**6. Combination of phage therapy and antibiotic to treat lung infections**

## **6.1 Mechanisms of phage-antibiotic synergy**

With the emergence of phage-resistant bacteria [67], the combination therapy of antibiotics and phages has drawn increasing attention. Synergistic effect of antibiotic and phage against *S. aureus* was first reported by Himmelweit et al. back in 1945 [68]. Similar synergistic antibacterial effects have also been observed in a number of subsequent studies [69–79]. In 2007, Comeau et al., coined the term phage-antibiotic synergy (PAS) corresponding to an incident where the killing effect of bacterial strains considerably higher when phage production increases by the sublethal concentrations of particular antibiotics [80]. While many antibiotics exhibit synergistic effect in combination with phages, two specific classes of antibiotics (namely beta-lactams and fluoro-quinolones) were shown to produce a more consistent and pronounced antibacterial synergistic effect with phage therapy. The precise mechanisms contributing to phageantibiotic synergy remain largely unknown. A few possible mechanisms have been proposed (**Figure 2**): (1) Antibiotic causes cell elongation or filamentation, thus subsequently promoting phage production; (2) Degradation of the extracellular membrane of bacteria by phage facilitates internalization of antibiotic into cells; (3) Auto-aggregation of bacterial cells leads to synergism; (4) Bacteria containing complete prophages could be induced by antibiotics which further kill bacteria [81]. The capacity of phage in resensitizating bacteria to certain antibiotics have also been reported as the host bacteria cannot develop resistance to phage and antibiotic simultaneously [82–84]. As a result, the phage-antibiotic combination can kill both phage-sensitive and antibiotic-sensitive pathogens with the phage lysing cells resistant to antibiotics and antibiotic mediated killing of phage-resistant bacterial cells and eventually inhibit the infections.

Interestingly, the sequence of phage and antibiotic administration was found to be critical in the overall antibacterial effect from the combination treatment. Chaudhry et al. showed the efficiency of removing *P. aeruginosa* PA14 biofilm was higher when the biofilm was treated with phages before antibiotics [85]. A similar observation was also reported in another study evaluating phage-antibiotics combination therapy against *S. aureus* biofilms [86]. However, the observed synergistic effects were found to be dependent on the class of antibiotics used. Pre-treatment

**Figure 2.** *Possible mechanisms responsible for phage-antibiotic synergy.*

with phage led to favorable antibacterial effect when combined with linezolid or tetracycline, whereas antagonism was observed between the phage and dicloxacillin or cefazolin. Furthermore, it is noteworthy that an antagonistic effect was observed when the bacterial biofilm was treated with antibiotics preceding the phage therapy, irrespective of which class of antibiotics used [86].

### **6.2 Novel tools for selection of optimum phage-antibiotic combination**

Since the exact mechanisms responsible for PAS are still unclear and the choice of the combinations is mostly empirical, it is not surprising that mixed results were reported in the literature [72, 82]. Also, the concentration of antibiotics used in previous studies was limited to one or two levels, which is not enough to predict the efficacious concentration when applied in clinical treatment. To solve these problems, Liu et al. developed a high-throughput platform called synogram by combining an optically based real-time microtiter plate readout with a matrix-like heat map to quickly assess the effects of various phage and antibiotic concentrations on bacterial growth [87]. They concluded that PAS is highly dependent on the antibacterial mechanism of action for antibiotic and phage pairs and their stoichiometry.

To guide the choice of phage-antibiotic combination, Rodriguez-Gonzalez et al. [88] developed an *in silico* nonlinear population dynamics model taking into account the systemic interactions between bacteria, phage and antibiotics to mimic *in vivo* application by given an immune response against bacteria. Using two *P. aeruginosa* strains, one phage-sensitive (resistant to antibiotic) and one antibiotic sensitive (resistant to phage), as the model bacteria, the phage-antibiotic combination therapy was confirmed to outperform the monotherapy. The role of the host immune response was also evaluated and the model predicted that the phageantibiotic combination failed to eliminate the infection when innate immunity was removed or severely reduced. Their findings confirmed the clearance of infection is depending on the nonlinear synergistic interactions between phage, antibiotic, and innate immunity. The *in silico* prediction was consistent with previous experimental results obtained *in vitro* and *in vivo*. While this model is a valuable tool in

identifying potential phage-antibiotic combinations, further modification of the model to yield high-resolution temporal data in addition to the final results will be useful for quantitative comparison of the model-based predictions with experimental results.
