Bacteriophages: The promising therapeutic approach for enhancing ciprofloxacin efficacy against bacterial infection

Abstract Background The emergence of ciprofloxacin‐resistant bacteria is a serious challenge worldwide, bringing the need to find new approaches to manage this bacterium. Bacteriophages (phages) have been shown inhibitory effects against ciprofloxacin‐resistance bacteria; thus, ciprofloxacin resistance or tolerance may not affect the phage's infection ability. Additionally, researchers used phage‐ciprofloxacin combination therapy for the inhibition of multidrug‐resistant bacteria. Results The sublethal concentrations of ciprofloxacin could lead to an increase in progeny production. Antibiotic treatments could enhance the release of progeny phages by shortening the lytic cycle and latent period. Thus, sublethal concentrations of antibiotics combined with phages can be used for the management of bacterial infections with high antibiotic resistance. In addition, combination therapy exerts various selection pressures that can mutually decrease phage and antibiotic resistance. Moreover, phage ciprofloxacin could significantly reduce bacterial counts in the biofilm community. Immediate usage of phages after the attachment of bacteria to the surface of the flow cells, before the development of micro‐colonies, could lead to the best effect of phage therapy against bacterial biofilm. Noteworthy, phage should be used before antibiotics usage because this condition may have allowed phage replication to occur first before ciprofloxacin interrupted the bacterial DNA replication process, thereby interfering with the activity of the phages. Furthermore, the phage‐ciprofloxacin combination showed a promising result for the management of Pseudomonas aeruginosa infections in mouse models. Nevertheless, low data are existing about the interaction between phages and ciprofloxacin in combination therapies, especially regarding the emergence of phage‐resistant mutants. Additionally, there is a challenging and important question of how the combined ciprofloxacin with phages can increase antibacterial functions. Therefore, more examinations are required to support the clinical usage of phage‐ciprofloxacin combination therapy.


| INTRODUC TI ON
Ciprofloxacin, a member of the fluoroquinolone drug class, is used to treat various Gram-negative bacteria such as Pseudomonas aeruginosa, Proteus mirabilis, Klebsiella pneumoniae, and Escherichia coli and Grampositive bacteria such as Staphylococcus aureus. This antibiotic suppresses the activities of DNA-gyrase and DNA topoisomerase during DNA replication, recombination, and repair. 1 Ciprofloxacin resistance in different bacteria has been increasingly reported worldwide because of mutations in DNA gyrase genes (gyrA), efflux pumps, or plasmidmediated-quinolone resistance in bacterial plasmids or chromosomes. 2 In this regard, applying a combination of different substances such as nanoparticles, natural compounds, and bacteriophages with ciprofloxacin to enhance the efficacy of this antibiotic against multi-drug resistant (MDR) bacteria has received much attention. 3 Bacteriophages (phages) are viruses that could lyse bacteria while are not harmful to animal and human health. The resistance mechanisms to phages are different from antibiotics; therefore, phages have been widely used to treat MDR bacteria. 4 Furthermore, phage-antibiotic combination therapy could re-sensitize resistant bacteria to antibiotics. 5,6 A phage cocktail, a mixture containing two or more phages with various host ranges in a single suspension, could kill bacteria more effectively. 7,8 Phage cocktails could lead to a better decrease in bacterial density and enhancement of the phages' activities. 9 From this point of view, previous studies have shown that phage cocktail causes a higher reduction in bacterial infections.
Noteworthy, phages can destroy biofilm structure and improve the penetration of the antibiotic to the dipper layers of biofilm by inducing the synthesis of enzymes such as polysaccharide depolymerase. 10 This enzyme can particularly destroy the macromolecule carbohydrates that exist in the envelope of the bacterial host and help the phage attach, penetrate, and lyse the bacterial cells. 11 Additionally, phages could inhibit bacterial biofilm by inhibiting bacterial attachment, interference with quorum sensing, and the degradation of the exopolysaccharide matrix. 12 Therefore, phages not only could kill bacteria but also could destroy the biofilm community of these microorganisms.
Although U.S. Food and Drug Administration has not yet approved, phages-antibioticscombination therapy has been applied in different emergency clinical situations tomanage MDR infections. 13 To this end, phage-ciprofloxacin combination therapy is considered by scientists for enhancement of the ciprofloxacin efficacy, especially against MDR bacteria. In this review article, we will specifically discuss various aspects of this combination therapy to clarify the advantage and disadvantages of this combination therapy and promote its possible widespread use in the clinical setting.

| THE US E OF PHAG E S FOR CIPROFLOX ACIN -RE S IS TANT BAC TERIA
As mentioned, resistance to ciprofloxacin is becoming an increasingly serious public health concern in the clinical setting. Recently published studies reported phages as a good therapeutic choice for the inhibition of ciprofloxacin-resistant bacteria and in this section, we will evaluate these studies.
Ciprofloxacin is suggested for the treatment of salmonellosis; but, overuse of this antibiotic could lead to drug resistance, infection treatment failure, and severe clinical outcomes. Resistance to this antibiotic has been increasingly detected in different parts of the world. 14 To this end, phages were used in different studies as an alternative approach for the inhibition of ciprofloxacinresistant Salmonella. Pelyuntha  However, P22 showed the lowest lytic efficacy against the clinical isolates of this bacterium with antibiotic resistance. 16 It's noteworthy to mention that bacteria could develop different mechanisms for phage resistance that could hinder the interactions between bacterial hosts and phages at multiple levels. These include mechanisms that prevent phage attachment to the bacterial surface, inhibition of phage-DNA penetration into the bacterial cell, or phage-DNA cleavage once inside the cell ( Figure 1). 17,18 To this end, the specificity of phages against bacterial cells is mostly related to the binding affinity between receptor-binding proteins in phages and receptors in the host. Hence, the alterations in receptors of the host cell surface are responsible for phage resistance, resulting in decreased lytic activity and this phenomenon should be considered in future studies. 19,20 Collectively, the recent WHO priority list of antibiotic-resistant bacteria has shown fluoroquinolone-resistant and ESBL-producing In addition to Salmonella, phages were also used for the inhibition of S. aureus. In this regard, the results of in vitro evaluation of a recent study showed that initial incubation of S. aureus with the minimum inhibitory concentrations (MICs) of commonly used antibiotics such as ciprofloxacin and mupirocin, could induce tolerance in this bacterium. Therefore, S. aureus tolerance to antibiotics might be easily induced clinically, for example when the uptake of orally administered antibiotics is hindered or when patients used antibiotics inappropriately for a long time. On the other hand, the authors reported that their Sa87 and Sa83 phages that were received from AmpliPhi Australia (Brookvale, NSW, Australia) lysed approximately 70% S. aureus clinical isolates and that the performance of phages was independent of antibiotic resistance profiles.
Moreover, these phages destroyed the planktonic and biofilm community of antibiotic-sensitive CI3 and ATCC51650 even after antibiotic tolerance was induced. Hence, the authors suggested that antibiotic resistance or tolerance does not affect the phages' infection ability. 21 Therefore, recently published studies introduced phage therapy as a promising alternative treatment to manage MDR bacterial infections. However, unfortunately, the mentioned studies did not evaluate molecular interactions of phages and ciprofloxacin-resistant bacteria; hence, this issue should be considered in future studies.

| THE COMB INED USAG E OF PHAG E S A ND CIPRO FLOX ACIN
In the management of bacterial infections, the use of phages or antibiotics is restricted because of bacterial ability to develop resistance and the frequency of resistance is related to exposure to antibacterial agents. Two or more antibacterial agents, in combination therapy, using various mechanisms of action that could enhance each other's performance, decrease the chance of resistance evolution, and broaden activity spectra. 22,23 Phages have the specific ability to replication at the infection site, enhancing their density locally, at the expense of bacteria. However, bacteria by using different mechanisms could resist phages. Nevertheless, since the main mechanisms related to phage and antibiotics resistance do not overlap, the use of phage cocktails or phage-antibiotics combination therapy has been recently suggested and evaluated to enhance the treatment's efficacy. [24][25][26] In this regard, the researchers used different methods to study the inhibitory effects of phage-ciprofloxacin combination therapy.
P. aeruginosa, a Gram-negative bacillus, is naturally resistant to different antibiotic classes. Additionally, the continuous usage of antibiotics in patients with P. aeruginosa chronic infections, such as cystic fibrosis patients, could lead to additional antibiotic resistance.
Mutations in gyrA or genes that are associated with the efflux pump expression can lead to resistance to different members of the fluoroquinolones family, such as ciprofloxacin. 27,28 The emergence of ciprofloxacin-resistant in this bacterium is a serious challenge worldwide, bringing the need to find new approaches to manage this bacterium. To this end, phage-antibiotic combination was reported as a promising alternative to antibiotics for the inhibition of MDR P.

aeruginosa.
Ferran et al. developed a creative use of the hollow fiber infection model (HFIM) to assess the potential benefit of phage-ciprofloxacin combination therapy. 29 The HFIM mimics concentration profiles observed in patients and is a preclinical closed system that allows the culturing of microbial cultures in an enclosed compartment. This compartment is usually a discrete cartridge that in turn is threaded with semi-permeable fibers. 30 The results of this study showed that each treatment selects for phage or antibiotic-resistant clones in less than 30 h. On the other hand, the administration of ciprofloxacin after (4 h) the usage of phages, significantly removes the bacteria from the HFIM at 72 h. Phages-ciprofloxacin combination therapy, based on the clinical regimens, suppressed the growth of resistant clones, providing opportunities to downscale the use of multiple antibiotics. 29 In line with these results, another examination also reported that phage ELY-1-ciprofloxacin combination suppressed Escherichia coli regrowth and caused less bacterial resistance than ciprofloxacin and phage used alone. Noteworthy, the usage of antibiotics in MIC and 6 h after phage therapy, showed the optimum inhibitory effect. 31 Actually, a high amount of antibiotic may inhibit the DNA replication in bacterial cells, kill the host and prevent the replication of phages. This phenomenon may decrease the per-host cell output of phage; thus, when the phages are added at a low MOI of 1, we do have not enough phage titer to significantly reduced bacterial cell count. 31 Therefore, the decrease of the bacterial population size by the phages eradicates the minor population of spontaneous mutants less-susceptible to ciprofloxacin that could thus not be selected afterward.
In another investigation also optical density-based 'lysis-profile' assays were used to evaluate the impact of colistin and ciprofloxacin on the bacteriolytic, bactericidal, and new-virion-production performance of three P. aeruginosa phages. Colistin significantly interferes with virion production and phage bacteriolytic function even at its MIC (1× MIC). Nevertheless, ciprofloxacin at 1× or 3 × MIC showed little inhibitory effect against phages. Therefore, the authors proposed ciprofloxacin as a promising antibacterial agent for combination therapy with phages especially when phage replication is required for treatment success. 32 Furthermore, recently published studies also reported that the combination treatment of phages with ciprofloxacin significantly increases in susceptibility of MDR P. aeruginosa to antibiotics. 13,33,34 In one of these studies, the presence of phage increased the number of P. aeruginosa strains susceptible to this antibiotic by 81%. Hence, the use of phage-ciprofloxacin combination therapy, especially when we have MDR strains, could result in efficacious outcomes by re-sensitizing the bacterial strain to the antibiotic. 13 Furthermore, Holger et al. also reported that triple combination regimens including phage-ciprofloxacin-colistin lead to a remarkable reduction in MDR P. aeruginosa numbers. Besides, phage resistance was prevented or reduced in the presence of several classes of antibiotics. 34 In addition to mentioned bacteria, the combination uses of phages and ciprofloxacin showed promising results for the inhibition of S. Typhimurium. The results of the study published in 2020 showed that the lytic activities of phage P22 were significantly increased in the presence of ciprofloxacin at pH 7 and the authors suggested that antibiotics play an important role in the host-phage interaction, specifically in the adsorption process. 35 Another study also reported that this phage combined with ciprofloxacin significantly suppressed the S. Typhimurium growth depending on the treatment order and time. 36 The sub-lethal concentrations of ciprofloxacin could lead to an increase in progeny production.
Actually, antibiotic treatments could enhance the release of progeny phages by shortening the lytic cycle and latent period. Thus, combination therapy with phages and antibiotics (sub-lethal concentrations) can be used for the management of infection caused by MDR bacteria. 3 In addition, combination therapy exerts various selection pressures that can mutually decrease phage and antibiotic resistance. Furthermore, phages could block ciprofloxacin expulsion by modifying the efflux pump, leading to the accumulation of this antibiotic in S. Typhimurium. 23 Finally, sub-inhibitory concentrations of antibiotics could lead to the alteration in bacterial cell morphology which permits rapid phage maturation and accelerated cell lysis. 37 Taken together, the combination usage of phages and ciprofloxacin is not only applicable but also synergistic in the decrease of bac-  Table 1.

TA B L E 1 Studies used of phage-ciprofloxacin combination for inhibition of different bacterial growth.
Year of publication  improvement was only detected when pretreatment with depolymerase was followed by ciprofloxacin administration. These authors also reported a similar potential for two phages, non-producing and producing depolymerase, in inhibition of the planktonic community of K. pneumoniae. On the other hand, phage non-producing depolymerase, contrary to depolymerase producer, was not able to decrease the sessile colony count. 61 In line with this study, in a recently published study, different antibiofilm agents such as cipro- Other studies that used phage-ciprofloxacin staggered treatment for inhibition of bacterial biofilm are presented in Table 2.

| IN VIVO USAG E OF PHAG E-CIPROFLOX ACIN COMB INATION
As fully discussed in the previous sections, the combination use of ciprofloxacin and phages to manage MDR bacteria and biofilm community has been reported in many in vitro studies; nonetheless, this combination therapy has not been well received in clinical andin vivo research. However, in this section, we will review in vivo and clinical experiments that have used phage-antibiotic staggered treatment for the management of bacterial infections.
In this regard, different studies used inhalable powder by cospray drying Pseudomonas phage PEV20 with ciprofloxacin. In one of these studies, lung infection was established in neutropenic mice then these animals were treated with ciprofloxacin, phage, and PEV20-ciprofloxacin treatment using a dry powder insufflator. PEV20-ciprofloxacin combination powder remarkably decreased inflammation and the load of MDR P. aeruginosa in mouse lungs, while no obvious reduction in the bacterial load was observed when the animals were treated only with ciprofloxacin or PEV20. 67 The findings of Chan et al. also demonstrated that PEV20 and ciprofloxacin-PEV20 powders remained stable over long-term storage and showed remarkable antibacterial activities against lung mouse lung infection caused by P. aeruginosa. 68 Other studies also reported that ciprofloxacin can sufficiently stabilize phage through verification and/or hydrogen bonding at 4°C and co-spray dried phage PEV20-ciprofloxacin combination dry powder formulations could lead to the synergistic antibacterial effect against MDR P.
aeruginosa isolates. 69,70 In addition to lung infection, rats with aortic experimental endocarditis were treated with an anti-Pseudomonas phage cocktail alone or combined with ciprofloxacin. The results showed that phage/ciprofloxacin combinations were highly synergistic, successfully managed 64% (7/11) of rats, and suppressed the regrowth of phage-resistant mutants. 71 In another interesting study, the authors reported an 88-year-old man with a relapsing P.aeruginosa prosthetic knee infection. The  The co-administration of phage with antibiotics, especially after staggered exposure, enhanced the performance of antibiotics against biofilm. [55] Abbreviations: EPS, Exopolysaccharide, STEC, Shiga toxin-producing E. coli.
yet, and the data from in vivo studies are very limited. Thus, more and more animal and in vivo evaluations are needed to evaluate molecular interactions of phages with ciprofloxacin; additionally, pharmacokinetics and chronic safety of phage-ciprofloxacin staggered treatment should be considered in future studies.

AUTH O R CO NTR I B UTI O N S
AS, MN, and ZC conceived and designed the study, contributed to comprehensive research, and wrote the manuscript. Notably, all authors have read and approved the manuscript.

ACK N OWLED G M ENTS
We greatly appreciate the input from the BioRender team (BioRe nder.com) for their collaboration with us in figure design.

CO N FLI C T O F I NTE R E S T S TATE M E NT
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.

DATA AVA I L A B I L I T Y S TAT E M E N T
The authors confirm that the data supporting the findings of this study is available within the article.