In Vitro Evaluation of a Phage Cocktail Controlling Infections with Escherichia coli

Worldwide, poultry industry suffers from infections caused by avian pathogenic Escherichia coli. Therapeutic failure due to resistant bacteria is of increasing concern and poses a threat to human and animal health. This causes a high demand to find alternatives to fight bacterial infections in animal farming. Bacteriophages are being especially considered for the control of multi-drug resistant bacteria due to their high specificity and lack of serious side effects. Therefore, the study aimed on characterizing phages and composing a phage cocktail suitable for the prevention of infections with E. coli. Six phages were isolated or selected from our collections and characterized individually and in combination with regard to host range, stability, reproduction, and efficacy in vitro. The cocktail consisting of six phages was able to inhibit formation of biofilms by some E. coli strains but not by all. Phage-resistant variants arose when bacterial cells were challenged with a single phage but not when challenged by a combination of four or six phages. Resistant variants arising showed changes in carbon metabolism and/or motility. Genomic comparison of wild type and phage-resistant mutant E28.G28R3 revealed a deletion of several genes putatively involved in phage adsorption and infection.

[1]  R. Kaas,et al.  ResFinder 4.0 for predictions of phenotypes from genotypes , 2020, The Journal of antimicrobial chemotherapy.

[2]  G. Klein,et al.  Impact of Bacteriophage-Supplemented Drinking Water on the E. coli Population in the Chicken Gut , 2020, Pathogens.

[3]  I. Sampers,et al.  Occurrence and characterisation of biofilms in drinking water systems of broiler houses , 2019, BMC Microbiology.

[4]  John M. Fairbrother,et al.  Colibacillosis , 2019, Diseases of Swine.

[5]  Yue Xu,et al.  Identification of novel bacteriophage vB_EcoP-EG1 with lytic activity against planktonic and biofilm forms of uropathogenic Escherichia coli , 2018, Applied Microbiology and Biotechnology.

[6]  T. Miyamoto,et al.  Application of bacteriophages in simultaneously controlling Escherichia coli O157:H7 and extended-spectrum beta-lactamase producing Escherichia coli , 2018, Applied Microbiology and Biotechnology.

[7]  D. Nielsen,et al.  A bacteriophage cocktail targeting Escherichia coli reduces E. coli in simulated gut conditions, while preserving a non-targeted representative commensal normal microbiota , 2018, Gut microbes.

[8]  Stan J. J. Brouns,et al.  Targeting mechanisms of tailed bacteriophages , 2018, Nature Reviews Microbiology.

[9]  P. Schellenberger,et al.  University of Birmingham Development of a high-throughput ex-vivo burn wound model using porcine skin, and its application to evaluate new approaches to control wound infection , 2018 .

[10]  A. Coffey,et al.  Selection of Potential Therapeutic Bacteriophages that Lyse a CTX-M-15 Extended Spectrum β-Lactamase Producing Salmonella enterica Serovar Typhi Strain from the Democratic Republic of the Congo , 2018, Viruses.

[11]  J. Zanuncio,et al.  Bacteriophage Isolated from Sewage Eliminates and Prevents the Establishment of Escherichia Coli Biofilm , 2018, Advanced pharmaceutical bulletin.

[12]  M. Rohde,et al.  Identification and Characterization of T5-Like Bacteriophages Representing Two Novel Subgroups from Food Products , 2018, Front. Microbiol..

[13]  M. Skapas,et al.  Molecular analysis of the low-temperature Escherichia coli phage vB_EcoS_NBD2 , 2017, Archives of Virology.

[14]  W. Fokkens,et al.  Activity of Bacteriophages in Removing Biofilms of Pseudomonas aeruginosa Isolates from Chronic Rhinosinusitis Patients , 2017, Front. Cell. Infect. Microbiol..

[15]  Tian Ding,et al.  Evaluation of lytic bacteriophages for control of multidrug-resistant Salmonella Typhimurium , 2017, Annals of Clinical Microbiology and Antimicrobials.

[16]  A. Nowaczek,et al.  Bacteriophage therapy to combat bacterial infections in poultry , 2017, Virology Journal.

[17]  S. Parveen,et al.  Reduction of Salmonella in ground chicken using a bacteriophage , 2017, Poultry science.

[18]  A. González-Robles,et al.  Isolation and Characterization of phiLLS, a Novel Phage with Potential Biocontrol Agent against Multidrug-Resistant Escherichia coli , 2017, Front. Microbiol..

[19]  C. Khursigara,et al.  A Genotypic Analysis of Five P. aeruginosa Strains after Biofilm Infection by Phages Targeting Different Cell Surface Receptors , 2017, Front. Microbiol..

[20]  L. Goodridge,et al.  Phage Therapy Approaches to Reducing Pathogen Persistence and Transmission in Animal Production Environments: Opportunities and Challenges. , 2017, Microbiology spectrum.

[21]  S. Abedon,et al.  Editorial: Phage Therapy: Past, Present and Future , 2017, Front. Microbiol..

[22]  T. Riedel,et al.  Genome Sequence of Escherichia coli E28, a Multidrug-Resistant Strain Isolated from a Chicken Carcass, and Its Spontaneously Inducible Prophage , 2017, Genome Announcements.

[23]  G. Klein,et al.  The use of bacteriophages as One-Health approach to reduce multidrug-resistant bacteria , 2017 .

[24]  U. Roesler,et al.  The Efficacy of Isolated Bacteriophages from Pig Farms against ESBL/AmpC-Producing Escherichia coli from Pig and Turkey Farms , 2017, Front. Microbiol..

[25]  B. Dreiseikelmann,et al.  Characterization and genome comparisons of three Achromobacter phages of the family Siphoviridae , 2017, Archives of Virology.

[26]  A. R. Costa,et al.  Bacteriophages and their derivatives for the treatment and control of food-producing animal infections , 2017, Critical reviews in microbiology.

[27]  Hakdong Shin,et al.  Characterization and Genomic Study of the Novel Bacteriophage HY01 Infecting Both Escherichia coli O157:H7 and Shigella flexneri: Potential as a Biocontrol Agent in Food , 2016, PloS one.

[28]  G. Węgrzyn,et al.  Biodiversity of bacteriophages: morphological and biological properties of a large group of phages isolated from urban sewage , 2016, Scientific Reports.

[29]  J. Dennehy,et al.  Prophylactic Bacteriophage Administration More Effective than Post-infection Administration in Reducing Salmonella enterica serovar Enteritidis Shedding in Quail , 2016, Front. Microbiol..

[30]  S. Monecke,et al.  Surveillance of Extended-Spectrum Beta-Lactamase-Producing Escherichia coli in Dairy Cattle Farms in the Nile Delta, Egypt , 2016, Front. Microbiol..

[31]  T. Yonesaki,et al.  Characterization of the interactions between Escherichia coli receptors, LPS and OmpC, and bacteriophage T4 long tail fibers , 2016, MicrobiologyOpen.

[32]  G. Salmond,et al.  CRISPR-Cas gene-editing reveals RsmA and RsmC act through FlhDC to repress the SdhE flavinylation factor and control motility and prodigiosin production in Serratia , 2016, Microbiology.

[33]  M. Wagner,et al.  Tracking Foodborne Pathogenic Bacteria in Raw and Ready-to-Eat Food Illegally Sold at the Eastern EU Border. , 2016, Foodborne pathogens and disease.

[34]  P. Katharios,et al.  Isolation and Characterization of Two Lytic Bacteriophages, φSt2 and φGrn1; Phage Therapy Application for Biological Control of Vibrio alginolyticus in Aquaculture Live Feeds , 2016, PloS one.

[35]  A. Metcalfe,et al.  Bacteriophage Can Prevent Encrustation and Blockage of Urinary Catheters by Proteus mirabilis , 2015, Antimicrobial Agents and Chemotherapy.

[36]  L. Hansen,et al.  A novel bacteriophage cocktail reduces and disperses P seudomonas aeruginosa biofilms under static and flow conditions , 2015, Microbial biotechnology.

[37]  H. Berg,et al.  Mutations That Stimulate flhDC Expression in Escherichia coli K-12 , 2015, Journal of bacteriology.

[38]  M. Wiedmann,et al.  Selection and Characterization of Phage-Resistant Mutant Strains of Listeria monocytogenes Reveal Host Genes Linked to Phage Adsorption , 2015, Applied and Environmental Microbiology.

[39]  F. Breidt,et al.  Escherichia coli O157:H7 bacteriophage Φ241 isolated from an industrial cucumber fermentation at high acidity and salinity , 2015, Front. Microbiol..

[40]  A. Buckling,et al.  Quality and Safety Requirements for Sustainable Phage Therapy Products , 2015, Pharmaceutical Research.

[41]  S. Chhibber,et al.  Essential role of calcium in the infection process of broad‐spectrum methicillin‐resistant Staphylococcus aureus bacteriophage , 2014, Journal of basic microbiology.

[42]  H. De Greve,et al.  A cocktail of in vitro efficient phages is not a guarantee for in vivo therapeutic results against avian colibacillosis. , 2014, Veterinary microbiology.

[43]  Ole Lund,et al.  Real-Time Whole-Genome Sequencing for Routine Typing, Surveillance, and Outbreak Detection of Verotoxigenic Escherichia coli , 2014, Journal of Clinical Microbiology.

[44]  Jan P. Meier-Kolthoff,et al.  First genome sequences of Achromobacter phages reveal new members of the N4 family , 2014, Virology Journal.

[45]  B. Dreiseikelmann,et al.  Isolation and Characterization of Numerous Novel Phages Targeting Diverse Strains of the Ubiquitous and Opportunistic Pathogen Achromobacter xylosoxidans , 2014, PloS one.

[46]  P. Speck,et al.  Bacteriophage Reduces Biofilm of Staphylococcus Aureus Ex Vivo Isolates from Chronic Rhinosinusitis Patients , 2014, American journal of rhinology & allergy.

[47]  R. Lavigne,et al.  Predicting In Vivo Efficacy of Therapeutic Bacteriophages Used To Treat Pulmonary Infections , 2013, Antimicrobial Agents and Chemotherapy.

[48]  S. Abedon,et al.  Phage cocktails and the future of phage therapy. , 2013, Future microbiology.

[49]  Hong Sun,et al.  Effects of dietary inclusion of fermented cottonseed meal on growth, cecal microbial population, small intestinal morphology, and digestive enzyme activity of broilers , 2013, Tropical Animal Health and Production.

[50]  B. Koskella,et al.  Understanding Bacteriophage Specificity in Natural Microbial Communities , 2013, Viruses.

[51]  Hans-Peter Klenk,et al.  Visualization and Curve-Parameter Estimation Strategies for Efficient Exploration of Phenotype Microarray Kinetics , 2012, PloS one.

[52]  Helga Thorvaldsdóttir,et al.  Integrative Genomics Viewer (IGV): high-performance genomics data visualization and exploration , 2012, Briefings Bioinform..

[53]  K. Stanford,et al.  Genomic, Proteomic and Physiological Characterization of a T5-like Bacteriophage for Control of Shiga Toxin-Producing Escherichia coli O157:H7 , 2012, PloS one.

[54]  E. Bingen,et al.  Efficacy of Bacteriophage Therapy in Experimental Sepsis and Meningitis Caused by a Clone O25b:H4-ST131 Escherichia coli Strain Producing CTX-M-15 , 2012, Antimicrobial Agents and Chemotherapy.

[55]  A. Manges,et al.  Bacteriophages with the Ability to Degrade Uropathogenic Escherichia Coli Biofilms , 2012, Viruses.

[56]  A. Buckling,et al.  Introducing yesterday's phage therapy in today's medicine , 2012 .

[57]  W. Han,et al.  A Method for Generation Phage Cocktail with Great Therapeutic Potential , 2012, PloS one.

[58]  É. Oswald,et al.  Diagnostic Strategy for Identifying Avian Pathogenic Escherichia coli Based on Four Patterns of Virulence Genes , 2012, Journal of Clinical Microbiology.

[59]  I. Connerton,et al.  Application of a bacteriophage cocktail to reduce Salmonella Typhimurium U288 contamination on pig skin. , 2011, International journal of food microbiology.

[60]  D. Kang,et al.  Characterization and Comparative Genomic Analysis of a Novel Bacteriophage, SFP10, Simultaneously Inhibiting both Salmonella enterica and Escherichia coli O157:H7 , 2011, Applied and Environmental Microbiology.

[61]  H. Bao,et al.  Isolation and characterization of bacteriophages of Salmonella enterica serovar Pullorum. , 2011, Poultry science.

[62]  T. Wood,et al.  IS5 inserts upstream of the master motility operon flhDC in a quasi-Lamarckian way , 2011, The ISME Journal.

[63]  Romain Gallet,et al.  Effects of bacteriophage traits on plaque formation , 2011, BMC Microbiology.

[64]  H. Krisch,et al.  The gp38 Adhesins of the T4 Superfamily: A Complex Modular Determinant of the Phage’s Host Specificity , 2011, Genome biology and evolution.

[65]  P. Savelkoul,et al.  Extended-Spectrum β-Lactamase Genes of Escherichia coli in Chicken Meat and Humans, the Netherlands , 2011, Emerging infectious diseases.

[66]  S. Abedon,et al.  Phage treatment of human infections , 2011, Bacteriophage.

[67]  Mitchell J. Sullivan,et al.  Easyfig: a genome comparison visualizer , 2011, Bioinform..

[68]  S. Ryu,et al.  Characterization of a T5-Like Coliphage, SPC35, and Differential Development of Resistance to SPC35 in Salmonella enterica Serovar Typhimurium and Escherichia coli , 2011, Applied and Environmental Microbiology.

[69]  J. Azeredo,et al.  In vivo efficiency evaluation of a phage cocktail in controlling severe colibacillosis in confined conditions and experimental poultry houses. , 2010, Veterinary microbiology.

[70]  A. Jalila,et al.  Efficacy of a bacteriophage isolated from chickens as a therapeutic agent for colibacillosis in broiler chickens. , 2010, Poultry science.

[71]  Laura Kalinienė,et al.  Low-temperature T4-like coliphages vB_EcoM-VR5, vB_EcoM-VR7 and vB_EcoM-VR20 , 2010, Archives of Virology.

[72]  Ken Chen,et al.  VarScan: variant detection in massively parallel sequencing of individual and pooled samples , 2009, Bioinform..

[73]  S. Sillankorva,et al.  Isolation and characterization of bacteriophages for avian pathogenic E. coli strains , 2009, Journal of applied microbiology.

[74]  R. Sereno,et al.  The influence of the mode of administration in the dissemination of three coliphages in chickens. , 2009, Poultry science.

[75]  T. Ross,et al.  Use of bacteriophages as biocontrol agents to control Salmonella associated with seed sprouts. , 2009, International journal of food microbiology.

[76]  A. Donoghue,et al.  Bacteriophages for prophylaxis and therapy in cattle, poultry and pigs , 2008, Animal Health Research Reviews.

[77]  L. Wieler,et al.  Intestine and Environment of the Chicken as Reservoirs for Extraintestinal Pathogenic Escherichia coli Strains with Zoonotic Potential , 2008, Applied and Environmental Microbiology.

[78]  I. Sutherland,et al.  The use of phages for the removal of infectious biofilms. , 2008, Current pharmaceutical biotechnology.

[79]  F. Dziva,et al.  Colibacillosis in poultry: unravelling the molecular basis of virulence of avian pathogenic Escherichia coli in their natural hosts , 2008, Avian pathology : journal of the W.V.P.A.

[80]  J. Hinrichs,et al.  Environmental factors for phage-induced fermentation problems: replication and adsorption of the Lactococcus lactis phage P008 as influenced by temperature and pH. , 2007, Food microbiology.

[81]  L. Wieler,et al.  Avian pathogenic, uropathogenic, and newborn meningitis-causing Escherichia coli: how closely related are they? , 2007, International journal of medical microbiology : IJMM.

[82]  Paul S. Cohen,et al.  Role of Motility and the flhDC Operon in Escherichia coli MG1655 Colonization of the Mouse Intestine , 2007, Infection and Immunity.

[83]  R. Raya,et al.  Isolation and Characterization of a New T-Even Bacteriophage, CEV1, and Determination of Its Potential To Reduce Escherichia coli O157:H7 Levels in Sheep , 2006, Applied and Environmental Microbiology.

[84]  R. P. Ross,et al.  The newly isolated lytic bacteriophages st104a and st104b are highly virulent against Salmonella enterica , 2006, Journal of applied microbiology.

[85]  S. Iyoda,et al.  The long polar fimbriae genes identified in Shiga toxin-producing Escherichia coli are present in other diarrheagenic E. coli and in the standard E. coli collection of reference (ECOR) strains. , 2006, Research in microbiology.

[86]  Traci Haddock,et al.  Mouse Intestine Selects Nonmotile flhDC Mutants of Escherichia coli MG1655 with Increased Colonizing Ability and Better Utilization of Carbon Sources , 2005, Infection and Immunity.

[87]  Y. Tseng,et al.  Isolation and Characterization of Novel Giant Stenotrophomonas maltophilia Phage φSMA5 , 2005, Applied and Environmental Microbiology.

[88]  H. Brüssow,et al.  In Vitro and In Vivo Bacteriolytic Activities of Escherichia coli Phages: Implications for Phage Therapy , 2004, Antimicrobial Agents and Chemotherapy.

[89]  Joe E Grissom,et al.  Carbon nutrition of Escherichia coli in the mouse intestine. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[90]  K. Miyanaga,et al.  Toward rational control of Escherichia coli O157:H7 by a phage cocktail , 2004, Applied Microbiology and Biotechnology.

[91]  Dean Laslett,et al.  ARAGORN, a program to detect tRNA genes and tmRNA genes in nucleotide sequences. , 2004, Nucleic acids research.

[92]  S. Levy Antibiotic resistance: consequences of inaction. , 2001, Clinical infectious diseases : an official publication of the Infectious Diseases Society of America.

[93]  J. Mcneil,et al.  Prediction of rho-independent transcriptional terminators in Escherichia coli. , 2001, Nucleic acids research.

[94]  P. Babitzke,et al.  Positive regulation of motility and flhDC expression by the RNA‐binding protein CsrA of Escherichia coli , 2001, Molecular microbiology.

[95]  M. Rohde,et al.  The multicellular morphotypes of Salmonella typhimurium and Escherichia coli produce cellulose as the second component of the extracellular matrix , 2001, Molecular microbiology.

[96]  Kim Rutherford,et al.  Artemis: sequence visualization and annotation , 2000, Bioinform..

[97]  C. Whitfield,et al.  Distribution of Core Oligosaccharide Types in Lipopolysaccharides from Escherichia coli , 2000, Infection and Immunity.

[98]  S. Eddy,et al.  tRNAscan-SE: a program for improved detection of transfer RNA genes in genomic sequence. , 1997, Nucleic acids research.

[99]  R. Foschino,et al.  Characterization of two virulent Lactobacillus fermentum bacteriophages isolated from sour dough , 1995 .

[100]  U. Henning,et al.  Receptor-recognizing proteins of T-even type bacteriophages. The receptor-recognizing area of proteins 37 of phages T4 TuIa and TuIb. , 1990, Journal of molecular biology.

[101]  And,et al.  Roles of lipopolysaccharide and outer membrane protein OmpC of Escherichia coli K-12 in the receptor function for bacteriophage T4 , 1982, Journal of bacteriology.

[102]  J. Gill,et al.  Phage choice, isolation, and preparation for phage therapy. , 2010, Current pharmaceutical biotechnology.

[103]  E. Lingohr,et al.  Enumeration of bacteriophages by double agar overlay plaque assay. , 2009, Methods in molecular biology.

[104]  Torsten Seemann,et al.  Prokka: rapid prokaryotic genome annotation , 2014, Bioinform..

[105]  H. Ackermann Frequency of morphological phage descriptions in the year 2000 , 2001, Archives of Virology.