PHARMACEUTICAL MICROBIOLOGY - V. A. Galynkin - 2015

PART II. ANTIMICROBIAL AGENTS

CHAPTER 15. RESISTANCE OF MICROORGANISMS TO CHEMOTHERAPEUTIC AGENTS

15.1 Cellular and MOLECULAR MECHANISMS OF resistance

Resistance to chemotherapeutic agents (CTAs) is determined by a combination of the structural and metabolic characteristics of a microorganism. Natural (intrinsic) and acquired resistance are distinguished, with the latter being of paramount importance in the practice of antimicrobial therapy. Following the Introduction of a new CTA, there is typically a brief period during which the drug is highly and widely effective; however, an increasing number of resistant microorganisms subsequently emerge, causing the clinical value of the agent to drop sharply. For instance, shortly after the introduction of penicillin into clinical practice, only 8% of Staphylococcus aureus strains were resistant to it; today, this figure exceeds 75%. Resistant microbial populations generally emerge among pathogens responsible for hospital-acquired (nosocomial) infections. The rate atictions at which resistance develops and its underlying mechanisms depend on the specific microbial species.

The Mechanisms of microbial resistance to CTAs may be associated with the following factors:

1) presence of permeability barriers;

2) active efflux of the xenobiotic from The Cell;

3) presence of inactivating Enzymes;

4) absence or Modification of the target site.

15.1.1 Differences in cell membrane permeability

Differences in cell membrane permeability account for the varying susceptibility of Gram-positive and Gram-negative Bacteria to biocides. The latter are generally more resistant due to the protective function of a poorly permeable outer membrane. Membrane pores and their constituent Proteins, known as porins, allow the free diffusion of hydrophilic molecules with molecular weights up to 600-700 Da. Conversely, the diffusion of hydrophobic Antibiotics through these pores is restricted. The resistance of Pseudomonas aeruginosa even to hydrophilic biocides is attributed to the unique properties of its outer membrane, where lipopolysaccharides in the outer leaflet shield the pores.

Resistance may also result from impaired transport systems delivering the biocidal agent into the cell, or it can be mediated by specialized xenobiotic efflux systems. Such systems exist in mammalian Cells and have been identified in bacteria as specific pump proteins. These comprise either single cytoplasmic membrane transporter proteins or functionally linked groups of transporters, periplasmic proteins, and outer membrane porins.

A more complex system exists in Gram-negative bacteria due to the necessity of translocating compounds across the outer membrane. Membrane transport systems responsible for the efflux of antimicrobial compounds from the cell are driven in most cases by the energy of the transmembrane proton gradient and require ATP participation.

15.1.2 Enzymes inactivating antibiotics

Many microorganisms produce enzymes that specifically inactivate antibiotics by degrading or chemically modifying them. β-lactamases hydrolyze Penicillins and Cephalosporins (Fig. 24, 27). They are synthesized by numerous Gram-positive and Gram-negative bacteria. The enzyme can be constitutive (in Proteus spp., Pseudomonas aeruginosa, Klebsiella spp., Neisseria gonorrhoeae) or inducible (in Staphylococcus aureus, Serratia spp., etc.). β-lactamases of diverse origins vary significantly in molecular weight and Amino Acid Composition.

Certain penicillin and cephalosporin derivatives (see above) are resistant to β-lactamases [29]. Furthermore, resistance can be overcome by administering antibiotics in combination with β-lactamase inhibitors, such as clavulanic acid (Fig. 67).

Class="center">Fig. 67. Clavulanic acid.

Chloramphenicol is inactivated by resistant bacterial strains (Salmonella spp., Haemophilus influenzae, etc.) via Acetylation (Fig. 68). The acetyltransferase catalyzing this reaction is inducible in Gram-positive bacteria and constitutive in Gram-negative bacteria.

Fig. 68. Inactivation of chloramphenicol via two-step acetylation involving acetyltransferase.

Aminoglycoside antibiotics are inactivated by the acetylation of amino groups, as well as by the adenylylation or phosphorylation of specific hydroxyl groups. Most Aminoglycosides serve as substrates for more than one inactivating enzyme located in the periplasmic space or on the outer surface of the cytoplasmic membrane. The semisynthetic aminoglycoside amikacin is resistant to many, though not all, of these inactivating enzymes.

Target modification, which leads to a loss of susceptibility to an antibiotic, is determined by The Nature of the interacting structures. In E. coli, the substitution of just a single amino acid in a protein of the 30S ribosomal subunit renders the microorganism resistant to streptomycin. A similar resistance mechanism has been described in Streptococcus faecalis and Staphylococcus aureus.

Resistance to methicillin, cephalosporins, and Monobactams is associated with the presence of penicillin-binding proteins in microbial cells that also exhibit affinity for other β-lactams, which in turn induce their synthesis.

Erythromycin resistance is driven by modifications to the 50 S ribosomal subunit—specifically certain constituent proteins or 23 S rRNA—which lower the antibiotic's affinity for the ribosome.

Trimethoprim, frequently combined with the sulfonamide sulfamethoxazole in clinical practice, acts as an inhibitor of bacterial Dihydrofolate Reductase, a key enzyme in Folic acid METABOLISM. Resistant strains synthesize a form of dihydrofolate reductase that is insensitive to the antibiotic.

Trimethoprim resistance often co-occurs with sulfonamide resistance, the latter stemming from alterations in their molecular target: dihydropteroate synthase (DHPS). Furthermore, certain resistant microorganisms (such as Staphylococcus aureus) overproduce $p$-aminobenzoic acid, which competitively displaces the sulfonamide from the Active Site of DHPS.

Resistance to rifamycin is determined by modifications to its target—DNA-dependent RNA polymerase—whereas resistance to nalidixic acid results from alterations in DNA gyrase.

15.2 GENETIC BASIS OF Resistance

Genes conferring resistance to chemotherapeutic agents may form part of the bacterial chromosome or reside on Plasmids and Transposons. Plasmids carrying these genes are designated as R-factors (R-plasmids). They exhibit both intraspecies and interspecies transmissibility, meaning they are capable of Horizontal Gene Transfer, which spreads resistance across different genera, species, or strains of bacteria. R-plasmids confer multidrug resistance because they harbor genes encoding resistance factors against A wide variety of chemotherapeutic agents. Transposons, as Mobile Genetic Elements, facilitate the exchange of Genetic information between plasmids and the chromosome. The subcellular localization of resistance genes is in a state of constant flux: the more vital a gene is for cell viability, the less likely it is to persist extrachromosomally for extended periods. Moreover, the localization of resistance genes partly reflects their underlying biochemical mechanisms. For instance, pneumococcal resistance to sulfonamides is linked to a chromosomal mutation in the DHPS gene, and streptomycin resistance results from a mutation in a chromosomal gene encoding a specific ribosomal protein, whereas R-plasmids carry genes that direct the synthesis of antibiotic-inactivating enzymes (such as $eta$-lactamases, acetylases, phosphorylases, and adenylases).

Bacterial genomes are frequently mosaic in Structure, meaning they contain foreign DNA inserts of varying lengths. This mosaicism arises via horizontal gene transfer through genetic recombination (transformation, Transduction, or conjugation). Such mosaic patterns are most commonly found among genes encoding virulence factors and drug resistance, and are closely associated with gene cassettes. Antibiotic Resistance cassettes represent the smallest mobile genetic elements. They comprise a single gene alongside a specific sequence that Functions as a recombination site. These cassettes operate as discrete genetic units integrated into larger mobile structures known as integrons. Cassette genes typically lack their own promoter, relying instead on the integron's promoter for expression.

Resistance genes can persist stably even when unexpressed—that is, in the absence of selective pressure—as evidenced by the presence of biocide-resistant strains in natural environments devoid of chemotherapeutic agents. However, alternative selective factors cannot be ruled out, such as heavy metals, given that resistance genes may be integrated into gene cassettes (clusters) structured as operons that link resistance across multiple adverse factors. The persistence of resistance genes in nature is aided by their broad host range; their capacity for interaction is so pronounced that adaptive Selection must have occurred long before the human-made antibiotic era (for instance, within soil biocenoses containing antibiotic producers).

Alongside previously established pathways of horizontal gene transfer, a novel mechanism of gene transmission termed retransport has been described. In The First stage, the donor's transmissible plasmid enters the recipient cell and integrates with a plasmid mobilized by the donor; In the second stage, it returns to the donor cell enriched with novel genes. Mobilizable elements are not restricted to plasmids alone but can also include chromosomal segments excised through the action of conjugative transposons.

15.3 Strategies and Methods for Preventing Microbial Resistance to Chemotherapeutic Agents

The emergence and widespread dissemination of microbial resistance necessitate an ongoing search for novel antimicrobial agents. Intensive research conducted since the dawn of the antibiotic era has shown that screening for new producers frequently leads only to the rediscovery of "old" antibiotics. Consequently, contemporary research is branching into several innovative directions:

1) developing agents that target sites altered by prior exposure to "old" antibiotics;

2) designing inhibitors against enzymes that inactivate antibiotics;

3) identifying substances that suppress the active efflux of antibiotics from pathogen cells;

4) targeting novel structures, such as enzymes involved in protein and peptidoglycan synthesis;

5) employing cutting-edge "in vivo Gene Expression technology" (IVCET) screening techniques targeting microbial processes that occur exclusively within the host Organism rather than in vitro, such as the Synthesis of specific Polysaccharides, adhesion factors, and pathogenicity enzymes. To identify the genes driving these processes, RNA transcripts generated in vivo are compared with those produced in vitro. Total DNA is synthesized using Reverse Transcriptase and analyzed via Hybridization against a gene library of the given microorganism. The ultimate goal of this screening is to identify novel genes and uncover ways to inhibit their expression. The most promising targets—which lack eukaryotic homologs—are virulence factors and The regulatory mechanisms governing their expression. These regulatory pathways encompass environmental sensing (reception), signal transduction from the receptor to The Genome, and the subsequent Synthesis of the relevant virulence factor.

Equally promising are developments focused on therapeutics that block adhesion, secretion systems, and transcriptional regulatory pathways. One universal regulatory mechanism of the latter is quorum sensing in microorganisms. Its core principle involves the transcriptional activation of specific genes once a threshold binding level between a Transcription-activating protein and a low-molecular-weight autoinducer is reached. This mechanism facilitates rapid culture growth at high inoculation densities and plays a key role in the expression of virulence factors. Inhibitors of virulence determinants are expected to exhibit low in vitro activity and—crucially—will spare microorganisms lacking virulence factors, thereby preserving the normal microbiota.

As alternative chemotherapeutic agents, researchers propose utilizing fragments of innate immune protein factors that are species-specific and therefore non-immunogenic. One such candidate is a recombinant fragment of a human neutrophil protein capable of neutralizing Gram-negative bacterial endotoxin while exhibiting intrinsic antimicrobial activity.

The quest for novel antimicrobial agents involves characterizing not only the genome but also the proteome—the complete Complement of cellular proteins—to determine the cell's physiological state at any given moment. Proteomics characterizes the cellular phenotype across various Cell Cycle stages and environmental conditions, thereby providing the foundational insights required for target-based screening aimed at specific molecular sites.