Isolation of Bacteriophages of the Anaerobic Bacteria Bacteroides

Abstract

Here we describe the detection, enumeration, and isolation of bacteriophages infecting Bacteroides. The method is based on the infection of Bacteroides host strains and the production of visible plaques in a confluent lawn of the host strain using the double-layer agar method. This is a straightforward methodology that can be applied for the detection, enumeration and isolation of bacteriophages for other anaerobic bacteria, using an appropriate host strain and culture conditions. In the case of bacteriophages of Bacteroides the results can be obtained in less than 24 h, although the time could vary depending on the growth rate of the host strain.

Morphological and Genetic Characterization of Eggerthella lenta Bacteriophage PMBT5

“…There are only few studies describing successful methods of virulent phage isolation for intestinal bacteria under anaerobic conditions [ 29 , 30 ]. One reason for this could be the lack of a suitable isolation standard [ 28 ] and of appropriate isolation protocols.…”

“…Most studies on gut phages are based on metagenomic analyses of phages, rather than on the isolation of the phages themselves or knowledge of their hosts [ 15 , 23 , 24 , 25 , 26 , 27 ] One reason for this could be the lack of a clear standard method for isolating gut phages [ 28 ]. The few studies which so far have described the isolation of phages capable to form visible plaques under anaerobic conditions were focused on phages of the genera Bacteroides [ 25 , 29 , 30 ] and Clostridium [ 31 ], and the phages infecting the latter genera are almost exclusively temperate and were induced from the bacterial host genome. A member of the most abundant human-associated crAssphage group, phage ΦCrAss001, was only recently isolated for Bacteroides intestinalis [ 30 ].…”

Inflammation and bacteriophages affect DNA inversion states and functionality of the gut microbiota

“…After incubation, an aliquot of the culture was treated with chloroform (1:10 (v;v), vigorously mixed for 5 min and centrifuged at 16,000xg for 5 min. 88 The supernatant containing the phage suspensions were further filtered through low protein binding 0.22 μm pore size polyethersulfone (PES) membrane filters (Millex-GP, Millipore, Bedford, Massachusetts), diluted and plated as indicated in the previous paragraph to verify the uniformity of the plaques. Then, one well differentiated plaque was stabbed and the whole operation was repeated to obtain a high titer, over 1x10 9 plaque forming units (PFU), phage suspensions.…”

Inflammation and bacteriophages affect DNA inversion states and functionality of the gut microbiota

“…Then 1 ml of a culture of B. fragilis NCTC 9343 in exponential growth was inoculated into the tube, which was then incubated for 18h at 37 °C. After incubation, an aliquot of the culture was treated with chloroform (1:10 (v;v), vigorously mixed for 5 minutes and centrifuged at 16,000×g for 5 minutes 75 . The supernatant containing the phage suspensions were further filtered through low protein binding 0.22 µm pore size polyethersulfone (PES) membrane filters (Millex-GP, Millipore, Bedford, Massachusetts), diluted and plated as indicated in the previous paragraph to verify the uniformity of the plaques.…”