MICROBIOLOGY - M.H. Serhiichuk - 2008

Chapter 11. ANTIBIOTICS. PROBIOTICS

Antibiotics and Their Classification

The history of Antibiotics dates back to prehistoric times. The Maya Indians used green mold growing on corn kernels to heal wounds. They treated malaria with medicines derived from the bark of the cinchona tree (Cinchona officinalis) (Fig. 11.1).

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Fig. 11.1. Cinchona officinalis

This empirical knowledge was not lost and was later used by Europeans to produce cinchona, the first systemic medicinal remedy in Western medicine. However, The Challenge of complex and hitherto incurable infectious diseases (such as Sexually Transmitted Infections and fevers) spurred the search for more effective remedies.

Advances in chemical synthesis not only enabled The production of synthetic analogs of natural medicines—such as quinine, the most abundant alkaloid in cinchona—but also drove the search for novel synthetic drugs. Thus, in 1912, German chemist P. Ehrlich (1854–1915) introduced salvarsan (aminophenylarsonic acid), the first agent with specific activity against trypanosomes and the CAUSATIVE AGENT OF Syphilis. The second modern synthetic antimicrobial drug, introduced in 1935, was prontosil, which contained sulfanilamide. It was implemented as a Treatment against gram-positive cocci in various infections by the German bacteriologist G. Domagk (1895–1964), for which he was awarded the Nobel Prize in 1939 (Fig. 11.2).

Fig. 11.2. Chemical Structure of the first natural and synthetic antimicrobial drugs

P. Ehrlich laid the foundations of Chemotherapy, in particular, formulating METABOLISM/2.html">THE CONCEPT OF selective drug toxicity and advancing The Study of antimetabolites—structural analogs of natural cellular metabolites. These scientific principles guided The Development of several generations of modern synthetic chemotherapeutic agents: sulfonamides, nitrofurans, imidazoles, and fluoroquinolones.

Another fundamental doctrine, The Theory of antagonism, paved the way for a second direction in chemotherapy: the production and application of biosynthetic antimicrobial substances. Attempts to use mold Fungi date back to the Middle Ages. For instance, British chronicles describe the successful treatment of wounds with "moss from skulls." In the East, the philosopher and physician Avicenna recommended using mold for purulent lesions.

However, the first scientific study on the Practical Application of antagonistic interactions between Molds and infectious pathogens was published in 1871 by Russian physicians V. Manassein (1841–1901) and A. Polotebnov (1838–1908). Unfortunately, it remained relatively obscure, meaning the systematic study of microbial antagonism was largely catalyzed by the research of L. Pasteur, N. Gamaleya, and I. Mechnikov.

The scientific rationale for the antagonism of the mold fungus genus Penicillium against staphylococci was established by British microbiologist A. Fleming in 1928, who pointed out the potential of obtaining an antibacterial substance produced by the fungus. Subsequent research on Penicillium fungi, initiated by E. Chain (1906–1979) and H. Florey (1898–1968), not only enabled the isolation of this substance but also led to the creation of the first antibiotic—penicillin (1940). For this discovery, Fleming, Chain, and Florey were awarded the Nobel Prize in 1945.

Following this discovery, recognition grew regarding the antibacterial activity of Bacteria of the genus Streptomyces, established in the late 1930s by American microbiologist S. Waksman (1888–1973), Russian microbiologist M. Krasilnikov (1896–1973), and other researchers. Building on these findings, S. Waksman introduced the second modern antibiotic, streptomycin, in 1943, for which he received the Nobel Prize in 1952 (Fig. 11.3).

Fig. 11.4. Nobel laureates for the discovery of antibiotics S. Waksman and A. Fleming

The subsequent decades became known as the era of antibiotics, characterized by large-scale screening and production of antibiotics from fungi, bacteria, and other organisms.

The term "antibiotics," proposed by S. Waksman in 1942, originally referred to "chemical substances produced by microorganisms that are capable of inhibiting the growth of, or even destroying, bacteria and other microorganisms."

As our understanding of antibiotics has expanded and deepened, the definition of this concept has also evolved.

Antibiotics are chemotherapeutic substances produced by various organisms during their life processes—differing from regular metabolites by their Specificity and high activity—as well as synthetic analogs of these substances, which are capable of selectively inhibiting the growth of pathogens (Viruses, bacteria, fungi, Algae, Protozoa, helminths) and/or halting the development of malignant tumors.

Approaches to the Classification of antibiotics depend on professional Perspectives. Academic classifications are typically based on chemical structure and MECHANISM OF ACTION (Fig. 11.4, 11.5). Classification by spectrum of action, much like classification by biological origin, serves practical purposes in medical and scientific fields.

Fig. 11.4. Classification of antibiotics by chemical structure

Fig. 11.5. Targets of antibacterial antibiotics

The first antibiotic, penicillin, was obtained from the strain Penicillium chrysogenum (Fig. 11.6). This antibiotic marked the beginning of the family of classic β-lactam antibiotics.

Fig. 11.6. Colony and conidiophore of the fungus Penicillium chrysogenum

Natural Penicillins are a mixture of 4–5 structurally and functionally related compounds based on the penicillin core molecule: 6-aminopenicillanic acid (6-APA).

6-Aminopenicillanic acid (6-APA) is a β-lactam-thiazolidine heterobicyclic system, The structure of which was established in 1959 (Fig. 11.7).

Fig. 11.7. STRUCTURE OF THE penicillin core molecule

The synthesis of β-lactam antibiotics, particularly penicillins, occurs via Amino acid metabolism reactions as secondary metabolites. Aminoadipic acid and Cysteine form a dipeptide, which, with the participation of valine, is converted into a tripeptide, followed by Condensation into the cyclic structure of isopenicillin N (L-AAA-6-APA). The Diversity of natural penicillins is provided by the subsequent acylation reaction,

catalyzed by penicillin acylase in the presence of specific penicillin precursors—such as phenylacetic acid, phenoxyacetic acid, phenylethylamine, etc. These precursors determine the Structural Features of the side chain.

The technology of penicillin production became foundational for many Other Antibiotics. Initially, until 1946, surface cultivation of producers was carried out in liquid media. While this had the advantage of maximum oxygen supply, it made it impossible to remove excess heat and metabolic products, was labor-intensive, and involved high costs—one dose of antibiotic was obtained from 10–20 liters of culture liquid. Submerged cultivation overcame the drawbacks of surface cultivation, stimulated the development of aeration facilities, and currently prevails as the primary cultivation method.

The antibiotic is extracted from the producer's culture liquid by precipitation Methods to yield highly purified salt forms. The potency of the preparations is expressed in activity units or weight units.

Natural penicillins are narrow-spectrum antibiotics active against gram-positive cocci, predominantly staphylococci. Some penicillins (benzathine penicillins) are active against gonococci and treponemes (Table 11.1).

Table 11.1. Classic β-lactam antibiotics

In the early years of antibiotic therapy, there was an urgent need for broad-spectrum antibiotics—that is, those effective against both gram-positive and gram-negative bacteria. This drove the development of semi-synthetic derivatives based on a chemically modified biosynthetic antibiotic core.

Ampicillin was the first semi-synthetic penicillin. Like other semi-synthetic penicillins, it has a broad spectrum of activity and is acid-resistant, particularly in the gastric environment, unlike most natural penicillins which are administered by injection (Table 11.1).

Overall, over 50,000 semi-synthetic penicillins have been created, of which only 35–40 are used in clinical practice. The major drawback of both natural and semi-synthetic penicillins is their high allergenicity.

The strategy of developing semi-synthetic drugs became leading for another group of classic β-lactam antibiotics: the cephalosporins. The producer of the biosynthetic cephalosporin core, the microscopic fungus Cephalosporium acremonium (syn. Acremonium strictum), was first isolated in 1946. Due to the very low antimicrobial activity of the natural compound, the development of semi-synthetic drugs was initiated.

The Biosynthesis pathway of natural cephalosporins is similar to that of penicillins.

The chemical similarity of cephalosporins lies in the presence of a β-lactam ring. The core of the cephalosporin molecule is 7-aminocephalosporanic acid (7-ACA), a β-lactam-thiazine heterobicyclic system (Fig. 11.8).

Fig. 11.8. Structure of the cephalosporin core molecule

The molecule of the biosynthetic cephalosporin core offers greater modification possibilities; therefore, about 60,000 semi-synthetic derivatives have been created, of which more than 100 are used clinically.

Today, half of the antibiotics produced worldwide are cephalosporins, making β-lactams the leading class among all antibiotics.

It has also been established that cephalosporins can be obtained not only from fungi, but also from bacteria of the genus Streptomyces, which led to the development of cephalosporin analogs known as cephamycins.

Cephalosporins and cephamycins are classic β-lactams—broad-spectrum, highly active antibiotics spanning several generations, characterized by low toxicity and allergenicity, and ease of use (Table 11.1).

The Mechanism of action of classic β-lactam antibiotics (penicillins, cephalosporins, cephamycins) is linked to peptidoglycan synthesis. During the final Stages of peptidoglycan synthesis, transpeptidases catalyze The formation of cross-links within the unfinished polymer. It is these Enzymes with transpeptidase activity that serve as targets for penicillin, which is why they are called PBPs (penicillin-binding Proteins). By binding to the Active Site of the enzyme, they form an inactive penicilloyl-enzyme complex, thereby disrupting the completion of The Cell wall. A cell wall with unfinished peptidoglycan is defective and unable to perform its Functions,

while simultaneously activating The Cell's autolytic enzymes, ultimately leading to cell death.

β-lactams are not the only antibiotics capable of disrupting peptidoglycan synthesis. The Initial Stages of its synthesis are inhibited by D-cycloserine and fosfomycin, which act as structural analogs of Amino Acids. The synthesized peptidoglycan precursors can be blocked directly or via a carrier by peptide antibiotics such as vancomycin, ristocetin, or bacitracin, respectively.

β-lactam antibiotics, like other inhibitors of Introduction/4.html">Prokaryotic Cell wall synthesis, effectively target Gram-positive bacteria, whose peptidoglycan layer is not protected by an outer membrane. Their efficacy against Gram-negative bacteria depends on the molecular structure of the semi-synthetic β-lactams and the density of the outer membrane.

The declining efficacy of both natural and semi-synthetic β-lactam antibiotics is primarily associated with the action of β-lactamases (penicillinases and cephalosporinases). These are protective enzymes produced by microorganisms capable of cleaving the β-lactam ring within the antibiotic structure.

Carbapenems and Monobactams represent a major breakthrough in the development of modern antibiotics. These are non-classic β-lactam antibiotics (Table 11.2).

Table 11.2. Non-classic β-lactam antibiotics

Carbapenems feature a biheterocyclic structure incorporating a β-lactam ring. Monobactams possess a monocyclic core structure containing a β-lactam ring.

The non-classic nature of carbapenems and monobactams as β-lactams is related not only to their structural features, but also to their biological activity. Their primary mechanism of action is considered to be the inhibition of β-lactamases, which is why they are typically included in potentiated formulations of β-lactam antibiotics. Well-known Examples include Augmentin, a complex of amoxicillin and clavulanic acid, and Unasyn, a complex of ampicillin and sulbactam sodium.

The second known antibiotic after penicillin was streptomycin, which also marked the beginning of a new family of antibiotics—the Aminoglycosides. Obtained in 1943 from Streptomyces griseus, streptomycin was the first highly effective biosynthetic antituberculosis agent.

With the discovery of aminoglycoside antibiotics, streptomycetes became the leading producers of various antibiotics (Fig. 11.9).

Fig. 11.9. Branched Cells and a colony of bacteria of the genus Streptomyces

The modern family of aminoglycosides includes over 100 natural compounds synthesized by actinomycetes and bacteria of the genus Bacillus, as well as their semi-synthetic derivatives.

An aminoglycoside molecule consists of a core of a six-membered carbocyclic amino alcohol (aminocyclitol) linked via a glycosidic bond to one or more common or specific amino sugars (Fig. 11.10).

Fig. 11.10. Structure of the streptomycin molecule

An Analysis of the biosynthetic pathways of aminoglycoside antibiotics has demonstrated that they are closely linked to the Carbohydrate Metabolism of the producer cells. Ion-exchange sorption methods are the primary techniques used for Processing the culture broth to obtain aminoglycoside preparations.

Aminoglycosides are antibiotics produced by various bacteria, possessing a broad spectrum of activity with distinct effects on specific groups of microorganisms (Table 11.3).

The mechanism of action of aminoglycosides involves the formation of an aberrant initiation complex by binding to the 30S ribosomal subunit, which blocks the initiation of Translation. Aminoglycosides may also inhibit Translation termination.

Despite their widespread use, aminoglycoside antibiotics are administered exclusively via injection due to low gastrointestinal absorption, high toxicity, and a rapid rate of resistance development, placing them only fourth among other antibiotic classes.

Ranking second in terms of usage volume are non-polyene macrolide antibiotics.

The first non-polyene macrolide antibiotic, erythromycin, was isolated in 1952 from the culture broth of Streptomyces erythreus (synonym Saccharopolyspora erythraea). Soon after, oleandomycin and other natural non-polyene macrolides were obtained.

Table 11.3. Characteristics of aminoglycoside antibiotics

A structural feature of these compounds is the presence of a 14- or 16-membered lactone ring with sugar residues (Fig. 11.11).

Fig. 11.11. Structure of the erythromycin molecule

The synthesis of compounds with a lactone structure occurs via fatty acid metabolism pathways involving propionyl-CoA and malonyl-CoA, followed by polyketo acid condensation.

Not only natural antibiotics are obtained, but also the biosynthetic bases for numerous semisynthetic macrolide derivatives, which differ in both their structure and spectrum of activity (Table 11.4).

The mechanism of action of non-polyene macrolides is associated with Protein Synthesis AND involves blocking 23S rRNA within the peptidyl transferase region of the large ribosomal subunit, which inhibits peptide translocation from the aminoacyl center to the peptidyl center of the ribosome.

Non-polyene macrolide antibiotics are characterized by low toxicity, high bioavailability, and activity against antibiotic-resistant bacteria. Azalides, pioneered by azithromycin—a highly active, long-acting antibiotic—are considered the most promising macrolides.

Among Protein Synthesis Inhibitors, Lincosamides (pyranosides) are well known. These antibiotics have been studied since 1962, when the production of lincomycin by a strain of Streptomyces lincolniensis was established.

The molecular structure of these antibiotics is characterized by the presence of a pyrrolidine ring linked via a glycosidic bond to a sugar moiety (Fig. 11.12).

Table 11.4. Natural, synthetic, and semisynthetic macrolide antibiotics

In addition to the natural antibiotic lincomycin, its chloro-deoxy derivative, clindamycin, is used; it is 2–10 times more active than lincomycin, particularly against Peptococcus and Peptostreptococcus species. Lincosamides exhibit activity against most Gram-positive cocci (staphylococci, pneumococci, and other streptococci), with the exception of enterococci, as well as non-spore-forming anaerobic bacteria (bacteroides, eubacteria, fusobacteria, etc.). These antibiotics are not active against bacteria of the genera Haemophilus, Neisseria, and Clostridium.

The mechanism of action of lincosamides involves blocking the formation of bacterial polyribosomes, which disrupts the translation process. The widespread use of lincosamides is limited by potential dysbiosis resulting from the elimination of the anaerobic flora.

Tetracycline antibiotics are widely used in medical practice. They compete with macrolide antibiotics in terms of frequency of use.

Fig. 11.12. Structure of the lincomycin molecule

The first tetracycline was obtained in 1948 from the culture broth of Streptomyces aureofaciens. This antibiotic was chlortetracycline and was named aureomycin. Currently, over 30 biosynthetic Tetracyclines are known.

The tetracycline molecule consists of one aromatic and three hydroaromatic rings (Fig. 11.13).

Fig. 11.13. Structure of the tetracycline molecule

The biosynthesis of tetracyclines follows the polyketide pathway involving malonyl-CoA and malonamyl-CoA, followed by decarboxylation, methylation, and cyclization.

Unlike other antibiotics, the search for semisynthetic tetracyclines has proven less fruitful. Nevertheless, the derivatives obtained have already found widespread application (Table 11.5).

Table 11.5. Natural and semisynthetic tetracyclines

The mechanism of action of tetracycline antibiotics involves the inhibition of both 30S and 40S ribosomal subunits by blocking the binding of aminoacyl-tRNA to the aminoacyl site of the ribosome.

Tetracyclines are broad-spectrum bacteriostatic antibiotics. High toxicity, the development of microbial resistance to these drugs, and the creation of more effective and safer antibiotics from other groups have narrowed the clinical indications for tetracyclines. Semisynthetic antibiotics of this family possess improved pharmacokinetic properties and activity against bacteria resistant to natural tetracyclines.

Among protein synthesis inhibitor antibiotics, aromatic antibiotics—represented by chloramphenicol and levomycetin—also have a significant history of clinical use.

Chloramphenicol, isolated from the culture broth of the actinomycete Streptomyces venezuelae and investigated as early as 1947, became the first highly effective agent against rickettsiae. Structurally, chloramphenicol is a nitrophenyl dichloroacetamido propanediol (Fig. 11.14).

Fig. 11.14. Structure of the chloramphenicol molecule

The biosynthesis of this antibiotic is closely linked to the biosynthesis of aromatic amino acids.

Following the determination of its structure in 1950, the production of the synthetic analog of chloramphenicol, levomycetin, was established. Levomycetin represents the D-threo-form within the racemic drug synthomycin. Chemical modification of chloramphenicol yielded thiamphenicol (thiocymetin).

Due to their relatively simple chemical structure, aromatic antibiotics are produced on an industrial scale via total chemical synthesis.

Chloramphenicol (levomycetin) exhibits a broad spectrum of activity. It acts bactericidally against bacteria of the genera Haemophilus, Neisseria, and Streptococcus, and bacteriostatically against Shigella, Proteus, Salmonella, Escherichia, Mycoplasma, Rickettsia, Chlamydia, Bacteroides, Fusobacterium, and Clostridium.

Among the complications associated with aromatic antibiotics, Bone Marrow suppression is the primary concern; however, The Use of thiamphenicol helps avoid anemic states. The development of microbial resistance to these antibiotics remains another challenge in their clinical application.

The mechanism of action of these antibiotics involves the inhibition of the 50S ribosomal subunit by blocking the region that catalyzes peptide chain elongation during protein synthesis.

Members of related antibiotic families can also affect various organisms. Specifically, non-polyene macrolide antibiotics are known as inhibitors of bacterial protein synthesis, whereas polyene macrolides act as inhibitors of fungal Membrane Functions.

Polyenes contain from three to eight double bonds within their lactone ring. Depending on the number of these bonds, they are classified as trienes, pentaenes, hexaenes, and heptaenes. Their molecular structure features a hydrophilic region bearing hydroxyl groups and a hydrophobic region (Fig. 11.15).

Fig. 11.15. Structure of the nystatin molecule

Producers of polyene antibiotics have been known since 1950, when nystatin was isolated from the culture fluid of Streptomyces noursei. In 1955, amphotericin was obtained from the culture broth of

Streptomyces nodosus. Amphotericin B is widely recognized as the gold standard for the spectrum and level of antifungal activity.

The best-known examples include the hexaene nystatin, and the heptaenes levorin, amphotericin, and mycoheptin (Table 11.6).

Table 11.6. Polyene antibiotics

The mechanism of action of polyene antibiotics involves the formation of pores within fungal cell membranes. The hydrophobic Regions of the antibiotic molecules form complexes with membrane ergosterol, while the hydrophilic regions are oriented toward the interior of the channel pore. Such channels disrupt fungal membrane function by reducing its selectivity and increasing permeability.

Polyenes are capable of binding to both ergosterol and Cholesterol, which accounts for the significant side effects associated with these drugs. These adverse effects limit their widespread clinical use in fungal infections and drive the search for more effective and safer alternatives.

High toxicity is also characteristic of other membrane-active peptide antibiotics. For this reason, their medical use is limited, though they are widely used in scientific research to study membrane transport.

The formation of channels in bacterial membranes can be facilitated by gramicidin A, a linear peptide antibiotic synthesized by the bacterium Bacillus brevis. Depsipeptide antibiotics, consisting of Amino Acids and hydroxy acids, form a closed-chain structure—an "ion trap"—in nonpolar environments. The cavity size of this trap matches the size of the corresponding ion, which ensures the selectivity of the antibiotic's action. By their mechanism of action, these antibiotics act as ionophores, or ion carriers. The outer sphere of the antibiotic is hydrophobic, allowing it to cross the lipid layer of the membrane. Such antibiotics include valinomycin, enniatins, nonactin, monactin, etc. Their producers are bacteria of the genus Streptomyces and fungi of the genus Fusarium.

Most peptide antibiotics have a cyclic structure and act as membrane disruptors (Table 11.7).

Table 11.7. Cyclopeptide antibiotics

The mechanism of action of these antibiotics involves the formation of complexes with Phospholipids, which alters the cationic permeability of the membrane.

Structurally, cyclic peptide antibiotics differ in their amino acid residue content and structural configuration. For instance, gramicidin S is a cyclodecapeptide, polymyxins have a linear-cyclic structure, and subtilin and nisin are peptide-polycyclopeptides.

The spectrum of activity of these antibiotics covers predominantly Gram-positive bacteria, with the exception of polymyxins, which affect Gram-negative bacteria.

The medical application of cyclic peptide antibiotics is limited by their high toxicity; however, they remain drugs of choice when pathogens are resistant to other antibiotics. Some antibiotics, such as subtilin and nisin, are well-known as preservatives in the food industry.

Antibacterial agents also include the cyclic peptide bacitracin and the polycycleglycopeptides vancomycin and ristomycin, which were mentioned in the Discussion of peptidoglycan synthesis-inhibiting antibiotics.

Among peptide antibiotics, There are also those capable of affecting Eukaryotic cells—particularly tumors—in addition to microorganisms. These include chromopeptides (actinomycins) and glycopeptides (bleomycins).

Nucleotropic antibiotics are synthesized by various species of actinomycetes and are represented by various compounds (Table 11.8).

Table 11.8. Nucleotropic antibiotics

By their mechanism of action, mitosanes act as DNA strand "cross-linkers," which disrupts strand Separation during Replication. Glycopeptides and picolinic acid derivatives cause DNA breaks. Anthracyclines, aureolic acid derivatives, and chromopeptides are capable of intercalation—insertion into the DNA molecule—thanks to their chromophore, which is a complex of 6-membered rings.

A classic intercalator is actinomycin D. The actinomycin molecule contains a chromophoric phenoxazine group and two peptide lactones consisting of five protein and non-protein amino acids (Fig. 11.16).

Upon interaction with DNA, the chromophoric group of actinomycin localizes between Base Pairs, while the peptide regions of the antibiotic molecule stabilize this complex by forming Hydrogen Bonds with the DNA strands. In this manner, actinomycins inhibit DNA-dependent nucleic acid synthesis.

Fig. 11.16. Structure of the actinomycin D molecule

In addition to direct interaction with DNA, synthesis inhibition can occur by targeting the enzymes responsible for it. For example, anthracyclines and glycopeptides are capable of inhibiting topoisomerases in tumor cells.

Ansamycin antibiotics are inhibitors of bacterial DNA-dependent RNA polymerase, which disrupts Messenger RNA synthesis.

The production of ansamycin compounds is known in plants, but it is most efficient in bacteria of the genera Streptomyces and Nocardia, which produce rifamycin antibiotics.

A structural feature of these antibiotics is the presence of an aliphatic lactam chain linked to a naphthalene or benzene ring (Fig. 11.17).

Fig. 11.17. Structure of the rifamycin molecule

The synthesis of Rifamycins is similar to that of macrolide antibiotics. Semisynthetic derivatives—such as ansamycin, rifamide, and rifampicin—have been developed from biosynthetic rifamycins through chemical modification. These are broad-spectrum antibiotics active against bacteria of the genera Mycobacterium, Staphylococcus, Streptococcus, Neisseria, Haemophilus, and Legionella.

Rifampicin is widely used as an antituberculosis agent, as well as against pathogens resistant to other antibiotics.

The extensive list of antibiotic agents representing various classes and Applications demonstrates the development of a powerful arsenal primarily aimed at treating infectious and oncological diseases. The potential of microorganisms as antibiotic producers has enabled the establishment of a biotechnological industry for the production of drugs

for chemotherapy. Due to their specific and selective action, antibiotics have become indispensable tools in academic research.

The widespread use of antibiotics for decades has not only yielded significant success in combating infections but has also created numerous problems. Aside from adverse effects (such as allergies, dysbiosis, nephro-, hepato-, and ototoxicity), a major challenge in their application is the development of resistance in both microbial and host cells.

Resistance is the ability of bacteria to withstand the action of antibiotics.

The Emergence of resistant forms is driven by the heterogeneity of natural microorganism and tumor cell populations—where antibiotic-sensitive and resistant cells coexist—alongside mutation-Selection processes. The primary mechanisms of resistance formation are divided into three levels.

At the level of protective enzymes, microorganisms inactivate antibiotic molecules before they reach their target. Known protective enzymes include β-lactamases, chloramphenicol Hydrolases, macrolide esterases, macrolide transferases, aminoglycoside transferases, and lincosamide transferases.

At the target level in macrolide-resistant bacteria, the conformation of the peptidyl transferase center is altered due to the methylation of a specific adenine residue in the 23S rRNA by a specific methylase.

When resistance to β-lactams develops in methicillin-resistant staphylococci or streptococci, the cells synthesize transpeptidase 2a (PBP 2a), which can substitute for the functions of all other transpeptidases (penicillin-binding proteins) while remaining insensitive to antibiotics.

In vancomycin-resistant enterococci, a change occurs in the peptidoglycan target—the pentapeptide is replaced by a depsipeptide terminating in D-lactate instead of D-Alanine. This prevents the antibiotic from recognizing its target.

At the level of transport systems, resistance develops through the active expulsion of the antibiotic from the cell, a phenomenon known as efflux.

The active efflux of antibiotics from Mycobacterium tuberculosis cells provides resistance alongside target-level resistance. A more efficient resistance mechanism is the efflux of tetracyclines by various microorganisms.

The resistance of eukaryotic cells to chemotherapeutic drugs correlates with the overexpression of P-glycoprotein and the MRP protein, both of which mediate efflux.

All mechanisms of resistance have genetic foundations. Enzymes that modify antibiotic targets, transport systems, and enzymes that

inactivate antibiotics are encoded by both chromosomal and, predominantly, plasmid genes. For example, the methylase that modifies the macrolide target is encoded by the erm Gene (Erythromycin Ribosome Methylation), vancomycin resistance by the van gene, macrolide glycosyltransferase as the product of the mgt gene, and macrolide esterase as the product of the plasmid ere gene.

Plasmid resistance genes are known as R-factors. Other Mobile Genetic Elements—such as Transposons and IS-elements—also contribute to the development of resistance. All of these can be transferred between cells of different species and genera of Gram-negative bacteria via conjugation, and in Gram-positive bacteria via Transduction, which accounts for a slower rate of transfer (Fig. 11.18).

Fig. 11.18. Genetic pathways of resistance development

Multidrug resistance refers to simultaneous resistance to multiple antibiotics belonging to different families. For instance, resistance to macrolides, lincosamides, and Streptogramins defines the MLS phenotype.

Cross-resistance is the acquisition of resistance to one antibiotic that confers resistance to other antibiotics within the same family.

Strategies to slow down the rise of Antibiotic Resistance include:

1. Excluding antibiotics from medical regimens as prophylactic agents.

2. Eliminating the repeated use of the same antibiotics in practice, with a return to older drugs after 10–15 years.

3. Modifying therapeutic regimens, increasing therapeutic doses of the antibiotic directly at the site of inflammation, and shortening the duration of treatment.

4. Establishment of Databases and control systems for the spread of resistant microbial strains.

Practical measures to combat resistance involve the development of potentiated formulations and the modification of antibiotics with a proven track record of clinical efficacy.

Potentiated forms of β-lactam antibiotics, developed by incorporating β-lactamase inhibitors—carbapenems and monobactams—into the formulations, have significantly enhanced the efficacy of β-lactam antibiotic therapy. Additionally, inhibitors of 23S rRNA methylase have been developed for inclusion in potentiated macrolide antibiotic formulations.

The development of semi-synthetic derivatives resistant to β-lactams has led to the introduction of novel penicillins, namely isoxazolylpenicillins. Dibekacin (bidesoxykanamycin) is a derivative that exhibits resistance to aminoglycoside phosphotransferases.

An example of developing derivatives that recognize modified targets is the class of glycylcyclines (semi-synthetic tetracyclines).

Inhibition of tetracycline efflux pumps is achieved using 13-cyclopentyl-5-hydroxytetracyclines.

The discovery and development of novel antibiotics remains a vital objective in chemotherapy. Traditional screening approaches are no longer sufficient to achieve this successfully. Modern drug discovery strategies rely heavily on advancements in Genomics. Research efforts begin not with the identification of antibiotics as inhibitory substances, but rather with their specific targets. Bacterial-specific genes and their expression products, which are absent in eukaryotes, serve as potential targets for the next generation of antimicrobial agents.