Novel Antibacterial Approaches and Therapeutic Strategies

1. Introduction

The failure to effectively treat infectious diseases owing to an increasing trend in antimicrobial resistance (AMR) stands out as an emerging threat to public health, resulting in increased morbidity and mortality rates, extended hospitalization time, and greater financial burden. In a recent analysis, it was estimated that drug-resistant infections by 88 pathogen-drug combinations caused 4.95 million deaths worldwide, 1.27 million (25%) out of which were due to AMR [1] Particularly, the widespread use of antibiotics in human and veterinary medicine together with environmental pollution by residual antibacterial pharmaceuticals generate a serious concern in relation to the emergence and spread of multi-drug resistant (MDR) pathogens. Recently, the World Health Organization (WHO) has alerted of a "post-antibiotic era" where simple infections and minor injuries might once again become deadly threats [2] Moreover, antibiotics used to fight these infections can also have negative side effects [3], as the drugs used to eliminate the infectious agents also affect beneficial microbiota leaving an urgent need to re-evaluate treatments in terms of their ability to specifically target pathogenic bacteria while conserving the necessary microbiota [3].

In hospitals, broad-spectrum antibacterials and invasive procedures contribute to the high prevalence of MDR microbes, particularly among immuno-compromised patients, creating unique surveillance challenges. Medical devices like catheters and ventilators act as colonization sites for bacteria that can cause hospital-associated infections [4]. Moreover, the nature of resistant strains capable of forming biofilms should be considered, as they are hard to eliminate and lead to spread and repeated infections [5]. Horizontal gene transfer (HGT) adds even more complexity to this problem since it enables the sharing of resistant genes among bacterial species, thereby facilitating the spread of resistance in healthcare settings and ecosystems [6,7,8]. To overcome this, there is a need for novel therapeutic strategies with better long-term effects.

Exploration for new antibacterial agents is turning the attention towards previously unexplored bacterial mechanisms such as riboswitches and caseinolytic protease (ClpP) systems in the fight against bacterial resistance. Their goal is to inhibit those pathogen survival pathways at lower risk for developing resistance [9,10]. In addition, repurposing existing or investigational drugs approved for non-antibacterial use has been an effective strategy for discovering new therapies [11]. Combination therapies (pairing antibiotics with antivirulence agents—quorum-sensing inhibitors or efflux pump blockers) can also enhance the therapeutic effect [12,13]. This structural barrier has converted the development of effective drugs against pathogenic Gram-negative bacteria into a real challenge. Most antibiotics are limited by their ability to penetrate cells effectively because they face a protective outer membrane (OM) present in these bacteria. To overcome this challenge, novel approaches are under development to either alter and render the bacterial membrane more permeable or facilitate targeted drug uptake [14,15,16].

Advancements in genomic technologies have opened avenues for novel antibacterial drug discovery by improving the identification of targets within bacterial genomes. The rapid evolution of molecular biology and sequencing technologies, such as whole-genome sequencing (WGS) and metagenomics, have paved the way for identifying new drug targets, anticipating resistance patterns, and designing personalized therapies [17].

However, despite having made significant strides in antibacterial research, there has been a substantial decline in novel antibiotic development and a simultaneous rise in bacterial resistance over the past three decades [18]. The economic disincentives to the pharmaceutical industry have decreased its R&D investment in the field. Antibacterial treatments are for temporary use only, leading to reduced long-term profitability compared with medications for chronic diseases. Also, new government regulations and high clinical trial costs make it harder to enter the market. While initiatives, including public funding for early-stage research and market-entry rewards for antibacterial agents, have been suggested to incentivize development, they have not yet promoted sufficient investment in novel R&D within the industry [19,20].

Here, we review current antibacterial approaches, recent therapeutic developments, and some of the future strategies needed to fight the worldwide problem of AMR.

2. Factors Contributing to the Increase in Bacterial Infections

One of the major causes managed by human medicine is antibiotic overuse, which is widely documented as a driver of bacterial resistance to antibiotics. Overusing antibiotics for viral infections, the non-judicious use of broad-spectrum antibiotics, and patients not completing their antibiotic course all create selective pressure, giving resistant bacteria a survival advantage [6]. Even low doses of antibiotics can act as a selective pressure for adaptation in bacteria, raising the likelihood of mutations in resistance genes and expediting the emergence of MDR bacteria in healthcare settings [4]. Infections acquired in these environments are considered the third leading cause of human death after cancer and heart disease. Horizontal gene transfer (HGT) is the primary biological mechanism contributing to the rising resistance in bacteria and plays a major role in the spread of antibiotic resistance genes (ARGs). Mobile genetic elements serve as vehicles for HGT in both hospital and environmental settings, increasing the burden of resistance [5]. The most common way for HGT to disseminate ARGs in bacteria is conjugation. In the conjugation process, a donor bacterium can transfer plasmids containing its resistance genes directly into a recipient through contact between the two, especially in settings with high bacterial density, such as hospital or agricultural environments [6]. Conjugative plasmids transport several ARGs originating MDR microbes [6]. Through transformation, bacteria can take up free DNA from their surroundings and acquire resistance genes. This frequently occurs in sites with a high abundance of dead bacterial cells, allowing ARGs to be taken up and incorporated into the genomes of living bacteria [7]. Another biological mechanism of HGT is transduction, where bacteriophages transport ARGs from one bacterium to another. Transduction is especially important in environments rich in bacteriophages, such as the human gut microbiome or wastewater treatment plants [8]. High levels of antibiotics favor the conjugation frequency and expression of plasmid-carried conjugation-related genes (as seen in wastewater or agricultural runoffs). Sub-minimal inhibitory concentration (MIC) levels of antimicrobials such as meropenem and ciprofloxacin have been shown to markedly facilitate the horizontal plasmid-mediated transfer of resistance from Klebsiella pneumoniae and Escherichia coli [21].

While we understand the significance of HGT on increased resistance, managing its effects is still challenging. Microbial communities are diverse in environments such as wastewater treatment plants, hospitals, and, most importantly, the human gut, making tracking and controlling ARG transfers out of complex microbiomes extremely difficult. In addition, biofilms on biotic and abiotic surfaces help resistant bacteria survive and spread due to the persistence of ARGs in biofilms.

The application of antibiotics to livestock generates pools of resistant bacteria that can transfer ARGs to human pathogens through HGT. Animal health products and antibiotics are widely used in livestock as growth promoters or to prevent infection, creating reservoirs of resistant bacteria. These resilient microorganisms can spread to humans through direct contact, food items, and environmental contamination. Antibiotic-resistant microbes can spread across water and soil through agricultural runoffs containing resistant bacteria and antibiotics [22]. Resistant bacteria reservoirs are even made more complex as migratory birds, soil, and air [23]

Nosocomial infections are considered the third leading cause of death, after cancer and cardiovascular disease. Catheters and ventilators are common sources of contamination from bacteria. Thus, bacteria-harboring resistance genes survive and disseminate rapidly to immunocompromised patients in such environments [4]. More recently, a study monitored the transfer of plasmids that harbored ARGs between bacterial species within a hospital setting [7]. This movement of plasmids between bacteria is a significant factor in the rapid spread of resistance in hospitals [7]

HGT is also facilitated to a significant extent by the presence of non-antibiotic compounds like heavy metals, food preservatives, and pharmaceuticals. Silver nanoparticles commonly used in personal care products release silver ions into the environmental system and induce oxidative stress response of bacteria, further stimulating the transfer of plasmid [24] and facilitating conjugative transmission of ARGs. Likewise, well-known active pharmaceutical ingredients (APIs) such as the antidepressant fluoxetine and synthetic analgesics like acetaminophen promote ARG transfer with mechanisms involving increased permeability of the cell membrane and SOS response induction, activating DNA repair pathways that facilitate plasmid integration [21]. The increased membrane permeability, the stress responses, and an increased probability for intact HGT promote the dissemination of resistant microbes to environments beyond health care [25].

3. Search for Novel Targets and Therapies

Classical antibiotics act on essential bacterial processes, including cell wall synthesis (e.g., β-lactams), protein synthesis (e.g., aminoglycosides), and DNA replication (e.g., fluoroquinolones). Although effective, the rampant overprescription and abuse of these medications result in the emergence of resistant strains due to the selective pressure applied to the bacterial population owing to the transfer of the HGT elements [7]. Furthermore, such antibiotics commonly have toxicity and off-target effects when given in high doses or for a prolonged time, threatening their long-term effectiveness [26].

Instead, the new antibiotics are based on original bacterial processes that are important for survival and with less likely development of resistance. Riboswitches are non-coding RNA domains that detect specific target ligands and regulate gene expression in bacteria. A recent study targeting flavin mononucleotide (FMN), thiamine pyrophosphate (TPP), and glmS riboswitches with chimeric antisense oligonucleotides effectively resulted in the inhibition of bacterial growth but without exhibiting toxicity on human cell lines [9], representing a rational and selective action against bacteria such as Staphylococcus aureus and Escherichia coli that are substantial resistant pathogens [10]. Antibiotics in development target the bacterial caseinolytic protease (ClpP), essential for maintaining proper levels of protein homeostasis within bacteria, promoting their death. This class of antibiotics, known as activators of self-compartmentalizing proteases (ACPs), has demonstrated activity against Gram-negative pathogens for which traditional antibiotics are ineffective, including the Neisseria species [10].

Combination therapies are one way to improve the effectiveness of both old and new antibiotics. Combined treatment of gallium (a siderophore quencher) and furanone C-30 (a quorum-sensing blocker) with antibiotics colistin and ciprofloxacin have been demonstrated to restore sensitivity against Pseudomonas aeruginosa [12]. However, β-lactams are still ineffective in combatting resistant bacteria due to the production of β-lactamase by these microorganisms. A combination of meropenem-vaborbactam, consisting of a carbapenem and a β-lactamase inhibitor, can overcome this resistance mechanism and restore activity against carbapenem-resistant Enterobacterales [13].

Antibacterials that target bacterial membranes represent an alternative mode of action with the potential to avert resistance development. PQ401, a new class of neutral diarylurea compounds, displays broad-spectrum activity against antibiotic-resistant and antibiotic-tolerant S. aureus. PQ401 circumvents an antibiotic limitation where cross-resistance to membrane-active agents like polymyxins has been observed [27] by causing select disruption of bacterial membranes.

Novel antibiotics with less toxicity and improved safety profiles are being rolled out. The replacement of l-Val with d-Val significantly enhanced the stability and safety of engineered antimicrobial peptides (AMPs) by presenting a potent activity against MDR bacteria in respiratory infections while exhibiting a 4-fold improvement in safety compared to earlier versions, showing less toxicity to human cells [28].

4. Challenges in Developing Antibiotics Targeting Gram-Negative Outer Membranes

The unique structure of the cell envelope in Gram-negative bacteria contains an extra outer membrane (OM) that restricts drug permeability. This prevents many antibiotics from accessing their intracellular targets and plays an important role in intrinsic and acquired antibiotic resistance, primarily linked to permeability barriers, efflux pumps, and resistance mechanisms.

4.1. The Outer Membrane as a Permeability Barrier

The outer leaflet of the outer membrane contains lipopolysaccharides (LPS), whereas the inner leaflet is composed of phospholipids, both of which form a hydrophobic and impermeable barrier to many antibiotics against Gram-negative bacteria. This low permeability is a key characteristic by which Gram-negative bacteria are inherently more resistant to antibiotics than Gram-positive [29]. For antibiotics to reach their intracytoplasmic targets, they must cross this OM. Yet only a narrow range of chemical entities can do so due to the barrier’s preference for small, hydrophilic compounds that permeate through porins.

4.2. Porins and Antibiotic Entry

Porins (e.g., E. coli OmpF) function as channels that allow for passive diffusion of hydrophilic molecules, including some antibiotics, into the cell. Nonetheless, porin mutations result in a decrease in expression and/or conformational alteration, limiting the entry of antibiotics. Furthermore, porins have dynamic open-closed states to control antibiotic permeability, and thus, drug molecules cannot reach target sites inside the cell stably [30].

4.3. Efflux Pumps

Furthermore, even when antibiotics penetrate into OM, Gram-negative bacteria can express efflux pumps-membrane proteins that maintain their intracellular concentration of antibiotics at a non-lethal level. Pumps in this group (e.g., AcrAB-TolC system in E. coli) decrease the intracellular concentration of antibiotics, especially the lipophilic or bulkier ones, and contribute to multidrug resistance [15].

4.4. Targeting Outer Membrane Proteins

Despite being an efficient approach to overcome resistance, antibiotics that act on essential players in the OM, for example, β-barrel assembly machinery (BAM complex) and lipopolysaccharide(s) transport machinery (Lpt), are relatively new. The BAM complex inserts β-barrel proteins into the OM, whereas Lpt transports lipopolysaccharide (LPS) to the OM. These systems are also the target of inhibitors that permeabilize membranes and increase the susceptibility of bacteria to antibiotics [14]. A primary challenge in designing these inhibitors is that they must circumvent efflux pumps and reach high local concentrations at their sites of action. A range of compounds that repress OM assembly are non-soluble by efflux systems, necessitating combination procedures or adjustments to increase their intracellular retention [31].

A promising broad-spectrum antibacterial active against Gram-negative bacteria is darobactin A, which acts on the OM protein BamA, binding this target in the periplasm and impairing protein folding and integration. Thus, a major advantage of this antibacterial is to circumvent the need to pass over the permeability barrier [32]. It was recently shown that natural darobactin A, obtained from an entomopathogenic bacterium, can be processed to yield darobactins D (D22) and 69 (D69). D22 was active in vivo against murine infections of Pseudomonas aeruginosa and E. coli and rescued zebrafish embryos infected with A. baumannii [33]

Chemical perturbants such as MAC-0568743 disrupt the OM while selectively preserving the inner membrane, enabling antibiotics, such as vancomycin, to penetrate resistant Gram-negative bacteria [34]. These OM disruptors work in tandem with common antibiotics, improving their effectiveness against resistant strains.

Novel approaches target the OM to breach its integrity, permitting antibiotics to permeate and exert their effects more effectively [35]. SPR741 is a novel polymyxin B analog that permeabilizes explicitly the outer membrane while leaving the inner membrane intact, enabling antibiotics to act on their intracellular targets more efficiently [36]. OM disruptors and Gram-positive active antibacterials have been reported to be effective against murine infections caused by A. baumannii, Klebsiella pneumoniae, and E. coli [35]. Due to the significance of LPS in the outer membrane of Gram-negative bacteria as an outer component, it maintains membrane integrity and is antibiotic penetration-resistant. Disruption of the OM has been proposed as a promising strategy by targeting LPS biosynthesis and transport, specifically the enzymes involved in both biological processes [37].

4.5. Importance of Disrupting Transport and Synthesis of Lipoproteins as an Antibacterial Strategy

This emerging approach reduces the robustness of MDR Gram-negative bacteria by disrupting OM lipoprotein transport and biogenesis. These proteins are acylated, anchored into the inner membrane, and transported to the OM by dedicated transport systems, such as the ATP-binding cassette (ABC) transporter LolCDE. Lipoproteins play fundamental roles in OM assembly, nutrient uptake, and resistance to environmental stress and antibiotics [38]. Consequently, pathogenic bacteria have developed decreased reliance on the lipoprotein transport pathway, which is now gaining traction as a valuable target for novel antibacterial therapies [31].

The Lol (Localization of lipoproteins) pathway constituting the LolCDE transporter is required to extract lipoproteins from the inner membrane and convey them to the OM. Key components of the Lol pathway include:

• LolCDE Complex — The ATP-binding cassette (ABC) transporter LolCDE extracts lipoproteins from the inner membrane and is critical for their subsequent delivery to the OM. Recent structural studies have shed light on the transport mechanism of lipoproteins by LolCDE, involving significant rearrangements in the transmembrane helices causing large-scale "extrusion" movement of lipoprotein from the membrane [36]. Muñoz et al. recently designed a novel class of selective and potential gram-negative pathogenic bacteria active antibiotic called lolamicin [3]. For drug design, they targeted the essential LolCDE complex that mediates the transport of OM-specific lipoproteins to the periplasm via the cytoplasmic membrane [39]. Lolamicin displayed activity against an MDR-isolates collection of 130 strains via efficacy against multiple murine pneumonia and septicemia models as well as protection against C. difficile gut infection in a mouse model of microbiome [5]. They suggested that lolamicin is active against Gram-negative pathogenic bacteria but inactive toward commensals (both Gram-positive and -negative) based on low sequence homology between the two classes of organisms. Thus, lolamicin seems to be at least a potential candidate for testing and possibly application against human infections.
• Chaperones: LolA and LolB are periplasmic chaperone proteins that transfer lipoproteins across the periplasm and insert them into OM. If either LolA or LolB is disrupted, lipoproteins become mislocalized, and defects in OM assembly arise [40].
• Inhibition of Lipoprotein Diacylglyceryl Transferase (Lgt): It catalyzes the first step in lipoprotein biogenesis, where it acylates lipoproteins in the inner membrane. Lgt inhibitors like G2824 mess up OM permeability to make bacteria more susceptible to serum killing and antibiotics. Importantly, these inhibitors show activity against strains resistant to other lipoprotein pathway inhibitors [41].

While interfering with lipoprotein transport provides a powerful approach, resistance mechanisms developed by bacteria counteract these inhibitors. Deleting the lpp gene, which encodes Braun's lipoprotein in Gram-negative bacteria, circumvents inhibiting downstream steps in lipoprotein biosynthesis. On the other hand, moving down the path and targeting later steps, as with inhibitors against Lgt itself, seems less susceptible to this resistance mechanism [42], making them more favorable for clinical development.

Inhibiting lipoprotein biogenesis or transport induces bacteria's sensitivity to current antibiotics and may repurpose previously inadequate drugs for use against MDR pathogens. This method is particularly encouraging for infections caused by P. aeruginosa, A. baumannii, and K. pneumoniae, which have shown resistance to traditional antibiotics [31].

Stress response systems regulate lipoprotein trafficking in bacteria. In E. coli, the Cpx system monitors the delivery of OM lipoproteins and activates a stress response to promote protection against the so-called trafficking defects. Targeting this system can make lipoprotein transport inhibitors more efficacious, as this is essential for bacterial survival in the presence of lipoprotein trafficking indisposition [42].

4.6. Chemical Space Limitations

The process of discovering new antibiotics is especially hindered now with the limited diversity in chemical libraries traditionally used for drug discovery. Most compounds are optimized for Gram-positive pathogens and lack the required physicochemical properties for penetrating the OM of Gram-negative pathogens. New strategies attempt to broaden the chemical space by finding matrix molecules that can go in through the OM and not get extruded out by efflux pumps. Optimizing these parameters for higher permeability, coupled with the need to maintain optimal hydrophobicity and charge so the compound can pass through porins and enter into cells, has been an avenue pursued in recent work [15].

4.7. These Complexities Pose Their Own Challenges in Clinical Development

The potential for OM disruptors to cause toxicity is one of the key challenges in developing these findings into clinically relevant treatments. Murepavadin, a Lpt system inhibitor that showed promise in preventing P. aeruginosa pathogenesis, was shown to be effective but discontinued due to the observed nephrotoxicity during clinical trials [34]. This provides a rationale for why these two considerations must change — namely, all mechanisms that disrupt bacterial membranes may also adversely affect mammalian cell membranes, making it difficult to find drugs with enough specificity and safety.

Combination therapies against Gram-negative OM will be required in the future. Treatment with OM disruptors and intracellular-targeting antibiotics can synergistically increase drug efficacies while decreasing the likelihood of resistance emergence. This is also exemplified by other combination therapies that use OM perturbation to help antibiotics, such as β-lactams and glycopeptides, access their targets in Gram-negative bacteria [43]. Using efflux and permeability predictions in medicinal chemistry can enable the rational design of updated antibiotics [16]. Finally, the inhibitors of drugs that target resistance mechanisms (e.g., viz efflux pump, β-lactamase), when developing along with agents disrupting the OM, can reduce antibiotic resistance and retain current antibiotics viability, gaining time for novel alternative class antibacterial development [44].

5. Emerging Antibiotics and Strategies

In recent years, several new compounds and strategies have emerged. These new antibiotics include novel classes and derivatives targeting resistant pathogens, innovative delivery systems, and combination therapies (Table 1).

5.1. Derivatives of Tetracycline (Eravacycline)

Eravacycline, a new synthetic tetracycline derivative recently approved for treating complicated intra-abdominal infections, has broad-spectrum activity against Gram-positive and Gram-negative bacteria, including MDR strains of A. baumannii and E. coli. Eravacycline was designed to avoid the most common tetracycline resistance mechanisms, such as efflux pumps and ribosomal protection proteins [45,46].

5.2. Siderophore Cephalosporins (Cefiderocol)

Cefiderocol: A novel cephalosporin that has a siderophore entry mechanism whereby it hijacks the bacterial iron transport system to enter through the outer membrane of Gram-negative bacteria. Cefiderocol avoids existing resistance mechanisms such as porin mutations and efflux pumps, making it highly effective for carbapenem-resistant Enterobacterales and P. aeruginosa [47,48].

5.3. Aminoglycoside Derivatives (Plazomicin)

Plazomicin is a next-generation aminoglycoside with reduced susceptibility to aminoglycoside-modifying enzymes, the most prevalent resistance mechanism to this class of antibiotics. It is indicated for complicated urinary tract infections (UTIs) and demonstrates in vitro potency against MDR Enterobacterales, including carbapenemase-producing strains [49,50,51].

5.4. Fluoroquinolones(Delafloxacin)

Delafloxacin (DLX) is a novel broad-spectrum fluoroquinolone recently discovered to have activity against Gram-positive and Gram-negative pathogens, including methicillin-resistant S. aureus (MRSA). It displays enhanced activity in acidic environments, such as those found at infection sites, and is effective for skin and soft tissue infections [52,53].

5.5. New β-Lactam/β-Lactamase Inhibitor Combinations (Meropenem-Vaborbactam)

Meropenem-vaborbactam is a combination therapy of the carbapenem meropenem with the β-lactamase inhibitor vaborbactam that inhibits serine carbapenemases and restores activity to meropenem for use against carbapenem-resistant Enterobacterales. It is indicated for complicated UTIs, and it is a last-resort therapy against carbapenem-resistant bacteria [13].

5.6. New Glycopeptides (Corbomycin)

Corbomycin is a new glycopeptide that binds the bacterial cell wall to inhibit autolysins-mediated peptidoglycan remodeling during bacterial growth. Such action is distinguishable from typical glycopeptides such as vancomycin and has low rates of resistance emergence. It has been proven effective against MRSA and other resistant Gram-positive pathogens [54].

5.7. Nanomaterials in the Context of the Drug Delivery

Recently, nanotechnology has emerged as a novel approach for optimal antibiotic delivery and combatting resistance. Nanoparticles would enhance antibiotics' pharmacokinetics, improve bacterial cell internalization, and facilitate biofilm penetration. These drug delivery platforms decrease the development of resistance by improving effective targeting, leading to a lower dose requirement of antibiotics. Recent developments in nanotechnology involve using polymer-based and inorganic nanoparticles as carriers for traditional antibiotics, improving their efficacy against resistant pathogens [55].

5.8. MD-124: Antibiotic Potentiator

MD-124 is an oral small-molecule adjuvant, re-establishing (in the context of current antibiotic therapies) Gram-negative drug resistance to clinically relevant antibiotics. MD-124 acts synergistically with antibiotics such as colistin against bacteria and restores susceptibility in carbapenem-resistant strains by selectively permeabilizing the OM. Antibiotic potentiators like MD-124 fight resistance without developing new antibiotics [43].

5.9. Bacteriophage Therapy

Viruses that infect bacterial pathogens, known as bacteriophages, are an alternative to traditional antibiotics, which still need advancement via design implementing in vivo and ex vivo-based approaches. The one that has gotten some attention is phage therapy, which shows promise in the fight against antibiotic-resistant bacteria, especially under personalized medicine circumstances. Similar studies are being conducted with phages against other pathogens, such as the therapeutic use of phages to treat drug-resistant P. aeruginosa infections in cystic fibrosis patients [56].

6. Post-Genomic Era Antibacterial Drug Discovery

The post-genomics era has uncovered many new targets for antibiotic drug discovery and developed strategies to combat the growing threat of resistance against current antibacterials. Three of these targets, discovered through advances in bacterial genomics and molecular biology, included bacterial signal transduction systems, lipid biosynthesis pathways, and structural components essential for bacterial viability (Table 2).

6.1. Histidine Kinases in Two-Component (TCS) and Quorum Sensing Systems

Bacterial two-component systems (TCSs), with histidine kinases (HKs) as integral components, play a critical role in bacterial adaptation to environmental stress, such as antibiotic exposure. Histidine kinases are involved in bacterial signaling pathways that mediate antibiotic resistance, and they can be targeted to disrupt these pathways. Due to the absence of TCSs in humans, HKs are an attractive target for selective inhibition of bacterial growth without threat to human cells. Recent work has shown that HK inhibition can affect bacterial signaling to render pathogens less virulent. HKs possess an ATP-binding domain, which is highly conserved between bacterial species and offers a promising target for broad-spectrum antimicrobial activity [57]. Moreover, HKs also serve as the target of novel specific drugs to treat infections caused by MDR organisms, such as S. aureus and A. baumannii [58].

Other protein kinases, notably those that play roles in bacterial signaling pathways, provide attractive drug targets. Serine/threonine kinases are important pathogen virulence factors that regulate bacterial growth, and inhibitors of these enzymes in Mycobacterium tuberculosis may be useful candidates for treating tuberculosis. Most recent work has identified compounds that impede these kinases from their function and kill the organism in animal models [59]. A comprehensive survey of histidine kinase (HK) inhibitors has been performed, providing information on structural classifications, chemical structures, IC50 & MIC values, antivirulence properties, and exploring domains for HK targets, including the ATP-binding domain, HK sensors, histidine phosphorylation domain, and dimerization [58].

Quorum sensing (QS) is achieved by a cell-to-cell communication system allowing Gram-positive and Gram-negative bacteria to regulate gene expression, including virulence factor production, as a function of population density. The inhibitory effect on the quorum sensing disrupts these processes and leads to less bacterial pathogenicity and biofilm formation, which is also generally resistant to antibiotics. Accordingly, it has been shown that inhibiting the AgrC histidine kinase in the quorum-sensing system of S. aureus reduces virulence and biofilm formation [60]. The VraTSR three-component system is another important S. aureus system that recognizes cell wall damage after antibiotic exposure. Compounds that inhibit the VraTSR system have decreased the survival of this bacterium exposed to cell wall-targeting antibiotics and could be useful for combination therapies [61].

6.2. Lipid Biosynthesis Pathways

The OM of Gram-negative bacteria contains an important element, lipid A. Targeting its biosynthetic pathway allows for the OM to be compromised, making the cells more susceptible to antibiotics. Over the past eight years, significant effort has been directed toward other enzymes in the lipid A biosynthesis, such as LpxC and LpxH, the second and fourth enzymes of the Raetz pathway. LpxC inhibitors, like LPC-233, are potent bactericidal agents against MDR pathogens without the cardiovascular toxicity of early LpxC inhibitors [62]. Inactivating other LpxC inhibitors has been shown to inhibit growth in clinically relevant Gram-negative pathogens, including P. aeruginosa and K. pneumoniae [63]. In addition, inhibition of LpxH accumulates toxic lipid A intermediates and thus kills the bacteria due to an essential step in the fatty acid mobilization of lipid A biosynthesis. Inhibition of both LpxC and LpxH, which work in tandem with lipid A biosynthesis, thus provides a dual-action inhibition strategy that represents a powerful tool against MDR infections [64].

One such strategy has targeted agents interfering with lipid II, an important component of bacterial cell wall biosynthesis. An example of such a synergistic approach is the combination of Lipid II-targeting antibiotics with outer membrane-acting peptides to potentiate their activity against Gram-negative pathogens [65].

6.3. Protein Synthesis Inhibition

Since protein synthesis is a fundamental process for bacterial life, the ribosome has proven to be a successful target for antibiotic development. However, the most recent work centers on spermine-conjugated short proline-rich lipopeptides that target and block protein synthesis in E. coli without compromising bacterial membrane integrity. These lipopeptides exhibit broad-spectrum activity and synergy with conventional antibiotics, particularly in MDR strains [66]. Recent work has also focused on designing antimicrobial peptides that target the prokaryotic ribosome, preventing protein synthesis and showing high potency against MDR S. aureus and E. coli [65].

6.4. Inhibitors of DNA Gyrase and Topoisomerase

During bacterial DNA replication, two key enzymes important for the process include DNA gyrase and topoisomerase IV. Fluoroquinolones, drugs that target these enzymes, have enjoyed great success but are now facing a vigorous counterattack in the form of resistance. New compounds are developing that inhibit DNA gyrase by novel mechanisms. The synthesis of 1,2,4-oxadiazole-chalcone hybrids was reported, and high Gram-positive and Gram-negative antibacterial activity was observed through DNA gyrase inhibition [67].

6.5. Targets in Bacterial Peptidoglycan and Membrane.

Work has thus far centered around synthetic oligomers that cause peptidoglycan disruption, targeting Gram-positive pathogens such as S. aureus. The materials' high antibacterial activity provides good reference significance for their use in functional materials against drug-resistant bacteria [68].

7. What's New in Bacterial Genome Sequencing

The emergence of bacterial genome sequencing has transformed our perception of how bacteria function biochemically and molecularly, how they respond to antibiotics (clinical resistances), and how they reveal novel targets for new antibiotics. Whole-genome sequencing (WGS) and metagenomics have since emerged as integral components of clinical diagnostics and drug discovery. Here, we discuss some major breakthroughs in bacterial genome sequencing and their relevance for tracking antibiotic resistance and developing new antibiotics.

7.1. Predicting Antibiotic Resistance Using Whole-Genome Sequencing

Whole-genome sequencing is now an essential tool for predicting bacterial pathogens' antibiotic-resistance genes (ARGs). Sequencing genomes of drug-resistant bacteria can detect ARGs, determine transmission potential, and predict resistance profiles at the clinical level.

• Antimicrobial Resistance Prediction: The use of WGS data to predict resistance phenotypes (particularly in Gram-negative bacteria) is currently being explored. Machine learning models developed on WGS data can predict resistance to multiple antibiotics with a high degree of accuracy, enabling real-time clinical decisions and potentially swift treatment tailored to the group's type and required level of therapy [69]. This could provide in silico antibiograms, thereby assisting the correlative management of resistant infections.
• Genomic Databases for Resistance Genes: Novel tools such as BacAnt and ARTS are available for genome annotation regarding antimicrobial resistance genes (ARGs) and mobile genetic elements (MGEs), which are crucial for monitoring the transmission of resistance genes. Such bioinformatics platforms automate the detection of ARGs and MGEs, aiding epidemiological studies and resistance monitoring [70].

7.2. Discovery of New Antibiotics Through Genome Mining

The advent of genome sequencing technology has unraveled biosynthetic gene clusters (BGCs) responsible for generating novel secondary metabolites, some with promising antibiotic activity. Identifying previously uncharacterized BGCs in mined bacterial genomes can lead to discovering potential new antibiotic compounds.

• Triggering of Silent Gene Clusters: Many genes responsible for producing antibiotics remain silent during normal laboratory conditions. With improvements in genome mining approaches such as CRISPR / gene editing technologies, these silent clusters have been induced for expression, producing, e.g., lignin or lactobacilli [17].
• Drug Discovery with Comparative Genomics: Over the years, based on comparative genomic approaches, highly conserved genes across bacterial species are essential as novel drug targets. Despite the inherent redundancy in secondary metabolite gene clusters, it has been possible to connect several novel chemical structures with antibacterial activity to their respective gene clusters for rational antibiotic design [71].

7.3. A Novel Approach for Antibiotic Resistance Surveillance - Metagenomics

Research based on metagenomic sequencing has been performed in various environmental contexts, including hospitals, soil, and the human gut, to investigate microbial communities and resistomes (the collective set of all ARGs). Such an approach is useful for tracing the environmental sources of resistance genes, as well as how human activity drives the dissemination of antibiotic-resistant bacteria.

• Environmental Resistome Studies: Most ARGs are present in environmental bacteria long before they can be recovered from clinical pathogens. Methods such as metagenomics and metatranscriptomics could further increase the detection of unknown ARGs in bacteria that may not be culturable [72].
• Predictive Surveillance for Emerging Resistance — High-throughput genomic sequencing integrated with functional genomics enables the observation of resistance genes across space and time. Such predictive surveillance is necessary to detect and mitigate emerging resistance threats early enough [73].

7.4. Third-Generation Sequencing and Producing Complete Genomes

The emergence of third-generation sequencing platforms (e.g., PacBio and Oxford Nanopore), which can assemble entire bacterial genomes (including plasmids and mobile genetic elements coding for ARGs), have facilitated a better resolution to unveil the resistance mechanisms involved in MDR organisms. Such technologies address the limitations of short-read sequencing, which fails to resolve repetitive elements and insertion sequences that are key to understanding resistance mechanisms. Recent advances have created gold-standard protocols for using third-generation sequencing to assemble bacterial genomes and identify ARGs within antibiotic-resistant strains. Methods based on DNA extraction with magnetic beads followed by long-read sequencing are suitable for plasmid reconstruction that carries resistance genes [74]. They help to reconstruct the genetic context and transmission dynamics of ARGs.

8. Applying Genomic Advances to Improve Public Health

Genome sequencing has been applied to rapidly detect resistant pathogens and guide treatment in clinical settings. Nowadays, WGS is utilized to build resistance profiles of clinical isolates, which can help with effective antibiotic selection and circumvent broad-spectrum antibiotics. The information on resistance mechanisms available through genome sequencing of clinical isolates will allow targeted treatment regimens. It is extremely useful in the case of MDR organisms, where rapid diagnostics are critical to patient outcomes. To this end, machine-learning models leveraging these genomic data are being trained to predict antimicrobial resistance and optimize therapy for patients infected with resistant pathogens [75].

8.1. Monitoring Antimicrobial Resistance Using Genomics

WGS has changed AMR surveillance, making it possible to discriminate accurately the resistance gene and its passing into bacterial populations. The genotyping approach aids in the detection of new resistant variants that can best help formulate effective containing strategies.

• AMR Surveillance platforms in clinical settings: Several bioinformatics platforms have been developed, such as ResFinder, CARD, and MEGARes, to catalog ARGs and their dissemination through clinical and environmental samples. These enable real-time tracking of resistance mechanisms and have become an integral aspect of global surveillance [76]. In vitro activity against P. aeruginosa and Citrobacter spp. demonstrated the potential of nanopore sequencing for monitoring plasmid dissemination and resistance gene development during hospital outbreaks, which allows rapid action to stop the spread of resistant strains [77].
• Metagenomics, applied to environmental samples and animal habitats: Metagenomic approaches for environmental and animal habitats are increasingly being used to identify reservoirs of ARGs that could transfer to human pathogens. Thus, understanding the environmental transmission routes/pathways of antibiotic resistance is essential, and surveillance programs that incorporate environmental sampling are needed for identifying potential community sources [78]. Genomics is also being utilized for the detection of emerging pathogens and their antibiotic resistance profiles in populations. Projecting genomic diversity onto genotype-based diagnostics provides a more streamlined pathogen surveillance strategy than random sampling and can improve the identification of new resistance variants in pathogens like Neisseria gonorrhoeae [79].

8.2. Improving Detection and Response to Outbreaks

Outbreak management needs genomic sequencing. It enables public health authorities to help identify sources of outbreaks and quickly trace the transmission of infections.

• Outbreak Investigations and Responses: WGS has been important to multiple outbreak investigations, allowing strain differentiation between outbreak and sporadic isolates. This ability provides for better identification of transmission chains, such as WGS-based tracking of Staphylococcus pseudintermedius infections in companion animals and their connections to pathogens in humans [80].
• Real-Time Genomic Tracking: With portable sequencing technologies like nanopore sequencing, bacterial infections can be tracked in real-time from a genomic perspective. These technologies are also advantageous in resource-limited settings and public health emergencies when genomic data can be used critically to guide interventions [77].

8.3. The advent of Personalized Medicine and Precision Public Health

Genomic tools have opened doors to personalized medicine in which the therapy can be adapted based on host genetics and pathogen identity. In a recent study, WGS, alongside machine learning, was employed to inform resistance-minimizing treatment optimization using patient-specific risk assessment for efficiency in the urinary tract and wound infections [81]. WGS now plays an integral role in precision public health by forecasting the antimicrobial resistance potential of clinical pathogens before therapy. Genomic prediction of resistance to key antibiotics such as meropenem and ciprofloxacin based on WGS improved treatment outcomes by ensuring appropriate patient therapy [69].

8.4. One Health Surveillance

The One Health approach acknowledges human, animal, and environmental health together so it can be used to battle antimicrobial resistance as part of the global public health challenges. Genomic technologies allow the interdisciplinary monitoring of pathogens and ARGs in the environment, fostering One Health approaches. For example, genomic surveillance of Salmonella and E. coli animal pathogens has demonstrated considerable genetic overlap between animal and human infections, suggesting the need for integrated One Health surveillance systems. Integrating veterinary diagnostic data into national surveillance programs has been shown to enhance the detection of resistance trends in companion and food animals that may have significant implications for human health [82].

9. AI for Antibiotic Discovery

AI has become a new tool with great potential to identify new drug targets and antibacterial agents against bacterial resistance [83,84], as this innovative approach allows us to analyze large datasets that outperform traditional methods and allow discovering complicated associations of biological information (e.g., genomic or transcriptomic) with chemical/physical data using learning algorithms and artificial neural networks [85]. Also, it helps to re-purpose the available drugs that were earlier developed for other diseases or infections as a treatment for drug-resistant infections [86]. For example, an MIT research team trained a deep-learning neural network to evaluate over 100 million compounds in their database [87]. Halicin, initially developed as a diabetes medication, was revealed by following this method. Halicin, a small molecule with a different backbone than traditional antibiotics, shows bactericidal activity against Gram-negative pathogens that would otherwise be resistant to all available antibiotics, like M. tuberculosis and Enterobacteriaceae. It has also proved effective against Clostridioides difficile and MDR A. baumannii in murine infection models, highlighting the advantages of deep-learning approaches for novel targets and antibiotics [86].

9.1. Small-Molecule Antibiotics

These AI-based screening models minimize the extensive and costly in vitro experiments that prevent scientists from comprehensively surveying existing and repurposing novel compounds, facilitating economic identification of viable antibiotics. AI has been particularly useful for optimizing the process of small molecule discovery – a mainstay in antibiotic development. Antimicrobial compounds have been identified using machine-learning models to screen large chemical libraries [88]. Novel Small Molecule Optimization using Generative Techniques such as Generative Adversarial Networks (GANs) and Variational Autoencoders (VAEs) are among some of the techniques employed to design novel small molecules specific to certain antimicrobial properties. Models that generate chemical structures based on specific activity (such as for activity against a given bacterial pathogen) can optimize molecules for bioavailability and toxicity [89].

9.2. Discovery of Antimicrobial Peptides (AMPs)

Several deep-learning models have been created to expedite AMP discoveries that are capable of predicting the antimicrobial activity of peptide sequences. The AMP predictive deep learning model (AMPs-Net) presented superior predictions of peptide activities than the traditional mechanisms and allowed the identification of new AMPs with potential strong antibacterial capabilities [90]. The pairing of AI with molecular dynamics simulations allows for discovering AMPs that show high therapeutic value and increased stability. In a recent study, a deep learning model was used to mine large metagenomic datasets and built AMPSphere — an exhaustive catalog of ~900,000 non-redundant peptides, many previously untested experimentally. This strategy has identified peptides with potent in vitro and in vivo activity against drug-resistant pathogens [91].

9.3. Predicting Antibiotic Resistance

Prediction of antibiotic susceptibility phenotypes through analysis of bacterial WGSs is a promising approach to facilitate the development of targeted therapies. Models to Predict Resistance in Salmonella spp. and N. gonorrhoeae show 90% accuracy [29]. Machine learning can discern subtle patterns in genomic datasets, identify novel genetic mutations associated with antibiotic resistance mechanisms, and help lay the groundwork for specific therapies [92].

9.4. AI and Antibacterial Combination Treatments

Other models give rise to synergistic drug combinations that boost antibiotic activity. They also can analyze databases of drug interactions and resistance mechanisms and explore combinations that could be more effective in collaboration than when acting alone [93]. This is especially useful for returning older antibiotics and other agents against resistant strains. Recent advances in system biology and artificial intelligence have made it possible to take metabolic pathways through which bacteria process drugs into account, thereby predicting and leading researchers toward the optimal combinations of drugs that will best impact bacterial growth and survival [94]. Computational models such as these facilitate the discovery of new drug pairs to lower resistance development [94].

9.5. Mechanism of Action (MOA) Prediction

Because drug combinations have complex interactions affecting multiple bacterial pathways simultaneously, they are excellent candidates for AI-based approaches to machine learning and clinical prediction that elucidate the MOA of such drugs. By analyzing transcriptomic and proteomic data, they can predict the mechanism of action of novel and existing antibiotics. These models explain the interactions of antibiotics with bacterial targets and the cellular behaviors of bacteria [95].

9.6. Advantages and Challenges

AI provides enormous value for drug development mainly due to its speed and cost-efficiencies. It analyzes vast amounts of data quickly and can find drug candidates significantly faster than conventional methods, thereby decreasing time and cost in the drug development cycle. However, the amount and quality of training data significantly restricts its effectiveness, showing that AI models may be less accurate when utilizing new biological systems [93]. Although antibiotic candidates are highly predictive in models, it is crucial to experimentally validate candidate molecules and demonstrate that they retain their efficacy and safety. To fulfill the promise of computational predictions, we require microbiologists and computational scientists to work together closely so that laboratory testing can provide insights into and confirm predictions. Lastly, many models (especially deep learning) are often seen as "black boxes, " making explaining how they generate predictions challenging. Higher transparency and interpretability of the models are essential for wide acceptance in antibiotic discovery [90].

10. Decreased Industry Investment into Antibacterial Drug Development

Even with all of the academic research efforts working towards improvements in antibacterial therapies, the lack of interest from the pharmaceutical industry in investing in the development of new antibacterial drugs is a quite significant hurdle in the fight against the increasing crisis of antimicrobial resistance. This has been related to a variety of factors, including financial barriers, scientific barriers, regulatory barriers, and the poor economic model of antibiotic development.

10.1. Unprofitable Antibiotics

Compared to drugs for chronic diseases, antibiotics have generally been viewed as less profitable to develop. Antibiotics are typically given over the short term, making them less suited for long-term revenue. By contrast, drugs for chronic diseases provide steady streams of income, as patients will take the drugs for life. In addition, new antibiotics are being used as last-line treatments, effectively lowering their market potential [20]. These are economic realities that discourage pharmaceutical companies from investing in antibiotics R&D, especially when taking the risks associated with the discovery and approval of these products into account [96].

10.2. Challenges in Science and Technology

The drug discovery of low-hanging fruits has mostly been exploited, so it is becoming increasingly difficult to identify novel compounds with broad efficacy against multiple targets. Moreover, bacteria develop resistance mechanisms on a regular basis, shrinking the therapeutic window available for recently discovered antibiotics before resistance arises [97]. The complexity of bacterial resistance also makes developing effective antibiotics even more difficult. Bacteria such as P. aeruginosa and A. baumannii harbor various layers of resistance like efflux pumps, biofilm formation and enzymatic degradation of drugs. These contribute to the challenge of identifying new compounds or drug combinations that can circumvent or overcome mechanisms of resistance [98].

10.3. Obstacles in Regulation and Market

The regulatory environment for antibiotics is affecting developers more heavily. Finally, the costs and time to produce clinical trial data are significant barriers, especially concerning antibiotics. Regulatory agencies often demand that extensive clinical trials be performed to demonstrate the efficacy and safety of a drug against resistant strains, potentially involving special patient populations that are challenging to recruit. Moreover, traditional non-inferiority trial designs for antibiotic development do not always deliver conclusive evidence of efficacy against resistant pathogens, and this complicates the approval process [99]. Moreover, profitability after market introduction is also uncertain, as antibiotic stewardship programs promote judicious use of new antibiotics in order to defeat resistance, limiting potential sales. Preserving the effectiveness and urgency of antibiotics versus potential profitability for drug companies creates a pressure imbalance deterring innovation [100].

Creating financial incentives to make antibiotic R&D more attractive to pharmaceutical companies has been one of the main areas of efforts to revive the antibiotic pipeline. Various mechanisms have been proposed and implemented in some territories, including public funding early-stage research (push incentives) and market entry rewards (pull incentives). For example, US legislation such as the Generating Antibiotic Incentives Now (GAIN) Act has offered financial rewards and longer exclusivity periods for companies working on new antibiotics [19]. Yet these incentives were little more than band-aids applied to a patient with declining investment vital signs.

New antibiotics for resistant pathogens tend to be derivatives of existing compounds rather than new chemical entities, thus primarily offering incremental health benefits [101]. In addition, the lack of a long-term sustainable business model for antibiotics has deterred pharmaceutical companies from participating in these programs. Potential solutions include innovative financial models that reward companies based on the efficacy of their antibiotics to maintain low levels of resistance (e.g., “antibiotic susceptibility bonus” [100]).

Due to the crisis of global antibiotic resistance, effective international collaboration is needed to pool resources for antibiotic funding, regulation, and stewardship initiatives [102]. Initiatives like the Wellcome Trust’s Drug-Resistant Infections Priority Program aim to develop a sustainable ecosystem for antibiotic R&D, promote public-private partnerships to provide financial incentives for sustainable, long-term investment into the R&D of new antibiotics [96].

11. Conclusions

The emergence of bacterial infections and MDR organisms is driven by a combination of factors, including antibiotic abuse and misuse, HGT, environmental contamination, and hospital dynamics. To respond to this worldwide threat, action is needed to ensure the appropriate use of antibiotics, enhance infection control practices, and reduce the transmission of bacteria-carrying resistance genes in healthcare, agriculture, and the environment. New antibiotics acting against bacterial growth and survival mechanisms may provide further benefits over classical antibiotics, such as improved activity against resistant pathogens, reduced toxicity, and the potential for resistance development to be delayed. Focusing either on previously unused bacterial processes or combination therapies, these new approaches underline the hope of circumventing the global antibiotic resistance crisis. However, difficulties remain with targeting of the Gram-negative OM in bacterial pathogens, including permeability barriers to macromolecules and efflux pumps that extrude small molecules to prevent their intracellular activity or even cytotoxicity towards bacteria. Figuring out better OM assembly systems, optimizing drug design, and combining therapies are pushing the boundaries toward overcoming these barriers.

In this post-genomic era, with the structure-function-based characterization of a plethora of novel molecular targets such as histidine kinases, protein and lipid biosynthesis enzymes, quorum-sensing systems, and DNA gyrase, such targets provide avenues to generate new therapeutics that evade recognized drug resistance mechanisms and can be used against MDR organisms with broader utility.

Whole-genome and metagenomic methods are central to predicting resistance, tracking its dissemination, and discovering new antibiotic agents. The ongoing development of bioinformatics tools and third-generation sequencing platforms support such tasks. In addition, these strategies can give public health agencies the ability to respond promptly to new threats, forecast resistance, and customize interventions. They will be crucial to tackling the increasing threat of antibiotic resistance and ultimately improving health at a global level with public health frameworks that integrate these technologies.

AI is transforming antibiotic discovery by providing capabilities for novel compound identification, resistance mechanism prediction, and drug combination optimization. But it continues to face challenges, especially around data availability and model explainability.

Economics, regulatory pathways, and scientific hurdles are the main reasons why the pharmaceutical industry has receded from developing antibiotics. Antibiotic treatments consist of a few doses over short periods of time, which means they have a very slim profit margin, even when such drugs are costly to develop, which makes companies reluctant to do antibiotic R&D in the first place. Although some financial incentives, such as market entry rewards and longer exclusivity periods, were introduced in 2005 to try to spur innovation using the profit motive, these developments have signified little actual successful innovation.

There is no panacea for developing new antibacterial drugs; a multifaceted approach, including resistance-resistant strategies, reviving older drugs, targeting bacterial metabolism, and utilizing advanced technologies such as nanotechnology and systems biology, etc., will be essential to stave off this public health crisis for the foreseeable future. The new strategies bring hope in the fight against these multidrug-resistant pathogens but will need more research, funding, and interdisciplinary cooperation to succeed.