Improving Phage-Biofilm In Vitro Experimentation

Abstract

Bacteriophages or phages, the viruses of bacteria, are abundant components of most ecosystems, including those where bacteria predominantly occupy biofilm niches. Understanding the phage impact on bacterial biofilms therefore can be crucial toward understanding both phage and bacterial ecology. Here, we take a critical look at the study of bacteriophage interactions with bacterial biofilms as carried out in vitro, since these studies serve as bases of our ecological and therapeutic understanding of phage impacts on biofilms. We suggest that phage-biofilm in vitro experiments often may be improved in terms of both design and interpretation. Specific issues discussed include (a) not distinguishing control of new biofilm growth from removal of existing biofilm, (b) inadequate descriptions of phage titers, (c) artificially small overlying fluid volumes, (d) limited explorations of treatment dosing and duration, (e) only end-point rather than kinetic analyses, (f) importance of distinguishing phage enzymatic from phage bacteriolytic anti-biofilm activities, (g) limitations of biofilm biomass determinations, (h) free-phage interference with viable-count determinations, and (i) importance of experimental conditions. Toward bettering understanding of the ecology of bacteriophage-biofilm interactions, and of phage-mediated biofilm disruption, we discuss here these various issues as well as provide tips toward improving experiments and their reporting.

Further Considerations on How to Improve Phage Therapy Experimentation, Practice, and Reporting: Pharmacodynamics Perspectives

“…Related to the problem of not describing dosing explicitly in terms of phage titers, in some cases authors seem to ambiguously describe the number of bacteria that phages have been applied to. 25 , 28 This number, however, is the denominator in most MOI calculations, resulting, as a consequence, in an inability for readers to estimate what phage titers have been used based on MOI information if titer information is not also explicitly supplied.…”

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

“…Phages can also affect host diversity, e.g., by “killing the winner”, and this keeps competitively dominant species or populations “in check” [ 7 ]. As such, they may be employed for the biological control of environments [ 8 , 9 ]. It is exactly this characteristic of phages that should be considered when “domesticating” them to control infecting or contaminating bacteria in patients, agriculture, or food processing.…”

“…There is a consensus that screening and selecting the right phages is of key importance for achieving successful therapeutic outcomes. Some suggest that the impact of phages on bacterial biofilms could be crucial toward understanding both phage and bacterial ecology [ 9 ]. However, the challenge is that there are no validated in vitro methods [ 31 , 32 ] to determine the phage characteristics that are important for predicting in vivo therapeutic efficacy [ 22 ] or performance [ 27 ], for instance, in view of future clinical trials that are desperately needed both to prove phage product efficacy and to determine the most effective phage therapy protocols [ 20 ].…”

Biological foundations of successful bacteriophage therapy

“…The finger‐like bacterial fimbriae and other adhesins that are important in biofilm initiation (Déziel et al , 2001; Pohlschroder & Esquivel, 2015; Maldarelli et al , 2016; Delerue & Ramamurthi, 2021) are also common phage receptors (Phage tail machines as sophisticated infection devices). Phages have proven useful against bacteria in biofilms (Abedon, 2019; Patey et al , 2019; Melo et al , 2020b; Petrovic Fabijan et al , 2021), but the study of these systems is difficult (Abedon et al , 2021b; Pires et al , 2021). Although phages often exhibit potent in vitro activity against bacteria in biofilms, effective biofilm eradication may require combination strategies (Verma et al , 2009; Seth et al , 2013; Tkhilaishvili et al , 2018; Henriksen et al , 2019; Morris et al , 2019), with failures attributed to difficulties in accessing target cells and the development of phage‐resistant subpopulations.…”