scholarly journals Target-Specific Assay for Rapid and Quantitative Detection of Mycobacterium chimaera DNA

2017 ◽  
Vol 55 (6) ◽  
pp. 1847-1856 ◽  
Author(s):  
Enrique Zozaya-Valdés ◽  
Jessica L. Porter ◽  
John Coventry ◽  
Janet A. M. Fyfe ◽  
Glen P. Carter ◽  
...  
Keyword(s):  
In Silico ◽  
Bacterial Species ◽  
Invasive Disease ◽  
Diagnostic Methods ◽  
Quantitative Detection ◽  
Content Type ◽  
Diagnostic Assays ◽  
Mycobacterial Genome ◽  
Increased Sensitivity

ABSTRACT Mycobacterium chimaera is an opportunistic environmental mycobacterium belonging to the Mycobacterium avium - M. intracellulare complex. Although most commonly associated with pulmonary disease, there has been growing awareness of invasive M. chimaera infections following cardiac surgery. Investigations suggest worldwide spread of a specific M. chimaera clone, associated with contaminated hospital heater-cooler units used during the surgery. Given the global dissemination of this clone, its potential to cause invasive disease, and the laboriousness of current culture-based diagnostic methods, there is a pressing need to develop rapid and accurate diagnostic assays specific for M. chimaera . Here, we assessed 354 mycobacterial genome sequences and confirmed that M. chimaera is a phylogenetically coherent group. In silico comparisons indicated six DNA regions present only in M. chimaera . We targeted one of these regions and developed a TaqMan quantitative PCR (qPCR) assay for M. chimaera with a detection limit of 100 CFU/ml in whole blood spiked with bacteria. In vitro screening against DNA extracted from 40 other mycobacterial species and 22 bacterial species from 21 diverse genera confirmed the in silico -predicted specificity for M. chimaera . Screening 33 water samples from heater-cooler units with this assay highlighted the increased sensitivity of PCR compared to culture, with 15 of 23 culture-negative samples positive by M. chimaera qPCR. We have thus developed a robust molecular assay that can be readily and rapidly deployed to screen clinical and environmental specimens for M. chimaera .

scholarly journals A target-specific assay for rapid and quantitative detection of Mycobacterium chimaera DNA in environmental and clinical specimens

2017 ◽  
Author(s):  
Enrique Zozaya-Valdés ◽  
Jessica L. Porter ◽  
John Coventry ◽  
Janet A. M. Fyfe ◽  
Glen P. Carter ◽  
...  
Keyword(s):  
In Silico ◽  
Bacterial Species ◽  
Invasive Disease ◽  
Diagnostic Methods ◽  
Quantitative Detection ◽  
Diagnostic Assays ◽  
Mycobacterial Genome ◽  
Increased Sensitivity ◽  
Taqman Qpcr Assay

AbstractMycobacterium chimaera is an opportunistic environmental mycobacterium, belonging to the Mycobacterium intracellulare complex. Although most commonly associated with pulmonary disease, there has been growing awareness of invasive M. chimaera infections following cardiac surgery. Investigations suggest world-wide spread of a specific M. chimaera clone, associated with contaminated hospital heater-cooler units used during the surgery. Given the global dissemination of this clone, its potential to cause invasive disease, and the laboriousness of current culture-based diagnostic methods, there is a pressing need to develop rapid and accurate diagnostic assays, specific for M. chimaera. Here, we assessed 354 mycobacterial genome sequences and confirmed that M. chimaera is a phylogenetically coherent group. In silico comparisons indicated six DNA regions present only in M. chimaera. We targeted one of these regions and developed a TaqMan qPCR assay for M. chimaera with a detection limit of 10 CFU in whole blood. In vitro screening against DNA extracted from 40 other mycobacteria and 22 bacterial species from 21 diverse genera confirmed in silico predicted specificity for M. chimaera. Screening 33 water samples from heater cooler units with this assay highlighted the increased sensitivity of PCR compared to culture, with 15 of 23 culture negative samples positive by M. chimaera qPCR. We have thus developed a robust molecular assay that can be readily and rapidly deployed to screen clinical and environmental specimens for M. chimaera.

scholarly journals Enhancing Terpene Yield from Sugars via Novel Routes to 1-Deoxy-d-Xylulose 5-Phosphate

2014 ◽  
Vol 81 (1) ◽  
pp. 130-138 ◽  
Author(s):  
James Kirby ◽  
Minobu Nishimoto ◽  
Ruthie W. N. Chow ◽  
Edward E. K. Baidoo ◽  
George Wang ◽  
...  
Keyword(s):  
Bacterial Species ◽  
Xylose Reductase ◽  
Pentose Phosphate ◽  
Terpene Biosynthesis ◽  
Content Type ◽  
E Coli ◽  
Pathway Expression ◽  
Terpene Synthesis ◽  
Selection For

ABSTRACTTerpene synthesis in the majority of bacterial species, together with plant plastids, takes place via the 1-deoxy-d-xylulose 5-phosphate (DXP) pathway. The first step of this pathway involves the condensation of pyruvate and glyceraldehyde 3-phosphate by DXP synthase (Dxs), with one-sixth of the carbon lost as CO2. A hypothetical novel route from a pentose phosphate to DXP (nDXP) could enable a more direct pathway from C5sugars to terpenes and also circumvent regulatory mechanisms that control Dxs, but there is no enzyme known that can convert a sugar into its 1-deoxy equivalent. Employing a selection for complementation of adxsdeletion inEscherichia coligrown on xylose as the sole carbon source, we uncovered two candidate nDXP genes. Complementation was achieved either via overexpression of the wild-typeE. coliyajOgene, annotated as a putative xylose reductase, or via various mutations in the nativeribBgene.In vitroanalysis performed with purified YajO and mutant RibB proteins revealed that DXP was synthesized in both cases from ribulose 5-phosphate (Ru5P). We demonstrate the utility of these genes for microbial terpene biosynthesis by engineering the DXP pathway inE. colifor production of the sesquiterpene bisabolene, a candidate biodiesel. To further improve flux into the pathway from Ru5P, nDXP enzymes were expressed as fusions to DXP reductase (Dxr), the second enzyme in the DXP pathway. Expression of a Dxr-RibB(G108S) fusion improved bisabolene titers more than 4-fold and alleviated accumulation of intracellular DXP.

scholarly journals Role of sapA and yfgA in Susceptibility to Antibody-Mediated Complement-Dependent Killing and Virulence of Salmonella enterica Serovar Typhimurium

2017 ◽  
Vol 85 (9) ◽  
Author(s):  
Edna M. Ondari ◽  
Jennifer N. Heath ◽  
Elizabeth J. Klemm ◽  
Gemma Langridge ◽  
Lars Barquist ◽  
...  
Keyword(s):  
Salmonella Enterica ◽  
Protein Translocation ◽  
Invasive Disease ◽  
Immune Serum ◽  
Insertion Mutant ◽  
Content Type ◽  
Serovar Typhimurium ◽  
Serum Killing ◽  
Increased Sensitivity

ABSTRACT The ST313 pathovar of Salmonella enterica serovar Typhimurium contributes to a high burden of invasive disease among African infants and HIV-infected adults. It is characterized by genome degradation (loss of coding capacity) and has increased resistance to antibody-dependent complement-mediated killing compared with enterocolitis-causing strains of S. Typhimurium. Vaccination is an attractive disease-prevention strategy, and leading candidates focus on the induction of bactericidal antibodies. Antibody-resistant strains arising through further gene deletion could compromise such a strategy. Exposing a saturating transposon insertion mutant library of S. Typhimurium to immune serum identified a repertoire of S. Typhimurium genes that, when interrupted, result in increased resistance to serum killing. These genes included several involved in bacterial envelope biogenesis, protein translocation, and metabolism. We generated defined mutant derivatives using S. Typhimurium SL1344 as the host. Based on their initial levels of enhanced resistance to killing, yfgA and sapA mutants were selected for further characterization. The S. Typhimurium yfgA mutant lost the characteristic Salmonella rod-shaped appearance, exhibited increased sensitivity to osmotic and detergent stress, lacked very long lipopolysaccharide, was unable to invade enterocytes, and demonstrated decreased ability to infect mice. In contrast, the S. Typhimurium sapA mutants had similar sensitivity to osmotic and detergent stress and lipopolysaccharide profile and an increased ability to infect enterocytes compared with the wild type, but it had no increased ability to cause in vivo infection. These findings indicate that increased resistance to antibody-dependent complement-mediated killing secondary to genetic deletion is not necessarily accompanied by increased virulence and suggest the presence of different mechanisms of antibody resistance.

scholarly journals RNA G-Quadruplex Structures Mediate Gene Regulation in Bacteria

mBio ◽  
2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Xiaolong Shao ◽  
Weitong Zhang ◽  
Mubarak Ishaq Umar ◽  
Hei Yuen Wong ◽  
Zijing Seng ◽  
...  
Keyword(s):  
Gene Regulation ◽  
Cell Envelope ◽  
Bacterial Species ◽  
Virulence Gene ◽  
Content Type ◽  
Cellular Processes ◽  
Wide Range ◽  
G Quadruplex ◽  
Eukaryotic Organisms

ABSTRACT Guanine (G)-rich sequences in RNA can fold into diverse RNA G-quadruplex (rG4) structures to mediate various biological functions and cellular processes in eukaryotic organisms. However, the presence, locations, and functions of rG4s in prokaryotes are still elusive. We used QUMA-1, an rG4-specific fluorescent probe, to detect rG4 structures in a wide range of bacterial species both in vitro and in live cells and found rG4 to be an abundant RNA secondary structure across those species. Subsequently, to identify bacterial rG4 sites in the transcriptome, the model Escherichia coli strain and a major human pathogen, Pseudomonas aeruginosa, were subjected to recently developed high-throughput rG4 structure sequencing (rG4-seq). In total, 168 and 161 in vitro rG4 sites were found in E. coli and P. aeruginosa, respectively. Genes carrying these rG4 sites were found to be involved in virulence, gene regulation, cell envelope synthesis, and metabolism. More importantly, biophysical assays revealed the formation of a group of rG4 sites in mRNAs (such as hemL and bswR), and they were functionally validated in cells by genetic (point mutation and lux reporter assays) and phenotypic experiments, providing substantial evidence for the formation and function of rG4s in bacteria. Overall, our study uncovers important regulatory functions of rG4s in bacterial pathogenicity and metabolic pathways and strongly suggests that rG4s exist and can be detected in a wide range of bacterial species. IMPORTANCE G-quadruplex in RNA (rG4) mediates various biological functions and cellular processes in eukaryotic organisms. However, the presence, locations, and functions of rG4 are still elusive in prokaryotes. Here, we found that rG4 is an abundant RNA secondary structure across a wide range of bacterial species. Subsequently, the transcriptome-wide rG4 structure sequencing (rG4-seq) revealed that the model E. coli strain and a major human pathogen, P. aeruginosa, have 168 and 161 in vitro rG4 sites, respectively, involved in virulence, gene regulation, cell envelope, and metabolism. We further verified the regulatory functions of two rG4 sites in bacteria (hemL and bswR). Overall, this finding strongly suggests that rG4s play key regulatory roles in a wide range of bacterial species.

scholarly journals Computational Chemogenomics Drug Repositioning Strategy Enables the Discovery of Epirubicin as a New Repurposed Hit for Plasmodium falciparum and P. vivax

2020 ◽  
Vol 64 (9) ◽  
Author(s):  
Letícia Tiburcio Ferreira ◽  
Juliana Rodrigues ◽  
Gustavo Capatti Cassiano ◽  
Tatyana Almeida Tavella ◽  
Kaira Cristina Peralis Tomaz ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
In Silico ◽  
Antimalarial Activity ◽  
Drug Repositioning ◽  
Dna Gyrase ◽  
Plasmodium Yoelii ◽  
Computer Assisted ◽  
Content Type

ABSTRACT Widespread resistance against antimalarial drugs thwarts current efforts for controlling the disease and urges the discovery of new effective treatments. Drug repositioning is increasingly becoming an attractive strategy since it can reduce costs, risks, and time-to-market. Herein, we have used this strategy to identify novel antimalarial hits. We used a comparative in silico chemogenomics approach to select Plasmodium falciparum and Plasmodium vivax proteins as potential drug targets and analyzed them using a computer-assisted drug repositioning pipeline to identify approved drugs with potential antimalarial activity. Among the seven drugs identified as promising antimalarial candidates, the anthracycline epirubicin was selected for further experimental validation. Epirubicin was shown to be potent in vitro against sensitive and multidrug-resistant P. falciparum strains and P. vivax field isolates in the nanomolar range, as well as being effective against an in vivo murine model of Plasmodium yoelii. Transmission-blocking activity was observed for epirubicin in vitro and in vivo. Finally, using yeast-based haploinsufficiency chemical genomic profiling, we aimed to get insights into the mechanism of action of epirubicin. Beyond the target predicted in silico (a DNA gyrase in the apicoplast), functional assays suggested a GlcNac-1-P-transferase (GPT) enzyme as a potential target. Docking calculations predicted the binding mode of epirubicin with DNA gyrase and GPT proteins. Epirubicin is originally an antitumoral agent and presents associated toxicity. However, its antiplasmodial activity against not only P. falciparum but also P. vivax in different stages of the parasite life cycle supports the use of this drug as a scaffold for hit-to-lead optimization in malaria drug discovery.

scholarly journals Regulation of OmpA Translation and Shigella dysenteriae Virulence by an RNA Thermometer

2019 ◽  
Vol 88 (3) ◽  
Author(s):  
Erin R. Murphy ◽  
Johanna Roßmanith ◽  
Jacob Sieg ◽  
Megan E. Fris ◽  
Hebaallaha Hussein ◽  
...  
Keyword(s):  
In Silico ◽  
Bacterial Species ◽  
Regulation Of Gene Expression ◽  
Structure And Function ◽  
Shigella Dysenteriae ◽  
Content Type ◽  
Bacterial Physiology ◽  
Wide Range ◽  
Rna Thermometer ◽  
And Function

ABSTRACT RNA thermometers are cis-acting riboregulators that mediate the posttranscriptional regulation of gene expression in response to environmental temperature. Such regulation is conferred by temperature-responsive structural changes within the RNA thermometer that directly result in differential ribosomal binding to the regulated transcript. The significance of RNA thermometers in controlling bacterial physiology and pathogenesis is becoming increasingly clear. This study combines in silico, molecular genetics, and biochemical analyses to characterize both the structure and function of a newly identified RNA thermometer within the ompA transcript of Shigella dysenteriae. First identified by in silico structural predictions, genetic analyses have demonstrated that the ompA RNA thermometer is a functional riboregulator sufficient to confer posttranscriptional temperature-dependent regulation, with optimal expression observed at the host-associated temperature of 37°C. Structural studies and ribosomal binding analyses have revealed both increased exposure of the ribosomal binding site and increased ribosomal binding to the ompA transcript at permissive temperatures. The introduction of site-specific mutations predicted to alter the temperature responsiveness of the ompA RNA thermometer has predictable consequences for both the structure and function of the regulatory element. Finally, in vitro tissue culture-based analyses implicate the ompA RNA thermometer as a bona fide S. dysenteriae virulence factor in this bacterial pathogen. Given that ompA is highly conserved among Gram-negative pathogens, these studies not only provide insight into the significance of riboregulation in controlling Shigella virulence, but they also have the potential to facilitate further understanding of the physiology and/or pathogenesis of a wide range of bacterial species.

scholarly journals Genome Sequence of Lipopeptide- and Antioxidant-Producing Strain Bacillus velezensis NWUMFkBS10.5

2019 ◽  
Vol 8 (28) ◽  
Author(s):  
Adetomiwa A. Adeniji ◽  
Olubukola O. Babalola
Keyword(s):  
Genome Sequence ◽  
In Silico ◽  
Metabolic Pathways ◽  
Biocontrol Agent ◽  
Bacillus Velezensis ◽  
Content Type ◽  
Genome Profiling ◽  
Antimicrobial Lipopeptides

Candidate biocontrol agent Bacillus velezensis NWUMFkBS10.5 possesses unique genomic characteristics revealed by antiSMASH analysis and in vitro metabolomic elucidation. Besides its capability to produce antimicrobial lipopeptides, further in silico genome profiling predicted the presence of metabolic pathways for synthesizing antioxidants like lampranthin-2, miraxanthin V, and 2-decarboxybetanidin.

scholarly journals Characterization of Guided Entry of Tail-Anchored Proteins 3 Homologues in Mycobacterium tuberculosis

2019 ◽  
Vol 201 (14) ◽  
Author(s):  
Kuan Hu ◽  
Ashley T. Jordan ◽  
Susan Zhang ◽  
Avantika Dhabaria ◽  
Amanda Kovach ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Structure Prediction ◽  
Bacterial Species ◽  
Normal Growth ◽  
Anion Transport ◽  
Content Type ◽  
Reading Frame ◽  
Anion Transporters ◽  
Anion Transporter

ABSTRACT We characterized an operon in Mycobacterium tuberculosis, Rv3679-Rv3680, in which each open reading frame is annotated to encode “anion transporter ATPase” homologues. Using structure prediction modeling, we found that Rv3679 and Rv3680 more closely resemble the guided entry of tail-anchored proteins 3 (Get3) chaperone in eukaryotes. Get3 delivers proteins into the membranes of the endoplasmic reticulum and is essential for the normal growth and physiology of some eukaryotes. We sought to characterize the structures of Rv3679 and Rv3680 and test if they have a role in M. tuberculosis pathogenesis. We solved crystal structures of the nucleotide-bound Rv3679-Rv3680 complex at 2.5 to 3.2 Å and show that while it has some similarities to Get3 and ArsA, there are notable differences, including that these proteins are unlikely to be involved in anion transport. Deletion of both genes did not reveal any conspicuous growth defects in vitro or in mice. Collectively, we identified a new class of proteins in bacteria with similarity to Get3 complexes, the functions of which remain to be determined. IMPORTANCE Numerous bacterial species encode proteins predicted to have similarity with Get3- and ArsA-type anion transporters. Our studies provide evidence that these proteins, which we named BagA and BagB, are unlikely to be involved in anion transport. In addition, BagA and BagB are conserved in all mycobacterial species, including the causative agent of leprosy, which has a highly decayed genome. This conservation suggests that BagAB constitutes a part of the core mycobacterial genome and is needed for some yet-to-be-determined part of the life cycle of these organisms.

scholarly journals Horizontal Spread of Rhodococcus equi Macrolide Resistance Plasmid pRErm46 across Environmental Actinobacteria

2020 ◽  
Vol 86 (9) ◽  
Author(s):  
Sonsiray Álvarez-Narváez ◽  
Steeve Giguère ◽  
Londa J. Berghaus ◽  
Cody Dailey ◽  
José A. Vázquez-Boland
Keyword(s):  
Bacterial Species ◽  
Low Frequency ◽  
Rhodococcus Equi ◽  
Macrolide Resistance ◽  
Conjugative Plasmid ◽  
Bacterial Populations ◽  
Prolonged Incubation ◽  
Content Type ◽  
Host Restriction

ABSTRACT Conjugation is one of the main mechanisms involved in the spread and maintenance of antibiotic resistance in bacterial populations. We recently showed that the emerging macrolide resistance in the soilborne equine and zoonotic pathogen Rhodococcus equi is conferred by the erm(46) gene carried on the 87-kb conjugative plasmid pRErm46. Here, we investigated the conjugal transferability of pRErm46 to 14 representative bacteria likely encountered by R. equi in the environmental habitat. In vitro mating experiments demonstrated conjugation to different members of the genus Rhodococcus as well as to Nocardia and Arthrobacter spp. at frequencies ranging from ∼10−2 to 10−6. pRErm46 transfer was also observed in mating experiments in soil and horse manure, albeit at a low frequency and after prolonged incubation at 22 to 30°C (environmental temperatures), not 37°C. All transconjugants were able to transfer pRErm46 back to R. equi. Conjugation could not be detected with Mycobacterium or Corynebacterium spp. or several members of the more distant phylum Firmicutes such as Enterococcus, Streptococcus, or Staphylococcus. Thus, the pRErm46 host range appears to span several actinobacterial orders with certain host restriction within the Corynebacteriales. All bacterial species that acquired pRErm46 expressed increased macrolide resistance with no significant deleterious impact on fitness, except in the case of Rhodococcus rhodnii. Our results indicate that actinobacterial members of the environmental microbiota can both acquire and transmit the R. equi pRErm46 plasmid and thus potentially contribute to the maintenance and spread of erm(46)-mediated macrolide resistance in equine farms. IMPORTANCE This study demonstrates the efficient horizontal transfer of the Rhodococcus equi conjugative plasmid pRErm46, recently identified as the cause of the emerging macrolide resistance among equine isolates of this pathogen, to and from different environmental Actinobacteria, including a variety of rhodococci as well as Nocardia and Arthrobacter spp. The reported data support the notion that environmental microbiotas may act as reservoirs for the endemic maintenance of antimicrobial resistance in an antibiotic pressurized farm habitat.

scholarly journals Antimicrobial Properties of a Chitosan Dextran-Based Hydrogel for Surgical Use

2011 ◽  
Vol 56 (1) ◽  
pp. 280-287 ◽  
Author(s):  
Manal A. Aziz ◽  
Jaydee D. Cabral ◽  
Heather J. L. Brooks ◽  
Stephen C. Moratti ◽  
Lyall R. Hanton
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Bacterial Species ◽  
Antimicrobial Properties ◽  
Sinus Surgery ◽  
Peptide Bonds ◽  
Content Type ◽  
E Coli ◽  
Transmission Electron

ABSTRACTA chitosan dextran-based (CD) hydrogel, developed for use in endoscopic sinus surgery, was tested for antimicrobial activityin vitroagainst a range of pathogenic microorganisms. The microdilution technique was used to determine minimum inhibitory, minimum bactericidal, and minimum fungicidal concentrations. In addition, the time-kill efficacy of CD hydrogel was determined for two bacterial species. Scanning and transmission electron microscopy were carried out to elucidate the antimicrobial mechanism of this compound. CD hydrogel was found to be effective againstStaphylococcus aureus,Streptococcus pyogenes,Escherichia coli, andClostridium perfringensat its surgical concentration of 50,000 mg/liter. Minimum bactericidal concentrations ranged from 2,000 to 50,000 mg/liter. Dextran aldehyde (DA) was found to be the antimicrobial component of the CD hydrogel with MBC ranging from 2,000 to 32,000 mg/liter.S. aureusappeared to be killed at a slightly faster rate thanE. coli. Candida albicansandPseudomonas aeruginosawere more resistant to CD hydrogel and DA. Scanning and transmission electron microscopy ofE. coliandS. aureusincubated with CD hydrogel and DA alone revealed morphological damage, disrupted cell walls, and loss of cytosolic contents, compatible with the proposed mode of action involving binding to cell wall proteins and disruption of peptide bonds. Motility and chemotaxis tests showedE. colito be inhibited when incubated with DA. The antibacterial activity of CD hydrogel may make it a useful postsurgical aid at other body sites, especially where there is a risk of Gram-positive infections.

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