Phage Production and Maintenance of Stocks, Including Expected Stock Lifetimes

Fortier, Louis‐Charles; Moineau, Sylvain

doi:10.1007/978-1-60327-164-6_19

Cited by 87 publications

(58 citation statements)

References 21 publications

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“…For long‐term storage, bacterial cultures were stored in a 20% glycerol solution at −80°C. Phage was stored at 4°C but for long‐term storage, phage was stored in a 50% glycerol solution at −80°C (Fortier & Moineau, ). S. aureus was grown in brain heart infusion (BHI) media and infected with phage K. For all experiments, E. coli was grown using LB media or LB agar.…”

Section: Methodsmentioning

confidence: 99%

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A scaled‐down model for the translation of bacteriophage culture to manufacturing scale

Ali

Rafiq

Ratcliffe

2019

Biotech & Bioengineering

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Therapeutic bacteriophages are emerging as a potential alternative to antibiotics and synergistic treatment of antimicrobial‐resistant infections. This is reflected by their use in an increasing number of recent clinical trials. Many more therapeutic bacteriophage is being investigated in preclinical research and due to the bespoke nature of these products with respect to their limited infection spectrum, translation to the clinic requires combined understanding of the biology underpinning the bioprocess and how this can be optimized and streamlined for efficient methods of scalable manufacture. Bacteriophage research is currently limited to laboratory scale studies ranging from 1–20 ml, emerging therapies include bacteriophage cocktails to increase the spectrum of infectivity and require multiple large‐scale bioreactors (up to 50 L) containing different bacteriophage–bacterial host reactions. Scaling bioprocesses from the milliliter scale to multi‐liter large‐scale bioreactors is challenging in itself, but performing this for individual phage‐host bioprocesses to facilitate reliable and robust manufacture of phage cocktails increases the complexity. This study used a full factorial design of experiments approach to explore key process input variables (temperature, time of infection, multiplicity of infection, agitation) for their influence on key process outputs (bacteriophage yield, infection kinetics) for two bacteriophage–bacterial host bioprocesses (T4 – Escherichia coli; Phage K – Staphylococcus aureus). The research aimed to determine common input variables that positively influence output yield and found that the temperature at the point of infection had the greatest influence on bacteriophage yield for both bioprocesses. The study also aimed to develop a scaled down shake‐flask model to enable rapid optimization of bacteriophage batch bioprocessing and translate the bioprocess into a scale‐up model with a 3 L working volume in stirred tank bioreactors. The optimization performed in the shake flask model achieved a 550‐fold increase in bacteriophage yield and these improvements successfully translated to the large‐scale cultures.

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Section: Methodsmentioning

confidence: 99%

“…Phage was stored at 4°C but for long-term storage, phage was stored in a 50% glycerol solution at −80°C(Fortier & Moineau, 2009). …”

mentioning

confidence: 99%

A scaled‐down model for the translation of bacteriophage culture to manufacturing scale

Ali

Rafiq

Ratcliffe

2019

Biotech & Bioengineering

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“…Prophages were induced from C. difficile isolates by UV irradiation (302 nm) (28). Briefly, aliquots of overnight cultures were diluted 10 Ϫ1 , 10 Ϫ2 , and 10 Ϫ3 and further spotted onto the surface of a BHI agar plate and bacteria were allowed to grow in an anaerobic atmosphere for 4 h. Bacteria were then exposed to UV light (302 nm) for 10 s with a transilluminator and overlaid with molten top agar containing a log-phase culture of a C. difficile test strain (optical density at 600 nm of ϳ0.3 to 0.5) (27,34). After overnight incubation under anaerobic conditions, a clearing zone in the bacterial lawn above a given spot was indicative of phage induction and further propagation on the test strain.…”

Section: Methodsmentioning

confidence: 99%

Characterization of Temperate Phages Infecting Clostridium difficile Isolates of Human and Animal Origins

Sekulović

Garneau

Néron

et al. 2014

Appl Environ Microbiol

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Clostridium difficile is a Gram-positive pathogen infecting humans and animals. Recent studies suggest that animals could represent potential reservoirs of C. difficile that could then transfer to humans. Temperate phages contribute to the evolution of most bacteria, for example, by promoting the transduction of virulence, fitness, and antibiotic resistance genes. In C. difficile, little is known about their role, mainly because suitable propagating hosts and conditions are lacking. Here we report the isolation, propagation, and preliminary characterization of nine temperate phages from animal and human C. difficile isolates. Prophages were induced by UV light from 58 C. difficile isolates of animal and human origins. Using soft agar overlays with 27 different C. difficile test strains, we isolated and further propagated nine temperate phages: two from horse isolates (CD481-1 and CD481-2), three from dog isolates (CD505, CD506, and CD508), and four from human isolates (CD24-2, CD111, CD146, and CD526). Two phages are members of the Siphoviridae family (CD111 and CD146), while the others are Myoviridae phages. Pulsed-field gel electrophoresis and restriction enzyme analyses showed that all of the phages had unique double-stranded DNA genomes of 30 to 60 kb. Phages induced from human C. difficile isolates, especially the members of the Siphoviridae family, had a broader host range than phages from animal C. difficile isolates. Nevertheless, most of the phages could infect both human and animal strains. Phage transduction of antibiotic resistance was recently reported in C. difficile. Our findings therefore call for further investigation of the potential risk of transduction between animal and human C. difficile isolates.

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“…When resistance against a given phage occurs, a new phage can be created to target and destroy the new strain. Some protocols on isolation of phage have been mentioned in literature (Garcia et al, 2009; Moineau and Fortier, 2009). The selection and screening a new phage is faster than the development of novel antibiotics which can take up to several years (Sulakvelidze et al, 2001).…”

Section: Introductionmentioning

confidence: 99%

The factors affecting effectiveness of treatment in phages therapy

2014

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In recent years, the use of lytic bacteriophages as antimicrobial agents controlling pathogenic bacteria has appeared as a promising new alternative strategy in the face of growing antibiotic resistance which has caused problems in many fields including medicine, veterinary medicine, and aquaculture. The use of bacteriophages has numerous advantages over traditional antimicrobials. The effectiveness of phage applications in fighting against pathogenic bacteria depends on several factors such as the bacteriophages/target bacteria ratio, the mode and moment of treatment, environmental conditions (pH, temperature...), the neutralization of phage and accessibility to target bacteria, amongst others. This report presents these factors and the challenges involved in developing phage therapy applications.

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Phage Production and Maintenance of Stocks, Including Expected Stock Lifetimes

Cited by 87 publications

References 21 publications

A scaled‐down model for the translation of bacteriophage culture to manufacturing scale

A scaled‐down model for the translation of bacteriophage culture to manufacturing scale

Characterization of Temperate Phages Infecting Clostridium difficile Isolates of Human and Animal Origins

The factors affecting effectiveness of treatment in phages therapy

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