Antibacterial agents are fundamental tools in microbiology, antimicrobial susceptibility testing, resistance mechanism studies, infection model construction, and antibacterial activity evaluation. When selecting antibacterial agents for research, priority should be given to the target of action, antibacterial spectrum, bactericidal or bacteriostatic properties, MIC/MBC, solubility, stability, model compatibility, and interference with detection readouts, rather than simply selecting based on “broad spectrum” or “high potency.”

Keywords: antibacterial agents; antibiotic classification; mechanism of action; antibacterial spectrum; MIC determination; antimicrobial susceptibility testing; resistance mechanism; infection model

1 Research Application Positioning of Antibacterial Agents

1.1 Basic Concepts

(1) Anti-infective agents

Anti-infective agents are a broad concept that covers drugs targeting bacteria, fungi, viruses, parasites, spirochetes, rickettsiae, mycoplasma, chlamydia, and other infectious agents. In research experiments, the target organism should first be clearly defined to avoid generalizing antibacterial agents as all anti-infective agents.

(2) Antibacterial agents

Antibacterial agents mainly refer to drugs that inhibit or kill bacteria, including natural antibiotics, semi-synthetic antibiotics, and fully synthetic antibacterial drugs. They are commonly used for antimicrobial susceptibility testing, inhibition of bacterial growth, time-kill kinetics, validation of resistance mechanisms, intervention in infection models, and positive control setup.

(3) Antibiotics

Antibiotics originally referred to substances produced by microorganisms that can inhibit or kill other microorganisms, such as penicillin, streptomycin, tetracycline, and erythromycin. In modern research contexts, antibiotics are often used interchangeably with antibacterial agents, but synthetic drugs such as quinolones, sulfonamides, and nitroimidazoles are not classical natural antibiotics.

1.2 Core Criteria for Research Selection

(1) Target of action

Antibacterial agents may act on cell wall synthesis, cell membrane integrity, protein synthesis, DNA replication, RNA transcription, or folate metabolism. The target of action determines the experimental readout and direction of mechanistic validation. For example, β-lactams are suitable for studies of cell wall injury and β-lactamase-mediated resistance, while fluoroquinolones are suitable for studies of DNA replication damage and gyrA/parC mutations.

(2) Antibacterial spectrum

The antibacterial spectrum determines whether a drug is suitable for experiments involving Gram-positive bacteria, Gram-negative bacteria, anaerobes, atypical pathogens, mycobacteria, or multidrug-resistant bacteria. In in vitro susceptibility testing, drugs should be selected according to the background of standard strains, clinical isolates, or engineered strains, and naturally resistant drugs should not be used as positive controls.

(3) Experimental concentration

Experimental concentrations are usually designed around MIC, MBC, time-kill curves, sub-inhibitory concentrations, mutant selection windows, or exposure levels in animal models. Cell experiments and animal models cannot directly copy in vitro MIC values; drug solubility, stability, cytotoxicity, tissue distribution, and endpoint interference must also be evaluated.

Table 1 Key Parameters for Selecting Antibacterial Agents in Research

2 Common Antibacterial Drug Classes and Experimental Characteristics

2.1 Cell Wall Synthesis Inhibitors

(1) β-Lactams

β-Lactams include penicillins, cephalosporins, cephamycins, carbapenems, monobactams, and β-lactamase inhibitor combinations. These drugs inhibit penicillin-binding proteins, block peptidoglycan cross-linking, and are typical bactericidal agents active during bacterial proliferation. In research, they are commonly used in studies of cell wall injury, β-lactamase-mediated resistance, PBP mutations, ESBL-producing strains, CRE strains, and drug combination sensitization. Experimental design should consider drug hydrolysis, culture medium conditions, inoculum effect, and activity restoration by β-lactamase inhibitors.

(2) Glycopeptides

Glycopeptides are represented by vancomycin, norvancomycin, and teicoplanin. They inhibit peptidoglycan elongation and cross-linking by binding to the D-Ala-D-Ala structure of cell wall precursors. These drugs mainly target Gram-positive bacteria and are ineffective against Gram-negative bacteria because of the outer membrane barrier. In research, they are suitable for studies of MRSA, MRCNS, resistant enterococci, Gram-positive cell wall thickening, vancomycin tolerance, and glycopeptide resistance phenotypes.

(3) Fosfomycin class

Fosfomycin inhibits an early step in cell wall synthesis, with a target upstream of β-lactams. In research, it is commonly used for studies of the MurA target, phosphosugar transport systems, urinary tract infection-related strains, transport mutations, and combination sensitization. Fosfomycin susceptibility testing is sensitive to medium and supplements, and some systems require control of conditions such as glucose-6-phosphate.

2.2 Protein Synthesis Inhibitors

(1) Aminoglycosides

Aminoglycosides act on the bacterial 30S ribosomal subunit, interfere with protein translation, and disrupt cell membrane integrity. They are concentration-dependent bactericidal agents. Representative drugs include streptomycin, gentamicin, tobramycin, amikacin, kanamycin, and neomycin. In research, they are commonly used for aerobic Gram-negative bacilli, Pseudomonas aeruginosa, combination regimens for Mycobacterium tuberculosis, ribosomal target mutations, and resistance mediated by aminoglycoside-modifying enzymes. These drugs are ineffective against anaerobes, and mitochondrial translation interference should be considered in cell experiments.

(2) Macrolides

Macrolides act on the 50S ribosomal subunit and inhibit protein elongation. They are usually bacteriostatic agents. Representative drugs include erythromycin, roxithromycin, clarithromycin, and azithromycin. In research, they are commonly used for studies of Mycoplasma, Chlamydia, Legionella, Streptococcus, intracellular pathogens, and biofilms. Drugs such as azithromycin can also affect inflammatory responses and immune cell function, so drug blank controls should be included when cytokines or host responses are investigated.

(3) Tetracyclines and glycylcyclines

Tetracyclines act on the 30S ribosomal subunit and prevent aminoacyl-tRNA from entering the ribosomal A site. Representative drugs include tetracycline, doxycycline, and minocycline. Tigecycline is the representative glycylcycline and retains activity against some resistant bacteria. In research, these drugs are commonly used for Mycoplasma, Chlamydia, Rickettsia, Brucella, multidrug-resistant Acinetobacter baumannii, tet resistance genes, efflux pumps, and ribosomal protection proteins. Doxycycline is also commonly used in Tet-On/Tet-Off gene expression regulation systems, in which its antibacterial activity may interfere with microbial models.

(4) Chloramphenicols

Chloramphenicols act on the peptidyl transferase center of the 50S ribosomal subunit and inhibit protein synthesis. Representative drugs include chloramphenicol and thiamphenicol. In research, they are commonly used for bacterial translation inhibition, cat resistance genes, plasmid selection markers, cerebrospinal fluid penetration-related models, and broad-spectrum bacteriostatic controls. When used in mammalian cells or animal models, mitochondrial translation interference and bone marrow suppression-related toxicity should be considered.

(5) Lincosamides

Lincosamides include lincomycin and clindamycin. They act on the 50S ribosomal subunit and show good activity against Gram-positive bacteria and anaerobes. In research, they are commonly used for anaerobes, skin and soft tissue infection models, toxin expression regulation, and Gram-positive bacterial protein synthesis inhibition studies. Clindamycin can inhibit toxin production in some bacteria; therefore, virulence factor experiments should distinguish antibacterial effects from toxin expression suppression.

(6) Oxazolidinones

The representative oxazolidinone is linezolid, which inhibits the initiation phase of bacterial protein synthesis and is active against Gram-positive bacteria such as MRSA, VRE, and resistant streptococci. In research, it is suitable for studies of resistant Gram-positive bacteria, ribosomal initiation complexes, VRE models, and control experiments for candidate drugs against resistant bacteria. This class is ineffective against most Gram-negative bacilli and is not suitable as a positive control in Gram-negative bacterial models.

2.3 Nucleic Acid Synthesis and Metabolic Intervention Agents

(1) Fluoroquinolones

Fluoroquinolones act on DNA gyrase and topoisomerase IV, blocking DNA replication. They are concentration-dependent bactericidal agents. Representative drugs include norfloxacin, ciprofloxacin, levofloxacin, and moxifloxacin. In research, they are commonly used for studies of DNA damage responses, SOS responses, gyrA/parC mutations, mutant selection windows, resistance evolution, and biofilm clearance. Systems containing metal ions such as calcium, magnesium, aluminum, and iron may reduce the effective drug concentration.

(2) Rifamycins

Rifamycins inhibit DNA-dependent RNA polymerase. Representative drugs include rifampicin, rifapentine, and rifabutin. In research, they are commonly used for studies of mycobacteria, tuberculosis models, RNA transcription inhibition, rpoB mutations, and combination therapy in biofilms. Rifampicin monotherapy can very easily select resistant mutants, so combination conditions are usually required in resistance experiments and infection models.

(3) Sulfonamides

Sulfonamides exert bacteriostatic effects by inhibiting the folate synthesis pathway. Trimethoprim-sulfamethoxazole enhances activity by dual blockade of folate metabolism through sulfamethoxazole and trimethoprim. In research, they are commonly used for folate metabolism studies, Stenotrophomonas maltophilia, Pneumocystis, Nocardia, some MRSA strains, and selective culture. Thymidine and folate-related components in the culture medium may affect pharmacodynamic results.

(4) Nitroimidazoles

Nitroimidazoles include metronidazole, tinidazole, and ornidazole. They are mainly reduced and activated under anaerobic conditions to produce metabolites that damage DNA. In research, they are suitable for anaerobic bacteria, mixed intra-abdominal flora, periodontal flora, Clostridioides difficile-related models, and bactericidal mechanism studies under anaerobic conditions. These drugs are ineffective against aerobic bacteria, and anaerobic conditions must be strictly controlled during experiments.

(5) Nitrofurans

Nitrofurans include nitrofurantoin, furazolidone, and nitrofurazone. They exert antibacterial effects through multi-target interference with bacterial metabolism and nucleic acid damage. In research, they are mainly used for urinary tract-related strains, intestinal bacterial models, and evaluation of topical antibacterial materials. Nitrofurantoin is more suitable for urine-related concentration models and is not suitable as a positive control in systemic infection models.

2.4 Cell Membrane-Targeting Agents

(1) Polymyxins

Polymyxins include polymyxin B and polymyxin E. They exert bactericidal effects by binding lipopolysaccharide in the outer membrane of Gram-negative bacteria and disrupting membrane integrity. In research, they are commonly used for studies of multidrug-resistant Gram-negative bacteria, carbapenem-resistant Enterobacterales, Acinetobacter baumannii, Pseudomonas aeruginosa, membrane permeability, and LPS structure. In vitro experiments should consider the effects of drug adsorption, protein binding, and inoculum effect on MIC results.

(2) Cyclic lipopeptides

The representative cyclic lipopeptide is daptomycin, which binds to the Gram-positive bacterial cell membrane, causes membrane potential depolarization, and kills bacteria. In research, it is commonly used for studies of MRSA, VRE, streptococci, infective endocarditis models, membrane depolarization, and pharmacodynamic evaluation of resistant Gram-positive bacteria. Daptomycin is ineffective against Gram-negative bacteria and can be inactivated by pulmonary surfactant, so it is not suitable as a positive drug in pneumonia models. In vitro experiments should consider the effect of calcium concentration on antibacterial activity.

Table 2 Representative Antibacterial Agents and CAS Numbers

3 Selection Scenarios for Antibacterial Agents in Research Experiments

3.1 In Vitro Antibacterial Activity Evaluation

(1) MIC and MBC determination

MIC is used to evaluate the lowest concentration that inhibits visible bacterial growth, while MBC is used to evaluate bactericidal activity. Bactericidal agents such as β-lactams, aminoglycosides, fluoroquinolones, polymyxins, and daptomycin are more suitable for further MBC determination and time-kill curve analysis. Bacteriostatic agents such as macrolides, tetracyclines, and sulfonamides are more suitable for integrated interpretation with growth curves, OD readings, and CFU counts.

(2) Time-kill curves

Time-kill curves are used to distinguish rapid killing, delayed killing, regrowth, and tolerance. Concentration-dependent drugs can be tested at multiple MIC-based gradients to observe killing rates. Time-dependent drugs should focus on exposure duration and maintained concentration. For persister cells, biofilm bacteria, and slow-growing bacteria, the observation period should be extended and combined with CFU counting.

(3) Combination therapy evaluation

Checkerboard dilution assays, FICI calculation, time-kill curves, and agar diffusion methods can be used to evaluate synergy, additivity, indifference, or antagonism. β-Lactams combined with β-lactamase inhibitors, β-lactams combined with aminoglycosides, and polymyxins combined with tigecycline or carbapenems are common combinations in resistant bacterial research.

3.2 Resistance Mechanism Studies

(1) Enzyme-mediated resistance

β-Lactamases, aminoglycoside-modifying enzymes, and chloramphenicol acetyltransferase can directly alter drug activity. In experiments, resistance mechanisms can be validated through inhibitor restoration assays, gene knockout/overexpression, susceptibility changes, and enzyme activity assays.

(2) Target mutations

Fluoroquinolones are commonly used for studies of gyrA, gyrB, parC, and parE mutations. Rifampicin is commonly used for rpoB mutation screening. Linezolid and macrolides can be used for studies of altered ribosomal targets. Target mutation experiments should combine sequencing, MIC changes, and growth fitness evaluation.

(3) Efflux pumps and membrane permeability

Tetracyclines, macrolides, fluoroquinolones, and polymyxins are commonly used in studies of efflux pumps, outer membrane proteins, and LPS structural changes. Efflux pump inhibitors, membrane permeability dyes, and membrane potential assays can help determine the source of resistance.

3.3 Cell Infection Models

(1) Intracellular pathogen models

Macrolides, tetracyclines, fluoroquinolones, and rifamycins are commonly used in intracellular pathogen studies. Experimental design should evaluate drug entry into cells, host cell cytotoxicity, and changes in intracellular bacterial burden, rather than judging antibacterial effects only from the drug concentration in the culture medium.

(2) Host response interference

Some antibacterial agents can affect mitochondrial function, cytokine release, ROS levels, and immune signaling pathways. When inflammatory factors, cell viability, apoptosis, or immune phenotypes are evaluated, drug blank controls, solvent controls, and sterile cell controls should be included.

3.4 Animal Infection Models

(1) Compatibility with infection site

Drugs in animal models should be selected according to infection site. Lung models require consideration of lung tissue and the alveolar environment; urinary tract models require consideration of urinary drug concentration; intra-abdominal, wound, and biofilm models require consideration of local exposure and mixed infections. Drug concentration design should be based on preliminary experiments, pharmacokinetic data, and model endpoints, and should not directly copy in vitro MIC values.

(2) Distinguishing pharmacodynamic effects from host effects

In animal models, drug effects may result from direct bactericidal activity, bacteriostatic activity, immunomodulation, or reduced inflammation. Experimental endpoints should include bacterial burden, histopathology, inflammatory factors, animal status, and drug exposure data to avoid directly equating improved host responses with enhanced antibacterial activity.

3.5 Biofilm Models

(1) Model compatibility

Biofilm bacteria are significantly less susceptible to antibacterial agents than planktonic bacteria. Polymyxins, aminoglycosides, fluoroquinolones, rifampicin, and some β-lactams are commonly used for biofilm killing and combination therapy evaluation.

(2) Result interpretation

Biofilm experiments should simultaneously assess total biofilm biomass, viable bacterial count, metabolic activity, and structural changes. Crystal violet staining only reflects total biofilm biomass and is not equivalent to viable bacterial killing. CFU counting, live/dead staining, and microscopic imaging can improve result reliability.

Table 3 Key Selection Points for Antibacterial Agents in Research Experiments

4 Common Experimental Questions and Result Interpretation

4.1 How should antibacterial drug concentration gradients be set in MIC experiments?

Concentration gradients are usually prepared as two-fold serial dilutions around the expected MIC. For strains with unknown susceptibility, a broader range can be used first, followed by a narrowed gradient according to preliminary screening results. For poorly soluble drugs, the maximum soluble concentration and final solvent concentration should first be confirmed to avoid precipitation, pH changes, or solvent toxicity affecting readouts.

4.2 How should positive control antibacterial agents be selected?

Positive controls should match the bacterial strain and experimental purpose. For Gram-positive bacterial models, vancomycin, linezolid, daptomycin, or suitable β-lactams can be selected. For Gram-negative bacterial models, ciprofloxacin, amikacin, meropenem, or polymyxins can be selected. For anaerobic bacterial models, metronidazole or clindamycin can be used. For atypical pathogen models, macrolides, tetracyclines, or fluoroquinolones can be selected.

4.3 How should in vitro efficacy with no effect in animal models be analyzed?

The first points to check are whether drug exposure is sufficient, whether effective concentrations can be reached at the infection site, whether the drug is restricted by protein binding or tissue barriers, whether biofilms or abscesses are present in the model, whether host immune status has changed, and whether the culture conditions used in vitro overestimated drug activity.

4.4 Can antibacterial agents interfere with cell viability assays?

Some antibacterial agents can affect mitochondria, redox reactions, fluorescent probes, absorbance readings, or cellular metabolic enzyme activity. In CCK-8, MTT, ATP luminescence, ROS, and membrane potential assays, drug blank wells, solvent controls, and bacteria-free cell controls should be included.

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