{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,26]],"date-time":"2026-10-26T18:24:58Z","timestamp":1793039098395,"version":"4.5.0"},"reference-count":55,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2021,3,29]],"date-time":"2021-03-29T00:00:00Z","timestamp":1616976000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Microbiol."],"abstract":"<jats:p>\n                    Antimicrobial resistance (AMR) is a major problem globally. The main bacterial organisms associated with urinary tract infection (UTI) associated sepsis are\n                    <jats:italic>E. coli<\/jats:italic>\n                    and\n                    <jats:italic>Klebsiella<\/jats:italic>\n                    along with\n                    <jats:italic>Enterobacter<\/jats:italic>\n                    species. These all have AMR strains known as ESBL (Extended Spectrum Beta-Lactamase), which are featured on the WHO priority pathogens list as \u201ccritical\u201d for research. Bacteriophages (phages), as viruses that can infect and kill bacteria, could provide an effective tool to tackle these AMR strains. There is currently no \u201cgold standard\u201d for developing a phage cocktail. Here we describe a novel approach to develop an effective phage cocktail against a set of ESBL-producing\n                    <jats:italic>E. coli<\/jats:italic>\n                    and\n                    <jats:italic>Klebsiella<\/jats:italic>\n                    largely isolated from patients in United Kingdom hospitals. By comparing different measures of phage efficacy, we show which are the most robust, and suggest an efficient screening cascade that could be used to develop phage cocktails to target other AMR bacterial species. A target panel of 38 ESBL-producing clinical strains isolated from urine samples was collated and used to test phage efficacy. After an initial screening of 68 phages, six were identified and tested against these 38 strains to determine their clinical coverage and killing efficiency. To achieve this, we assessed four different methods to assess phage virulence across these bacterial isolates. These were the Direct Spot Test (DST), the Efficiency of Plating (EOP) assay, the planktonic killing assay (PKA) and the biofilm assay. The final ESBL cocktail of six phages could effectively kill 23\/38 strains (61%), for\n                    <jats:italic>Klebsiella<\/jats:italic>\n                    13\/19 (68%) and for\n                    <jats:italic>E. coli<\/jats:italic>\n                    10\/19 (53%) based on the PKA data. The ESBL\n                    <jats:italic>E. coli<\/jats:italic>\n                    collection had six isolates from the prevalent UTI-associated ST131 sequence type, five of which were targeted effectively by the final cocktail. Of the four methods used to assess phage virulence, the data suggests that PKAs are as effective as the much more time-consuming EOPs and data for the two assays correlates well. This suggests that planktonic killing is a good proxy to determine which phages should be used in a cocktail. This assay when combined with the virulence index also allows \u201cphage synergy\u201d to inform cocktail design.\n                  <\/jats:p>","DOI":"10.3389\/fmicb.2021.613529","type":"journal-article","created":{"date-parts":[[2021,3,29]],"date-time":"2021-03-29T01:36:14Z","timestamp":1616981774000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":101,"title":["Analysis of Selection Methods to Develop Novel Phage Therapy Cocktails Against Antimicrobial Resistant Clinical Isolates of Bacteria"],"prefix":"10.3389","volume":"12","author":[{"given":"Melissa E. K.","family":"Haines","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Francesca E.","family":"Hodges","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Janet Y.","family":"Nale","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jennifer","family":"Mahony","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Douwe","family":"van Sinderen","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Joanna","family":"Kaczorowska","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Bandar","family":"Alrashid","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mahmuda","family":"Akter","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nathan","family":"Brown","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dominic","family":"Sauvageau","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Thomas","family":"Sicheritz-Pont\u00e9n","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Anisha M.","family":"Thanki","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Andrew D.","family":"Millard","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Edouard E.","family":"Galyov","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Martha R. J.","family":"Clokie","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2021,3,29]]},"reference":[{"key":"B1","doi-asserted-by":"publisher","first-page":"3","DOI":"10.1007\/s12250-014-3547-2","article-title":"Bacteriophage secondary infection.","volume":"30","author":"Abedon","year":"2015","journal-title":"Virol. Sin."},{"key":"B2","doi-asserted-by":"publisher","DOI":"10.3390\/v10030123","article-title":"\u201cFrench phage network\u201d-third meeting report.","volume":"10","author":"Ansaldi","year":"2018","journal-title":"Viruses"},{"key":"B3","doi-asserted-by":"publisher","first-page":"455","DOI":"10.1089\/cmb.2012.0021","article-title":"SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing.","volume":"19","author":"Bankevich","year":"2012","journal-title":"J. Comput. Biol."},{"key":"B4","doi-asserted-by":"publisher","first-page":"2114","DOI":"10.1093\/bioinformatics\/btu170","article-title":"Trimmomatic: a flexible trimmer for Illumina sequence data.","volume":"30","author":"Bolger","year":"2014","journal-title":"Bioinformatics"},{"key":"B5","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0133727","article-title":"Genomic analysis of the emergence and rapid global dissemination of the clonal group 258 Klebsiella pneumoniae pandemic.","volume":"10","author":"Bowers","year":"2015","journal-title":"PLoS One"},{"key":"B6","doi-asserted-by":"publisher","DOI":"10.1128\/AAC.01076-18","article-title":"Past and present perspectives on \u03b2-lactamases.","volume":"62","author":"Bush","year":"2018","journal-title":"Antimicrob. Agents Chemother."},{"key":"B7","doi-asserted-by":"publisher","DOI":"10.3390\/v10040163","article-title":"In vitro characteristics of phages to guide \u2018real life\u2019 phage therapy suitability.","volume":"10","author":"Casey","year":"2018","journal-title":"Viruses"},{"key":"B8","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1016\/B978-0-12-394805-2.00001-4","article-title":"Phage therapy pharmacology phage cocktails.","volume":"78","author":"Chan","year":"2012","journal-title":"Adv. Appl. Microbiol."},{"key":"B9","doi-asserted-by":"publisher","first-page":"769","DOI":"10.2217\/fmb.13.47","article-title":"Phage cocktails and the future of phage therapy.","volume":"8","author":"Chan","year":"2013","journal-title":"Future Microbiol."},{"key":"B10","doi-asserted-by":"publisher","DOI":"10.3389\/fmicb.2018.01476","article-title":"In Vitro design and evaluation of phage cocktails against Aeromonas salmonicida.","volume":"9","author":"Chen","year":"2018","journal-title":"Front. Microbiol."},{"key":"B11","first-page":"45","article-title":"Genome sequence assembly using trace signals and additional sequence information.","volume":"99","author":"Chevreux","year":"1999","journal-title":"Comput. Sci. Biol. Proc. Ger. Conf. Bioinform."},{"key":"B12","doi-asserted-by":"publisher","DOI":"10.1093\/ofid\/ofv213","article-title":"Risk factors and outcomes for carbapenem-resistant Klebsiella pneumoniae isolation, stratified by its multilocus sequence typing: ST258 versus non-ST258.","volume":"3","author":"Dhar","year":"2016","journal-title":"Open Forum Infect. Dis."},{"key":"B13","doi-asserted-by":"publisher","DOI":"10.1128\/AAC.00483-19","article-title":"Validating the AMRFINder tool and resistance gene database by using antimicrobial resistance genotype-phenotype correlations in a collection of isolates.","volume":"63","author":"Feldgarden","year":"2019","journal-title":"Antimicrob. Agents Chemother."},{"key":"B14","doi-asserted-by":"publisher","DOI":"10.1128\/AAC.02573-17","article-title":"Design of a broad-range bacteriophage cocktail that reduces Pseudomonas aeruginosa biofilms and treats acute infections in two animal models.","volume":"62","author":"Forti","year":"2018","journal-title":"Antimicrob. Agents Chemother."},{"key":"B15","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1038\/s41598-017-07910-5","article-title":"PhageTerm: a tool for fast and accurate determination of phage termini and packaging mechanism using next-generation sequencing data.","volume":"7","author":"Garneau","year":"2017","journal-title":"Sci. Rep."},{"key":"B16","doi-asserted-by":"publisher","DOI":"10.3390\/v10060288","article-title":"Phage therapy: what have we learned?","volume":"10","author":"Gorski","year":"2018","journal-title":"Viruses"},{"key":"B17","doi-asserted-by":"publisher","first-page":"1072","DOI":"10.1093\/bioinformatics\/btt086","article-title":"QUAST: quality assessment tool for genome assemblies.","volume":"29","author":"Gurevich","year":"2013","journal-title":"Bioinformatics"},{"key":"B18","doi-asserted-by":"publisher","first-page":"3836","DOI":"10.1128\/AEM.00241-10","article-title":"Abolition of biofilm formation in urinary tract Escherichia coli and klebsiella isolates by metal interference through competition for fur.","volume":"76","author":"Hancock","year":"2010","journal-title":"Appl. Environ. Microbiol."},{"key":"B19","doi-asserted-by":"publisher","DOI":"10.3390\/ph12010035","article-title":"Phages for phage therapy: isolation, characterization, and host range breadth.","volume":"12","author":"Hyman","year":"2019","journal-title":"Pharmaceuticals"},{"key":"B20","doi-asserted-by":"publisher","first-page":"217","DOI":"10.1016\/S0065-2164(10)70007-1","article-title":"Bacteriophage host range and bacterial resistance.","volume":"70","author":"Hyman","year":"2010","journal-title":"Adv. Appl. Microbiol."},{"key":"B21","doi-asserted-by":"publisher","first-page":"286","DOI":"10.1086\/653932","article-title":"Escherichia coli sequence type ST131 as the major cause of serious multidrug-resistant E. coli infections in the United States.","volume":"51","author":"Johnson","year":"2010","journal-title":"Clin. Infect. Dis."},{"key":"B22","doi-asserted-by":"publisher","DOI":"10.1186\/1471-2105-11-595","article-title":"BIGSdb: Scalable analysis of bacterial genome variation at the population level. BMC Bioinformatics","author":"Jolley","year":"2010"},{"key":"B23","doi-asserted-by":"publisher","first-page":"57","DOI":"10.4103\/0974-777X.52984","article-title":"Approach to a patient with urosepsis.","volume":"1","author":"Kalra","year":"2009","journal-title":"J. Glob. Infect. Dis."},{"key":"B24","doi-asserted-by":"publisher","first-page":"69","DOI":"10.1007\/978-1-60327-164-6_7","article-title":"Chapter 7: enumeration of bacteriophages by double agar overlay plaque assay.","volume":"501","author":"Kropinski","year":"2009","journal-title":"Methods Mol. Biol."},{"key":"B25","doi-asserted-by":"publisher","first-page":"3270","DOI":"10.1128\/JCM.01315-13","article-title":"Escherichia coli sequence type 131 as a prominent cause of antibiotic resistance among urinary Escherichia coli isolates from reproductive-age women.","volume":"51","author":"Kudinha","year":"2013","journal-title":"J. Clin. Microbiol."},{"key":"B26","doi-asserted-by":"publisher","first-page":"1547","DOI":"10.1093\/molbev\/msy096","article-title":"MEGA X: molecular evolutionary genetics analysis across computing platforms.","volume":"35","author":"Kumar","year":"2018","journal-title":"Mol. Biol. Evol."},{"key":"B27","doi-asserted-by":"publisher","first-page":"426","DOI":"10.1016\/j.medmal.2008.06.023","article-title":"Phage therapy experience at the Eliava Institute.","volume":"38","author":"Kutateladze","year":"2008","journal-title":"Med. Mal. Infect."},{"key":"B28","doi-asserted-by":"publisher","first-page":"141","DOI":"10.1007\/978-1-60327-164-6_14","article-title":"Chapter 14: phage host range and efficiency of plating.","volume":"501","author":"Kutter","year":"2009","journal-title":"Methods Mol. Biol."},{"key":"B29","doi-asserted-by":"publisher","first-page":"127","DOI":"10.1093\/bioinformatics\/btl529","article-title":"Interactive Tree Of Life (iTOL): an online tool for phylogenetic tree display and annotation.","volume":"23","author":"Letunic","year":"2007","journal-title":"Bioinformatics"},{"key":"B30","doi-asserted-by":"publisher","first-page":"1674","DOI":"10.1093\/bioinformatics\/btv033","article-title":"MEGAHIT: an ultra-fast single-node solution for large and complex metagenomics assembly via succinct de Bruijn graph.","volume":"31","author":"Li","year":"2015","journal-title":"Bioinformatics"},{"key":"B31","doi-asserted-by":"publisher","first-page":"439","DOI":"10.1007\/BF02013107","article-title":"Clinical significance of beta-lactamase induction and stable derepression in gram-negative rods.","volume":"6","author":"Livermore","year":"1987","journal-title":"Eur. J. Clin. Microbiol."},{"key":"B32","doi-asserted-by":"publisher","DOI":"10.3389\/fmicb.2019.00574","article-title":"Therapeutic characterization and efficacy of bacteriophage cocktails infecting Escherichia coli, Klebsiella pneumoniae, and Enterobacter Species. Front. Microbiol.","author":"Manohar","year":"2019"},{"key":"B33","doi-asserted-by":"publisher","DOI":"10.3389\/fmicb.2015.01271","article-title":"On-demand isolation of bacteriophages against drug-resistant bacteria for personalized phage therapy.","volume":"6","author":"Mattila","year":"2015","journal-title":"Front. Microbiol."},{"key":"B34","doi-asserted-by":"publisher","first-page":"2112","DOI":"10.1111\/1462-2920.14590","article-title":"Riding the wave of genomics to investigate aquatic coliphage diversity and activity.","volume":"21","author":"Michniewski","year":"2019","journal-title":"Environ. Microbiol."},{"key":"B35","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0118557","article-title":"Isolation of phages for phage therapy: a comparison of spot tests and efficiency of plating analyses for determination of host range and efficacy.","volume":"10","author":"Mirzaei","year":"2015","journal-title":"PLoS One"},{"key":"B36","doi-asserted-by":"publisher","DOI":"10.3389\/fpubh.2019.00355","article-title":"Characterization of lytic bacteriophages infecting multidrug-resistant shiga toxigenic atypical Escherichia coli O177 strains isolated from cattle feces.","volume":"7","author":"Montso","year":"2019","journal-title":"Front. Public Heal."},{"key":"B37","doi-asserted-by":"publisher","first-page":"968","DOI":"10.1128\/AAC.01774-15","article-title":"Bacteriophage combinations significantly reduce clostridium difficile growth in vitro and proliferation in vivo.","volume":"60","author":"Nale","year":"2015","journal-title":"Antimicrob. Agents Chemother."},{"key":"B38","article-title":"Antimicrobial resistance: tackling a crisis for the health and wealth of nations.","author":"O\u2019Neill","year":"2014","journal-title":"Rev. Antimicrob. Resist."},{"key":"B39","doi-asserted-by":"publisher","DOI":"10.1128\/aac.01148-20","article-title":"Emerging antimicrobial resistant high-risk clones among Klebsiella pneumoniae: ST307 and ST147.","volume":"3","author":"Peirano","year":"2020","journal-title":"Antimicrob. Agents Chemother."},{"key":"B40","doi-asserted-by":"publisher","first-page":"297","DOI":"10.1586\/eri.13.12","article-title":"Clinical relevance of the ESKAPE pathogens.","volume":"11","author":"Pendleton","year":"2013","journal-title":"Expert Rev. Anti. Infect. Ther."},{"key":"B41","doi-asserted-by":"publisher","DOI":"10.3390\/v11070586","article-title":"Two phages of the genera felixunavirus subjected to 12 hour challenge on Salmonella infantis showed distinct genotypic and phenotypic changes.","volume":"11","author":"Rivera","year":"2019","journal-title":"Viruses"},{"key":"B42","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0158039","article-title":"Sepsis caused by extended-spectrum beta-lactamase (ESBL)-positive K. pneumoniae and E. coli: comparison of severity of sepsis, delay of anti-infective therapy and ESBL genotype.","volume":"11","author":"Sakellariou","year":"2016","journal-title":"PLoS One"},{"key":"B43","doi-asserted-by":"publisher","DOI":"10.7717\/peerj.590","article-title":"Synergy as a rationale for phage therapy using phage cocktails.","volume":"2","author":"Schmerer","year":"2014","journal-title":"PeerJ"},{"key":"B44","doi-asserted-by":"publisher","first-page":"2068","DOI":"10.1093\/bioinformatics\/btu153","article-title":"Prokka: rapid prokaryotic genome annotation.","volume":"30","author":"Seemann","year":"2014","journal-title":"Bioinformatics"},{"key":"B45","author":"Seemann","year":"2020","journal-title":"mlst, Github."},{"key":"B46","doi-asserted-by":"publisher","first-page":"17","DOI":"10.1089\/phage.2019.0001","article-title":"The virulence index: a metric for quantitative analysis of phage virulence.","volume":"1","author":"Storms","year":"2019","journal-title":"Phage"},{"key":"B47","doi-asserted-by":"publisher","DOI":"10.3389\/fmicb.2016.00465","article-title":"Bacteriophages as potential treatment for urinary tract infections.","volume":"7","author":"Sybesma","year":"2016","journal-title":"Front. Microbiol."},{"key":"B48","doi-asserted-by":"publisher","first-page":"115","DOI":"10.1093\/jac\/2.2.115","article-title":"The beta-lactamases of gram-negative bacteria and their role in resistance to beta-lactam antibiotics.","volume":"2","author":"Sykes","year":"1976","journal-title":"J. Antimicrob. Chemother."},{"key":"B49","doi-asserted-by":"publisher","first-page":"17904","DOI":"10.1038\/s41598-018-36229-y","article-title":"Complete genome analysis of a Siphoviridae phage TSK1 showing biofilm removal potential against Klebsiella pneumoniae.","volume":"8","author":"Tabassum","year":"2018","journal-title":"Sci. Rep."},{"key":"B50","doi-asserted-by":"publisher","first-page":"318","DOI":"10.1016\/S1473-3099(17)30753-3","article-title":"Discovery, research, and development of new antibiotics: the WHO priority list of antibiotic-resistant bacteria and tuberculosis.","volume":"18","author":"Tacconelli","year":"2018","journal-title":"Lancet Infect. Dis."},{"key":"B51","doi-asserted-by":"publisher","DOI":"10.3389\/fmicb.2018.02127","article-title":"Bacteriophage ZCKP1: a potential treatment for Klebsiella pneumoniae isolated from diabetic foot patients.","volume":"9","author":"Taha","year":"2018","journal-title":"Front. Microbiol."},{"key":"B52","doi-asserted-by":"publisher","DOI":"10.3389\/fcimb.2018.00376","article-title":"Bacteriophage therapy: clinical trials and regulatory hurdles the challenge of multi-drug resistant bacteria.","volume":"8","author":"Tomas","year":"2018","journal-title":"Front. Cell. Infect. Microbiol."},{"key":"B53","doi-asserted-by":"publisher","first-page":"460","DOI":"10.1080\/21505594.2016.1222343","article-title":"The global epidemiology of carbapenemase-producing Enterobacteriaceae.","volume":"8","author":"van Duin","year":"2017","journal-title":"Virulence"},{"key":"B54","doi-asserted-by":"publisher","DOI":"10.3389\/fmicb.2020.00327","article-title":"Development of a bacteriophage cocktail to constrain the emergence of phage-resistant Pseudomonas aeruginosa.","volume":"11","author":"Yang","year":"2020","journal-title":"Front. Microbiol."},{"key":"B55","doi-asserted-by":"publisher","first-page":"2640","DOI":"10.1093\/jac\/dks261","article-title":"Identification of acquired antimicrobial resistance genes.","volume":"67","author":"Zankari","year":"2012","journal-title":"J. Antimicrob. Chemother."}],"container-title":["Frontiers in Microbiology"],"original-title":[],"link":[{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fmicb.2021.613529\/full","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2021,3,29]],"date-time":"2021-03-29T01:36:20Z","timestamp":1616981780000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fmicb.2021.613529\/full"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2021,3,29]]},"references-count":55,"alternative-id":["10.3389\/fmicb.2021.613529"],"URL":"https:\/\/doi.org\/10.3389\/fmicb.2021.613529","relation":{"has-preprint":[{"id-type":"doi","id":"10.1101\/2020.10.13.337345","asserted-by":"object"}]},"ISSN":["1664-302X"],"issn-type":[{"value":"1664-302X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2021,3,29]]},"article-number":"613529"}}