Reply to Kumari and Singh, "Antibiofilm Activity of Small Molecules Produced by Staphylococcus epidermidis against Staphylococcus aureus".

Abstract

We appreciated Kumari and Singh´s comments on our paper (11). Although we have continued trying to determine the identity of the bioactive molecules present in Staphylococcus epidermidis cell-free conditioned media (CFCM), we are yet to be successful. Initially, phenol-soluble modulins (PSMs) were a potential candidate. However, the protease treatment used in our study, which did not abrogate CFCM activity, was somewhat similar to the treatment used by Bojet et al. (1), where supernatant activity due to PSMs was abolished by addition of a Streptomyces griseus protease mixture at 1 mg/ml at 37°C for 30 min followed by heat inactivation (80°C for 20 min). In our study, we treated CFCM with proteinase K (20 mg/ml) for 1 h at 37°C and also performed heat inactivation (100°C for 40 min) without any reduction of antibiofilm activity. In an independent study, Li et al. (2) also abolished supernatant activity due to PSMs by treating it with proteinase K (100 μg/ml) at 37°C for 1 h. Although these conditions are similar to the ones used in our study, it is important to note that the proteinase K concentration employed was 200-fold lower than the one we used and was still sufficient to eliminate PSM activity. Nevertheless, Kumari and Singh are correct when they state that the conformation of PSM aggregates can affect protease susceptibility, and we agree that it might be worth checking the effect of purified S. epidermidis PSMs on Staphylococcus aureus biofilms. Even if PSMs are involved, we believe that, altogether, our data suggest that the bioactivity detected is multifactorial and may involve multiple molecules present in S. epidermidis CFCM. Our transcriptome sequencing (RNA-seq) results showed a huge impact of CFCM on gene expression, repressing the expression of genes associated with both biofilm formation and quorum sensing. These two activities are thought to be inversely regulated, and inhibition of quorum sensing is associated with increased biofilm formation (3–5). Previous work has shown that PSMs from the same species prevent biofilm formation and promote biofilm disruption in both S. aureus and S. epidermidis (6–9). If PSMs prove to be one of the antibiofilm molecules in S. epidermidis CFCM, it will be interesting to determine their mechanism of action and activity during various interspecies interactions, as has been shown by Kumari and Singh regarding Rhizopus arrhizus (10). We thank the authors for all the suggestions for experiments, and we believe that testing CFCM of an agr mutant strain of S. epidermidis will be a valuable addition to future work.