3.3. Biofilm Degradation and Killing of Bacteria in Biofilms by Phages
Colonization of wounds, surgically implanted materials or catheters by biofilm-producing bacteria such as P. aeruginosa and S. aureus is a common complication for modern surgical and orthopedic practices. Biofilm formation helps bacteria to evade the patient immune system and enhances antibiotic resistance. A biofilm is a community of microorganisms formed on a biotic or abiotic surface that consist of bacterial cells within an extracellular polymeric substance (EPS)-based milieu composed of polysaccharides, teichoic acids, proteins, and extracellular DNA [72]. Bacterial cells in biofilms are phenotypically different from their planktonic analogs, with reduced motility, distinct gene transcription patterns and a different spectrum of metabolic activity [73,74]. Bacteria within a biofilm EPS form a very tight tissue-like structure that is difficult to remove from a wounds or the surface of a medical device. Bacteria in biofilms are also significantly more resistant to antibiotics than planktonic bacteria, and this complicates the therapy of biofilm-associated infections [75]. The recalcitrance of bacterial biofilms to chemical antibiotic therapy presents a major obstacle to the successful treatment of the biofilm-associated infections that can complicate wound healing and manifest as chronic wounds that lead to amputation or death. Since biofilm formation complicates the treatment of wound infections, several approaches have been developed to control biofilm development. These approaches do not kill the bacteria in the biofilm but rather make the biofilm-bound bacteria planktonic and thus more susceptible to conventional antibiotic therapy. Killing bacteria within a biofilm can be achieved using various antibiotic agents that interfere with essential biological processes or by using different endolysins that disrupt the outer envelope of bacteria. A strategy for combating antibiotic resistant biofilm-associated infections such as those by P. aeruginosa or S. aureus is the use of lytic phages, since it has been demonstrated in a number of laboratories including ours that some phages can efficiently infect and lyse not only planktonic but also biofilm forms of host bacteria. To be effective against biofilms, phages must be able to propagate inside their hosts within biofilm matrix and release their progeny into the surrounding environment for dissemination [76]. This implies that phage candidates effective against biofilms should not only be able to disrupt the EPS matrix of the biofilm but should also provide a robust burst size upon infecting the biofilm bacteria. However, these interactions and the optimal characteristics required for phage anti-biofilm activity still need to be studied and determined. A number of recent publications reported the use of bacteriophages for the control of biofilms caused by P. aeruginosa and other bacteria [55,77]. In one publication, marked biofilm clearance was observed, with up to a five-log decrease in bacterial counts [78]. Other authors were less successful, reporting only a 2-log reduction in the number of bacteria in a biofilm formed by S. aureus using a specific bacteriophage in combination with sharp debridement in a rabbit wound punch model [79]. Additional approaches included the use of bacteriophages in conjunction with other agents, such as cobalt ion, to reduce biofilm formation or sub-lethal doses of antibiotics in combination with phages to control biofilms formed by E. coli in vitro [80]. By expressing polysaccharide depolymerases and lysins, phages penetrate the bacterial capsule or peptidoglycan layer to access host receptors and infect [81]. Since these enzymes could enable penetration and degradation biofilms, phages that encode such enzymes should be included in therapeutic cocktails. Challenges with using phages to degrade biofilms have also been described [82]; this is clearly an area that requires further investigation to enable rational inclusion of phage anti-biofilm capability in therapeutic approaches.
3.4. Phage Synergy with Antibiotics
Antibiotics will remain the clinical standard of care treatment of bacterial infections for the foreseeable future despite the challenges of rising antimicrobial resistance and MDR infections, so finding new antimicrobials that work synergistically with them is a key area of near-term focus for drug development. Combinations of phages with antibiotics in treatments can yield synergies that should be exploited to both potentiate the action of antibiotics and also to integrate viable combination therapies into the clinical arsenal, so understanding interactions between phages and antibiotics is an essential aim [83–85]. Phage-antibiotic synergy (PAS) has been demonstrated in both Gram-positive and Gram-negative bacteria; sub-lethal concentrations of several classes of antibiotics have a positive effect on the size of phage plaques and efficiency of phage propagation [86–89]. A mechanism of PAS was recently discovered as lysis delay and significant increase of phage burst size caused by excessive growth and filamentation of bacterial cells and relative shortage of holin supply in response to an antibiotic as a stress-inducing factor [89]. Another more important effect of PAS is synergistic killing of planktonic bacterial cultures [90–93], and bacterial biofilms in vitro [80,93–96], as well as enhanced efficacy of combined application of phage and antibiotic in the treatment of colibacillosis in broilers [97], and even therapy of S. aureus diabetic foot infections in humans [98]. In a more targeted approach, a lytic phage was selected for P. aeruginosa that uses an outer membrane porin as its receptor that is a component of a multidrug efflux system, placing pressure on the host to mutate toward increased drug sensitivity to evade the phage [99]. This represents an approach that aims to re-sensitize MDR pathogens to conventional antibiotics to extend their utility, and selected phages can be paired in combination therapies with the antibiotic(s) to which they increase sensitivity [29]. Notably, some combinations of phages and antibiotics do not provide synergy [87,88,92], thus potential interference must be considered in designing and applying combined antibiotic-phage therapy [100].
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