New approaches for combating polyresistant ESKAPE pathogens

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Introduction

In recent decades, the overuse and misuse of antibiotics, as well as social and economic factors, have accelerated the spread of antibiotic-resistant bacteria, making the etiologic therapy of infectious processes with antibacterial drugs ineffective. In 2024, in light of growing antibiotic resistance, the World Health Organization (WHO) published a list of pathogens designated by the acronym ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and members of the genus Enterobacter). It is these microorganisms that pose the greatest threat to global health [105]. Understanding the resistance mechanisms of these bacteria is a key step in the development of new antimicrobial agents to combat antibiotic-resistant bacteria [63].

Currently, at least 700 000 people worldwide die each year from bacterial infections due to antimicrobial resistance. WHO predicts that without new and more effective treatments, this number could rise to 10 million by 2050 [94]. The global problem of antibiotic resistance was greatly exacerbated during the COVID-19 pandemic. Despite the fact that antibiotics are not effective against viruses, including the causative agent of COVID-19, antibiotic use increased throughout the pandemic along with the number of new COVID-19 infections.

Of concern is the dramatic increase in resistance of ESKAPE strains to carbapenems, which has become a major public health problem due to the lack of effective alternative antibacterial agents, as well as significant difficulties in developing new antibiotics. In 2021, only six of the thirty-two antibiotics in clinical development had some efficacy against ESKAPE group bacteria and were categorized as novel. This situation prompted the search for alternative treatments for bacterial infections to avoid the emergence or spread of resistance in microorganisms [21].

Alternative modern therapies currently in practice or undergoing trials include the use of antibiotics in combination with adjuvants, bacteriophage therapy, the use of antimicrobial peptides and antibodies, phytochemicals, and nanoparticles as antibacterial agents [64].

Main part

Background

Researchers all over the world note that microorganisms of the ESKAPE group are the main cause of nosocomial infections [25]. Carbapenem-resistant A. baumannii and representatives of the Enterobacteriaceae family (K. pneumoniae, K. aerogenes, Enterobacter cloacae, etc.) resistant to 3rd generation cephalosporins and carbapenems are included by WHO in the list of pathogens with critical priority, while vancomycin-resistant E. faecium and methicillin-resistant E. faecium and methicillin-resistant S. aureus (MRSA) and carbopenem-resistant P. aeruginosa are listed as a high priority group [105].

One of the critical microorganisms is A. baumannii. It is defined as multidrug resistant when the pathogen is resistant to at least 3 classes of antibiotics (penicillins and cephalosporins including their combinations, fluoroquinolones and aminoglycosides) and as extensively drug resistant when it is resistant to more than 3 classes of antibiotics and to carbapenems. A strain resistant to all the above antibiotics as well as to polymyxin and tigecycline is referred to as panresistant [62]. The ability of A. baumannii to persist on surfaces and resist disinfectants helps the survival of the bacteria in healthcare settings [65].

A. baumannii  poses a major challenge to clinicians due to the presence of a number of resistance determinants: efflux pumps, internal ADC cephalosporinase, OXA-51 β-lactamase, and acquired carbapenemases such as OXA (Fig. 1) [10].

 

Figure 1. Global carbapenem resistance of A. baumannii

Note. Percentage of carbapenem-resistant A. baumannii strains from 1997 to 2022 worldwide.

 

P. aeruginosa  is an opportunistic microorganism with intrinsic resistance mechanisms including impaired cell wall permeability to drugs, efflux pumps for drug efflux from the cell, and cephalosporinase [55]. In addition, P. aeruginosa expresses many virulence factors [46]. The resistance of P. aeruginosa clinical isolates worldwide is increasingly complemented by acquired resistance determinants, including extended-spectrum beta-lactamases and carbapenemases (Fig. 2) [6].

 

Figure 2. Global carbapenem resistance of P. aeruginosa

Note. Percentage of carbapenem-resistant P. aeruginosa strains from 1997 to 2022 worldwide.

 

β-lactam antibiotics are the therapy of choice for infections caused by methicillin-sensitive S. aureus (MSSA). Meanwhile, methicillin-resistant S. aureus (MRSA) shows resistance to most β-lactams (Fig. 3).

Therefore, vancomycin remains the antibiotic of choice in the therapy of staphylococcal infections. Toxicity and increasing resistance to vancomycin require a reassessment of the treatment strategy for such infections. However, clinical data on the use of alternative agents do not provide reliable evidence for the complete replacement of vancomycin as the working antibiotic for MRSA infections [67].

Enterococci are commensals of the human gastrointestinal tract. Most E. faecalis isolates retain sensitivity to ampicillin with low resistance to vancomycin (5–10%) [68]. At the same time, hospital-acquired E. faecalis strains are resistant to ampicillin in most cases, and 0.3–3% of strains are resistant to vancomycin in Eastern Europe, 30–60% of strains in South America, and 20–50% of strains in the United States (Fig. 4) [1].

 

Figure 3. Global antibiotic resistance of MRSA (methicillin-resistant S. aureus)

Note. Percentage of antibiotic-resistant S. aureus strains from 1997 to 2023 worldwide.

 

Figure 4. Global vancomycin resistance of E. faecalis

Note. Percentage of vancomycin resistant E. faecalis from 1997 to 2022 worldwide.

 

Several members of the ESKAPE group belong to the Enterobacteriaceae family, including K. pneumoniae, Klebsiella aerogenes, and Enterobacter cloacae [97]. Escherichia coli is also a serious threat [79]. These microorganisms often manifest in urinary tract infections (UTIs), pneumonia, and bacteremia [78], and possess a genome complemented with conjugative plasmids carrying resistance genes: extended-spectrum β-lactamases and carbapenemases (e.g., KPC and OXA-48-like serine carbapenemases; NDM, VIM, and IMP metallo-β-lactamases) (Fig. 5) [85].

 

Figure 5. Global carbapenem resistance of Enterobacteriaceae

Note. Percentage of carbapenem resistant Enterobacteriaceae from 1997 to 2022 worldwide.

 

Thus, today there is an urgent need to develop new strategies for the therapy of bacterial infections, as existing drugs are increasingly ineffective. The search for new antibiotics seems to be the surest way out, as alternative therapies for bacterial infections have gained insufficient evidence and clinical trial base. But there are factors holding back the search for new antibiotics [21]:

1. High requirements for new antibiotics. Drugs including antibiotics undergo many tests and clinical trials to ensure their safety and efficacy. New antibiotics must meet strict criteria that are set by regulatory organizations. Recently approved antibiotics are delafloxacin, varobactam + meropenem (Vabomere), plazomicin, eravacycline, omadacycline, relabactam + imipenem (Recarbrio), lefamulin, pretomanid, lascufloxacin, cefiderocol, levonadifloxacin [2]. Of the 11 new antibiotics approved since 2017, including three newly approved antibiotics from 2019, only two, varobactam + meropenem and lefamulin, represent new classes. Resistance has already been detected to these antibiotics [14], as bacteria are forming resistance much faster than new antibacterial drugs entering the market. Other recently approved antibiotics are derivatives of existing classes whose clinical utility is limited and for which resistance mechanisms already exist.
2. High research costs, which are not always recouped due to the fact that most antibiotics are used for short courses of treatment, which may not be profitable for drug manufacturers in the long term. Because of this, many pharmaceutical companies are not interested in developing new antibiotics [2].

That is why our review emphasizes the need to develop modern alternative therapies for bacterial infections that will provide an opportunity to avoid the spread of global microbial resistance [104].

Alternative therapies currently in practice or undergoing clinical and preclinical trials include the use of antibiotics in combination or with adjuvants, phage therapy, the use of antimicrobial peptides, antibacterial antibodies, phytochemicals, and nanoparticles as antibacterial agents [64].

Phage therapy

With the increasing resistance of bacteria to antibiotics, bacteriophages have attracted the attention of researchers. The use of bacteriophage preparations has a significant advantage: phages have strict specificity without increasing the risk of opportunistic infections; the need for low doses to achieve a therapeutic effect; rapid proliferation within host bacteria and achievement of the necessary therapeutic concentrations [29]. In contrast to antibiotics, phages also have the advantage of being able to evolve and mutate with their host, circumventing emerging resistance [77]. Phages can become both therapeutic alternatives and adjuvants to traditional antibiotics [34].

A considerable amount of preclinical data and a growing body of clinical data indicate the enormous therapeutic potential of bacteriophages in a wide range of infectious diseases [58]. However, the use of phage therapy can be complicated by the development of resistance to bacteriophages and the need to tailor phage cocktails for the specific bacterial strain causing the infection, which confronts clinicians with strict public health legislation [99].

One of the limitations of using phages as stand-alone antimicrobials is that bacteria develop resistance to phages as well with high frequency [39]. The use of a combination of phages can limit resistance, but like antibiotics, combinations need to be carefully selected [41].

Various methods of administration of bacteriophages have been investigated and data from clinical studies have been reported, including topical, inhalation, oral, and injectable methods of administration (intravenous, intramuscular, subcutaneous, and directly into the lesion). When phages are administered orally, recombination between phage genomes in the intestine is possible [11], but intravenous delivery is effective in almost all known cases [24, 98] (Table 1).

 

Table 1. Studies on the use of bacteriophages in vivo

Target microorganism	Study model	Agent, doses	Effectiveness
P. aeruginosa	Clinical case, man, 76 years old, chronic aortic graft infection	1 × 108 PFU of personalized phage cocktail + ceftazidime intravenously 2 times daily for 21 days [15]	Elimination of infection
P. aeruginosa	Clinical case, man, 26 years old, cystic fibrosis, bacterial lung infection	4 × 109 PFU of personalized phage cocktail intravenously every 8 hours daily for 8 weeks [51]	Elimination of infection
P. aeruginosa	Clinical case, man, 67 years old, bacterial infection of urinary tract	2 × 107 PFU of personalized phage cocktail + colistin and meropenem into the bladder every 12 hours daily for 10 days [45]	Elimination of infection
K. pneumoniae	Clinical case, man, 62 years old, knee prothesis infection	6.3 × 1010 PFU of monovalent bacteriophage intravenously daily for 40 days [13]	Elimination of infection
A. baumannii	Clinical case, man, 77 years old, hospital-acquired bacterial infection after craniectomy	8.5 × 107 PFU of monovalent bacteriophage suspended in Ringer solution with lactate through the central catheter every 2 hours 98 times [50]	Elimination of infection
S. aureus	Clinical case, woman, 35 years old, trophic leg ulcer	3.2 × 1010 PFU of the phage cocktail topically on the wound surface daily for 7 days [48, 83]	Treatment failure, purulent inflammation of the wound
K. pneumoniae	Clinical case, woman, 40 years old, cystic fibrosis, bacterial lung infection	2-phage cocktail with 2 × 108 PFU by inhalation and 1.8 × 109 PFU daily via nasogastric tube for 4 days [86]	Elimination of the pathogen in bronchoalveolar lavage but presence in feces
E. faecalis	Clinical case, 3 men, 52, 61 and 68 years old, chronic bacterial prostatitis	2 × 109 PFU of personalized phage cocktail rectally 2 times a day for 1 month [53]	Elimination of infection
E. coli	Clinical case, man, 66 years, chronic bacterial prostatitis	Intesti and Ses phage cocktails orally and rectally daily for 30 days [45]	Elimination of infection

 

Phage-antibiotic combinations are a promising therapeutic alternative, especially when limited antibiotic options are available. Combination therapy has achieved success in the treatment of infectious diseases such as endocarditis, bacteremia, osteomyelitis and peritonitis [26]. Reports [94] describing the effects of phage-antibiotic combinations often demonstrate enhanced phage activity in the presence of sub-inhibitory concentrations of antibiotics. This phenomenon was named [22] phage-antibiotic synergy (PAS), which is characterized by an increase in the number of phages released after phage cell lysis in the presence of sublethal doses of β-lactam antibiotics.

The effectiveness of combinations of antibiotics and lytic bacteriophages was first shown in 1941 by the example of the combined use of bacteriophages with sulfonamide drugs against S. aureus and Escherichia coli [49]. Later, the positive effects of joint exposure were demonstrated in animal models [26]. Similar results were obtained with penicillin [42]. The term “synergism” (“synergistic effect”) was introduced in 2007. An increase in the size of lysis zones of E. coli culture under the action of bacteriophage in the presence of subinhibitory concentrations of antibiotics (aztreonam, cefotaxime, ticarcillin, piperacillin, ampicillin, nalidixic acid, mitomycin C) was described [22]. Over time, the term “synergism” acquired a broader meaning. It began to be understood as cases when the efficacy of the phage and antibiotic combination as a whole significantly exceeds the sum of individual effects [27, 89]. In one of the studies, positive effects are subdivided into additive effect, synergism and facilitation, where under additive effect the authors understand the result when the combined use of two agents leads to cell growth suppression equal to the sum of the effects of each component separately, under synergism — exceeding the additive effect, and under facilitation — the effect when the combined action gives a more significant suppression of bacterial growth than the most effective agent when administered separately, but less in comparison with additive effect [89].

Combination therapy

Combination therapy is the use of several antibiotics in combination to target different mechanisms of bacterial resistance simultaneously. Combination therapy can be effective in the treatment of bacterial infections because it targets several aspects of the pathogen’s infectious potential simultaneously. Combination antimicrobial therapy has become an option for the treatment of infections caused by multidrug-resistant bacteria due to its broader coverage of susceptible microorganisms and synergistic effect [12]. However, with such therapy, there is a risk of increased toxicity and development of multidrug resistance [70].

Combination of antibiotics has been tested as a treatment method by a number of researchers because the probability of pathogen resistance development to a combination of two drugs is much less than to a single drug. The combination of drugs also extends the spectrum of action [100] in severe infections caused by multidrug-resistant pathogens [3]. Gram-positive members of ESKAPE, E. faecium and S. aureus, have been tested against a combination of fosfomycin and daptomycin, which successfully eliminated the infection [23, 91]. Most combinations tested against S. aureus in vitro include daptomycin or vancomycin with other antibiotics, including ceftaroline, an antibiotic recently approved for use. The effects of these and other similar combinations have also been tested in various mouse models that eliminated staphylococcal infection with minimal toxicity [60]. The efficacy of combination therapy has also been demonstrated with combinations with colistin. Colistin (polymyxin E) is an antibiotic of last resort prescribed against Gram-negative bacteria. In recent years, studies on the treatment of infections caused by K. pneumoniae and A. baumannii using the combination of colistin or tigecycline with other antibiotics in vitro and in cohort studies have been conducted and promising results have been shown [5, 110].

The original β-lactam-β-lactamase β-lactamase inhibitor (BL-BLI) combinations (i.e., amoxicillin-clavulanic acid, ampicillin-sulbactam, cefoperazone-sulbactam, piperacillin-tazobactam, and ticarcillin-clavulanic acid) were highly active against class A serine β-lactamases [30, 74]. K. pneumoniae resistance to them evolved with the emergence of four structurally and functionally different groups of β-lactamases: class B metallo-β-lactamases (MBL), class C serine β-lactamases AmpC, oxacillinases (OXA)-class D serine β-lactamases, and novel class A carbapenemases (KPC) [30, 74].

As a result, BL-BLIs with activity against all clinically important β-lactamases (e.g., KPC-2, OXA-23, OXA-24/40, AmpC, and New Delhi MBL-1 [NDM-1]) have become less effective, but new combinations such as cefepime-taniborbactam and cefepime-zidebactam are being developed that cover a broad spectrum of these enzymes and may fulfill this need [74, 108].

Diazabicyclooctanes (DBOs) are non-β-lactam synthetic inhibitors of β-lactamases [30]. Most studies show that DBOs inhibit class A and C β-lactamases, while minor activity against class D β-lactamases has also been observed [74]. In February 2015, avibactam became the first DBO drug approved by the FDA, the Food and Drug Administration, which is responsible for protecting and promoting public health through the control and supervision of food, drugs, and cosmetics. The activity of avibactam depends on the partner (e.g., ceftazidime, ceftaroline, aztreonam, cefepime, or imipenem), β-lactam-avibactam combinations are potentially highly effective against many ESKAPE pathogens, including Enterobacteriales and P. aeruginosa [72]. Replacing the β-lactam partner antibiotic with a clinically available β-lactamase inhibitor is another approach to treat infections caused by strains carrying multiple classes of β-lactamases, such as combining tazobactam with the novel cephalosporin ceftolozane [106, 109].

Nevertheless, the increasing resistance of microorganisms every year requires testing more and more new combinations of antibiotics, which leads to an endless search. Therefore, antibiotic combinations are a temporary solution to preserve the use of existing drugs while alternative strategies are being developed and tested.

Nanoparticles

Nanomedicine is one of the emerging areas for the elimination of antibiotic-resistant pathogens. Various nanomaterials with intrinsic antibacterial properties are being developed: metal-based nanoparticles (NPs) (e.g. silver, gold, copper and zinc oxide). They are widely used not only to enhance the efficacy of already existing antibiotics but also to reduce bacterial drug resistance [35, 61]. At the nanoscale, the physical and chemical properties of metals change dramatically compared to bulk material due to size and shape effects and the high surface area to volume ratio of nanomaterials [95]. That is, several properties must be considered at once: ion release, hardness, plasmon and superparamagnetism [20].

Nanoparticles affect the cell in several ways at once. Physical contact of bacteria with nanoparticles leads to membrane damage due to their adsorption and penetration into the cell [96]. Adsorption of nanoparticles causes depolarization of the cell wall, changing its negative charge and making it more permeable. As a result, the cell wall is destroyed and reactive oxygen species are formed [80] causing DNA denaturation [90]. The antibacterial activity of nanoparticles can also be due to leaching of ions. These ions can diffuse inside the cell and interact with the cell membrane and wall, as well as with cell macromolecules such as proteins and nucleic acids [17, 52]. High concentrations of reactive oxygen species produced inside or outside the cell due to nanoparticles, damage the cell membrane [82], put bacterial cells into oxidative stress, carry out lipid peroxidation, and destroy the cell wall by disrupting the structure of peptidoglycan [43, 44], degrade proteins and nucleic acids [33], leading to cell death.

One of the most common applications of nanoparticles in modern medicine is implantable devices. Implants must have biocompatibility, corrosion resistance, and antibacterial properties that nanoparticles can provide [56]. Nanoparticles are used to treat catheters, dental implants, and are used as antibacterial additives in dressings to treat skin wounds and burns. Both Gram-positive and Gram-negative pathogenic bacteria can cause chronic infections associated with skin wounds. For example, silver nanoparticles significantly inhibit bacterial growth and increase the rate of wound healing when used in combination with polyvinyl alcohol and chitosan [19, 36, 57].

In the field of new antibacterial agents, nanoparticles represent a promising alternative to antibiotics. Due to the combination of different effects on the bacterial cell, they have a wide range of antibacterial activity, affecting also drug-resistant microorganisms [40]. Nevertheless, toxicity to eukaryotic cells at high dosages of nanoparticles, as well as acceleration of horizontal transfer of resistance genes at low dosages, sublethal for bacteria, requires further study of this area and refinement of existing methods of nanoparticle application [92].

Antibiotic adjuvants

Antibiotic adjuvants are compounds that are used in combination with antibiotics to enhance their action against bacterial infections. Some molecules are combined with antibiotics to make an ineffective drug effective. These molecules, called “adjuvants” or “resistance disruptors”, have little or no intrinsic antimicrobial activity [38], but can inhibit mechanisms that confer resistance, making pathogens susceptible to the action of antibiotics [8]. Adjuvants can effectively enhance the action of existing antibiotics by reducing the minimum inhibitory concentration of antibiotic required to kill bacteria, allowing the use of existing therapies that may have been ineffective for a particular patient [66].

Several classes of adjuvants are known such as efflux pump inhibitors, β-lactamase inhibitors, quorum sensing inhibitors and adjuvants that disrupt bacterial cell wall synthesis and membrane permeability [73]. Also, depending on the intended purpose and the tasks performed, adjuvants can be categorized into 2 classes: class I antibiotic adjuvants act directly on the resistance mechanisms of bacterial cells to help antibiotics regain their efficacy, while class II adjuvants enhance the activity of the antibiotic in the host [107]. Class I includes active resistance inhibitors (β-lactamase inhibitors) [38], passive resistance inhibitors (efflux pump inhibitors [87], quorum sensing inhibitors [37, 47], biofilm inhibitors [37] and cell membrane permeability enhancers [81]. Class II includes antibiotic action enhancers (antimicrobial peptides that stimulate immunity) [4].

The strategy of using antibiotic adjuvants also has certain limitations, such as the labor-intensive and expensive identification of compounds and substances with the required physicochemical properties that can be used as adjuvants and administered together with antibiotics. In addition, it is necessary to evaluate the possibility of side effects when using certain adjuvants in each patient [18].

Antimicrobial peptides

Antimicrobial peptides are short, positively charged defense oligopeptides produced by all living organisms including protozoa, bacteria, archaea, fungi, plants and animals [103]. They show a broad spectrum of activity against a large number of bacterial pathogens. The ability of antimicrobial peptides to interact with the bacterial cell membrane and thereby induce cell lysis makes them a potential alternative for combating multidrug-resistant pathogens [7]. In addition, unlike antibiotics, antimicrobial peptides physically damage the bacterial cell through electrostatic interactions, thereby making it difficult for bacteria to develop resistance to them [76].

Histatin 5 is a natural cationic peptide of human saliva that is rich in histidine. This peptide shows strong antibiofilm and bactericidal activity against ESKAPE in vitro [31]. The cationic peptide WLBU-2 and the natural antimicrobial peptide LL-37 demonstrated 90% biofilm inhibition compared to tobramycin, ciprofloxacin, ceftazidime and vancomycin [54].

Similar to the positive in vitro results, antimicrobial peptides also show promising in vivo activity against ESKAPE group bacteria. For example, the peptide HLR1r, a structural derivative of the human milk protein, lactoferrin, at a very low concentration (5 mg/kg) was found to exhibit antimicrobial activity against an MRSA-infected rat wound excision model, as well as anti-inflammatory and anti-cytotoxic effects in vitro, suggesting the use of HLR1r in topical application formulations for the treatment of skin infections [9].

However, the paucity of antimicrobial peptides seeking clinical approval makes them an underpowered alternative to antibiotics for widespread use in healthcare settings. Despite their high in vitro and in vivo activity, antimicrobial peptides have yet to be clinically tested. Also, cytotoxicity for mammalian cells, tendency to degradation by tissue proteases, loss of activity at low salt concentrations or in the presence of plasma proteins, and higher production costs compared to other antimicrobial agents make it difficult to implement this type of therapy [59, 84].

Conclusion

Economic incentives for pharmaceutical companies and private and public sector collaboration can help filling the gap in antimicrobial drug development. Continuous epidemiologic surveillance and monitoring of antibiotic prescribing and consumption can delay the spread of antibiotic-resistant microorganisms. In addition, other potential ways to reduce the incidence of resistance are the use of antibiotic combinations or the development of alternative therapies [102].

However, despite the large number of studies conducted on the efficacy of alternative therapies for bacterial infections, each of them has been found to have drawbacks that hinder their adoption into routine use by clinicians. When combinations of antibiotics are used, the phenomenon of antagonism may occur [16]. The toxicity of such a drug increases [32, 70]. The use of adjuvants is complicated by labor costs in the search for new representatives and insufficient base of clinical use [101]. When phage therapy is used, resistance is also formed due to alteration of phage receptors of the host cell, and patient side effects are also possible [70]. Antibacterial peptides lose their activity at low salt concentrations or in the presence of plasma proteins and also have cytotoxicity [60, 84]. Nanoparticles are not used in routine clinical practice due to the lack of research base to verify toxicity, immunomodulatory response and pharmacokinetics conducted in vivo [69].

Therefore, document No. 2045-r (dated September 25, 2017) “Strategy for the Prevention of the Spread of Antimicrobial Resistance” was adopted at the state level, according to which one of the main directions of public health will be to study the mechanisms of antibiotic resistance, create alternative drugs for treatment, and inform the population about the rational use of antibiotics.

About the authors

L. S. Konkova

St. Petersburg Pasteur Institute

Email: lykraeva@yandex.ru

Junior Researcher, Laboratory of Medical Bacteriology

Россия, St. Petersburg

E. V. Rogacheva

St. Petersburg Pasteur Institute

Email: lykraeva@yandex.ru

PhD (Biology), Junior Researcher, Laboratory of Medical Bacteriology

Россия, St. Petersburg

L. A. Kraeva

St. Petersburg Pasteur Institute; Military Medical Academy named after S.M. Kirov

Author for correspondence.
Email: lykraeva@yandex.ru

DSc (Medicine), Associate Professor, Head of the Laboratory of Medical Bacteriology, Professor of the Department of Microbiology

Россия, St. Petersburg; St. Peterburg

References

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