Development of a qPCR platform for quantification of the five bacteriophages within bacteriophage cocktail 2 (BFC2)

To determine phage titers accurately, reproducibly and in a non-laborious and cost-effective manner, we describe the development of a qPCR platform for molecular quantification of five phages present in bacteriophage cocktail 2 (BFC2). We compared the performance of this molecular approach, with regard to quantification and reproducibility, with the standard culture-based double agar overlay method (DAO). We demonstrated that quantification of each of the five phages in BFC2 was possible by means of qPCR, without prior DNA extraction, but yields were significantly higher in comparison to DAO. Although DAO is assumed to provide an indication of the number of infective phage particles, whereas qPCR only provides information on the number of phage genomes, the difference in yield (qPCR/DAO ratio) was observed to be phage-dependent and appeared rather constant for all phages when analyzing different (freshly prepared) stocks of these phages. While DAO is necessary to determine sensitivity of clinical strains against phages in clinical applications, qPCR might be a valid alternative for rapid and reproducible quantification of freshly prepared stocks, after initial establishment of a correction factor towards DAO

A case of phage therapy against pandrug-resistant Achromobacter xylosoxidans in a 12-year-old lung-transplanted cystic fibrosis patient

Bacteriophages are a promising therapeutic strategy among cystic fibrosis and lung-transplanted patients, considering the high frequency of colonization/infection caused by pandrug-resistant bacteria. However, little clinical data are available regarding the use of phages for infections with Achromobacter xylosoxidans. A 12-year-old lung-transplanted cystic fibrosis patient received two rounds of phage therapy because of persistent lung infection with pandrug-resistant A. xylosoxidans. Clinical tolerance was perfect, but initial bronchoalveolar lavage (BAL) still grew A. xylosoxidans. The patient's respiratory condition slowly improved and oxygen therapy was stopped. Low-grade airway colonization by A. xylosoxidans persisted for months before samples turned negative. No re-colonisation occurred more than two years after phage therapy was performed and imipenem treatment was stopped. Whole genome sequencing indicated that the eight A. xylosoxidans isolates, collected during phage therapy, belonged to four delineated strains, whereby one had a stop mutation in a gene for a phage receptor. The dynamics of lung colonisation were documented by means of strain-specific qPCRs on different BALs. We report the first case of phage therapy for A. xylosoxidans lung infection in a lung-transplanted patient. The dynamics of airway colonization was more complex than deduced from bacterial culture, involving phage susceptible as well as phage resistant strains

Evaluation of the Stability of Bacteriophages in Different Solutions Suitable for the Production of Magistral Preparations in Belgium

In Belgium, the incorporation of phages into magistral preparations for human application has been permitted since 2018. The stability of such preparations is of high importance to guarantee quality and efficacy throughout treatments. We evaluated the ability to preserve infectivity of four different phages active against three different bacterial species in five different buffer and infusion solutions commonly used in medicine and biotechnological manufacturing processes, at two different concentrations (9 and 7 log pfu/mL), stored at 4 °C. DPBS without Ca2+ and Mg2+ was found to be the best option, compared to the other solutions. Suspensions with phage concentrations of 7 log pfu/mL were unsuited as their activity dropped below the effective therapeutic dose (6-9 log pfu/mL), even after one week of storage at 4 °C. Strong variability between phages was observed, with Acinetobacter baumannii phage Acibel004 being stable in four out of five different solutions. We also studied the long term storage of lyophilized staphylococcal phage ISP, and found that the titer could be preserved during a period of almost 8 years when sucrose and trehalose were used as stabilizers. After rehydration of the lyophilized ISP phage in saline, the phage solutions remained stable at 4 °C during a period of 126 days