Isolation of novel lytic bacteriophages with the potential to be used for detection of viable Mycobacterium avium subsp. paratuberculosis

There has been considerable interest in phage-based detection of viable Mycobacterium avium subsp. paratuberculosis (MAP) over recent years because test results are obtained much more quickly than by culture. The aim of this study was to isolate novel lytic mycobacteriophages capable of infecting and lysing MAP cells that may have the potential to replace the D29 mycobacteriophage currently employed in such tests. Five environmental samples (soil, lake water and bovine faeces) were tested for the presence of lyticmycobacteriophages, directly and after enrichment. Initial plaque assays were performed with a lawn of fastgrowing Mycobacterium smegmatis, rather than MAP. Lytic phages were isolated from one enriched soil sample, and 10 plaques were randomly selected and individually amplified for further characterisation.Restriction enzyme digestion of phage DNAs with BamH1, Cla1, EcoR1, HaeIII and HindIII revealed that there may be up to four phage types amongst the 10 isolated. The host range was determined by performing plaqueassays with phage cocktails and 13 different Mycobacterium spp. Plaques were observed on lawns of M. smegmatis, M. fortuitum and M. marinum. Ability to infect and lyse slow-growing MAP cells could not be determined by plaque assay, so was demonstrated by coating magnetic beads with selected phage isolates and carrying out PhMS-PCR. A PCR product was obtained after phagomagnetic separation of a 3x10⁴ cfu/ml MAP solution and incubation for 1 hour at 37°C. Potentially several different lytic mycobacteriophages havebeen isolated and ongoing research is comparing their performance relative to the D29 mycobacteriophage. This will assess what benefits in terms of detection sensitivity or shorter time to results, if any, they could provide if incorporated into phage-based assays to rapidly detect viable MAP.