Phage on tap–a quick and efficient protocol for the preparation of bacteriophage laboratory stocks

Bonilla, Natasha; Rojas, María Isabel; Cruz, Giuliano Netto Flores; Hung, Shr-Hau; Rohwer, Forest; Barr, Jeremy J.

doi:10.7717/peerj.2261

Cited by 271 publications

(178 citation statements)

References 29 publications

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“…Bacteriophages were concentrated by centrifugation at 6,000 ϫ g for 10 h at 4°C, and phage pellets were gently resuspended in 10 ml of Dulbecco's phosphate-buffered saline and sterilized again by filtration. The sterilized phage suspensions were subjected to ultrafiltration and 1-octanol extraction to remove lipopolysaccharide (LPS) as previously described (30). In most cases, this final treatment was performed at the Laboratory for Viral Information at San Diego State University.…”

Section: A Baumannii Clinical Isolatesmentioning

confidence: 99%

Development and Use of Personalized Bacteriophage-Based Therapeutic Cocktails To Treat a Patient with a Disseminated Resistant Acinetobacter baumannii Infection

Schooley

Biswas

Gill

et al. 2017

Antimicrob Agents Chemother

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927

811

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Widespread antibiotic use in clinical medicine and the livestock industry has contributed to the global spread of multidrug-resistant (MDR) bacterial pathogens, including Acinetobacter baumannii. We report on a method used to produce a personalized bacteriophage-based therapeutic treatment for a 68-year-old diabetic patient with necrotizing pancreatitis complicated by an MDR A. baumannii infection. Despite multiple antibiotic courses and efforts at percutaneous drainage of a pancreatic pseudocyst, the patient deteriorated over a 4-month period. In the absence of effective antibiotics, two laboratories identified nine different bacteriophages with lytic activity for an A. baumannii isolate from the patient. Administration of these bacteriophages intravenously and percutaneously into the abscess cavities was associated with reversal of the patient's downward clinical trajectory, clearance of the A. baumannii infection, and a return to health. The outcome of this case suggests that the methods described here for the production of bacteriophage therapeutics could be applied to similar cases and that more concerted efforts to investigate the use of therapeutic bacteriophages for MDR bacterial infections are warranted.

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Section: A Baumannii Clinical Isolatesmentioning

confidence: 99%

Development and Use of Personalized Bacteriophage-Based Therapeutic Cocktails To Treat a Patient with a Disseminated Resistant Acinetobacter baumannii Infection

Schooley

Biswas

Gill

et al. 2017

Antimicrob Agents Chemother

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927

811

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“…This reveals some of the mechanism behind the PLE's successful anti-phage activity and lays the groundwork for asking classic questions about evolutionary theory in respect to optimal virulence and selective pressures in complicated parasitic relationships, all the while providing insight into an ongoing evolutionary arms race that impacts human health. Bacteriophages were propagated on PLE (-) V. cholerae hosts and prepped via polyethylene glycerol precipitation or concentration and media exchange on Amicon Ultra -15 (Millipore) centrifugal filters (73,74). Stocks were stored in sodium chloride-tris-ethylenediaminetetraacetic acid buffer (STE), and quantified via the soft agar overlay method (73).…”

Section: Discussionmentioning

confidence: 99%

DominantVibrio choleraephage exhibits lysis inhibition sensitive to disruption by a defensive phage satellite

Hays

Seed

2019

Preprint

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AbstractBacteriophages and their bacterial hosts are locked in a dynamic evolutionary arms race. Phage satellites, selfish genomic islands which exploit both host bacterium and target phage, further complicate the evolutionary fray. One such tripartite system involves the etiological agent of the diarrheal disease cholera – Vibrio cholerae, the predominant phage isolated from cholera patients – ICP1, and a phage satellite – PLE. When ICP1 infects V. cholerae harboring the integrated PLE genome, PLE accelerates host lysis, spreading the PLE while completely blocking phage production protecting V. cholerae at the population level. Here we identify a single PLE gene, lidI, sufficient to mediate accelerated lysis during ICP1 infection and demonstrate that LidI functions through disrupting lysis inhibition – an understudied outcome of phage infection when phages vastly outnumber their hosts. This work identifies ICP1-encoded holin and antiholin genes teaA and arrA respectively, that mediate this first example of lysis inhibition outside the T-even coliphages. Through lysis inhibition disruption, LidI is sufficient to limit the number of progeny phage produced from an infection. Consequently, this disruption bottlenecks ICP1 evolution as probed by recombination and CRISPR-Cas targeting assays. These studies link novel characterization of the classic phenomenon of lysis inhibition with a conserved protein in a dominant phage satellite, highlighting the importance of lysis timing during infection and parasitization, as well as providing insight into the populations, relationships, and evolution of bacteria, phages, and phage satellites in nature.ImportanceWith increasing awareness of microbiota impacting human health comes intensified examination of, not only bacteria and the bacteriophages that prey upon them, but also the mobile genetic elements (MGEs) that mediate interactions between them. Research is unveiling evolutionary strategies dependent on sensing the milieu: quorum sensing impacts phage infection, phage teamwork overcomes bacterial defenses, and abortive infections sacrifice single cells protecting populations. Yet, the first discovered environmental sensing by phages, known as lysis inhibition (LIN), has only been studied in the limited context of T-even coliphages. Here we characterize LIN in the etiological agent of the diarrheal disease cholera, Vibrio cholerae, infected by a phage ubiquitous in clinical samples. Further, we show that a specific MGE, the phage satellite PLE, collapses LIN with a conserved protein during its anti-phage program. The insights gleaned from this work add to our expanding understanding of microbial fitness in natural contexts beyond the canonical bacterial genome and into the realm of antagonistic evolution driven by phages and satellites.

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“…1A). We used the Phage-on-Tap protocol (36) to isolate and purify phages into high-titer preparations targeting the nine strains. Eight bacteriophages were isolated and used to establish an A. baumannii -specific phage library.…”

Section: Resultsmentioning

confidence: 99%

Bacteriophages targetingAcinetobacter baumanniicapsule induce antimicrobial resensitization

Altamirano

Forsyth

Patwa

et al. 2020

Preprint

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21Carbapenem-resistant Acinetobacter baumannii is responsible for frequent, hard-to-treat and often fatal 22 healthcare-associated infections. Phage therapy, the use of viruses that infect and kill bacteria, is an approach 23 gaining significant clinical interest to combat antibiotic-resistant infections. However, a major limitation is that 24 bacteria can develop resistance against phages. Here, we isolated phages with activity against a panel of A.25 baumannii strains and focused on clinical isolates AB900 and A9844 and their phages for detailed 26 characterization. As expected, coincubation of the phages with their hosts in vitro resulted in the emergence of 27 phage-resistant bacterial mutants. Genome sequence analysis revealed that phage-resistant mutants harbored 28 loss-of-function mutations in genes from the K locus, responsible for the biosynthesis of the bacterial capsule. 29Using molecular biology techniques, phage adsorption assays, and quantitative evaluation of capsule 30 production, we established that the bacterial capsule serves as the primary receptor for these phages. As a 31 collateral phenotype of impaired capsule production, the phage-resistant strains could not form biofilms, became 32 fully sensitized to the human complement system, showed increased susceptibility to beta-lactam antibiotics, 33 and became vulnerable to additional phages. Finally, in a murine model of bacteremia, the phage-resistant A. 34 baumannii demonstrated a diminished capacity to colonize blood and solid tissues. This study demonstrates 35 that phages can be used not only for their lytic activity but, if combined with a posteriori knowledge of their 36 receptors and the mechanism of bacterial resistance, for their potential synergy with other antimicrobial agents, 37 thus providing even broader clinical options for phage therapy. 39 Keywords 40Phage therapy, Acinetobacter baumannii, bacterial capsule, phage receptors, antimicrobial resistance. 41 42 2 Introduction 43In 2019, antimicrobial resistance was listed by the World Health Organization (WHO) as one of the top 44 ten threats to global health (1). Multidrug resistant (MDR) infections are consistently associated with poor clinical 45 outcomes and represent a significant financial burden on the healthcare system (2, 3). Also in 2019, the WHO 46 and Centers for Disease Control and Prevention (CDC) prioritized carbapenem-resistant Acinetobacter 47 baumannii as a pathogen critically requiring research and development of new antimicrobial strategies (4, 5). 48A. baumannii, a gram-negative coccobacillus, is a member of the ESKAPE group of pathogens, which are 49 prominent for causing frequent and hard-to-treat healthcare-associated infections (6). 50As a species, A. baumannii is highly resilient, and capable of surviving for months in biofilms on abiotic 51 surfaces (7, 8). It can cause pneumonia, bacteremia, urinary tract infections, meningitis and wound infections, 52 particularly in the context of intensive care units, and is frequently associated with indwelling med...

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Phage on tap–a quick and efficient protocol for the preparation of bacteriophage laboratory stocks

Cited by 271 publications

References 29 publications

Development and Use of Personalized Bacteriophage-Based Therapeutic Cocktails To Treat a Patient with a Disseminated Resistant Acinetobacter baumannii Infection

Development and Use of Personalized Bacteriophage-Based Therapeutic Cocktails To Treat a Patient with a Disseminated Resistant Acinetobacter baumannii Infection

DominantVibrio choleraephage exhibits lysis inhibition sensitive to disruption by a defensive phage satellite

Bacteriophages targetingAcinetobacter baumanniicapsule induce antimicrobial resensitization

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