Bacteriophage-Resistant Mutant of Enterococcus faecalis Is Impaired in Biofilm Formation

Abstract

Enterococcus faecalis is a common gram-positive non-spore-forming bacterium in nature and is found in the upper respiratory tract, intestine, and mouth of healthy people. E. faecalis is also one of the common pathogens causing nosocomial infections and is resistant to several antibiotics commonly used in practice. Thus, treating drug-resistant E. faecalis with antibiotics is challenging, and new approaches are needed. In this study, we isolated a bacteriophage named EFap02 that targets E. faecalis strain EFa02 from sewage at Southwest Hospital. Phage EFap02 belongs to the Siphoviridae family with a long tail of approximately 210 nm, and EFap02 can tolerate a strong acid and alkali environment and high temperature. Its receptor was identified as the capsular polysaccharide. Phage-resistant mutants had loss-of-function mutations in glycosyltransferase (gtr2), which is responsible for capsular polysaccharide biosynthesis, and this caused the loss of capsular polysaccharide and interruption of phage adsorption. Although phage-resistant mutants against EFap02 can be selected, such mutants are impaired in biofilm formation due to the loss of capsular polysaccharide, which compromises its virulence. Therefore, this study provided a detailed description of the E. faecalis EFap02 phage with the potential for treating E. faecalis infection.

Keywords: Enterococcus faecalis; bacteriophage; biofilm; capsular polysaccharide; phage receptor; phage resistance.

Figure 1

Phage EFap02 general biological characteristics. (A) Phage plaques on a double-layer agar plate of EFap02. (B) TEM image of phage EFap02. EFap02 has an icosahedral head (length 44 nm ± 5, width 40 nm ± 5) and a long tail (210 nm ± 5). The scale bar in the right corner is 50 nm. (C) Different multiplicities of infection (MOI) of EFap02. When the MOI was 0.0001, the EFap02 titer was the highest. (D) One-step growth curve of EFap02.

Figure 2

Stability of EFap02 under different conditions. (A) EFap02 is stable over a broad range of pH values (pH values from 4 to 11). (B) EFap02 is viable over a broad range of temperatures. (C) EFap02 is sensitive to chloroform, and the titer drops to approximately 5 × 10⁶ PFU/ml after treatment.

Figure 3

Genome characterization of phage EFap02. Genome map of phage EFap02 and genetic characteristics. Circular genome visualization of EFap02 was performed using the CGView server database. Annotation of the specific function of ORFs was conducted using RAST and the BLASTP database. The first inner circle with the red histogram indicates the GC content, while the second inner circle with the purple and green histograms indicates the GC skew. The outer black circle indicates the predicted ORFs of phage EFap02.

Figure 4

Phylogenetic relationships of phages infecting Enterococcus faecalis. Terminase large subunit and the neighbor-joining method were used to construct the phylogenetic tree.

Figure 5

(A) Growth curve of the phage–bacteria coculture. Thirty hours after phage addition, the OD₆₀₀ of the phage and bacteria coculture began to increase. (B) Adsorption rate of phage onto three strains. This result indicates that EFap02 can adsorb EFa02 and EFa02R::gtr2 but not EFa02R (**** p < 0.0001). (C) The mutation site of the glycosyltransferase Group 2 gene in EFa02R; the mutant site is G209T, which results in the amino acid change from serine to isoleucine. (D) EOP assay indicates that EFap02 can infect EFa02 and EFa02R::gtr2 but not EFa02R.

Figure 6

(A) Capsule staining of EFa02, EFa02R, and EFa02R::gtr2. Capsule staining showed that EFa02 and EFa02R::gtr2 have the capsule, but EFa02R lost the capsule. (B) The biofilm mass of EFa02R was less than those of EFa02 and EFa02R::gtr2 (*p < 0.05) at the given time points.