In Vitro Techniques and Measurements of Phage Characteristics That Are Important for Phage Therapy Success

Glonti, Tea; Pirnay, Jean‐Paul

doi:10.3390/v14071490

Cited by 61 publications

(49 citation statements)

References 107 publications

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“…Some authors underline the importance of using low titres of phage when investigating its capacity to both lyse the host strain and produce new virions ( de Melo et al., 2019 ). Other consider that the spot test should only be used as qualitative activity detection method that has to be confirmed by double agar overlay plaque assay ( Glonti and Pirnay, 2022 ).…”

Section: Current Methods For Bacteriophage Activity Testingmentioning

confidence: 99%

“…Furthermore, TD assays based on the OmniLog™ system also allow for more complex analyses, such as phage-antibiotic synergy ( Henry et al., 2012 ). This is nowadays a substantial asset, as experts are increasingly agreeing that phages will more probably be used in combination with antibiotics rather than totally replace those ( Glonti and Pirnay, 2022 ).…”

Section: Current Methods For Bacteriophage Activity Testingmentioning

confidence: 99%

“…By contrast, instead of monitoring bacterial growth, computer-assisted imagery techniques applied to solid agar plaque assay allow to index the number and the growth kinetics of viral plaque formation in a much shorter turnaround time (TAT) than the traditional plaque assays. Moreover, it allows the detection of phage-resistant bacterial microcolonies inside the boundaries of plaques and can thus track phage resistance, as bacterial re-growth of initially phage-sensitive bacteria still requires phage plaque and bacterial colony formation on agar ( Perlemoine et al., 2021 ; Glonti and Pirnay, 2022 ).…”

Section: Current Methods For Bacteriophage Activity Testingmentioning

confidence: 99%

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Determination of phage susceptibility as a clinical diagnostic tool: A routine perspective

Daubie

Chalhoub

Blasdel³

et al. 2022

Front. Cell. Infect. Microbiol.

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As the global burden of disease caused by multidrug resistant bacteria is a major source of concern, credible clinical alternatives to antibiotic therapy, such as personalized phage therapy, are actively explored. Although phage therapy has been used for more than a century, the issue of an easy to implement diagnostic tool for determining phage susceptibility that meets current routine clinical needs is still open. In this Review, we summarize the existing methods used for determining phage activity on bacteria, including the three reference methods: the spot test, the double agar overlay plaque assay, and the Appelmans method. The first two methods rely on the principle of challenging the overnight growth of a lawn of bacteria in an agar matrix to a known relative phage to bacteria concentration and represent good screening tools to determine if the tested phage can be used for a “passive” and or “active” treatment. Beside these methods, several techniques, based on “real-time” growth kinetics assays (GKA) have been developed or are under development. They all monitor the growth of clinical isolates in the presence of phages, but use various detection methods, from classical optical density to more sophisticated techniques such as computer-assisted imagery, flow-cytometry, quantitative real-time polymerase chain reaction (qPCR) or metabolic indicators. Practical considerations as well as information provided about phage activity are reviewed for each technique. Finally, we also discuss the analytical and interpretative requirements for the implementation of a phage susceptibility testing tool in routine clinical microbiology.

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Section: Current Methods For Bacteriophage Activity Testingmentioning

confidence: 99%

Section: Current Methods For Bacteriophage Activity Testingmentioning

confidence: 99%

Section: Current Methods For Bacteriophage Activity Testingmentioning

confidence: 99%

See 1 more Smart Citation

Determination of phage susceptibility as a clinical diagnostic tool: A routine perspective

Daubie

Chalhoub

Blasdel³

et al. 2022

Front. Cell. Infect. Microbiol.

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“…Ten-fold serial dilutions of the phage lysates were prepared in phage buffer (50 mM TRIS HCl pH 7.5; 0.1 M NaCl; 8 mM MgSO 4 ). The phages were enumerated on indicator strains using the MD/SP (Multiple Dilutions on Single Plates) method [ 37 ] on MRSΦ agar [ 18 ]. Tested strains were IOEBS277, CRBO1381, CRBO1384 and BL4 ( O. oeni ), CRBO2176 and DSM17330 ( O. kitaharae ), UCMA15228 ( O. sicerae ) and JP736 ( O. alcoholitolerans ).…”

Section: Methodsmentioning

confidence: 99%

Phage Encounters Recorded in CRISPR Arrays in the Genus Oenococcus

Barchi¹,

Philippe²,

Chaïb³

et al. 2022

Viruses

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The Oenococcus genus comprises four recognized species, and members have been found in different types of beverages, including wine, kefir, cider and kombucha. In this work, we implemented two complementary strategies to assess whether oenococcal hosts of different species and habitats were connected through their bacteriophages. First, we investigated the diversity of CRISPR-Cas systems using a genome-mining approach, and CRISPR-endowed strains were identified in three species. A census of the spacers from the four identified CRISPR-Cas loci showed that each spacer space was mostly dominated by species-specific sequences. Yet, we characterized a limited records of potentially recent and also ancient infections between O. kitaharae and O. sicerae and phages of O. oeni, suggesting that some related phages have interacted in diverse ways with their Oenococcus hosts over evolutionary time. Second, phage-host interaction analyses were performed experimentally with a diversified panel of phages and strains. None of the tested phages could infect strains across the species barrier. Yet, some infections occurred between phages and hosts from distinct beverages in the O. oeni species.

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“…They may be useful in combination with existing antibacterial agents (46,49). Furthermore, their ease of manipulation and greater understanding of the properties that favor phage therapy success have raised the possibility of uses beyond infectious disease (50)(51)(52).…”

Section: Introductionmentioning

confidence: 99%

EGFR-targeted bacteriophage lambda penetrates model stromal and colorectal carcinoma tissues, is taken up into carcinoma cells, and interferes with 3-dimensional tumor formation

et al. 2022

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IntroductionColorectal cancer and other adult solid cancers pose a significant challenge for successful treatment because the tumor microenvironment both hinders the action of conventional therapeutics and suppresses the immune activities of infiltrating leukocytes. The immune suppression is largely the effect of enhanced local mediators such as purine nucleosides and eicosanoids. Genetic approaches have the promise of interfering with these mechanisms of local immunosuppression to allow both intrinsic and therapeutic immunological anticancer processes. Bacterial phages offer a novel means of enabling access into tissues for therapeutic genetic manipulations.MethodsWe generated spheroids of fibroblastic and CRC cancer cells to model the 3-dimensional stromal and parenchymal components of colorectal tumours. We used these to examine the access and effects of both wildtype (WT) and epidermal growth factor (EGF)-presenting bacteriophage λ (WT- λ and EGF-λ) as a means of delivery of targeted genetic interventions in solid cancers. We used both confocal microscopy of spheroids exposed to AF488-tagged phages, and the recovery of viable phages as measured by plaque-forming assays to evaluate access; and measures of mitochondrial enzyme activity and cellular ATP to evaluate the outcome on the constituent cells.ResultsUsing flourescence-tagged derivatives of these bacteriophages (AF488-WT-λ and AF488-EGF-λ) we showed that phage entry into these tumour microenvironments was possible and that the EGF ligand enabled efficient and persistent uptake into the cancer cell mass. EGF-λ became localized in the intracellular portion of cancer cells and was subjected to subsequent cellular processing. The targeted λ phage had no independent effect upon mature tumour spheroids, but interfered with the early formation and growth of cancer tissues without the need for addition of a toxic payload, suggesting that it might have beneficial effects by itself in addition to any genetic intervention delivered to the tumour. Interference with spheroid formation persisted over the duration of culture.DiscussionWe conclude that targeted phage technology is a feasible strategy to facilitate delivery into colorectal cancer tumour tissue (and by extension other solid carcinomas) and provides an appropriate delivery vehicle for a gene therapeutic that can reduce local immunosuppression and/or deliver an additional direct anticancer activity.

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In Vitro Techniques and Measurements of Phage Characteristics That Are Important for Phage Therapy Success

Cited by 61 publications

References 107 publications

Determination of phage susceptibility as a clinical diagnostic tool: A routine perspective

Determination of phage susceptibility as a clinical diagnostic tool: A routine perspective

Phage Encounters Recorded in CRISPR Arrays in the Genus Oenococcus

EGFR-targeted bacteriophage lambda penetrates model stromal and colorectal carcinoma tissues, is taken up into carcinoma cells, and interferes with 3-dimensional tumor formation

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