Optimization of Phage-Antibiotic Combinations against Staphylococcus aureus Biofilms.

Phage therapy has gained attention due to the spread of antibiotic-resistant bacteria and narrow pipeline of novel antibiotics. Phage cocktails are hypothesized to slow the overall development of resistance by challenging the bacteria with more than one phage. Here, we have used a combination of plate-, planktonic-, and biofilm-based screening assays to try to identify phage-antibiotic combinations that will eradicate preformed biofilms of Staphylococcus aureus strains that are otherwise difficult to kill. We have focused on methicillin-resistant S aureus (MRSA) strains and their daptomycin-nonsusceptible vancomycin-intermediate (DNS-VISA) derivatives to understand whether the phage-antibiotic interactions are altered by the changes associated with evolution from MRSA to DNS-VISA (which is known to occur in patients receiving antibiotic therapy). We evaluated the host range and cross-resistance patterns of five obligately lytic S. aureus myophages to select a three-phage cocktail. We screened these phages for their activity against 24-h bead biofilms and found that biofilms of two strains, D712 (DNS-VISA) and 8014 (MRSA), were the most resistant to killing by single phages. Specifically, even initial phage concentrations of 107 PFU per well could not prevent visible regrowth of bacteria from the treated biofilms. However, when we treated biofilms of the same two strains with phage-antibiotic combinations, we prevented bacterial regrowth when using up to 4 orders of magnitude less phage and antibiotic concentrations that were lower than our measured minimum biofilm inhibitory concentration. We did not see a consistent association between phage activity and the evolution of DNS-VISA genotypes in this small number of bacterial strains. IMPORTANCE The extracellular polymeric matrix of biofilms presents an impediment to antibiotic diffusion, facilitating the emergence of multidrug-resistant populations. While most phage cocktails are designed for the planktonic state of bacteria, it is important to take the biofilm mode of growth (the predominant mode of bacterial growth in nature) into consideration, as it is unclear how interactions between any specific phage and its bacterial hosts will depend on the physical properties of the growth environment. In addition, the extent of bacterial sensitivity to any given phage may vary from the planktonic to the biofilm state. Therefore, phage-containing treatments targeting biofilm infections such as catheters and prosthetic joint material may not be merely based on host range characteristics. Our results open avenues to new questions regarding phage-antibiotic treatment efficiency in the eradication of topologically structured biofilm settings and the extent of eradication efficacy relative to the single agents in biofilm populations.

Eradication of Biofilm-Mediated Methicillin-Resistant Staphylococcus aureus Infections In Vitro: Bacteriophage-Antibiotic Combination.

Bacterial biofilms are difficult to eradicate and can complicate many infections by forming on tissues and medical devices. Phage+antibiotic combinations (PAC) may be more active on biofilms than either type of agent alone, but it is difficult to predict which PAC regimens will be reliably effective. To establish a method for screening PAC combinations against Staphylococcus aureus biofilms, we conducted biofilm time-kill analyses (TKA) using various combinations of phage Sb-1 with clinically relevant antibiotics. We determined the activity of PAC against biofilm versus planktonic bacteria and investigated the emergence of resistance during (24 h) exposure to PAC. As expected, fewer treatment regimens were effective against biofilm than planktonic bacteria. In experiments with isogenic strain pairs, we also saw less activity of PACs against DNS-VISA mutants versus their respective parentals. The most effective treatment against both biofilm and planktonic bacteria was the phage+daptomycin+ceftaroline regimen, which met our stringent definition of bactericidal activity (>3 log10 CFU/mL reduction). With the VISA-DNS strain 8015 and DNS strain 684, we detected anti-biofilm synergy between Sb-1 and DAP in the phage+daptomycin regimen (>2 log10 CFU/mL reduction versus best single agent). We did not observe any bacterial resensitization to antibiotics following treatment, but phage resistance was avoided after exposure to PAC regimens for all tested strains. The release of bacterial membrane vesicles tended to be either unaffected or reduced by the various treatment regimens. Interestingly, phage yields from certain biofilm experiments were greater than from similar planktonic experiments, suggesting that Sb-1 might be more efficiently propagated on biofilm. IMPORTANCE Biofilm-associated multidrug-resistant infections pose significant challenges for antibiotic therapy. The extracellular polymeric matrix of biofilms presents an impediment for antibiotic diffusion, facilitating the emergence of multidrug-resistant populations. Some bacteriophages (phages) can move across the biofilm matrix, degrade it, and support antibiotic penetration. However, little is known about how phages and their hosts interact in the biofilm environment or how different phage+antibiotic combinations (PACs) impact biofilms in comparison to the planktonic state of bacteria, though scattered data suggest that phage+antibiotic synergy occurs more readily under biofilm-like conditions. Our results demonstrated that phage Sb-1 can infect MRSA strains both in biofilm and planktonic states and suggested PAC regimens worthy of further investigation as adjuncts to antibiotics.