The Staphylococcus epidermidis biofilm matrix confers protection against a phage that is highly active against dormant cells

Abstract

The complex heterogeneous biofilm structure confers to bacteria a key survival strategy. Biofilms are microbial communities that can attach to both abiotic and biotic surfaces and are highly implicated in the development of several nosocomial infections. The increasing need of innovative and efficient treatments to target these complex structures has led to a rising relevance on bacteriophages (phages) as a strategy for biofilm control and prevention. Theoretically, it is expected that due to the closeness of cells, phage infection of biofilms would be very efficient. However, several reported of phage/biofilm interaction had demonstrated a poor efficacy. In fact, the biofilm phenotype protects the cells against phage predation due to several factors: the dense biofilm matrix, the low metabolic state of biofilm cells and the quick development/multiplication of phage resistant variants are some of the features that impair phage-biofilm interactions. Staphylococci are amongst the more prevalent isolates from different types of infection. They usually form thick biofilms and are very difficult to target with antibiotics, being consequently a nice model to study phage-biofilm interactions. Several staphylococcal phages were isolated and tested against biofilms. Although some studies have demonstrated the efficacy of phage against biofilms, only a few were successful against staphylococcal biofilms. In this work we isolated a novel Staphylococcus epidermidis-specific phage, named SEP1. SEP1 has a broad lytic spectrum of activity and the rare ability to infect stationary phase cells. Indeed, phage-host interactions were analyzed by flow cytometry that showed that stationary-phase cells responded immediately to SEP1 addition. Quantitative PCR experiments corroborate that phage genes are expressed within 5 min of contact with stationary phase cells. However, SEP1 was inefficient against intact S. epidermidis biofilms. To understand the underlying factors impairing SEP1 inefficacy against biofilms, this phage was tested against distinct biofilm-derived bacterial populations. Interestingly, SEP1 was able to lyse both active and dormant biofilm cells, suggesting that the inefficacy on biofilm control resulted from biofilm composition and architecture. To demonstrate this hypothesis, SEP1 was tested in scraped biofilms resulting in a 2 log reduction in the number of viable cells, after six hours of infection. Our results provide compelling evidence indicating that the biofilm matrix can work as a decoy, allowing the bacterium to hinder phage infection.