The Protective Effect of Staphylococcus epidermidis Biofilm Matrix against Phage Predation

Abstract

Staphylococcus epidermidis is a major causative agent of nosocomial infections, mainly associated with the use of indwelling devices, on which this bacterium forms structures known as biofilms. Due to biofilms’ high tolerance to antibiotics, virulent bacteriophages were previously tested as novel therapeutic agents. However, several staphylococcal bacteriophages were shown to be inefficient against biofilms. In this study, the previously characterized S. epidermidis-specific Sepunavirus phiIBB-SEP1 (SEP1), which has a broad spectrum and high activity against planktonic cells, was evaluated concerning its efficacy against S. epidermidis biofilms. The in vitro biofilm killing assays demonstrated a reduced activity of the phage. To understand the underlying factors impairing SEP1 inefficacy against biofilms, this phage was tested against distinct planktonic and biofilm-derived bacterial populations. Interestingly, SEP1 was able to lyse planktonic cells in different physiological states, suggesting that the inefficacy for biofilm control resulted from the biofilm 3D structure and the protective effect of the matrix. To assess the impact of the biofilm architecture on phage predation, SEP1 was tested in disrupted biofilms resulting in a 2 orders-of-magnitude reduction in the number of viable cells after 6 h of infection. The interaction between SEP1 and the biofilm matrix was further assessed by the addition of matrix to phage particles. Results showed that the matrix did not inactivate phages nor affected phage adsorption. Moreover, confocal laser scanning microscopy data demonstrated that phage infected cells were less predominant in the biofilm regions where the matrix was more abundant. Our results provide compelling evidence indicating that the biofilm matrix can work as a barrier, allowing the bacteria to be hindered from phage infection.

Understanding the Complex Phage-Host Interactions in Biofilm Communities

“…The protective role of the biofilm matrix to phage predation was clearly demonstrated in a recent study by Melo et al (20) that assessed the interaction of a Staphylococcus epidermidis phage with different biofilm-associated host cell populations. The observations of this study were corroborated by confocal laser scanning microscopy (CLSM) data, which demonstrated that phageinfected cells appeared only in certain regions of the biofilm where lower amounts of matrix were present, evidencing that the biofilm matrix can serve as a shield to protect the embedded bacteria from viral attack (20). In fact, the spatial organization of the biofilm can be a determinant to the success of phage infection, as it may lead to limited mobility of cells that tend to organize in localized niches with different nutrient availability (21).…”

Virulence Factors in Coagulase-Negative Staphylococci

“…This extracellular matrix is fundamental for structural and functional roles as it provides stability against mechanical forces and creates a unique environment that is essential for the biofilm lifestyle [82,84]. Importantly, the matrix also plays a part in protection against disinfectants, antibiotics, immune cells activity, and bacteriophage (phage) predation [36,85,86]. Nevertheless, to ensure a functional organization, where nutrients are distributed into the deeper layers of the biofilm, channels need to be molded.…”

“…More recently, a S. epidermidisspecific phage (SEP1) was shown to infect different S. epidermidis planktonic cells, namely on exponential and stationary phases [528]. Although not able to infect intact biofilms, SEP1 was able to infect scraped biofilms, persister and biofilm-released cells, suggesting that its activity was affected by the biofilm matrix [85].…”

Improving Phage-Biofilm In Vitro Experimentation

“…The structure of still-intact biofilms, however, may reduce phage access to constituting bacteria, such as due to bacteria being buried beneath other bacteria [ 84 , 135 , 136 , 137 ] or due to biofilm matrix serving as a virion-diffusion barrier [ 138 , 139 ]. As a consequence, disrupting biofilm structure to separate individual cells for enumeration could make those bacteria more susceptible to phage adsorption (illustrated in Figure 6 , top panel), and bacteria released by biofilm disruption indeed become more susceptible to adsorption to phages that are added to cultures following that disruption [ 139 , 140 ]. To accurately assess numbers of biofilm-associated CFUs, it therefore can be crucial to prevent phages from adsorbing bacteria following biofilm disruption, and this concern could be particularly relevant if those phages are present within biofilms in relatively high numbers.…”