1. Introduction

The advent of antimicrobial compounds stands among the most transformative developments in medical science [1]. Fleming's groundbreaking penicillin discovery in 1928 initiated a therapeutic revolution that fundamentally altered infection management protocols, substantially diminishing global disease burden and fatality rates. These medications became instrumental in treating previously fatal conditions including respiratory infections, mycobacterial diseases, and systemic sepsis, while enabling complex medical interventions such as organ replacement procedures, cancer treatments, and extensive surgical operations.

Nevertheless, this therapeutic success has generated unforeseen complications. Antimicrobial resistance (AMR) now constitutes a paramount global health emergency [2]. WHO data indicates that AMR directly causes approximately 1.27 million annual fatalities while contributing to nearly 5 million additional deaths globally, surpassing mortality rates associated with HIV/AIDS and malaria combined [2]. The proliferation of resistant organisms including methicillin-resistant Staphylococcus aureus (MRSA), carbapenem-resistant Klebsiella pneumoniae, and extended-spectrum β-lactamase (ESBL)-producing Escherichia coli has compromised the effectiveness of primary antimicrobial agents [3]. Furthermore, resistance determinants such as NDM-1 and MCR-1 have achieved inter-species and global distribution, signaling the potential onset of a post-antimicrobial era.

Simultaneously, novel antimicrobial development has encountered significant obstacles. Following the 1980s, the drug development pipeline has contracted substantially due to complex scientific, regulatory, and financial barriers. Pharmaceutical enterprises confront substantial research investments, insufficient economic returns, and intricate approval pathways, resulting in considerable delays in discovering and advancing new antimicrobials. While resistant pathogens continue evolving and current medications progressively lose potency, limited new antimicrobial options emerge to replace them.

This review systematically examines diverse bacterial resistance mechanisms and analyzes potential alternative therapeutic strategies. We will comprehensively investigate various resistance pathways including enzymatic degradation, active efflux systems, target site modifications, and protective biofilm formation. Additionally, we explore promising alternative treatments currently under development, including bacteriophage applications and antimicrobial peptide therapies.

2. Theoretical framework

2.1. Antimicrobial action mechanisms and classification systems

Antimicrobial agents prevent bacterial proliferation through bacteriostatic or bactericidal mechanisms that disrupt essential cellular processes. According to their primary molecular targets, antimicrobials are categorized as follows [4]:

(1) Cell Wall Biosynthesis Inhibitors

β-lactam compounds (encompassing penicillins, cephalosporins, carbapenems, and monobactams) function by binding penicillin-binding proteins (PBPs), which facilitate peptidoglycan cross-linking essential for cell wall structural integrity. Interference with this process results in osmotic cell lysis. Glycopeptide antibiotics like vancomycin similarly target cell wall synthesis but bind D-Ala-D-Ala peptidoglycan precursor termini.

(2) Protein Biosynthesis Inhibitors

These agents exploit differences between prokaryotic and eukaryotic ribosomal structures to selectively target bacterial protein synthesis. Aminoglycosides such as gentamicin bind irreversibly to 30S ribosomal subunits, causing translation errors. Tetracyclines like doxycycline block aminoacyl-tRNA binding at ribosomal A-sites. Macrolides, lincosamides, and chloramphenicol target 50S ribosomal subunits, preventing translocation and peptide bond formation.

(3) Nucleic Acid Synthesis Inhibitors

Quinolone antibiotics target DNA gyrase and topoisomerase IV enzymes, disrupting DNA supercoiling and replication processes. Rifamycin compounds interact with bacterial RNA polymerase, blocking transcription initiation.

(4) Cell Membrane Disruptors

Polymyxin antibiotics bind to phospholipid components of gram-negative bacterial outer membranes, compromising membrane integrity. Due to potential kidney toxicity, these agents are typically reserved for severe infections caused by extensively resistant organisms.

(5) Metabolic Pathway Inhibitors

Sulfonamides and trimethoprim target folate metabolism pathways, preventing nucleotide synthesis required for DNA and RNA production.

2.2. Resistance development principles

Antimicrobial resistance emergence involves complex genetic processes including spontaneous mutations, horizontal genetic transfer, and selective evolutionary pressures [4].

(1) Spontaneous Genetic Mutations

Random genomic alterations can confer survival advantages under antimicrobial selection pressure. Examples include quinolone resistance mutations in gyrA or parC genes, and ribosomal RNA modifications causing macrolide or aminoglycoside resistance.

(2) Horizontal Genetic Transfer Mechanisms

Resistance genes spread between bacterial species through transformation (external DNA uptake), transduction (bacteriophage-mediated transfer), and conjugation (direct cell-to-cell transfer). Mobile genetic elements including plasmids, transposons, and integrons facilitate resistance gene dissemination. Colistin-resistant E. coli and carbapenem-resistant Enterobacteriaceae (CRE) have raised global alarm due to horizontal genetic transfer [3].

(3) Active Efflux and Permeability Reduction

Bacterial efflux systems like AcrAB-TolC in E. coli reduce intracellular drug concentrations. Simultaneously, outer membrane protein modifications decrease drug uptake, contributing significantly to multi-drug resistance in gram-negative bacteria [5].

(4) Biofilm-Associated Resistance

Biofilms are structured microbial communities encased in self-produced polymeric matrices. These structures create physical barriers to antimicrobial penetration while inducing metabolic dormancy and altered gene expression patterns that reduce drug susceptibility.

3. Contemporary challenges in antimicrobial resistance

3.1. Multi-drug and extensively drug-resistant pathogen emergence

Current resistance patterns demonstrate alarming trends toward multi-drug resistance across all known antimicrobial classes. The reality is that resistance evolution outpaces new drug development, while pharmaceutical investment in antimicrobial research continues declining [6].

Multi-drug resistance develops through two primary pathways [3]. First, bacteria acquire multiple resistance genes, each conferring protection against specific drug classes, often carried on transferable plasmids. Second, organisms may upregulate genes encoding multi-drug efflux pumps, drug-inactivating enzymes, or target modification systems.

Bacterial strains resistant to three or more antimicrobial categories are classified as multi-drug resistant (MDR). Organisms resistant to all but one or two drug classes are designated extensively drug-resistant (XDR), while strains resistant to all available antimicrobials are termed pan-drug resistant (PDR).

3.2. Detailed resistance mechanism analysis

(1) Enhanced Efflux Pump Activity

Efflux pumps represent energy-dependent membrane transport systems that expel toxic compounds from bacterial cells, reducing intracellular drug concentrations. The initial tetracycline efflux pump was identified in E. coli during 1980, encoded on transferable plasmids that facilitated rapid dissemination [7].

While efflux mechanisms are chromosomally encoded and contribute to intrinsic resistance, clinical resistance typically results from mutations enhancing pump expression or efficiency.

(2) Target Site Modifications

Bacteria reduce antimicrobial effectiveness by altering, modifying, or protecting cellular targets, disrupting drug binding. Only mutations that reduce antimicrobial binding without compromising essential protein functions provide selective advantages.

Quinolone resistance frequently results from mutations in the quinolone-resistance-determining region (QRDR) of DNA gyrase and topoisomerase IV in both gram-positive and gram-negative organisms.

(3) Enzymatic Drug Inactivation

Bacterial production of drug-inactivating enzymes represents a clinically significant resistance mechanism. Primary enzyme categories include β-lactamases, aminoglycoside-modifying enzymes, and chloramphenicol acetyltransferases [4].

β-lactamases hydrolyze β-lactam antimicrobials by cleaving characteristic ring structures in penicillins, cephalosporins, monobactams, and carbapenems. Encoding genes may be chromosomal or located on mobile genetic elements.

(4) Biofilm-Mediated Drug Tolerance

Biofilms are surface-attached microbial communities surrounded by self-produced extracellular matrices. These structures impede antimicrobial penetration while creating concentration gradients that protect deeper cell layers from drug exposure.

Biofilm resistance stems from poor drug penetration combined with intrinsic resistance mechanisms. Surface bacteria may be eliminated while deeper organisms remain protected and viable.

(5) Genetic Material Exchange

Resistance traits transfer vertically through inheritance and horizontally through conjugation, transformation, and transduction. Resistance genes may confer protection against single drugs or multiple antimicrobial classes and are frequently located on transferable genetic elements.

4. Alternative therapeutic strategies

Confronting the intensifying resistance crisis, researchers actively investigate diverse alternative treatment approaches [6]:

4.1. Novel drug development approaches

(1) Combination Antibiotic-Adjuvant Systems

Antibiotic adjuvant combinations represent highly successful therapeutic strategies. Despite progress in β-lactamase inhibitor development, effective inhibitors for class B metallo-β-lactamases remain urgently needed. Currently, no inhibitors exist for these enzymes, whose prevalence among significant gram-negative pathogens continues increasing dramatically [3].

Augmentin exemplifies successful combination therapy, pairing amoxicillin with clavulanic acid. The β-lactamase inhibitor enhances amoxicillin effectiveness by preventing enzymatic inactivation.

(2) Advanced Drug Delivery Technologies

To overcome limited cellular permeability, innovative delivery systems enhance drug cellular entry capabilities. Synthetic siderophore conjugates represent promising approaches for improving antimicrobial uptake. Research demonstrates that ampicillin-siderophore conjugates achieve 100-fold enhanced efficacy against gram-negative enterobacteria and 1000-fold improved activity against P. aeruginosa [6].

4.2. Biological treatment alternatives

(1) Antimicrobial Peptides and Oxidative Therapies

Antimicrobial peptides function as innate immune system components with unique antimicrobial mechanisms. These molecules disrupt bacterial membranes, interfere with cell wall synthesis, or target intracellular components. Specific peptides like Plantaricin A analogs enhance antibiotic efficacy by increasing membrane permeability and synergizing with hydrophobic antibiotics.

Oxidative stress-generating compounds provide attractive strategies for overcoming resistance. Reactive oxygen species successfully eliminate resistant bacteria including multi-drug resistant S. aureus and carbapenemase-producing E. coli [8,9].

(2) Bacteriophage Applications

Rising antibiotic-resistant infections have renewed interest in bacteriophage therapy as conventional antibiotic alternatives. European clinical applications demonstrate successful therapeutic and preventive uses with minimal normal microflora disruption [10].

Recent clinical experiences provide valuable insights into phage therapeutics. Patients infected with resistant Mycobacterium abscessus and antibiotic-resistant Acinetobacter baumannii achieved cures through phage therapy. T4 coliphage treatment successfully managed infant diarrhea caused by enteropathogenic E. coli.

(3) Antibody-Antibiotic Conjugates

Therapeutic antibodies increasingly attract attention as alternative infectious disease treatments. High target antigen specificity makes these molecules attractive for therapeutic applications [11].

Antibody-antibiotic conjugates combine monoclonal antibodies, linker molecules, and cytotoxic drugs for efficient targeted delivery. By targeting pathogen-specific biomolecules while preserving microbiota and activating immune responses, antibacterial monoclonal antibodies demonstrate significant potential for addressing resistance and bacterial infections.

(4) CRISPR-Based Resistance Targeting

CRISPR-Cas systems constitute bacterial adaptive immune mechanisms representing well-characterized antiphage defense systems. Phages develop counter-strategies against bacterial defense mechanisms, though resistance trade-offs between phage and antibiotic susceptibility remain poorly understood [12].

Since phage resistance represents the primary obstacle to phage therapy, trade-offs between phage resistance and antimicrobial resistance (phage steering) can be leveraged clinically. Research demonstrates that targeting pleiotropic trade-offs effectively promotes antibiotic susceptibility and resensitizes resistant bacteria in synergistic phage-antibiotic applications.

4.3. Ecosystem-level management strategies

(1) Integrated One Health Approaches

WHO reports globally elevated bacterial resistance levels, emphasizing One Health approach necessity for addressing resistance crises. This strategy operates across local, national, and global levels through collaboration among policymakers, stakeholders, practitioners, and researchers [2].

One Health represents an integrated approach designed to optimize human, animal, and ecosystem health balance. This concept recognizes that human, domestic and wild animal, plant, and environmental health are interconnected and interdependent.

(2) Policy Development and Antimicrobial Stewardship

WHO, United Nations, and European Union initiatives aim to reduce and restrict antimicrobial use in animals. These include legislating bans on specific antibiotics in agricultural systems for growth promotion and promoting stewardship in treating food animals and companion animals.

WHO developed the Global Action Plan for managing antimicrobial resistance (GAP-AMR) and launched the Global Antimicrobial Resistance and Use Surveillance System (GLASS). These initiatives continuously address knowledge gaps while working toward GAP-AMR program objectives [2].

5. Conclusions

Antibiotic resistance complexity and severity constitute among the most pressing contemporary public health challenges. Through systematic analysis, we clearly observe that bacteria have evolved sophisticated mechanisms countering antibiotic action, including enhanced efflux, target modifications, enzymatic inactivation, biofilm formation, and horizontal gene transfer. These mechanisms frequently operate synergistically, enabling simultaneous resistance development to multiple antibiotics, forming multi-drug or pan-drug resistant strains.

Alternative therapy exploration demonstrates diversified, combinatorial, and precision-oriented development trends. From efflux inhibitors to bacteriophage therapy, from antimicrobial peptides to CRISPR systems, from nanomaterials to antibody-antibiotic conjugates, each strategy exhibits unique advantages and potential. These alternative approaches are not mutually exclusive but can achieve synergistic effects through combined applications, providing renewed hope for overcoming resistance challenges [13].

This analysis emphasizes that addressing resistance crises requires comprehensive strategies involving global collaboration and multidisciplinary integration. This includes accelerating anti-resistance technology clinical translation while considering human, animal, and environmental health relationships under One Health guidance. Policymakers, researchers, clinicians, and the public should collaborate in addressing this global challenge.

Looking forward, emerging platforms including CRISPR systems and nanotechnology are expected to become transformative therapeutic modality cores. As bacterial resistance mechanism understanding deepens and new technologies develop, sustained effort and investment will ultimately enable humanity to prevail against resistant bacteria, providing robust global public health security.