research Institute, Flagstaff, Arizona, United States of America ORCID http://orcid.org/0000-0002-8746-5307 Bruce A. Hungate Affiliations Center for Ecosystem Science and Society, Northern Arizona University, Flagstaff, Arizona, United States of America, Department of Biological Sciences, Northern Arizona University, Flagstaff, Arizona, United States of America ORCID http://orcid.org/0000-0002-7337-1887 Benjamin J. Koch Affiliations Center for Ecosystem Science and Society, Northern Arizona University, Flagstaff, Arizona, United States of America, Department of Biological Sciences, Northern Arizona University, Flagstaff, Arizona, United States of America Gregg S. Davis Affiliation: Milken Institute School of Public Health, George Washington University, Washington DC, United States of America ORCID http://orcid.org/0000-0001-7684-0426 Cindy M. Liu Affiliation: Milken Institute School of Public Health, George Washington University, Washington DC, United States of AmericaCitation: Price LB, Hungate BA, Koch BJ, Davis GS, Liu CM (2017) Colonizing opportunistic pathogens (COPs): The beasts in all of us. ...COPs lack predictable periods between colonization and infection, making their epidemiology cryptic. Because of this, COPs can cause insidious epidemics, where new clones transmit widely-even globally-among healthy populations before being recognized. ...antibiotics can select for antibiotic-resistant COPs, in addition to commensals within the host microbiome, regardless of whether or not the specific COP was the intended target of the antibiotic treatment. COPs require new active and integrative surveillance programs Enhanced surveillance of COPs can enable public health agencies to identify and control emerging COP clones more quickly than is currently possible. Because of the insidious nature of COP epidemics, active surveillance programs that monitor both COPs circulating among asymptomatic carriers in the community and COPs causing clinical infections are crucial.
We report a robust, versatile approach called CRISPR live-cell fluorescent in situ hybridization (LiveFISH) using fluorescent oligonucleotides for genome tracking in a broad range of cell types, ...including primary cells. An intrinsic stability switch of CRISPR guide RNAs enables LiveFISH to accurately detect chromosomal disorders such as Patau syndrome in prenatal amniotic fluid cells and track multiple loci in human T lymphocytes. In addition, LiveFISH tracks the real-time movement of DNA double-strand breaks induced by CRISPR-Cas9-mediated editing and consequent chromosome translocations. Finally, by combining Cas9 and Cas13 systems, LiveFISH allows for simultaneous visualization of genomic DNA and RNA transcripts in living cells. The LiveFISH approach enables real-time live imaging of DNA and RNA during genome editing, transcription, and rearrangements in single cells.
Bacteria grow and transform elements at different rates, and as yet, quantifying this variation in the environment is difficult. Determining isotope enrichment with fine taxonomic resolution after ...exposure to isotope tracers could help, but there are few suitable techniques. We propose a modification to stable isotope probing (SIP) that enables the isotopic composition of DNA from individual bacterial taxa after exposure to isotope tracers to be determined. In our modification, after isopycnic centrifugation, DNA is collected in multiple density fractions, and each fraction is sequenced separately. Taxon-specific density curves are produced for labeled and nonlabeled treatments, from which the shift in density for each individual taxon in response to isotope labeling is calculated. Expressing each taxon's density shift relative to that taxon's density measured without isotope enrichment accounts for the influence of nucleic acid composition on density and isolates the influence of isotope tracer assimilation. The shift in density translates quantitatively to isotopic enrichment. Because this revision to SIP allows quantitative measurements of isotope enrichment, we propose to call it quantitative stable isotope probing (qSIP). We demonstrated qSIP using soil incubations, in which soil bacteria exhibited strong taxonomic variations in (18)O and (13)C composition after exposure to (18)Owater or (13)Cglucose. The addition of glucose increased the assimilation of (18)O into DNA from (18)Owater. However, the increase in (18)O assimilation was greater than expected based on utilization of glucose-derived carbon alone, because the addition of glucose indirectly stimulated bacteria to utilize other substrates for growth. This example illustrates the benefit of a quantitative approach to stable isotope probing.
Circumcision is associated with significant reductions in HIV, HSV-2 and HPV infections among men and significant reductions in bacterial vaginosis among their female partners.
We assessed the penile ...(coronal sulci) microbiota in 12 HIV-negative Ugandan men before and after circumcision. Microbiota were characterized using sequence-tagged 16S rRNA gene pyrosequencing targeting the V3-V4 hypervariable regions. Taxonomic classification was performed using the RDP Naïve Bayesian Classifier. Among the 42 unique bacterial families identified, Pseudomonadaceae and Oxalobactericeae were the most abundant irrespective of circumcision status. Circumcision was associated with a significant change in the overall microbiota (PerMANOVA p = 0.007) and with a significant decrease in putative anaerobic bacterial families (Wilcoxon Signed-Rank test p = 0.014). Specifically, two families-Clostridiales Family XI (p = 0.006) and Prevotellaceae (p = 0.006)-were uniquely abundant before circumcision. Within these families we identified a number of anaerobic genera previously associated with bacterial vaginosis including: Anaerococcus spp., Finegoldia spp., Peptoniphilus spp., and Prevotella spp.
The anoxic microenvironment of the subpreputial space may support pro-inflammatory anaerobes that can activate Langerhans cells to present HIV to CD4 cells in draining lymph nodes. Thus, the reduction in putative anaerobic bacteria after circumcision may play a role in protection from HIV and other sexually transmitted diseases.
E. coli
ST131 is an important extraintestinal pathogen that can colonize the gastrointestinal tracts of humans and food animals. Here, we combined detection of accessory traits associated with avian ...adaptation (ColV plasmids) with high-resolution phylogenetics to quantify the portion of human infections caused by ST131 strains of food animal origin. Our results suggest that one ST131 sublineage—ST131-
H
22—has become established in poultry populations around the world and that meat may serve as a vehicle for human exposure and infection. ST131-
H
22 is just one of many
E. coli
lineages that may be transmitted from food animals to humans. Additional studies that combine detection of host-associated accessory elements with phylogenetics may allow us to quantify the total fraction of human extraintestinal infections attributable to food animal
E. coli
strains.
Escherichia coli
sequence type 131 (ST131) has emerged rapidly to become the most prevalent extraintestinal pathogenic
E. coli
clones in circulation today. Previous investigations appeared to exonerate retail meat as a source of human exposure to ST131; however, these studies focused mainly on extensively multidrug-resistant ST131 strains, which typically carry allele 30 of the
fimH
type 1 fimbrial adhesin gene (ST131-
H
30). To estimate the frequency of extraintestinal human infections arising from foodborne ST131 strains without bias toward particular sublineages or phenotypes, we conducted a 1-year prospective study of
E. coli
from meat products and clinical cultures in Flagstaff, Arizona. We characterized all isolates by multilocus sequence typing,
fimH
typing, and core genome phylogenetic analyses, and we screened isolates for avian-associated ColV plasmids as an indication of poultry adaptation.
E. coli
was isolated from 79.8% of the 2,452 meat samples and 72.4% of the 1,735 culture-positive clinical samples. Twenty-seven meat isolates were ST131 and belonged almost exclusively (
n
= 25) to the ST131-
H
22 lineage. All but 1 of the 25 H22 meat isolates were from poultry products, and all but 2 carried poultry-associated ColV plasmids. Of the 1,188 contemporaneous human clinical
E. coli
isolates, 24 were ST131-
H
22, one-quarter of which occurred in the same high-resolution phylogenetic clades as the ST131-
H
22 meat isolates and carried ColV plasmids. Molecular clock analysis of an international ST131-
H
22 genome collection suggested that ColV plasmids have been acquired at least six times since the 1940s and that poultry-to-human transmission is not limited to the United States.
Bacterial colonization is hypothesized to play a pathogenic role in the non-healing state of chronic wounds. We characterized wound bacteria from a cohort of chronic wound patients using a 16S rRNA ...gene-based pyrosequencing approach and assessed the impact of diabetes and antibiotics on chronic wound microbiota.
We prospectively enrolled 24 patients at a referral wound center in Baltimore, MD; sampled patients' wounds by curette; cultured samples under aerobic and anaerobic conditions; and pyrosequenced the 16S rRNA V3 hypervariable region. The 16S rRNA gene-based analyses revealed an average of 10 different bacterial families in wounds--approximately 4 times more than estimated by culture-based analyses. Fastidious anaerobic bacteria belonging to the Clostridiales family XI were among the most prevalent bacteria identified exclusively by 16S rRNA gene-based analyses. Community-scale analyses showed that wound microbiota from antibiotic treated patients were significantly different from untreated patients (p = 0.007) and were characterized by increased Pseudomonadaceae abundance. These analyses also revealed that antibiotic use was associated with decreased Streptococcaceae among diabetics and that Streptococcaceae was more abundant among diabetics as compared to non-diabetics.
The 16S rRNA gene-based analyses revealed complex bacterial communities including anaerobic bacteria that may play causative roles in the non-healing state of some chronic wounds. Our data suggest that antimicrobial therapy alters community structure--reducing some bacteria while selecting for others.
Fungal load quantification is a critical component of fungal community analyses. Limitation of current approaches for quantifying the fungal component in the human microbiome suggests the need for ...new broad-coverage techniques.
We analyzed 2,085 18S rRNA gene sequences from the SILVA database for assay design. We generated and quantified plasmid standards using a qPCR-based approach. We evaluated assay coverage against 4,968 sequences and performed assay validation following the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines.
We designed FungiQuant, a TaqMan® qPCR assay targeting a 351 bp region in the fungal 18S rRNA gene. Our in silico analysis showed that FungiQuant is a perfect sequence match to 90.0% of the 2,617 fungal species analyzed. We showed that FungiQuant's is 100% sensitive and its amplification efficiencies ranged from 76.3% to 114.5%, with r(2)-values of >0.99 against the 69 fungal species tested. Additionally, FungiQuant inter- and intra-run coefficients of variance ranged from <10% and <20%, respectively. We further showed that FungiQuant has a limit of quantification 25 copies and a limit of detection at 5 copies. Lastly, by comparing results from human-only background DNA with low-level fungal DNA, we showed that amplification in two or three of a FungiQuant performed in triplicate is statistically significant for true positive fungal detection.
FungiQuant has comprehensive coverage against diverse fungi and is a robust quantification and detection tool for delineating between true fungal detection and non-target human DNA.
Programmable control of spatial genome organization is a powerful approach for studying how nuclear structure affects gene regulation and cellular function. Here, we develop a versatile CRISPR-genome ...organization (CRISPR-GO) system that can efficiently control the spatial positioning of genomic loci relative to specific nuclear compartments, including the nuclear periphery, Cajal bodies, and promyelocytic leukemia (PML) bodies. CRISPR-GO is chemically inducible and reversible, enabling interrogation of real-time dynamics of chromatin interactions with nuclear compartments in living cells. Inducible repositioning of genomic loci to the nuclear periphery allows for dissection of mitosis-dependent and -independent relocalization events and also for interrogation of the relationship between gene position and gene expression. CRISPR-GO mediates rapid de novo formation of Cajal bodies at desired chromatin loci and causes significant repression of endogenous gene expression over long distances (30–600 kb). The CRISPR-GO system offers a programmable platform to investigate large-scale spatial genome organization and function.
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•CRISPR-GO is a versatile system for targeting genome loci to nuclear compartments•An inducible and reversible approach to study spatial genome organization•CRISPR-GO mediates rapid and reversible de novo Cajal body (CB) formation•CB-chromatin colocalization represses distal (30–600 kb) gene expression
An engineered CRISPR-based platform for inducible recruitment of specific genomic loci to distinct nuclear compartments reveals positional effects on gene expression and cellular function.
Dolosigranulum pigrum-a lactic acid bacterium that is increasingly recognized as an important member of the nasal microbiome. Currently, there are limited rapid and low-cost options for confirming D. ...pigrum isolates and detecting D. pigrum in clinical specimens. Here we describe the design and validation of a novel PCR assay targeting D. pigrum that is both sensitive and specific. We designed a PCR assay targeting murJ, a single-copy core species gene identified through the analysis of 21 D. pigrum whole genome sequences. The assay achieved 100% sensitivity and 100% specificity against D. pigrum and diverse bacterial isolates and an overall 91.1% sensitivity and 100% specificity using nasal swabs, detecting D. pigrum at a threshold of 1.0 × 10
D. pigrum 16S rRNA gene copies per swab. This assay adds a reliable and rapid D. pigrum detection tool to the microbiome researcher toolkit investigating the role of generalist and specialist bacteria in the nasal environment.
Bacterial load quantification is a critical component of bacterial community analysis, but a culture-independent method capable of detecting and quantifying diverse bacteria is needed. Based on our ...analysis of a diverse collection of 16 S rRNA gene sequences, we designed a broad-coverage quantitative real-time PCR (qPCR) assay--BactQuant--for quantifying 16 S rRNA gene copy number and estimating bacterial load. We further utilized in silico evaluation to complement laboratory-based qPCR characterization to validate BactQuant.
The aligned core set of 4,938 16 S rRNA gene sequences in the Greengenes database were analyzed for assay design. Cloned plasmid standards were generated and quantified using a qPCR-based approach. Coverage analysis was performed computationally using >670,000 sequences and further evaluated following the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines.
A bacterial TaqMan® qPCR assay targeting a 466 bp region in V3-V4 was designed. Coverage analysis showed that 91% of the phyla, 96% of the genera, and >80% of the 89,537 species analyzed contained at least one perfect sequence match to the BactQuant assay. Of the 106 bacterial species evaluated, amplification efficiencies ranged from 81 to 120%, with r2-value of >0.99, including species with sequence mismatches. Inter- and intra-run coefficient of variance was <3% and <16% for Ct and copy number, respectively.
The BactQuant assay offers significantly broader coverage than a previously reported universal bacterial quantification assay BactQuant in vitro performance was better than the in silico predictions.
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