Infections caused by multidrug-resistant (MDR) bacteria have highlighted the importance of the development of new antimicrobial agents. While bacteriophages (phages) are widely studied as alternative agents to antibiotics, combined treatments using phages and antibiotics have exhibited Phage–Antibiotic Synergy (PAS), in which antibiotics promote phage replication and extraordinary antimicrobial efficacy with reduced development of bacterial resistance. This review paper on the current progress of phage–antibiotic therapy includes aspects of the mechanisms of PAS and the therapeutic performance of PAS in combating multidrug-resistant bacterial infections. The choice of phages and antibiotics, the administration time and sequence, and the concentrations of the two agents impact the bacterial inhibitory effects to different extents. Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species (the ESKAPE pathogens) have developed resistance to oxazolidinones, lipopeptides, macrolides, fluoroquinolones, tetracyclines, β-lactams, β-lactam–β-lactamase inhibitor combinations, polymyxins, glycopeptides, and carbapenems [A. baumannii, P. aeruginosa, and Enterobacteriaceae are listed as the most critical pathogens that require the development of new antibiotics because they have developed resistance to carbapenem, which is the last-line drug for combating resistant bacteria [Acinetobacter, carbapenem-resistant Enterobacteriaceae, and drug-resistant Neisseria gonorrhoeae are listed as urgent threats [Antibiotics are one of the greatest discoveries in modern medicine and have extensively decreased morbidity and mortality caused by different types of bacterial infections, improving the quality of life and life span of patients . Howeverbapenems . In the bacteria ,3. In An threats . This anBacteriophages (or phages) are viruses that specifically target bacteria . Phage tVirulent phages (or lytic phages), which are unable to lysogenise their hosts, multiply in bacterial hosts, and lead to bacterial lysis after each infection cycle, are only used for therapeutic investigation against bacterial infections. Either wild-type lytic phages ,8 or engAntibiotics are chemical compounds that have a specific mechanism for disrupting either the cell wall or the intracellular pathways. For example, carbapenem inhibits cell wall formation, and ciprofloxacin inhibits DNA replication. Because the mechanisms for phages to inhibit bacteria are different from those of antibiotics, phages can treat MDR bacterial infections. However, bacteria can still evolve an antiphage system in a number of ways: (1) mutating bacterial cell surface receptors, preventing the entry of phage DNA ,14; (2) Phage–antibiotic therapy has been successfully shown to reduce the emergence of phage-resistant and antibiotic-resistant strains. An often-cited example is the fact that the degeneration of cell surface receptors caused by phages restores antibiotic sensitivity, as those receptors are responsible for the efflux of antibiotics . SeveralAs a promising therapeutic strategy for bacterial infection, the combination therapy using phages and antibiotics has now been studied extensively, not only because of its improved performance in reducing phage and antibiotic resistance but also for the synergistic antibacterial effects achieved by phage replication enhancement in the presence of antibiotics  ,23.This review focuses on the PAS response and its mechanisms; the development of resistance in bacteria in response to phage–antibiotic combined treatment; and the application of phage–antibiotic treatment in vitro, in vivo, and in clinical case studies.PAS has been observed between phages and antibiotics. This has raised interest in the therapeutic potential of PAS to improve bacterial killing ,25.The term PAS initially described the phenomenon of the improved antimicrobial effect caused by stimulated phage replication in the presence of sublethal concentrations of antibiotics. Nowadays, the term PAS is used in circumstances where synergistic antimicrobial effects occur. In this review, we use the term PAS to describe the phenomenon of increased phage activity in the presence of sublethal concentrations of antibiotics. The terms synergistic effects or synergism are used herein to describe the significant improvement in bacterial growth inhibition with phage–antibiotic co-treatment.Escherichia coli clinical isolate MFP; on T4-like phages against standard laboratory E. coli strains; on T3 and T7 phages against laboratory E. coli strain AS19; and on T4-type Yersinia phage PST against Yersinia pseudotuberculosis in the presence of sublethal β-lactams such as aztreonam, cefotaxime, ticarcillin, piperacillin, ampicillin, and quinolones such as nalidixic acid. The plaque size enlargement implies enhanced phage production during plaque formation, resulting in increased bacteria growth inhibition and/or lysis. Other than E. coli phage and Y. pseudotuberculosis phage [P. aeruginosa [S. aureus [Bacillus cereus [Enterococcus faecalis [Burkholderia cepacian [PAS was first studied by Comeau et al. , where sis phage , plaque ruginosa ,27, S. a. aureus ,29, Bacicepacian .Plaque size can be affected by multiple factors, including the intrinsic traits of the phage and the infected host, as well as extrinsic factors such as incubation time, temperature, agar density, and bacterial density . As thesP. aeruginosa PA01 from an average of 1.15 ± 0.18 μm to 1.8 ± 0.18 μm  [The PAS response in lytic phages is considered directly related to bacterial filamentation, which increases the bacterial size and cellular surface area ,27,37,38.6-fold) .S. aureus ATCC 12201; PBEF7 and PBEF9 against E. faecalis KCTC 2011; PBBC03 against B. cereus KCTC 1012; T4 against E. coli K-12 strain ATCC 700926; PBEC22, PBEC24, and PBEC82 against E. coli Crooks strain ATCC 8739; and PA26, PA22, and PA25 against P. aeruginosa ATCC 13388. Of the 88 tested phage and antibiotic combinations, 56 exhibited an increase in plaque size along with antibiotic-induced bacterial filamentation in rods or bacterial swelling in cocci [P. aeruginosa phage PA22, in which PAS did not occur , yet the bacteria filamented. However, the reason remains unclear.In another PAS study, 11 phages in combination with 8 antibiotics  were tested against their hosts: phage SA11 against in cocci . The preE. coli strains, PBP1s are involved in bacterial elongation, PBP2s are responsible for cell elongation and rod shape maintenance, and PBP3s are responsible for septal wall formation during cell division. Blocking PBP1s leads to direct cell lysis, blocking PBP2s leads to ovoid bacterial cells, and blocking PBP3s leads to bacterial filamentation [Most of the antibiotic-induced filamentation can be explained by two mechanisms: the β-lactam-induced and SOS-response-mediated mechanisms. β-lactams alter the morphology of bacteria by binding to penicillin-binding proteins (PBPs) on the bacterial surface. PBPs are key peptidoglycan-synthesizing enzymes that play an integral role in constructing the bacterial cell wall. For example, in entation .E.coli and P. aeruginosa cells [E. coli and P. aeruginosa can form filaments in 3–4 h in the presence of sublethal concentrations of cefuroxime and ceftazidime (0.008 × MIC~1 × MIC) [E. coli and P. aeruginosa cells, β-lactams such as imipenem and mecillinam have higher affinity for PBP2s and are reported to cause ovoid cells; ampicillin has similar affinity for PBP2s and PBP3s and is reported to cause localised swelling [β-lactams vary in their affinity for different PBPs, which induce different alterations in bacterial shapes to different extents or result in direct cell lysis . β-lactasa cells ,41. In tsa cells ,41,42. E1 × MIC) ,41. On tswelling  as well swelling . Other tswelling , PAS resConsequently, the choice of a specific β-lactam antibiotic affects the degree of filamentation, which in turn may affect the adsorption rate of phages by increasing phage targeting receptors on the bacterial cell wall  was detected in the presence of a sublethal dose of ciprofloxacin, whereas other tested bacteria  exhibited both recA expression and filamentation [recA deletion E. coli mutant strain showed filamentation and PAS in the presence of ciprofloxacin even with no recA expression. These results suggest that the SOS response may not necessarily be responsible for bacterial filamentation induced by DNA synthesis inhibitors or DNA disrupting agents; filamentation could also be triggered through other pathways.It is generally believed that the observed PAS response with quinolones is due to filamentation induced by the SOS response. However, exceptions were reported by Kim et al. . P. aeruentation . MoreoveS. aureus, E. faecalis, B. cereus, E. coli K-12, E. coli Crooks, or P. aeruginosa, or in trimethoprim against E. coli K-12. One possible reason is that SOS-response-mediated filamentation did not happen at the tested antibiotic concentration or within the exposure period (not specified in the article) as filamentation is affected not only by the antibiotic class but also by the antibiotic concentration and duration of exposure [recA expression was only tested in ciprofloxacin, it is unknown whether the SOS response is triggered by trimethoprim and sulfamethoxazole.Moreover, although SOS-response-mediated filamentation has been reported in studies with mono-treatment of trimethoprim and sulfamethoxazole , conflicexposure . As the The SOS response was also found to improve temperate phage activity because it triggers temperate phage assembly and release due to the cleavage of phage repressors by RecA ,48. We hBacterium coli [P. aeruginosa [E. coli [Although PAS and filamentation have rarely been observed with sublethal concentrations of antibiotics other than β-lactam and DNA-disrupting antibiotics ,27, filaium coli , aminoglruginosa ) or RNA [E. coli ). Althou[E. coli ,24,27.In addition to structural changes in bacterial cells , some alterations in the phage lytic cycle were found in the presence of filamentation and the PAS response. To date, three main alterations in the phage lytic cycle have been reported to induce the PAS response . It is uPAS may be due to bacterial filamentation and resultant changes in the phage lytic cycle , but filFilamentation occurred either when PBPs were inhibited by β-lactam antibiotics (β-lactam-induced) or as a survival mechanism when DNA was damaged by antibiotics (SOS-response-mediated). When bacteria remain filamented after antibiotic treatment, genome replication and expression continue without separation into daughter cells. Once antibiotics are depleted or the DNA is repaired, bacterial division and replication resume. If, before the antibiotics are depleted, the phages lyse all bacteria cells with only limited numbers of the lytic cycle, no significant phage replication is observed. This does not provide an advantage to phage multiplication, hence no PAS. However, due to early bacterial clearance, phage–antibiotic co-therapy is still beneficial because the phages promote bacterial lysis.Since phage–antibiotic therapies are mainly used for combating MDR infection, it is expected that MDR bacteria cannot be fully killed/inhibited by antibiotics alone and that they continue to grow in the presence of antibiotics; consequently, the phages undergo replication. In addition, if the antibiotic dose is too low for a subinhibitory effect against the bacteria, or the bacteria are highly resistant to the antibiotics and filamentation cannot be induced, PAS may not occur. Thus, PAS may hinge on a balance between the bacterial replication rate in the presence of a sublethal concentration of antibiotics and the phage replication rate to lyse the host. As a result, the choice of the type and dose of antibiotics for a given phage is a critical determinant of the PAS response.Phage adsorption to bacteria, as the first stage of the lytic cycle, is one of the critical steps for phage multiplication. Phage adsorption includes reversible and irreversible binding. Phage adsorption rate refers to irreversible binding.E. coli, S. aureus, and P. aeruginosa in the presence of sublethal concentrations of antibiotics. By pre-treating E. coli B/r H266 with a low concentration of penicillin, the adsorption rate of T4 phages was significantly increased (4.68 × 107 phage/mL/min) compared to the control (3.36 × 107 phage/mL/min) [An improvement in phage adsorption efficiency was found in /mL/min) .50), 75% (T75), and 100% (T100) of phage MR-5 to be adsorbed to S. aureus ATCC 43300 pre-treated with antibiotics for 90 min  and a control bacteria culture. Pre-treatment with linezolid and tetracycline significantly decreased T50 , T75 , and T100 , while clarithromycin and telithromycin significantly decreased T50  and T75 .Kaur et al.  measuredP. aeruginosa PA01 in the presence of 1.06 µg/mL aztreonam lysine was conducted using transmission electron microscopy (TEM). The cell significantly increased in length  and width . Meanwhile, approximately a two-fold increase in phage attachment was detected in the presence of aztreonam lysine: an average of 2 ± 1 phage E79 adsorbed to each P. aeruginosa PA01 cell in the control, while 5 ± 2 phages per cell were detected in an aztreonam lysine-treated culture [Another assessment of the filamentation and improved attachment of phage E79 to  culture . However culture . MicroscIn filamentation-associated PAS, some have proposed that the increase in adsorption rate proportional to the increased bacterial surface area corresponded to the increased number of receptors on the bacterial surface ,52. EvidE. coli B/r outer membranes regardless of cell sizes. This was surmised from the proportional increase in the irreversible phage adsorption rate and surface area [P. aeruginosa cells was reported [LPS density was reported to be consistent on ace area . A reducreported , but wheChanges in the bacterial cell wall receptors during filamentation are important because they affect PAS. However, limited investigations have been conducted on phage receptor densities during filamentation.E. coli bacteria cells [Cell lysis is the last stage in the infection cycle of lytic phages . The peria cells ,27.E. coli AS19 [E. coli ATCC 11303 [S. aureus ATCC 43300 decreased from 33.3 min to 16.6, 18.3, 23.4, and 23.4 min in the presence of sublethal 1 μg/mL linezolid, 0.25 μg/mL tetracycline, 4 μg/mL clarithromycin, and 4 μg/mL telithromycin, respectively [P. aeruginosa PA01 from around 9 to 6 min [Accelerated bacterial lysis has been reported to be accompanied by PAS in many studies. The addition of 0.030 µg/mL cefotaxime reduced the latent period of T4 and two other T4-type phages (RB33 and RB49) from 2 h to 75–90 min in oli AS19 . MoreoveCC 11303 . The latectively . Aztreonto 6 min .Bacterial filamentation is an intermediate state where bacterial chromosomes continuously replicate without cell division . Hence, E. coli. A 2.1-fold increase in green fluorescence was detected using a fluorometer [Further confirmation of holin expression was measured using engineered T4 phage with an enhanced green fluorescent gene in filamented orometer . In addiorometer ,26.E. coli K-12 strain with subinhibitory concentrations of cefotaxime and ciprofloxacin, which was due to a decreased holin concentration in the filamented bacterial cell [Although holin is increased in filamented cells, its concentration depends on not only the protein expression rate but also the cell dimensions . Conflicial cell . While Kial cell  detectedial cell .Both accelerated and delayed lysis have been found to be associated with PAS. A shortened latent period or accelerated lysis time increases the rate of repeating the infective cycle, while delayed lysis may allow more time for phage assembly and consequently enlarges the phage burst size .The burst size  refers tE. coli B/r cells induced by pre-treatment with a low concentration of penicillin. Compared with the normal non-treated cells, the filamented cells increased around 4-fold in size along with a 2.5- to 4-fold increase in single burst sizes, suggesting a correlation between bacterial cell size and burst size. Hadas et al. [Hadas et al.  compareds et al.  proposedE. coli K-12 cells induced by sublethal ciprofloxacin treatment. They postulated that the increased burst size was related to the increased availability of viral components. Further quantification of mRNAs, DNA, and proteins was conducted in these filamented E. coli cells, showing a 2-fold increase in the mRNA that encodes T4 phage DNA polymerase, which resulted in also a 2-fold increase in the T4 phage DNA. In addition, a 1.5-fold increase was observed in the mRNA that encodes the phage major capsid protein, but no increase was detected in the capsid protein production. Thus, not all viral components  are necessarily increased in the filamented cells. Overall, the relationship between the increased bacterial cell size and increased viral components or other factors is still not well established.Kim et al.  also obsE. coli culture from 20 min to 28 min. The single burst size was enhanced from 270 to 700 plaque-forming units per cell [Another hypothesis is that delayed cell lysis see  allows aper cell . Kim et The increased burst size is one of the contributors to PAS, but it is not the only factor determining the overall phage production. Other factors, such as the latent period, should also be considered for PAS investigation.Other than filamentation-induced PAS in lytic phages, a recent in vitro study also reported temperate phage–antibiotic synergy, so-called “tPAS” . TemperaE.coli K-12. Eradication (≥8 log reduction) was achieved by HK97 (MOI ≥ 10) with either MIC or ½ × MIC ciprofloxacin after overnight incubation, whereas HK97 alone was ineffective against E. coli K-12. The study suggests that the latent period and burst size were not affected by the addition of ciprofloxacin. One of the underlying mechanisms was improved prophage induction triggered by ciprofloxacin via the recA-mediated SOS response. As mentioned in recA with gene recA deletion, which did not exhibit synergy with ciprofloxacin [Al-Anany et al.  examinedfloxacin . Anotherfloxacin .The antimicrobial effects achieved by HK97 with a sublethal concentration of ciprofloxacin are significant. However, “tPAS” is as of yet poorly studied. More investigations with other bacteria species with in vitro and in vivo models are needed for implementing its use therapeutically.P. aeruginosa phages with ciprofloxacin in the absence of bacterial filamentation [P. aeruginosa phage PA22 with tetracycline showed no significant plaque size enlargement even when bacterial filamentation occurred [PAS has been assessed by bacterial filamentation and plaque size enlargement; however, these effects may not always occur. The PAS response was found in entation . Moreoveoccurred .In another study tested with ampicillin, penicillin G, kanamycin, rifampicin, and tetracycline, tetracycline was found to produce the greatest plaque size increase, yet the bacterial cell size increase was the smallest , suggestDue to the large number of available antibiotics and the variations between different phages and bacterial hosts, it is very difficult and time-consuming to systematically assess the PAS response as well as the corresponding characteristics of the phage lytic cycle. This has hindered researchers in further exploring the underlying mechanisms.To date, there are various marketed antibiotics for treating bacterial infections. However, after prolonged exposure to the same or similar antimicrobial agents, bacteria can evolve and develop resistance by reducing drug uptake, increasing drug efflux, modifying drug target sites, and inactivating the drug ,64.Various approaches to using phages and antibiotics, with different mechanisms of inhibiting MDR infections, have been widely investigated. While a single-agent treatment kills its sensitive population and selects resistant bacteria, treatments with two or more agents with different mechanisms of action cover larger host populations. For instance, the combined use of phages and antibiotics leads to the simultaneous selection of phage-sensitive and antibiotic-sensitive populations, resulting in a very small or non-existent population resistant to both agents .In addition, with lethal concentrations of antibiotics and phages, bacteria are unlikely to rapidly evolve and acquire resistance to multiple agents that target different pathways in a short period of time. In particular, when the development of resistance to either agent adversely alters the bacterial components/functions involved in the attacking pathways of the other agent, adaptation trade-offs happen .For example, one of the known adaptation trade-offs between antibiotic resistance and phage resistance is multidrug efflux pump development and alleviated phage attachment. Some bacteria developed a resistance to antibiotics by expressing multidrug efflux pumps that remove the drug from the cells, hence the bacteria become multidrug- or pan-drug-resistant . On the P. aeruginosa phage OMKO1 binds with hosts at the outer membrane porin M (OprM) of the multidrug efflux pump. OprM was knocked out in engineered phage-resistant P. aeruginosa strain to force phage resistance. The engineered strain then regained sensitivity to antibiotics such as ciprofloxacin, tetracycline, ceftazidime, and erythromycin. The minimal inhibitory concentrations (MICs) of these antibiotics decreased by 12-fold compared to non-gene-modified strains [Pseudomonas-targeting phage PEV31, phage-resistant isolates of P. aeruginosa regained sensitivity to ciprofloxacin [Several studies observed gene mutations or reduced protein expression of bacterial drug efflux pumps after combined treatment with phages and bacteria ,21. This strains . The MIC strains ,20. In afloxacin ,67.Phage–antibiotic therapy has promising therapeutic potential for enhancing antimicrobial effects due to PAS and decreasing the likelihood of resistance development. Moreover, it reduces the required antibiotic concentration compared to mono-antibiotic treatment.Pseudomonas-targeting phage PEV20 and ciprofloxacin enhanced P. aeruginosa biofilm eradication, highlighting the potential for reducing the antibiotic concentration required to combat highly recalcitrant infections associated with biofilms [P. aeruginosa biofilms were treated with phage EPA1 and ciprofloxacin, meropenem, and gentamicin, synergism was observed at 1 × MIC with a 4.7, 4.1, and 2.6 log reduction in biofilm density, respectively. However, antagonism of antimicrobial activity was observed in ciprofloxacin and meropenem at 8 × MIC [Decreased antibiotic MIC when used in combination with phages was observed in several studies ,69,70,71biofilms . Althoug 8 × MIC . This isE. coli in combination with phage ELY-1, without compromising the antimicrobial effect [A. baumannii phage Ab105-2jDCI was combined with meropenem and imipenem at 1/8 × MIC. On the other hand, synergism occurred with no regrowth of bacteria when the antibiotic concentration was increased to ¼ × MIC [In contrast, increasing the ciprofloxacin dose from a sublethal dose (1/10 × MIC and 1/5 × MIC) to a lethal dose up to 1 × and 2 × MIC successfully inhibited the regrowth of l effect . In anot ¼ × MIC . Thus, dSeveral studies ,70,72,73Aminoglycosides, which inhibit protein synthesis, did not show significant synergism with phages in simultaneous administration but showed better bacterial killing and enhanced phage production in sequential administration ,74,75,76P. aeruginosa strains were initially treated with phage LUZ7 (105 pfu/mL). Simultaneous treatment strongly suppressed the growth of P. aeruginosa with no regrowth over 70 h. The most significant synergistic bactericidal effect (p = 0.027) occurred when streptomycin was administered 12 h after phage treatment.Torres-Barcelo et al.  conductep < 0.04). Synergism occurred when the antibiotics were administered 24 h after pre-treatment with the phage cocktail, showing a significant reduction (from 8 to 2 log) in P. aeruginosa biofilm density. In another study, two aminoglycosides (kanamycin and neomycin) were compared with another protein synthesis inhibitor antibiotic, tetracycline, in co-treatment with phage T3 on E. coli biofilm. Interestingly, tetracycline showed no inhibition of phage infectivity even though it has a similar antibacterial mechanism (inhibiting protein synthesis) to kanamycin and neomycin, which decreased the phage burst size, replication, and efficiency of plating [Moreover, co-treatment with an anti-pseudomonal phage cocktail (phage NP1 and phage NP3) combined with gentamicin and tobramycin (8 × MIC)  hampered plating . This anAminoglycosides led to a misreading of different cistrons or genes on the 30 s ribosomal subunit, which eventually inhibited the initiation of different proteins. For example, kanamycin and gentamicin inhibited the translation of coat proteins, whereas kasugamycin inhibited maturation proteins . HoweverBased on the above findings, a delay in the administration of antibiotics (about 6–12 h) after phage pre-treatment led to stronger antimicrobial activity; this observation was more prevalent with aminoglycosides than other antibiotic classes . To maxiP. aeruginosa, E. coli, and Salmonella typhimurium.DNA synthesis inhibitor ciprofloxacin seemed to yield contradictory results, as a phage–ciprofloxacin combination can exert similar killing effects regardless of the administration time. Both simultaneous and sequential treatment (antibiotics administered after phages) exerted synergism against bacteria P. aeruginosa was observed despite titer reduction (1.8 log) as compared with sequential treatment, although it was not statistically significant (p > 0.05) [E. coli [E. coli. The bacterial killing was maximised when ciprofloxacin (1 × MIC) was added 6 h after the phage pre-exposure. Bacterial density was significantly reduced  as compared to other administration times , with no bacterial regrowth observed at the end of the experiment. The phage titer in all delayed ciprofloxacin treatments increased significantly (1.7–2.0 log) compared to simultaneous administration. These results agreed with those from another study [S. typhimurium pre-treated with the phage for 6 h and followed by ciprofloxacin showed the highest reduction (3 log) in bacteria count as compared with using antibiotics or phages alone. Contradictory results were observed with tetracycline, where simultaneous treatment with phages showed antagonism in S. aureus [E. coli [When ciprofloxacin (1 × MIC) was given simultaneously with phage PA14, a synergistic effect against  > 0.05) . This de[E. coli . However[E. coli . Ciprofler study , in whic. aureus  but not [E. coli , showingRecently, Wang et al.  indicateS. aureus reference strain and five rifampin-resistant S. aureus (RRSA) strains, three of which were methicillin-susceptible and two were methicillin-resistant. Synergistic effects on biofilm reduction were compared between simultaneous and staggered administrations of the antibiotics and phage. Most strains were susceptible to the antibiotics, whereas two strains showed resistance only to doxycycline and levofloxacin. No synergistic effects were observed in the inhibition of anti-biofilm activity in any of the combined treatments with simultaneous application  on all the strains, except the phage–rifampin combination on the reference strain. However, in sequential treatment, synergism was observed in doxycycline (16–128 µg/mL) and linezolid  after 24 h of phage (106 PFU/mL) pre-exposure. Biofilm density  was strongly reduced in all six strains, including the resistant strains, by the phage–levofloxacin (16–128 µg/mL) and phage–clindamycin (64–256 µg/mL) treatments, with five and four strains, respectively, out of the six showing synergistic effects on reducing biofilm density. Rifampin was the only exception; the combination therapy had no bactericidal synergism in any RRSA strain. Rifampin was the only antibiotic that showed minimal antimicrobial activity in all RRSAs, and this was due to the high resistance of the clinical strain, as the binding site of rifampin was altered by the mutation of rpoB on RNA polymerase. The binding affinity of rifampin to bacterial RNA polymerase was reduced [Phage Sb-1 and an antibiotic  were used together on an  reduced .Although staggered administration is an essential factor in exerting synergism, the host strain is also important. Synergism was observed in some RRSA strains but not others, showing that biofilm inhibitory effects were different even when using the same phage–antibiotic (levofloxacin) combinations. The above results demonstrated that host strains can show different inhibition profiles and synergistic effects , but theP. aeruginosa and S. aureus [P. aeruginosa biofilms, whereas S. aureus biofilms only had a 2 log decrease. This was due to the specificity of the phage, as EPA1 is an anti-pseudomonal phage. The reduction in S. aureus biofilms was caused by the administration of a high concentration of gentamicin (8 × MIC).These studies only demonstrated the phage–antibiotic treatment on a single bacterial species. When phage EPA1 and gentamicin were used on biofilms of dual bacterial species, . aureus , no syneE. coli infection with phage ϕHP3 and ceftazidime in human pooled urine and heat-inactivated serum showed completely different results compared to that in Luria-Bertani (LB) medium [9 PFU/mL of phage, which showed an additive effect. Similar results were found in urine in which only high doses of phage (109 PFU/mL) and ceftazidime (256 µg/mL) produced synergism. In contrast, synergism was observed in LB medium at low doses of ceftazidime (2 µg/mL) and phage (104 PFU/mL). The overall reduction in the bacterial level in serum and urine may not be due to the high doses, but the low starting bacterial inoculum, as untreated bacteria gradually decrease in the presence of urine and serum. This phenomenon was also observed in another study with E. coli isolates in urine samples [p < 0.001). It was found that urea was the main factor for reduced E. coli growth. Increased urea concentration corresponds to reduced bacteria count [As the pharmacokinetics of antibiotics in physiological media such as serum and urine can vary, phage–antibiotic efficiency is altered accordingly. Treating ) medium . The ant samples : bacteriia count .Overall, due to the specificity of phages and the diverse pharmacokinetics of antibiotics, phage–antibiotic efficiency depends on the host strains and physiological media.Galleria mellonella were extensively used for testing a wide range of bacterial infections.Although phage–antibiotic treatment is widely studied in vitro, little research focuses on preclinical in vivo trials to evaluate the efficacy of combination treatment. Promising results in treating bacterial infection showed the potential of phage–antibiotic treatment in vivo . Mice anK. pneumoniae in larvae of moth Galleria mellonella. The survival rate of the larvae significantly increased to 50% and 75% when co-treated with phage and mitomycin C or imipenem, respectively, compared to either antibiotic or phage monotherapy, except phage–imipenem co-treatment of the imipenem-resistant strain. This was due to the hydrolysis of imipenem by β-lactamase in the resistant strain. Nonetheless, the mortality rate of the larvae significantly decreased with all other combined treatments [Galleria mellonella models, achieving a 75% survival rate in treating E. coli infection when phage ΦWL-3 was used in combination with fosfomycin [A. baumannii increased the survival rate by approximately 30% (p < 0.05) compared to phage monotherapy [Galleria mellonella model has enabled high numbers of replicates, but their physiological characteristics are much different to those of mammals, so rodent infection models would be more relevant.Mitomycin C and imipenem were combined with lytic phage vB_KpnM-VAC13 to treat imipenem-resistant strain K2534 and persisted strain K3325 of Gram-negative bacteria eatments . Additiosfomycin . In addiotherapy . UtilisaP. aeruginosa lung infection. Co-spray dried phage and ciprofloxacin powder administrated intratracheally significantly reduced the bacterial density compared to single treatment with either agent [P. aeruginosa, a phage cocktail combined with ciprofloxacin was highly synergistic, resulting in negative vegetation in 7 out of 11 rats with more than a 6 log reduction in bacterial concentration, as opposed to zero rats with either phage or ciprofloxacin monotherapy [S. aureus (MRSA) ATTC 43300 strain to induce prosthetic joint and diabetic foot infections [A neutropenic mouse model was used to determine the efficacy of phage PEV20 combined with ciprofloxacin on er agent . In addiotherapy . Bacterifections ,88. The These findings on animal studies of phage–antibiotic combined treatments demonstrated a reduction in bacterial burden caused by both Gram-positive and Gram-negative bacteria, and the ability to recover from the infections increased considerably.Phage therapy was applied to patients in the early years of its discovery, but its use was decreased due to the lack of regulation or standardised methodology, and for safety reasons . In viewK. pneumoniae in a 63-year-old female with Type-2 diabetes and hypertension. Phage monotherapy failed twice, as phage resistance caused re-emergent bacterial isolates. When trimethoprim-sulfamethoxazole was administered orally twice a day with the phage cocktail once a day through bladder irrigation, the resistant strain was completely inhibited, and the urinary tract infection did not recur 6 months after discharge from hospital [A phage cocktail that consisted of six lytic phages combined with trimethoprim-sulfamethoxazole was used to treat a recurrent urinary tract infection with drug-resistant hospital .A. baumannii (CRAB). Phage Ab_SZ3 was administrated with tigecycline and polymyxin E. The phage was given by a vibrating mesh nebuliser once daily, with gradually increasing doses from 5 × 106 PFU to 5 × 1010 PFU on Day 13. Tigecycline was given intravenously for the first 5 days of treatment, followed by oral polymyxin E for another 5 days. In a total of 16 days of treatment, no CRAB cultures were detected in the patient’s sputum from the 7th day onwards, with only one exception on the 15th day, when a positive CRAB culture was found. One month after phage treatment, the patient developed sepsis caused by E. faecium and S. haemolyticus. During treatment with vancomycin, colonisation by P. aeruginosa was also found in the patient’s bronchoalveolar lavage fluid (BALF) culture. Even with the occurrence of different types of bacterial infection and the pressure of antibiotic use, CRAB showed no reappearance after the combination treatment [However, not all patients with chronic infections had the same recovery profile after treatment. An 86-year-old female patient with chronic obstructive pulmonary disease was infected by carbapenem-resistant reatment .As an intact immune system plays an important role in suppressing bacterial infections with opportunistic pathogens , chronicAchromobacter species, the combination therapy of phage Ax2CJ45f2 and two antibiotics (cefiderocol and meropenem/vaborbactam) by intravenous administration improved forced expiratory volume in one second  from 33% to 60%. The symptoms were alleviated with decreased sputum production and reduced cough. No adverse effects were observed with the combined treatment [In a pediatric patient with cystic fibrosis female infected with pan-drug-resistant reatment .Above all, phage–antibiotic combinations have produced encouraging outcomes in treating multi-drug-resistant bacterial infections. However, standardisation of the treatment method is difficult, as patients have different medical histories and varying levels of immunity. The administration route, time, and dose of phage and antibiotics differed for each patient. Formulating personalised treatments is time-consuming and expensive, but standardised regimens may not exert the same efficiency in all patients.While most studies focused on the synergistic effects of phages and antibiotics against one bacterial strain or species, there is a demand for investigations of the phage–antibiotic treatment of polymicrobial systems. In clinical settings, bacterial infections are usually associated with multiple strains or species. As a result, the genetic pool is enlarged in these polymicrobial systems, and there are more opportunities for mutations to occur in bacteria as well as phages. These uncertainties may produce varying efficacies in phage–antibiotic therapy.P. aeruginosa phage 14/1 with gentamycin reduced bacteria density in biofilm for the first 12 days, but then regrowth occurred due to resistance development [Another challenge of polymicrobial systems is the typical formation of biofilm. The spatial structure of biofilm may prevent antimicrobials from reaching sensitive populations. Hence, their efficacy against biofilms is reduced compared to planktonic cells. Biofilms also allow more time for co-evolution between phage- and antibiotic-selected populations under prolonged exposure to phage and antibiotics. For instance, an in vitro study reported that co-administration of elopment .P. fluorescens SBW25 was found to be faster than that of bacteriophage SBW25Φ2 [It is believed that phages have the advantage of co-adapting with bacteria; hence, they can overcome the development of bacteria resistance to phages in recurrent or chronic infections. However, the mutation rate of bacteria  SBW25Φ2 .P. fluorescens SBW25 was the lowest after 66-days of co-treatment with SBW25Φ2 (MOI = 0.01) and streptomycin  compared to SBW25Φ2 with streptomycin at 0.2 µg/mL or without streptomycin. The phage titer decreased more significantly with a higher concentration of streptomycin, and no phage was detected with 2 µg/mL (1/10 × MIC) streptomycin by Day 14, whereas only a small decrease of around 1 log in phage titer was observed on Day 66 without streptomycin. The phage titer started to decline as bacteria hosts developed resistance to the phage [p < 0.001 for 0.2 and 2 µg/mL streptomycin compared with no streptomycin. No statistical significance was detected between streptomycin treatments of different concentrations) [Furthermore, Cairns et al. reported that the bacterial density of he phage . Anotherrations) . This obrations) .As there is only limited research that investigated the long-term mutation dynamics of bacteria and phages in the presence of antibiotics, it is unknown whether the presence of antibiotics promotes the mutation rate of bacteria and/or phages, or whether the changes in mutation rates vary depending on the bacteria species, antibiotic class, or phage type.P. aeruginosa lung infection [So far, phage-based clinical therapies are mainly personalised. Unlike antibiotics, the specificity of phage has hindered its wide use as predefined formulations on the market. In response to that, strategies commonly considered include the modification of the antimicrobial spectrum of phages through genetic engineering , the expnfection ,99 and hnfection  have shonfection ,98. Stilnfection ,101.As antibiotic resistance is a life-threatening danger to global health, phage therapy has attracted attention over the past few decades as a potentially efficient alternative solution. The abundance, specificity, and versatility of phages are their advantages. Since antibiotics have been found to enhance phage production and further increase antimicrobial activity, phage–antibiotic combination treatments have been extensively studied. In the presence of antibiotics, instead of dividing, bacteria undergo elongation (filamentation), which facilitates phage adsorption, modifies bacterial cell lysis time, and/or increases phage burst size, which in turn results in enhanced phage replication. In vitro experiments showed synergistic antimicrobial effects with phage–antibiotic co-treatment, resulting in a significant reduction in bacterial growth. Moreover, antibiotic doses and administration sequence are two factors contributing to the variability in synergism. High antibiotic doses do not necessarily produce better bactericidal effects with phages, as inhibition driven by antibiotics may adversely affect phage production. The antibiotic suppression of phage replication in simultaneous administration was demonstrated in several studies, while sequential treatment with phages followed by antibiotics showed better antibacterial effects. The order of administration depends on the specific antibiotic. In vivo efficiency generally agreed with in vitro findings. Promising outcomes with better survival and faster healing have been presented in rodent models and larvae models when combination therapy was given. Further investigations with other animal models or systems need to be developed for a better understanding of phage and antibiotic co-therapy. In clinical case studies, phages combined with antibiotics improved treatment outcomes and reduced bacterial growth. Nevertheless, recurrent bacterial infections in immunocompromised patients are difficult to treat.Successful clinical cases of personalised phage–antibiotic treatment in tackling persistent MDR infections have been reported, but much progress needs to be made before it becomes popular. Firstly, as phages are bacteria-specific, a rapid diagnosis should be developed for precise phage identification. Secondly, world-wide phage banks are needed for the quick selection of suitable phages. Thirdly, dose regimens, including dosage and administration sequence, must be optimised for safe and efficient phage–antibiotic treatment for infections of different severity. Current or completed clinical trials on bacteriophages lack data for their combined use with sublethal dose of antibiotics. Although phage with sublethal doses of antibiotic reported more benefits than those with lethal doses of antibiotics in in vitro models, it may be not ethical for clinicians to prescribe sublethal doses of antibiotics based on existing clinical trials. Hence, further dose optimisation and clinical trials are needed. Fourthly, to prevent a regrowth of uncleared MDR bacteria, researchers should develop a strategy or dose regimen to achieve full pathogen eradication. In addition, phage–antibiotic therapy in association with innate immune system responses is another possible and promising method for eradicating MDR infections. Finally, rapidly formulating methods must be developed for improved delivery to sites of infections.While individualised treatment is the current mainstream of phage-based therapy, it is time-consuming and expensive. Predetermined phage–antibiotic formulations are considered for improved cost-effectiveness, but selecting suitable phage–antibiotic combinations is another challenge because they are not always synergistic against all bacterial strains. Extensive foundational work in synergy screening is needed for selecting phage–antibiotic combinations against different species. Moreover, formulating methods must be developed or optimised for improving stability for convenient storage and transport, and for adjusting the release of phages and antibiotics to achieve either simultaneous or sequential treatments. We believe that with increasing evidence from in vitro and in vivo works, as well as future clinical trials, it is feasible to develop predetermined formulations.Current methods in treating bacterial infections are not necessarily limited to conventional phage–antibiotic combined treatments; genetically engineered phages and phage-coded enzymes have also attracted recent attention. By modifying the tail fibre genes of phages to increase the host range for multiple targeting, the bactericidal effects would be significantly enhanced. Endolysin is an example of a phage-coded enzyme. Its rapid and potent killing of bacteria without apparent resistance development is attractive and may become a potential treatment for MDR infections. By combining new techniques and agents with antibiotics, phage–antibiotic combined treatment is clearly more promising for treating resistant bacterial infections than mono-phage therapy.


Salmonella spp. is a relevant foodborne pathogen with worldwide distribution. To mitigate Salmonella infections, bacteriophages represent an alternative to antimicrobials and chemicals in food animals and food in general. Bacteriophages (phages) are viruses that infect bacteria, which interact constantly with their host. Importantly, the study of these interactions is crucial for the use of phages as a mitigation strategy. In this study, experimental coevolution of Salmonella Enteritidis (S. Enteritidis) and a lytic phage was conducted in tryptic soy broth for 21 days. Transfer to fresh media was conducted daily and every 24 hours, 2 mL of the sample was collected to quantify Salmonella OD600 and phage titter. Additionally, time-shift experiments were conducted on 20 colonies selected on days 1, 12, and 21 to evaluate the evolution of resistance to past (day 1), present (day 12), and future (day 21) phage populations. The behavior of the dynamics was modeled and simulated with mathematical mass-action models. Bacteria and phage from days 1 and 21 were sequenced to determine the emergence of mutations. We found that S. Enteritidis grew for 21 days in the presence and absence of the phage and developed resistance to the phage from day 1. Also, the phage was also able to survive in the media for 21 days, however, the phage titer decreased in approx. 3 logs PFU/mL. The stability of the lytic phage population was consistent with the leaky resistance model. The time-shift experiments showed resistance to phages from day 1 of at least 85% to the past, present, and future phages. Sequencing of S. Enteritidis showed mutations in genes involved in lipopolysaccharide biosynthesis genes rfbP and rfbN at day 21. The phage showed mutations in the tail phage proteins responsible for recognizing the cell surface receptors. These results suggest that interactions between bacteria and phage in a rich resource media generate a rapid resistance to the infective phage but a fraction of the population remains susceptible. Interactions between Salmonella and lytic phages are an important component for the rational use of phages to control this important foodborne pathogen. Salmonella spp. is a relevant zoonotic pathogen transmitted to humans through food and contact with animals   arms race dynamics where new alleles allow bacterial resistance and phage infectivity over time, in which bacteria become more resistant to phages from the past than to present and future phages ; and ii)s SBW25) . To our ublished . TherefoSalmonella Enteritidis and a lytic phage in a high level of nutrients media. To assess the effect of the interactions between S. Enteritidis and the phage; we studied the abundance of S. Enteritidis and the phage, modelled their interactions, characterized the evolutionary model, and identified genomic changes in S. Enteritidis and the phage upon 21 days of coevolution.In the present study, we performed a coevolution assay for Salmonella Enteritidis strain DR016 was isolated from chicken feces in backyard farms of central Chile . Salmonella was grown in TSB for 16 h at 37°C and stored with 20% of glycerol at -80°C. Phage vB_Sen_STGO-35-1 (STGO-35-1) was isolated from a backyard chicken flock, using a strain of S. Enteritidis as host, according with the protocol described by al Chile . This isS. Enteritidis DR016 and phage STGO-35-1 at MOI 0.01 .The coevolution experiment one-host-one-phage was conducted using MOI 0.01 . The bacMOI 0.01 . Four reS. Enteritidis was measured with a spectrophotometer using OD600nm . The phage titer was determined using unevolved S. Enteritidis and titer was conducted using the “spot-test”, as previously described  with binomial errors and the logit- link function. Replicates were included as a random effect and both bacterial or phage presence and time sample were included as fixed factors. The analysis was conducted using RStudio statistical software, version 3.5.2. Model was run with the ‘glmer’ function in the ‘lme4’ package in R.During the experiment, daily samples of 2 mL were collected in each replicate. The estimation of escribed  , present (day 12), and future (day 21). For the phage, 1 mL of lysate was stored as described above. For S. Enteritidis, cultures were serially diluted to 1·106 CFU/mL, a total of 100 µL were streaked on TSA plates and 20 colonies were randomly selected for purification and frozen with 20% glycerol at -80°C. The proportions of S. Enteritidis resistance to phage and phage infectivity in S. Enteritidis was calculated from the three times by cross streaking the isolated 20 colonies across lines crossed by streaked phages, as previously described  with binomial errors and the logit- link function to test whether phage local adaptation was affected by the time sample in which bacteria was collected using RStudio (version 3.5.2). In this analysis, replicates were included as a random effect and both bacterial and phage time samples were included as fixed factors. As the replicates had a similar behavior, we used a generalized linear mixed model (GLMM) to examine variation in bacterial resistance where replicates were included as a random effect and both bacterial and phage time samples were included as fixed factors.To evaluate bacterial resistance and phage infectivity, generated through the coevolution experiment, phage and escribed   a reference genome represented by S. Enteritidis before the coevolution assays, ii) S. Enteritidis coevolved from day 1 from all 4 replicates, iii) S. Enteritidis coevolved from day 21 from all 4 replicates, and iv) controls  from days 1 and 21 (S. Enteritidis before coevolution (reference genome) were assembled using the ‘isolate’ mode in SPAdes v3.14.0. Then, scaffolds with length <1kb were removed from the assembly. Blasting resulting scaffolds against an NCBI database of Salmonella enterica, showed that all scaffolds presented >95% of identity. One scaffold with 100% identity to Salmonella enterica plasmid pSJTUF10978 (CP015525) was removed. The final assembly had a consensus length of 4,647,970 bp spanning 24 scaffolds, with an N50 value of 490,728 bp. The average genome coverage was 134X with a GC % of 52.1%. Finally, collinearity with S. enterica (CP050716) was evaluated using D-GENIES (S. enterica genome (98.79%) was covered by the assembly. Gene annotation of this assembly was performed with RASTtk  reference phage represented by STGO-35-1 before the coevolution, ii) coevolved phage from day 1 from replicate 1, and iii) coevolved phage at day 21 from replicate 1. Phenol/chloroform extraction of DNA and precipitation with ethanol were performed for phage STGO-35-1, as previously described . DNA conSalmonella and phage genomes, we used Bowtie2 (version 2.4.1), using default parameters , which splits adjacent SNPs into individual SNPs, left-aligns indels, and regularizes the representation of complex variants. SnpEff (version 4.0e)   was usedS. Enteritidis showed that it survived in the presence of the phage for 21 days . In general, the four replicates behaved homogeneously, with an exponential multiplication on day one and then maintaining an optical density between 1.3 and 1.6 until day 21. In the control, where only S. Enteritidis was inoculated, the optical density was similar among the replicates, with no significant changes. These results suggest that the phage STGO-35-1 in the media did not affect the abundance of S. Enteritidis using a MOI of 0.01. We also observed a homogeneous behavior among the replicates in terms of phage populations. On day one, the phage had a higher titer and then declined steadily over time but did not go extinct. The control with only phage inoculation was indetectable by PFU quantification after day 3 . Our results could indicate that S. Enteritidis of day 1 (past) developed resistance to the phage. Still, a minor population of S. Enteritidis remains susceptible and allows the phage to replicate for 21 days. Our results indicate that a subpopulation of Salmonella is still phage-susceptible in all the replicates tested. We observed that the phage titer decreased over time (104 PFU/mL) . Similar behaviors of host-phage coexistence were reported for Streptococcus thermophilus and a lytic phage (The quantifications of  21 days . In gene 21 days . In geneic phage .R) at 24 hours, which explains why the phage titer decreased . This result is repeated when different rates for the transition of susceptible to resistant bacteria (μN) are used in the simulations. Interestingly, in all the cases, a minor population of coexisting susceptible bacteria (N) is present in the culture, which could explain why the phage survive for the 21 days, even when the dominant population of Salmonella are resistant. The latter observation could support the hypothesis of the cohabiting population of susceptible bacteria being able to host the phage multiplication and its maintenance in the culture; however, the density of this population (1·102 CFU/mL) might be too low to satisfy the probability of infection (E. coli (R) but maintaining the resistance generation rate constant over time. The results indicate that dominance of resistance at 24 hours occurs in most cases . In this model, a considerable rate of genetic and/or phenotypic reversion of resistant to susceptible bacteria occurs, and then populations can find stability through low rates of multiplication . The leaky resistance model was described for E. coli and a virulent mutant of phage Lambda; and in Salmonella, a previous study that coevolved S. Enteritidis for 12 days with a lytic phage, did not show population density for susceptible bacteria  the past (day 1), ii) the present (day 12), and iii) the future (day 21); (S. Enteritidis developed a high resistance proportion (0.75 – 1.00) against the phages for all the different times  in all four replicates, without significant differences between the different phage times or replicates  . We also tested phage infectivity of isolated Salmonella obtained from i) the past (day 1), ii) the present (day 12), and iii) the future (day 21). Conversely, phage infectivity was found in low proportions (0.00 – 0.25) in all three times and replicates as well, similarly, without significant differences between Salmonella time or replicates  . Overall, our results show a bacterial resistance mechanism emergence during the first day of coevolution that lasted through the 21 days of the experiments. Over time, high resistance to phage was reported for other bacterial genera such as Vibrio sp. and E. coli with lytic phages . Importantly, the interactions between bacteria and lytic phages depend on the host and the environment with controlled parameters as temperature, resources, and multiplicity of infection  . We alsoc phages . But thiis s25pp . Importanfection .S. Enteritidis could have an advantage over phages in this model of TSB, suggesting, for instance, that it could be easier for Salmonella to mutate the phage receptor than for the phage to find the exact mutation for the new receptor under these experimental conditions   . This inovement) . Then, eruginosa . Thus, fS. Enteritidis populations from days 1 and 21 in all four replicates to determine SNPs in coevolved Salmonella with the lytic phage. We found 31 SNPs distributed in the replicates for days 1 and 21 . SNPs were classified into four categories according to nucleotide changes and putative impact. Specifically, in i) missense presenting a moderate impact (13 SNPs), ii) frameshift presenting a high impact (3 SNPs), iii) stop gained presenting a high impact (1 SNP), and iv) intergenic mutations presenting a lower impact (9 SNPs). At day one, 6 SNPs were identified, most of them being missense (5 SNPs), followed by intergenic (1 SNP). These SNPs, were found in genes encoding membrane proteins as the oxaloacetate decarboxylase and undecaprenyl-phosphate-galactosyltransferase, involved in the synthesis of antigen-O for the LPS , suggesting that the mechanism for resistance is dynamic and can change through, and between, replicates. It would be interesting to evaluate if mutations observed at day 21 are more stable or can still be subjected to further modifications. In a previous study conducted in S. Enteritidis, most of the SNP at coevolved Salmonella with a lytic phage were associated with cell wall proteins and capsule proteins (Pseudomonas after exposure to DSM3 were also on membrane receptor. Additionally, different studies report that mutation in LPS genes induces phage resistance to Salmonella (In general, identified SNPs were present in genes encoding proteins involved in different processes, such as metabolism, virulence factors, LPS biosynthesis, and membrane protein biosynthesis proteins . Other mlmonella . This inS. Enteritidis. However, our results indicate a potential counter resistance mechanism in phage STGO-35-1 to overcome the bacteria resistance developed.To determine SNPs in the lytic phage STGO-35-1, we sequenced phage isolated from day 1 and 21. A comprehensive report of genomic characteristics of this phage was recently reported  and a shSalmonella Enteritidis and a lytic phage vB_Sen_STGO-35-1 in a rich nutrient media for 21 days. Our study highlights the potential of S. Enteritidis to co-evolve with lytic phages in a high nutrient environment by rapidly developing a resistant population but maintaining a low frequency of susceptible bacteria available to the phage for replication. The genomics data showed that Salmonella have different SNPs in response to the phage presence and the phage had less SNPs than the bacteria, but in important proteins described as bacterial receptors While most of the changes occurred during the first 24 hours, initial resistance mechanisms could change throughout the 21 days as observed in our sequencing results. Understanding these interactions contributes to the responsible use of the phages as biocontrol tools.Results presented here contribute to expand our current knowledge on coevolution dynamics using a model of The datasets presented in this study can be found in online repositories. The name of the repository and accession number can be found below: NCBI; PRJNA821546.RB-M, DR, FPÁ, and AM-S: conceived the idea, designed and conducted the experiments; MS, RG, JB, FA, SR, and CH-W: analyzed and interpreted data; RB-M, DR, DÁ, RG, and AM-S: wrote the manuscript, RB: discussed the results and critically revised the manuscript. All authors contributed to the article and approved the submitted version.We acknowledge the funding sources: 1 [ANID FONDECYT 1181167 to AIMS], 2 [ANID Millennium Science Initiative/ Millennium Initiative for Collaborative Research on Bacterial Resistance NCN17_081 to AM-S], and ANID FONDECYT 3210317 to DA.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Mycobacterium abscessus lung disease previously developed anti-phage neutralizing antibodies while receiving intravenous phage therapy. Subsequent phage nebulization resulted in transient weight gain, decreased C-reactive protein, and reduced Mycobacterium burden. Weak sputum neutralization may have limited the outcomes, but phage resistance was not a contributing factor.An elderly man with refractory  Mycobacterium abscessus is an emerging opportunistic pathogen of increasing clinical significance [M abscessus lung disease is often complicated due to limited numbers of effective antibiotics, prolonged duration of therapy, and frequent treatment-related adverse events [M abscessus infections have prompted exploration of mycobacteriophage therapy for these organisms, although the optimal patient selection, dosing, and route of phage administration for nontuberculous mycobacteria (NTM) remain unclear [ificance . The mede events . Bacterie events . Poor tr unclear , 6.M abscessus. The first patient was a 15-year-old girl with cystic fibrosis (CF) whose post–lung transplant course was complicated by treatment-refractory disseminated M abscessus infection, which was successfully controlled with the use of an IV mycobacteriophage cocktail [M abscessus subspecies massiliense lung disease [M abscessus treatment failure and led to its discontinuation. Here we report the outcomes of this patient after switching the route of phage administration to aerosolized delivery.Two recent case reports , 8 descrcocktail . A secon disease ; howeverM abscessus lung disease was treated for 6 months with an IV cocktail of 3 mycobacteriophages  in addition to a multidrug antibiotic regimen, as previously described [M abscessus burden in sputum at month 1 (C), with subsequent rebound in mycobacterial colony-forming units (CFUs) that temporally correlated with the emergence of a robust anti-phage immunoglobulin M and immunoglobulin G (IgG) neutralizing antibody response, specific to the phages used therapeutically [An 81-year-old man with non-CF bronchiectasis and refractory macrolide-resistant  month 1 C, with sutically . After aH), the phage cocktail was administered twice daily via nebulization using a Philips Respironics InnoSpire Essence Compressor Nebulizer System at a dose of 1 × 109 plaque-forming units in 3 mL of 0.9% normal saline. Safety and efficacy monitoring during nebulized therapy included serial clinical and microbiological assessments, in-home spirometry with the Microlife PF 100 digital peak flow and forced expiratory volume in 1 second (FEV1) meter, and serial chest computed tomography (CT). Standing weight was recorded regularly by the patient (A) and quantitative assessment of M abscessus counts in expectorated sputum was calculated approximately monthly as the mean CFUs/mL−1 (C). Serial M abscessus isolates were assessed for changes in antibiotic susceptibility and for acquisition of phage resistance.To overcome serum neutralization and to increase phage delivery to the site of infection, the patient was started on nebulized administration of the same triple phage cocktail. Regulatory and institutional approval was obtained from the United States Food and Drug Administration and the Johns Hopkins Institutional Review Board and written informed consent was obtained prior to initiation. While continuing multidrug antibiotic therapy for NTM lung disease H, the ph patient A and quaFUs/mL−1 C. Serial1 of 1.71 L (A). Following initiation of nebulized phage therapy, FEV1 transiently increased to 2.13 L on day 3, and later remained relatively stable over time, with a 0.12 L decline during the 9-month nebulized treatment period (B). Changes in companion antibiotics during phage therapy were motivated by adverse events suspected to be antibiotic-related. These events included development of eosinophilia, prompting discontinuation of IV imipenem, and nausea and mild liver function test abnormalities, leading to discontinuation of omadacycline and clofazimine . Parenchymal lung abnormalities on chest CT appeared relatively unchanged over time, with only minor interval changes . C-reactive protein (CRP) decreased from 69.3 mg/L prenebulized phage to a nadir of 23.9 mg/L 4 months into nebulized phage (B). While M abscessus burden in sputum fluctuated (C), weight gain during nebulized phage therapy correlated temporally with a relative quantitative reduction in M abscessus in sputum and a decrease in CRP—consistent with an improved systemic response to infection with the use of nebulized phage.With initiation of nebulized phage therapy, there was a reduction in subjective sputum production. By approximately 3.5 months into nebulized phage therapy, the patient’s body weight increased by 8.7% for a total weight gain of 4.3 kg (9.5 lbs) from a prenebulized phage baseline weight of 49.7 kg (109.5 lb) A. C-reaced phage B. While uctuated C, weightA–1C). Because of concerns for potential physical destruction and phage loss with the jet compression-type nebulizer [Approximately 4 months after initiation of nebulized phage delivery, these apparent benefits from phage administration dissipated, and the subsequent 7 weeks of treatment were accompanied by weight loss, increase in CRP, and rising sputum bacterial counts A–1C. Becebulizer , 11, admM abscessus isolates recovered 7 and 8 months after the start of phage nebulization were tested for phage susceptibility; these specimens had similar profiles to earlier isolates and remained fully sensitive to all 3 phages in the regimen (A). The emergence of bacterial resistance to phage therapy was not observed, nor were there changes in antibiotic susceptibilities , and a weak but notable response to all 3 phages prior to nebulization was observed, which fluctuated after the start of nebulization but did not increase consistently or substantially; sputum IgA titers remained about 100-fold lower than serum IgG levels to the same phages . This sputum IgA reactivity may have occurred, in part, as a response to the 9 months of prior IV phage administration. However, sputum samples showed only weak phage neutralization , which was about 4 logs lower than observed with serum samples 8 months post–phage nebulization .To determine if treatment failure was caused by the emergence of phage resistance,  regimen A. The embilities . Sputum B and 1C). After the start of aerosolized delivery, weight gain, fall in CRP, and decline in mycobacterial load were initially encouraging. These clinical improvements lasted longer (3–4 months) than those observed after start of IV therapy (1–2 months). However, these gains with nebulized phage were again only transient, and the mechanisms underlying the limited duration of the benefits of phage aerosolization in this patient are unclear. We anticipated that emergence of phage resistance might occur, but after 8 months of nebulization—and 15 months of total phage therapy—the strain remained fully sensitive to phages Muddy and BPsΔ33HTH_HRM10, and with only a slight reduction in sensitivity to ZoeJΔ45 (A), which was also observed after IV treatment alone [M abscessus clinical isolates is consistent with in vitro studies [Mycobacterium infections. Aerosolized treatment failure did not result from antibody-mediated phage neutralization either, as only relatively weak sputum IgA responses to the phages were observed, and only mild neutralization (E and 1G). However, neutralization did increase at later times (7 and 8 months) after the start of nebulization, and it is plausible that this contributed to limitation of treatment effect.The clinical progression following the switch to aerosolized phage administration mirrors what was observed after the start of IV administration: some benefits initially, followed by subsequent dissipation of those clinical gains. For IV administration, the emergence of strong neutralizing antibody response to the phages was temporally correlated with increases in mycobacterial burden and rises in CRP B and 1C. ZoeJΔ45 A, which  studies  and is alization E and 1G.Mycobacterium infections. Some patients may see clinical improvement and long-term benefits [This case study highlights the challenges with using aerosolized bacteriophages for controlling benefits  whereas Open Forum Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.ofac194_Supplementary_DataClick here for additional data file.


Antibiotic resistance is one of the major challenges that humankind shall face in the short term. (Bacterio)phage therapy is a valuable therapeutic alternative to antibiotics and, although the concept is almost as old as the discovery of phages, its wide application was hindered in the West by the discovery and development of antibiotics in the mid-twentieth century. However, research on phage therapy is currently experiencing a renaissance due to the antimicrobial resistance problem. Some countries are already adopting new ad hoc regulations to favor the short-term implantation of phage therapy in clinical practice. In this regard, the Phage Therapy Work Group from FAGOMA (Spanish Network of Bacteriophages and Transducing Elements) recently contacted the Spanish Drugs and Medical Devices Agency (AEMPS) to promote the regulation of phage therapy in Spain. As a result, FAGOMA was asked to provide a general view on key issues regarding phage therapy legislation. This review comes as the culmination of the FAGOMA initiative and aims at appropriately informing the regulatory debate on phage therapy. The emergence of multi-drug-resistant (MDR) bacteria seriously undermines our ability to control bacterial infectious diseases. A recent study shows that MDR pathogens already cause more than one million deaths a year, and the prospects are even more concerning ,2. There(a)Activity against antibiotic-resistant bacteria. Phages can infect and kill bacteria, including MDR strains . This is(b)Specificity. The high specificity of many phages towards their host bacterial strains makes them a highly selective therapy that prevents the dysbiosis of the healthy microbiota. Contrary to antibiotics, only strains of the same genus or species—and often just one or very few strains within a species—are susceptible to infection by a given phage, protecting the normal microbiota and reducing side effects . The spe(c)Multiplication at the site of the infection (auto-dosage). Phages can multiply at the site of the infection. Once the phages reach the targeted bacteria, they will replicate and generate progeny. Therefore, if sufficient phage particles are able to reach the infection site, phage therapy can be considered as auto-dosage treatment. Furthermore, once the infection is successfully controlled, phages would be eliminated in the absence of bacterial hosts. Thus, whenever an auto-dosed, “active” treatment is achieved, it can elicit the infection eradication by only a single administration .(d)Ubiquity and diversity. Phages can be found in virtually any environment , and the(e)Evolvability. Phages are evolving entities and, therefore, can be optimized using directed evolution techniques. This opens up many possibilities compared to conventional treatments, which are stable chemical compounds. Phage evolvability can be exploited in many ways, such as increasing lytic capability, improving particle stability, expanding the host range, or counteracting bacterial resistance. For instance, the Appelmans’ protocol uses spontaneous mutation and recombination among phages present in a cocktail to produce phage variants capable of infecting initially non-susceptible bacterial strains ,20.(f)Safety. Humans are carriers of many different phages forming the phageome ,22. Thei(a)Phage-resistance. In the same way that resistance to antibiotics emerges, bacteria can become resistant to phage infection. The most common solution to address this involves the use of cocktails of different phages, rather than a single phage, and/or the “à la carte” selection of phages for each particular infectious isolate. This makes it much less likely that the host will become resistant to all phages at the same time . The so-(b)Specificity. This feature can be a double-edged sword. Phage specificity requires careful susceptibility testing of each bacterial pathogen before treatment, which may be viewed as an issue for certain acute infections that require urgent action. In addition, this specificity may require either the development of large phage libraries and/or extensive sampling and screening efforts to provide sufficient coverage of bacterial diversity. This can be a daunting task and has posed major regulatory issues, since, according to the current framework, each individual phage should undergo review and approval. In addition, the eventual need to develop a different phage preparation for each bacterial pathogen, as a personalized medicine, reduces business profitability and can be viewed as a serious drawback by pharmaceutical companies. Again, phage cocktails targeting different receptors or different bacterial strains would be a potential solution.(c)In vivo phage activity. There is not necessarily a correlation between the in vitro and in vivo behavior of a phage, particularly regarding its propagation ability. This is due both to the complexity of body fluids and the ecological in vivo interactions ,30. In a(d)Immune response. Since phages are made of biomacromolecules, they are potentially capable of eliciting an immune reaction upon administration . General(e)Gene transfer. Phages potentially have the ability to modify the genome of the host bacteria, which may increase their virulence or dissemination of antibiotic resistance genes . Indeed,The main steps for obtaining phage suspensions suitable for use in clinical settings are summarized in 31 particles [The process of phage therapy begins with the identification and selection of phages and bacterial strains suitable for phage production. Phages are found in any environment, even under extreme conditions, being the most abundant biological entities on our planet, with a current total estimate of 10articles . Phage “articles , since particles .Mycobacterium smegmatis is strongly preferred for the isolation of mycobacteriophages over the pathogenic, slowly growing Mycobacterium tuberculosis [Once samples are collected, phage screening parameters will depend on the target bacterium. In general, phages can be isolated using reference strains or clinical isolates as hosts for propagation . In somerculosis ). If a brculosis . Apart frculosis ,48. In gPhage production is typically carried out by the double-layer agar method, which allows isolation of individual phages, and then liquid culture of single plaques . A pure ® (CIM) columns [The main goal of this process is to avoid the presence of bacterial toxins, lipopolysaccharide, or other cellular debris, in the phage suspensions . In addi columns . These m2+ and Ca2+ ions (around 10 mM in the form of CaCl2 or MgSO4) are the most used supplements since they are added to the culture medium before infection to facilitate adsorption and are then present in the recovered lysate [Depending on the phage, phages can be stored at different temperatures, commonly at 4 °C, −80 °C, or in liquid nitrogen (−196 °C), or can be freeze-dried ,55. Protd lysate ,56. Othed lysate ,58. Adsod lysate .Phages are basically protein structures and, therefore, they are susceptible to proteases, certain chemical compounds, high temperatures, pH, and ionic strength. Thus, it is important to use an appropriate formulation to ensure that the phage titer remains stable both during formulation and storage and in the in vivo environment where they are applied. Again, the optimal formulation conditions may vary depending on the specific phage, so this deserves careful consideration in the case of phage cocktails, as each type of phage may require individually tailored conditions . However(a)Oral administration is appropriate for gastrointestinal infectious diseases. In some cases, oral phages given without additional protection, as water-based liquid suspensions, reportedly survived gastric passage and were recovered in the feces ,61,62. T(b)Topical administration of phages is chosen for skin infections, wounds, burns, ulcers, and osteoarticular infections . Phages (c)The local phage treatment of respiratory infections requires preparing phages either as stable liquid formulations for intranasal instillation or nebulization, or as a solid powder in an inhalable form . The mos(d)Intravenous administration is recommended in the treatment of systemic infections . In this(e)Intravesical instillation of liquid phage preparations has also been used to treat genitourinary tract infections ,78.(a)Phage identity. The identity of each phage is defined by its specific genomic sequence ,80. Meta(b)Phage Titer. The titer of each individual phage is classically assessed by the double-layer agar method. An alternative is lethality curves, in which the kinetics of phage-induced lysis are assessed by measuring the optical density of phage-infected bacterial cultures . Other m(c)General Purity. For biopharmaceuticals, the purity and correct composition is classically assessed by high-performance liquid chromatography, combined with mass spectrometry if necessary. These methods can be used to identify phage capsid proteins, toxins, or other bacterial proteins. Because of the potential risk posed by the necessary production with pathogenic bacterial hosts, quality criteria should specify maximum levels for contaminants such as toxins or bacterial DNA, which normally must be tested with specific and appropriate molecular biology methods as specified below.(d)Toxins. Several in vitro methods have been developed for endotoxins quantification: gel-clot, turbidimetric, and chromogenic tests. Among the latter, the limulus amebocyte lysate assay is the most widely used . When th(e)Contaminating Nucleic Acids. Quality controls may also be required to determine the concentration of contaminating nucleic acids . The presence and concentration of residual nucleic acids are typically checked by qPCR.(f)Other Quality Controls. Current regulations on sterility or general quality parameters in pharmaceutical products should also apply to phage-based pharmaceuticals . Some paAs products intended for human therapy, phage preparations must comply with certain quality criteria that ensure their safety for clinical use, as well as exhaustive traceability documentation. The production and delivery parameters must be set up by the phage preparation supplier in accordance with the applicable regulations see  and to mPerhaps the greatest hurdle to the implementation of phage therapy in Western medicine is the lack of appropriate regulation. If phage preparations are considered “classical” medicinal products, they must comply with the corresponding legislation concerning medicinal products production and quality. This essentially means that they should follow GMP (good manufacturing practices) requirements, which has imposed an important problem for the development of medicinal phage preparations in terms of increasing costs or complicating management for developing large-scale production .Although therapeutic phages shall be considered indeed a medicinal product, the nature itself of the phage makes it essentially different from common antimicrobial chemotherapy. Indeed, phage specificity, virus–host co-evolution, or a complex in vivo pharmacological behavior have negatively influenced the outcome of many of the phage therapy clinical trials conducted in contemporary times ,89,90,91A recently adopted strategy in Belgium has allowed a more systematic and practical approach to personalized phage therapy, which also provides production and handling flexibility . The key to this approach is to consider phage products as magistral preparations, since, unlike medicinal products, these are subject to less strict regulation in terms of their production and marketing ,94. In tRegardless of the sub-optimal regulatory status of phage therapy in most countries, including Spain, phage preparations for therapy are available either commercially  or through the request to academic or clinical institutions devoted to phage therapy research and promotion.Pseudomonas aeruginosa) and testing the identity and quantity of bacteria present in every patient [Although potentially controversial , an adap patient . Further(a)In the case of a severe infection produced by an MDR bacterium.(b)When the infection occurs in an area reluctant to the use of antibiotics, such as in prosthetics.(c)Or, in general, whenever there is no standard of care option available, such as patients suffering from hypersensitivity to the antibiotic treatment.In our opinion, the use of phage therapy would be advisable:In these situations, i.e., in the absence of any other satisfactory therapy and when there is a real risk to the patient’s life or significant deterioration of their quality of life, the use of phages, possibly in combination with standard-of-care antibiotics, would be advisable. Conversely, whenever the patient’s infection can be satisfactorily treated with antibiotics, those will always be the therapy of choice. Additionally, a proper diagnostic of the infection-causing bacteria must be conducted see , as wellHaemophilus influenzae, P. aeruginosa, Stenotrophomonas maltophilia, S. aureus, Achromobacter spp., Burkholderia cepacia, and Mycobacterium abscessus, usually resistant to many antimicrobials. Among them, the main pathogens in Spain are S. aureus and P. aeruginosa , with the latter being significantly associated with worse pulmonary function. According to a Spanish multicenter study, 55% of P. aeruginosa isolates from CF patients were MRD, while 16% were extensively drug-resistant [P. aeruginosa, is expected to have a remarkable impact on the life quality and expectancy of these patients.Cystic fibrosis (CF) is a rare disease . Althouesistant . In addiesistant ,105. CF P. aeruginosa and S. aureus resistant to methicillin, fluoroquinolones, and rifampicin. In these cases, treatment options are often limited to antibiotics of last resort, such as polymyxins which have been shown to have higher relapse rates and higher frequency of complications.These are a particular form of deep, localized infections with poor response to antibiotic therapy. The diffusion of antibiotics into bone tissue is often low and is negatively affected by the presence of bacterial biofilms at the contact between bone and prosthetic material. Repeated surgeries to eradicate the biofilm mechanically, i.e., removing the implant and/or resecting the infected bone, and sometimes amputation, is the only infection control option . Most ofThere are some recent examples of clinical cases (CF and osteoarticular infections) treated with phages that support the feasibility of this therapy .In addition to phages, their lytic enzymes have also been explored as antimicrobials . Phage lSince peptidoglycan is present exclusively in bacteria and not in mammalian cells, the risk of cytotoxic effects to humans and animals is minimized. However, since they are proteins, they may induce an immune response. Indeed, in vitro and in vivo studies have shown that neutralizing antibodies can be generated upon repeated exposure to lysins. However, although these antibodies reduce the antibacterial activity of the enzybiotics, they do not completely neutralize them ,123, whiSystemic administration of enzybiotics may also release cellular debris from lysed bacteria, which may induce a proinflammatory response. These bacterial cell debris include lipopolysaccharides, (lipo)teichoic acids, and peptidoglycan through membrane fragmentation and can potentially lead to severe complications such as septic shock. However, allergic or severe inflammatory reactions have not been described so far upon administration of enzybiotics, even in clinical trials with human subjects ,131. The® SAL200 (Tonabacase) [S. aureus bacteremia without observing any adverse effects. Tonabacase is currently in phase II [S. aureus [In recent years, the way has been paved for recombinant lysins to enter different phases in preclinical and clinical trials. Accelerated clinical advances and their high technical feasibility make them a good alternative therapy to replace or to be used in combination with conventional antibiotics in the short term, even with some authors pointing out that lysins may be able to enter the clinic in a shorter term than phages . Some coabacase)  and Exebabacase) , againstphase II , and Exe. aureus .As discussed in this review, the application of phage therapy is experiencing a considerable boom in recent years. Since 2017, the global phage therapy market reached USD 567.9 million and growth expectations at compound annual growth rate (CAGR) are of 3.9% for the period from 2018 to 2026 . In 2016However, some potential hurdles may still be found in the way towards a successful market entrance, such as the already mentioned regulatory issues , the quaIn this context and given that large pharmaceutical companies have practically stopped investing in new antimicrobial compounds, many scientific startups are focusing on studying and characterizing phages and their products for their therapeutic application. These new companies are located mainly in the USA, India, Korea, Canada, and some European countries. In Spain, the first company focused on the development of endolysins (Telum Therapeutics) has recently been created . An optiBacterial resistance is a major global threat and a leading cause of mortality worldwide. The lack of effective antibiotics against MDR strains urgently requires therapeutic alternatives, and phages or their derivatives can be a promising approach with sufficient scientific and technological know-how in place. However, the lack of both specific regulation for their clinical use and public awareness stands out, in our opinion, as the major hurdles to be presently faced. With this review, we would like to emphasize the advantages of phages as therapeutic tools and how they can be used to combat MDR bacteria. Moreover, despite some foreseeable drawbacks also covered here, phages are nowadays the only short-term solution for many patients. Taking this into account, a suitable regulatory initiative from the competent authorities would be welcomed in Spain in the short term. Both scientific literature and the lead examples of other countries prove that phage therapy is already mature to be translated into a regulation that must ease, protect, and help developing the phage therapy market and infrastructure.