scholarly journals Computational Basis for On-Demand Production of Diversified Therapeutic Phage Cocktails

mSystems ◽  
2020 ◽  
Vol 5 (4) ◽  
Author(s):  
Catherine M. Mageeney ◽  
Anupama Sinha ◽  
Richard A. Mosesso ◽  
Douglas L. Medlin ◽  
Britney Y. Lau ◽  
...  
Keyword(s):  
Galleria Mellonella ◽  
Bacterial Pathogens ◽  
Rapid Development ◽  
Opportunistic Pathogen ◽  
Near Neighbor ◽  
Computational Molecular Biology ◽  
Integrase Gene ◽  
Content Type ◽  
Technology Platform ◽  
On Demand

ABSTRACT New therapies are necessary to combat increasingly antibiotic-resistant bacterial pathogens. We have developed a technology platform of computational, molecular biology, and microbiology tools which together enable on-demand production of phages that target virtually any given bacterial isolate. Two complementary computational tools that identify and precisely map prophages and other integrative genetic elements in bacterial genomes are used to identify prophage-laden bacteria that are close relatives of the target strain. Phage genomes are engineered to disable lysogeny, through use of long amplicon PCR and Gibson assembly. Finally, the engineered phage genomes are introduced into host bacteria for phage production. As an initial demonstration, we used this approach to produce a phage cocktail against the opportunistic pathogen Pseudomonas aeruginosa PAO1. Two prophage-laden P. aeruginosa strains closely related to PAO1 were identified, ATCC 39324 and ATCC 27853. Deep sequencing revealed that mitomycin C treatment of these strains induced seven phages that grow on P. aeruginosa PAO1. The most diverse five phages were engineered for nonlysogeny by deleting the integrase gene (int), which is readily identifiable and typically conveniently located at one end of the prophage. The Δint phages, individually and in cocktails, killed P. aeruginosa PAO1 in liquid culture as well as in a waxworm (Galleria mellonella) model of infection. IMPORTANCE The antibiotic resistance crisis has led to renewed interest in phage therapy as an alternative means of treating infection. However, conventional methods for isolating pathogen-specific phage are slow, labor-intensive, and frequently unsuccessful. We have demonstrated that computationally identified prophages carried by near-neighbor bacteria can serve as starting material for production of engineered phages that kill the target pathogen. Our approach and technology platform offer new opportunity for rapid development of phage therapies against most, if not all, bacterial pathogens, a foundational advance for use of phage in treating infectious disease.

scholarly journals Computationally-guided technology platform for on-demand production of diversified therapeutic phage cocktails

2020 ◽  
Author(s):  
Catherine M. Mageeney ◽  
Anupama Sinha ◽  
Richard A. Mosesso ◽  
Douglas L. Medlin ◽  
Britney Y. Lau ◽  
...  
Keyword(s):  
Galleria Mellonella ◽  
Bacterial Pathogens ◽  
Rapid Development ◽  
Opportunistic Pathogen ◽  
Near Neighbor ◽  
Computational Molecular Biology ◽  
Integrase Gene ◽  
Technology Platform ◽  
On Demand

ABSTRACTNew therapies are necessary to combat increasingly antibiotic-resistant bacterial pathogens. We have developed a technology platform of computational, molecular biology, and microbiology tools which together enable on-demand production of phages that target virtually any given bacterial isolate. Two complementary computational tools that identify and precisely map prophages and other integrative genetic elements (IGEs) in bacterial genomes are used to identify prophage-laden bacteria that are close relatives of the target strain. Phage genomes are engineered to disable lysogeny, through use of long amplicon PCR and Gibson assembly. Finally, the engineered phage genomes are introduced into host bacteria for phage production. As an initial demonstration, we used this approach to produce a phage cocktail against the opportunistic pathogen Pseudomonas aeruginosa PAO1. Two prophage-laden P. aeruginosa strains closely related to PAO1 were identified, ATCC 39324 and ATCC 27853. Deep sequencing revealed that mitomycin C treatment of these strains induced seven phages that grow on P. aeruginosa PAO1. The most diverse five of these were engineered for non-lysogeny by deleting the integrase gene (int), which is readily identifiable and typically conveniently located at one end of the prophage. The Δint phages, individually and in cocktails, showed killing of P. aeruginosa PAO1 in vitro as well as in a waxworm (Galleria mellonella) model of infection.SIGNIFICANCE STATEMENTThe antibiotic-resistance crisis in medicine and agriculture has led to renewed interest in phage therapy as an alternative means of treating infection. However, conventional methods for isolating pathogen-specific phage are slow, labor-intensive, and frequently unsuccessful. We have demonstrated that prophages carried by near-neighbor bacteria can serve as starting material for production of engineered phages that kill the target pathogen. Our approach and technology platform offer new opportunity for rapid development of phage therapies against most, if not all, bacterial pathogens, a foundational advance for use of phage in treating infectious disease.

scholarly journals Evolutionary Model of Cluster Divergence of the Emergent Marine PathogenVibrio vulnificus: From Genotype to Ecotype

mBio ◽  
2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Mario López-Pérez ◽  
Jane M. Jayakumar ◽  
Jose M. Haro-Moreno ◽  
Asier Zaragoza-Solas ◽  
Geethika Reddi ◽  
...  
Keyword(s):  
Population Dynamics ◽  
Genetic Factors ◽  
Vibrio Vulnificus ◽  
Bacterial Pathogens ◽  
Evolutionary Model ◽  
Opportunistic Pathogen ◽  
Secretion Systems ◽  
Carbohydrate Utilization ◽  
Content Type ◽  
Pathogenic Vibrios

ABSTRACTVibrio vulnificus, an opportunistic pathogen, is the causative agent of a life-threatening septicemia and a rising problem for aquaculture worldwide. The genetic factors that differentiate its clinical and environmental strains remain enigmatic. Furthermore, clinical strains have emerged from every clade ofV. vulnificus. In this work, we investigated the underlying genomic properties and population dynamics of theV. vulnificusspecies from an evolutionary and ecological point of view. Genome comparisons and bioinformatic analyses of 113 V. vulnificusisolates indicate that the population ofV. vulnificusis made up of four different clusters. We found evidence that recombination and gene flow between the two largest clusters (cluster 1 [C1] and C2) have drastically decreased to the point where they are diverging independently. Pangenome and phenotypic analyses showed two markedly different lifestyles for these two clusters, indicating commensal (C2) and bloomer (C1) ecotypes, with differences in carbohydrate utilization, defense systems, and chemotaxis, among other characteristics. Nonetheless, we identified frequent intra- and interspecies exchange of mobile genetic elements (e.g., antibiotic resistance plasmids, novel “chromids,” or two different and concurrent type VI secretion systems) that provide high levels of genetic diversity in the population. Surprisingly, we identified strains from both clusters in the mucosa of aquaculture species, indicating that manmade niches are bringing strains from the two clusters together. We propose an evolutionary model ofV. vulnificusthat could be broadly applicable to other pathogenic vibrios and facultative bacterial pathogens to pursue strategies to prevent their infections and emergence.IMPORTANCEVibrio vulnificusis an emergent marine pathogen and is the cause of a deadly septicemia. However, the genetic factors that differentiate its clinical and environmental strains and its several biotypes remain mostly enigmatic. In this work, we investigated the underlying genomic properties and population dynamics of theV. vulnificusspecies to elucidate the traits that make these strains emerge as a human pathogen. The acquisition of different ecological determinants could have allowed the development of highly divergent clusters with different lifestyles within the same environment. However, we identified strains from both clusters in the mucosa of aquaculture species, indicating that manmade niches are bringing strains from the two clusters together, posing a potential risk of recombination and of emergence of novel variants. We propose a new evolutionary model that provides a perspective that could be broadly applicable to other pathogenic vibrios and facultative bacterial pathogens to pursue strategies to prevent their infections.

scholarly journals Effect of Incubation Temperature on Antibiotic Resistance and Virulence Factors of Acinetobacter baumannii ATCC 17978

2017 ◽  
Vol 62 (1) ◽  
Author(s):  
P. Malaka De Silva ◽  
Patrick Chong ◽  
Dinesh M. Fernando ◽  
Garrett Westmacott ◽  
Ayush Kumar
Keyword(s):  
Acinetobacter Baumannii ◽  
Galleria Mellonella ◽  
Incubation Temperature ◽  
Opportunistic Pathogen ◽  
Effect Of Temperature ◽  
Content Type ◽  
Content Model ◽  
Key Factor ◽  
Plastic Surfaces ◽  
Increased Susceptibility

ABSTRACT Acinetobacter baumannii is a notorious opportunistic pathogen that is prevalent mainly in hospital settings. The ability of A. baumannii to adapt and to survive in a range of environments has been a key factor for its persistence and success as an opportunistic pathogen. In this study, we investigated the effect of temperature on the clinically relevant phenotypes displayed by A. baumannii at 37°C and 28°C. Surface-associated motility was significantly reduced at 28°C, while biofilm formation on plastic surfaces was increased at 28°C. Decreased susceptibility to aztreonam and increased susceptibility to trimethoprim-sulfamethoxazole were observed at 28°C. No differences in virulence, as assayed in a Galleria mellonella model, were observed. Proteomic analysis showed differential expression of 629 proteins, of which 366 were upregulated and 263 were downregulated at 28°C. Upregulation of the Csu and iron uptake proteins at 28°C was a key finding for understanding some of the phenotypes displayed by A. baumannii at 28°C.

scholarly journals Functional Analysis of the Collagen Binding Proteins of Streptococcus parasanguinis FW213

mSphere ◽  
2020 ◽  
Vol 5 (5) ◽  
Author(s):  
Yi-Ywan M. Chen ◽  
Pei-Hua Tsai ◽  
Zong-Sian Ye ◽  
Yu-Wen Huang ◽  
Hui-Ru Shieh ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Binding Proteins ◽  
Type I Collagen ◽  
Galleria Mellonella ◽  
Opportunistic Pathogen ◽  
Type I ◽  
Collagen Binding ◽  
Content Type ◽  
Extracellular Matrix Molecules ◽  
Streptococcus Parasanguinis

ABSTRACT Streptococcus parasanguinis is a dominant isolate of dental plaque and an opportunistic pathogen associated with subacute endocarditis. As the expression of collagen binding proteins (CBPs) could promote the establishment of S. parasanguinis in the host, the functions of three putative CBP-encoding loci, Spaf_0420, Spaf_1570, and Spaf_1573, were analyzed using isogenic mutant strains. It was revealed that S. parasanguinis FW213 bound effectively to fibronectin and type I collagen, but the strain’s affinity for laminin and type IV collagen was quite low. By using various deletion derivatives, it was found that these three loci mediated the binding of S. parasanguinis to multiple extracellular matrix molecules, with type I collagen as the common substrate. Derivative strains with a deletion in any of the three loci expressed reduced binding to trypsin-treated swine heart valves. The deletion of these loci also reduced the viable count of S. parasanguinis bacteria within macrophages, especially the loss of Spaf_0420, but only strains with deletions in Spaf_0420 and Spaf_1570 expressed reduced virulence in the Galleria mellonella larva model. The deletion of Spaf_1570 and Spaf_1573 affected mainly the structure, but not the overall mass, of biofilm cultures in a flow cell system. Thus, CBPs are likely to be more critical for the initial colonization of S. parasanguinis on host tissues during the development of endocarditis. IMPORTANCE Bacteria generally can utilize multiple adhesins to establish themselves in the host. We found that Streptococcus parasanguinis, a dominant oral commensal and an opportunistic pathogen for subacute endocarditis, possesses at least three collagen-binding proteins that enable S. parasanguinis to successfully colonize damaged heart tissues and escape innate immune clearance. The binding specificities of these three proteins for extracellular matrix molecules differ, although all three proteins participate in biofilm formation by S. parasanguinis. The “multiligand for multisubstrate” feature of these adhesins may explain the high adaptability of this microbe to different tissue sites.

scholarly journals Biological Impact of a Large-Scale Genomic Inversion That Grossly Disrupts the Relative Positions of the Origin and Terminus Loci of theStreptococcus pyogenesChromosome

2019 ◽  
Vol 201 (17) ◽  
Author(s):  
Dragutin J. Savic ◽  
Scott V. Nguyen ◽  
Kimberly McCullor ◽  
W. Michael McShan
Keyword(s):  
Dna Sequences ◽  
Parental Strain ◽  
Large Scale ◽  
Galleria Mellonella ◽  
Acute Infection ◽  
Relative Length ◽  
Published Data ◽  
Rich Medium ◽  
Content Type

ABSTRACTA large-scale genomic inversion encompassing 0.79 Mb of the 1.816-Mb-longStreptococcus pyogenesserotype M49 strain NZ131 chromosome spontaneously occurs in a minor subpopulation of cells, and in this report genetic selection was used to obtain a stable lineage with this chromosomal rearrangement. This inversion, which drastically displaces theorisite relative to the terminus, changes the relative length of the replication arms so that one replichore is approximately 0.41 Mb while the other is about 1.40 Mb in length. Genomic reversion to the original chromosome constellation is not observed in PCR-monitored analyses after 180 generations of growth in rich medium. Compared to the parental strain, the inversion surprisingly demonstrates a nearly identical growth pattern in the first phase of the exponential phase, but differences do occur when resources in the medium become limited. When cultured separately in rich medium during prolonged stationary phase or in an experimental acute infection animal model (Galleria mellonella), the parental strain and the invertant have equivalent survival rates. However, when they are coincubated together, bothin vitroandin vivo, the survival of the invertant declines relative to the level for the parental strain. The accompanying aspect of the study suggests that inversions taking place nearoriCalways happen to secure the linkage oforiCto DNA sequences responsible for chromosome partition. The biological relevance of large-scale inversions is also discussed.IMPORTANCEBased on our previous work, we created to our knowledge the largest asymmetric inversion, covering 43.5% of theS. pyogenesgenome. In spite of a drastic replacement of origin of replication and the unbalanced size of replichores (1.4 Mb versus 0.41 Mb), the invertant, when not challenged with its progenitor, showed impressive vitality for growthin vitroand in pathogenesis assays. The mutant supports the existing idea that slightly deleterious mutations can provide the setting for secondary adaptive changes. Furthermore, comparative analysis of the mutant with previously published data strongly indicates that even large genomic rearrangements survive provided that the integrity of theoriCand the chromosome partition cluster is preserved.

A framework for programmatically designing user interfaces in JavaScript

2015 ◽  
Vol 11 (3) ◽  
pp. 254-269
Author(s):  
Henry Larkin
Keyword(s):  
User Interface ◽  
User Interfaces ◽  
Design Methodology ◽  
Rapid Development ◽  
Program Code ◽  
Web Based ◽  
Content Type ◽  
Mobile Web ◽  
Mobile Interfaces ◽  
New Framework

Purpose – The purpose of this paper is to investigate the feasibility of creating a declarative user interface language suitable for rapid prototyping of mobile and Web apps. Moreover, this paper presents a new framework for creating responsive user interfaces using JavaScript. Design/methodology/approach – Very little existing research has been done in JavaScript-specific declarative user interface (UI) languages for mobile Web apps. This paper introduces a new framework, along with several case studies that create modern responsive designs programmatically. Findings – The fully implemented prototype verifies the feasibility of a JavaScript-based declarative user interface library. This paper demonstrates that existing solutions are unwieldy and cumbersome to dynamically create and adjust nodes within a visual syntax of program code. Originality/value – This paper presents the Guix.js platform, a declarative UI library for rapid development of Web-based mobile interfaces in JavaScript.

scholarly journals BrpA Is Involved in Regulation of Cell Envelope Stress Responses in Streptococcus mutans

2012 ◽  
Vol 78 (8) ◽  
pp. 2914-2922 ◽  
Author(s):  
J. P. Bitoun ◽  
S. Liao ◽  
X. Yao ◽  
S.-J. Ahn ◽  
R. Isoda ◽  
...  
Keyword(s):  
Streptococcus Mutans ◽  
Stress Responses ◽  
Antimicrobial Agents ◽  
Galleria Mellonella ◽  
Cell Envelope ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Content Type ◽  
Lipid Ii ◽  
The Impact

ABSTRACTPrevious studies have shown that BrpA plays a major role in acid and oxidative stress tolerance and biofilm formation byStreptococcus mutans. Mutant strains lacking BrpA also display increased autolysis and decreased viability, suggesting a role for BrpA in cell envelope integrity. In this study, we examined the impact of BrpA deficiency on cell envelope stresses induced by envelope-active antimicrobials. Compared to the wild-type strain UA159, the BrpA-deficient mutant (TW14D) was significantly more susceptible to antimicrobial agents, especially lipid II inhibitors. Several genes involved in peptidoglycan synthesis were identified by DNA microarray analysis as downregulated in TW14D. Luciferase reporter gene fusion assays also revealed that expression ofbrpAis regulated in response to environmental conditions and stresses induced by exposure to subinhibitory concentrations of cell envelope antimicrobials. In aGalleria mellonella(wax worm) model, BrpA deficiency was shown to diminish the virulence ofS. mutansOMZ175, which, unlikeS. mutansUA159, efficiently kills the worms. Collectively, these results suggest that BrpA plays a role in the regulation of cell envelope integrity and that deficiency of BrpA adversely affects the fitness and diminishes the virulence of OMZ175, a highly invasive strain ofS. mutans.

scholarly journals Ceftaroline Activity against Bacterial Pathogens Frequently Isolated in U.S. Medical Centers: Results from Five Years of the AWARE Surveillance Program

2015 ◽  
Vol 59 (4) ◽  
pp. 2458-2461 ◽  
Author(s):  
Helio S. Sader ◽  
Robert K. Flamm ◽  
Jennifer M. Streit ◽  
David J. Farrell ◽  
Ronald N. Jones
Keyword(s):  
Staphylococcus Aureus ◽  
Streptococcus Pneumoniae ◽  
Bacterial Pathogens ◽  
Surveillance Program ◽  
Broth Microdilution ◽  
Methicillin Resistant ◽  
Content Type ◽  
Medical Centers ◽  
Resistant Strains

ABSTRACTA total of 84,704 isolates were collected from 191 medical centers in 2009 to 2013 and tested for susceptibility to ceftaroline and comparator agents by broth microdilution methods. Ceftaroline inhibited allStaphylococcus aureusisolates at ≤2 μg/ml and was very active against methicillin-resistant strains (MIC at which 90% of the isolates tested are inhibited [MIC90], 1 μg/ml; 97.6% susceptible). AmongStreptococcus pneumoniaeisolates, the highest ceftaroline MIC was 0.5 μg/ml, and ceftaroline activity against the most commonEnterobacteriaceaespecies (MIC50, 0.12 μg/ml; 78.9% susceptible) was similar to that of ceftriaxone (MIC50, ≤0.25 μg/ml; 86.8% susceptible).

Factors impacting BIM application in prefabricated buildings in China with DEMATEL-ISM

2021 ◽  
Vol ahead-of-print (ahead-of-print) ◽  
Author(s):  
Cheng Gong ◽  
Hongyu Xu ◽  
Feng Xiong ◽  
Jian Zuo ◽  
Na Dong
Keyword(s):  
Rapid Development ◽  
Information Modeling ◽  
Critical Factors ◽  
Interpretive Structural Modeling ◽  
Content Type ◽  
Expert Interview ◽  
Improvement Strategies ◽  
Building Information ◽  
Share Data ◽  
Strategic Goals

Purpose Some papers have investigated the complex factors impacting building information modeling (BIM) application in prefabricated buildings (PBs), but few paid attention to their interaction relationships. Ignoring the fact that different factors are not isolated may lead to some key factors being overlooked without appropriate improvement strategies being proposed. This paper aims to analyze those factors and their inter-relationships, with the view to identify the critical factors and their interaction relationships so as to derive constructive strategies that would effectively facilitate BIM adoption in Chinese prefabrication. Design/methodology/approach First, factors influencing BIM application in prefabrication are extracted and collated by literature review, expert interview and analysis of PBs characteristics. Thereafter, an evaluation laboratory (decision-making trial and evaluation laboratory) and interpretive structural modeling are used to explore the relationships and hierarchy among the factors. Based on the degree of cause and centrality, critical factors are extracted and the interaction relationship are investigated. Findings The results show that BIM policies and standards for PBs are the main causal factors. The maturity of BIM software and BIM data interface for PBs, willingness to share data, the strategic goals of the enterprise, BIM law and BIM input and benefit are the main transitional factors while BIM staff and workflow, enterprise attitude, distribution of BIM liability and cooperation of participants are the main direct factors. Originality/value Based on the above findings, corresponding improvement strategies are proposed so as to promote BIM application in prefabrication and the rapid development of China’s PBs efficiently.

Zie ik het of zie ik het niet?

2021 ◽  
Vol 49 (1) ◽  
pp. 51-83
Author(s):  
Jorre Vannieuwenhuyze ◽  
Karen Donders ◽  
Ike Picone
Keyword(s):  
European Union ◽  
Government Regulation ◽  
Video On Demand ◽  
The European Union ◽  
Content Type ◽  
Consumption Behavior ◽  
Local Content ◽  
Positive Effects ◽  
On Demand ◽  
The Impact

Abstract Do I see or not? A study on the impact of placement on program consumption in an on-demand environment The European Union (2018) stipulates that Member States can implement rules to ensure the findability and visibility of local content in video- on- demand environments. Indeed, several countries are concerned that their own audiovisual programs or journalistic products will be consumed less in such environments. It is argued that, in such environments, media users completely decide themselves about their consumption agency, but such statements are also contested. In this research we analyze the impact of placement on the consumption of audiovisual programs in the video-on-demand environments of the Flemish broadcasters VRT and DPG. From experimental research we conclude that there is indeed a significant impact of placement on consumption behavior and that, in other words, manipulations by intermediary gatekeepers can have potentially negative and positive effects on the consumption of local content. Government regulation would therefore be a useful tool to safeguard the importance of proximity of content.

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