REVIEW | doi:10.20944/preprints202005.0232.v1
Subject: Life Sciences, Microbiology Keywords: phage resistance; phage therapy; vConTACT 2.0; phage resistance ecology; bacteriophages
Online: 14 May 2020 (08:09:04 CEST)
: As antibiotic resistance undermines efforts to treat bacterial infections, phage therapy is being increasingly considered as an alternative in clinical settings and agriculture. However, a major concern in using phages is that pathogens will develop resistance to the phage. Due to the constant evolutionary pressure by phages, bacteria have evolved numerous mechanisms to block infection. If we determine the most common among them, we could use this knowledge to guide phage therapeutics. Here we compile data from 88 peer-reviewed studies where phage resistance was experimentally observed and linked to a bacterial gene, then assessed these data for patterns. In total, 141 host genes were identified to block infection against one or more of 80 phages (representing five families of the Caudovirales) across 16 microbial host genera. These data suggest that bacterial phage resistance is diverse, but even well-studied systems are understudied, and there are gaping holes in our knowledge of phage resistance across lesser-studied regions of microbial and viral sequence space. Fortunately, scalable approaches are newly available that, if broadly adopted, can provide data to power ecosystem-aware models that will guide harvesting natural variation towards designing effective, broadly applicable phage therapy cocktails as an alternative to antibiotics.
ARTICLE | doi:10.20944/preprints201910.0025.v1
Subject: Life Sciences, Microbiology Keywords: Acinetobacter baumannii; multiresistant; mutant lytic phage; phage therapy; antibiotic-phage synergy.
Online: 2 October 2019 (08:42:31 CEST)
The global health emergency caused by multi-drug resistant bacteria has led to the search for and development of new antimicrobial agents. Phage therapy is an abandoned antimicrobial therapy that has been resumed in recent years. In this study, we mutated a lysogenic phage from Acinetobacter baumannii into a lytic phage (Ab105-2phiΔCI) showing antimicrobial activity against A.baumannii clinical strains (such as Ab177_GEIH-2000 which showed MICs to meropenem and imipenem of 32 µg/ml and 16 µg/ml, respectively as well as belonging to GEIH-REIPI Spanish Multicenter A. baumannii Study II 2000/2010, Umbrella Genbank Bioproject PRJNA422585). We then enhanced the time kill curves (in vitro) and in Galleria mellonella survival assays (in vivo) antimicrobial activity of the new lytic phage by combining it with carbapenem antibiotics (meropenem and imipenem). We observed in vitro, an antimicrobial synergistic effect (from 4 log to 7 log CFU/ml) with meropenem plus lytic phage in all combinations analysed (0.1, 1 and 10 MOI of Ab105-2phiΔCI mutant as well as 1/4 and 1/8 MIC of meropenem). Moreover, we had a decrease in bacterial growth of 8 log CFU/ml for the combination of imipenem at 1/4 MIC plus lytic phage (Ab105-2phiΔCI mutant) and of 4 log CFU/ml for the combination of imipenem at 1/8 MIC plus lytic phage (Ab105-2phiΔCI mutant) in both MOI 1 and 10. These results were confirmed in in vivo (G. mellonella) obtaining a higher effectiveness in the combination of imipenem and Ab105-2phiΔCI mutant (P<0.05 by Log Rank-Matel Cox test). This approach could help to reduce the emergence of phage resistant bacteria and restore sensitivity to the antibiotics when used to combat multiresistant strains of Acinetobacter baumannii.
TECHNICAL NOTE | doi:10.20944/preprints202102.0410.v2
Subject: Life Sciences, Biochemistry Keywords: Phage Purification; phage isolation; phage characterization; jumbo bacteriophages; top agar; double agar overlay; high throughput sequencing
Online: 19 February 2021 (09:53:32 CET)
In the post antimicrobial era, increasing attention is paid towards using bacteriophage (phage in short) therapy to control antibiotic-resistant bacteria. The first step in phage therapy applications is isolating highly efficient lytic phages or phage cocktails from various sources. When a double-layer- agar with around 0.7% agar in top agar is employed, it results in a low number of phage isolation with a poor resolution, and in many cases, you miss the phage. To address this problem, a low concentration of agar in top agar is examined for better phage isolation. Here, our results proved the efficiency of isolating phage upon formulating a double-layer agar with 0.3% agar in top agar. A sewage sample was collected then phages were isolated, purified, and spotted on a top layer agar with 0.3% agar. The results showed the possibility of isolating a higher number of phages on 0.3% top agar than 0.7%. The finding advocates using 0.3% top agar for the double-layer agar, as it will provide fast, better, and easy phage screening and isolation.
ARTICLE | doi:10.20944/preprints202104.0045.v1
Subject: Life Sciences, Biochemistry Keywords: S. mutans; Phage therapy; Dental caries; S. mutans phage; Bacteriophage; Biofilm.
Online: 2 April 2021 (11:15:54 CEST)
Streptococcus mutans is a key bacterium in dental caries- one of the most prevalent chronic infectious diseases. Conventional treatment both fails to specifically target the pathogenic bacteria and at-tempts to eradicate commensal bacteria as well. Thus, caries remains one of most common and challenging diseases. The use of bacterial viruses as anti-bacterial agents, is gaining interest worldwide. Hardly any phages were described against S. mutans. The objective of this study was to isolate anti-S. mutans phages and to characterize their antimi-crobial properties. Human saliva samples were filtered and screened for potential phages. Standard double-layered agar method was used for isolation. Whole genome sequence analysis and morphology visualization by TEM, were used for anti-S. mutans phage identification. Antibacterial properties were evaluated using clinical strains and ATCC strains of S. mutans in various states. Antibacterial effect was also tested on human cariogenic dentin. One phage against S. mutans was isolated and termed SMHBZ8. This phage showed effective lytic activity in vitro against both planktonic and biofilm S. mutans cultures. Moreover, the phage showed antibacterial effect when used on cariogenic dentin. The isolation and characterization of SMHBZ8 may be the first step in developing a potential phage therapy for dental caries.
ARTICLE | doi:10.20944/preprints201809.0340.v1
Subject: Life Sciences, Virology Keywords: bacteriophage; phage-host interactions; phage receptors; high-throughput screen; tail fibers
Online: 18 September 2018 (10:00:35 CEST)
Factors affecting the host-virus interaction must be understood for the effective application of bacteriophages to combat bacterial pathogens. Two novel E. coli phages, the T1-like siphophage LL5 and the rV5-like myophage LL12, were subjected to forward genetic screens against the Keio collection, a library of single non-essential gene deletions in E.coli str. BW25113. These genome-wide screens and subsequent experiments identified eight genes required for efficient propagation of phage LL5 and six genes required for propagation of LL12. The majority of the genes identified were involved in production of the phage receptors. E. coli mutants deficient in heptose II and the phosphoryl substituent of heptose I of the inner core lipopolysaccharide (LPS) were unable to propagate phage LL5, as were mutants deficient in the outer membrane protein TolC. Mutants lacking glucose I of the LPS outer core failed to propagate LL12. Two cytoplasmic chaperones, PpiB and SecB, were found to be required for efficient propagation of phage LL5 but not LL12. This approach may be useful for identifying phage receptors and required host factors in other phages, which would provide valuable information for their potential use as therapeutics and for phage engineering.
ARTICLE | doi:10.20944/preprints202108.0397.v1
Online: 19 August 2021 (10:30:53 CEST)
Background: The dairy industry heavily relies on fermentation processes driven in high proportion by Lactococcus lactis. The fermentation process can be perturbed or even stopped by bacteriophage activity leading to complete loss of fermentation batch or decreased quality product. Monitoring of the phage diversity and dynamics in the process allows to implement protective measures (e.g. starter rotation) in order to maintain unperturbed production.; Methods: Universal primers were used to amplify sequences of the 936, c2, and P335 Lactococcus phage types. The amplicons were sequences with Sanger method and obtained degenerate sequences were analyzed using simple bioinformatic pipeline in R environment.; Results: The most prevalent phage type is 936, followed by P335, whereas c2 type is less frequent.; Conclusions: Curd cheeses prepared on non-pasteurized milk based on native milk microbiota had higher diversity of phages distinct of these found in dairy plants. Sanger sequencing of heterogenous amplicons generated on metagenome DNA can be used to asses low-complexity microbiota diversity.
REVIEW | doi:10.20944/preprints202010.0607.v1
Subject: Life Sciences, Biochemistry Keywords: immunohistochemistry; phage display; monoclonal antibody
Online: 29 October 2020 (10:25:14 CET)
Immunohistochemistry is a widely used technique for research and diagnostic purposes that relies on the recognition by antibodies of antigens expressed in tissues. However, tissue processing and particularly formalin fixation affect the conformation of these antigens through the formation of methylene bridges. Although antigen retrieval techniques can partially restore antigen immunoreactivity, it is difficult to identify antibodies that can recognize their target especially in formalin-fixed paraffin-embedded tissues. Most of the antibodies currently used in immunohistochemistry have been obtained by animal immunization; however, in vitro display techniques represent alternative strategies that have not been fully explored yet. This review provides an overview of phage display-based antibody selections using naïve antibody libraries on various supports (fixed cells, dissociated tissues, tissue fragments, and tissue sections) that have led to the identification of antibodies suitable for immunohistochemistry.
REVIEW | doi:10.20944/preprints202209.0037.v1
Subject: Biology, Animal Sciences & Zoology Keywords: Multidrug-resistant Acinetobacter baumannii; Phage therapy
Online: 2 September 2022 (09:51:24 CEST)
Acinetobacter baumannii is a multidrug-resistant and invasive pathogen associated with the etiopathology of both an increasing number of nosocomial infections and of relevance to poultry production systems. Multidrug-resistant Acinetobacter baumannii has been reported in connection to severe challenges to clinical treatment, mostly due to an increase rate of resistance to carbapenems. Amid the possible strategies aiming to reduce the insurgence of antimicrobial resistance, phage therapy has gained particular importance for the treatment of bacterial infections. This review summarises the different phage-therapy approaches currently in use for multiple-drug resistant Acinetobacter baumannii, including single phage therapy, phage cocktails, phage -antibiotic combination therapy, phage coding Acinetobacter baumannii and the novel phage enzyme treatment. Although phage therapy represents a potential treatment solution for multidrug-resistant Acinetobacter baumannii, further research is needed to unravel some unanswered questions especially in regard to its in vivo applications, before possible routine clinical use.
ARTICLE | doi:10.20944/preprints202111.0560.v1
Subject: Life Sciences, Microbiology Keywords: Renaturation; Denaturation; Phage; Fusion protein; Podoviruses
Online: 30 November 2021 (11:35:53 CET)
In antimicrobial-peptide/protein engineering, understanding the peptide/protein’s adaptability to harsh environmental conditions such as urea, proteases, fluctuating temperatures, high salts provide enormous insight into the pharmacokinetics and pharmacodynamics of the engineered peptide/protein and its ability to survive the harsh internal environment of the human body such as the gut or the harsh external environment to which they are applied. A previous work in our laboratory demonstrated that our cloned Eɛ34 TSP showed potent antimicrobial activity against Salmonella newington, and more so, could prevent biofilm formation on decellularized tissue. In this work, the effects of urea-acid on the Eɛ34 stability is studied, and the results demonstrates that at lower pHs of 3 and 4 with urea the protein was denatured into monomeric species. However, the protein withstood urea denaturation above pH of 5 and thus remained as trimeric protein. The mechanism of denaturation of Eɛ34 TSP seems to show that urea denatures proteins by depleting hydrophobic core of the protein by directly binding to the amide units via hydrogen bonds. The results of our in-silico investigation determined that urea binds with Eɛ34 TSP with relative free energies range of -3.4 to -2.9 kcal/mol at the putative globular head binding domain of the protein. The urea molecules interacts with with the protein’s predicted hydrophobic core, thus, disrupting and exposing the shielded hydrophobic moieties of Eɛ34 TSP to the solvent. We further showed that after the unfolding of Eɛ34 TSP via urea-acid, renaturation of the protein to its native conformation was possible within few hours. This unique characteristic of refolding of Eɛ34 TSP which is similar to that of the P22 phage tailspike protein is of special interest to protein scientists and can also be exploited in antimicrobial-protein engineering.
ARTICLE | doi:10.20944/preprints202110.0308.v1
Subject: Life Sciences, Microbiology Keywords: Salmonella newington; ɛ34 phage; Lipopolysaccharides; Antibacterial
Online: 21 October 2021 (12:47:13 CEST)
Salmonella can cause acute and chronic infections in humans. Salmonella species are known to cause food poisoning and other diseases in developing countries. Their role in the pathogenesis of these diseases has received increased international attention. Despite numerous advances in sanitation, they still can infect humans and cause outbreaks in developed countries. For example, Salmonella causes about 1.2 million illnesses in the US each year with over 450 deaths. Additionally, Salmonella outbreaks cause significant losses to chicken producers globally. The Salmonella species is also prone to acquiring resistance to various classes of antibiotics. Hence, the need for a paradigm shift from antibiotics to bacteriophages to manage, control and treat bacterial infections. The ɛ34 phage belongs to Podoviruses and categorized into the P22-like phages. The P22-like phages include ɛ34, ES18, P22, ST104, and ST64T. In this work, we investigated the antibacterial property of ɛ34 phage tailspike protein against Salmonella newington (S. newington). We demonstrate here that, the phage’s tailspike protein enzymatic property as a LPS hydrolase synergizes with Vero Cell culture supernatant in killing S. newington. Using decellularized cartilage scaffold as an ex vivo tissue model, the ɛ34 TSP protected the scaffold from S. newington biofilm formation. Computational analysis of the ɛ34 TSP interaction with membrane proteins of S. newington demonstrated a higher probability (0.7318) of binding to ompA of S. newington, and when docked to ompA extracellular component, it produced a high free energy of -11.3kcal/mol. We also demonstrate the resistance/sensitivity of the tailspike to the digestive enzyme trypsin. The data obtained in this work indicates that the trypsin resistant tailspike protein of Ɛ34 phage can be formulated as a novel antibacterial agent against S. newington.
ARTICLE | doi:10.20944/preprints202104.0411.v1
Subject: Medicine & Pharmacology, Allergology Keywords: pseudomonas aeruginosa; nosocomial infections; decontamination; phage
Online: 15 April 2021 (12:23:49 CEST)
Nosocomial infections can be transmitted by contaminated hospital surfaces with resistant pathogens. conventional sanitations are not efficiently contributing to removing resistant pathogens. Bacteriophages suggest as decontaminating agents, safe, their selective ability to kill specific bacteria. This work aimed to assess the efficiency of a phage in removing Pseudomonas aeruginosa from different hard surfaces. The decontamination ability of phages w was tested in vitro against Pseudomonas aeruginosa strain. Cystoviridae Phages with titer (2 × 1012 PFU/mL) can efficiently reduce viable bacterial cells on contaminated surfaces. The treated surfaces with alcohol 70% and phage showed an evident drop of bacterial cell number from 1 h to 24 h. These results suggest that bacteriophages are biocontrol agents removing nosocomial infection pathogens transmitted by contaminated surfaces in the hospital environment.
ARTICLE | doi:10.20944/preprints201906.0125.v1
Online: 13 June 2019 (13:33:30 CEST)
The human gut microbiome (GM) plays an important role in human health and diseases. However, while substantial progress has been made in understanding the role of bacterial inhabitants of the gut, much less is known regarding the viral component of the GM. Bacteriophages (phages) are viruses attacking specific host bacteria and likely play important roles in shaping the GM. Although metagenomic approaches have led to the discoveries of many new viruses, they largely remain uncultured as their hosts have not been identified, which hampers our understanding of their biological roles. Existing protocols for isolation of viromes generally require relatively high input volumes and are generally more focused on extracting nucleic acids of good quality and purity for down-stream analysis and less on purification of still infective viruses. Here we report the development of an efficient protocol requiring low sample input yielding purified viromes containing still infective phages which also are of sufficient purity for genome sequencing. We validated the method through spiking of known phages followed by plaque assays, qPCR and metagenomic sequencing. The protocol should facilitate the culturing of novel viruses from the gut as well as large scale studies on gut viromes.
ARTICLE | doi:10.20944/preprints202102.0357.v1
Subject: Mathematics & Computer Science, Algebra & Number Theory Keywords: Cholera; Bacteriophage; phage; Vibrio Cholerae; Sensitivity analysis; epidermic
Online: 17 February 2021 (09:44:31 CET)
Cholera is an acute enteric infectious disease caused by the Gram-negative bacterium Vibrio Cholerae. Despite a huge body of research, the precise nature of its transmission dynamics has yet to be fully elucidated. Mathematical models can be useful to better understand how an infectious agent can spread and be properly controlled. We develop a compartmental model describing a Human population, a bacterial population as well as a phage population. We show that there might be eight equilibrium points; one of which is a disease free equilibrium point. We carry out numerical simulations and sensitivity analyses and we show that the presence of phage can reduce the number of infectious individuals. Moreover, we discuss the main implications in terms of public health management and control strategies.
REVIEW | doi:10.20944/preprints201906.0094.v1
Subject: Life Sciences, Microbiology Keywords: Bacteriophages, Septic wound infection, MDR, Biofilms, Phage Cocktails
Online: 11 June 2019 (10:08:07 CEST)
Lytic bacteriophages have the efficacy to act and eradicate pathogenic bacteria as an attractive tool in the near future. Bacteriophages specifically kill multidrug-resistant bacteria even which have the capacity to form biofilms. The present review mainly focused on the efficacy of bacteriophages and cocktails as therapeutic agents against predominate MDR-bacteria and their biofilms which are isolated from septic wound infections. The body of evidence includes data from studies investigating bacteriophages from sewage samples as novel antibacterial and antibiofilm agents against pathogenic bacteria. The goal of this review is to present an overview on predominant bacteria from septic wound infection, the biofilm-forming capacity of bacteria, lytic effect of bacteriophages and phage cocktails with an emphasis on the application of bacteriophages against septic wound causing bacteria.
ARTICLE | doi:10.20944/preprints201703.0232.v1
Subject: Life Sciences, Virology Keywords: Filamentous phage; M13; gp1; Zonula occludens toxin (Zot); phage assembly; assembly 27 complex; ATPase; membrane protein; molecular hinge; secretion; Walker motifs
Online: 31 March 2017 (11:13:40 CEST)
In contrast to lytic phages, filamentous phages are assembled in the inner membrane and secreted across the bacterial envelope without killing the host. For assembly and extrusion of the phage across the host cell wall, filamentous phages code for membrane-embedded morphogenesis proteins. In the outer membrane of E. coli, the protein gp4 forms a pore-like complex, while gp1 and gp11 form a complex in the inner membrane of the host. By comparing sequences with other filamentous phages, we identified putative Walker A and B motifs in gp1 with a conserved lysine in the Walker A motif (K14), and a glutamic and aspartic acid in the Walker B motif (D88, E89). In this work we demonstrate that both, Walker A and Walker B, are essential for phage production. The crucial role of these key residues suggest that gp1 is likely to be a molecular motor driving phage assembly. We further identified essential residues for the function of the assembly complex. Mutations in three out of six cysteine residues abolish phage production. Similarly, two out of six conserved glycine residues are crucial for gp1 function. We hypothesise that the residues represent molecular hinges allowing domain movement for nucleotide binding and phage assembly.
ARTICLE | doi:10.20944/preprints202205.0319.v1
Subject: Life Sciences, Virology Keywords: phage display; epitope mapping; COVID-19; genomic library; NGS
Online: 24 May 2022 (04:20:15 CEST)
The development of antibody therapies against SARS-CoV-2 remains a challenging task during the ongoing COVID-19 pandemic. All approved therapeutic antibodies are directed against the receptor binding domain (RBD) of Spike and lost neutralization efficacy against continuously emerging SARS-CoV-2 variants, which especially mutate in the RBD region. Previously, phage display has been used to identify epitopes of antibody responses against several diseases. Such epitopes have been applied to design vaccines or neutralizing antibodies. Here, we constructed an ORFeome phage display library for the SARS-CoV-2 genome. Open reading frames (ORFs) representing the SARS-CoV-2 genome were displayed on the surface of phage particles in order to identify enriched immunogenic epitopes from COVID-19 patients. Library quality was assessed by both, NGS and epitope mapping of a monoclonal antibody with known binding site. The most prominent epitope captured represented parts of Spike´s fusion peptide (FP). It is associated with the cell entry mechanism of SARS-CoV-2 into the host cell and the serine protease TMPRSS2 cleaves Spike within this sequence. Blocking of this mechanism could be a potential target for non-RBD binding therapeutic anti-SARS-CoV-2 antibodies. As mutations within the FP amino acid sequence were rather rare among SARS-CoV-2 variants so far, this may be an advantage in the fight against future virus variants.
COMMUNICATION | doi:10.20944/preprints202201.0345.v1
Subject: Biology, Other Keywords: Africa Phage Forum; Phages; Research; Collaboration; Network; Capacity development
Online: 24 January 2022 (11:14:10 CET)
The problem of antimicrobial resistance has created a new need for alternative/ complementary treatments. To this end, bacteriophages offer an exciting prospect, as they can infect and kill specific bacteria without harming the host. This survey aimed to evaluate the state of applied phage research in Africa, among the members of the Africa phage Forum (APF). This was a cross-sectional survey whereby a google form was created for the members of the Africa Phage forum to fill so as to access the stage of phage research in Africa. Data was collected between June and July 2021 using a structured questionnaire form. A total of 65 out of a total of 101 forum members completed the questionnaire. The survey indicated that a majority 68% of phage researchers in Africa were at the training stages of their career. Some available participants were limited (8%). Most of the members identified funding, lack of skill set, near absence of adequate laboratory infrastructure as major hurdles for phage research. Despite these challenges, 73.3% of APF members work with the ESKAPE group with the majority of its members carrying out research in Phage in Biocontrol (80%), whereas others perform research related to human phage therapy (60%). However, it appeared this research has not yet reached the stage of commercialization. Overall, Phage research is in its infancy in Africa. Key challenges included poor laboratory infrastructure, lack of capacity building in the phage field, and lack of local awareness on the significance of phages for policymakers and governments. APF could, therefore, play a role in creating phage awareness in Africa; mobilizing resources; enhancing networks and collaborations amongst APF members and beyond, especially with more experienced phage mentors in the Western countries, to greatly reduce the gap in knowledge and enhance phage research in Africa.
ARTICLE | doi:10.20944/preprints202112.0127.v1
Subject: Biology, Agricultural Sciences & Agronomy Keywords: plant pathogenic bacterium; phage; bacterial wilt; biological control; lyophilization
Online: 8 December 2021 (14:12:11 CET)
Ralstonia solanacearum is a pathogen that causes bacterial wilt producing severe damage in staple solanaceous crops. Traditional control has low efficacy and/or environmental impact. Recently, the bases of a new biotechnological method by lytic bacteriophages vRsoP-WF2, vRsoP-WM2 and vRsoP-WR2 with specific activity against R. solanacearum were established. However, some aspects remain unknown, such as the survival and maintenance of the lytic activity after submission to a preservation method as the lyophilization. To this end, viability and stability of lyophilized vRsoP-WF2, vRsoP-WM2 and vRsoP-WR2 and their capacity for bacterial wilt biocontrol have been determined against one pathogenic Spanish reference strain of R. solanacearum in susceptible tomato plants in different conditions and making use of various cryoprotectants. The assays carried out have shown satisfactory results with respect to the viability and stability of the bacteriophages after the lyophilization process, maintaining high titres throughout the experimental period, also with respect to the capacity of the bacteriophages for the biological control of bacterial wilt, controlling this disease in more than 50% of the plants. The results offer good prospects for the use of lyophilization as a conservation method for the lytic bacteriophages of R. solanacearum in view of their commercialization as biocontrol agents.
ARTICLE | doi:10.20944/preprints202209.0004.v1
Subject: Biology, Other Keywords: phage Rih21; MRSA; novel bacteriophage; S. aureus; bacteriophage; genome analysis
Online: 1 September 2022 (04:11:06 CEST)
From the hospital waste-water, a novel bacteriophage was isolated and characterized. According to characterization properties, this bacteriophage belongs to the Siphoviridae family, the maximum bacteriophage titer was recorded at 37°C and a pH of 7.2, had a 44,789 bp linear double-strand DNA genome, and within the genome sequence, there are 61 genes, all of which are encoded into proteins. Although this bacteriophage does not have any virulence factors or antimicrobial resistance genes and had specific lytic activity against some antimicrobial resistance S. aureus clinical isolates.
Subject: Life Sciences, Microbiology Keywords: Klebsiella pneumoniae; nonmucoid; O-antigen; outer membrane protein; phage receptor
Online: 25 July 2020 (18:55:02 CEST)
The use of bacteriophage is reemerging as a tool for combatting multi-drug resistant bacterial infections. In our previous study, we showed that colistin resistant carbapenem-resistant Klebsiella pneumoniae (ColR-CRKP) is more susceptible to killing by lytic tailed phages, including ФNJS1 specific for nonmucoid K. pneumoniae. Although we demonstrated that alteration on surface charges of ColR-CRKP promotes phage adherence and infection, the receptor for ФNJS1 was still unknown. In current study, we identified O-antigen was involved in the reversible adsorption, and outer membrane protein (OMP) FepA may be served as one of the irreversible receptors for ФNJS1. We firstly found accelerated reversible phage adsorption to ColR-CRKP cells, and that periodate treatment of bacteria inhibited the phage binding, indicating LPS may be involved in phage reversible adsorption. ФNJS1-resistant bacterial mutants screening revealed that mutants in ∆wecG(mTn5) and ∆wecA(mTn5), two genes responsible for LPS biosynthesis, affected phage adsorption capacity and phage infectivity. The loss of wzyE encoding O-antigen polymerase showed no significant difference in phage adsorption but increased phage infectivity, suggesting the long chain length of O-antigen may also be a barrier for bacteriophage infection. Among four OMP mutants including ∆fepA, ∆fhuA, ∆ompA and ∆ompC, only ∆fepA slowed phage lysis rate, suggesting FepA may be as one of irreversible receptors for ФNJS1. The results are helpful to better understand why ColR-CRKP sensitizes phage infection and to combat multi-drug resistant K. pneumoniae infections in the future.
REVIEW | doi:10.20944/preprints201807.0091.v1
Subject: Medicine & Pharmacology, Other Keywords: bacterial infection; antibiotic resistance; bacteriophage; antibiotic therapy; phage therapy; review
Online: 5 July 2018 (10:09:09 CEST)
Bacteriophages, viruses that are widespread throughout the world, are highly specific for bacteria, usually of a single species and often of a particular strain. After being discovered and isolated 100 years ago, their use, called phage therapy, was instituted in medicine two years later and quickly used around the world to treat various bacterial infections. In the West, phage therapy was overshadowed in the second half of the 20th century by antibiotic therapy, which was then thought to be the definitive solution. But because of the increase in bacterial resistance to antibiotics, the idea of using bacteriophages in medicine has been reawakened. The innumerable observations reported over the years in the literature constitute an invaluable experience. We and some of our colleagues have, in the last decade treated some patients compassionately. With the available documentation and our own experience we discuss the potential indications and limitations of phage therapy. The observation of the increasing number of therapeutic failures in the announced perspective of a post-antibiotic era, we believe, that the introduction of bacteriophages into the therapeutic arsenal seems conceivable today to two preconditions: that their production as biologic drug meets current regulatory standards and that the benefit-risk assessment was conducted in a modern setting. Phage therapy could be applied as a substitution or supplement to antibiotic therapy under multiple circumstances in different modes, precise indications and limits.
ARTICLE | doi:10.20944/preprints202206.0105.v1
Subject: Biology, Other Keywords: Smear-ripened cheese; virulent phages; rind bacteria; phage reservoirs; viral genomics
Online: 7 June 2022 (10:37:21 CEST)
Smear-ripened cheeses host complex microbial communities that play a crucial role in the ripening process. Although bacteriophages have been frequently isolated from dairy products, their diversity and ecological role in such type of cheese remain underexplored. In order to fill this gap, the main objective of this study was to isolate and characterize bacteriophages from the rind of a smear-ripened cheese. Thus, viral particles extracted from cheese rind were tested against a collection of bacterial isolates through a spot assay. In total, five virulent bacteriophages infecting Brevibacterium aurantiacum, Glutamicibacter arilaitensis, Leuconostoc falkenbergense and Psychrobacter aquimaris species were obtained. All exhibit a narrow host range, being only able to infect a few cheese-rind isolates within the same species. The complete genome of each phage was sequenced using both Nanopore and Illumina technologies, assembled and annotated. Sequence comparison with known phages revealed that four of them may represent at least new genera. The distribution of the five virulent phages into the dairy-plant environment was also investigated by PCR and three potential reservoirs were identified. This work provides new knowledge on the cheese rind viral community and an overview of the distribution of phages within a cheese factory.
ARTICLE | doi:10.20944/preprints202201.0234.v1
Subject: Life Sciences, Microbiology Keywords: AMR, Surveillance; One Health Approach; Alternative Antibiotics; Comparative Medicine; Phage Therapy
Online: 17 January 2022 (14:46:22 CET)
Antibiotics are in excessive use that has extensively increased antimicrobial resistance worldwide which has become the major public concern among the countries. To control this threat proper monitoring of the antimicrobial usage along with the increasing rate of antimicrobial resistance (AMR) is required. Further, surveillance of both the parameters is highly recommended for comparing the differences in distinct countries. Moreover, alternatives for antibiotics are also surveyed and are being researched for quick use in the near future. AMR is an issue that needs immense attention from various sectors. Thus, intervention of multisector is highly encouraged for better outcomes. One Health is one of the approaches that play a vital role in resolving this issue. In this research paper, six different European countries are discussed in terms of antimicrobial usage and AMR in the human and livestock sectors with the help of literature study and various reports published by different organizations. Data study has been conducted to collect the data for comparison study. Data sources of AMR and antimicrobial usage are analyzed and a thorough comparison of both antimicrobial use and AMR are conducted. Also, the application of One Health is studied for a balanced system. This article provides about various surveillance systems that are formed only to keep a track on the upcoming situation of AMR and the consumption of antimicrobials by the humans as well as animals. The article does not provide about all the details required to monitor the AMR issue but firmly allow the readers to get acknowledged with the broad information about the antimicrobial resistance across the six countries of Europe. The regular data collected by the different organizations play a vital role in monitoring the status of AMR and antimicrobial usage by humans and in live stocks. These annual reports have highly helped the government to decide for alternatives and have focused in many training activities to combat the AMR situation globally. AMR prevention is linked to the One Health concept. As antibiotic resistance genes persist on an interface between environment and animal and animal health, an approach is required in all three areas that stress the concept of 'One Approach to Health.'
REVIEW | doi:10.20944/preprints202105.0231.v1
Subject: Medicine & Pharmacology, Allergology Keywords: Phage therapy; clinical trials; animal models; safety and toxicity; immune activation
Online: 11 May 2021 (10:30:59 CEST)
Increasing rates of infection by antibiotic resistant bacteria have led to a resurgence of interest in bacteriophage (phage) therapy. Several phage therapy studies in animals and humans have been completed over the last two decades. We conducted a systematic review of safety and toxicity data associated with phage therapy in both animals and humans reported in English-language publications from 2008 – 2021. Overall, 69 publications met our eligibility criteria including 20 animal studies, 35 clinical case reports or case series, and 14 clinical trials. After summarizing safety and toxicity data from these publications, we discuss potential approaches to optimizing safety and toxicity monitoring with the therapeutic use of phage moving forward. In our systematic review of the literature, we found few, but no serious, adverse events associated with phage therapy. Comprehensive and standardized reporting of potential toxicities associated with phage therapy has generally been lacking in the published literature. Structured safety and tolerability endpoints are necessary when phages are administered as anti-infective therapeutics.
ARTICLE | doi:10.20944/preprints202102.0075.v1
Subject: Life Sciences, Biochemistry Keywords: bat; monoclonal antibodies; lyssaviruses; neutralization; glycoprotein; ABLV; rabies; RABV; phage display
Online: 2 February 2021 (08:27:58 CET)
Australian bat lyssavirus (ABLV) is a rhabdovirus that circulates in four species of pteropid bats (ABLVp) and the yellow-bellied sheath-tailed bat (ABLVs) in mainland Australia. In the three confirmed human cases of ABLV, rabies illness preceded fatality. As with rabies virus (RABV), post-exposure prophylaxis (PEP) for potential ABLV infections consists of wound cleansing, ad-ministration of the rabies vaccine and injection of rabies immunoglobulin (RIG) proximal to the wound. Despite the efficacy of PEP, the inaccessibility of human RIG (HRIG) in the developing world and the high immunogenicity of equine RIG (ERIG) has led to consideration of human monoclonal antibodies (hmAbs) as a passive immunization option that offers enhanced safety and specificity. Using a recombinant vesicular stomatitis virus (rVSV) expressing the glycoprotein (G) protein of ABLVs and phage display, we identified two hmAbs, A6 and F11, which completely neutralize ABLVs/ABLVp, and RABV at concentrations ranging from 0.19-3.12 µg/mL and 0.39-6.25 µg/mL respectively. A6 and F11 recognize overlapping epitopes in the lyssavirus G protein, ef-fectively neutralizing phylogroup 1 lyssaviruses, while having little effect on phylogroup 2 and non-grouped diverse lyssaviruses. These results suggest A6 and F11 could be effective therapeutic and diagnostic tools for phylogroup 1 lyssavirus infections.
ARTICLE | doi:10.20944/preprints201812.0026.v2
Subject: Life Sciences, Virology Keywords: Lactobacillus plantarum; phage; new genus; annotation; comparative genomics; phylogenetics; isolation; diversity
Online: 11 June 2019 (09:54:23 CEST)
Lactobacillus plantarum is a bacterium with promising applications to the food industry and agriculture and probiotic properties. So far, bacteriophages of this bacterium have been moderately addressed. We examined the diversity of five new L. plantarum phages via whole genome shotgun sequencing and in silico protein predictions. Moreover, we looked into their phylogeny and their potential genomic similarities to other complete phage genome records through extensive nucleotide and protein comparisons. These analyses revealed a high degree of similarity among the five phages, which extended to the vast majority of predicted virion-associated proteins. Based on these, we selected one of the phages as a representative and performed transmission electron microscopy and structural protein sequencing tests. Overall, the results suggested that the five phages belong to the family Myoviridae, they have a long genome of 137.973-141.344 bp, a G/C content of 36,3-36,6% that is quite distinct from their host’s, and, surprisingly, seven to 15 tRNAs. Only an average 41/174 of their predicted genes were assigned a function. The comparative analyses unraveled considerable genetic diversity for the five L. plantarum phages of this study. Hence, the new genus “Semelevirus” was proposed, which comprises exclusively the five phages. This novel lineage of Lactobacillus phages provides further insight into the genetic heterogeneity of phages infecting Lactobacillus sp.. The five new Lactobacillus phages have a potential value for the development of more robust starters through, for example, the selection of mutants insensitive to phage infections. The five phages could also form part of phage cocktails, which producers would apply in different stages of L. plantarum fermentations in order to create a range of organoleptic outputs.
ARTICLE | doi:10.20944/preprints202112.0049.v1
Subject: Biology, Agricultural Sciences & Agronomy Keywords: bacterial wilt; biological control; phage; microscopy; sequencing; molecular characterization; genomic characterization; depolymerase
Online: 3 December 2021 (10:36:47 CET)
Ralstonia solanacearum is the causative agent of bacterial wilt, one of the most destructive plant diseases. While chemical control has an environmental impact, biological control strategies can allow sustainable agrosystems. Three lytic bacteriophages (phages) of R. solanacearum with biocontrol capacity in environmental water and plant were isolated from river water in Europe but not fully characterized, their genomic characterization being fundamental to understand their biology. In this work, the phage genomes were sequenced and subjected to bioinformatic analysis. The morphology was also observed by electron microscopy. Phylogenetic analyses were performed with a selection of phages able to infect R. solanacearum and the closely related phytopathogenic species R. pseudosolanacearum. The results indicated that the genomes of vRsoP-WF2, vRsoP-WM2 and vRsoP-WR2 range from 40,688 to 41,158 bp with almost 59% GC-contents, 52 ORFs in vRsoP-WF2 and vRsoP-WM2, and 53 in vRsoP-WR2 but, with only 22 or 23 predicted proteins with functional homologs in databases. Among them, two lysins and one exopolysaccharide (EPS) depolymerase, this type of depolymerase being identified in R. solanacearum phages for the first time. These three European phages belong to the same novel species within the Gyeongsanvirus, Autographiviridae family (formerly Podoviridae). These genomic data will contribute to a better understanding of the abilities of these phages to damage host cells and, consequently, to an improvement in the biological control of R. solanacearum.
REVIEW | doi:10.20944/preprints202103.0338.v1
Subject: Keywords: Wolbachia; phage WO; cytoplasmic incompatibility; male killing; feminization; parthenogenesis; evolution; vector control
Online: 12 March 2021 (09:17:01 CET)
The most widespread intracellular bacteria in the animal kingdom are maternally-inherited endosymbionts of the genus Wolbachia. Their prevalence in arthropods and nematodes worldwide and a stunning arsenal of parasitic and mutualistic adaptations make these bacteria a biological archetype for basic studies of symbiosis and applied outcomes for curbing human and agricultural diseases. Here, we conduct a summative, centennial analysis of living in the Wolbachia world. We synthesize literature on Wolbachia’s host range, phylogenetic diversity, genomics, cell biology, and applications to filarial, arboviral, and agricultural diseases. We also review the mobilome of Wolbachia including phage WO and its essentiality to hallmark phenotypes in arthropods. Finally, the Wolbachia system is an exemplar for discovery-based science education using biodiversity, biotechnology, and bioinformatics lessons. As we approach a century of Wolbachia research, applications, and education, the interdisciplinary science and knowledge from this symbiosis stand as a model for consolidating and teaching the integrative rules of endosymbiotic life.
ARTICLE | doi:10.20944/preprints201907.0044.v1
Subject: Life Sciences, Virology Keywords: Bacteriophage; Lactococcus lactis; receptor-binding protein; carbohydrate binding module; phage-host interactions
Online: 2 July 2019 (11:45:19 CEST)
Abstract: With the availability of an increasing number of 3D structures of bacteriophage components, combined with powerful in silico predictive tools, it has become possible to decipher the structural assembly and functionality of phage adhesion devices. In the current study, we examined 113 members of the 936 group of lactococcal siphophages, and identified a number of Carbohydrate Binding Modules (CBMs) in the neck passage structure and major tail protein, on top of evolved Dit proteins as recently reported by us. The binding ability of such CBM-containing proteins was assessed through the construction of green fluorescent protein fusion proteins and subsequent binding assays. Two CBMs, one from the phage tail and another from the neck, demonstrated definite binding to their phage-specific host. Bioinformatic analysis of the structural proteins of 936 phages reveals that they incorporate binding modules which exhibit structural homology to those found in other lactococcal phage groups and beyond, indicating that phages utilize common structural “bricks” to enhance host binding capabilities. The omnipresence of CBMs in Siphophages supports their beneficial role in the infection process, as they can be combined in various ways to form appendages with different shapes and functionalities, ensuring their success in host detection in their respective ecological niches.
ARTICLE | doi:10.20944/preprints201806.0039.v1
Subject: Engineering, Biomedical & Chemical Engineering Keywords: Protein A; Heptapeptide; M13 Phage-Display; ELISA; Fc; purification; docking; molecular dynamics simulations
Online: 4 June 2018 (12:03:52 CEST)
Purification of biologically-derived therapeutics is a major cost contributor to the production of this rapidly growing class of pharmaceuticals. Monoclonal antibodies comprise a large percentage of these products therefore new antibody purification tools are needed. Small peptides, as opposed to traditional antibody affinity ligands such as Protein A, may have advantages in stability and production costs. Multiple heptapeptides that demonstrate Fc binding behavior that have been identified from a combinatorial peptide library using M13 Phage Display are presented herein. Seven unique peptide sequences of diverse hydrophobicity and charge were identified. All seven peptides showed strong binding to the four major human IgG isotypes, human IgM, as well as binding to canine, rat, and mouse IgG. These seven peptides were also shown to bind human IgG4 from DMEM cell culture media with 5% FCS and 5 g/L ovalbumin present. These peptides may be useful as surface ligands for antibody detection and purification purposes. Molecular docking and classical molecular dynamics (MD) simulations were conducted to elucidate the mechanisms and energetics for the binding of these peptides to the Fc region. The binding site was found to be located between the two glycan chains inside the Fc fragment. Both hydrogen bonding and hydrophobic interactions were found to be crucial for the binding interactions. Excellent agreement for the binding strength was obtained between experimental results and simulations.
REVIEW | doi:10.20944/preprints201803.0227.v1
Subject: Life Sciences, Biotechnology Keywords: Antivenom; snakebite; small molecule toxin inhibitors; oligonucleotides; antibodies; phage display; next generation antivenom; recombinant antivenom
Online: 27 March 2018 (13:41:46 CEST)
With the inclusion of snakebite envenoming on the World Health Organisation’s list of Neglected Tropical Diseases, an incentive has been established to promote research and development effort in novel snakebite antivenom therapies. Different technological approaches are being pursued by different research groups, including the use of small molecule inhibitors against enzymatic toxins, as well as peptide and oligonucleotide-based aptamers and antibody-based biotherapeutics against both enzymatic and non-enzymatic toxins. In this article, the most recent advances in these fields are presented, and the advantages, disadvantages, and feasibility of using different toxin-neutralizing molecules are reviewed. Particular focus within small molecules is directed towards the inhibitors, varespladib, batimastat, and marimastat, while in the field of antibody-based therapies, novel recombinant polyclonal plantivenom technology is discussed.
Subject: Life Sciences, Biochemistry Keywords: continuous evolution; protein design; protein engineering; phage; bacterial one-hybrid; plaque assay; mutational analysis; DNA sequencing
Online: 5 November 2019 (02:19:01 CET)
Directed evolution methods are becoming increasingly popular, as they are extremely powerful toward developing new biomolecules with altered/novel activities, e.g., proteins with new catalytic functions or substrate specificities, and nucleic acids that recognize an intended target. Especially useful are systems that have incorporated continuous evolution, where the protein to be evolved undergoes continuous mutagenesis to evolve a desired trait with little to no input from the researcher once the system is started. However, continuous evolution methods can be challenging to implement in the lab and daunting for researchers to invest time and resources. Our intent is to provide basic information and helpful suggestions that we have gained from our experience with bacterial phage-assisted continuous evolution (PACE). Specifically, we review factors to consider before adopting PACE for a given evolution scheme, different types of selection circuits that can be utilized with particular focus on the PACE-B1H selection system, what optimization of a PACE selection circuit may look like using directed evolution of ME47 as a case study, and additional techniques that may be incorporated into a PACE experiment. With this information, researchers will be better equipped to determine if PACE is a valid strategy to use to evolve their proteins and how to set up a valid selection circuit.
ARTICLE | doi:10.20944/preprints202208.0478.v1
Subject: Life Sciences, Microbiology Keywords: antibacterial proteins; encapsulating protein; high molecular-weight bacteriocins; insect patho-genic bacterium; phage tail-like protein; purification methods
Online: 29 August 2022 (09:00:10 CEST)
Brevibacillus laterosporus (Bl) is a Gram-positive and spore-forming bacterium belonging to the Brevibacillus brevis phylogenetic cluster. Globally, insect pathogenic strains of the bacterium have been isolated, characterised, and some activities patented. Two isolates, Bl 1821L and Bl 1951, exhibiting pathogenicity against the diamondback moth and mosquitoes, are under development as a biopesticide in New Zealand. However, due to the suspected activity of putative antibacterial proteins (ABPs), the endemic isolates often grow erratically. Various purification methods including size exclusion chromatography, sucrose density gradient centrifugation, polyethylene glycol precipitation, and ammonium sulphate precipitation employed in this study enabled the isolation of two putative antibacterial proteins of ~30 kD and ~48 kD from Bl 1821L and one putative antibacterial protein of ~30 kD from Bl 1951. Purification of the uninduced cultures of Bl 1821L and Bl 1951 also yielded the protein bands of ~30 kD and ~48 kD on SDS-PAGE which indicated their spontaneous induction. Disc diffusion assay was used to determine the antagonistic activities of the putative ABPs. Subsequent transmission electron microscope (TEM) examination of purified putative antibacterial protein-containing solution showed the presence of encapsulin (~30 kD) and polysheath (~48 kD) like structures. Although only the ~30 kD protein was purified from Bl 1951, both structures were seen in this strain under TEM. Furthermore, while assessing the antibacterial activity of some fractions of Bl 1951 against Bl 1821L in size exclusion chromatography method, population of Bl 1821L persister cells was noted. Overall, this work added a wealth of knowledge for the purification of the HMW proteins (bacteriocins) of the Gram-positive bacteria including Bl.
ARTICLE | doi:10.20944/preprints202008.0633.v1
Subject: Biology, Other Keywords: giant phage; phiKZ; Pseudomonas aeruginosa; nucleoid; pseudo-nucleus; analytical electron microscopy; electron tomography; fluorescent in situ hybridization; stress response
Online: 28 August 2020 (10:29:39 CEST)
Bacteria develop various defense mechanisms against stresses, including the bacteriophage infection. The giant phiKZ phage infection induced the appearance of a pseudo-nucleus inside the bacterial cytoplasm. Here, we used FISH, electron tomography and analytical electron microscopy to study the morphology of this unique nucleus-like shell and to demonstrate the distribution of phiKZ and bacterial DNA in infected P. aeruginosa cells. The maturation of the pseudo-nucleus was traced in short intervals for 40 min after infection. This study was accompanied by the identification of phiKZ and bacterial DNA by real-time RCR. We demonstrated that phage DNA that isolated from the cytoplasm during all infection stages were compacted within the pseudo-nucleus in a specific structure. Bacterial DNA was diminished in the course of infection, but did not completely degrade until at least 40 min after phage application. The content of the total phage DNA, on the other hand, increased. EDX analysis confirmed these results and revealed that, during the infection, Sulfur content in the bacterial cytoplasm increased, which suggests the increase of DNA-binding Met-reach proteins synthesis, which could protect bacterial DNA from stress.
ARTICLE | doi:10.20944/preprints202111.0558.v1
Subject: Life Sciences, Microbiology Keywords: Yersinia pestis; vaccine; guinea pigs; bubonic plague; inactivated vaccine; phage; bacterial ghost; protection; protein-E-mediated lysis, holin-endolysin system
Online: 30 November 2021 (11:08:18 CET)
To develop a modern plague vaccine, we used hypo-endotoxic Yersinia pestis bacterial ghosts (BGs) with combinations of genes encoding the bacteriophage ɸX174 lysis-mediating protein E and/or holin-endolysin systems from λ or L-413C phages. Expression of the protein E gene resulted in the BGs that retained the shape of the original bacterium. Co-expression of this gene with genes coding for holin-endolysin system of the phage L-413C caused formation of structures resembling collapsed sacs. Such structures, which have lost their rigidity, were also formed as a result of the expression of only the L-413C holin-endolysin genes. Similar holin-endolysin system from phage λ containing mutated holin gene S and intact genes R-Rz coding for the endolysins caused generation of mixtures of BGs that had (i) practically preserved and (ii) completely lost their original rigidity. The addition of protein E to the work of this system shifted the equilibrium in the mixture towards the collapsed sacs. The collapse of the structure of BGs can be explained by endolysis of peptidoglycan sacculi. Immunizations of laboratory animals with the variants of BGs followed by infection with a wild-type Y. pestis strain showed that bacterial envelopes protected only cavies. BGs with peptidoglycan maximally hydrolyzed had a greater protectivity compared to BGs with preserved peptidoglycan skeleton.