Isolation of a Novel Phage with Activity against Streptococcus mutans Biofilms

Abstract

Streptococcus mutans is one of the principal agents of caries formation mainly, because of its ability to form biofilms at the tooth surface. Bacteriophages (phages) are promising antimicrobial agents that could be used to prevent or treat caries formation by S. mutans. The aim of this study was to isolate new S. mutans phages and to characterize their antimicrobial properties. A new phage, ɸAPCM01, was isolated from a human saliva sample. Its genome was closely related to the only two other available S. mutans phage genomes, M102 and M102AD. ɸAPCM01 inhibited the growth of S. mutans strain DPC6143 within hours in broth and in artificial saliva at multiplicity of infections as low as 2.5x10-5. In the presence of phage ɸAPCM01 the metabolic activity of a S. mutans biofilm was reduced after 24 h of contact and did not increased again after 48 h, and the live cells in the biofilm decreased by at least 5 log cfu/ml. Despite its narrow host range, this newly isolated S. mutans phage exhibits promising antimicrobial properties.

Fig 1. Transmission electron micrograph of phage ɸAPCM01, stained with uranyl acetate.

Fig 2. One-step growth curve of phage ɸAPCM01 with S. mutans strain DPC6143 in BHI broth at 37°C.

Three independent experiments were carried out. Error-bars indicate standard deviation.

Fig 3. Effect of phage ɸAPCM01 on a growing culture of S. mutans DPC6143.

(a) Phage activity was assessed by OD_600nm measures after 18 h of contact in BHI broth at 37°C. Experiments were performed in a 96-well microplate, and each condition was tested in 8 wells of the microplate. Enumerations were performed in triplicate for each tested MOI (x), with a detection threshold of 20 cfu/ml. (b) Killing curves were assessed by OD_600nm measures every 15 min for 18 h at MOI of 2.5x10^-5 (○), 2.5x 10⁻⁴ (■), 2.5x 10⁻³ (□), 2.5x 10⁻² (◆), 0.25 (◇), 2.5 (☓), 25 (▲), 250 (△), no phage (●), and sterile medium ($•$). (c) Phage activity was assessed by OD_600nm measures and by bacterial counts (x) performed in triplicate after 18 h of contact in artificial saliva. MOI: multiplicity of infection; AS: sterile artificial saliva. Error-bars indicate standard deviation. ***p<0.001.

Fig 4. Effect of phage ɸAPCM01 on a 48 h-biofilm formed by S. mutans DPC6143, after 24 h (■) and 48 h (■) of contact between phage and biofilm.

(a) Biofilm metabolic activity was assessed by OD_492nm measures after treatment with XTT supplemented with menadione. Experiments were performed in 96-well microplates, and each condition was tested in 8 wells of the microplate. (b) Bacterial counts in biofilms were performed in triplicate after contact with the phage. *p<0.05; **p<0.01; ***p<0.001. Error-bars indicate standard deviation.

Fig 5. Genomic organization of ɸAPCM01 compared with that of phages M102 and M102AD.

Each arrow represents an ORF, with the colour representing the putative function of the encoded protein indicated on the right. Percent amino acid identity between adjacent genomes is colour coded as outlined to the left.

Fig 6. Molecular phylogenetic analysis by Maximum Likelihood method of endolysins in S. mutans phage ɸAPCM01.

Comparison of ORF19 (a) and ORF20 (b) of ɸAPCM01 to other endolysins in streptococcal phages. Numbers indicate branches support based on 1000 bootstrap replications.