CkP1 bacteriophage, a S16-like myovirus that recognizes Citrobacter koseri lipopolysaccharide through its long tail fibers

Abstract

Citrobacter koseri is an emerging Gram-negative bacterial pathogen, which causes urinary tract infections. We isolated and characterized a novel S16-like myovirus CKP1 (vB_CkoM_CkP1), infecting C. koseri. CkP1 has a host range covering the whole C. koseri species, i.e., all strains that were tested, but does not infect other species. Its linear 168,463-bp genome contains 291 coding sequences, sharing sequence similarity with the Salmonella phage S16. Based on surface plasmon resonance and recombinant green florescence protein fusions, the tail fiber (gp267) was shown to decorate C. koseri cells, binding with a nanomolar affinity, without the need of accessory proteins. Both phage and the tail fiber specifically bind to bacterial cells by the lipopolysaccharide polymer. We further demonstrate that CkP1 is highly stable towards different environmental conditions of pH and temperatures and is able to control C. koseri cells in urine samples. Altogether, CkP1 features optimal in vitro characteristics to be used both as a control and detection agent towards drug-resistant C. koseri infections. KEY POINTS: • CkP1 infects all C. koseri strains tested • CkP1 recognizes C. koseri lipopolysaccharide through its long tail fiber • Both phage CkP1 and its tail fiber can be used to treat or detect C. koseri pathogens.

Keywords: Bacterial infection; Bacteriophage; Citrobacter; Control; Diagnostics; Long tail fiber.

Fig. 1

Phage CkP1 biophysical stability. Effect of different temperature (left) and pH (right) conditions on CkP1 phage survival after 24-h exposure by enumerating the number of phages. Averages and standard deviations of three repeated experiments are given. Significance was determined by Student t test (*P < 0.05)

Fig. 2

Phage CkP1 electron micrographs. Phage particles of CkP1 negatively stained with 2% uranyl acetate observed at TEM. Long tail fibers folded along the tail sheath (white arrow) or extended (black arrow) are indicated

Fig. 3

Phage CkP1 multiple genome alignment. A Whole-genome pairwise comparisons between the phage CkP1 (GenBank accession no. MW239124) and the Salmonella phage S16 (GenBank accession no. HQ331142). B Pairwise comparison of the long tail fiber region between the prototype E. coli phage T4 (GenBank accession no. NC_000866), C. koseri phage CkP1, and Salmonella phage S16. All comparisons were made using tBLASTX and visualized with EasyFig. CDSs are drawn to scale and colored according to their predicted function

Fig. 4

Phage CkP1 LFT binding of C. koseri cells. C. koseri cells (CK#1) suspended in 10 mM Tris–HCl (pH = 7) were incubated with GPF, GFP-gp267, GFP-gp267/gp268, or GFP-gp267trunc/gp268, washed twice and visualized under epifluorescence microscopy, in bright field or using the FITC filter

Fig. 5

Control of C. koseri in urine with CkP1. Antibacterial effect of CkP1 on urine samples artificially contained with C. koseri (CK#1). Phage was applied at a MOI of 0.1 and the antibacterial effect measured by reduction of bacterial CFUs/ml at 4 h and 24 h. Significance was determined by the Student t test (*P < 0.05). Averages and standard deviations of four repeated experiments are given