A rapid and simple protocol for the isolation of bacteriophages from coastal organisms

Graphical abstract


Background information
Although large numbers of bacteriophages are reported from seawater, the successful recovery of bacteriophages capable of killing specific pathogenic bacteria is dependent on host density and surrounding environmental conditions. Most existing protocols to recover lytic bacteriophages from coastal zones require large volumes of water inoculated with host bacteria to enrich the target bacteriophages followed by the concentration of viral particles through tangential flow filtration or by ultracentrifugation [1,2]. These methods lack efficiency when bacteriophage numbers are low. Conversely, filter feeding bivalves such as mussels concentrate a large number of microorganisms, including bacteria and bacteriophages due to their constant filtration activity, the large volume of water filtered over time and their internal environmental stability. Our method takes advantage of the natural ability of filter-feeders to concentrate microorganisms to allow the recovery of large numbers of bacteriophages using a quick and easy protocol (See Fig. 1). We have validated our method by searching for specific bacteriophages capable of lysing the bacterium Vibrio ordalii from the Chilean coastal zone. We selected an abundant filter-feeding intertidal mussel (Perumytilus purpuratus) and recovered a high number of bacteriophages. Vibrio ordalii was grown at 20 C in tryptone-soy agar (TSA) or tryptone-soy broth (TSB) (Thermo, Oxoid) in which 50 % of the water was replaced with filtersterilized seawater.

Bacterial counts
To count the bacterial cells in the suspension, the sample was treated with DAPI fluorescent dye (4 0 ,6-diamine-2-phenylindole-dilactate, Merck) [3]. After DAPI staining, three 10 mL serial dilutions (1/10, 1/100 and 1/1000) were performed and 4 % paraformaldehyde was added to each. The solutions were filtered using 0.22 mm pore size polycarbonate membranes (Merck, Millipore), and the filters were allowed to dry for 15 min over absorbent paper. 15 mL of a 2 mg/L DAPI solution, was placed on a slide, covered with the filters and incubated in a dark damp box for 15 min at room temperature. The filters were removed and washed in 96 % ethanol for 10 s and further dried on absorbent paper for one hour at 37 C in the dark. Dry filters were mounted on a slide with 10 mL of Vecta Shield mounting medium (Vector Labs) and covered with a clean cover glass. Stained cells were observed in a fluorescence microscope with a filter set of $359 nm l excitation and $461 nm l emission, using a 100x lens with a standardized 10 Â 10 mm quadrant in the eyepiece as counting grid. A minimum of 500 cells was counted for each sample and the dilution was chosen that allow the required fields to be stated. The total number of cells per sample was estimated by establishing the ratio between counting area and the total effective filtration area of the polycarbonate filter. This value was multiplied by the number of counted cells and the dilution factor and divided by the total filtered volume as showed in the equation: where, A is the filtration area; X is the number of counted cells; f is the dilution factor, and a is the area of the field observed under the microscope.

Isolation of bacteriophages
Benthic filter-feeder mollusks (mytilid mussels) were collected from the coastal zone of Antofagasta, Chile. These were placed in sterile containers and processed on the same day of collection. First, 100 g of soft tissue were extracted from the shells and homogenized, adding 100 mL of seawater into a sterile blender for 1 min. The product was centrifuged for 10 min at 4000 g and $ 50 mL of supernatant was carefully collected, avoiding the movement or inclusion of solid debris. At this stage, a viscous supernatant was obtained due to the presence of elevated amounts of suspended solids, in addition to microorganisms and viral particles. Bacterial counts were conducted during this stage. Two mL of supernatant was sequentially filtered through nitrocellulose membranes of 5 mm, 0.45 mm, and 0.22 mm pore size (Millipore) to eliminate major particles. 100 mL of chloroform was added to 1 mL of the filtrate to remove the remaining bacterial remnants. After 30 s of vortex shaking, the tubes were centrifuged for 5 min at 10,000 g. Finally, 500 mL of the aqueous phase corresponding to the virion suspension was recovered, carefully avoiding the uptake of chloroform.

Bacteriophage counting
Specific lytic phages were isolated by the double layer agar culture technique [1,4]. For this, 10 mM of MgCl 2 was added to the culture medium (TSB), to provide the divalent cations required for phage multiplication [1]. Two-layer gel-plates were prepared using Bacto-Agar (Difco) as a gelling agent at two different concentrations: 1.5 % for the lower layer and 0.7 % for the upper layer. The lower layer media was autoclaved and poured into Petri dishes. The upper layer media was autoclaved and maintained in glass tubes at 45 C prior to use (for no more than 1 h). For the cultivation of phages, a bacterial inoculum in the exponential phase was prepared. Subsequently, three serial dilutions of the enriched (putative) virion samples (1/10, 1/100 and 1/1000) were prepared and 1 mL of each of them was mixed with 0.1 mL of the bacterium culture in the early exponential phase and 3 mL of the 0.7 % agar. The mixtures were homogenized and poured onto previously prepared plates with the solid (low layer) agar. Finally, the plates were incubated overnight at 20 C, after this time, the lysis plates counts were performed, establishing the number of plates forming units (PFU) for each dilution.

Selection of lytic phages
At least 30 clearly defined lysis plates were selected by gently picking them with sterile wooden sticks (toothpicks), and used to inoculate 1 mL of early exponential bacterial culture containing 10 mM MgCl 2 . They were incubated overnight at 20 C and the isolation process was repeated at least three times to ensure the presence of a single lytic phage per tube. The phages obtained from the process were named according to the nomenclature norms proposed by Kropinski et al. [5], following the detail published in the REBASE database (http://rebase.neb.com/rebase/rebase.html). Presence of genes associated with a temperate life cycle (i.e. integration/excision genes) must be evaluated by molecular techniques (PCR-Sequencing) to finally ensure that our isolated is an obligately lytic bacteriophage [6].

Enrichment and phages selection
As an optional step to increase the number of specific phages for a bacterium, 200 mL of the suspension of (putative) virions could be added to 20 mL of early exponential bacterial culture, supplemented with 10 mM of MgCl2. Then, keep it at 20 C until observing a medium clarification, for a maximum period of 48 h. To recover the phages, follow the procedure described previously.

Validation
We undertook a search for lytic bacteriophages for the bacterial strain Vibrio ordalii ATCC-33509 in the intertidal zone of San Jorge bay (Antofagasta, Chile, 23 38 0 00 00 S 70 24 0 00 00 W). We sampled at five points along a 50 m transect, with each point separated by 10 m. At each point, 5 L of sea water and 20 bivalve mussel (Perumytilus purpuratus) were collected and subsequently processed to assess bacterial counts and lytic phage recovery for Vibrio ordalii, following the protocol detailed above. Water samples were subject to a prior enrichment stage to increase the number of lytic specific phages for Vibrio ordalii. Mean (AE SD) cell counts were 1E + 09 (AE 2E + 08) cell/mL in Perumytilus and 7E + 5 (AE3E+05) cell/mL in the surrounding sea water.
Lytic activity against Vibrio ordalii was observed for all the extracts from Perumytilus, with a mean (AESD) total count of lysis plaques, of 4E + 4 (AE8E+03) PFU/mL (Fig. 2). No lysis plaques were observed from seawater samples (Fig. 2b). A total of 30 well-defined lysis plates were selected and separated from the rest (Fig. 2a). Seven of the selected plaques (putative phages) still showed lytic activity after the third re-inoculation. We selected one of these re-inoculated plaques to be sequenced and analyzed, resulting in the identification of a lytic bacteriophage belonging to the Siphoviridae family [7]. Phages with the capacity to lyse other native bacterial strains previously isolated from mussels were also found and are currently being evaluated.