A New Insight into Phage Combination Therapeutic Approaches Against Drug-Resistant Mixed Bacterial Infections

Abstract

Rising antibiotic resistance among bacterial pathogens has become a substantial health issue for human civilization. The emergence of these pathogens in high-risk diseases such as cystic fibrosis (CF) has led to financial and nonfinancial losses, necessitating alternative therapies. This study presents an overview of such approaches, including phage therapy, antimicrobial peptides (AMPs), nanotechnology, monoclonal antibodies (mAbs), microbial therapies (probiotic therapy), clustered regularly interspaced short palindromic repeats technology (CRISPR), and aptamers focusing on their mechanisms of action and exploring the impact of combining phage and phage derivatives with the mentioned approaches. Although alternative approaches and their combinations with phages show promise, the phage–antibiotic combination has been the subject of most studies, and It has been proven to be highly effective in combating antibiotic-resistant infections. Other combinations also appear promising, but further studies are needed to determine their effectiveness. This emphasizes the need for more thorough research into different phage combination treatments beyond the well-established phage–antibiotic strategy.

Introduction

Antimicrobial resistance (AMR) has become one of the most troublesome issues of hygiene that can make current medicine insufficient. With the continuing indiscriminate use of antibiotics, it is estimated that AMR will continue to grow, resulting in reaching AMR casualties more than 10 million deaths per year by 2050.¹ The consistent increase in AMR and the declaration of emergency for this situation by the World Health Organization (WHO) make the future of antibiotics as a proper therapeutic approach for bacterial infections unclear.² The situation even gets worse by emerging persistent bacterial infections. Among bacterial pathogens, ESKAPE bacteria are considered the most problematic pathogens with drug-resistant features and the leading cause of persistent infections. Moreover, based on WHO predictions, antibiotic-resistant microorganisms, with ESKAPE pathogens playing a major role, could cause 10 million deaths per year by 2050. The WHO anticipates this scenario if no action is taken against AMR.³

The prevalence of mixed bacterial infections (MBIs) in high-risk diseases such as cystic fibrosis (CF) patients⁴ with the ability to survive against frequent antibiotic exposure raises concerns regarding the effectiveness of existing antibiotic treatment approaches and demand for innovative antibacterial therapeutics other than antibiotics due to the complicated situations that the patients can face. For instance, CF patients usually risk a high prevalence of persistent MBI due to high mucus density in the airways.⁵

Bacteriophages (phages),⁶ antimicrobial peptides (AMPs),⁷ nanotechnology,⁸ monoclonal antibodies (mAbs),⁹ probiotics,¹⁰ clusterd regularly interspaced short palindromic repeats (CRISPR) technology,¹¹ and aptamers¹² are treatment approaches that have been studied as alternative treatments recently. Since phages are safe and have high potential antimicrobial activity against drug-resistant bacteria, this treatment method can be evaluated against persistent bacterial infections.¹³ In addition, AMPs have notable antimicrobial activity (membrane disruption,¹⁴ targeting polysaccharides¹⁵ and intracellular components,¹⁶ and antibiofilm activity¹⁷), are broadly expressed in eukaryotic and prokaryotic organisms, and are considered an alternative way to combat drug-resistant bacteria.¹⁸ In recent years, nanotechnology has become a well-known strategy widely used in various biological approaches, including antibacterial drug development.¹⁹ Applications of nanotechnology in tackling AMR include utilizing nanoparticles (NPs) as drug carriers²⁰ or using them as direct antibacterial agents due to their antibacterial activity via disrupting the cell wall and cell membrane, reactive oxygen species production, targeting intracellular composition, and antibiofilm activity.²¹ Another promising treatment approach is mAbs. Due to specificity and high potential antibacterial activity by four different mode of actions, including neutralizing bacterial toxins, antibody-dependent cell-mediated cytotoxicity (ADCC)-killing, opsonophagocytosis, and inhibition of bacterial virulence factors, mAbs have gained researchers’ interest as an alternative therapeutic approach.^22,23 The antibacterial activity of probiotics, which is originated from their immunomodulatory properties, production of antimicrobial substances, showing competitive exclusion with pathogen bacteria, enhancement of mucosal barrier function,²⁴ and antibiofilm activity,²⁵ as well as their positive impact in increasing patients’ quality of life, draws researchers’ attention to these approaches to develop novel antibacterial medicine.^26,27 Another example of novel treatment approaches to eliminate bacterial pathogens is targeting specific genes such as virulence or antibiotic resistance genes, which can be done via nucleic acid-based therapeutics such as aptamers or CRISPR as a direct therapeutic approach.^11,12

The prevalence of MBI with AMR features in high-risk patients makes treating procedures complicated, which can increase the mortality rate of individuals. Thus, combating these microorganisms with the optimal treatment choice that can be applied alone or in combination is crucial to avoid AMR. This review sheds light on recent advances in possible phage and phage combinations with other alternative therapeutic combinations against drug-resistant bacteria. The mechanisms of action of these methods are also discussed. However, the main goal of this study is to highlight phage therapy and combination phage therapy.

Antibiotic Therapy for Bacterial Infections

The first antibiotic in history, salvarsan, was introduced in 1910 (Fig. 1); since then antibiotics have significantly altered modern medicine and increased the average life expectancy of humans. The most successful period of natural-based antibiotic discovery began in 1928 with the discovery of penicillin. Despite the mentioned advantages, the indiscriminate use of antibiotics and the reduction of their development gradually resulted in the emergence of AMR.²⁸

FIG. 1.

Illustration showcasing the timeline of the alternative therapeutic approaches. These events are significant as they mark the first incidents in history, such as the discovery of the initial substance of its kind. Within the figure, the gray line denotes critical historical events related to antibiotics.^28–31 Additional events, such as those connected to ABNPs,^32,33 nanocarriers,^8,34,35 and CRISPR technology,³⁶ are marked on the gray line, with colored sections placed just above it. Furthermore, events associated with AMPs^37–40 and probiotics^26,41–44 can be found on the gray line, and colored segments are placed in the middle of the figure to highlight their importance. As for aptamers, their related events^45–47 are depicted on the gray line alongside colored sections at the top of the figure. Phage therapy events⁴⁸ can be observed in the colored parts at the figure’s top. Finally, events concerning mAbs^22,35,49 are presented on the gray line and in the colored sections beneath it. ABNPs, antibacterial nanoparticles; CRISPR, clustered regularly interspaced short palindromic repeats; AMPs, antimicrobial peptides; mAbs, monoclonal antibodies.

AMR prevalence is not the only cons of antibiotics. Although they are essential for treating bacterial infections, antibiotics can have several adverse effects on their consumers. Along with neurological and psychiatric side effects such as confusion, headaches, seizures, and anxiety,⁵⁰ these side effects also include gastrointestinal disturbances such as nausea, vomiting, and diarrhea.^51,52 Furthermore, the use of antibiotics can change the gut microbiota, which can lead to antibiotic-associated diarrhea and worse disorders such as toxic megacolon and colitis.⁵³ Also, in a recent study, Klingel et al. reported that the CF patients who received azithromycin as a treatment had an inferior pulmonary function in comparison with patients who did not receive azithromycin as a treatment (azithromycin nonconsumers); additionally, they were coinfected with methicillin-resistant Staphylococcus aureus (MRSA) more than azithromycin nonconsumers and had lesser relative FEV₁ feedback.⁵⁴ Furthermore, antibiotics have an antibacterial activity against nonpathogenic and symbiotic bacteria, resulting in a decline in lung function, changing the microbial ecology of the lungs, and the prevalence of some ESKAPE pathogens such as Enterococcus spp. and Klebsiella pneumoniae in CF patients.^55,56 Therefore, despite the urgent demand for new antibacterial treatments, many pharmaceutical companies have pulled out of new antibiotic research projects in favor of focusing their development and research efforts on alternative therapeutic areas.⁵⁷

Phage Therapy

Phages are 50–200 nm viruses that attack and infect bacterial hosts.⁵⁸ Phages are widely spread throughout the biosphere because they are obligatory bacterial hosts and can be found practically everywhere, even in the human body.⁵⁹ Due to abundant alternative treatments for patients infected with antibiotic-resistant bacteria and the promising outcomes from current case studies using personalized phages, interest in using phages as a therapeutic agent has recently increased.⁶⁰ Utilizing phages as a therapeutic agent has proven efficient and secure for treating infectious disorders brought on by various species of bacteria, including drug-resistant strains.⁶¹ Notwithstanding, phage therapy still has cons, including the high cost of phage screening, time-consuming procedure within diagnosis and treatment, the emergence of phage-resistant bacteria, fewer clinical trial data, and induction of phage-neutralizing antibodies in the body.⁶² Nevertheless, by adopting appropriate solutions and optimization, the negative aspects of phage therapy can be adjusted to some extent. For example, establishing novel high-throughput screening techniques can lower the cost of phage screening.⁶³ Rapid diagnostic tools can shorten the time it takes to diagnose and treat a patient.⁶⁴ Phage cocktails can be used to fight bacteria that are resistant to phages.⁶⁵ Phage therapy’s acceptability and dependability will increase with the collection of more clinical trial data and the standardization of methods. Lastly, methods such as encapsulated phages to avoid or decrease phage-neutralizing antibodies can improve the effectiveness of treatment.⁶⁶

Lytic Activities of Phages

The lytic activity of phages originated from their life cycle. After completing the prelysis stages of the single life cycle of phages, including attachment, infection, multiplication, and assembly, host cell lysis occurs thanks to lysis-related proteins of phages.⁶⁷ The use of phages in planktonic form, which uses the mechanism of host lysis, goes back a hundred years to treat bacterial infections. Nonetheless, due to the emergence of antibiotics, using phages as medicine was limited to a few countries.⁴⁸

Due to increasing studies with promising results about the therapeutic potential of lytic phages against drug-resistant bacterial pathogens⁶⁸ and MBIs,⁶⁹ phage therapy is a high-potential alternative treatment for complicated bacterial infections. Although planktonic phages are not the only phage therapy option, phage derivative substances such as lysis-related agents (such as enzymes and proteins) can be another promising route to fight against AMR.

Endolysins are phage-coded enzymes that can degrade the bacterial host’s peptidoglycan. Due to peptidoglycan degradation activity similar to penicillin’s antibacterial mechanism, these enzymes are so-called enzybiotics.⁷⁰ Owing to the broader spectrum and lower resistance rate development, quick action, and supreme efficiency compared with phages, endolysins are considered a reasonable alternative phage-derived approach to combat antibiotic-resistant bacteria.⁷¹

Virion-associated peptidoglycan hydrolases (VAPGHs) are another phage-related peptidoglycan-degraded enzyme that belongs to the enzybiotics.⁷² VAPGHs consider endolysin analogs and show peptidoglycan degrading activity similar to endolysins except for lytic transglycosylase activity.⁷³ Moreover, they are generally larger and structurally more diverse than endolysins.⁷⁴ In addition to antibacterial activity, due to other properties such as its mode of action, which causes low resistance occurrence to these enzymes, specificity, stability to high thermal conditions, and feasibility of modular designing, make these enzymes a good representative of a suitable enzybiotic and a potential solution to AMR.^73,75

Depolymerases are another phage-encoded enzyme with antibacterial activity. Thanks to their polysaccharide degradation features, these enzymes gain researchers’ attention by degrading extracellular polymeric substances. These antipolysaccharide features help phages penetrate biofilms and invade bacterial host cells by degrading their capsules via targeting capsular polysaccharides.⁷⁶ Specific and high-potential anticapsule activities of depolymerase against various K. pneumoniae⁷⁷ and Acinetobacter baumannii⁷⁸ capsular types were reported in previous studies. Moreover, these enzymes can target various substrates, including sialic acid, rhamnogalacturonan, galacturonate, and alginate, making depolymerases promising alternative therapeutic approaches against antibiotic-resistant bacteria.⁷⁹

Holins and pinholins are other examples of phage-related proteins with potential antibacterial activity. Holins can form 200–400-nm holes in the plasma membrane of bacteria. These holes are formed to assist endolysins in being released and hydrolyze cell walls, and it is necessary for endolysins to reach peptidoglycan.⁸⁰ Holin’s antibacterial potential alone and fused with endolysin were reported in previous studies.⁸¹ In addition, the antibacterial potential of holins against both gram-positive and gram-negative bacteria suggests that holins may have a wider variety of applications than endolysins. Moreover, holins can be modified to include a supplementary peptide that selectively attaches and targets specific bacteria. Hence, holins can be applied in a wide range of biotechnological applications, such as gene therapy and bacterial infection control.⁸²

Pinholins are lytic proteins of phages considered members of holins class II.⁸³ These proteins make pinholes less than 2 nm in diameter in the host bacteria’s inner membrane. The pinholes’ size prevents endolysin from passing through them. However, it does allow ions to get through, which depolarizes the membrane, enabling the signal-anchor-release (SAR) endolysins to be folded back into an active form and target the peptidoglycan.⁸³ In addition, despite holins, there is no evidence of pinholins’ antibacterial activity alone or in combination with other substances. Nonetheless, pinholins have an essential role in the phage lysis cycle despite their mechanism of action still needing to be fully understood.⁸⁴

While other phage-related substances degrade the cell wall and inner membrane of bacteria, an interruption of the outer membrane of gram-negative bacteria is also crucial. The specific phage-encoded proteins known as spanins⁸⁵ make this interruption activity possible. Without spanins, the lysis procedure is expected to be prevented, and the release of virus particles from the bacterial cells will be inhibited.⁸⁶ In addition, it is believed that the peptidoglycan meshwork inhibits spanin’s action. Hence, peptidoglycan disruption mediated by holins and endolysins is necessary for the spanin mechanism of action.⁸⁴ The outer membrane interruption should have occurred following the endolysins’ peptidoglycan hydrolysis,⁸⁴ showcasing the interconnectedness of these processes in the degradation of bacteria and the possible combination of the three phage-encoded proteins as a therapeutic approach. The antibacterial mechanisms of action of phage and phage derivatives are provided in Figure 2.

FIG. 2.

Antibacterial mechanisms of phages and phage derivatives. Lytic phage’s antibacterial mechanism consists of injecting genetic material by phage, then the molecular machine of bacteria is stolen, and phage progenies are released following the disruption of the bacterial cell wall. Cell wall disruption can be mediated by several phage-related enzymes and proteins that target the capsule, outer membrane, peptidoglycan, and inner membrane.

Why Using Combination Phage Therapy Is Necessary?

With the emergence of antibiotic-resistant bacterial species in the current age, using phage therapy as an alternative therapeutic agent in place of antibiotics is recommended. Although the high specificity of phages is considered one of these viruses’ most challenging features.⁸⁷ Owing to the prevalence of unusual MBIs (bacterial infections with more than one bacterial species) with drug resistance characteristics that increase the mortality and morbidity of patients,⁸⁸ using phages as a therapy has become a sophisticated therapeutic approach. This complicated situation can be prevalent in different types of disorders, such as urinary tract infections,⁸⁹ chronic osteomyelitis,⁹⁰ various types of infections in individuals with diabetes,⁹¹ HIV,⁹² and CF.⁹³ Hence, to combat drug-resistant MBIs in the mentioned patients, phages as a monotherapy must be combined with other therapies to increase the chance of curing patients. In addition, combining therapeutic agents decreases the chance of acquired resistance among bacteria.⁹⁴ Nonetheless, choosing proper agents with some features such as nontoxicity, high antibacterial activity, and lack of antagonistic behavior is crucial to having highly efficient medicine (Table 1).

Table 1.

Comparison of Combined Agents Based on Key Characteristics for Enhanced Efficiency in Combination Therapy

Agent	Advantages	Disadvantages	Limitations in phage combination	Overcoming limitations
Antibiotics	Broad-spectrum activity²⁸	Prevalence of resistance, side effects, antibacterial activity against the beneficial symbiotic bacteria²⁸	Antibiotics’ rapid bactericidal action can interfere with phage replication, underscoring the need for meticulous dosing. Certain antibiotics may target bacterial components crucial for phage replication, rapid antibacterial activity	Reducing antibiotic dose, avoid choosing antibiotics with direct effect on phages
AMPs	Broad-spectrum activity^37,95	Immunogenicity, inactivation due to ionic-based effects, cytotoxicity, immediate degradation^37,95	AMPs can disrupt viral particles and interfere with viral entry and replication, modulating the host immune response, which can have a potential impact on phage efficacy	Appropriate dosing and encapsulation with nanoparticles
ABNPs	Manipulable, lacks severe side effects, resistance prevalence rarely happens, durable, affordable, high-temperature tolerance⁹⁶	Toxicity, antibacterial activity against the beneficial symbiotic bacteria⁹⁶	Possible antiviral activity	Selecting the appropriate nanoparticle type based on size and surface properties, determining the optimal concentration and dosage, ensuring stability in formulation to prevent compromising activity
Nanocarriers	Prolonged drug serum half-life, consistent and uniform dispersion in the site of action, sustained release, reduced side effects of medicines, synergistic effect with drugs, withholding any interference with nonloaded therapeutics, the ability to be designed as multifunction medicine^20,97–99	Challengeable NPs choosing due to physicochemical and biological issues¹⁰⁰	Possible loss of function of phages in encapsulation process, challenging to reach encapsulation efficiency due to size and structure incompatibility, complex manufacturing processes	Choose nanocarriers that provide stability, controlled release, and compatibility with phages. Optimize encapsulation techniques
mAbs	High specificity and antibacterial potential²²	Toxicity, high cost, side effects¹⁰¹	Target specificity of both agents may affect antibacterial efficacy in mixed bacterial infections, limited knowledge, and research gaps	Understand the distinct mechanisms of action of phages and mAbs. Ensure both are effective against the diagnosed bacterial pathogen
Probiotics	Boosts immune system, prevents dysbiosis, improves QOL, anti-inflammatory potential²⁶	Narrow spectrum, environmental sensitivity, limited clinical data^24,102	Immune response boosting by probiotics that may interfere with phage activity	Ensure compatibility between phages and probiotics in the formulation. Determine the optimal timing and sequence of administration. It may be beneficial to administer phage derivatives first to reduce pathogen load, followed by probiotics
CRISPR technology	Specific and programmable¹⁰³	High specificity can limit the impact of a drug, resistance can emerge¹⁰³	Lack of proper delivery systems, possible antagonistic effect by phage derivatives, limited knowledge, and research gaps	Develop effective delivery systems for both CRISPR components and phages. Investigate the best administration timing to avoid disrupting CRISPR entry sites by phage derivatives. Dosing and timing are crucial to prevent interference
Aptamers	High stability, inexpensive manufacturing, intrinsic amplification capabilities¹⁰⁴	Toxicity, inferior pharmacokinetics compared with mAbs¹⁰⁵	Possible disruption of phage entry routes, limited knowledge, and research gaps	Timing and dosing are essential to avoid disrupting phage entry routes
Phage derivatives	Specificity, low bacterial resistance rate, manipulable^106,107	Limited efficiency against stationary-phase bacteria, stability issues, cost^106,107	Possible disruption of phage entry routes, rapid antibacterial activity	Ensure phage derivatives do not disrupt phage binding sites. Consider rapid bacterial killing by phage derivatives, adjust dosing and timing

AMPs, antimicrobial peptides; ABNPs, antibacterial nanoparticles; CRISPR, clustered regularly interspaced short palindromic repeats; mAbs, monoclonal antibodies.

Combination Phage Therapy

Generally, combination phage therapy, known as treating infections caused by drug-resistant bacteria with phages in conjunction with antibiotics,¹⁰⁸ can cause a synergistic effect between antibiotics and phages and even resensitize bacteria to antibiotics in some cases.¹⁰⁹ However, according to the recent advances in phage field studies, this definition can change due to promising results of recent studies evaluating the combination of phage therapy or phage-related antibacterial substances with other promising therapies.

Phage cocktails and phage–antibiotic combination

Among other phage combination therapies, phage cocktails and phage–antibiotic combinations have been studied well. The amalgamation of at least two phages, known as a phage cocktail, has emerged as a prospective avenue for combined phage therapies, displaying encouraging outcomes in both in vitro and in vivo experiments.¹¹⁰ The application of phage therapy as a phage cocktail has several advantages, including the possibility of the synergism effect of different phage particles with each other,¹¹¹ reducing phage resistance occurrence, increasing the long-term therapeutic efficacy of phages, and providing various selection pressures to the host cell.¹¹² Also, it can increase host range and show better antibiofilm activity.⁶⁵ The way that phages and antibiotics work together largely depends on their distinctive modes of action. Antibiotics belong to several classes and target bacterial processes, such as DNA replication, protein synthesis, and cell wall construction. The antibacterial effect can be enhanced when combined with phages exploiting these vulnerabilities. For example, bacterial membranes and cell wall disruption by phages can increase the effectiveness of antibiotic treatment.^113,114 Moreover, it is shown that utilizing proper antibiotics can facilitate phage progeny release from bacteria.¹¹⁴

For instance, the antibacterial procedure of the combined phage cocktail with colistin and meropenem was recently evaluated.⁶⁹ In the mentioned study, a 42-year-old patient with mixed bacterial (A. baumannii and K. pneumoniae) osteomyelitis was successfully cured by phage cocktails and antibiotics.⁶⁹ Also, it is reported that phage-ceftazidime and phage-imipenem/relebactam combination can improve treatment outcomes for drug-resistant P. aeruginosa infections in Siamese cat¹¹⁵ and a 52-year-old male patient, respectively.¹¹⁶ Thus, using phages as a cocktail and combined with antibiotics in phage therapy should be prioritized over mono-phage therapy. Nonetheless, the antagonistic effect of some antibiotics, such as β-lactams with some phages, was reported in recent studies.¹¹⁷ For instance, the antagonistic effect of ciprofloxacin and ΦHP3 due to topoisomerase II inhibition activity of ciprofloxacin against phage and bacteria was detected by Liu et al.¹¹³ Hence, choosing suitable antibiotics to combine with phages based on the phage type and the antibiotic mechanism is crucial for effective combination therapy and avoiding any possible antagonistic effects.

Phage-derivative combinations

Similar to phages, antibiotics can also be combined with phage derivatives. One of the notable combinations in this regard is a combination of Dpo71 (depolymerase) and colistin, which was examined by Chen et al.¹¹⁸ In the mentioned study, the combination approach shows more antibiofilm effect due to the antibiofilm activity of depolymerase, resulting in high accessibility of colistin to target bacteria.¹¹⁸ Furthermore, an enhanced antibacterial effect between phage, depolymerase, and antibiotic,¹¹⁹ as combination therapy consisting of phage KP15, depolymerase KP34p57, and ciprofloxacin, was reported in previous studies,¹²⁰ emphasizing the crucial role of KP34p57 in combined phage therapeutics as a biofilm degrader, which provides accessibility of KP15 and ciprofloxacin against target bacteria. Moreover, unlike ΦHP3, no antagonistic effects were detected between KP15 and ciprofloxacin in the mentioned study, suggesting that ciprofloxacin is ineffective or has little effect on phage KP15 topoisomerase II. Also, the study highlights preliminary studies on phage biology before combining them with other antibacterial agents.

As described in previous sections, phage derivatives are complete packages of antibacterial agents targeting the inner membrane, peptidoglycan, outer membrane, and bacteria capsule. In addition to the synergism that can be seen between them,⁸¹ the functionality of some of these derivatives (spanins) is dependent on other derivatives (holins and endolysins).⁸⁴ Thus, using these substances in combination seems to be more logical. Notwithstanding, as far as the authors’ knowledge, there is no evidence of using spanins as an antibacterial agent alone or in combination or fused with other phage derivatives, while the synergistic effect of endolysin with holin,⁸¹ VAPGHs,¹²¹ and depolymerase¹²² was detected in previous surveys.

Phage-derivative-AMP combination

AMPs are essential elements of the intrinsic immunological defense system. In most living species, AMPs have been produced by the host organism to strengthen the immune response and protect against pathogens. Due to their broad-spectrum antibacterial activity, different pharmacodynamics compared with antibiotics, synergistic effect with antibiotics,^37,95 and high potential in degrading biofilms,¹²³ AMPs are considered high-potential antibacterial agents.

According to previous studies, AMP and phage-derivative combination can be a proper therapeutic approach due to the synergistic effects of these components. Nisin with antibacterial activities, such as creating holes in the membrane and inhibiting cell wall synthesis,¹²⁴ and cecropin A with cell membrane interrupting features¹²⁵ are among AMPs used as combined agents with endolysins. For instance, it is reported that there is an anti-staphylococcal synergistic effect between LysH5 and nisin.¹²⁶ Furthermore, the fused endolysin-AMP agent (ST01-cecropin A) described by Lim et al. shows high antibacterial activity against gram-negative bacteria compared with ST01 alone.¹²⁷ The synergistic mechanisms of these peptides with endolysins may be related to their ability to weaken the bacterial membrane, which leaves bacteria more vulnerable to the action of endolysins, which engage the cell wall.

Also, the triple combined therapeutic, including AbEndolysin fused with cecropin A, and cefotaxime, ceftazidime, and aztreonam antibiotics, is another combined therapeutic that showed a synergistic antibacterial effect against A. baumannii. In this study, the combined therapeutic successfully rescued infected mice, emphasizing the pivotal role of phage-derivative-AMP-based combination therapies in combating drug-resistant bacterial infections.¹²⁸ In the case of using such a combination, high cell wall accessibility to beta-lactams and endolysin provided by cecropin A in addition to anticell wall activity of beta-lactams and endolysins with two different procedures, including targeting penicillin-binding proteins (PBPs) to cell wall generation inhibition¹²⁹ and degrading the peptidoglycan layer of cell walls, respectively,¹³⁰ may enhance the antibacterial activity of the agents. However, the antiviral activity of AMPs that can also impact phages is one of the limitations of using these viruses and AMPs as a combination therapy.¹³¹ Thus, the feasibility of amalgamating AMPs with phages that can turn into combined therapy demanded further studies for possible solutions in this manner. Also, investigating the mechanisms of action of AMPs and phage biology is highly recommended to avoid antagonistic effects when used as combination therapies.

Phage and nanotechnology approaches

Nanotechnology can be used to combat drug-resistant bacteria in two different ways. The first method uses antibacterial nanoparticles (ABNPs) as direct antibacterial material. On the contrary, NPs are used as carrier vehicles to deliver medicines to target.¹³² NPs with antibacterial activity can be the satisfactory definition for ABNPs. They have many advantages over antibiotics, such as not causing immediate, severe side effects and resistance against them rarely happens.

Moreover, ABNPs are maintained in the body for a notably extended period compared with relatively small antibiotics, which may be advantageous for achieving long-lasting therapeutic effects. In addition, antimicrobial ABNP preparation could be more affordable than synthesizing antibiotics. Also, ABNPs can tolerate the high temperatures used in sterilizing without losing any characteristics that cause conventional antibiotics to become inactive.⁹⁶

In previous studies, researchers evaluated the combination of endolysins with various ABNPs, including poly(propylene imine) dendrimers,¹³³ AgNPs,¹³⁴ and carbosilane dendrimers combined therapeutic with endolysin.¹³⁵ Additionally, they examined the potential combination of liposome-based ABNPs with a phage cocktail.¹³⁶ In the mentioned cases, except carbosilane dendrimers that show synergistic antibacterial effects with endolysin, any other ABNPs enhance the performance of phage cocktails or endolysins. The enhanced antibacterial activity of each mentioned combined therapy might originate from the unique mechanism of action of each ABNP, including the cell membrane disrupting activity¹³⁷ of poly(propylene imine) dendrimers, interfering with vital bacterial processes and membrane disruption of AgNPs,¹³⁸ and biofilm disruption and interfering with crucial bacterial processes of carbosilane dendrimers (particularly those modified with metal ions such as copper(II) and ruthenium(II)).^139,140

Drug carriers can play a pivotal role in unraveling drug-resistant MBI issues. Utilizing NPs as drug carriers has many advantages, such as withholding any interference with nonloaded drugs,⁹⁷ the ability to be designed as multifunction medicine,⁹⁸ sustained release of a drug,⁹⁹ elevating drug serum half-life, making eliminating bacteria possible even at low doses of medicine, consistent and uniform dispersion in the site of action, and reduced side effects of medicines^20,141 that put NPs in the solution list of antisuperbug approaches. Moreover, nanodrug carriers can be used depending on disease-specific conditions to evade such conditions and successfully target the infective agent. In the case of CF, there are three notable biological barriers, including high mucus density in the lungs, formation of biofilm by infecting bacteria, and intracellular infection by some pathogens in the way of effective antibacterial therapy against bacterial infections.^142,143 To bypass these barriers, liposomes, polymer NPs, nanostructured lipid carriers, and nanodrug crystals are the most critical carriers that have been used in pulmonary delivery of encapsulated antibacterial agents by dry powder inhalers (Fig. 3).^144,145

FIG. 3.

Notable barriers to CF patients’ bacterial infection cure. The biofilms formed by pathogenic bacteria, intracellular infections, and the dense consistency of mucus contribute to the complexities encountered in effectively treating these infections. Nevertheless, utilizing nanocarriers for precise delivery of the desired therapeutic substance to the site of infection is a valuable approach. CF, cystic fibrosis.

Liposomes are one of the NPs used to deliver phage cocktails and enhance their efficacy over free ones.¹⁴⁶ Polymer NPs such as poly(lactic-co-glycolic acid)¹⁴⁷ and hydrogels, including sodium alginate hydrogel and carboxymethylcellulose hydrogel for encapsulation of phages, have also been evaluated in previous studies.¹⁴⁸ The studies’ promising results are one step closer to the highly efficient target delivery of phages for drug-resistant infections.

Phage–probiotic combination therapy

In the modern era, probiotics have become more popular and are being more successfully marketed.²⁶ Bifidobacterium and Lactobacillus genera, as well as Lactococcus species, Streptococcus thermophilus, Escherichia coli Nissle 1917, and Saccharomyces boulardii, are the most commonly used microorganisms as probiotics.²⁴ Probiotics can boost the immune system, prevent dysbiosis,²⁶ show antibacterial activity alone or in combination with other antibacterial agents,¹⁴⁹ and increase patients’ quality of life.¹⁵⁰

Phage and probiotic combination can be an alternative antibacterial solution for three reasons. First, phages are highly specific and do not negatively impact commensal bacteria or probiotics; in some cases, they can improve probiotics’ function.¹⁵¹ Second, combining phages and probiotics can enhance the antibacterial activity of probiotics and phages.¹⁵² Third, in addition to antibacterial activity, some probiotics can restore the proper balance of the gut flora by elevating intestinal acidity and inhibiting the growth of several harmful bacteria.²⁴ By using several mechanisms of action, the combined phage–probiotic therapy approach helps avoid acquired resistance by making it more difficult for bacteria to establish resistance simultaneously. Probiotics, for instance, can prevent the growth of pathogens by competitive exclusion, the synthesis of antimicrobial compounds, and the augmentation of host immune systems, whereas phages directly lyse microorganisms. Therefore, probiotics can be a promising composition to phage combination therapies and a proper supplementary material to increase patients’ quality of life. Various investigated phage combination therapies and examples are provided in Figure 4 and Table 2.

FIG. 4.

Various combination therapies in the case of combating antibiotic-resistant bacteria.

Table 2.

Examples of Phage or Phage Derivative-Based Combination Therapy

Type of combination	Therapeutic components	Target bacteria	Type of study	Main results	Reference
Phage cocktail	Three, two, or one phage of 19 different phages was combined and examined.	E. faecium	In vitro	Phage cocktail restricted target bacterial growth longer than monophages	¹⁵³
Phage cocktail	Ab105-2phiDCI Ab105-2phiDCI404ad vB_AbaP_B3	A. baumannii	In vitro	The synergistic effect of the cocktail against the biofilm of strains was detected	¹⁵⁴
Phage cocktail	vB_KpnS_FZ10 vB_KpnP_FZ12 vB_KpnM_FZ14 vB_KpnS_FZ41	K. pneumoniae	In vitro	Combination phages increased the spectrum of phage lytic activity	¹⁵⁵
Phage cocktail	CM_Kpn_HB132952 CM_Kpn_HB143742 CM_Kpn_HB154724	K. pneumoniae	In vitro and in vivo	The phage cocktail was more effective at lysis bacteria in comparison with monophage	¹¹⁰
Phage cocktail	Ten different phages were combined and examined in 3 groups	A. baumannii	Case report	A patient with a drug-resistant A. baumannii infection was rescued	¹⁵⁶
Phage cocktail or mono phage + antibiotics	Phages: NV-497 NV-503–01 Antibiotics: daptomycin ceftaroline	E. faecium	Ex vivo	Synergistic effect of combined NV-497 with daptomycin and ceftaroline and combined NV-497 with NV-503–01 was detected	¹⁵⁷
Phage cocktail + antibiotic	Phages: vB_PaeP_PYO2 vB_PaeP_DEV vB_PaeM_E215 vB_PaeM_E217 Antibiotic: ciprofloxacin	Pseudomonas aeruginosa	In vivo	The treatment effect improved by combining a phage cocktail with ciprofloxacin	¹⁵⁸
Phage + antibiotic	Phage: PEV20 Antibiotic: ciprofloxacin	P. aeruginosa	In vitro	The synergistic antibiofilm effect of PEV20 and ciprofloxacin was detected	¹⁵⁹
Phage + antibiotic	Phage: SAFA EPA-1 Antibiotic: gentamicin	P. aeruginosa and S. aureus	In vitro	SAFA + EPA-1 + gentamicin therapy regimen declared as the most successful antibiofilm regimen	¹⁶⁰
Phage + depolymerase	Phage: Phage 731 Phage 13 Depolymerase: B1Dep	K. pneumoniae	In vitro	Synergistic effect between mono phage and B1Dep detected	¹¹⁹
Phage + depolymerase + antibiotic	Phage: KP34 KP15 Depolymerase: KP34p57 Antibiotic: ciprofloxacin	K. pneumoniae	In vitro	Combined KP15 with KP34p57 and ciprofloxacin declared as the finest antibiofilm composition, along with mono usage of KP34 or combined KP34 with ciprofloxacin or KP15	¹²⁰
Depolymerase + antibiotic	Depolymerase: Dpo71 Antibiotic: colistin	A. baumannii	In vitro and in vivo	Colistin antibacterial activity in an in vivo model enhanced by Dpo71, also, the Dpo71 and colistin combination has higher antibiofilm activity in comparison with mono phage or colistin usage	¹¹⁸
Endolysin + AMP	Endolysin: LysH5 AMP: nisin	S. aureus	In vitro	Synergism antibacterial effect between LysH5 and nisin detected	¹²⁶
Endolysin + AMP	Endolysin: Lys11 AMP: R8K vAMP059	S. aureus	In vitro	Synergism antibacterial effect between Lys11 and R8K detected	¹⁶¹
Endolysin + AMP + antibiotic	Endolysin: AbEndolysin AMP: Cecropin A Antibiotics: Cefotaxime Ceftazidime Aztreonam Meropenem Imipenem	A. baumannii	In vitro and in vivo	Fused AbEndolysin with cecropin A (eAbEndolysin) rescued infected mice; synergistic effect between eAbEndolysin with cefotaxime, ceftazidime, and aztreonam detected	¹²⁸
Endolysin + AMP	Endolysin: ST01 AMP: Cecropin A	P. aeruginosa, K. pneumoniae, A. baumannii, Enterobacter cloacae	In vitro	The antibacterial effect of ST01 fused with cecropin A increased	¹²⁷
Endolysin + antibiotic	Endolysin: LNT113 Antibiotics: Colistin Meropenem Tigecycline Chloramphenicol Azithromycin Ciprofloxacin Ceftazidime Kanamycin	P. aeruginosa, K. pneumoniae, A. baumannii, E. cloacae	In vitro	A synergistic effect of LNT113 with colistin was detected. Also, LNT113 enhanced meropenem, tigecycline, chloramphenicol, azithromycin, and ciprofloxacin antibacterial activity.	¹⁶²
Endolysin + depolymerase	Endolysin: LysK Depolymerase: DA7	S. aureus	In vitro	The synergistic effect of LysK with DA7 detected	¹²²
Endolysin + endolysin	LysB4 LysSA11 LysB4EAD	S. aureus	In vitro	Fused LysB4EAD-LysSA11 showed higher antibacterial activity in comparison with mono usage of LysSA11 and LysB4EAD	¹⁶³
Endolysin + holin	Endolysin: LysSMP Holin: HolSMP	S. aureus	In vitro	The synergistic effect of LysSMP and HolSMP detected	⁸¹
Endolysin + holin	RL_Hlys	P. aeruginosa, K. pneumoniae, S. aureus	In vitro	Holin-fused endolysin compound shows a better antibacterial effect in comparison with endolysin	¹⁶⁴
Endolysin + ABNPs	Endolysin: The name of endolysin is not mentioned ABNPs: poly(propylene imine) dendrimers	P. aeruginosa	In vitro	Antibacterial activity of endolysin enhanced by combination with dendrimer	¹³³
Endolysin + nanocarrier	Endolysin: LysRODI Nanocarrier: Liposome-based nanocarrier	S. aureus	In vitro	Liposomes can be used as an encapsulation component	¹⁶⁵
Endolysin + ABNPs	Endolysin: The name of endolysin is not mentioned ABNPs: AgNPs	P. aeruginosa	In vitro	AgNPs increased the antibacterial activity of endolysin	¹³⁴
Endolysin + ABNPs	Endolysin: The name of endolysin was not mentioned ABNPs: Carbosilane dendrimers	P. aeruginosa	In vitro	The synergistic effect of endolysin and carbosilane dendrimers detected	¹³⁵
Endolysin + VAPGHs	Endolysin: LysH5 VAPGHs: HydH5	S. aureus	In vitro	A synergistic effect between LysH5 and HydH5 detected	¹²¹
Phage cocktail + ABNPs	Phage cocktail: CKɸ ABNPs: Liposome-based ABNPs	P. aeruginosa	In vitro	Combined therapy was more efficient in comparison with monotherapies	¹³⁶
Phage + nanocarrier	Phages: Phage K Phage 134 Phage 135 Phage 136 Nanocarrier: Poly(lactic-co-glycolic acid)-based microparticles	S. aureus and P. aeruginosa	In vitro and in vivo	Phage-loaded carriers were cytocompatible with lung-derived epithelial cells; these combined therapeutics showed potential antibacterial activity	¹⁴⁷
Phage cocktail + antibiotic + nanocarriers	Phage cocktail: PB10 PA19 Nanocarrier: Sodium alginate hydrogel Carboxymethylcellulose hydrogel Antibiotic: Ciprofloxacin	P. aeruginosa	In vitro and in vivo	The phage cocktail and ciprofloxacin-loaded hydrogel accelerated wound healing and reduced bacterial loads. The synergistic effect between the phage cocktail and ciprofloxacin was detected.	¹⁴⁸
Phage cocktail + probiotic	Phages: vB_SenS-EnJE1 vB_SenS-EnJE6 Probiotic: L. reuteri	Salmonella enterica serovar Typhimurium	In vitro and in vivo	The combination therapy successfully reduced inflammation in mice	¹⁶⁶
Phage cocktail + probiotic	Phages: Names of phages are not mentioned Probiotic: E. coli Nissle 1917	E. coli	In vivo	E. coli gut colonization inhibited via combined therapy in mice	¹⁶⁷

Prospects of Combination Phage Therapies

According to the studies that assessed the efficacy of combination therapies, the complementary components that affected a common target together or had direct or indirect antibacterial activity increased the effectiveness and synergism (Fig. 5). For instance, a combination of KP15, KP34p57, and ciprofloxacin shows the highest antibiofilm activity.¹¹⁸ On the contrary, using phage cocktails can be a proper example of standard target components, and probiotics are among the components that can have direct or indirect antibacterial activity. In addition to any combination therapies assessed in previous works, many other combinations can still be evaluated in future studies to achieve the most proper medicine against MBIs. Agents such as aptamers, spanins, mAbs, and CRISPR-based therapeutics also may hold significant potential for investigation as combination therapies. By investigating their combined effects with the previously stated agents, scientists might discover the possible antibacterial efficacy of these agents.

FIG. 5.

Complementary mechanisms of action to combat persistent polymicrobial infections.

Spanin-based combination therapies

As a phage-encoded protein, there is a lack of information about the antibacterial activity of these proteins due to the possible inactivity of spanins with the presence of peptidoglycan meshwork.⁸⁴ Notwithstanding, owing to the potential of spanins to fuse with other proteins,¹⁶⁸ it seems that a fusion of spanin with endolysin and holin can enhance the antibacterial activity of the components on account of the antipeptidoglycan activity of endolysin and holin. Fusion of spanin with AMPs with antipeptidoglycan activity (e.g., oritavancin, nisin, ramoplanin, and friulimicin¹⁶⁹) or using spanins as an adjuvant to an antipeptidoglycan antibiotic such as β-lactam antibiotics¹⁷⁰ can be a proper option. Hence, exploring the potential fusion of spanins with other proteins such as endolysin, holin, or AMPs, as well as utilizing spanins as an adjuvant to antipeptidoglycan antibiotics, could offer promising options for enhancing the antibacterial activity of these approaches.

Phage-target-specific therapeutic combination

Aptamers, CRISPR-based therapeutics, and mAbs are target-specific therapeutics with high-potential antibacterial activities. Aptamers are synthetic RNA or ssDNA oligonucleotides that are incredibly organized and bind to their corresponding targets with high affinity and specificity.¹⁷¹ Aptamers’ antibacterial features consist of neutralizing bacterial toxins, depolarization of cell wall, biofilm formation inhibition, opsonization, controlling the propagation of pathogen bacteria, inhibition of antibiotic resistance genes, attachment to the cell surface, and degradation and blocking antibiotic resistance enzymes.^172,173

mAbs are target-specific laboratory-produced antibodies cloned from single immune cells.¹⁰¹ These types of medicines can have an antibacterial effect by neutralizing bacterial toxins by blocking their binding to host cell receptors, ADCC, opsonophagocytosis, inhibition of bacterial virulence factors,^22,23 and antibiofilm activity.¹⁷⁴

The biofilm inhibitory potential of aptamers and mAbs is a marvelous feature that can combine with depolymerase to achieve high antibiofilm activity and provide accessibility to other therapeutics. Also, it seems the studies that can build on the inhibition of resistance genes by aptamers and, in parallel, use antibiotics as combined therapy have become attractive. In the case of the other antibacterial activities of mAbs and aptamers, it demanded further studies to evaluate the feasibility of mAbs and aptamers as combined therapy with phages and uncover any possible antagonistic effects between them and phages or other substances.

The CRISPR-Cas system-based therapeutics’ antibacterial activity cleaves the pathogen’s virulence factor and antibiotic resistance genes.¹¹ These types of medicine are typically delivered by phages or phage-related agents such as phagemid or genetically modified phages. In addition, encapsulation of phage to deliver CRISPR-related agents into the infected cells by intracellular pathogens and CRISPR-Cas bearer plasmid can be suitable for this manner.¹⁷⁵ The proper delivery of these therapeutics is pivotal to achieving highly efficient medicine, and the role of phages in this manner is undeniable. When it is clear that CRISPR-Cas system-based therapeutics have high potential in combating drug-resistant bacteria, especially their gene editing potential to eliminate resistance genes in target bacteria, efficient delivery of these therapeutics is still a big concern because, without efficient delivery, target bacteria remain unharmed.¹⁷⁶ Hence, it is crucial to wipe out barriers such as biofilms in the way of CRISPR delivery systems to achieve highly efficient antibacterial compounds, and it is not possible without using previously discussed antibiofilm agents in combination with CRISPR-Cas system-based therapeutics.

Current Gaps and Future Perspective

According to the current resistance status, the emergence of MBIs and future antibiotic resistance prospects, utilizing combination therapies, is inevitable. Nevertheless, there needs to be more information about the exact mechanism of action and antibacterial activity of spanins, holins, and pinholins alone or in combination with other agents. Moreover, various combinations, such as targeted molecular therapeutics with phages or phage derivatives, antibiotics, and ABNPs, are yet to be assessed. Another huge gap in combination therapies is the lack of in vivo studies that make these types of therapy not reliable enough. In the case of CF, the need for proper in vivo models with CF-mimicking characteristics¹⁷⁷ is another issue that must be addressed in future studies. Furthermore, regulatory and ethical challenges related to these therapies are another difficulty scientists may face. Thus, to gain acceptance as a treatment for combination therapy, appropriate regulatory requirements must be created, and ethical concerns must be resolved.

Identifying any single bacterial species or subspecies associated with MBI, identifying any confounding factor such as biofilms or high mucus density, and choosing proper agents as combination therapies according to the situation are pivotal to achieving a highly efficient combination therapy. While these therapies are considered practical therapeutic approaches, performing the mentioned steps can be time-consuming. Therefore, the following steps must be taken: using sophisticated novel tools to reduce the time of these processes.

The emergence of artificial intelligence (AI) is evolving and revolutionizing life sciences. AI tools have a high potential for predicting infection occurrence,¹⁷⁸ biofilm occurrence, choosing antibiofilm agents,¹⁷⁹ and discovering new antibacterial agents,¹⁸⁰ developed or developing. Also, AI can play a significant role in choosing the optimal therapeutics for patients in personalized conditions.¹⁸¹ With increased data from biofilm formation chemicals, infection occurrence percentage, and interaction of various antibacterial agents with their target, the AI tools grow and will be more accurate than ever. In addition, developing AI tools that can design proteins that never existed¹⁸² and predict biofilm chemicals developing proteins with antibiofilm activity will be available soon.

To put all this together, AI will accelerate the discovery of new treatments and brighten the future of biological sciences. In particular, AI will diagnose infections, recommend possible combinations of treatments based on the properties of the agents, and even create new protein designs with antibacterial activity for use in mono or combination treatments. AI can speed up identifying therapeutic combinations that work, cutting down on the time needed for experimental validation, and enabling the creation of novel medicines. It is expected that AI will play a vital role in the advancement of therapeutics as we advance, especially in the areas of combination therapies and phage therapy. The future of biomedicine will not just be based on combination therapies but also on combination AIs, servers, and robots to combat complex infections that will threaten humans’ future.

Conclusion

In the modern era, AMR in bacterial pathogens is a significant hygiene problem in the developing and developed countries. Since these pathogens resist antibiotics, fighting against these bacteria requires alternative therapeutic approaches. Hence, many alternative approaches have been investigated by researchers to deal with antibiotic-resistant bacterial pathogens in recent years.

Therapeutic agents based on mAbs, CRISPR technology, nanotechnology, probiotics, aptamers, AMPs, and phages have shown promising results. Despite phage utilization as a planktonic form, phage derivatives are also considered a promising alternative therapeutic approach due to their broad-spectrum antibacterial activity compared with planktonic phages. Enhancing the efficacy of phages or phage-relative derivatives, especially in treating MBIs, is another challenging phage therapy case. The easiest and most applicable method for addressing this challenge is combination therapies. However, most combination therapies are limited to phage cocktails, phage with antibiotic combination, and endolysin with antibiotics, and the other combined methods are limited to the in vitro efficacy evaluation studies or need more reliable data. Hence, subsequent investigations must expand our understanding of phage combinations beyond the widely recognized phage–antibiotic approach. To create comprehensive and robust treatments against drug-resistant bacterial infections, we must investigate the synergies and possible antagonists among multiple therapies. The combination of phage and antibiotics continues to be a mainstay of current research efforts in this sector, even though it is impossible to conclusively determine which technique has the highest or most minor potential efficacy due to a lack of evidence in other combination therapies.

In conclusion, the use of phage therapy has a long way to go until it is accepted as a universal drug against antibiotic-resistant bacteria since there are many challenges facing this type of treatment process currently. Notwithstanding, recent clinical studies have brought phage therapy closer to the market in Western countries. The use of phage derivative agents or combination therapies is in the early stages of research, and except for endolysins, which have been studied in vivo, other methods are currently undergoing the in vitro process. Meanwhile, with the continuation of in vitro research, many questions arise about various aspects of combination therapies, leading to a more protracted process of their acceptance as the primary treatment.

Footnotes

Acknowledgments

We want to extend our gratitude and appreciation to Freepik (), Margot Riggi (licensed under CC-BY 4.0 Unported), and Servier Medical Art (licensed under CC-BY 3.0 Unported) for their remarkable contribution in assisting us with the creation of the pictures featured in this article.

Authors’ Contributions

All authors contributed to the study’s conception and design. M.R.: Conceptualizing and writing and reviewing the original draft. D.J.-G. and E.M.: Writing and reviewing the original draft. N.Z.: Reviewing and editing the original draft and supervising. All authors read and approved the final article.

Disclosure Statement

The authors declare that they have no known competing interests.

Funding Information

No funding was received for this article.

References

Dillon

, Dimonaco

, Creevey

. Accessory genes define species-specific routes to antibiotic resistance. Life Sci Alliance, 2024; 7(4):e202302420; doi: 10.26508/lsa.202302420

Medrano

, Lee

, Young

, et al. Surveillance of antimicrobial resistance in Escherichia coli, Salmonella, and Campylobacter recovered from laying hens, their environment and products in Canada indicated a stable level of resistance to critically important antimicrobials, in varying time periods between 2007 and 2021. Int J Food Microbiol, 2024; 412:110541; doi: 10.1016/j.ijfoodmicro.2023.110541

Agrawal

, Patel

. Antibiotic resistance profile and detection in ESKAPE pathogens. In: ESKAPE Pathogens: Detection, Mechanisms and Treatment Strategies. ( Busi

, Prasad

. eds.) Springer Nature: Singapore; 2024; pp. 33–77; doi: 10.1007/978-981-99-8799-3_2

Yellepeddi

, Lindley

, Radetich

, et al. Population pharmacokinetics and target attainment analysis of vancomycin after intermittent dosing in adults with cystic fibrosis. Antimicrob Agents Chemother, 2024; 68(1):e0099223; doi: 10.1128/aac.00992-23

De Boeck

. Cystic fibrosis in the year 2020: A disease with a new face. Acta Paediatr, 2020; 109(5):893–899; doi: 10.1111/apa.15155

Rahimi

, Bakht

, Javadi

, et al. Characterization of novel bacteriophage PSKP16 and its therapeutic potential against β-lactamase and biofilm producer strain of K2-Hypervirulent Klebsiella pneumoniae pneumonia infection in mice model. BMC Microbiol, 2023; 23(1):233; doi: 10.1186/s12866-023-02979-7

Mechkarska

, Cunning

, Taggart

, et al. Identification of an antimicrobial peptide from the venom of the trinidad Thick-Tailed Scorpion Tityus trinitatis with potent activity against ESKAPE Pathogens and clostridioides difficile. Antibiotics (Basel), 2023; 12(9):1404; doi: 10.3390/antibiotics12091404

Hawthorne

, Bernuci

, Bortolanza

, et al. Chapter 29 - Clinical developments in antimicrobial nanomedicine: toward novel solutions. In: Nanostructures for Antimicrobial Therapy. ( Ficai

, Grumezescu

. eds.). Micro and Nano Technologies Elsevier; 2017; pp. 653–668; doi: 10.1016/B978-0-323-46152-8.00029-9

, Shahriar

SSM

, Andrabi

, et al. It takes two to tangle: Microneedle patches co-delivering monoclonal antibodies and engineered antimicrobial peptides effectively eradicate wound biofilms. Macromol Biosci, 2024; 24(5):e2300519; doi: 10.1002/mabi.202300519

10.

Bresee

, Lamont

, Ocampo

, et al. Implementation strategies for hospital-based probiotic administration in a stepped-wedge cluster randomized trial design for preventing hospital-acquired Clostridioides difficile infection. BMC Health Serv Res, 2023; 23(1):1386; doi: 10.1186/s12913-023-10350-9

11.

Buckner

MMC

, Ciusa

, Piddock

LJV

. Strategies to combat antimicrobial resistance: Anti-plasmid and plasmid curing. FEMS Microbiol Rev, 2018; 42(6):781–804; doi: 10.1093/femsre/fuy031

12.

Soundy

, Day

. Selection of DNA aptamers specific for live Pseudomonas aeruginosa. PLoS One, 2017; 12(9):e0185385; doi: 10.1371/journal.pone.0185385

13.

Abedon

. Use of phage therapy to treat long-standing, persistent, or chronic bacterial infections. Adv Drug Deliv Rev, 2019; 145:18–39; doi: 10.1016/j.addr.2018.06.018

14.

Ramos-Martín

, Herrera-León

, Antonietti

, et al. Antimicrobial peptide K11 selectively recognizes bacterial biomimetic membranes and acts by twisting their bilayers. Pharmaceuticals (Basel), 2020; 14(1):1; doi: 10.3390/ph14010001

15.

Bugli

, Martini

, Di Vito

, et al. Antimicrobial peptides for tackling cystic fibrosis related bacterial infections: A review. Microbiol Res, 2022; 263:127152; doi: 10.1016/j.micres.2022.127152

16.

C-F

, Fang

C-M

, Sekaran

. Intracellular targeting mechanisms by antimicrobial peptides. Antimicrob Agents Chemother, 2017; 61(4):e02340-16; doi: 10.1128/AAC.02340-16

17.

Batoni

, Maisetta

, Esin

. Therapeutic potential of antimicrobial peptides in polymicrobial biofilm-associated infections. Int J Mol Sci, 2021; 22(2):482; doi: 10.3390/ijms22020482

18.

Gera

, Kankuri

, Kogermann

. Antimicrobial peptides - Unleashing their therapeutic potential using nanotechnology. Pharmacol Ther, 2022; 232:107990; doi: 10.1016/j.pharmthera.2021.107990

19.

McNeil

. Nanotechnology for the biologist. J Leukoc Biol, 2005; 78(3):585–594; doi: 10.1189/jlb.0205074

20.

Chakraborty

, Jha

, Roy

, et al. Nanobiotics against antimicrobial resistance: Harnessing the power of nanoscale materials and technologies. J Nanobiotechnology, 2022; 20(1):375; doi: 10.1186/s12951-022-01573-9

21.

Hetta

, Ramadan

, Al-Harbi

, et al. Nanotechnology as a promising approach to combat multidrug resistant bacteria: A comprehensive review and future perspectives. Biomedicines, 2023; 11(2):413; doi: 10.3390/biomedicines11020413

22.

Wang

, Chen

, Lu

. Anti-bacterial monoclonal antibodies: Next generation therapy against superbugs. Appl Microbiol Biotechnol, 2022; 106(11):3957–3972; doi: 10.1007/s00253-022-11989-w

23.

Kharga

, Kumar

, Patel

SKS

. Recent advances in monoclonal antibody-based approaches in the management of bacterial sepsis. Biomedicines, 2023; 11(3):765; doi: 10.3390/biomedicines11030765

24.

Suez

, Zmora

, Segal

, et al. The pros, cons, and many unknowns of probiotics. Nat Med, 2019; 25(5):716–729; doi: 10.1038/s41591-019-0439-x

25.

Kang

M-S

, Park

G-Y

, Lee

A-R

. In vitro Preventive effect and mechanism of action of Weissella cibaria CMU against Streptococcus mutans Biofilm formation and periodontal pathogens. Microorganisms, 2023; 11(4):962; doi: 10.3390/microorganisms11040962

26.

O’Toole

, Marchesi

, Hill

. Next-generation probiotics: The spectrum from probiotics to live biotherapeutics. Nat Microbiol, 2017; 2(5):17057; doi: 10.1038/nmicrobiol.2017.57

27.

Singh

, Shadan

, Ma

. Biotechnological applications of probiotics: A multifarious weapon to disease and metabolic abnormality. Probiotics Antimicrob Proteins, 2022; 14(6):1184–1210; doi: 10.1007/s12602-022-09992-8

28.

Hutchings

, Truman

, Wilkinson

. Antibiotics: Past, present and future. Curr Opin Microbiol, 2019; 51:72–80; doi: 10.1016/j.mib.2019.10.008

29.

Okusanya

, Bhavnani

, Hammel

, et al. Pharmacokinetic and pharmacodynamic evaluation of liposomal amikacin for inhalation in cystic fibrosis patients with chronic pseudomonal infection. Antimicrob Agents Chemother, 2009; 53(9):3847–3854; doi: 10.1128/AAC.00872-08

30.

Ramsey

, Dorkin

, Eisenberg

, et al. Efficacy of aerosolized tobramycin in patients with cystic fibrosis. N Engl J Med, 1993; 328(24):1740–1746; doi: 10.1056/NEJM199306173282403

31.

Ventola

. The antibiotic resistance crisis. P T, 2015; 40(4):277–283.

32.

Makabenta

JMV

, Nabawy

, Li

C-H

, et al. Nanomaterial-based therapeutics for antibiotic-resistant bacterial infections. Nat Rev Microbiol, 2021; 19(1):23–36.

33.

Nowack

, Krug

, Height

. 120 years of nanosilver history: Implications for policy makers. Environ Sci Technol, 2011; 45(4):1177–1183; doi: 10.1021/es103316q

34.

Kim

, Park

J-E

, Im

, et al. Recent advances in nanotechnology with nano-phytochemicals: Molecular mechanisms and clinical implications in cancer progression. Int J Mol Sci, 2021; 22(7):3571; doi: 10.3390/ijms22073571

35.

Bayda

, Adeel

, Tuccinardi

, et al. The history of nanoscience and nanotechnology: From chemical-physical applications to nanomedicine. Molecules (Basel), 2019; 25(1):112; doi: 10.3390/molecules25010112

36.

Chowdhry

, Lu

, Lee

, et al. Enhancing CRISPR/Cas systems with nanotechnology. Trends Biotechnol, 2023; 41(12):1549–1564; doi: 10.1016/j.tibtech.2023.06.005

37.

Nakatsuji

, Gallo

. Antimicrobial peptides: Old molecules with new ideas. J Invest Dermatol, 2012; 132(3 Pt 2):887–895; doi: 10.1038/jid.2011.3870 2

38.

Pleasants

, Michalets

, Williams

, et al. Pharmacokinetics of vancomycin in adult cystic fibrosis patients. Antimicrob Agents Chemother, 1996; 40(1):186–190; doi: 10.1128/AAC.40.1.186

39.

Scopp

, Gillette

, Kumar

, et al. Treatment of oral lesions with topically applied vancomycin hydrochloride. Oral Surg Oral Med Oral Pathol, 1967; 24(5):703–706; doi: 10.1016/0030-4220(67)90220-4

40.

Waterer

, Lord

, Hofmann

, et al. Phase I, Dose-Escalating Study of the safety and pharmacokinetics of inhaled dry-Powder Vancomycin (AeroVanc) in volunteers and patients with cystic fibrosis: A new approach to therapy for methicillin-resistant Staphylococcus aureus. Antimicrob Agents Chemother, 2020; 64(3):e01776-19; doi: 10.1128/AAC.01776-19

41.

Almeida-Santos

, Novais

, Peixe

, et al. Enterococcus spp. as a producer and target of bacteriocins: A double-edged sword in the antimicrobial resistance crisis context. Antibiotics (Basel), 2021; 10(10):1215; doi: 10.3390/antibiotics10101215

42.

Bruzzese

, Raia

, Spagnuolo

, et al. Effect of Lactobacillus GG supplementation on pulmonary exacerbations in patients with cystic fibrosis: A Pilot Study. Clin Nutr, 2007; 26(3):322–328; doi: 10.1016/j.clnu.2007.01.004

43.

Shin

, Gwak

, Kamarajan

, et al. Biomedical applications of Nisin. J Appl Microbiol, 2016; 120(6):1449–1465; doi: 10.1111/jam.13033

44.

Tang

, Zhao

. Commercial strains of lactic acid bacteria with health benefits. In: Lactic Acid Bacteria. ( Chen

. eds.) Springer Nature Singapore: Singapore; 2019; pp. 297–369; doi: 10.1007/978-981-13-7832-4_10

45.

Ding

, Gan

, Ho

. Single-stranded DNA oligoaptamers: Molecular recognition and LPS antagonism are length- and secondary structure-dependent. J Innate Immun, 2009; 1(1):46–58; doi: 10.1159/000145542

46.

Group

. Preclinical and phase 1A clinical evaluation of an anti-VEGF pegylated aptamer (EYE001) for the treatment of exudative age-related macular degeneration. Retina, 2002; 22(2):143–152; doi: 10.1097/00006982-200204000-00002

47.

Haßel

, Mayer

. Aptamers as therapeutic agents: Has the initial euphoria subsided? Mol Diagn Ther, 2019; 23(3):301–309; doi: 10.1007/s40291-019-00400-6

48.

Gordillo Altamirano

, Barr

. Unlocking the next generation of phage therapy: The key is in the receptors. Curr Opin Biotechnol, 2021; 68:115–123; doi: 10.1016/j.copbio.2020.10.002

49.

Kollberg

, Carlander

, Olesen

, et al. Oral administration of specific yolk antibodies (IgY) may prevent Pseudomonas aeruginosa infections in patients with cystic fibrosis: A phase I feasibility study. Pediatr Pulmonol, 2003; 35(6):433–440; doi: 10.1002/ppul.10290

50.

Bangert

, Hasbun

. Neurological and psychiatric adverse effects of antimicrobials. CNS Drugs, 2019; 33(8):727–753; doi: 10.1007/s40263-019-00649-9

51.

Dahiya

, Nigam

. Antibiotic-therapy-induced gut dysbiosis affecting gut microbiota—brain axis and cognition: Restoration by intake of probiotics and synbiotics. Int J Mol Sci, 2023; 24(4):3074; doi: 10.3390/ijms24043074

52.

Cunha

. Antibiotic side effects. Medi Clin N, 2001; 85(1):149–185; doi: 10.1016/S0025-7125(05)70309-6

53.

Gao

, Liu

, Zhang

, et al. Alterations in gut microbiota and inflammatory cytokines after administration of antibiotics in mice. Microbiol Spectr, 2024; 12(8):e0309523; doi: 10.1128/spectrum.03095-23

54.

Klingel

, Stanojevic

, Tullis

, et al. Oral azithromycin and response to pulmonary exacerbations treated with intravenous tobramycin in children with cystic fibrosis. Ann Am Thorac Soc, 2019; 16(7):861–867; doi: 10.1513/AnnalsATS.201811-774OC

55.

Kristensen

, Prevaes

SMPJ

, Kalkman

, et al. Development of the gut microbiota in early life: The impact of cystic fibrosis and antibiotic treatment. J Cyst Fibros, 2020; 19(4):553–561; doi: 10.1016/j.jcf.2020.04.007

56.

Scialo

, Amato

, Cernera

, et al. Lung microbiome in cystic fibrosis. Life (Basel), 2021; 11(2):94; doi: 10.3390/life11020094

57.

Vila

, Moreno-Morales

, Ballesté-Delpierre

. Current landscape in the discovery of novel antibacterial agents. Clin Microbiol Infect, 2020; 26(5):596–603; doi: 10.1016/j.cmi.2019.09.015

58.

Hatfull

, Dedrick

, Schooley

. Phage therapy for antibiotic-resistant bacterial infections. Annu Rev Med, 2022; 73:197–211; doi: 10.1146/annurev-med-080219-122208

59.

Ganeshan

, Hosseinidoust

. Phage therapy with a focus on the human microbiota. Antibiotics, 2019; 8(3):131; doi: 10.3390/antibiotics8030131

60.

Grecu

, Henea

M-E

, Rîmbu

, et al. The bacteriophages therapy of interdigital pyoderma complicated by cellulitis with antibiotic-resistant Pseudomonas aeruginosa in a Dog—Case Report. Vet Sci, 2023; 10(11):642; doi: 10.3390/vetsci10110642

61.

Johri

, Johri

, Hoyle

, et al. Case report: Successful treatment of recurrent E. coli infection with bacteriophage therapy for patient suffering from chronic bacterial prostatitis. Front Pharmacol, 2023; 14:1243824; doi: 10.3389/fphar.2023.1243824

62.

, Zhu

. Potential of therapeutic bacteriophages in nosocomial infection management. Front Microbiol, 2021; 12:638094; doi: 10.3389/fmicb.2021.638094

63.

Doss

, Barekzi

, Gauthier

. Improving high-throughput techniques for bacteriophage discovery in multi-well plates. J Microbiol Methods, 2022; 200:106542; doi: 10.1016/j.mimet.2022.106542

64.

Bayat

, Hilal

, Thirugnanasampanthar

, et al. High throughput platform technology for rapid target identification in personalized phage therapy. Nat Commun, 2024; 15(1):5626; doi: 10.1038/s41467-024-49710-2

65.

Forti

, Roach

, Cafora

, et al. Design of a broad-range bacteriophage cocktail that reduces Pseudomonas aeruginosa biofilms and treats acute infections in two animal models. Antimicrob Agents Chemother, 2018; 62(6):e02573-17; doi: 10.1128/AAC.02573-17

66.

Pinto

, Silva

, Pastrana

, et al. The clinical path to deliver encapsulated phages and lysins. FEMS Microbiol Rev, 2021; 45(5):fuab019; doi: 10.1093/femsre/fuab019

67.

Harper

, et al. Introduction to bacteriophages. In: Bacteriophages: Biology, Technology, Therapy. ( Harper

, Abedon

, Burrowes

. eds) Springer International Publishing: Cham; 2020; pp. 1–14; doi: 10.1007/978-3-319-40598-8_48-2

68.

Fedorov

, Samokhin

, Kozlova

, et al. Short-term outcomes of phage-antibiotic combination treatment in adult patients with periprosthetic hip joint infection. Viruses, 2023; 15(2):499; doi: 10.3390/v15020499

69.

Nir-Paz

, Gelman

, Khouri

, et al. Successful treatment of antibiotic-resistant, poly-microbial bone infection with bacteriophages and antibiotics combination. Clin Infect Dis, 2019; 69(11):2015–2018; doi: 10.1093/cid/ciz222

70.

Pantiora

, Georgakis

, Premetis

, et al. Metagenomic analysis of hot spring soil for mining a novel thermostable enzybiotic. Appl Microbiol Biotechnol, 2024; 108(1):163; doi: 10.1007/s00253-023-12979-2

71.

Rahman

, Wang

, Sun

, et al. Endolysin, a promising solution against antimicrobial resistance. Antibiotics (Basel), 2021; 10(11):1277; doi: 10.3390/antibiotics10111277

72.

Elshayeb

, Ahmed

, Marmar

, et al. Potentiality of lytic bacteriophages and their virolysins in lysing multi-drug resistant Salmonella typhi. Afr J Biotechnol, 2017; 16(41):2014–2023.

73.

Drulis-Kawa

, Majkowska-Skrobek

, Maciejewska

. Bacteriophages and phage-derived proteins–application approaches. Curr Med Chem, 2015; 22(14):1757–1773; doi: 10.2174/0929867322666150209152851

74.

Oliveira

, São-José

, Azeredo

. Phage-derived peptidoglycan degrading enzymes: Challenges and future prospects for in vivo therapy. Viruses, 2018; 10(6):292; doi: 10.3390/v10060292

75.

Heselpoth

, Swift

, Linden

, et al., et al. Enzybiotics: endolysins and bacteriocins. In: Bacteriophages: Biology, Technology, Therapy. ( Harper

, Abedon

, Burrowes

. eds.) Springer International Publishing: Cham; 2017; pp. 1–42; doi: 10.1007/978-3-319-40598-8_34-1

76.

Topka-Bielecka

, Dydecka

, Necel

, et al. Bacteriophage-derived depolymerases against bacterial biofilm. Antibiotics (Basel), 2021; 10(2):175; doi: 10.3390/antibiotics10020175

77.

Hua

, Wu

, Guo

, et al. Characterization and functional studies of a novel depolymerase against K19-Type Klebsiella pneumoniae. Front Microbiol, 2022; 13:878800; doi: 10.3389/fmicb.2022.878800

78.

Drobiazko

, Kasimova

, Evseev

, et al. Capsule-targeting depolymerases derived from Acinetobacter baumannii prophage regions. Int J Mol Sci, 2022; 23(9):4971; doi: 10.3390/ijms23094971

79.

Pires

, Oliveira

, Melo

LDR

, et al. Bacteriophage-encoded depolymerases: Their diversity and biotechnological applications. Appl Microbiol Biotechnol, 2016; 100(5):2141–2151; doi: 10.1007/s00253-015-7247-0

80.

Saier

, Reddy

. Holins in bacteria, eukaryotes, and archaea: Multifunctional xenologues with potential biotechnological and biomedical applications. J Bacteriol, 2015; 197(1):7–17; doi: 10.1128/JB.02046-14

81.

Shi

, Li

, Yan

, et al. Combined antibacterial activity of phage lytic proteins holin and lysin from Streptococcus suis bacteriophage SMP. Curr Microbiol, 2012; 65(1):28–34; doi: 10.1007/s00284-012-0119-2

82.

Abril

, Carrera

, Notario

, et al. The use of bacteriophages in biotechnology and recent insights into proteomics. Antibiotics (Basel), 2022; 11(5):653; doi: 10.3390/antibiotics11050653

83.

Grabowski

, Łepek

, Stasiłojć

, et al. Bacteriophage-encoded enzymes destroying bacterial cell membranes and walls, and their potential use as antimicrobial agents. Microbiol Res, 2021; 248:126746; doi: 10.1016/j.micres.2021.126746

84.

Cahill

, Young

. Chapter Two – phage lysis: multiple genes for multiple barriers. In: Advances in Virus Research. ( Kielian

, Mettenleiter

, Roossinck

. eds.) Academic Press; 2019; pp. 33–70; doi: 10.1016/bs.aivir.2018.09.003

85.

Khan

, Rotich

, Morris

, et al. Probing the structural topology and dynamic properties of gp28 using continuous wave electron paramagnetic resonance spectroscopy. J Phys Chem B, 2023; 127(43):9236–9247; doi: 10.1021/acs.jpcb.3c03679

86.

Aslam

, Arshad

, Aslam

, et al. Bacteriophage proteome: Insights and potentials of an alternate to antibiotics. Infect Dis Ther, 2021; 10(3):1171–1193; doi: 10.1007/s40121-021-00446-2

87.

Magaziner

, Salmond

GPC

. A novel T4- and λ-based receptor binding protein family for bacteriophage therapy host range engineering. Front Microbiol, 2022; 13:1010330; doi: 10.3389/fmicb.2022.1010330

88.

Shehi

, Ghazanfar

, Fortuzi

, et al. An unusual case of polymicrobial bacteremia from methicillin-resistant Staphylococcus Aureus and Shigella. Cureus, n.d; 12(12):e12011; doi: 10.7759/cureus.12011

89.

Dragounová

, Ryabchykov

, Steinbach

, et al. Identification of bacteria in mixed infection from urinary tract of patient’s samples using Raman analysis of dried droplets. Analyst, 2023; 148(16):3806–3816; doi: 10.1039/D3AN00679D

90.

Jerzy

, Francis

. Chronic Osteomyelitis – Bacterial flora, antibiotic sensitivity and treatment challenges. Open Orthop J, 2018; 12:153–163; doi: 10.2174/1874325001812010153

91.

Cooke

. Infections in people with diabetes. Medicine, 2022; 50(11):729–732; doi: 10.1016/j.mpmed.2022.08.006

92.

Tessema

, Ali

, Zenebe

. Bacterial associated urinary tract infection, risk factors, and drug susceptibility profile among adult people living with HIV at Haswassa University Comprehensive Specialized Hospital, Hawassa, Southern Esthiopia. Sci Rep, 2020; 10(1):10790; doi: 10.1038/s41598-020-67840-7

93.

Lindgren

, McDaniel

, Novak

, et al. Acute polymicrobial airway infections: Analysis in cystic fibrosis mice. Microbiology (Reading) 2023; 169(1):001290; doi: 10.1099/mic.0.001290

94.

Shariati

, Noei

, Chegini

. Bacteriophages: The promising therapeutic approach for enhancing ciprofloxacin efficacy against bacterial infection. J Clin Lab Anal, 2023; 37(9–10):e24932; doi: 10.1002/jcla.24932

95.

Nielsen

, Alford

, Yung

DBY

, et al. Self-assembly of antimicrobial peptoids impacts their biological effects on ESKAPE bacterial pathogens. ACS Infect Dis, 2022; 8(3):533–545; doi: 10.1021/acsinfecdis.1c00536

96.

Borthagaray

, Mondelli

, Facchin

, et al. Chapter 8 – Silver-containing nanoparticles in the research of new antimicrobial agents against ESKAPE pathogens. In: Inorganic Frameworks as Smart Nanomedicines. ( Grumezescu

. eds.) William Andrew Publishing; 2018; pp. 317–386; doi: 10.1016/B978-0-12-813661-4.00008-0

97.

Jadid

MFS

, Jafari-Gharabaghlou

, Bahrami

, et al. Enhanced anti-cancer effect of curcumin loaded-niosomal nanoparticles in combination with heat-killed Saccharomyces cerevisiae against human colon cancer cells. J Drug Deliv Sci Technol, 2023; 80:104167; doi: 10.1016/j.jddst.2023.104167

98.

Mousazadeh

, Khorsandi

, Zarghami

. Stimulus-responsive nanocarrier from star-shaped polyethyleneimine-β-cyclodextrin coated mesoporous silica for targeted combination cancer therapy. J Drug Deliv Sci Technol, 2023; 88:104940; doi: 10.1016/j.jddst.2023.104940

99.

Mofarrah

, Jafari-Gharabaghlou

, Dadashpour

, et al. Fabricating ZSM-5 zeolite/polycaprolactone nano-fibers co-loaded with dexamethasone and ascorbic acid: Potential application in osteogenic differentiation of human adipose-derived stem cells. J Drug Deliv Sci Technol, 2023; 79:103999; doi: 10.1016/j.jddst.2022.103999

100.

Ezike

, Okpala

, Onoja

, et al. Advances in drug delivery systems, challenges and future directions. Heliyon, 2023; 9(6):e17488; doi: 10.1016/j.heliyon.2023.e17488

101.

Bayer

. An overview of monoclonal antibodies. Semin Oncol Nurs, 2019; 35(5):150927; doi: 10.1016/j.soncn.2019.08.006

102.

Williams

. Probiotics. Am J Health Syst Pharm, 2010; 67(6):449–458; doi: 10.2146/ajhp090168

103.

Park

H-M

, Park

, Berani

, et al. In silico optimization of RNA-protein interactions for CRISPR-Cas13-based antimicrobials. Biol Direct, 2022; 17(1):27; doi: 10.1186/s13062-022-00339-5

104.

Park

. Nucleic acid aptamer-based methods for diagnosis of infections. Biosens Bioelectron, 2018; 102:179–188; doi: 10.1016/j.bios.2017.11.028

105.

Sola

, Menon

, Moreno

, et al. Aptamers against live targets: Is in vivo selex finally coming to the edge? Mol Ther Nucleic Acids, 2020; 21:192–204; doi: 10.1016/j.omtn.2020.05.025

106.

Liu

, Hu

, Li

, et al. Therapeutic potential of bacteriophage endolysins for infections caused by Gram-positive bacteria. J Biomed Sci, 2023; 30(1):29; doi: 10.1186/s12929-023-00919-1

107.

Khan

, Rasheed

, Yang

, et al. Endolysins: A new antimicrobial agent against antimicrobial resistance. Strategies and opportunities in overcoming the challenges of endolysins against Gram-negative bacteria. Front Pharmacol, 2024; 15:1385261; doi: 10.3389/fphar.2024.1385261

108.

Osman

A-H

, Kotey

FCN

, Odoom

, et al. The potential of bacteriophage-antibiotic combination therapy in treating infections with multidrug-resistant bacteria. Antibiotics (Basel), 2023; 12(8):1329; doi: 10.3390/antibiotics12081329

109.

Canfield

, Chatterjee

, Espinosa

, et al. Lytic bacteriophages facilitate antibiotic sensitization of Enterococcus faecium. Antimicrob Agents Chemother, 2023; 65(5):e00143–e21; doi: 10.1128/AAC.00143-21

110.

Liang

, Han

, Shi

, et al. Effective of phage cocktail against Klebsiella pneumoniae infection of murine mammary glands. Microb Pathog, 2023; 182:106218; doi: 10.1016/j.micpath.2023.106218

111.

Schmerer

, Molineux

, Bull

. Synergy as a rationale for phage therapy using phage cocktails. Peer J, 2014; 2:e590; doi: 10.7717/peerj.590

112.

, Shi

, Sun

, et al. A novel method to create efficient phage cocktails via use of phage-resistant bacteria. Appl Environ Microbiol, 2022; 88(6):e0232321; doi: 10.1128/aem.02323-21

113.

Gu Liu

, Green

, Min

, et al. Phage-antibiotic synergy is driven by a unique combination of antibacterial mechanism of action and stoichiometry. mBio, 2020; 11(4):e01462-20; doi: 10.1128/mBio.01462-20

114.

Manohar

, Madurantakam Royam

, Loh

, et al. Synergistic effects of phage-antibiotic combinations against Citrobacter amalonaticus. ACS Infect Dis, 2022; 8(1):59–65; doi: 10.1021/acsinfecdis.1c00117

115.

Braunstein

, Hubanic

, Yerushalmy

, et al. Successful phage-antibiotic therapy of P. aeruginosa implant-associated infection in a Siamese cat. Vet Q, 2024; 44(1):1–9; doi: 10.1080/01652176.2024.2350661

116.

Teney

, Poupelin

J-C

, Briot

, et al. Phage therapy in a burn patient colonized with extensively drug-resistant Pseudomonas aeruginosa responsible for relapsing ventilator-associated pneumonia and bacteriemia. Viruses, 2024; 16(7):1080; doi: 10.3390/v16071080

117.

, Li

, Han

, et al. The antagonistic interactions between a polyvalent phage SaP7 and β-lactam antibiotics on combined therapies. Vet Microbiol, 2022; 266:109332; doi: 10.1016/j.vetmic.2022.109332

118.

Chen

, Liu

, Zhang

, et al. Phage-derived depolymerase as an antibiotic adjuvant against multidrug-resistant Acinetobacter baumannii. Front Microbiol, 2022; 13:845500; doi: 10.3389/fmicb.2022.845500

119.

Pertics

, Kovács

, Schneider

. Characterization of a lytic bacteriophage and demonstration of its combined lytic effect with a K2 depolymerase on the hypervirulent Klebsiella pneumoniae Strain 52145. Microorganisms, 2023; 11(3):669; doi: 10.3390/microorganisms11030669

120.

Latka

, Drulis-Kawa

. Advantages and limitations of microtiter biofilm assays in the model of antibiofilm activity of Klebsiella phage KP34 and its depolymerase. Sci Rep, 2020; 10(1):20338; doi: 10.1038/s41598-020-77198-5

121.

Rodríguez-Rubio

, Martínez

, Rodríguez

, et al. Enhanced staphylolytic activity of the Staphylococcus aureus Bacteriophage vB_SauS-phiIPLA88 HydH5 Virion-Associated Peptidoglycan Hydrolase: Fusions, Deletions, and Synergy with LysH5. Appl Environ Microbiol, 2012; 78(7):2241–2248; doi: 10.1128/AEM.07621-11

122.

Olsen

NMC

, Thiran

, Hasler

, et al. Synergistic removal of static and dynamic Staphylococcus aureus biofilms by combined treatment with a bacteriophage endolysin and a polysaccharide depolymerase. Viruses, 2018; 10(8):438; doi: 10.3390/v10080438

123.

Ramalho

, de Cássia Orlandi Sardi

, Júnior

, et al. The synthetic antimicrobial peptide IKR18 displays anti-infectious properties in Galleria mellonella in vivo model. Biochim Biophys Acta Gen Subj, 2022; 1866(12):130244; doi: 10.1016/j.bbagen.2022.130244

124.

Prince

, Sandhu

, Ror

, et al. Corrigendum: Lipid-II independent antimicrobial mechanism of nisin depends on its crowding and degree of oligomerization. Sci Rep, 2017; 7:41346; doi: 10.1038/srep41346

125.

, Li

, Zhang

, et al. Cecropin A–melittin mutant with improved proteolytic stability and enhanced antimicrobial activity against bacteria and fungi associated with gastroenteritis in vitro. Biochem Biophys Res Commun, 2014; 451(4):650–655; doi: 10.1016/j.bbrc.2014.08.044

126.

García

, Martínez

, Rodríguez

, et al. Synergy between the phage endolysin LysH5 and nisin to kill Staphylococcus aureus in pasteurized Milk. Int J Food Microbiol, 2010; 141(3):151–155; doi: 10.1016/j.ijfoodmicro.2010.04.029

127.

Lim

, Hong

, Jung

, et al. Bactericidal effect of cecropin a fused endolysin on drug-resistant gram-negative pathogens. J Microbiol Biotechnol, 2022; 32(6):816–823; doi: 10.4014/jmb.2205.05009

128.

Islam

, Kim

, et al. Engineering of lysin by fusion of antimicrobial peptide (cecropin A) enhances its antibacterial properties against multidrug-resistant Acinetobacter baumannii. Front Microbiol, 2022; 13:988522; doi: 10.3389/fmicb.2022.988522

129.

Kitano

, Tomasz

. Triggering of autolytic cell wall degradation in Escherichia coli by beta-lactam antibiotics. Antimicrob Agents Chemother, 1979; 16(6):838–848.

130.

Escobedo

, Pérez de Pipaon

, Rendueles

, et al. Cell wall modifications that alter the exolytic activity of lactococcal phage endolysins have little impact on phage growth. Front Microbiol, 2023; 14:1106049; doi: 10.3389/fmicb.2023.1106049

131.

Watts

, Hänni

, Smith

, et al. Human antimicrobial peptide inactivation mechanism of enveloped viruses. J Colloid Interface Sci, 2024; 657:971–981; doi: 10.1016/j.jcis.2023.11.055

132.

Wang

, Yang

, Shi

, et al. Antibiotic-free antibacterial strategies enabled by nanomaterials: Progress and perspectives. Adv Mater, 2020; 32(18):e1904106; doi: 10.1002/adma.201904106

133.

Ciepluch

, Maciejewska

, Gałczyńska

, et al. The influence of cationic dendrimers on antibacterial activity of phage endolysin against P. aeruginosa cells. Bioorg Chem, 2019; 91:103121; doi: 10.1016/j.bioorg.2019.103121

134.

Ciepluch

, Skrzyniarz

, Barrios-Gumiel

, et al. Dendronized silver nanoparticles as bacterial membrane permeabilizers and their interactions with P. aeruginosa lipopolysaccharides, lysozymes, and phage-derived endolysins. Front Microbiol, 2019; 10:2771; doi: 10.3389/fmicb.2019.02771

135.

Quintana-Sanchez

, Gómez-Casanova

, Sánchez-Nieves

, et al. The antibacterial effect of PEGylated carbosilane dendrimers on P. aeruginosa alone and in combination with phage-derived endolysin. Int J Mol Sci, 2022; 23(3):1873; doi: 10.3390/ijms23031873

136.

Cafora

, Poerio

, Forti

, et al. Evaluation of phages and liposomes as combination therapy to counteract Pseudomonas aeruginosa infection in wild-type and CFTR-null models. Front Microbiol, 2022; 13:979610; doi: 10.3389/fmicb.2022.979610

137.

Wrońska

, Felczak

, Zawadzka

, et al. Poly(Propylene Imine) Dendrimers and Amoxicillin as dual-action antibacterial agents. Molecules, 2015; 20(10):19330–19342; doi: 10.3390/molecules201019330

138.

Rodríguez-Barajas

, de Jesús Martín-Camacho

, Pérez-Larios

. Mechanisms of metallic nanomaterials to induce an antibacterial effect. Curr Top Med Chem, 2022; 22(30):2506–2526; doi: 10.2174/1568026622666220919124104

139.

Rodríguez-Prieto

, Popp

, Copa-Patiño

, et al. Silver (I) N-Heterocyclic carbenes carbosilane dendritic systems and their imidazolium-terminated analogues as antibacterial agents: Study of Their Mode of Action. Pharmaceutics, 2020; 12(10):968; doi: 10.3390/pharmaceutics12100968

140.

Llamazares

, Sanz del Olmo

, Ortega

, et al. Antibacterial effect of carbosilane metallodendrimers in planktonic cells of gram-positive and gram-negative bacteria and Staphylococcus aureus biofilm. Biomolecules, 2019; 9(9):405; doi: 10.3390/biom9090405

141.

Devrim

, Bozkır

. Chapter 7 – Nanocarriers and their potential application as antimicrobial drug delivery. In: Nanostructures for Antimicrobial Therapy. ( Ficai

, Grumezescu

. eds.) Elsevier; 2017; pp. 169–202; doi: 10.1016/B978-0-323-46152-8.00007-X

142.

Jiang

, Lin

, Taggart

, et al. Nanodelivery strategies for the treatment of multidrug‐resistant bacterial infections. J Interdiscip Nanomed, 2018; 3(3):111–121; doi: 10.1002/jin2.48

143.

Huang

, Kłodzińska

, Wan

, et al. Nanoparticle-mediated pulmonary drug delivery: State of the art towards efficient treatment of recalcitrant respiratory tract bacterial infections. Drug Deliv Transl Res, 2021; 11(4):1634–1654; doi: 10.1007/s13346-021-00954-1

144.

Kirtane

, Verma

, Karandikar

, et al. Nanotechnology approaches for global infectious diseases. Nat Nanotechnol, 2021; 16(4):369–384.

145.

Tang

, Ouyang

, Li

, et al. Nanomaterials for delivering antibiotics in the therapy of Pneumonia. Int J Mol Sci, 2022; 23(24):15738; doi: 10.3390/ijms232415738

146.

Chadha

, Katare

, Chhibber

. Liposome loaded phage cocktail: Enhanced therapeutic potential in resolving Klebsiella pneumoniae mediated burn wound infections. Burns, 2017; 43(7):1532–1543; doi: 10.1016/j.burns.2017.03.029

147.

Kalelkar

, Moustafa

, Riddick

, et al. Bacteriophage-Loaded Poly(lactic-co-glycolic acid) Microparticles Mitigate Staphylococcus aureus Infection and Cocultures of Staphylococcus aureus and Pseudomonas aeruginosa. Adv Healthc Mater, 2022; 11(10):e2102539; doi: 10.1002/adhm.202102539

148.

Shafigh Kheljan

, Sheikhzadeh Hesari

, Aminifazl

, et al. Design of Phage-Cocktail-Containing Hydrogel for the Treatment of Pseudomonas aeruginosa-Infected Wounds. Viruses, 2023; 15(3):803; doi: 10.3390/v15030803

149.

Safarpour-Dehkordi

, Chabok

, Asgari

, et al. A comprehensive investigation of the medicinal efficacy of antimicrobial fusion peptides expressed in probiotic bacteria for the treatment of Pan Drug-Resistant (PDR) infections. Arch Microbiol, 2024; 206(3):93; doi: 10.1007/s00203-023-03823-2

150.

Chan

, Xu

, Cheng

, et al. A novel infant microbiome formula (SIM03) improved eczema severity and quality of life in preschool children. Sci Rep, 2024; 14(1):3168; doi: 10.1038/s41598-024-53848-w

151.

Grubb

, Wrigley

, Freedman

, et al. PHAGE-2 Study: Supplemental Bacteriophages Extend Bifidobacterium animalis subsp. lactis BL04 benefits on gut health and microbiota in healthy adults. Nutrients, 2020; 12(8):2474; doi: 10.3390/nu12082474

152.

D’Accolti

, Soffritti

, Bini

, et al. Potential use of a combined bacteriophage–probiotic sanitation system to control microbial contamination and AMR in healthcare settings: A Pre-Post Intervention Study. Int J Mol Sci, 2023; 24(7):6535; doi: 10.3390/ijms24076535

153.

Wandro

, Ghatbale

, Attai

, et al. Phage cocktails constrain the growth of Enterococcus. mSystems, 2022; 7(4):e00019-22; doi: 10.1128/msystems.00019-22

154.

Blasco

, Bleriot

, González de Aledo

, et al. Development of an anti-Acinetobacter baumannii biofilm phage cocktail: Genomic adaptation to the host. Antimicrob Agents Chemother, 2022; 66(3):e0192321; doi: 10.1128/AAC.01923-21

155.

Zurabov

, Zhilenkov

. Characterization of four virulent Klebsiella pneumoniae bacteriophages, and evaluation of their potential use in complex phage preparation. Virol J, 2021; 18(1):9; doi: 10.1186/s12985-020-01485-w

156.

Schooley

, Biswas

, Gill

, et al. Development and use of personalized bacteriophage-based therapeutic cocktails to treat a patient with a disseminated resistant Acinetobacter baumannii Infection. Antimicrob Agents Chemother, 2017; 61(10):e00954-17; doi: 10.1128/AAC.00954-17

157.

Kunz Coyne

, Stamper

, El Ghali

, et al. Phage-antibiotic cocktail rescues daptomycin and phage susceptibility against daptomycin-nonsusceptible Enterococcus faecium in a simulated endocardial vegetation ex vivo model. Microbiol Spectr, 2023; 11(4):e0034023; doi: 10.1128/spectrum.00340-23

158.

Cafora

, Deflorian

, Forti

, et al. Phage therapy against Pseudomonas aeruginosa infections in a cystic fibrosis zebrafish model. Sci Rep, 2019; 9(1):1527; doi: 10.1038/s41598-018-37636-x

159.

Chang

RYK

, Das

, Manos

, et al. Bacteriophage PEV20 and ciprofloxacin combination treatment enhances removal of P. aeruginosa biofilm isolated from cystic fibrosis and wound patients. AAPS J, 2019; 21(3):49; doi: 10.1208/s12248-019-0315-0

160.

Akturk

, Melo

LDR

, Oliveira

, et al. Combining phages and antibiotic to enhance antibiofilm efficacy against an in vitro dual species wound biofilm. Biofilm, 2023; 6:100147; doi: 10.1016/j.bioflm.2023.100147

161.

Gouveia

, Pinto

, Veiga

, et al. Synthetic antimicrobial peptides as enhancers of the bacteriolytic action of staphylococcal phage endolysins. Sci Rep, 2022; 12(1):1245; doi: 10.1038/s41598-022-05361-1

162.

Hong

H-W

, Kim

, Jang

, et al. Combination effect of engineered endolysin EC340 with antibiotics. Front Microbiol, 2022; 13:821936; doi: 10.3389/fmicb.2022.821936

163.

Son

, Kong

, Cha

, et al. Simultaneous control of Staphylococcus aureus and Bacillus cereus using a hybrid endolysin LysB4EAD-LysSA11. Antibiotics (Basel), 2020; 9(12):906; doi: 10.3390/antibiotics9120906

164.

Basit

, Qadir

, Qureshi

, et al. Cloning and expression analysis of fused holin-endolysin from RL bacteriophage; Exhibits broad activity against multi drug resistant pathogens. Enzyme Microb Technol, 2021; 149:109846; doi: 10.1016/j.enzmictec.2021.109846

165.

Portilla

, Fernández

, Gutiérrez

, et al. Encapsulation of the antistaphylococcal endolysin LysRODI in pH-Sensitive liposomes. Antibiotics (Basel), 2020; 9(5):242; doi: 10.3390/antibiotics9050242

166.

Wang

, Ji

, Qiu

, et al. A phage cocktail combined with the enteric probiotic Lactobacillus reuteri ameliorated mouse colitis caused by S. typhimurium. Food Funct, 2022; 13(16):8509–8523.

167.

Porter

, Johnston

, Kisiela

, et al. Bacteriophage cocktail and microcin-producing probiotic Escherichia coli Protect Mice against gut colonization with multidrug-resistant Escherichia coli Sequence Type 131. Front Microbiol, 2022; 13:887799; doi: 10.3389/fmicb.2022.887799

168.

Berry

, Rajaure

, Young

. Spanin function requires subunit homodimerization through intermolecular disulfide bonds. Mol Microbiol, 2013; 88(1):35–47; doi: 10.1111/mmi.12167

169.

Malanovic

, Lohner

. Antimicrobial peptides targeting gram-positive Bacteria. Pharmaceuticals (Basel), 2016; 9(3):59; doi: 10.3390/ph9030059

170.

Lahiri

, Johnstone

, Ross

, et al. Avibactam and Class C β-Lactamases: Mechanism of inhibition, conservation of the binding pocket, and implications for resistance. Antimicrob Agents Chemother, 2014; 58(10):5704–5713; doi: 10.1128/AAC.03057-14

171.

Duan

, Chang

, Su

, et al. Generation of a specific aptamer for accurate detection of sarafloxacin based on fluorescent/colorimetric/SERS triple-readout sensor. Biosens Bioelectron, 2024; 249:116022; doi: 10.1016/j.bios.2024.116022

172.

Wan

, Liu

, Zu

. Oligonucleotide aptamers for pathogen detection and infectious disease control. Theranostics, 2021; 11(18):9133–9161; doi: 10.7150/thno.61804

173.

Bruno

. Predicting the uncertain future of aptamer-based diagnostics and therapeutics. Molecules, 2015; 20(4):6866–6887; doi: 10.3390/molecules20046866

174.

Lyu

, Shang

, Wang

, et al. Monoclonal antibodies specific to the extracellular domain of histidine Kinase YycG of Staphylococcus epidermidis Inhibit Biofilm Formation. Front Microbiol, 2020; 11:1839; doi: 10.3389/fmicb.2020.01839

175.

Zhou

, Yang

, Li

, et al. Exploiting a conjugative endogenous CRISPR-Cas3 system to tackle multidrug-resistant Klebsiella pneumoniae. EBioMedicine, 2023; 88:104445; doi: 10.1016/j.ebiom.2023.104445

176.

Huan

, Torraca

, Brown

, et al. P1 Bacteriophage-enabled delivery of CRISPR-Cas9 Antimicrobial activity against Shigella flexneri. ACS Synth Biol, 2023; 12(3):709–721; doi: 10.1021/acssynbio.2c00465

177.

, Tai

, Chang

, et al. Overcoming challenges to make bacteriophage therapy standard clinical treatment practice for cystic fibrosis. Front Microbiol, 2020; 11:593988; doi: 10.3389/fmicb.2020.593988

178.

Hada-Neeman

, Weiss-Ottolenghi

, Wagner

, et al. Domain-scan: Combinatorial sero-diagnosis of infectious diseases using machine learning. Front Immunol, 2020; 11:619896; doi: 10.3389/fimmu.2020.619896

179.

Rajput

, Bhamare

, Thakur

, et al. Anti-biofilm: Machine learning assisted prediction of IC50 activity of chemicals against biofilms of microbes causing antimicrobial resistance and implications in drug repurposing. J Mol Biol, 2023; 435(14):168115; doi: 10.1016/j.jmb.2023.168115

180.

Lluka

, Stokes

. Antibiotic discovery in the artificial intelligence era. Ann N Y Acad Sci, 2023; 1519(1):74–93; doi: 10.1111/nyas.14930

181.

Briganti

, Le Moine

. Artificial intelligence in medicine: Today and tomorrow. Front Med (Lausanne), 2020; 7:27; doi: 10.3389/fmed.2020.00027

182.

, Kaplan

, Buehler

. Generative design of de novo proteins based on secondary structure constraints using an attention-based diffusion model. Chem, 2023; 9(7):1828–1849; doi: 10.1016/j.chempr.2023.03.020