Advances in Phage Therapy: Targeting the Burkholderia cepacia Complex

Abstract

The increasing prevalence and worldwide distribution of multidrug-resistant bacterial pathogens is an imminent danger to public health and threatens virtually all aspects of modern medicine. Particularly concerning, yet insufficiently addressed, are the members of the Burkholderia cepacia complex (Bcc), a group of at least twenty opportunistic, hospital-transmitted, and notoriously drug-resistant species, which infect and cause morbidity in patients who are immunocompromised and those afflicted with chronic illnesses, including cystic fibrosis (CF) and chronic granulomatous disease (CGD). One potential solution to the antimicrobial resistance crisis is phage therapy-the use of phages for the treatment of bacterial infections. Although phage therapy has a long and somewhat checkered history, an impressive volume of modern research has been amassed in the past decades to show that when applied through specific, scientifically supported treatment strategies, phage therapy is highly efficacious and is a promising avenue against drug-resistant and difficult-to-treat pathogens, such as the Bcc. In this review, we discuss the clinical significance of the Bcc, the advantages of phage therapy, and the theoretical and clinical advancements made in phage therapy in general over the past decades, and apply these concepts specifically to the nascent, but growing and rapidly developing, field of Bcc phage therapy.

Keywords: Bcc phage therapy; Burkholderia cepacia complex (Bcc); antibiotic resistance; bacteria; bacteriophages; pathogenesis; phage therapy; phage therapy treatment strategies; phages.

Figure 1

Environmental and pathogenic roles of Burkholderia species. Members of the genus Burkholderia have a wide range of roles in both environmental and clinical settings. Although many species directly promote plant growth, secrete antibacterial and antifungal agents to protect plants from infection from other organisms, and contribute heavily to soil nutrient flux, they are also known to cause disease in a number of crops including onions, tobacco, rice, as well as several species of flowering plants. Due to their impressive versatility with respect to carbon and energy sources, Burkholderia species have been investigated for use in bioremediation, and are known to be capable of breaking down several difficult-to-degrade contaminants. Unfortunately, several species within this genus are known to cause severe diseases in both human and non-human animals—including B. mallei—which causes glanders in horses and other animals; B. pseudomallei, the causative agent of melioidosis in humans and other animals; and members of the Bcc, which cause persistent, difficult-to-treat and often fatal lung infections in susceptible human populations.

Figure 2

Antibiotic resistance mechanisms of the Burkholderia cepacia complex. Bcc members are resistant to a wide range of antibiotic compounds as a result of their large repertoire of both innate and acquired resistance mechanisms. Resistance to $\beta$-lactam antibiotics is achieved through a combination of reduced membrane permeability (A), mutated penicillin-binding proteins (B), and chromosomally encoded $\beta$-lactamases (C). Extrusion via porins (D) and efflux pumps (E) are responsible for resistance to a large number of compounds, including trimethoprim, chloramphenicol, tetracyclines and certain quinolones, while mutation of dihydrofolate reductase provides additional resistance to trimethoprim (F). At the outer membrane, LPS modifications prevent aminoglycoside binding and therefore block the trafficking of these compounds into the cell (G), while the reduced negative charge of the outer membrane reduces the binding of polymyxins and cationic antimicrobial peptides (H), thereby rendering Bcc species less vulnerable to these compounds.

Figure 3

Replication cycle of Caudovirales bacteriophages. All phages of the order Caudovirales replicate through two complementary cycles. Prior to infection, phage particles are propelled through medium by random electrostatic interactions with nearby molecules (1) until they reach a bacterial host cell, whereupon they adsorb to its surface by using their tail fibers and subsequently utilize the spontaneous binding and unbinding of these tail fibers to engage in a random walk across the cell surface (2). Upon interacting with its cognate secondary receptor, the phage infects the cell by injecting its genetic material (3) and is then compelled to proceed with one of two possible replication cycles. In the lytic cycle, which is normally employed when host cell density is high, the phage genome is replicated (4), transcribed and translated into the components of the phage particles (5), which are then assembled within the cell (6). Finally, the host cell is lysed (7), and liberated progeny phages are able to subsequently infect additional cells. In the alternative lysogenic cycle, which is often employed when host cell density is low, the phage genome is either integrated into the bacterial genome (8a) or circularized into a phagemid (8b), and this prophage then forms replicates passively along with its lysogenized host (9). Deterioration of host cell conditions due to starvation or other stressors may cause induction of the prophage, whereupon it returns to the lytic cycle (10) and ultimately destroys its host cell via lysis.

Figure 4

Interactions between bacteriophages and the mammalian immune system. In addition to their direct anti-bacterial action via killing of target bacterial cells, phages can contribute to bacterial killing indirectly by stimulating the host immune system—a multifactorial process that can be divided into two main pathways. At the infection site, phage-mediated lysis of bacteria induces the release of bacterial pathogen-associated molecular patterns (b-PAMPs), which trigger the activation of tissue-resident macrophages (1). Upon activation, these macrophages use cytokine signaling to activate circulating macrophages and neutrophils and recruit them to the infection site (2), where they indiscriminately eliminate phages and bacteria alike via phagocytosis followed by ROS-mediated degradation (3). Furthermore, cytokine signaling by b-PAMP-activated tissue-resident macrophages leads to the activation and recruitment of B-lymphocytes (4), which subsequently initiate a specific humoral response against invading bacteria (5). Simultaneously, phage particles (and fragments thereof) introduced through treatment and reproduced via the lytic cycle stimulate the activation of other tissue-resident macrophages (6), which similarly recruit circulating macrophages and neutrophils (7) that subsequently eliminate both bacteria and phages via indiscriminate internalization and degradation (8). Finally, tissue-resident macrophages may activate circulating B cells through presentation of phage peptides (9), leading to a specific humoral response against phage particles (10).

Figure 5

Phage therapy 2.0. Top panel: strategies for the circumvention of problematic phage resistance. One major therapeutic approach is the "Anti-virulence strategy", in which phages are utilized as a selection pressure favoring avirulent bacterial phenotypes. To achieve this, clinicians may employ phages that utilize virulence structures, such as pili, as primary receptors (A). When such a phage is employed, susceptible cells are destroyed and the survivors, although invulnerable to infection, lack critical virulence factors (B). These avirulent survivors (red) may grow normally in vitro (C), but are unable to evade the host immune system in vivo and therefore grow poorly (D). Targeting a single virulence factor may be insufficient to eliminate in vivo virulence and thus survival, however, and the Anti-virulence strategy should therefore be combined with the Multiple-targets strategy, in which a cocktail of at least two phages—which target distinct virulence factors as receptors—is employed (E). Such treatments leave a small number of survivors, including rare mutants resistant to both phages (F). By combining this treatment with an antibiotic regimen, to create a polyphage–antibiotic cocktail, clinicians can reduce the bacterial population even further—to a point at which the host immune system, even if partially compromised, is able to eliminate the infection (G). Bottom panel: mechanisms of phage–antibiotic synergy. Although polyphage–antibiotic cocktails are efficacious even when the effects of their constituents are merely additive, several combinations are now known to produce synergistic killing effects through at least three putative mechanisms of action. In the Delayed lysis mechanism (I), co-treatment with phage and subinhibitory doses of antibiotics that target the bacterial cell wall, such as $\beta$-lactams, causes swelling and elongation of effected cells. Although this delays the time to lysis, it causes an increased buildup of phage particles inside the cell leading to increased burst size, which can allow for more rapid overall killing of the bacterial population. In the Induction mechanism (II), treatment with a temperate phage is followed by treatment with a DNA-damaging agent, such as ciprofloxacin, which induces the prophage to the lytic cycle. Since most of the survivors of the initial phage attack are lysogens, their induction to the lytic cycle destroys the vast majority of the overall bacterial population. Similar to the Anti-virulence strategy, the Phage–antibiotic catch-22 mechanism (III) utilizes a particular combination of phage and antibiotics as opposing selection pressures. Treatment with tetracycline, for instance, against which a major mechanism of resistance is efflux, selects for a bacterial population that possesses the efflux pump. Subsequent treatment with a virulent phage utilizing the efflux pump as a receptor therefore allows most of the population to be infected and eliminated.