scholarly journals Universal microbial diagnostics using random DNA probes

2016 ◽  
Vol 2 (9) ◽  
pp. e1600025 ◽  
Author(s):  
Amirali Aghazadeh ◽  
Adam Y. Lin ◽  
Mona A. Sheikh ◽  
Allen L. Chen ◽  
Lisa M. Atkins ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Pathogenic Bacteria ◽  
Disease Outbreaks ◽  
Dna Probes ◽  
Signal Recovery ◽  
Human Pathogens ◽  
Infectious Disease Outbreaks ◽  
Target Dna ◽  
Detection Schemes

Early identification of pathogens is essential for limiting development of therapy-resistant pathogens and mitigating infectious disease outbreaks. Most bacterial detection schemes use target-specific probes to differentiate pathogen species, creating time and cost inefficiencies in identifying newly discovered organisms. We present a novel universal microbial diagnostics (UMD) platform to screen for microbial organisms in an infectious sample, using a small number of random DNA probes that are agnostic to the target DNA sequences. Our platform leverages the theory of sparse signal recovery (compressive sensing) to identify the composition of a microbial sample that potentially contains novel or mutant species. We validated the UMD platform in vitro using five random probes to recover 11 pathogenic bacteria. We further demonstrated in silico that UMD can be generalized to screen for common human pathogens in different taxonomy levels. UMD’s unorthodox sensing approach opens the door to more efficient and universal molecular diagnostics.

scholarly journals Antimicrobial Activity of Chitosan Oligosaccharides with Special Attention to Antiparasitic Potential

Marine Drugs ◽  
2021 ◽  
Vol 19 (2) ◽  
pp. 110
Author(s):  
Nayara Sousa da Silva ◽  
Nathália Kelly Araújo ◽  
Alessandra Daniele-Silva ◽  
Johny Wysllas de Freitas Oliveira ◽  
Júlia Maria de Medeiros ◽  
...  
Keyword(s):  
Disease Outbreaks ◽  
Biological Properties ◽  
Enzymatic Production ◽  
Infectious Disease Outbreaks ◽  
Chitosan Oligosaccharides ◽  
Lower Viscosity ◽  
Biomedical Industry ◽  
Parasite Aggregation ◽  
Hydrolysis Of

The global rise of infectious disease outbreaks and the progression of microbial resistance reinforce the importance of researching new biomolecules. Obtained from the hydrolysis of chitosan, chitooligosaccharides (COSs) have demonstrated several biological properties, including antimicrobial, and greater advantage over chitosan due to their higher solubility and lower viscosity. Despite the evidence of the biotechnological potential of COSs, their effects on trypanosomatids are still scarce. The objectives of this study were the enzymatic production, characterization, and in vitro evaluation of the cytotoxic, antibacterial, antifungal, and antiparasitic effects of COSs. NMR and mass spectrometry analyses indicated the presence of a mixture with 81% deacetylated COS and acetylated hexamers. COSs demonstrated no evidence of cytotoxicity upon 2 mg/mL. In addition, COSs showed interesting activity against bacteria and yeasts and a time-dependent parasitic inhibition. Scanning electron microscopy images indicated a parasite aggregation ability of COSs. Thus, the broad biological effect of COSs makes them a promising molecule for the biomedical industry.

Enhancing microbiological safety of water and food

2014 ◽  
Author(s):  
◽  
Zhenyu Shen
Keyword(s):  
16S Rdna ◽  
In Silico ◽  
Mung Bean ◽  
Disease Outbreaks ◽  
Laboratory Strain ◽  
Human Pathogens ◽  
University Of Missouri ◽  
Enterohaemorrhagic Escherichia Coli ◽  
Wastewater Samples

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT AUTHOR'S REQUEST.] Human and animal feces are the main sources of pathogens associated with foodborne and waterborne disease outbreaks. The studies in Volume I proposed ribosomal intervening sequences (IVSs) can be used as host-specific genetic markers for tracking fecal sources in polluted food and water. In Chapter 3, using bioinformatics tools, one IVS in Faecalibacterium 16S rDNA specific to poultry (chicken and turkey) was identified in silico. A PCR assay was then developed to confirm the IVS was only present in fecal or wastewater samples from poultry. The study in Chapter 4 comprehensively examined host specificities of IVSs in 16S rDNA of 73 genera of fecal bacteria. A total of 13 IVSs were identified to be associated particular host sources in silico and the host-specificities of eight of them were confirmed by PCR reactions. Furthermore, a novel beef cattle-specific IVS was identified with next generation sequencing (NGS). Internalization of human pathogens in produce poses high risks to human health because the surface-sanitization method fail to inactivate the pathogens. The study in Volume II proposed to control the pathogen internalization with endophytic Bacillus subtilis strains isolated from vegetables. Twelve endophytic B. subtilis strains isolated from inner tissue of lettuce stems and mung bean seeds displayed inhibitory activity against a Salmonella enterica indicator in vitro and two of them (LCA1 and M24) showed broad inhibitory spectrums. LCA1, M24, and a laboratory strain B. subtilis 168 could internalize in mung bean sprouts and significantly reduced the internalization of S. enterica and enterohaemorrhagic Escherichia coli (EHEC).

scholarly journals Widespread Distribution in Pathogenic Bacteria of Di-Iron Proteins That Repair Oxidative and Nitrosative Damage to Iron-Sulfur Centers

2008 ◽  
Vol 190 (6) ◽  
pp. 2004-2013 ◽  
Author(s):  
Tim W. Overton ◽  
Marta C. Justino ◽  
Ying Li ◽  
Joana M. Baptista ◽  
Ana M. P. Melo ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Pathogenic Bacteria ◽  
Nitrosative Stress ◽  
Human Pathogens ◽  
Paramagnetic Resonance ◽  
Widespread Distribution ◽  
Iron Proteins ◽  
E Coli

ABSTRACT Expression of two genes of unknown function, Staphylococcus aureus scdA and Neisseria gonorrhoeae dnrN, is induced by exposure to oxidative or nitrosative stress. We show that DnrN and ScdA are di-iron proteins that protect their hosts from damage caused by exposure to nitric oxide and to hydrogen peroxide. Loss of FNR-dependent activation of aniA expression and NsrR-dependent repression of norB and dnrN expression on exposure to NO was restored in the gonococcal parent strain but not in a dnrN mutant, suggesting that DnrN is necessary for the repair of NO damage to the gonococcal transcription factors, FNR and NsrR. Restoration of aconitase activity destroyed by exposure of S. aureus to NO or H2O2 required a functional scdA gene. Electron paramagnetic resonance spectra of recombinant ScdA purified from Escherichia coli confirmed the presence of a di-iron center. The recombinant scdA plasmid, but not recombinant plasmids encoding the complete Escherichia coli sufABCDSE or iscRSUAhscBAfdx operons, complemented repair defects of an E. coli ytfE mutant. Analysis of the protein sequence database revealed the importance of the two proteins based on the widespread distribution of highly conserved homologues in both gram-positive and gram-negative bacteria that are human pathogens. We provide in vivo and in vitro evidence that Fe-S clusters damaged by exposure to NO and H2O2 can be repaired by this new protein family, for which we propose the name repair of iron centers, or RIC, proteins.

Application of DNA microarray technology for detection, identification, and characterization of food-borne pathogens

2006 ◽  
Vol 52 (1) ◽  
pp. 1-8 ◽  
Author(s):  
M Kostrzynska ◽  
A Bachand
Keyword(s):  
Dna Microarray ◽  
Dna Sequences ◽  
Pathogenic Bacteria ◽  
Dna Microarrays ◽  
Simultaneous Detection ◽  
Microarray Technology ◽  
Food Borne Pathogens ◽  
Food Borne ◽  
Target Dna

DNA microarrays represent the latest advance in molecular technology. In combination with bioinformatics, they provide unparalleled opportunities for simultaneous detection of thousands of genes or target DNA sequences and offer tremendous potential for studying food-borne microorganisms. This review provides an up-to-date look at the application of DNA microarray technology to detect food-borne pathogenic bacteria, viruses, and parasites. In addition, it covers the advantages of using microarray technology to further characterize microorganisms by providing information for specific identification of isolates, to understand the pathogenesis based on the presence of virulence genes, and to indicate how new pathogenic strains evolved epidemiologically and phylogenetically.Key words: DNA microarrays, food-borne pathogens, detection.

scholarly journals In-vitro pathogen inhibition: Comparing the inhibitory effects of a complex multistrain synbiotic with simple probiotics containing the yeast Saccharomyces boulardii or Lactobacillus rhamnosus bacteria

2019 ◽  
Author(s):  
Jacek Piatek ◽  
Henning Sommermeyer ◽  
Arleta Ciechelska-Rybarczyk ◽  
Malgorzata Bernatek
Keyword(s):  
Bacterial Infections ◽  
Pathogenic Bacteria ◽  
Lactobacillus Rhamnosus ◽  
Saccharomyces Boulardii ◽  
Human Pathogens ◽  
Inhibitory Effects ◽  
Probiotic Strains ◽  
Pathogen Inhibition ◽  
Different Strains

AbstractSupplementation with probiotics is considered as alternative treatment or adjuvant therapy for a number of bacterial infections for which the use of antibiotics is either not recommended or emerging antibiotic resistance is a major concern. Inhibition of the growth of pathogenic bacteria has been related to a number of different activities of probiotic bacteria or yeasts, some of which are very specific for particular strains of probiotics. As the different inhibition activities might act additively or even synergistically, probiotic multistrain products are discussed as potentially being more effective in pathogen inhibition than products containing one or a small number of probiotic strains. The present study investigated the in vitro inhibition of Escherichia (E.) coli, Shigella spp., Salmonella (S.) typhimurium and Clostridum (Cl.) difficile, all being human pathogens of significant worldwide healthcare concerns. The probiotic containing the yeast Sacharomyces (S.) boulardii inhibited all four pathogens. Similar inhibitions were observed with a bacterial probiotic containing three different strains (Pen, E/N and Oxy) of Lactobacillus (Lc.) rhamnosus. Compared to the inhibition found for these probiotics, the inhibitory effects of a complex multistrain synbiotic, containing nine different probiotic strains (6 Lactobacilli and 3 Bifidobacteria) and the prebiotic fructooligosaccharide (FOS), were significantly stronger. The stronger inhibition by the complex multistrain synbiotic was observed for all four tested pathogens. Our findings support a hypothesis that complex synbiotic products containing a larger number of different strains combined with a prebiotic component might be more attractive candidates for further clinical characterization than simpler probiotics containing one or only few probiotic strains.

scholarly journals Purification of specific DNA species using the CRISPR system

2019 ◽  
Vol 4 (1) ◽  
Author(s):  
Toshitsugu Fujita ◽  
Hodaka Fujii
Keyword(s):  
Dna Sequences ◽  
Chromatin Immunoprecipitation ◽  
In Vitro Method ◽  
Guide Rna ◽  
Crispr System ◽  
Target Dna ◽  
Genomic Regions ◽  
Generation Sequencing ◽  
Subsequent Identification

AbstractIn 2013, we developed a new method of engineered DNA-binding molecule-mediated chromatin immunoprecipitation that incorporates the clustered regularly interspaced short palindromic repeats (CRISPR) system to purify specific DNA species. This CRISPR-mediated purification can be performed in-cell or in vitro; CRISPR complexes can be expressed to tag target DNA sequences in the cells to be analyzed, or a CRISPR ribonucleoprotein complex consisting of recombinant nuclease-dead Cas9 (dCas9) and synthetic guide RNA can be used to tag target DNA sequences in vitro. Both methods enable purification of specific DNA sequences in chromatin structures for subsequent identification of molecules (proteins, RNAs, and other genomic regions) associated with the target sequences. The in vitro method also enables enrichment of purified DNA sequences from a pool of heterogeneous sequences for next-generation sequencing or other applications. In this review, we outline the principle of CRISPR-mediated purification of specific DNA species and discuss recent advances in the technology.

scholarly journals Bacterial biodiversity drives the evolution of CRISPR-based phage resistance in Pseudomonas aeruginosa

2019 ◽  
Author(s):  
Ellinor O Alseth ◽  
Elizabeth Pursey ◽  
Adela M Luján ◽  
Isobel McLeod ◽  
Clare Rollie ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Dna Sequences ◽  
Bacterial Species ◽  
Opportunistic Pathogen ◽  
Phage Resistance ◽  
Natural Environments ◽  
Human Pathogens ◽  
Resistance Evolution ◽  
Trade Offs

Approximately half of all bacterial species encode CRISPR-Cas adaptive immune systems1, which provide immunological memory by inserting short DNA sequences from phage and other parasitic DNA elements into CRISPR loci on the host genome2. Whereas CRISPR loci evolve rapidly in natural environments3, bacterial species typically evolve phage resistance by the mutation or loss of phage receptors under laboratory conditions4,5. Here, we report how this discrepancy may in part be explained by differences in the biotic complexity of in vitro and natural environments6,7. Specifically, using the opportunistic pathogen Pseudomonas aeruginosa and its phage DMS3vir, we show that coexistence with other human pathogens amplifies the fitness trade-offs associated with phage receptor mutation, and therefore tips the balance in favour of CRISPR-based resistance evolution. We also demonstrate that this has important knock-on effects for P. aeruginosa virulence, which became attenuated only if the bacteria evolved surface-based resistance. Our data reveal that the biotic complexity of microbial communities in natural environments is an important driver of the evolution of CRISPR-Cas adaptive immunity, with key implications for bacterial fitness and virulence.

scholarly journals Cas12a trans-cleavage can be modulated in vitro and is active on ssDNA, dsDNA, and RNA

2019 ◽  
Author(s):  
Ryan T. Fuchs ◽  
Jennifer Curcuru ◽  
Megumu Mabuchi ◽  
Paul Yourik ◽  
G. Brett Robb
Keyword(s):  
Dna Sequences ◽  
Nuclease Activity ◽  
Target Sequence ◽  
Sequence Composition ◽  
Guide Rna ◽  
Dna Targeting ◽  
Single Turnover ◽  
Target Dna ◽  
Buffer Composition

ABSTRACTCRISPR-Cas12a (Cpf1) are RNA-guided nuclease effectors of acquired immune response that act in their native organisms by cleaving targeted DNA sequences. Like CRISPR-Cas9 RNA-guided DNA targeting enzymes, Cas12a orthologs have been repurposed for genome editing in non-native organisms and for DNA manipulationin vitro. Recent studies have shown that activation of Cas12a via guide RNA-target DNA pairing causes multiple turnover, non-specific ssDNA degradation intrans, after single turnover on-target cleavage incis. We find that the non-specifictransnuclease activity affects RNA and dsDNA in addition to ssDNA, an activity made more evident by adjustment of reaction buffer composition. The magnitude of thetransnuclease activity varies depending on features of the guide RNA being used, specifically target sequence composition and length. We also find that the magnitude oftransnuclease activity varies between the three most well-studied Cas12a orthologs and that the Cas12a fromLachnospiraceaebacterium ND2006 appears to be the most active.

scholarly journals Reversible regulation of Cas12a activities by AcrVA5-mediated acetylation and CobB-mediated deacetylation

2021 ◽  
Author(s):  
Xiaoman Kang ◽  
Lei Yin ◽  
Songkuan Zhuang ◽  
Tianshuai Hu ◽  
Zhile Wu ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Wide Spectrum ◽  
Defense System ◽  
Post Translational Modification ◽  
Defense Systems ◽  
Target Dna ◽  
Short Palindromic Repeat ◽  
Type V

The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR associated (Cas) system protects bacteria and archaea from the invasion of foreign genetic elements. To cope with the host CRISPR systems, phages have evolved many strategies, including the anti-CRISPR (Acr) proteins, to inactivate the Cas nucleases. Recently, it has been reported that the type V-A Cas12a effector can be acetylated and inactivated by AcrVA5, which is a GNAT-family acetyltransferase. However, it is unclear whether the host has any coping strategies to reactivate the defense system. Here we show that the AcrVA5-acetylated Cas12a can be deacetylated by bacterial deacetylase CobB, reactivating Cas12a for both in vitro cleavage of target DNA sequences and in vivo protection of the host from invasion of foreign nucleic acids. Therefore, this study not only shows the reversible regulation of Cas12a activities by post-translational modification but also reveals CobB as a secondary safeguard to bacterial CRISPR defense systems. In addition, we demonstrate that AcrVA5 is a wide-spectrum acetyltransferase, acetylating a large number of target proteins besides Cas12a, and the AcrVA5-acetylated targets can also be deacetylated by CobB.

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