Application of a bacteriophage lysin to disrupt biofilms formed by the animal pathogen Streptococcus suis

Abstract

Bacterial biofilms are crucial to the pathogenesis of many important infections and are difficult to eradicate. Streptococcus suis is an important pathogen of pigs, and here the biofilm-forming ability of 32 strains of this species was determined. Significant biofilms were completely formed by 10 of the strains after 60 h of incubation, with exopolysaccharide production in the biofilm significantly higher than that in the corresponding planktonic cultures. S. suis strain SS2-4 formed a dense biofilm, as revealed by scanning electron microscopy, and in this state exhibited increased resistance to a number of antibiotics (ampicillin, amoxicillin, ciprofloxacin, kanamycin, and rifampin) compared to that of planktonic cultures. A bacteriophage lysin, designated LySMP, was used to attack biofilms alone and in combination with antibiotics and bacteriophage. The results demonstrated that the biofilms formed by S. suis, especially strains SS2-4 and SS2-H, could be dispersed by LySMP and with >80% removal compared to a biofilm reduction by treatment with either antibiotics or bacteriophage alone of less than 20%; in addition to disruption of the biofilm structure, the S. suis cells themselves were inactivated by LySMP. The efficacy of LySMP was not dose dependent, and in combination with antibiotics, it acted synergistically to maximize dispersal of the S. suis biofilm and inactivate the released cells. These data suggest that bacteriophage lysin could form part of an effective strategy to treat S. suis infections and represents a new class of antibiofilm agents.

Fig. 1.

Capacity of 32 strains of S. suis to form biofilms as determined by 24-well microplate crystal violet assay. Ten strains were identified as forming significant biofilms on this basis. All assays were performed in triplicate, and the data are expressed as means ± standard deviations.

Fig. 2.

Scanning electron micrograph of S. suis SS2-4 biofilm formed after 72 h of growth. Aggregates and microcolonies of S. suis SS2-4 covered the surface of the coverslip to produce a compact and dense biofilm structure. Panels A to D show different magnifications.

Fig. 3.

SDS-PAGE, zymogram analysis, and Western blotting of purified LySMP. The size of LySMP is ca. 55 kDa as shown on the right. (A) SDS-PAGE stained with Coomassie brilliant blue stain. (B) Zymogram analysis. A boiled cell suspension of S. suis SS2-4 was overlaid on the gel for the band to act against; lytic activity appears as a translucent band on the opaque background. (C) Western blotting with mouse LySMP antibody.

Fig. 4.

(A) Effect of purified LySMP on S. suis biofilms 12 h after treatment. (B) Viable counts of biofilm-grown bacteria 12 h after LySMP treatment and without removal of suspension. (C) Viable counts of biofilm-grown bacteria 12 h after LySMP treatment with the free cells removed from suspension. The viable counts of washed residual bacteria are lower than those in panel B, showing that free viable cells remain in suspension after treatment with LySMP. (D) Turbidity reduction assay for planktonic bacteria after treatment with LySMP. (E) Viable counts of planktonic bacteria after treatment with LySMP. Comparison of these data with those in panels B and C shows that LySMP alone or mixed with antibiotics had a more significant bactericidal effect on planktonic bacteria than on biofilm-grown bacteria. All assays were performed in triplicate, and the data are expressed as means ± standard deviations.

Fig. 5.

Analysis of the effect of LySMP on S. suis SS2-4 biofilm disruption. Up to 80% biofilm degradation could be achieved by LySMP within 4 h. LySMP control (□), which is the product expressed by the plasmid lacking the lysin gene insert, had little influence on the biofilm. Purified LySMP (▴) eliminated almost all of the biofilms, and elution buffer (+) had little or no effect on the biofilms. Amoxicillin (×), ampicillin (▵), ciprofloxacin (⋄), kanamycin (○), and rifampin (♦) all had minimal effects. THB (−) had no effect. All assays were performed in triplicate, and the data are expressed as means ± standard deviations.

Fig. 6.

Photomicrographs of S. suis SS2-4 biofilms stained with crystal violet at different times after treatment with purified LySMP or elution buffer as a control. The preformed biofilm was progressively dispersed by LySMP, while the control showed little change.

Fig. 7.

Dose-effect relationship between LySMP and S. suis SS2-4 biofilm integrity. All assays were performed in triplicate, and the data are expressed as means ± standard deviations. LySMP was effective at relatively low doses; there was no reduction in efficacy between 400 and 50 IU under the conditions in these experiments.