Targeted dual-receptor phage cocktail against Cronobacter sakazakii: insights into phage-host interactions and resistance mechanisms

Abstract

Introduction: Cronobacter sakazakii is a notorious foodborne pathogen, frequently contaminating powdered infant formula and causing life-threatening diseases in infants. The escalating emergence of antibiotics-resistant mutants has led to increased interest in using bacteriophage as an alternative antimicrobial agent.

Methods: Two phages, CR8 and S13, were isolated from feces and soil samples and their morphology, physiology, and genomics were characterized. Phage receptor was determined using deletion mutants lacking flgK, rfaC, fhuA, btuB, lamb, or ompC genes, followed by complementation. Phage-resistant mutants were analyzed for phenotypic changes and fitness trade-offs using motility assays and Caco-2 cell invasion models.

Results: CR8 and S13 were identified as members of Caudoviricetes. Phage CR8 and phage S13 utilize flagella and LPS, respectively, to adhere to host cells. Bacterial challenge assay demonstrated delayed emergence of the resistant mutant as well as stronger lytic activity of a phage cocktail consisting of CR8 and S13 than the single phage treatment. Phenotypic analysis of the phage cocktail resistant strain, designated as CSR strain, revealed that the resistance resulted from the impaired receptor proteins for phage, such as defects in motility and alteration in LPS structure. CSR strain exhibited significant attenuation in invading human intestinal epithelial Caco-2 cells compared to WT cells.

Conclusion: This study demonstrates that the development of the phage cocktail targeting distinct host receptors can serve as a promising antimicrobial strategy to effectively control C. sakazakii.

Keywords: Cronobacter sakazakii; bacteriophage therapy; fitness trade-off; flagella; lipopolysaccharides; phage cocktail; phage resistance.

Figure 1

Morphology of phage CR8 and S13. TEM images of phage CR8 (A), S13 (B) with contractile tail. Scale bar, 100 nm.

Figure 2

Genomic features of phage CR8 and S13. Genome maps of CR8 (A) and S13 (B). Predicted ORFs in the genome were annotated and colored by functional groups as follows: phage structure and packaging (green), nucleotide metabolism (magenta), DNA replication/modification (blue), host lysis (red), RNA modification (pink), transcription (teal), cell signal (purple) and additional function (orange).

Figure 3

Flagella and LPS are receptors for CR3 and S13, respectively. The sensitivity of the bacteria to CR3 (A) and S13 (B) phages was determined by spotting assay. Complementation of flagella and LPS completely restored the phage susceptibility. Noted strains are WT + p, wild type harboring pBAD18; ΔflgK + p, ΔflgK harboring pBAD18; ΔflgK + pflgK, ΔflgK expressing flgK, ΔrfaC + p, ΔflgK harboring pBAD18; ΔrfaC + prfaC ΔrfaC expressing rfaC. (C,D) adsorption kinetics of phage CR8 (C) and S13 (D) against C. sakazakii ATCC 29544 (circle), ΔflgK (square) and ΔrfaC (triangle). All bacterial strains grown exponentially were challenged with each phage at an MOI of 0.01. Data displayed are mean ± SEM for three biological replicates. P_t, phage titer (PFU/ mL) at the indicated time; P₀, initial phage titer.

Figure 4

Bacterial challenge assay with phages CR8 and S13 against C. sakazakii ATCC 29544. Each phage or a phage cocktail was added at a MOI of 1 to the bacterial culture after 1.5 h incubation. Bacterial growth was determined by measuring absorbance at 600 nm hourly. Data shown and error bars are means ± SEM for three biological replicates at 1 h increments.

Figure 5

Characterization of CSR strain resistant to a phage cocktail infection. (A) Motility of C. sakazakii strains on semisolid agar plates: WT, ΔflgK, ΔrfaC and CSR, respectively. (B) TEM analysis. Scale bars, 500 nm. (C) LPS profiles. LPS fractions were extracted using hot phenol-water method, fractionated by DOC-PAGE and fluorescently stained. (D) Autoaggregation of the CSR strain. Bacterial cultures of WT, ΔflgK, ΔrfaC, and CSR strain were diluted at an OD₆₀₀ of 1.5 using fresh TSB broth and incubated statically at room temperature. The data displayed are a representative of three biological replicates.

Figure 6

Attenuation in invasion resulted from the acquisition of phage resistance. Invasive ability into human epithelial cells, Caco-2. Confluent monolayers of Caco-2 epithelial cells were challenged with WT C. sakazakii or the CSR strain, incubated for 1.5 h, followed by gentamicin treatment at a concentration of 100 μg/mL for a further 1.5 h. The Caco-2 cells were lysed with 1% TritonX-100 to harvest the intracellular bacteria. The data shown and error bars are mean ± SEM for three biological replicates. Statistical significance was conducted using unpaired two- tailed Student’s t test and significance displayed as ****p < 0.0001.