Phage susceptibility testing and infectious titer determination through wide-field lensless monitoring of phage plaque growth

Perlemoine, Prisca; Marcoux, Pierre R.; Picard, E.; Hadji, E.; Zelsmann, M.; Mugnier, Grégoire; Marchet, A.; Resch, Grégory; O’Connell, Larry; Lacot, Éric

doi:10.1371/journal.pone.0248917

Cited by 15 publications

(15 citation statements)

References 36 publications

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“…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%

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%

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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“…Infectious titre (IT) is measured through the observation and counting of lysis plaques in a host bacterial lawn in soft agar (Perlemoine et al, 2021). IT decay during storage or processing can have profound effects on downstream applications if the reduction is not mitigated or compensated for.…”

Section: Introductionmentioning

confidence: 99%

Container material dictates stability of bacteriophage suspensions: Light scattering and infectivity measurements reveal mechanisms of infectious titre decay

O’Connell

Roupioz

Marcoux

2022

Journal of Applied Microbiology

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Aims: To measure the infectious titre (IT) decay rate for various bacteriophages as a function of storage container material. Additionally, parallel light scattering and infectious titre measurements reveal distinct mechanisms for IT loss, depending on phage. Methods and Results: Suspensions of bacteriophages 44AHJD, P68 and gh-1 were stored in various labware. IT of each suspension was repeatedly measured over the course of 2 weeks. Large variability in IT decay was observed, with >4 log 10 loss in glass and low-binding polypropylene. Incubation of polymer containers with Bovine Serum Albumin (BSA) resulted in a consistent reduction in IT decay. Aggregation state of phage suspensions was studied by nanoparticle tracking analysis (NTA), revealing highest aggregation in glass-stored suspensions and lowest after storage in BSA-treated containers. Conclusions: Glass and 'low-binding' containers may aggravate IT decay while BSA treatment may present an easy mitigation strategy. IT versus NTA titre diagrams highlight the importance of phage inactivation in combination with aggregation.Significance and impact of the study: Container material is a significant determinant of bacteriophage IT decay. It is therefore essential to confirm IT following storage and tailor choice of phage storage containers accordingly. Aggregation of phages and adsorption onto labware surfaces are not only the mechanisms accounting for IT loss but also biological instability.

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“…For example, Low et al described a method that identifies phage-host interactions by fluorescence tagging of phages with subsequent tracking of the infection using flow cytometry and confocal fluorescence microscopy, along with live/dead assay for phage susceptibility (Low et al, 2020). There are also studies where the prediction of phage infectivity was approached through machine learning and imaging (Perlemoine et al, 2021;Lood et al, 2022;O'Connell et al, 2022). The study by Perlemoine et al introduced a custom wide-field lens-less imaging device, which can be used for phage plaque detection (Perlemoine et al, 2021).…”

Section: Introductionmentioning

confidence: 99%

“…There are also studies where the prediction of phage infectivity was approached through machine learning and imaging (Perlemoine et al, 2021;Lood et al, 2022;O'Connell et al, 2022). The study by Perlemoine et al introduced a custom wide-field lens-less imaging device, which can be used for phage plaque detection (Perlemoine et al, 2021). The methodology can detect phage-resistant bacterial micro-colonies within 3 hours, however, the scalability to screen multiple phages has not yet been demonstrated.…”

Section: Introductionmentioning

confidence: 99%

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Rapid hydrogel-based phage susceptibility test for pathogenic bacteria

Patpatia

Schaedig

Dirks

et al. 2022

Front. Cell. Infect. Microbiol.

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Phage therapy is one alternative to cure infections caused by antibiotic resistant bacteria. Due to the narrow host range of phages, hundreds to thousands of phages are required to cover the diversity of bacterial pathogens. In personalized phage therapy, fast selection of the phages for individual patients is essential for successful therapy. The aims of this study were to set up a rapid hydrogel-based liquid phage susceptibility assay (PST) for the selection of phages for therapeutic use and to establish a “ready-to-screen” plate concept, where phages are readily stored in hydrogel as small droplets in microtiter plate wells. We first tested four commercially available hydrogels (GrowDex, Askina, Purilon, and Intrasite) for their suitability as phage matrices in PSTs with four phages, two of which infecting Escherichia coli and two Staphylococcus aureus. Of these four hydrogels, GrowDex was the best matrix for PST, as it did not inhibit bacterial growth, released phages quickly when mixed with bacterial culture, and maintained phage viability well. We then optimized the assay for both optical density and microscopy readers using GrowDex as matrix with 23 bacterial strains representing 10 different species and 23 phages possessing different morphologies and genome sizes. When the bacterial growth was monitored by microscopy reader, the PST was executed in just 3 hours, and there was no need for overnight culturing bacterial cells prior to the assay, whereas using optical density reader, bacteria had to be pre-cultured overnight, and the assay time was five hours. Finally, we evaluated the effect of three different chemical stabilizers (trehalose, hyaluronic acid, and gelatin) in a six-month stability assay with six model phages. These phages assay behaved very differently in respect to the chemical stabilizers, and there was not a single stabilizer suitable for all phages. However, when gelatin (0.01%) or hyaluronic acid (0.2 mg/ml) was used as stabilizer, all tested phages were still considered as positives in PST after a six-month storage in 1 ml volume. In “ready-to-screen” plates, the differences in phage stabilities were even more profound, varying from two to six months for the most and least stable phages, respectively.

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Phage susceptibility testing and infectious titer determination through wide-field lensless monitoring of phage plaque growth

Cited by 15 publications

References 36 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

Container material dictates stability of bacteriophage suspensions: Light scattering and infectivity measurements reveal mechanisms of infectious titre decay

Rapid hydrogel-based phage susceptibility test for pathogenic bacteria

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