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Review

Novel Antimicrobials, Drug Delivery Systems and Antivirulence Targets in the Pipeline—From Bench to Bedside

by
Oana Săndulescu
1,2,
Ioana Viziteu
1,
Anca Streinu-Cercel
1,2,
Victor Daniel Miron
1,3,*,
Liliana Lucia Preoțescu
1,2,
Narcis Chirca
1,
Simona Elena Albu
1,
Mihai Craiu
1,3 and
Adrian Streinu-Cercel
1,2
1
Faculty of Medicine, Carol Davila University of Medicine and Pharmacy, 050474 Bucharest, Romania
2
National Institute for Infectious Diseases “Prof. Dr. Matei Balș”, 021105 Bucharest, Romania
3
National Institute for Mother and Child Health “Alessandrescu-Rusescu”, 20382 Bucharest, Romania
*
Author to whom correspondence should be addressed.
Appl. Sci. 2022, 12(22), 11615; https://doi.org/10.3390/app122211615
Submission received: 19 September 2022 / Revised: 7 November 2022 / Accepted: 10 November 2022 / Published: 16 November 2022
(This article belongs to the Special Issue Antibacterial Strategies in Biomaterials)
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Abstract

:
In a fast-paced medical reality, biosciences and bioengineering have become essential components in medical research and development. The aim of this paper is to characterize the recent progresses made in fighting antimicrobial resistance, particularly in relation to WHO’s priority pathogens, by providing an in-depth review of novel antimicrobials, drug delivery systems for targeted antimicrobial action and novel antivirulence targets. We systematically searched the ClinicalTrials.gov database to identify clinical trials targeting WHO’s priority 1 (critical) pathogens: carbapenem-resistant Acinetobacter baumannii, carbapenem-resistant Pseudomonas aeruginosa, and carbapenem-resistant ESBL-producing Enterobacteriaceae. We identified a limited number of clinical trials, specifically for: one novel betalactamase inhibitor for Acinetobacter spp., one anti-virulence human monoclonal antibody for Pseudomonas spp. and no novel antimicrobials for carbapenem-resistant Enterobacteriaceae. We also performed a review of field literature to exemplify the main applications of drug delivery systems in infectious diseases, particularly in achieving targeted antibiotic distribution, in enhancing local activity with reduced off-target effects, triggered antibiotic release and triggered antibacterial photodynamic therapy. We conclude by presenting novel targets for antivirulence therapeutics that act by disrupting quorum sensing, inhibiting bacterial adherence and biofilm formation, silencing virulence traits and neutralizing bacterial toxins. Furthermore, the main principles of rational antimicrobial use are highlighted, in an effort to describe potential areas for targeted intervention, from diagnostic stewardship to antimicrobial stewardship.

1. Introduction

In a fast-paced medical reality, biosciences and bioengineering have now become essential components in medical research and development. The drug discovery process is complex; it starts with high-throughput screening, then moves into preclinical testing and, eventually, first in-human phase 1 clinical trials to establish safety in healthy volunteers. If no safety signals emerge at this point, phase 2 trials are subsequently performed to further confirm safety, to establish pharmacokinetics and to look at preliminary efficacy data in the actual target patient population. These are followed by larger, more inclusive phase 3 pivotal clinical trials, which should be adequately powered to establish efficacy against the main clinically relevant endpoints. Only based on an extensive review of all this preclinical and clinical data are regulatory agency approvals provided. Even after the final approval of a new drug, research continues into phase 4 post-marketing clinical trials and independent studies to ensure that the real-world safety and efficacy data match those from the pivotal clinical trials [1].
Antimicrobial resistance is part of the World Health Organization’s (WHO’s) list of 10 global public health threats that humanity is facing [2] and the rates and burden of antimicrobial resistance are increasing globally [3,4,5]. With the advent of multidrug resistance, the WHO has defined a set of priority pathogens for which drug development should be fast tracked [2,6]. These are classified based on priority into:
  • “Priority 1 (critical): carbapenem-resistant Acinetobacter baumannii, carbapenem-resistant Pseudomonas aeruginosa and carbapenem-resistant ESBL-producing Enterobacteriaceae
  • Priority 2 (high): vancomycin-resistant Enterococcus faecium, methicillin-resistant, vancomycin-intermediate and resistant Staphylococcus aureus, clarithromycin-resistant Helicobacter pylori, fluoroquinolone-resistant Campylobacter spp., fluoroquinolone-resistant Salmonellae, cephalosporin-resistant, fluoroquinolone-resistant Neisseria gonorrhoeae
  • Priority 3 (medium): penicillin-non-susceptible Streptococcus pneumoniae, ampicillin-resistant Haemophilus influenzae and fluoroquinolone-resistant Shigella spp.” [2]
The aim of this paper is to characterize the recent progress made in fighting antimicrobial resistance, particularly in relation to WHO’s priority pathogens, by providing an in-depth review of novel antimicrobials, novel materials with biomedical applications used for drug delivery systems for targeted antimicrobial action and novel targets.
To ascertain the development of novel antimicrobial agents, we performed three systematic searches of all clinical trials registered in the ClinicalTrials.gov database (https://clinicaltrials.gov/) in July 2022, targeting WHO’s priority 1 (critical) pathogens. The search applied the following search terms: “Acinetobacter”, “resistant Pseudomonas” or “carbapenem-resistant Enterobacteriaceae”. For inclusion in the literature review, we selected only (1) interventional trials of (2) novel antimicrobial agents and (3) ongoing or completed within the past 5 years. Exclusion criteria were: (1) trials that were terminated early and did not list any results and (2) trials for novel applications of already approved antimicrobials.

2. Results of Systematic Search for Clinical Trials

The search for “Acinetobacter” on clinicaltrials.gov yielded 41 records, of which only 9 were interventional trials, 6 for ETX2514, which is a broad-spectrum diazabicyclooctenone betalactamase inhibitor [7], 2 for phage therapy and 1 for polymyxin B, administered alone or combined with imipenem (Table 1).
A search for “resistant Pseudomonas” on clinicaltrials.gov yielded 15 records, of which only 2 were interventional, both for MEDI3902, an anti-virulence human monoclonal antibody targeting anti-P. aeruginosa PcrV and Psl (a phase 1 [8] and a phase 2 trial).
The search for “carbapenem-resistant Enterobacteriaceae” (CRE) yielded 40 results, of which only 16 were of interventional trials. Of these, 4 had been withdrawn/terminated/suspended and 12 were ongoing or had been completed within the past 5 years. No novel antimicrobial agent was identified for CRE based on this search, as the interventional medicinal product was fecal microbiota in 8 instances, probiotics in 2 instances and antimicrobial agents that had since received approval in 2 other instances (meropenem plus vaborbactam and imipenem/cilastatin plus relebactam).
We thus identified a major gap in clinical trials for agents targeting WHO’s critical pathogens, with no novel antimicrobial classes having reached clinical testing. The main research area currently in clinical development appears to be that of novel betalactamase inhibitors, which, if successful, could restore the antimicrobial activity of selected betalactam drugs. However, betalactamase production is only one of many bacterial resistance mechanisms and a multifaceted approach is needed to comprehensively block more of the potential resistance pathways.
This highlights the fact that there is an important need for the further development of novel antimicrobials, alternative drug delivery systems to increase efficacy of existing antimicrobials or antivirulence therapeutics.

3. Antimicrobial Resistance—Mechanisms and Mitigation Strategies

Shortly after the discovery of the first antibiotics, bacterial resistance was also documented. This led to a race in drug discovery, paralleled or surpassed by a bacterial race in development of resistance.
Mechanisms of antimicrobial resistance are varied in nature, being either intrinsic or acquired, and multiple mechanisms can be present in the same bacterial species at the same time. Different classifications have been proposed for mechanisms of antimicrobial resistance, but the most common one includes [9]: limiting drug uptake, target modification, drug inactivation and active drug efflux (Figure 1).

3.1. Limiting Drug Uptake

Depending on where in the bacterial cell the antimicrobial exerts its effect, for most antibiotics, the drug needs to be taken up by the bacteria as an initial step. However, this process can be hindered intrinsically, as is the case with certain Gram-negative lipidic cell membranes that constitutively do not allow the diffusion of hydrophilic drugs [10] or by acquiring a thickened cell wall in Gram-positive bacteria [9].
For drugs that are not chemically capable of diffusing through the bacterial outer membrane, small porin channels are required for drug uptake and this is especially true for betalactam antibiotics or fluoroquinolones. Mechanisms of porin-induced resistance seen in Gram-negative bacteria include a down-regulation in the number of porins expressed on the outer membrane as well as chemical or conformational changes in the porin structure that render it incompatible with binding and/or transport of the antimicrobial drug [10].

3.2. Target Modification

Another important mechanism of bacterial resistance prevents the antimicrobial from binding to its target and this is dependent on where in the cell or when during the cell life cycle this target is positioned. For example, for cell-wall-acting agents, Gram-positive germs change their penicillin-binding proteins and, therefore, betalactams no longer recognize the target where they were supposed to bind.
For drugs that act by inhibiting protein synthesis, i.e., aminoglycosides, mutations can occur in the 30S ribosomal binding site used by the drug [11], while for macrolides, streptogramin and lincosamides, which normally bind to the 50S ribosomal site, methylation of the 23S ribosomal RNA subunit by methyltransferases leads to resistance [12].
Antimicrobials that act by inhibiting nucleic acid synthesis can also be affected by target modification, e.g., alteration in the alpha subunit of DNA gyrase (gyrA gene), mediating quinolone resistance [11]. For drugs such as trimethoprim, which has antimetabolite activity and acts by inhibiting dihydrofolate reductase, thus preventing synthesis of folic acid, resistance can occur through decreased affinity of dihydrofolate reductase [13].

3.3. Drug Inactivation

Bacterial enzymes can induce the inactivation of certain antimicrobials. Classical examples include betalactamases that hydrolyze betalactam drugs, aminoglycoside modifying enzymes, such as acetyltransferases, adenyltransferases and phosphotransferases, that can induce resistance to many drugs in the aminoglycosides class [11] but may also inactivate certain fluoroquinolones, as is the case with acetyl-CoA-dependent acetyltransferases, such as AAC(6′)-Ib, which also targets ciprofloxacin or norfloxacin, but spares levofloxacin, which does not have a nitrogen available for acylation [14]. Furthermore, enzyme-catalyzed modification has also been reported for lincosamides through adenylylation [14].

3.4. Active Drug Efflux

Once the antimicrobial has entered the bacterial cell and reached the periplasm or cytoplasm, the drug can be actively pumped outside of the bacterial cell through efflux pumps, which are embedded in the bacterial plasma membrane [15]. These efflux pumps are responsible for much of the intrinsic or constitutive resistance of Gram-negative bacilli to certain antimicrobials but can also be seen in Gram-positive germs and can be determinants of acquired drug resistance. Specifically, efflux pumps can induce high-level resistance to tetracycline [15], resistance to fluoroquinolones, macrolides, streptogramin and lincosamides, glycylcyclines, aminoglycosides, oxazolidinones, metronidazole and betalactams [16].
Efflux pumps can be drug-specific or multidrug pumps. The former are encoded on mobile genetic elements, while the latter, which can transport several different molecules out of the bacterial cell, are mainly encoded on chromosomes.

3.5. Strategies for Mitigating the Occurrence of Antimicrobial Resistance

As briefly described above, antimicrobial resistance mechanisms are varied in nature and can occur at any step along the drug’s action pathway, from accessing its target to performing its chemical functions. As part of the natural law of “survival of the fittest”, the biological pressure posed by the administration of antimicrobial drugs leads to selection of resistant microbial variants. By finding therapeutic approaches for silencing microbial virulence without harming the bacteria per se, the selection of resistant variants could theoretically be averted. Furthermore, by applying drug delivery systems, a more targeted effect of existing antimicrobials could be obtained directly at the site of infection, while avoiding off-site effects and the induction of resistance pressure in the remaining microbiome. These approaches are described below.

4. Drug Delivery Systems—Applications in Infectious Diseases

Drug delivery systems (DDSs) can be applied to enhance the different pharmacokinetic properties of antimicrobial agents, from absorption to distribution, metabolism and excretion. Direct applications of DDS include liposomal formulations that increase effectiveness while decreasing toxicity, as is the case of liposomal amphotericin B used in the treatment of severe fungal infections [17]. A DDS also allows for the conditioning of antimicrobials for extended release, ensuring optimal drug levels over a prolonged length of time, which is essential for antibiotics with time-dependent killing patterns, such as beta-lactams. Furthermore, the release of antimicrobials can be specifically targeted to the site of infection or triggered only in the presence of the pathogen or of particular environmental conditions or external stimuli, thus enhancing local activity with reduced off-target effects.

4.1. Targeted Antibiotic Release

While certain infections are hard to treat due to direct antimicrobial resistance of the pathogenic agent, others pose therapeutical concern because the antimicrobial to which the pathogen is otherwise susceptible is not able to reach the infectious site. To mitigate this issue, a DDS applying nanoparticle formulations can use targeting ligands to guide the drug in reaching and concentrating in specific infectious foci, ensuring the drug’s good local efficacy while avoiding off-target toxic effects, such as toxicity or dysmicrobism. Furthermore, nanocarriers can be used to ensure that higher drug concentrations are reached in infectious sites, thus repotentiating drugs and restoring activity of antibiotics for which the pathogenic agent displays increased minimum inhibitory concentrations (MICs). Such examples include vancomycin-nanoparticle conjugates, which show enhanced target avidity, leading to membrane permeabilization and 13-fold to >100-fold improvement in MIC for vancomycin-resistant S. aureus, E. faecium and E. faecalis strains [18].

4.2. Sustained Local Release for Bone Applications

DDSs can also facilitate the local targeted use of agents that would otherwise have high toxicity if systemically administered. For example, tobramycin-loaded poly(DL-lactic-co-glycolicacid) microspheres have been tested in in vivo models for potential future application as depot systems in the treatment of osteomyelitis, showing a sustained linear release of the antibiotic over the course of 4 weeks [19]. An injectable chitosan-based thermosensitive hydrogel engineered to include vancomycin-loaded nanoparticles showed promising results in a rabbit osteomyelitis model, with sustained antibiotic release for 26 days, confirmed antimicrobial activity and stimulation of osteoblast proliferation [20].
Polymeric microspheres entrapping other aminoglycosides, such as streptomycin, have also been proposed as coatings on implants [21], allowing for the slow release of the antimicrobial for sustained durations following the surgical insertion of the implant, with the aim of reducing biofilm formation on the newly implanted foreign body. Antibiotic-loaded poly(methyl methacrylate)-based bone cement, functionalized with hollow nanostructured titanium-dioxide nanotubes, allows for better local release of gentamicin or vancomycin, with 50% released over the course of two months, compared to only a 5% release achieved in the absence of tubule functionalization [22].

4.3. Stimuli-Responsive DDS for Triggered Antibiotic Release

Coatings with “smart” materials can ensure that the active drug is released only in tissues displaying particular endogenous environmental conditions (based on pH or temperature), specific microbial recognition patterns or conditioning by an exogenous stimulus (such as light or therapeutic ultrasound).

4.3.1. pH-Sensitive DDS

One of the most important cues in the intracellular and extracellular milieu is the pH. Examples of DDS responsive to pH changes include rifampin-based macromolecules with poly(acrylic acid) side chains that present a coiled conformation, limiting the antibiotic release, under acidic conditions, and a stretched conformation, potentiating the antibiotic release, under alkaline pH conditions [23].

4.3.2. Pathogen-Sensitive DDS

An application of an infection-responsive antibacterial agent is the coating of medical implants with vancomycin conjugated to an S. aureus-sensitive peptide sequence. In the absence of S. aureus, the antibiotic is not released, whereas in the presence of S. aureus in the area surrounding the implant, this sensor peptide will be recognized and then cleaved by a staphylococcal enzyme, leading to the release of the antibiotic only in the presence of the pathogen [24].
Daptomycin-loaded polydopamine-coated gold nanocages conjugated to antibodies against S. aureus lipoproteins have been shown to be effective in in vivo MRSA biofilm models. A similar type of gentamicin-loaded nanocages were effective against P. aeruginosa biofilms when conjugated to an antibody against a P. aeruginosa outer membrane protein. However, the same nanocage formulation was not efficient when loaded with ceftaroline or vancomycin and conjugated to anti-Spa antibodies against S. aureus [25].

4.4. Ultrasound- and Light-Sensitive DDS

A combination of prolonged antibiotic elution with on-demand release of ceftriaxone when triggered with therapeutic ultrasounds was demonstrated for “smart” polylactic acid films with ceftriaxone-containing microchamber arrays [26]. Thiol chitosan-wrapped gold nanoshells, triggered by near-infrared laser, have been tested as photothermal antibacterial agents for Gram-positive (S. aureus) and Gram-negative (P. aeruginosa and E. coli) antibiotic-resistant pathogens [27]. A photodynamic nano-assembly of chlorin e6 covalently conjugated with chitosan exhibited a bactericidal action against methicillin-resistant S. aureus (MRSA) and Acinetobacter baumannii, with therapeutic efficacy comparable to that of vancomycin in MRSA infection murine models [28]. Gold nanoshells functionalized with carboxylate-terminated organosulfur ligands have been shown to be effective in killing E. faecalis on silicone-based catheters [29].
Furthermore, photothermal destruction of bacteria has also been tested in combination with triggered antibiotic release. Specifically, an injectable hydrogel-based drug reservoir containing ciprofloxacin-loaded photothermal polydopamine nanoparticles mixed with glycol chitosan was tested in a mouse skin defect S. aureus infection model. The hydrogel combines the trapping of bacteria on its surface and the release of the antibiotic while also inducing local hyperthermia when triggered with near-infrared light irradiation [30].

4.5. Enhanced Antimicrobial Activity

Teicoplanin-loaded chitosan–polyethylene oxide nanofibers allow for sustained release of teicoplanin over an interval of 12 days, leading to up to a two-fold enhancement in antibacterial activity, also confirmed through better wound closure in an in vivo rat full-thickness wound model [31].
Tetracycline-loaded mesoporous silica nanospheres have displayed bactericidal efficacy against otherwise resistant strains of E. coli [32]. Tetracycline-conjugated carbon nanoparticles have been shown to inhibit bacterial efflux pumps, leading to 10-fold higher antibacterial activity compared to the free form of the antibiotic, when tested against tetracycline-resistant K. pneumoniae [33].

4.6. “Trojan Horse” Strategies in DDS for Infectious Diseases

Siderophore-dependent iron uptake pathways have been explored as potential “Trojan horse” enhancers of antibiotic bacterial cell penetration, particularly in Gram-negative pathogens [34]. As such, the natural siderophore enterobactin has been shown to transport repurposed antimicrobial molecules into E. coli isolates [35] and two synthetic siderophore mimetics, 1,3,5-N,N′,N″-tris-(2,3-dihydroxybenzoyl)-triaminomethylbenzene (MECAM) and 1,4,7,10-tetrakis(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane (DOTAM), have the ability to transport antimicrobials into Pseudomonas aeruginosa isolates [34]. Other cargo-delivery systems, such as cell-penetrating peptides (CPPs), are also being studied for their ability to transport antimicrobials across cell membranes [36].
Another potential application of “Trojan horse” strategies for DDSs in infectious diseases consists of the conjugation of antimicrobials or antivirulence therapeutics to antibacterial antibodies. This technique has shown promise in the selective and guided delivery of antimicrobials and particular examples would be the delivery of polymyxin B to Klebsiella aerogenes strains [37] or of the G2637 antibiotic to P. aeruginosa by antibody-antibiotic conjugates [38].
Naturally occurring stabilized antimicrobial peptides, such as histatins, defensins or cathelicidins, as well as synthetic antimicrobial peptides, can display antibacterial or antifungal properties [39], as well anti-biofilm action and local tissue immunomodulatory and regenerative activity [40] and can be delivered through microparticles or nanoparticles as part of DDS strategies [39,40]. While showing potential promise for future research, naturally occurring peptides display relatively poor metabolic stability and a short half-life, requiring stabilization in order to allow them to evade the cell enzymatic degradation pathways [41].

4.7. Bench-to-Bedside and Technology Readiness in DDS for Infectious Diseases

Tremendous progress has been made in the field of DDS in general, and pre-clinical research is developing at a faster pace than ever. However, few applications of DDS for the treatment of infectious diseases have so far reached clinical practice. One classical example is liposomal amphotericin B [17], which is now the main formulation of this antifungal drug that is being used for systemic administration as induction therapy for cryptococcosis or histoplasmosis. Other current applications of DDS include prevention of post-operative prosthesis infection by using premixed antibiotic bone cements [42], antimicrobial wound dressings based on metals, such as silver or bismuth, based on polymers, such as polyhexamethylene biguanide, quaternary ammonium compounds, oxidizing agents, chlorhexidine or locally acting antimicrobials (for example, bacitracin) [43].

5. Novel Targets—Antivirulence Therapeutics

An entire new field of therapeutics is currently being developed, with the aim of silencing bacterial virulence without killing the pathogen, thus avoiding the induction of antimicrobial resistance. The use of common bacteriostatic or bactericidal drugs elicits an important survival pressure, particularly among residual bacteria from the existing flora, which have been exposed to varying therapeutic or sub-therapeutic antibiotic concentrations. Theoretically, by silencing the pathogens without harming them, this resistance pressure could be diminished.
Antivirulence therapeutics can act by disrupting bacterial communication pathways, i.e., quorum sensing, by inhibiting bacterial adherence or by directly silencing particular virulence genes or traits. As extensive reviews on the topic have already been performed [44,45,46], we will only concentrate below on one or two examples of therapeutics blocking each major virulence mechanism for clinically relevant Gram-positive and Gram-negative bacteria.

5.1. Disrupting Quorum Sensing

Quorum sensing is the mechanism of chemically mediated inter-bacterial communication. This communication leads to downstream signaling and gene regulation, as a direct response to signals regarding bacterial cell population density. Thus, quorum sensing can modulate the transition from the pro-adhesive status involved in initial colonization to the activation of virulence genes required to induce a clinically significant infection, once a certain critical bacterial population density has been reached.
In Gram-positive pathogens, such as S. aureus, a series of quorum sensing regulators have been described, among which perhaps the best characterized is the accessory gene regulator (agr) system. Specific inhibitors of different types of Agr proteins have been shown to inhibit quorum sensing and decrease virulence in S. aureus. For example, the repurposed drug bumetanide specifically binds to AgrA and decreases the expression of virulence genes, including alpha-hemolysin, phenol-soluble modulins and Panton-Valentine leukocidin in vitro, with confirmed in vivo efficacy from a murine dermonecrosis infection model, where ulcer development was significantly reduced [47].
Ajoene, a naturally occurring sulfur-rich molecule, has been described to inhibit small regulatory RNAs (sRNA) in both S. aureus and P. aeruginosa, thus interfering with quorum sensing and downregulating the expression of virulence genes coding for hemolysins and proteases in S. aureus and the rsmY and rsmZ genes in P. aeruginosa, which are responsible for the synthesis of virulence factors, polysaccharides and motility factors. Thus, ajoene has been described as a broad-spectrum quorum sensing inhibitor [48].

5.2. Inhibiting Bacterial Adherence

Adherence is the first and most important step in establishing bacterial colonization of host tissues. Only after bacteria have successfully adhered to tissues do they start to replicate and then activate their virulence traits or their biofilm-forming ability. By blocking adhesion, infections can be prevented or treated and, therefore, anti-adhesion therapeutics have been intensely studied.
N-ethyl maleimide and its analogs were tested together with protamine sulfate to ascertain their inhibiting activity on the bifunctional enzyme N-acetylglucosamine-1-phosphate uridyltransferase (GlmU), thus, preventing bacterial adhesion of Gram-positive germs, such as S. aureus or Enterococcus spp., by interfering with peptidoglycan and lipopolysaccharide biosynthesis [49].
For Gram-negative germs, an example is the application of high-affinity mannoside antagonists of type 1 pilus adhesin FimH against uropathogenic E. coli strains, with promising results in treating urinary tract infections by blocking the binding of the FimH adhesin to the mannose on the bladder’s epithelium and by reducing the intestinal reservoir of uropathogenic E. coli [50].

5.3. Inhibiting Biofilm Formation

While the inhibition of adhesion can also lead to inhibition of biofilm formation, there are several steps in biofilm formation that can be specifically targeted by anti-biofilm agents. For example, the small-molecule SYG-180-2-2 decreases bacterial adhesion and polysaccharide intercellular adhesin production while downregulating the expression of biofilm-related genes, such as icaA, in clinical MRSA isolates [51]. Furthermore, inhibition of MRSA biofilm has been demonstrated for the natural compound punicalagin [52] and for the naturally occurring flavonol glycoside kaempferitrin [53]. The latter has also been studied for delivery by silver or copper nanoparticles and in a zebrafish infection model [53]. For Gram-negative bacteria, such as P. aeruginosa, biofilm formation can be blocked by 6-methylcoumarin, which has been studied for potential application on solid surface coatings together with polyurethane [54].

5.4. Silencing Virulence Traits

Certain molecules can serve as multi-purpose agents, blocking multiple virulence pathways at once. For example, punicalagin, described above for its anti-biofilm action on MRSA, can also inhibit sortase A (SrtA) as an antivirulence therapeutic [52], while ClpP peptidase and ClpXP serine protease can be inhibited by tailored phenyl esters [55]. Small molecules that lack antimicrobial activity, such as SYG-180-2-2, described above for its anti-biofilm activity, can also inhibit the production of important virulence factors, such as hemolysin and staphyloxanthin, and reduce the formation of skin abscesses in murine models [56].
In Gram-negative bacteria, 6-methylcoumarin, also described for its anti-biofilm properties, also inhibits a set of virulence factors specific to P. aeruginosa (e.g., pyocyanin, siderophore, exopolysaccharide, elastase, proteases) and its combined antivirulence action has been demonstrated in a Caenorhabditis elegans infection model [54]. Another anti-virulence therapeutic, RESP-X, is a novel humanized monoclonal antibody that silences virulence in P. aeruginosa and which has recently been approved to proceed to a phase 1 first in-human clinical trial.

5.5. Inhibiting Betalactamases

Inhibition of betalactamases is an important mechanism for restoring activity of existing betalactam drugs against otherwise resistant organisms. Examples of novel betalactamase inhibitors include the orally bioavailable β-lactamase inhibitor ledaborbactam, which restores ceftibuten activity against Enterobacterales producing Ambler class A-, C- and D betalactamases in murine models [57]. For Ambler class B-producing Enterobacterales, a series of metallo-betalactamase (MBL) inhibitors are currently in preclinical development, showing potential in restoring carbapenem efficacy, with the most active MBL inhibitors decreasing the MIC of meropenem by up to 128-fold in vitro in clinical isolates [58].

5.6. Neutralization of Toxins—Bench-to-Bedside in Antivirulence Therapeutics

Rabbit-derived anti-staphylococcus hyperimmune products have been tested for their neutralization activity against staphylococcal toxins such as IBT-V02, leukotoxins such as HlgAB, HlgCB and LukED, and superantigens such as SEC1, SED, SEK and SEQ, with in vivo confirmation of efficacy in murine models of bacteremia and pneumonia [59]. Human monoclonal antibodies such as suvratoxumab target S aureus alpha-hemolysin and have shown promising results in pre-clinical research, but still need further clinical testing, as suvratoxumab has confirmed its safety profile in clinical trials, but it failed to prevent pneumonia in mechanically ventilated patients colonized with S. aureus in the lower respiratory tract based on preliminary data [60].
Antibodies have also been tested for neutralization of Clostridioides difficile toxins A and/or B, with good results for bezlotoxumab, which significantly reduced the rate of recurrence of Clostridioides difficile-associated diarrhea in phase 3 clinical trials [61], leading to its approval by the US Food and Drug Administration in 2016 and by the European Medicines Agency in 2017.
The topic of toxin neutralization has been an intense focus of study over the past 4 decades, with articles dating back to 1976 describing the neutralizing activity of convalescent sera against P. aeruginosa exotoxin [62]. Recent studies have also looked at single-chain variable fragment antibodies against the P. aeruginosa exotoxin A domain I [63] or human single-chain antibodies to neutralize the elastolytic activity of the LasB elastase/pseudolysin [64]. However, despite many years of intense study and the advent of novel technologies that can now easily identify and purify monoclonal neutralizing antibodies, research is still far from reaching the patient’s bedside, with only one anti-virulence human monoclonal antibody targeting the P. aeruginosa V-antigen (PcrV) and the exopolysaccharide Psl, MEDI3902 [8], having reached phase 2 clinical trials.

5.7. Theragnostics in Infectious Diseases

Theragnosis represents a developing field in precision medicine, which combines molecular techniques for diagnosis and targeted therapy. It has important applications in oncology, where specific cancer cells can be recognized and targeted based on their unique molecular prints. Emerging data also show that tissues experiencing an infectious process can also be recognized based on their expression of particular cytokine and cell-mediator profiles and bacterial pathogens also exhibit specific recognition patterns, thus allowing the advancement of theragnostic options in this clinical area.
Short DNA oligonucleotides such as aptamers have been explored for their high-affinity target binding and a specific example is that of Porphyromonas gingivalis-specific DNA aptamers, used to functionalize nanographene oxide and tested in vitro as potentiators for antimicrobial photodynamic therapy (aPDT). Sustained release over a period of 240 h was confirmed, along with a reduction in 48-h mature biofilm and in the metabolic activity of P. gingivalis, without induction of apoptosis in normal human gingival fibroblast cells, and these results are particularly relevant for the field of biofilm-mediated chronic inflammatory diseases such as periodontitis [65].
In vitro data are also available for aptamer-functionalized emodin nanoparticles loaded with the dermcidin-derived peptide DCD-1L, which, in combination with blue laser light, have demonstrated anti-biofilm activity against Enterococcus faecalis, with potential application in the treatment of root canal treatment failures [66].
Linear peptides or peptide aptamers against anti-ferric uptake regulators have also been explored for their potential in disrupting the regulation of genes involved in iron homeostasis, virulence traits and oxidative stress in vitro and in vivo in a fly E. coli infection model [67].
While these approaches are still far from reaching clinical practice, there is promise in the development of precision medicine in the field of infectious or infectious-mediated diseases.

6. Rational Antimicrobial Use Practices

Despite small but incremental advances in research regarding novel antimicrobials, drug delivery systems and novel antivirulence targets, antimicrobial resistance cannot be mitigated without consistent application of rational use practices. This refers, on the one hand, to diagnostic and antimicrobial stewardship (AMS) as well as rational prescribing practices among all medical specialties and across all care levels, for inpatients as well as outpatients [68,69], but also to rational use, particularly patient-driven antimicrobial use in settings where antibiotics can be accessed either without a prescription or by requesting a prescription, phenomenon described as “pressure to prescribe” [70]. Thus, education regarding appropriate and inappropriate antimicrobial use and judicious differentiation of viral from bacterial infections [71] are of utmost importance and should not only be provided for clinicians but also for the general population [72].
Numerous opportunities exist for targeted intervention to reduce avoidable antibiotic use. We illustrate such opportunities for intervention throughout the continuum of care, at patient level, pharmacy level and healthcare level in Figure 2. Specifically, when a patient presents fever, he or she can go to the pharmacy, to the general practitioner or to the hospital. In each of these settings, a differential diagnosis will be considered, whether the fever is due to a viral or a bacterial infection and the resulting decision will lead to an appropriate or an inappropriate prescription (Figure 2—upper panel). At each of these steps, there is room for improvement (Figure 2—lower panel) and by integrating patient education, peer education and implementation of AMS and diagnostic stewardship, appropriate prescribing can be facilitated. These different interventions are described in more detail below.

6.1. Antimicrobial Stewardship

Antimicrobial stewardship (AMS) programs promote and supervise rational antimicrobial use within healthcare institutions and can include different core elements, depending on the setting [73]. For example, the focus of the AMS program can shift slightly, for hospitals managing inpatients [73,74], for outpatient departments [73,75] or for long-term nursing facilities [73,76]. According to the US Centers for Disease Control and Prevention, core components of hospital AMS programs should include: leadership commitment to dedicate the required resources, accountability by appointing one leader of the AMS team, pharmacy expertise by appointing a clinical pharmacist as co-leader, action through implementing interventions to control antimicrobial use, tracking and surveillance of antimicrobial prescribing practices, reporting surveillance data and AMS program outputs, as well as education of healthcare staff at all levels on optimal prescribing practices [77].
Different toolkits have been designed and are readily available, to aid in implementing AMS programs throughout healthcare facilities [78] and bedside tools can be used to predict the risk of Gram-negative drug-resistant infection in hospitalized patients [79]. Furthermore, clinical decision support systems can facilitate therapeutic choices, by discriminating between cases that might indeed require antibiotic treatment and those that would not, and by allowing the choice of the most appropriate agent based on patient risk profiles [80,81].

6.2. Diagnostic Stewardship

Diagnostic stewardship is a relatively recent concept, compared to AMS, but it is gaining more and more consideration with the advent of novel diagnostic techniques. It has become extremely relevant with the increased use of multiplex polymerase chain reaction (PCR) tests. When ordering a test from the laboratory, one should always consider what added benefit that particular information will bring. Ideally, by confirming the viral etiology of an infection, unnecessary antibiotic use could be averted. However, positive findings for bacterial agents in multiplex PCR panels should be treated with caution, and a comprehensive case analysis should assess whether that particular bacterial organism is, indeed, likely to be the causative pathogen in the clinical infectious syndrome, or rather a simple bystander that only colonized the site of sample collection, for example, the nasopharynx [82].

6.3. Education on Rational Antimicrobial Use

As mentioned above, “pressure to prescribe” is the phenomenon where patients or their relatives either directly request an antibiotic prescription from their healthcare provider or indirectly try to influence the provider’s decision on whether or not to prescribe an antibiotic, at times through an exaggeration of signs and symptoms or by leading the medical conversation towards the point where an antibiotic is otherwise prescribed [70,82]. This phenomenon has been described in almost all types of medical settings, from the general practitioner’s office to tertiary care hospitals, to community pharmacies and it is extremely common in pediatric practice, where the parents’ perception regarding the severity of their child’s illness can potentially influence therapeutic decisions [82,83]. To mitigate this pressure, it is extremely important to deploy at least two types of educational interventions in the general community at large. One is response driven, i.e., when being asked to prescribe an antibiotic for what is most likely a viral infection, the practitioner can reassure the patient and offer information regarding the correct use of antimicrobials [82,84]. The other is preemptive education of the general population, either in the healthcare sector [85] or through outreach campaigns. Both are equally important and patients and their relatives should be regarded as partners in shared medical decisions and, particularly, for this reason, they should have access to the best available knowledge, translated into non-medical language that would be more easily understandable to them.

6.4. International Standards on Rational Antimicrobial Use

Rational antimicrobial use is a very important guiding principle for antimicrobial stewardship. However, multiple definitions of “rational use” coexist and are implemented in different settings. For example, as mentioned above, the US CDC has proposed a clear set of core elements to be included in hospital AMS programs [78]. This was based on an assessment of appropriateness of antimicrobial use in US hospitals from 2017 to 2022, which showed that 55.9% of antibiotic hospital-based prescriptions were not in line with recommended prescribing practices; particularly worrisome was the fact that 50.1% of patients being prescribed antibiotics for suspected urinary tract infection did not in fact present any signs or symptoms of infection [86].
The model proposed by the UK of performing regular clinical audits of prescribing practices should further be implemented in cross-border settings, to ensure that the best quality of healthcare is consistently being delivered. This approach does, however, come with its own caveats, as it can only be done in settings where clinical guidelines exist and are consistently implemented.
Meanwhile, in the EU, the European Center for Disease Control and Prevention proposed, in 2017, a set of EU guidelines for the prudent use of antimicrobials [87,88]. As important as these rules and regulations are, their effectiveness and impact on healthcare-related outcomes still need to be assessed in order to better inform implementation actions.

7. Conclusions

In conclusion, antimicrobial resistance is a very important phenomenon requiring complex targeted interventions to limit the resistance pressure while also promoting the development process for novel antimicrobial targets across all levels of in vitro and in vivo research and throughout the clinical development phases leading up to introduction in clinical practice. Furthermore, multiple opportunities for intervention exist, to facilitate appropriate antimicrobial prescribing practices throughout the continuum of care.

Author Contributions

O.S., I.V., A.S.-C. (Anca Streinu-Cercel), V.D.M., L.L.P., N.C., S.E.A., M.C., and A.S.-C. (Adrian Streinu-Cercel) contributed equally to the article, in: conceptualization, methodology, formal analysis, data curation, writing—original draft preparation, writing—review and editing, visualization, supervision. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable.

Data Availability Statement

All data supporting this review are publicly available and can be retrieved from repositories and platforms such as ClinicalTrials.gov or PubMed.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Main mechanisms of antimicrobial resistance (created with BioRender.com): 1. limiting drug uptake (e.g., thickened cell wall, porin mutation, porin downregulation.); 2. target modification (e.g., penicillin-binding protein modification in the bacterial membrane, target modification during nucleic acid synthesis or ribosomal methylation); 3. drug inactivation (e.g., by betalactamases); 4. drug efflux through active pumps.
Figure 1. Main mechanisms of antimicrobial resistance (created with BioRender.com): 1. limiting drug uptake (e.g., thickened cell wall, porin mutation, porin downregulation.); 2. target modification (e.g., penicillin-binding protein modification in the bacterial membrane, target modification during nucleic acid synthesis or ribosomal methylation); 3. drug inactivation (e.g., by betalactamases); 4. drug efflux through active pumps.
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Figure 2. Opportunities for intervention to ensure appropriate antimicrobial prescribing practices throughout the continuum of care (created with BioRender.com). AMS = antimicrobial stewardship; CDSS = clinical decision support system.
Figure 2. Opportunities for intervention to ensure appropriate antimicrobial prescribing practices throughout the continuum of care (created with BioRender.com). AMS = antimicrobial stewardship; CDSS = clinical decision support system.
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Table
Table 1. Novel antimicrobials currently or recently evaluated in clinical trials.
Table 1. Novel antimicrobials currently or recently evaluated in clinical trials.
Site of Infection or Study PopulationNovel AgentPhase of the
Clinical Trial		Expected
Completion of the Clinical Trial (Year)		ClinicalTrials.gov Reference
Indication: carbapenem-resistant Acinetobacter baumannii
Healthy volunteersETX2514 and 14C-ETX2514Phase 1 (excretion and metabolism)2019 (completed)NCT04018950
Healthy volunteersETX2514 vs. placebo vs. moxifloxacinPhase 1 (cardiac repolarization)2019 (completed)NCT03985410
Volunteers with and without renal impairmentETX2514 plus sulbactamPhase 1 (renal safety)2018 (completed)NCT03310463
Healthy volunteersETX2514 vs. ETX2514 plus sulbactam vs. ETX2514 plus sulbactam plus imipenem/cilastatinPhase 1 (safety, tolerability and PK)2017 (completed)NCT02971423
Healthy volunteersETX2514 plus sulbactam1 (plasma, epithelial lining fluid and alveolar macrophage concentrations)2017 (completed)NCT03303924
HABP, VABP, bacteremiaETX2514/sulbactam + imipenem/cilastin
vs.
colistin + imipenem/cilastin		Phase 32021 (completed)NCT03894046
Pneumonia or bacteremia/septicemiaPhage treatmentExpanded access study2020-not specifiedNCT04636554
Diabetic foot ulcersLocal bacteriophage therapy TP-102(Local study: Israel)2021-recruitingNCT04803708
Critical illness with multi-drug resistant pathogensPolymyxin B vs. polymyxin B plus imipenemPhase 3 (local study: Puerto Rico)2019 (completed)NCT03159078
Resistant Pseudomonas aeruginosa
Healthy volunteersMEDI3902Phase 1 dose-escalation study2018 (completed)NCT02255760
Prevention of nosocomial pneumonia caused by P. aeruginosa in mechanically ventilated patientsMEDI3902 vs. placeboPhase 22016-2020 (completed)NCT02696902
Carbapenem-resistant Enterobacteriaceae (CRE)
Colonization and clinical infection with CRECapsulized FMTLocal study: IsraelApril 2023NCT04790565
Clinical infection with CRECapsulized FMTPhase 2, 3 June 2022NCT04146337
Colonization with CRE or VREFMT with frozen stool from donorsNot mentionedMarch 2022NCT04583098
Colonization with CRE or VREFMT with donor stool samples from stool bankPhase 2December 2021NCT03479710
Colonization with CREFMT from donorsPhase 1, 2February 2023NCT04759001
Colonization with CRECapsulized FMTPhase 1, 2December 2019NCT03167398
Colonization with KPCCapsulized FMTPhase 2September 2024NCT04760665
Colonization with MDR organisms after infection, in renal transplant recipientsFMT using allogeneic human stool in glycerolPhase 1December 2022NCT02922816
Prevention and decolonization of MDR bacteria Probiotics (Lactobacillus casei, Lactobacillus plantarum,
Streptococcus faecalis,
Bifidobacterium brevis)		Not mentionedDecember 2019NCT03967301
Colonization with MDR Gram-negative bacilliProbiotic: 4 Lactobacillus strains, 3 bifidobacteria strains, and Streptococcus
thermophilus 24,731 vs. capsulized FMT from healthy donor		Not mentionedJuly 2023NCT04431934
Serious infections due CRE (complicated UTI, acute pyelonephritis, HABP, VABP, bacteremia, abdominal infection)Meropenem + vaborbactam vs. best available therapyPhase 3July 2017NCT02168946
Colonization with MDR
bacteria		Colistin sulphate + neomycin sulphate days 1–5 then capsulized FMT days 7 and 8Phase 2March 2018NCT02472600
CRE or KPC infectionsImipenem/cilastatin/relebactamPhase 4August 2022NCT04785924
CRE = carbapenem-resistant Enterobacteriaceae; FMT = fecal microbiota transplantation; MDR = multidrug-resistant; HABP = hospital-acquired bacterial pneumonia; KPC = carbapenemase-producing Klebsiella spp.; PK = pharmacokinetics; VABP = ventilator-associated bacterial pneumonia; VRE = vancomycin-resistant enterococci.
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Săndulescu, O.; Viziteu, I.; Streinu-Cercel, A.; Miron, V.D.; Preoțescu, L.L.; Chirca, N.; Albu, S.E.; Craiu, M.; Streinu-Cercel, A. Novel Antimicrobials, Drug Delivery Systems and Antivirulence Targets in the Pipeline—From Bench to Bedside. Appl. Sci. 2022, 12, 11615. https://doi.org/10.3390/app122211615

AMA Style

Săndulescu O, Viziteu I, Streinu-Cercel A, Miron VD, Preoțescu LL, Chirca N, Albu SE, Craiu M, Streinu-Cercel A. Novel Antimicrobials, Drug Delivery Systems and Antivirulence Targets in the Pipeline—From Bench to Bedside. Applied Sciences. 2022; 12(22):11615. https://doi.org/10.3390/app122211615

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Săndulescu, Oana, Ioana Viziteu, Anca Streinu-Cercel, Victor Daniel Miron, Liliana Lucia Preoțescu, Narcis Chirca, Simona Elena Albu, Mihai Craiu, and Adrian Streinu-Cercel. 2022. "Novel Antimicrobials, Drug Delivery Systems and Antivirulence Targets in the Pipeline—From Bench to Bedside" Applied Sciences 12, no. 22: 11615. https://doi.org/10.3390/app122211615

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