1,512 research outputs found
Machine learning in the service of phage analysis: advancements, applications, and future prospects
Bacteriophages have shown great potential to address the threat to public health, posed by the emergence of antibiotic-resistant bacterial strains, as well as food safety, and biocontrol. With the advent of machine learning (ML) techniques, researchers have been able to leverage the power of computational algorithms to enhance the analysis and understanding of phages in various aspects, which emerged as powerful tools for unraveling the complexities of phage genomes and proteins, enabling rapid and accurate analysis.
Here we present two ML-based tools, PhagePromoter and PhageDPO, designed to facilitate phage analysis and enhance our understanding of their functionality. PhagePromoter is a state-of-the-art ML model specifically developed for predicting promoter regions within phage genomes. By leveraging a vast dataset of annotated phage genomes, PhagePromoter achieves high accuracy in identifying potential promoter sequences, providing valuable insights into phage gene expression and regulation.
Likewise, PhageDPO represents an innovative ML-based tool for predicting phage depolymerase proteins, which are crucial for the degradation of bacterial polysaccharides. By training on comprehensive datasets of known depolymerase sequences, PhageDPO demonstrates exceptional performance in identifying potential depolymerase candidates within phage genomes. This tool contributes to our understanding of phage-host interactions and their implications for therapeutic and biotechnological applications.
The development of PhagePromoter and PhageDPO highlights the growing significance of ML techniques in the field of phage analysis. By leveraging vast genomic datasets and employing advanced ML algorithms, these tools provide researchers with efficient means to explore the intricate characteristics of phages and their associated proteins. The integration of these tools into existing phage analysis pipelines enables comprehensive and accurate investigations, ultimately driving advancements in phage-based applications.info:eu-repo/semantics/publishedVersio
Molecular studies on bacteriophage endolysins and their potential to control gram-negative bacteria
Thesis for PhD degree in Chemical and Biological EngineeeringBacteriophages are viruses that specifically infect bacterial hosts to reproduce. At the end of the infection cycle, progeny virions are confronted with a rigid cell wall that impedes their release into the environment. Consequently, bacteriophages encode hydrolytic enzymes, called endolysins, to digest the peptidoglycan and cause bacteriolysis.
In contrast to their extensively studied counterparts, active against Gram-positives, endolysins from bacteriophages from a Gram-negative background remain less explored. This knowledge gap is largely due to their limited potential as an antimicrobial, which is related to the presence of an impermeable outer membrane in Gram-negatives that blocks the exogenous endolysin action. The experimental work developed in the scope of this thesis aimed at developing efficient strategies to potentiate the endolysin action against these pathogens.
An extensive in silico analysis was performed to provide new insights about endolysins structure and function and bacteriophage-endolysin-host ecology. It was possible to identify and analyze 723 putative endolysins sequences from 5 distinct bacteriophages families, infecting 64 different bacterial genera. These endolysins are tremendously diverse in terms of enzymatic function (24 different enzymatic and 13 binding domains), architecture arrangements (89 different types with either globular or modular design) and length (72 to 578 amino acid residues).
Three different novel endolysins (Lys68, ABgp46 and PVP-SE1gp146) were studied in detail. Biochemical characterization of Lys68 (from a Salmonella-infecting bacteriophage) showed that it is highly thermostable, withstanding temperatures up to 100°C, and able to refold to its original conformation upon thermal denaturation. Lys68 was able to lyse a wide panel of Gram-negative bacteria in combination with outer membrane permeabilizers. While the Lys68/EDTA combination could only inactivate Pseudomonas strains, the use of citric or malic acid as permeabilizer broadened and increased its antibacterial effect. Particularly against Salmonella, the combinatory effect of malic or citric acid with Lys68 led to approximately 3 to 5 log reductions after 2 hours, respectively. During an acid-promoted effect, weak acids permeabilized the lipopolysaccharide of most bacteria to Lys68, which retained a relative high activity under these acidic conditions. In case of EDTA, its chelation effect was only observed against Pseudomonas membranes, where ionic interactions are crucial stabilizing forces.
The endolysin ABgp46 (from an Acinetobacter-infecting bacteriophage) was shown to naturally inactivate 1 log of certain Acinetobacter strains. Tests in the presence of a number of weak acids (citric, malic, lactic, benzoic and acetic acid) resulted in a powerful antibacterial effect when combined with Abgp46. Higher bactericidal activity was consistently obtained when ABgp46 was combined with citric and malic acid, reducing all planktonic Cronobacter, Klebsiella and E. coli O157 (reductions of 1 to 3 logs) and Pseudomonas, Acinetobacter and Salmonella (reduction of more than 4 logs) species tested. It can be speculated that the major weak acid differences observed are related to their acid dissociated constant, that seems to favor compounds (with lower pKa values) that tend be more ionized. The same combinations did not have significant antibacterial activity when applied against Pseudomonas and Acinetobacter biofilms.
To enhance the activity of endolysins against Gram-negative cells, modified endolysins where constructed by fusing PVP-SE1gp146 (from a Salmonella-infecting bacteriophage) with different LPS-destabilizing peptides of polycationic, hydrophobic and amphipathic nature. This strategy resulted in an improvement of the activity of the modified endolysin compared to the native one (1 log reduction on Pseudomonas and Salmonella cells was obtained). The bactericidal activity of all modified variants was increased further in the presence of EDTA. A polycationic nonapeptide was the most efficient tag (maximum reduction of 5 logs). With a different purpose, attempts of increasing the endolysin (in this case Lys68) action against Listeria monocytogenes cells, by inserting species-specific peptidoglycan-binding peptides, did not result in a higher activity.
From the moment their genetic identity became known, endolysins have sparked the interest as alternatives for existing antibiotics. Here it was shown that endolysins can be used to kill not only Gram-positive, but also Gram-negative bacterial pathogens. These obtained results underline the great potential of using an endolysin-based strategy for prevention and/or control of Gram-negative pathogens in foodstuff, food processing surfaces, veterinary and medical applications.Fundação para a Ciência e a Tecnologia grant SFRH/BD/63734/2009 and the projects FCOMP-01-0124-FEDER-019446, FCOMP-01-0124-FEDER-027462 and PEst-OE/EQB/LA0023/2013. Project "Bio-Health - Biotechnology and Bioengineering approaches to improve health quality", ref. NORTE-07-0124-FEDER-000027, co-funded by de Programa Operacional Regional do Norte (ON.2 - O Novo Norte), QREN, FEDER
Bacteriophages and their role in food safety
The interest for natural antimicrobial compounds has increased due to alterations in consumer positions towards the use of chemical preservatives in foodstuff and food processing surfaces. Bacteriophages fit in the class of natural antimicrobial and their effectiveness in controlling bacterial pathogens in agro-food industry has led to the development of different phage products already approved by USFDA and USDA. The majority of these products are to be used in farm animals or animal products such as carcasses, meats and also in agricultural and horticultural products. Treatment with specific phages in the food industry can prevent the decay of products and the spread of bacterial diseases and ultimately promoting safe environments in animal and plant food production, processing and handling.
This is an overview of recent work carried out with phages as tools to promote food safety, starting with a general introduction describing the prevalence of foodborne pathogens and bacteriophages and a more detailed discussion on the use of phage therapy to prevent and treat experimentally induced infections of animals against the most common foodborne pathogens, the use of phages as biocontrol agents in foods, and also their use as biosanitizers of food contact surfaces(undefined
Bacteriophage endolysins as a response to emerging foodborne pathogens
Continuous reports on foodborne outbreaks and increasing prevalence of antibiotic-resistant bacteria call for the development of novel preservation techniques that assure the safety of food products. Bacteriophage endolysins are highly active antibacterial peptidoglycan hydrolases that have evolved over millions of years to become the ultimate weapon against bacteria, with potential to be used as a food preservative. We here give an overview of all distinct endolysins encountered so far, we discuss their inherent qualities and review their role in preventing and controlling foodborne pathogens, divulging their potential for future applications.This work was supported by a grant from the Portuguese Foundation for Science and Technology in the scope of the Projects PTDC/AGR-ALI/100492/2008 and PTDC/AGR-ALI/121057/2010. Hugo Oliveira is paid by the FCT grant SFRH/BD/63734/2009
Characterization of MSlys, the endolysin of Streptococcus pneumoniaephage MS1
Despite the use of pneumococcal conjugate vaccines, the number of infections related to Streptococcus pneumoniae continues to be alarming.
Herein, we identified, characterized the MSlys endolysin encoded in the phage MS1. We further tested its antimicrobial efficacy against planktonic and biofilm cells, assessing the culturability of cells and biofilm structure by scanning electron microscopy, and confocal laser scanning microscopy.
The modular MSlys endolysin consists of an amidase catalytic domain and a choline-binding domain. MSlys is active against isolates of children with otitis media, and conditions close to those found in the middle ear. Treatment with MSlys (2h, 4 µM) reduced planktonic cultures by 3.5 log10 CFU/mL, and 24- and 48-h-old biofilms by 1.5 and 1.8 log10 CFU/mL, respectively. Imaging of the biofilms showed thinner and damaged structures compared to control samples.
The recombinantly expressed MSlys may be a suitable candidate for treating pneumococcal infections, including otitis media.MDS acknowledges the Portuguese Foundation for Science and Technology (FCT) grant (SFRH/BD/128825/2017). This study was supported by the Portuguese Foundation for Science and
Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2019 unit and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte. This project also received funding from the
European Union's Horizon 2020 research and innovation programme under grant agreement No 713640. This study was also supported by the grant PTDC/CVT-CVT/29628/2017 [POCI-01-0145-FEDER-029628].info:eu-repo/semantics/publishedVersio
Combined treatment with bacteriophages and antibiotics as a strategy to control biofilm associated infections
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Genome sequences of four potentially therapeutic bacteriophages infecting Shiga toxin-producing Escherichia coli
Four phages infecting Shiga toxin-producing Escherichia coli (STEC) strains of different serotypes were isolated from wastewater samples. Their virion DNAs range from 51 to 170 kbp, are circularly permuted or have defined terminal repeats, and can encode 82 to 279 proteins. Despite their high similarity to other phages, only about 30\% of their genes have a predicted function.This study was supported by the Portuguese Foundation for Science and Technol-ogy (FCT) under the scope of the strategic funding of unit UIDB/04469/2020 and theBioTecNorte operation (NORTE-01-0145-FEDER-000004), funded by the European Regional Development Fund under the scope of Norte 2020–Programa Operacional Regional do Norte. This study was supported by grants PTDC/CVT-CVT/29628/2017 (POCI-01-0145-FEDER-029628) and POCI-01-0247-FEDER-033679.info:eu-repo/semantics/publishedVersio
Are endolysins promising agents in controlling E. coli associated post-weaning diarrhea in piglets?
Enterotoxigenic Escherichia coli (ETEC) associated infections are the major cause of
piglets mortality in weaning and post-weaning period, resulting in significant economic
losses to the swine industry. Furthermore, the increase of multidrug resistant ETECs have
been recognized a public health danger due to the potential transfer of resistance into the
food chain. Bacterio(phages) endolysins are enzymes produced in the end of phage lytic
cycle that are responsible for cell lysis. So far, no resistance has been reported, which
make endolysins an attractive alternative to antibiotics. In the present work, endolysins
were exploited to tackle ETECs in piglets. Two enzymes previously cloned were tested
against ETEC SP23 strain exponential cells. First, Lys68 (used herein as proof of concept)
was tested alone or in combination with 5 different organics acids (already implemented
in the piglets diet) citric, malic, formic, lactic and sorbic acids in 20 mM HEPES,
149 mM PBS and 140.33 mM simulated intestinal fluid (SIF). Later, PlyF307 endolysin
was also tested with malic and citric acids in SIF. Results demonstrated that both enzymes
reduced ETEC concentration in more than 4 orders of magnitude in HEPES. Lys68
together with malic acid displayed the best antibacterial activity in PBS, being able to
reduce approximately 1 order of magnitude. However, enzymes efficacy was drastically
reduced when tested in buffers that mimicked physiological conditions. In SIF, Lys68
and PlyF307 did not display antibacterial activity. In summary, phage endolysins revealed
to be ineffective to treat ETEC bacterial load in more complex environments.info:eu-repo/semantics/publishedVersio
The influence of P. fluorescens cell morphology on the lytic performance and production of phage ϕIBB-PF7A
This study aims at assessing the influence of Pseudomonas fluorescence cell morphology on the effectiveness and production of the lytic bacteriophage /IBBPF7A. P. fluorescens were cultured as rods or as elongated cells by varying the temperature and rotary agitation conditions. Cells presented rod shape when grown at temperatures up to 25C and also at 30C under static conditions, and elongated morphology only at 30C when cultures were grown under agitation. Elongated cells were 0.4 up to 27.9 lm longer than rod cells. Rod-shaped hosts were best infected by phages at 25C which resulted in an 82% cell density reduction. Phage infection of elongated cells was successful, and the cell density reductions achieved was statistically similar (P[0.05) to those obtained at the
optimum growth temperature of P. fluorescens. Phage burst size varied with the cell growth conditions and was approximately 58 and 153 PFU per infected rod and elongated cells, grown at 160 rpm, at 25C (the optimal temperature) and 30C, respectively. Phage adsorption was
faster to elongated cells, most likely due to the longer length of the host. The surface composition of rod and elongated cells is similar in terms of outer membrane proteins and lipopolysaccharide profiles. The results of this study suggest that the change of rod cells to an elongated morphology does not prevent cells from being attacked by phages and also does not impair the phage infection.This work was supported by a grant (SFRH/BD/18485/2004) from the Portuguese Foundation for Science and Technology (FCT)
Antibacterial activity on opportunistic Pseudomonas aeruginosa pathogen by a novel Salmonella phage endolysin
The Gram-negative pathogen Pseudomonas aeruginosa can cause severe infections of burn wound or cystic fibrosis on patients. Bacteriophage endolysin based strategy can offer a new alternative antimicrobial therapy. Endolysins are lytic enzymes that break down the peptidoglycan of bacterial cell wall at the late phage lytic cycle, however they are inactive on their own against Gram-negative bacteria when applied exogenously as recombinant proteins due to the peptidoglycan (endolysin substrate) protective outer membrane.We propose an innovative strategy to target Gram-negative Ps. aeruginosa based on the combination of endolysin enzymes and an outer membrane permeabilizing agent - ethylenediamine tetraacetic acid (EDTA).To validate this approach, we have isolated a novel Salmonella phage endolysin (68gpLys). Cloning this gene into E. coli expression system and subsequent large scale protein expression led to a high soluble yield of 14,3 mg/L of expression culture. In order to characterized it, muralytic assays on chloroform/Tris-HCl pretreated Ps. aeruginosa strain PAO1k (to remove the outer membrane) were made to check activity levels on substrate (398.05 Units/mM). The pH range was also determined with pH 7 being the optimum for the endolysin activity. For antimicrobial test, in vitro assays showed that incubation of 106 Ps. aeruginosa cells/mL with 0.5 mM EDTA and 5000 nM of 68gpLys, led to a strain inactivation of 3.42 ± 0.02 logarithmic reduction units in a time-frame of 30 min.Here we prove that the synergistic effect of endolysin 68gpLys with EDTA can significantly reduce Ps. aeruginosa contamination. These results suggests, the great potential of this strategy for prevention and/or control of other Gram-negative pathogens.Current work has been also development to engineer new endolysins with incorporated cell penetrating peptides (CPP), employing sited- and random-mutagenesis molecular techniques, to further enhance outer membrane permeabilization
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