Effective removal of staphylococcal biofilms by the endolysin LysH5

Abstract

Staphylococcal biofilms are a major concern in both clinical and food settings because they are an important source of contamination. The efficacy of established cleaning procedures is often hindered due to the ability of some antimicrobial compounds to induce biofilm formation, and to the presence of persister cells, a small bacterial subpopulation that exhibits multidrug tolerance. Phage lytic enzymes have demonstrated antimicrobial activity against planktonic and sessile bacteria. However, their ability to lyse and/or select persister cells remains largely unexplored so far. In this work, the lytic activity of the endolysin LysH5 against Staphylococcus aureus and Staphylococcus epidermidis biofilms was confirmed. LysH5 reduced staphylococcal sessile cell counts by 1-3 log units, compared with the untreated control, and sub-inhibitory concentrations of this protein did not induce biofilm formation. LysH5-surviving cells were not resistant to the lytic activity of this protein, suggesting that no persister cells were selected. Moreover, to prove the lytic ability of LysH5 against this subpopulation, both S. aureus exponential cultures and persister cells obtained after treatment with rifampicin and ciprofloxacin were subsequently treated with LysH5. The results demonstrated that besides the notable activity of endolysin LysH5 against staphylococcal biofilms, persister cells were also inhibited, which raises new opportunities as an adjuvant for some antibiotics.

Figure 1. Removal of 24 h-old biofilms of S. aureus and S. epidermidis.

Biofilms of S. aureus 15981, ISP479r, V329, 132, IPLA1 and IPLA16 and S. epidermidis B, YLIC17and DG2n were treated with 0.15 µM of LysH5 (light grey) and 0.2 µM of lysostaphin (white) during 6 hours. Alternatively, biofilms were treated with 0.15 µM of LysH5 during 6 hours, followed by another treatment with the endolysin for 12 hours (gross line in X axis). Control biofilms are represented in black. Adhered cell counts were expressed as log CFU/well. Bacteria detection threshold (<10 CFU/ml). Means and standard deviations were calculated from two biological replicates. Bars having an asterisk are significantly different from the control (ANOVA; P<0.05) and bars with a lower case ‘a’ indicates a significantly difference between the lysostaphin treatment and the treatment with LysH5 (ANOVA; P<0.05).

Figure 2. LTSEM micrographs of 24 h-old biofilms formed by S. aureus and S. epidermidis.

Not treated biofilms of S. aureus 15981 and S. epidermidis YLIC17 are represented in A and C, respectively; biofilms after treatment with LysH5 are represented in B and D.

Figure 3. Behavior of 24 h-old biofilms formed by S. aureus and S. epidermidis strains, grown in the presence of sub-inhibitory concentrations of LysH5.

Three strains were selected as representative of biofilm behavior (A) Prevention of the biofilm formation (S. aureus 15981); (B) Biofilm reduction (S. aureus ISP479r) and (C) no effect in biofilm formation (S. aureus IPLA16). Biofilm formation was expressed as relative absorbance (595 nm) of crystal violet stained cultures (treated/untreated cultures)(•). LysH5 concentration is expressed in µM. Each value is the mean ± standard deviation of two biological replicates.

Figure 4. S. aureus persister cells selection and elimination by LysH5.

(A) Activity of different concentrations of LysH5 against exponential cultures of S. aureus 15981. Means and standard deviations were calculated from two independent assays. Bacteria detection threshold (<10 CFU/ml). (B) Exponential cultures of S. aureus 113 treated with 2 µg/ml of rifampicin (•), and 3 µg/ml of ciprofloxacin (▴) for 4 h to select persister cells and subsequent treatment with 0.5 µM LysH5 (○ and Δ, respectively). Bacteria detection threshold (<10 CFU/ml). Each value represents the mean ± standard deviation of two biological replicates.