29.05.2025
research

The CEPEST phage collection

A collection of environmental phages infecting Pseudomonas putida isolated from Estonia (CEPEST).

P. putida is a biotechnologically relevant bacterium that can be used in bioremediation processes or bioproduction of polymers due to its robust and varied metabolism and high stress tolerance. The collection started when the need for research on the phage defence of P. putida necessitated the first isolation of phages.

Cover photo: TEM picture of the morphology of the nine first genera of CEPEST phages.

Photo is from the article: Isolation and characterization of a phage collection against Pseudomonas putida.

Figure 1. Graphical summary of phage isolation and the CEPEST phylogenietic tree of the first nine genera
Figure 1. Graphical summary of phage isolation and the CEPEST phylogenietic tree of the first nine genera (author: Environmental Microbiology)

Scientist collecting environmental samples.
About the collection:
The collection started in 2021 with the first few phages isolated in the Department of Genetics in IMCB and took off in 2022, when environmental samples for isolation were gathered by many students and staff of the IMCB. The phages were isolated by the enrichment method at 20 °C. The phage dsDNA was isolated and sequenced.

The first set of CEPEST phages was published in 2024, but the isolation is ongoing. In February 2024, the collection contained 67 phage isolates of 23 spp (95% genome identity) that belong to nine genera (70% genome identity). The phages are obligately lytic and have a very narrow host range. The environmental samples used for the isolation originated from Estonia, mainly from Tartu surroundings.

On the picture (by Andres Ainelo): scientist collecting environmental samples.

Accumulating knowledge on how P. putida defends itself against phages allows the use of sensitized strains for phage isolations, which has greatly increased the variety of our collection. In May 2025, the continuously updated collection already contains over 100 phages from 46 species cluster and 23 genera of phages. Mainly Estonian environmental samples have been used for the „phage hunt“, with the addition of some samples from Finland, Latvia and Poland. The origin of the environmental samples from Estonia is shown on the map (Firgure 2).

Figure 2. Map showing the locations of the environmental samples from Estonia of CEPEST collection as of March 2025
Figure 2. Map showing the locations of the environmental samples from Estonia of CEPEST collection as of March 2025 (author: Hedvig Tamman)

Figure 3. Plate with the bacterial lawn (dark surface) of wild-type P. putida PaW85 with see-through spots (plaques of phages, light)
Figure 3. Plate with the bacterial lawn (dark surface) of wild-type P. putida PaW85 with see-through spots (plaques of phages, light) (author: Alicja Lewańczyk)

Isolation procedure

For isolation, only a small sample size is needed. Just 50 – 100 mL of water or 20 g of soil is enough to use for isolation procedures. The sample needs to originate close to where the host could inhabit (for P. putida – soil and plant rhizosphere). Besides enrichment, also precipitation method is sometimes used (to isolate phages that have an inefficient infection cycle). After the enrichment or precipitation, phages are visualized by their ability to lyze bacteria, and thus, create clear spots (phage plaques) on a layer of bacteria (Firgure 3).

Published phage genomes are accessible under the NCBI BioProject #PRJNA1067406

Figure 4. Schematic representation of main steps of phage collection procedure
Figure 4. Schematic representation of main steps of phage collection procedure (author: Hedvig Tamman)
  • Brauer, A. et al. Isolation and characterization of a phage collection against Pseudomonas putida. Environmental Microbiology 26, e16671 (2024). DOI: 10.1111/1462-2920.16671
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