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Find video protocols related to scientific articles indexed in Pubmed.
Large-scale genomic sequencing of extraintestinal pathogenic Escherichia coli strains.
Genome Res.
PUBLISHED: 11-06-2014
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Large-scale bacterial genome sequencing efforts to date have provided limited information on the most prevalent category of disease: sporadically acquired infections caused by common pathogenic bacteria. Here, we performed whole genome sequencing and de novo assembly of 312 blood- or urine-derived isolates of extraintestinal pathogenic (ExPEC) Escherichia coli, a common agent of sepsis and community-acquired urinary tract infections, obtained during the course of routine clinical care at a single institution. We find that ExPEC E. coli are highly genomically heterogeneous, consistent with pan-genome analyses encompassing the larger species. Investigation of differential virulence factor content and antibiotic resistance phenotypes reveals markedly different profiles among lineages and among strains infecting different bodily sites. We use high-resolution molecular epidemiology to explore the dynamics of infections at the level of individual patients, including identification of possible person-to-person transmission. Notably, a limited number of discrete lineages caused the majority of bloodstream infections, including one sub-clone (ST131-H30) responsible for 28% of bacteremic E. coli infections over a three-year period. We additionally use a microbial genome-wide-association study (GWAS) approach to identify individual genes responsible for antibiotic resistance, successfully recovering known genes but notably not identifying any novel factors. We anticipate that in the near future, whole genome sequencing of microorganisms associated with clinical disease will become routine. Our study reveals what kinds of information can be obtained from sequencing clinical isolates on a large scale, even well-characterized organisms such as E. coli, and provides insight into how this information might be utilized in a healthcare setting.
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Mutually repressing repressor functions and multi-layered cellular heterogeneity regulate the bistable Salmonella?fliC census.
Mol. Microbiol.
PUBLISHED: 10-10-2014
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Bistable flagellar and virulence gene expression generates specialized Salmonella subpopulations with distinct functions. Repressing flagellar genes allows Salmonella to evade caspase-1 mediated host defenses and enhances systemic colonization. By definition, bistability arises when intermediate states of gene expression are rendered unstable by the underlying genetic circuitry. We demonstrate sustained bistable fliC expression in virulent Salmonella 14028 and document dynamic control of the distribution, or single-cell census, of flagellar gene expression by the mutually repressing repressors YdiV and FliZ. YdiV partitions cells into the fliC-OFF subpopulation, while FliZ partitions cells into the fliC-HIGH subpopulation at late time points during growth. Bistability of ?fliZ populations and ydiV-independent FliZ control of flagellar gene expression provide evidence that the YdiV-FliZ mutually repressing repressor circuit is not required for bistability. Repression and activation by YdiV and FliZ (respectively) can shape the census of fliC expression independently, and bistability collapses into a predominantly intermediate population in the absence of both regulators. Metered expression of YdiV and FliZ reveals variable sensitivity to these regulators and defines conditions where expression of FliZ enhances fliC expression and where FliZ does not alter the fliC census. Thus, this evolved genetic circuitry coordinates multiple layers of regulatory heterogeneity into a binary response.
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Performance Comparison of Illumina and Ion Torrent Next-Generation Sequencing Platforms for 16S rRNA-Based Bacterial Community Profiling.
Appl. Environ. Microbiol.
PUBLISHED: 09-26-2014
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High-throughput sequencing of the taxonomically informative 16S rRNA gene provides a powerful approach for exploring microbial diversity. Here we compare the performances of two common "benchtop" sequencing platforms, Illumina MiSeq and Ion Torrent Personal Genome Machine (PGM), for bacterial community profiling by 16S rRNA (V1-V2) amplicon sequencing. We benchmarked performance by using a 20-organism mock bacterial community and a collection of primary human specimens. We observed comparatively higher error rates with the Ion Torrent platform and report a pattern of premature sequence truncation specific to semiconductor sequencing. Read truncation was dependent on both the directionality of sequencing and the target species, resulting in organism-specific biases in community profiles. We found that these sequencing artifacts could be minimized by using bidirectional amplicon sequencing and an optimized flow order on the Ion Torrent platform. Results of bacterial community profiling performed on the mock community and a collection of 18 human-derived microbiological specimens were generally in good agreement for both platforms; however, in some cases, results differed significantly. Disparities could be attributed to the failure to generate full-length reads for particular organisms on the Ion Torrent platform, organism-dependent differences in sequence error rates affecting classification of certain species, or some combination of these factors. This study demonstrates the potential for differential bias in bacterial community profiles resulting from the choice of sequencing platform alone.
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Whole genome sequencing indicates Corynebacterium jeikeium comprises 4 separate genomospecies and identifies a dominant genomospecies among clinical isolates.
Int. J. Med. Microbiol.
PUBLISHED: 07-25-2014
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Corynebacterium jeikeium is an opportunistic pathogen which has been noted for significant genomic diversity. The population structure within this species remains poorly understood. Here, we explore the relationships among 15 clinical isolates of C. jeikeium (reference strains K411 and ATCC 43734, and 13 primary isolates collected over a period of 7 years) through genetic, genomic, and phenotypic studies. We report a high degree of divergence among strains based on 16S ribosomal RNA (rRNA) gene and rpoB gene sequence analysis, supporting the presence of genetically distinct subgroups. Whole genome sequencing indicates genomic-level dissimilarity among subgroups, which qualify as four separate and distinct Corynebacterium species based on an average nucleotide identity (ANIb) threshold of <95%. Functional distinctions in antibiotic susceptibilities and metabolic profiles characterize two of these genomospecies, allowing their differentiation from others through routine laboratory testing. The remaining genomospecies can be classified through a biphasic approach integrating phenotypic testing and rpoB gene sequencing. The genomospecies predominantly recovered from patient specimens does not include either of the existing C. jeikeium reference strains, implying that studies of this pathogen would benefit from examination of representatives from the primary disease-causing group. The clinically dominant genomospecies also has the smallest genome size and gene repertoire, suggesting the possibility of increased virulence relative to the other genomospecies. The ability to classify isolates to one of the four C. jeikeium genomospecies in a clinical context provides diagnostic information for tailoring antimicrobial therapy and may aid in identification of species-specific disease associations.
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Whole-genome sequencing for high-resolution investigation of methicillin-resistant Staphylococcus aureus epidemiology and genome plasticity.
J. Clin. Microbiol.
PUBLISHED: 05-21-2014
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Methicillin-resistant Staphylococcus aureus (MRSA) infections pose a major challenge in health care, yet the limited heterogeneity within this group hinders molecular investigations of related outbreaks. Pulsed-field gel electrophoresis (PFGE) has been the gold standard approach but is impractical for many clinical laboratories and is often replaced with PCR-based methods. Regardless, both approaches can prove problematic for identifying subclonal outbreaks. Here, we explore the use of whole-genome sequencing for clinical laboratory investigations of MRSA molecular epidemiology. We examine the relationships of 44 MRSA isolates collected over a period of 3 years by using whole-genome sequencing and two PCR-based methods, multilocus variable-number tandem-repeat analysis (MLVA) and spa typing. We find that MLVA offers higher resolution than spa typing, as it resolved 17 versus 12 discrete isolate groups, respectively. In contrast, whole-genome sequencing reproducibly cataloged genomic variants (131,424 different single nucleotide polymorphisms and indels across the strain collection) that uniquely identified each MRSA clone, recapitulating those groups but enabling higher-resolution phylogenetic inferences of the epidemiological relationships. Importantly, whole-genome sequencing detected a significant number of variants, thereby distinguishing between groups that were considered identical by both spa typing (minimum, 1,124 polymorphisms) and MLVA (minimum, 193 polymorphisms); this suggests that these more conventional approaches can lead to false-positive identification of outbreaks due to inappropriate grouping of genetically distinct strains. An analysis of the distribution of variants across the MRSA genome reveals 47 mutational hot spots (comprising ? 2.5% of the genome) that account for 23.5% of the observed polymorphisms, and the use of this selected data set successfully recapitulates most epidemiological relationships in this pathogen group.
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Coinfection of Fusobacterium nucleatum and Actinomyces israelii in mastoiditis diagnosed by next-generation DNA sequencing.
J. Clin. Microbiol.
PUBLISHED: 02-26-2014
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Some bacterial infections involve potentially complex mixtures of species that can now be distinguished using next-generation DNA sequencing. We present a case of mastoiditis where Gram stain, culture, and molecular diagnosis were nondiagnostic or discrepant. Next-generation sequencing implicated coinfection of Fusobacterium nucleatum and Actinomyces israelii, resolving these diagnostic discrepancies.
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Temporal and anatomical host resistance to chronic salmonella infection is quantitatively dictated by nramp1 and influenced by host genetic background.
PLoS ONE
PUBLISHED: 01-01-2014
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The lysosomal membrane transporter, Nramp1, plays a key role in innate immunity and resistance to infection with intracellular pathogens such as non-typhoidal Salmonella (NTS). NTS-susceptible C57BL/6 (B6) mice, which express the mutant Nramp1D169 allele, are unable to control acute infection with Salmonella enterica serovar Typhimurium following intraperitoneal or oral inoculation. Introducing functional Nramp1G169 into the B6 host background, either by constructing a congenic strain carrying Nramp1G169 from resistant A/J mice (Nramp-Cg) or overexpressing Nramp1G169 from a transgene (Nramp-Tg), conferred equivalent protection against acute Salmonella infection. In contrast, the contributions of Nramp1 for controlling chronic infection are more complex, involving temporal and anatomical differences in Nramp1-dependent host responses. Nramp-Cg, Nramp-Tg and NTS-resistant 129×1/SvJ mice survived oral Salmonella infection equally well for the first 2-3 weeks, providing evidence that Nramp1 contributes to the initial control of NTS bacteremia preceding establishment of chronic Salmonella infection. By day 30, increased host Nramp1 expression (Tg>Cg) provided greater protection as indicated by decreased splenic bacterial colonization (Tg
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Molecular diagnosis of Actinomadura madurae infection by 16S rRNA deep sequencing.
J. Clin. Microbiol.
PUBLISHED: 10-09-2013
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Next-generation DNA sequencing can be used to catalog individual organisms within complex, polymicrobial specimens. Here, we utilized deep sequencing of 16S rRNA to implicate Actinomadura madurae as the cause of mycetoma in a diabetic patient when culture and conventional molecular methods were overwhelmed by overgrowth of other organisms.
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Predictive diagnostics for Escherichia coli infections based on the clonal association of antimicrobial resistance and clinical outcome.
J. Clin. Microbiol.
PUBLISHED: 07-10-2013
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The ability to identify bacterial pathogens at the subspecies level in clinical diagnostics is currently limited. We investigated whether splitting Escherichia coli species into clonal groups (clonotypes) predicts antimicrobial susceptibility or clinical outcome. A total of 1,679 extraintestinal E. coli isolates (collected from 2010 to 2012) were collected from one German and 5 U.S. clinical microbiology laboratories. Clonotype identity was determined by fumC and fimH (CH) sequencing. The associations of clonotype with antimicrobial susceptibility and clinical variables were evaluated. CH typing divided the isolates into >200 CH clonotypes, with 93% of the isolates belonging to clonotypes with ? 2 isolates. Antimicrobial susceptibility varied substantially among clonotypes but was consistent across different locations. Clonotype-guided antimicrobial selection significantly reduced "drug-bug" mismatch compared to that which occurs with the use of conventional empirical therapy. With trimethoprim-sulfamethoxazole and fluoroquinolones, the drug-bug mismatch was predicted to decrease 62% and 78%, respectively. Recurrent or persistent urinary tract infection and clinical sepsis were significantly correlated with specific clonotypes, especially with CH40-30 (also known as H30), a recently described clonotype within sequence type 131 (ST131). We were able to clonotype directly from patient urine samples within 1 to 3 h of obtaining the specimen. In E. coli, subspecies-level identification by clonotyping can be used to significantly improve empirical predictions of antimicrobial susceptibility and clinical outcomes in a timely manner.
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Rapid 16S rRNA next-generation sequencing of polymicrobial clinical samples for diagnosis of complex bacterial infections.
PLoS ONE
PUBLISHED: 01-01-2013
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Classifying individual bacterial species comprising complex, polymicrobial patient specimens remains a challenge for culture-based and molecular microbiology techniques in common clinical use. We therefore adapted practices from metagenomics research to rapidly catalog the bacterial composition of clinical specimens directly from patients, without need for prior culture. We have combined a semiconductor deep sequencing protocol that produces reads spanning 16S ribosomal RNA gene variable regions 1 and 2 (?360 bp) with a de-noising pipeline that significantly improves the fraction of error-free sequences. The resulting sequences can be used to perform accurate genus- or species-level taxonomic assignment. We explore the microbial composition of challenging, heterogeneous clinical specimens by deep sequencing, culture-based strain typing, and Sanger sequencing of bulk PCR product. We report that deep sequencing can catalog bacterial species in mixed specimens from which usable data cannot be obtained by conventional clinical methods. Deep sequencing a collection of sputum samples from cystic fibrosis (CF) patients reveals well-described CF pathogens in specimens where they were not detected by standard clinical culture methods, especially for low-prevalence or fastidious bacteria. We also found that sputa submitted for CF diagnostic workup can be divided into a limited number of groups based on the phylogenetic composition of the airway microbiota, suggesting that metagenomic profiling may prove useful as a clinical diagnostic strategy in the future. The described method is sufficiently rapid (theoretically compatible with same-day turnaround times) and inexpensive for routine clinical use.
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Regulation of phenotypic heterogeneity permits Salmonella evasion of the host caspase-1 inflammatory response.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 12-05-2011
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Sensing and adapting to the environment is one strategy by which bacteria attempt to maximize fitness in an unpredictable world; another is the stochastic generation of phenotypically distinct subgroups within a genetically clonal population. In culture, Salmonella Typhimurium populations are bistable for the expression of flagellin. We report that YdiV controls this expression pattern by preventing transcription of the sigma factor that recruits RNA polymerase to the flagellin promoter. Bistability ensues when the sigma factor is repressed in a subpopulation of cells, resulting in two phenotypes: flagellin expressors and flagellin nonexpressors. Although the ability to swim is presumably a critical survival trait, flagellin activates eukaryotic defense pathways, and Salmonella restrict the production of flagellin during systemic infection. Salmonella mutants lacking YdiV are unable to fully repress flagellin at systemic sites, rendering them vulnerable to caspase-1 mediated colonization restriction. Thus, a regulatory mechanism producing bistability also impacts Salmonella virulence.
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Coordinated host responses during pyroptosis: caspase-1-dependent lysosome exocytosis and inflammatory cytokine maturation.
J. Immunol.
PUBLISHED: 07-29-2011
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Activation of caspase-1 leads to pyroptosis, a program of cell death characterized by cell lysis and inflammatory cytokine release. Caspase-1 activation triggered by multiple nucleotide-binding oligomerization domain-like receptors (NLRs; NLRC4, NLRP1b, or NLRP3) leads to loss of lysosomes via their fusion with the cell surface, or lysosome exocytosis. Active caspase-1 increased cellular membrane permeability and intracellular calcium levels, which facilitated lysosome exocytosis and release of host antimicrobial factors and microbial products. Lysosome exocytosis has been proposed to mediate secretion of IL-1? and IL-18; however, blocking lysosome exocytosis did not alter cytokine processing or release. These studies indicate two conserved secretion pathways are initiated by caspase-1, lysosome exocytosis, and a parallel pathway resulting in cytokine release, and both enhance the antimicrobial nature of pyroptosis.
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Optimization of periprosthetic culture for diagnosis of Propionibacterium acnes prosthetic joint infection.
J. Clin. Microbiol.
PUBLISHED: 05-04-2011
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Propionibacterium acnes is increasingly recognized as an important agent of prosthetic joint infection (PJI). However, the optimum culture conditions for recovery of this organism from PJI specimens have not been determined. By applying a prolonged 28-day culture incubation to all periprosthetic specimens received for bacterial culture from 198 revision arthroplasty procedures, we retrospectively determined that a 13-day culture incubation period is necessary for the recovery of P. acnes from patients with PJI. Incubation beyond this period was associated with increasing recovery of nondiagnostic isolates: 21.7% of P. acnes isolates believed to be clinically unimportant were recovered after 13 days of incubation. Importantly, a diagnosis of P. acnes PJI would have been missed in 29.4% of patients had extended culture incubation been applied only to anaerobic culture media. Although specimens from P. acnes PJIs were more commonly associated with the presence of ? 2 culture media positive for growth, acute inflammation (? 5 neutrophils/high-power field) was observed in only 40% of patients with PJIs that had more than one specimen submitted for bacterial culture. These results support the need for a minimum culture incubation period of 13 days to be applied to both aerobic and anaerobic culture media for all periprosthetic specimens. Optimal recovery of infecting organisms from PJI specimens will be an important component in generating a universal definition for PJI due to indolent agents of infection, such as P. acnes.
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Molecular diagnosis of cystoisosporiasis using extended-range PCR screening.
J Mol Diagn
PUBLISHED: 01-04-2011
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The differential diagnosis of diarrhea in immunocompromised patients encompasses many intestinal parasites including the coccidian Cystoisospora belli. Gastrointestinal infection with C. belli leads to cystoisosporiasis with diarrhea and, depending on host immune status, can cause extraintestinal disease. C. belli is usually diagnosed by examination of stool or intestinal biopsy specimens; however, the organism may be undetected using these test methods. Thus, more sensitive molecular tools for detection of pathogenic parasites are desirable. Herein is described a patient with AIDS who had persistent diarrhea of unknown cause. Microscopic examinations of stool and ileal biopsy specimens were initially unremarkable for any specific pathogen. Screening of DNA extracted from biopsy material using extended-range PCR primers recognizing conserved DNA sequences found in many fungi and parasites revealed infection with C. belli, which was confirmed at repeat histologic analysis. Extended-range PCR screening was used because the differential diagnosis was broad and other tools were not applied, yet this molecular approach led to the appropriate diagnosis and treatment of the condition. Thus, this approach offers a promising test for diagnosis of parasitic diseases that elude diagnosis using conventional methods.
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Ureaplasma urealyticum continuous ambulatory peritoneal dialysis-associated peritonitis diagnosed by 16S rRNA gene PCR.
J. Clin. Microbiol.
PUBLISHED: 08-25-2010
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In some patients with peritonitis complicating continuous ambulatory peritoneal dialysis (CAPD), a causative organism is never identified. We report a case of Ureaplasma urealyticum CAPD-associated peritonitis diagnosed by 16S rRNA gene PCR. Ureaplasma may be an underrecognized cause of peritonitis because it cannot be recovered using routine culture methods.
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Humanized nonobese diabetic-scid IL2rgammanull mice are susceptible to lethal Salmonella Typhi infection.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 08-16-2010
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Salmonella enterica serovar Typhi, the cause of typhoid fever, is host-adapted to humans and unable to cause disease in mice. Here, we show that S. Typhi can replicate in vivo in nonobese diabetic (NOD)-scid IL2rgamma(null) mice engrafted with human hematopoietic stem cells (hu-SRC-SCID mice) to cause a lethal infection with pathological and inflammatory cytokine responses resembling human typhoid. In contrast, S. Typhi does not exhibit net replication or cause illness in nonengrafted or immunocompetent control animals. Screening of transposon pools in hu-SRC-SCID mice revealed both known and previously unknown Salmonella virulence determinants, including Salmonella Pathogenicity Islands 1, 2, 3, 4, and 6. Our observations indicate that the presence of human immune cells allows the in vivo replication of S. Typhi in mice. The hu-SRC-SCID mouse provides an unprecedented opportunity to gain insights into S. Typhi pathogenesis and devise strategies for the prevention of typhoid fever.
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Proteomic profiling of lipopolysaccharide-activated macrophages by isotope coded affinity tagging.
J. Proteome Res.
PUBLISHED: 03-05-2010
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Lipopolysaccharide (LPS), a glycolipid component of the outer membranes of Gram-negative bacteria, initiates proinflammatory, proapoptotic, and antiapoptotic pathways upon binding to macrophage TLR4. Macrophages that are exposed to LPS become activated and exhibit altered morphology and response to infection. We performed isotope coded affinity tagging (ICAT), multidimensional liquid chromatography, and mass spectrometry to identify proteins that are differently expressed between naive and LPS-activated macrophages. We performed replicate ICAT analyses on RAW 264.7 cultured mouse macrophages as well as C57BL/6 bone marrow derived mouse macrophages. We identified and obtained relative abundances for 1064 proteins, of which we identified 36 as having significantly different expression levels upon activation by LPS. We also compared our results with a two color microarray gene expression assay performed by the Institute for Systems Biology and observed approximately 75% agreement between mRNA transcription and protein expression regarding up- or down-regulation of gene products. We used Western blot analysis to confirm the findings of ICAT and mRNA for one protein, sequestosome 1, the cellular concentration of which was observed to increase upon activation by LPS.
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SeqSharp: A general approach for improving cycle-sequencing that facilitates a robust one-step combined amplification and sequencing method.
J Mol Diagn
PUBLISHED: 03-04-2010
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Sequencing a specific DNA element within a genome or a complex mixture of DNA by the Sanger sequencing method generally involves PCR-mediated amplification of target DNA with forward and reverse primers, followed by a sequencing reaction directed from a single primer. To minimize the contribution of fluorescent signal due to the extension products originating from primers carried over from the amplification step, an intermediate step is routinely incorporated to remove the excess primers before proceeding to the sequencing reaction. We have developed a method called SeqSharp that removes noise in the sequencing data by enzymatically removing chain termination products originating from one or both of the amplification primers. This method substantially improves the quality of sequence information even without an intermediate primer removal step. Importantly, we show that SeqSharp significantly improves the sequence quality from a combined (one-step) amplification/sequencing protocol and provides a more robust method that, unlike previously described one-step sequencing methods, yields high quality sequence data from a single reaction by using equimolar primer concentrations. One-step SeqSharp is generally applicable and produced excellent sequence data from bacterial, fungal, and human DNA.
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Quantification of green fluorescent protein in cellular supernatant by capillary electrophoresis with laser-induced fluorescence detection for measurement of cell death.
Talanta
PUBLISHED: 01-05-2010
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A common method for quantifying cell death is measuring the concentration of lactate dehydrogenase (LDH) released by cells as their membranes become unstable. In cells expressing green fluorescent protein (GFP), degradation of the cell membrane also results in the release of GFP into the surrounding supernatant. In this study, we used capillary electrophoresis with laser-induced fluorescence detection to measure the levels of GFP in supernatants of UBIGFP/BL6 primary macrophages that had been infected with Salmonella typhimurium, treated with staurosporine, or exposed to H(2)O(2), all known inducers of cell death. We also used a standard LDH assay to measure the release of LDH into supernatants. We observed the rate of cell death quantified by release of GFP and LDH into supernatant to be essentially identical, demonstrating that GFP release is at least as good as an indicator of macrophage cell death as the established LDH release method.
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Innate immune response during Yersinia infection: critical modulation of cell death mechanisms through phagocyte activation.
J. Leukoc. Biol.
PUBLISHED: 09-04-2009
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Yersinia pestis, the etiological agent of plague, is one of the most deadly pathogens on our planet. This organism shares important attributes with its ancestral progenitor, Yersinia pseudotuberculosis, including a 70-kb virulence plasmid, lymphotropism during growth in the mammalian host, and killing of host macrophages. Infections with both organisms are biphasic, where bacterial replication occurs initially with little inflammation, followed by phagocyte influx, inflammatory cytokine production, and tissue necrosis. During infection, plasmid-encoded attributes facilitate bacterial-induced macrophage death, which results from two distinct processes and corresponds to the inflammatory crescendo observed in vivo: Naïve cells die by apoptosis (noninflammatory), and later in infection, activated macrophages die by pyroptosis (inflammatory). The significance of this redirected cell death for the host is underscored by the importance of phagocyte activation for immunity to Yersinia and the protective role of pyroptosis during host responses to anthrax lethal toxin and infections with Francisella, Legionella, Pseudomonas, and Salmonella. The similarities of Y. pestis and Y. pseudotuberculosis, including conserved, plasmid-encoded functions inducing at least two distinct mechanisms of cell death, indicate that comparative studies are revealing about their critical pathogenic mechanism(s) and host innate immune responses during infection. Validation of this idea and evidence of similar interactions with the host immune system are provided by Y. pseudotuberculosis-priming, cross-protective immunity against Y. pestis. Despite these insights, additional studies indicate much remains to be understood concerning effective host responses against Yersinia, including chromosomally encoded attributes that also contribute to bacterial evasion and modulation of innate and adaptive immune responses.
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Biogenesis of bacterial membrane vesicles.
Mol. Microbiol.
PUBLISHED: 05-08-2009
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Membrane vesicle (MV) release remains undefined, despite its conservation among replicating Gram-negative bacteria both in vitro and in vivo. Proteins identified in Salmonella MVs, derived from the envelope, control MV production via specific defined domains that promote outer membrane protein-peptidoglycan (OM-PG) and OM protein-inner membrane protein (OM-PG-IM) interactions within the envelope structure. Modulation of OM-PG and OM-PG-IM interactions along the cell body and at division septa, respectively, maintains membrane integrity while co-ordinating localized release of MVs with distinct size distribution and protein content. These data support a model of MV biogenesis, wherein bacterial growth and division invoke temporary, localized reductions in the density of OM-PG and OM-PG-IM associations within the envelope structure, thus releasing OM as MVs.
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Pyroptosis: host cell death and inflammation.
Nat. Rev. Microbiol.
PUBLISHED: 01-17-2009
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Eukaryotic cells can initiate several distinct programmes of self-destruction, and the nature of the cell death process (non-inflammatory or proinflammatory) instructs responses of neighbouring cells, which in turn dictates important systemic physiological outcomes. Pyroptosis, or caspase 1-dependent cell death, is inherently inflammatory, is triggered by various pathological stimuli, such as stroke, heart attack or cancer, and is crucial for controlling microbial infections. Pathogens have evolved mechanisms to inhibit pyroptosis, enhancing their ability to persist and cause disease. Ultimately, there is a competition between host and pathogen to regulate pyroptosis, and the outcome dictates life or death of the host.
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Diagnosis of neurocysticercosis by detection of Taenia solium DNA using a global DNA screening platform.
Clin. Infect. Dis.
PUBLISHED: 01-03-2009
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Neurocysticercosis is caused by Taenia solium infection of the brain. Diagnosis is most often made by visualization of the parasitic scolex by magnetic resonance imaging of the brain or by characteristic neuroimaging findings with serologic test results positive for T. solium. A patient who presents with a solitary brain lesion usually poses a diagnostic dilemma, because the differential diagnosis often includes neurocysticercosis and other infections or neoplasm. Although the sensitivity of serologic testing for T. solium approaches 100% in patients with multiple intraparenchymal cysts, the sensitivity of testing for patients with solitary cysts is <50%, which makes serologic testing a less useful diagnostic tool for patients with solitary central nervous system (CNS) lesions. We describe 2 patients with solitary CNS lesions who received a neurocysticercosis diagnosis after identification of T. solium DNA in brain biopsy tissue with use of a global DNA screening platform. Global screening is a promising tool for the diagnosis of CNS infection, especially when traditional diagnostic tools are insensitive.
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Isolation of Bordetella avium and novel Bordetella strain from patients with respiratory disease.
Emerging Infect. Dis.
PUBLISHED: 01-01-2009
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Bordetella avium is thought to be strictly an avian pathogen. However, 16S rRNA gene sequencing identified 2 isolates from 2 humans with respiratory disease as B. avium and a novel B. avium-like strain. Thus, B. avium and B. avium-like organisms are rare opportunistic human pathogens.
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The Yersinia virulence effector YopM binds caspase-1 to arrest inflammasome assembly and processing.
Cell Host Microbe
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Inflammasome assembly activates caspase-1 and initiates the inflammatory cell death program pyroptosis, which is protective against numerous pathogens. Consequently, several pathogens, including the plague causing bacterium Yersinia pestis, avoid activating this pathway to enhance their virulence. However, bacterial molecules that directly modulate the inflammasome have yet to be identified. Examining the contribution of Yersinia type III secretion effectors to caspase-1 activation, we identified the leucine-rich repeat effector YopM as a potent antagonist of both caspase-1 activity and activation. YopM directly binds caspase-1, which both inhibits caspase-1 activity and sequesters it to block formation of the mature inflammasome. Caspase-1 activation antagonizes Yersinia survival in vivo, and consequently YopM inhibition of caspase-1 is required for Yersinia pathogenesis. Thus, a bacterium obstructs pyroptosis utilizing a direct mechanism of caspase-1 inhibition that is distinct from known viral or host inhibitors.
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Non-genetic diversity shapes infectious capacity and host resistance.
Trends Microbiol.
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The spontaneous generation of distinct phenotypes within a clonal population of cells allows for both bet-hedging at the population level and the division of labor among subpopulations. This is emerging as an important theme in bacterial pathogenesis, because bacterial pathogens exhibit phenotypic heterogeneity with respect to characteristics that impact virulence. The phenomenon of persister cells and models of Salmonella enterica serovar Typhimurium (S. Typhimurium) pathogenesis illustrate the importance of non-genetic diversity in the disease process. Such heterogeneity can arise from specific genetic architectures amplifying stochastic fluctuations in factors affecting gene expression, and this also drives variation in eukaryotic cells. Thus reproducible variation in both host and pathogen processes affects the outcome of infection.
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Membrane vesicle release in bacteria, eukaryotes, and archaea: a conserved yet underappreciated aspect of microbial life.
Infect. Immun.
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Interaction of microbes with their environment depends on features of the dynamic microbial surface throughout cell growth and division. Surface modifications, whether used to acquire nutrients, defend against other microbes, or resist the pressures of a host immune system, facilitate adaptation to unique surroundings. The release of bioactive membrane vesicles (MVs) from the cell surface is conserved across microbial life, in bacteria, archaea, fungi, and parasites. MV production occurs not only in vitro but also in vivo during infection, underscoring the influence of these surface organelles in microbial physiology and pathogenesis through delivery of enzymes, toxins, communication signals, and antigens recognized by the innate and adaptive immune systems. Derived from a variety of organisms that span kingdoms of life and called by several names (membrane vesicles, outer membrane vesicles [OMVs], exosomes, shedding microvesicles, etc.), the conserved functions and mechanistic strategies of MV release are similar, including the use of ESCRT proteins and ESCRT protein homologues to facilitate these processes in archaea and eukaryotic microbes. Although forms of MV release by different organisms share similar visual, mechanistic, and functional features, there has been little comparison across microbial life. This underappreciated conservation of vesicle release, and the resulting functional impact throughout the tree of life, explored in this review, stresses the importance of vesicle-mediated processes throughout biology.
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Real-time quantitative reverse transcription PCR for monitoring of blood-stage Plasmodium falciparum infections in malaria human challenge trials.
Am. J. Trop. Med. Hyg.
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To detect pre-patent parasitemia, we developed a real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) for the asexual 18S ribosomal RNA (rRNAs) of Plasmodium falciparum. Total nucleic acids extracted from whole blood were combined with control RNA and tested by qRT-PCR. The assay quantified > 98.7% of parasite-containing samples to ±0.5 log(10) parasites/mL of the nominal value without false positives. The analytical sensitivity was ? 20 parasites/mL. The coefficient of variation was 0.6% and 1.8% within runs and 1.6% and 4.0% between runs for high and low parasitemia specimens, respectively. Using this assay, we determined that A-type 18S rRNAs are stably expressed at 1 × 10(4) copies per ring-stage parasite. When used to monitor experimental P. falciparum infection of human volunteers, the assay detected blood-stage infections 3.7 days earlier on average than thick blood smears. This validated, internally controlled qRT-PCR method also uses a small (50 ?L) sample volume requiring minimal pre-analytical handling, making it useful for clinical trials.
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High-resolution two-locus clonal typing of extraintestinal pathogenic Escherichia coli.
Appl. Environ. Microbiol.
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Multilocus sequence typing (MLST) is usually based on the sequencing of 5 to 8 housekeeping loci in the bacterial chromosome and has provided detailed descriptions of the population structure of bacterial species important to human health. However, even strains with identical MLST profiles (known as sequence types or STs) may possess distinct genotypes, which enable different eco- or pathotypic lifestyles. Here we describe a two-locus, sequence-based typing scheme for Escherichia coli that utilizes a 489-nucleotide (nt) internal fragment of fimH (encoding the type 1 fimbrial adhesin) and the 469-nt internal fumC fragment used in standard MLST. Based on sequence typing of 191 model commensal and pathogenic isolates plus 853 freshly isolated clinical E. coli strains, this 2-locus approach-which we call CH (fumC/fimH) typing-consistently yielded more haplotypes than standard 7-locus MLST, splitting large STs into multiple clonal subgroups and often distinguishing different within-ST eco- and pathotypes. Furthermore, specific CH profiles corresponded to specific STs, or ST complexes, with 95% accuracy, allowing excellent prediction of MLST-based profiles. Thus, 2-locus CH typing provides a genotyping tool for molecular epidemiology analysis that is more economical than standard 7-locus MLST but has superior clonal discrimination power and, at the same time, corresponds closely to MLST-based clonal groupings.
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