Browsing by Author "Dirks, Anna"

Antibiotic resistance is an increasing, terrible threat to human health, leading to a growing need for alternative therapies. Phage therapy, using bacterial viruses to fight infections, is a promising alternative to antibiotic therapy. However, several obstacles need to be overcome. Regrettably, phage therapy remains inaccessible to many laboratories worldwide due to the need for expensive machinery to establish sensitivity of bacteria to phage. Moreover, shipping phages between laboratories remains challenging. In the current study a device-free bacteriophage typing PhagoGramAssay was developed. In the assay bacteria suspended in soft agar were poured onto a 60-well Terasaki plate containing phages suspended in fibrillated nanocellulose separated from the bacteria by a seal. Phages were released into the bacterial agar layer by puncturing the seal to test for sensitivity observable with the naked eye. Contrast between lysis zone and bacterial lawn was enhanced using 2,3,5-triphenyltetrazolium chloride. Optimized parameters included the amount of bacteria and phage added, volume of phage suspension, agar percentage and thickness and puncturing tool size. In addition, a prototype of such a puncturing tool was developed. The optimized PhagoGramAssay was tested using several bacteria-phage combinations. Moreover, infectivity and stability of phages stored on Terasaki plates was followed over the course of 4 weeks. The optimal bacterial amount added was found to be a 1:300 dilution in soft agar taken from a OD600 = 1 culture. Phage suspensions used in the assay were found to need to have a titer of at least 108 PFU/ml in the original lysate, with 8 µl of 1:10 dilution in fibrillated nanocellulose present in the wells. Optimal agar conditions were found to be 0.4% – 0.5% (w/v) with a thickness of 2 mm – 3 mm. The optimal puncturing tool shape was found to be a slit with a thickness of 0.5 mm. When using these conditions sensitivity could be established for a vast number of bacteria-phage combinations. All phages remained stable and infective over the course of 4 weeks . The newly developed PhagoGramAssay can be further developed into a kit-like phage typing assay that would enable laboratories to test for sensitivity on site whenever a multi-drug resistant bacterial strain is isolated from a patient sample, effectively making phage therapy accessible to laboratories that cannot afford expensive machinery. Additionally, the use of fibrillated nanocellulose should enable laboratories to exchange phages. The final form of such a kit, however, is dependent on manufacturers and investors and may need to be adjusted accordingly.